Advances in Insect Physiology, Volume 05

Advances in

INSECT PHYSIOLOGY

VOLUME 5

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Advances in

Insect Physiology Edited by J . W. L. BEAMENT, J. E. TREHERNE and V. B. WIGGLESWORTH Depurttnetit

of Zoology, The University, Cnmbridse, England

VOLUME 5

1968 ACADEMIC PRESS London and New York

ACADEMIC PRESS INC. (LONDON) LTD

24-28

OVAL ROAD

LONDON NWI

I/ S. Edition published by ACADEMIC PRESS INC.

11 1

FIFTH AVENUE

NEW YORK. NEW YORK

Copyright

10003

8 1968 By Academic Press Inc. (London) Ltd Second printing 1974

All Rights Resewed NO PART OF THIS

nom

MAY RE REPRODUCED IN A N Y FORM, ny

PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS

Library of Congress Catalog Card Number: 63-14039 SBN 12-024205-2

Printed in Great Britain by Unwin Brothers Limited The Gresham Press, Old Woking, Surrey A member of the Staples Printing Group

List of Contributors to Volume 5 J . BOISTEL,Laboratoire de Physiologie Animale, Facult6 des Sciences de Rennes, France (p. 1) H. E. HINTON,Department of Zoology, University of Bristol, England (p. 65) J. W. S. PRINGLE, Department of Zoology, Unicersity of Oxford, England (P. 163) G . P. WALDBAUER, Department of Entomology, Unicersity of Ilhois, Urbana, Illinois, U.S.A. (p. 229) DONALD M. WILSON,Department of Biological Sciences, Stanford Unirersity, Stanford, California, U.S.A. (p. 289)

Acknowledgements The Editors wish to thank the publishers of Z . vergl. Physiol., J. Exp. Biol., Zoo1 Jb., Science, N . Y . , Philosophical Transactions of The Royal Society and Cambridge University Press for their kind permission to reproduce figures from their publications. The authors in this volume have been instructed to obtain necessary permissions for use of published figures and the Editors sincerely regret the omission in case any of the sources were overlooked. J. W. L. Beament J. E. Treherne V. B. Wigglesworth

Cot i tent s LISTOF CONTRIBUTORS TO VOLUME 5

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THESYNAPTIC TRANSMISSION A N D RELATEDPHENOMENA I N INSECTS J . ROISTEL

I. Introduction

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. 11. Some Features of the Organization of Insect Ganglia A. The Structure of Synaptic Regions . . B. Acetylcholine and Acetycholinesterase Content of Ganglia . . C. Some General Considerations . . III. The Electrical Activity of Ganglia . . A. Description of Phenomena . B. Physical Factors which Modify the Rhythmical Activities of Ganglia . C. Effects of Various Chemical and Pharmacological Substances . . IV. Properties of Synaptic Transmission A. General Characteristics of the Responses . B. Synaptic Delay . C. Refractory Period . . D. After-Discharge . E. Effects of Repetitive Stimulation . F. The Mechanism of Synaptic Transmission . G. Inhibitory Processes . V. General Conclusions . References .

1 2 2 7 9 11

II 18 21 28 28 33 33 35 36 38 52 54 57

SPIRACULAR GILLS H . E. HINTON

Introduction . The Plastron and the Environment. . Definition of Stages during Metamorphosis . Interrelation of Pupal and Adult Respiratory Systems Polyphyletic Origin of Spiracular Gills . A. Origin from Spiracles . B. Origin from Respiratory Horns . VI. Isolation of Tissue in Spiracular Gills . A. Origin of Isolated Tissue. . B. Significance of Differences in Time of Isolation of Gill Tissue C. Attributes of Isolated Tissue . D. Function of Isolated Tissue . E. Tissue Reservoirs . . VII. The Plastron . A . Structure . vii

I. 11. 111. IV. V.

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65 66 68 71 74 82 84 84 84 90 92 97 101

104 104

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CONTENTS

Vlll

105 112 114 I I4 120 122 123 133 141 1 44 146

B. Respiratory Efficiency . VIII. Resistance of Gill to High Pressures . IX. Spiracular Gills of Pupae A. Psephenidae . B. Torridincolidae . C. Tanyderidae . D. Tipulidae . E. Simuliidae . F. Blepharoceridae . G. Deuterophlebiidae . . H. Empididae . 1. Dolichopodidae . J. Canaceidae . . X. Spiracular Gills of Larvae A . Torridincolidae . B. Sphaeriidae and Hydroscaphidae References . COMPARATIVE PHYSIOLOGY OF THE J . W. S. PRINGLE

148 152 156 156

158 159

FLIGHT MOTOR

1. Introduction . I[. The Generation of Lift and Thrust. . A. General . B. The Flight of Coleoptera . C. Gliding Flight of Lepidoptera . . D. The Flight of Small Diptera . 111. The Kinematics of Wing Motion . A. Diptera. . B. Apis Mellifkra . IV. Stability in Flight. . A. Diptera. . B. Other Insects. . V. The Motor Mechanism of Flight Reflexes A . List of Reflexes . B. Initiation, Maintenance and Termination of Flight C. Control of Amplitude, Frequency and Power . D. Control of Velocity . E. Control of Lift . F. Control of Attitude . VI. Comparative Studies . A. Axioms. . B. Differentiation of the Flight Muscles . References .

THECONSUMPTION A N D UTILIZATION OF FOODB Y

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164 164 166 171 173 179 179 186 190

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190 195 198 198 199 200 206 209 21 1 217 217 219 223

~NSECTS

G . P. WALDBAUER

I. Introduction

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11. Consumption, Growth and Utilization Indices.

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229 23 I

ix

CONTENTS

A. Consumption and Growth . B. Digestibility and Efficiency of Conversion. . 111. Measuring Consumption and Utilization by Weight A. General Considerations . . B. The Gravimetric Method . C. Indirect Methods using Markers . IV. Food Consumption . V. Digestion and Conversion of Fresh and Dry Matter. A. Limitations of the Data . B. Comparison of Species . C. Comparison of Foods . D. Effects of Environmental Factors . E. Variations with Age and Sex . 'VI. Utilization of Dietary Constituents. . V I I . Utilization of Energy . Acknowledgement. . References .

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232 23 3 236 236 238 242 246 250 250 263 264 265 267 272 278 282 282

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THENERVOUS CONTROL OF INSECT FLIGHT AND RELATEDBEHAVIOUR DONALD M . WILSON

289 290 294 296 300 301 309 309 309 314 315 317 318 318 322 33 1 334

I. Introduction . It. Kinematics and Aerodynamics

. A. Wingbeat Frequency and Size .

111. Neurogenic Flyers

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A. Locusts. B. Dragonflies . C. Lepidopterans . IV. Myogenic Flyers . A. Motor Patterns . B. Significance of the Multiphasic and Multistable Patterns C. Hypothesis on Coordination in Flies . V. General Model for Flight Control . VI. Related Behaviour . A. Temperature and Flight . . B. Sound Production using the Wings . VII. General Discussion . References .

AUTHOR INDEX

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CUMULATIVE LISTOF AUTHORS .

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The Synaptic Transmission and Related Phenomena in Insects J. BOISTEL Laboratoire de Physiologie Animale, FncultP des Sciences de Rennes, France I. Introduction 11. Some Features of the Organization of Insect Ganglia . A. The structure of the synaptic regions . B. Acetylcholine and acetylcholinesteras- content of ganglia . C. Some general considerations . 111. The Electrical Activity of Ganglia . A. Description of phenomena . B. Physical factors which modify the rhythmical activities of ganglia C. Effects of various chemical and pharmacological substances . . IV. Properties of Synaptic Transmission A . General characteristics of the responses . B. Synaptic delay . C. Refractory pariod . D. After-discharge . E. Effects of repetitive stimulation . F. The mechanism of synaptic transmission . G. Inhibitory processes . V. General Conclusions . References .

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21 28 28 33 33 35 36 38 52 54 57

I. INTRODUCTION Cajal(1934), in the later years of the last century, developed a theory that, contrary to the general belief at that time, the neurones of the central nervous system are connected by zones of contiguity without cytoplasmic continuity; this provided the structural basis for the experimental studies of Sherrington (1897), who gave the name of “synapses” to these regions of neuronal contact. A synapse may be defined as the point at which the membrane of one neurone (the presynaptic neurone) comes into such close proximity to the membrane of a second (postsynaptic neurone), whether this be on the cell body, the axon or a dendrite, that the electrical activity, whether propagated or not, developing at the level of the presynaptic neurone, induces in the postsynaptic neurone either 1

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an increase or a decrease in its excitability or a propagated response along its axon. An essential characteristic of a synapse is the constant polarity of this transmission. The physiological study of the synapses involves combined researches in the fields of microanatomy, pharmacology, histochemistry and electrophysiology. Much information exists about these different aspects in vertebrates, in crustacea and in molluscs. Using these data as a basis for comparison, the present review will deal with the electrical phenomena in insect ganglia and particularly with synaptic transmission. Such a study has been made possible by the detailed information already available about the physiology of insect nerve fibres, as reviewed by Narahashi (1963). Information also exists concerning a number of pathways in the ganglionic nerve cord. The main notions concerning the microscopic and ultramicroscopic structure of the synaptic regions will first be recalled; an excellent survey of these matters is given by Smith and Treherne (1963) in an earlier volume of this series. Then follows a brief account of the identification, localization and quantities of the main chemical substances involved. All the electrical phenomena occurring in the ganglia will then be reviewed in detail, together with the physical and chemical factors by which they are influenced. Finally, the ideas that emerge from these different sections will be turned to account in order to explain the mechanism of synaptic transmission and to decide what factors may be responsible for triggering the impulses at the level of the postsynaptic membranes. 11. S O M E F E A T U R EOSF T H E O R G A N I Z A T I O N OF I N S E C T

GANGLIA A. T H E S T R U C T U R E OF S Y N A . P l ' I C R E G I O N S

A ganglion of an insect is surrounded by a fibrous sheath, the neural lamella, under which is situated an epithelial layer, the perineurium (cf. Wigglesworth (1960); Smith and Treherne (1963)). Beneath the perineurium are the pear-shaped cell bodies of the motor and internunciary neurones, together with the glial cells and their cytoplasmic processes. The axons of these cells send their extensions into the central region of the ganglion, the neuropile. As was shown by Cajal and Sanchez (1915), the neurones of insects have a single process which can be divided into three parts : a proximal intermediary segment situated in the cortical layer of the ganglion; a middle portion which gives off

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dendritic extensions; and a distal segment similar to an axon. Thus by analogy with other classes of animals the following types of synapses could conceivably exist : soma-somatic, axo-somatic, axo-dendritic and axo-axonic contacts. The existence of soma-somatic interneural junctions has recently been postulated in the corpora pedunculata of the wood ant (Formica lugubris, Zett.) (Landolt and Ris, 1966). These soma are separated by ultrathin glial sheaths which are occasionally interrupted by circular holes with an average surface area of 2.64 p2.These glial windows could represent regions of synaptic contact, although there were no accumulation of vesicles in the cytoplasm associated with these soma-somatic junctions. According to the work of Leghissa (1942), axo-somatic synapses exist in the nervous system of Carausius, where some fibres end as knobs on the nerve cell bodies. Such an arrangement has, however, not been described in the cockroach. In particular Hess (1958a) has shown that in this insect a network of glial processes embeds the nerve cells so that contact between the presynaptic endings and the cell bodies is precluded. I t follows from this that the regions of synaptic contacts are likely to be confined to the neuropile (Trujillo-Cenoz, 1962). Within the neuropile there are no glyocytic cell bodies and there are only tenuous glial branches present between nerve fibres, especially those of small diameter (Trujillo-Cenoz, 1962; Smith and Treherne, 1963). The possibility cannot be excluded that in some places these glial extensions may be completely absent, thus allowing adjacent axon profiles to come into very close apposition (Smith and Treherne, 1963) (Fig. I). On the other hand Hess (1958a) has shown that the larger axons are frequently invested with a narrow glial sheath. The following types of synaptic connections have been observed within the neuropile of insect ganglia: (a) Contacts exist between fibres where the end of one of them (probably the presynaptic one) seems to push into the other, as was described by Hess (1958a) in the case of the sixth abdominal ganglion of the cockroach (Fig. 2). Hess (1958a) has also shown that longitudinal synaptic contacts exist between fibres, one of them projecting into the other one. This aspect has been seen in the case of the ganglia of Peripluneta americana. Similar results have been obtained by Trujillo-Cenoz (1959) with the abdominal ganglia of Pholus labruscoe. (b) Cross-contacts between nerve fibres have been demonstrated in

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Periplanetu and Pholus ganglia (Hess, 1958a ; Trujillo-Cenoz, 1959). In some cases, Trujillo-Cen6z (1962) has described a deep groove in the hrgest fibre in which the other one is set, so increasing the contact surface. There is, however, no cytoplasmic continuity.

FIG. I. Electron micrograph showing two axons profiles (axl, axz) within the neuropile in a ganglion of Periplanrtu. Their membranes are separated by an intercellular gap of about 100 A. In axl large numbers of synaptic vesicles are concentrated along the inner surface of the membrane which shows a higher density than elsewhere in the axoplasm; these regions are “mirrored” in axz by foci of increased density (arrows). It is likely that these compleotes represent sites of synaptic contact. x 76,000.(From Smith and Treherne, 1963).

(c) Contacts by terminal knobs, in which one fibre (the supposed presynaptic one) protrudes in the other one (generally of smaller diameter), have been described in Curausius and Pholus (Leghissa, 1942; Trujillo-Cenoz, 1959). Furthermore, the same knob can make contact with several postsynaptic fibres. On the other hand

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Hess (1958a) has shown that a single fibre can be connected with synaptic knobs from several nerve fibres (Fig. 3). From these anatomical features, and also from those, essentially similar, obtained by Osborne (1966) for synapses in the brain neuropile of the blowfly, Phormia terrae-nouue, it can be seen that insect synapses and those of invertebrates generally are mainly of the axo-axonic type, whereas in vertebrates they are the most frequently either axo-somatic

FIG.2. Electron micrograph showing two nerve fibres in the neuropile of an abdominal ganglion of Peripluneta; one of them (A) is indenting another one (8) x 24,000. (From Hess, 1958a).

or axo-dendritic (Cajal, I934), although axo-axonic synapses have been described by Beccari (1920). In the regions of synaptic contacts described above, the electronmicroscope has shown that synaptic vesicles are present together with many mitochondria. The diameters of these vesicles are between 200 and 600 8, in the case of fholus (Trujillo-Cenoz, 1959, 1962) and between 300 and 1 l O O A for feripluneta (Smith and Treherne, 1963). These vesicles seem to be localized in the preterminal and the terminal

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nerve fibres and also occur along the length of the latter one. Landolt and Sandri ( I 966) have pointed out such vesicles in the corpora pedunculata of the wood ant (Formica luguhris, Zett.): in most of synaptic knobs there is a multitude of small vesicles (300-600 8, diameter); in some others one finds few larger vesicles (700-1000 A).

FIG.3. Electron micrograph of a portion of the sixth abdominal ganglion of Periplunetu showing several terminations of small fibres, indicated by arrows, containing granules pushing into a nerve fibre. x 3650. (From Hess, 1958a).

Electron-microscope investigation has shown that in some cases the synaptic vesicles are present in only one of the two fibres of the supposed synaptic region, the axon containing the vesicles being larger than the postsynaptic one (Smith and Treherne, 1963; Smith, 1965) (Fig. 1). However, in other cases apparent synaptic vesicles are found in both

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fibres in some regions of contact (Smith and Treherne, 1963). Similar results have been obtained by Osborne (1 966) in the brain neuropile of a blowfly larva, Phormia terrae-novae. The above vesicles are essentially similar to those which have been described in other animal species. In the earthworm, De Robertis and Bennett ( 1955) have shown that their diameters were between 200 and 400 A, while in the central nervous system of vertebrates, many authors have described oval vesicles whose diameters lie between 200 and 650 8, (these vesicles seem to be situated only in the synaptic knobs). It is generally assumed that these vesicles contain molecules of the transmitter substance, most probably acetylcholine. Electron-microscope pictures have also shown a few vesicles apparently discharging their contents into the synaptic cleft (see Eccles, 1964). The essential similarity between the conditions in these animals and in insects suggests that a similar chemical basis for synaptic transmission must be considered in the latter group. It should be emphasized that in the regions of synaptic contacts the plasma membranes are separated by a cleft of 100 to 200 8, in the case of the cockroach (Smith and Treherne, 1963), approximately 130 8, in the wood-ant (Landolt and Sandri, 1966). It might be supposed (see Eccles, 1964) that a cleft of such dimensions, which is also found in vertebrates (Palay, 1958; De Robertis, 1958), might indicate the existence of chemical transmission mechanism. With electrical synapses, on the other hand (for example the giant synapses of the abdominal ganglion of the crayfish), the interval between the fibre membranes seems to lie between a negligible value and 150 A, with an average value of only 60 8, (De Lorenzo, 1959; Robertson, 1961). Examples of such narrow cleft between pre- and postsynaptic membranes have also been pointed out in insects: in soma-somatic interneuronal junctions of the corpus pedunculatum of the wood ant, Formica lugubris, Zett. (Landolt and Ris, 1966); in the central nervous system of the blowfly larva, fhormia terrae-noaae (Osborne, 1966),where “tight junctions” between axons have been found. B. ACETYLCHOLINE A N D ACETYLCHOLINESTERASE CONTENT 9 F GANGLIA

t. Acetylcholine (ACh.).This substance has been identified in many insect species. I t occurs in various parts of the body but is mainly concentrated in the nervous system. Augustinson and Graham (1954) have shown that bees’ heads contain an ester having thechemicaland pharmacological properties of the ACh. I n the cockroach, ACh. and also other

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elements of the cholinergic system seem to be confined to the nervous tissue (Colhoun, 1958a). Mikalonis and Brown (1941) have also demonstrated ACh. in the eserinated nerve cord of Periplaneta americana. Tobias, Kollros and Savit (1946) have in addition shown that this substance is synthetized in extracts of the cockroach nerve cord and that, moreover, the ACh. of the nervous tissue varies appreciably after different treatments. These authors demonstrated, for example, that the ACh. content increased following the application of DDT to the nervous tissue of Periplaneta americana. According to Colhoun (1958a) the ACh. content of the thoracic and abdominal ganglia of Periplanera americana varies as a function of the acclimatization temperature, while a treatment with COz brings about an appreciable loss of ACh. In Periplaneta again, Chang and Kearns (1955) have shown that ACh. was the only choline ester present in appreciable amounts, an observation which has been confirmed by Colhoun and Spencer (1959). The ACh. content of the cockroach nerve cord has been estimated by several authors. For example Roeder (1948~)found a concentration of 32 pg/g while Colhoun (1958a) has estimated that the sixth abdominal ganglion of the cockroach contains 63 pg/g. These values are much higher than those obtained with other animal species. Thus, for example, a snail brain contains between 1 and 5 pg ACh./g wet weight (Kerkut and Cottrell, 1963) as compared with 58 pg/g for cockroach ganglia (Tobias et al., 1946), while in the nervous tissue of vertebrates there is from 5 to 50 times less ACh. than in insect nervous tissue. 2. Acetylcholinesterase ( AChE.).This enzyme has been studied by biochemical methods in several insect species (cf. Chadwick, 1963; Treherne, 1966), while some notable histochemical investigations have been carried out by Wigglesworth (1958) in Rhodnius and by Iyatomi and Kanehisa (1958) in Periplaneta. In addition Smith and Treherne ( I 965) have studied the localization of AChE. in the neuropile of Periplaneta using the electron-microscope. The reactive zones of acetylcholinesterase activity, demonstrated after treatment with thiolacetic acid, were associated with closely apposed axon membranes, in situations where intercalated glial processes were absent. In some cases, synaptic vesicles were shown to be localized along the membrane of an axon, the adjacent axon or axons containing few or no vesicles (Fig. 4). In these regions the restricted areas of esterase activity were frequently associated with foci of synaptic vesicles which suggests that these do represent synaptic zones. The reaction to AChE. was abolished in the presence of anticholinesterases, such as eserine. These results, and also similar ones obtained with the same techniques by Landolt and Sandri

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(1966) in the corpora pedunculata of the wood ant (Formica lugubris, Zett.), are in essential agreement with those obtained for vertebrate synapses. The precise mechanism by which the molecules of ACh. are discharged into the postsynaptic membrane is obscure. I t seems clear, however, that activation of the presynaptic neurone causes the rupture of synaptic vesicles, so as to release ACh. molecules into spaces between the pre- and postsynaptic membranes.

FIG.4. Electron micrograph of a region within the neuropile of Periplanefa ganglion showing the focal accumulation of synaptic vesicles (SV) in the axoplasm of one neurone (N) alongside localized regions of the axon membranes exhibiting cholinesterase activity (1-5); the presumed postsynaptic fibre (PO) contains no synaptic vesicles. x 61,700. The inset represents an enlarged part of region 3. The arrow shows a narrow gap between pre- and postsynaptic membranes. x 100,OOO. (From Smith and Treherne, 1965).

C. S O M E G E N E R A L C O N S I D E R A T I O N S

This survey of the ultrastructure of the supposed synaptic zones of the neuropile serves to emphasize the similarities which exist between these structures and the equivalent ones in other invertebrate and vertebrate central nervous tissues. From these studies, it is possible to erect

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several criteria which should be fulfilled in order to conclusively identify a cholinergic synapse. These criteria may be listed as follows: (a) That definite spaces exist between' closely applied adjacent axon membranes. (b) That glial cells or their processes must be absent from these regions. (c) That characteristic vesicles should be situated in the terminal axoplasm of one member of an axon pair. (d) That ACh. must be present in the appropriate region of the neuropile. (e) That AChE. must be strategically placed (and available for release) into the synaptic cleft. From the available evidence it would appear that these conditions are fulfilled in the case of ganglia of the cockroach nerve cord. The electrophysiological properties of this preparation will be considered in the remainder of this article. Essentially two synaptic systems have been studied, using fine electrophysiological techniques, in two species of cockroaches. One system is situated in the sixth abdominal ganglion, the other in the third thoracic ganglion of Periplunetu and have been studied by Callec and Boistel (l965a, b; 1966a, b), and by Rowe (l963), respectively. The choice of these preparations has been justified because their anatomy and a number of their neuronal circuits are relatively well known, although the extremely small dimensions of the nervous structures make their microelectrophysical study rather difficult. The synaptic system of the sixth abdominal ganglion has been described by Roeder (1948a), who has shown that many sensory fibres originating in the cercus receptors terminate in the ganglion (Fig. 5 ) . These fibres are coiinected by synapses with a few giant fibres which pass along the connectives of fhe nerve cord (first system). According to Roeder et a/. (1947) and Roeder (1948a), the giant fibres have a multicellular origin, the cell bodies being arranged in clusters of three to six towards the periphery of the ganglion close to the point of entry of the cercal nerves. The very tenuous axons penetrate into the neuropile and then point at right angles and continue to form the giant fibres. It is on this pathway that these axons make synaptic connection with !he endings of the cercal fibres. As there are many cercal fibres and very few giant fibres, several cercal fibres are probably connected with the same giant fibre. It is not clear from the anatomical results whether these synaptic contacts occur on the individual axons before they merge

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or whether connection is made on the fused portion which gives rise to the giant fibre (Fig. 5). This point is of some importance in the interpretation of synaptic processes (see pp. 17 and 18). In the third thoracic ganglion (the metathoracic) the above giant fibres make synaptic connections with motor neurones whose fibres supply the leg muscles (Roeder, 1948a, 1953; Hess, 1958b) (second system).

FIG.5. Diagram of the "evasion reflex arc" of feriplrtneru. Abdominal ganglia 1-5 have been omitted. Ascending giant fibres (only one is shown, G.F.) are synaptically connected on one side (sixth abdominal ganglion. A6) with cercal nerve fibres (Cer.) and on the other one (third thoracic ganglion. T3) with motor fibres M.F.). (From Roeder, 1953).

Thus it can be seen that the cercal fibres, the giant fibres and the motor axons make up the neurone chain responsible for the evasion reflex of the cockroach which can, for example, be produced by a brief puff of air on the cercus (Roeder, I948a). 111. T H E E L E C T R I C A LA C T I V I T YO F G A N G L I A A. D E S C R I P T I O N O F P H E N O M E N A

The electrical activity in nerve cord preparations has been studied in several species of insects during the past 30 years also, In 1930 Adrian recorded asynchronous discharges of action potentials in the isolated

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nerve cord of a caterpillar and of a water-beetle (Dytiscus) (Adrian, 1931). He suggested that these discharges originated in respiratory centres. A similar activity was demonstrated in the isolated nerve cord of the cockroach by Roeder and Roeder (1939) and Roeder (1948b). This activity was found to last for as long as 40 to 50 h r in a physiological solution containing glucose. Records were not obtained from isolated ganglia and, as no action potentials were observed in isolated connectives, Roeder suggested that the activity originates either from the cell bodies or from the synaptic regions, being a reflection of fluctuations in the excitability of these structures. Roeder, Tozian and Weiant (1960) have recorded the electrical activity of the phallus nerve in the Mantis when all the other nerves reaching the last abdominal ganglion had been severed, to eliminate afferent impulses. In these experiments the connectives joining this ganglion to the other parts of the cord were left temporarily intact. Under these conditions rhythmical impulses were observed in different fibres of the phallus nerve. When the activity of this nerve had become steady the connectives were cut just above the ganglion. After a transient increase in activity, due to injury, electrical activity occurred which showed that fibres which had previously been at rest started to fire (Fig. 6). It seems reasonable to suppose that section eliminated centres located at the cephalic level and which inhibited the last abdominal ganglion. Similar conclusions have also been obtained by the same authors for the last abdominal ganglion of Peripluneta when recording activity in the phallic nerve. The isolation of this ganglion by section of the connectives also induces an increased activity, mainly in the phallic nerve. These different results agree with the concept of intrinsic activity of ganglia. They also give information about the inhibitory influence of the cephalic centres. This latter point will be dealt with later in connection with synaptic transmission phenomena. Mill (1963), using metallic microelectrodes of about 0.5 p tip diameter, has observed regular discharges, which are probably spontaneous, in the eighth abdominal ganglion of A ~ s c h n alarvae, at a depth of between 50 and 200 p. The activities of several units could be simultaneously recorded, each of which showed a definite frequency (between 7 and 25 impulses/sec). The corresponding region was located in the “anterior dorsal transnerve tract”, which contained axons from motor cell bodies located between the first and second nerve roots. A spontaneous electrical activity was also recorded in a more central region, close to the posterior dorsal transnerve tract, in which the axons originate from groups of ventral and lateral motor cell bodies. Some bursts

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of spontaneous impulses were associated with, but not elicited by, respiratory movements. Using extracellular electrodes, Fielden and Hughes (1962) have recorded unitary activities from small bundles of the connectives, situated between the sixth and seventh abdominal ganglia of the dragonfly nymph. They considered that it was only possible to observe authentic spontaneous activity when the nervous system was completely isolated from the animal, for the peripheral receptors were shown to be capable of spontaneous discharges which also induced

Fici. 6. Efferent impulses recorded from phallic nerve or Nerve

X cf male

Manfis

wligiosn. The nerve has been cut distal to the electrodes and also all nerves arising from the

last abdominal ganglion. In (b) and (c), respectively 3 and 7 min after transection of the abdominal connectives, the electrical activity is higher than in (a), before transection. Time marker: l00c/sec. (From Roeder c f d.:1960).

such activity. In these conditions, interneurones exhibited a spontaneous activity which occurred in many cases as irregular discharges. Moreover, Fielden (1963) has shown that afferent volleys can produce either excitation or inhibition in interneurones with a background discharge. “Silent units” usually responded by repetitive discharges following a single nerve volley. According to Hunt and Kuno (1959) this may be an important factor in prolonging activities in chains of interneurones. Similar results. obtained by Svidersky ( 1967) from single

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neurones of the metathoracic ganglion in Locustu inigrutoriu migratorioides, suggest that the spontaneous activity of a region of this ganglion may be concerned in t h e maintenance of a certain background of nerve impulses involved in the mechanism of the flight control system. According to Roeder (1953), such a background could maintain some degree of tonus under conditions approaching zero sensory input. Essentially similar spontaneous activities were recorded using similar

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F I G . 7. Electrical activities obtained with an intracellular microelectrode in the sixth abdominal ganglion of feripluneio a/nericunu. A : A series of action potentials from a nerve cell body. B: One action potential which had the same origin as in A, but was recorded at a slower sweep speed. One can see clearly the wave of depolarization which precedes it. C: Rhythmical response of a nerve fibre situated inside the neuropile. (From Callec and Boistel. 1966a).

techniques in Crustacea as, for example, in the abdominal ganglia of the crayfish (Prosser, 1934; Hichar, 1960a). Repetitive and frequently rhythmical discharges, of amplitudes up to 30 mV, have been recorded from the third thoracic ganglion (Rowe, 1963) and the sixth abdominal ganglion (Callec and Boistel, 1965b) of the cockroach using intracellular capillary micro-electrodes. The direction of potential variation meant that records were obtained either as a result of direct penetration of the neurones or as a result of their injury

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(in this case the electrode tip would be in close contact with the membrane, thus creating an injury zone which would give access to the inner part of the neurone). This type of record was obtained from a depth of about 50 p (Callec and Boistel, 1965b). The low amplitude and the frequent fugacity of these responses, probably indicating some degree of injury, have led Callec and Boistel (l966a) to use microelectrodes of smaller diameter (0.1 p*). In these conditions they obtained repetitive responses of higher amplitude (about 100 mV) (Figs 7 and 8). There are three possible origins for these responses. Some of them, which were

FIG.8. Other types of electrical activities recorded in the same conditions as in Fig. 7. A : Anarchical action potentials from a postsynaptic fibre. B: Oscillations of potential recorded in a postsynaptic element. C: Rhythmical activity of a postsynaptic fibre. (From Callec and Boistel. 1966a).

nearly rhythmical and which were preceded by a slow wave of depolarization whose duration could reach 100 msec, could have originated in cell bodies situated in the ventro-median region of the ganglion at a depth between 0 and 50 p (Fig. 7A, B). Others, which were recorded at a depth of about 120 and 250 p, could have arisen from postsynaptic fibres in the neighbourhood of a synaptic zone. I n the latter case the action potentials were not always rhythmical but occurred randomly at the crests of the waves of depolarization only when the excitability * This (I965).

diameter was measured with an electron microscope by Boisseau and Boistel

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threshold was reached (Fig. 8A, C). These waves (Fig. 9B) were probably produced by impulses originated in presynaptic fibres. Rhythmical responses were also obtained from axom under conditions when the tip of the microelectrode was probably far from the synapses (Fig. 7C). The above supposition is supported by the observation that no appreciable previous waves of depolarization were observed in the latter case. In these different situations the amplitudes of the action potentials remained steady for periods of approximately 1 hr. This would indicate that the recorded activities were not caused by injury resulting from damage caused to the neurones by the microelectrodes. In these experiments a marking technique (see p. 47) was used to localize the position of the microelectrode tips using histological methods. These different types of responses should be compared to those obtained by Arvanitaki and Chalazonitis (l955), Tauc (1960) and Chalazonitis (1963) in the giant ganglion cells of Aplysia and by Preston and Kennedy (1960) in the caudal ganglion of the crayfish. The question again arises as to the origin of the activity outlined above. Does it, for example, arise in the cell itself and if so what is its rale? Is it, on the other hand, associated with a particular excitable region of the axon, or is it possible that afferent connections still exist? This last possibility seems to be excluded, at least in the preparations described by Callec and Boistel, for all the afferent nerves to the ganglion were cut, although the connections to other ganglia still remained. I n addition it was found to be impossible to stimulate, either antidromically or synaptically, cells penetrated by microelectrodes, although these cells could be excited directly (Callec and Boistel, 1966a). Injection of 2 x ACh. in the vicinity of a cell with a microelectrode produced either rhythmical impulses in a cell previously silent or an increased activity in a cell initially firing (Callec and Boistel, 1967), each action potential being induced by a depolarizing wave. The electrical activity could be stopped by more massive injection of ACh., most probably as a result of an excessive depolarization either of the soma itself or of the adjacent sensitive regions (Fig. 9). These results are similar to those ones obtained by Tauc and Gerschenfeld (1960) for the type D neurone of Apljvsia and by Kandel and Tauc (1966) for giant cells of Helix. In the case of Aplysia, solutions of ACh. at a concentration lower than were still effective. In the experiments carried out with the cockroach it is difficult to say whether the active zone was always the cell body membrane, although the ACh. injection experiments suggest that this is likely on account of the close position of the ACh. microelectrode to the cells. It could also

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be a region close to it, for example the synaptic one, from which the activity could propagate to the soma and also to the giant fibre, the slow wave of depolarization above mentioned being electrotonically transmitted. It is relevant in this connection to recall the interpretation of Tauc and Hughes concerning the giant neurones of Aplysiu. By stimulating with microelectrodes at difierent positions in a neurone T a w (1962a) has shown that the soma is the least excitable part of the

FIG.9. Same preparation as in Fig. 7. Changes in electrical activities of two nervc cell bodies intracellularly recorded, elicited by an injection of ACh. in the immediate vicinity of these cells. A: A cell body, localized in R, and previously silent, shows a transient elecACh. B: Increased electrical activity trical activity immediately after the injection of of a cell, situated in RP,induced by 10 ACh. In both cases it must be noticed that a slight depoiarization immediately follows the injection of ACh. (From Callec and Boistel, 1967).

cell, the most excitable one being located close to the synaptic zone. It has, in addition, been shown that an action potential occurs in a neurone whose cell body has been destroyed or made inexcitable by hyperpolarization. Thus the action potential arises at a distance from the soma and moves simultaneously towards the axon terminal and to the cell body. As the action potential approaches the soma, its amplitude decreases as a result of the loading effect of the non-excited somatic membrane so that the safety factor becomes too small. The spike is therefore blocked close to the soma. After a certain delay a new action

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potential (pseudospike) can occur in the resting part of the axon and in the axon hillock and which then invades the soma (Tauc, 1962a, b); Hagiwara and Morita (1962) on the other hand have shown that the somata of the leech segmental ganglion are directly invaded by spikes but that their amplitudes do not exceed 30 mV. In addition to propagation into the soma an action potential can, in some circumstances, also invade the axon branches following stimulation at some point on the neurone ( T a w and Hughes, 1963). Even if this action potential is not propagated in this way it will be able to facilitate, by electrotonic effect, responses in the other branches along which several synaptic zones are distributed. These effects will summate so that the response of a few branches would induce activity in all of the neurone branches. Although the above structures are not the same as those in insects it is possible, nevertheless, to transpose these conclusions to the cercal nerves-giant fibres system. Thus an action potential, generated in one of the nervous elements whose junction connects with a giant fibre, might be propagated along the giant fibre; on the other hand, it might either move antidror'nically along the other fibres or induce there a facilitation to the responses. In this latter case it might increase the frequency of the action potentials along the giant fibre if the fibre was not in a refractory period. These considerations emphasize the difficulties involved in identifying the origin and nature of the rhythmical activities recorded in different parts of the insect nervous system. I t appears that the cell bodies and the synaptic regions are especially excitable and, perhaps, spontaneously active, thus inducing activities along the axons. It seems likely that. as in Apljxiu, the cell bodies in insect ganglia may play an accessory part in the processes of synaptic transmission. An understanding of the various rhythmical activities in insect ganglia also requires a knowledge of those factors which induce or change these activities. This study which is dealt with in the next section is also likely to elucidate some details of the mechanism of the synaptic transmission in insects. U. P H Y S I C A L F A C T O R S W H I C H M O D I F Y T H E RHYTHMICAL ACTIVITIES OF G A N G L I A

1. Temperature

Kerkut and Taylor (1958) studied the effects of temperature changes on the electrical activity of cockroach nerve cords. In the first series of

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experiments the rate of spontaneous activity was recorded at different temperatures, after an equilibration period of 3-5 minutes. Under these conditions the frequency of impulses increased to a maximum as temperature was raised and then decreased. This maximum corresponded to the temperature at which the animals had been maintained before the experiment. The curve was found to be symmetrical about this maximum (Fig. 10). When sudden temperature changes were applied to the above preparation, the results differed according to whether an increase or a decrease in temperature was applied. In many cases the activity fell off Cockroach 31'

Cockroach 22" 18001

I

Temperature C

Tem perat u re "C

FIG.10. Changes in impulse rate for the isolated ganglia of the cockroach at different temperatures. The animals were kept at 22 C (left curve) and at 31 C fright curve) for four weeks before the experiments. I t must be noticed that the optimum temperature is higher on the right curve than on the left one. (From Kerkut and Taylor, 1958).

rapidly when temperature was increased from 13°C to 29'C. After about 10 sec this activity recovered and even exceeded the initial level. On the other hand a temperature decrease from 13°C to 4°C was found to induce an immediate increase in the rate of activity, although several seconds later this fell off to a lower level than the initial rate. Similar observations have also been made by Laverack (1961) in the case of

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Lumbricus. Hbwever, in the cockroach, some units were observed in which a persistent increase in activity occurred when the temperature was lowered, while a sustained decrease in activity was recorded in other ones. Finally, some units were observed which were not affected by temperature changes. Kerkut and Taylor tried to explain these anomalous variations in activity, which also occur in slug and crayfish ganglia by assuming that choline esterification has a higher Qlo than choline acetylation in insect ganglia. Under these conditions, increases in temperature would lead to a faster breakdown of ACh. by AChE. and, hence, to a decrease in nervous activity. On the other hand, a decrease in temperature would lead to a decline in the activity of the AChE., so as to cause an increase in the concentration of ACh. and thus increase nervous activity. It was assumed that compensatory processes must occur which re-establish the normal temperature rate relationship. These interpretations agree with those of Colhoun ( 1958b) which showed that the temperature of acclimatization of Periplaneta affected the ACh. content of the thoracic nerve cord.

2 . Electrical polarization of the abdominal ganglion of' the cockroach It is well known that the electrical activity of axons, somata or synaptic regions is related to the polarization level of their membranes. Thus according to Yamasaki and Narahashi (1954) a depolarization (catelectrotonus) applied to the sixth abdominal ganglion of the cockroach (Periplaneta umericana) either induces or increases the electrical activity of the giant fibres, while a hyperpolarization (anelectrotonus) decreases it. This study was resumed by Pichon and Boistel (1966) using microelectrodes which were inserted in the giant fibres of the connectives between the fifth and sixth abdominal ganglia of Periplanetu americana. Using this technique the authors were able to specify which events immediately followed either the commencement or the termination of polarization. Under these conditions, the rate of impulses became higher and reached a maximum (about 500/sec) as the electrical depolarization was increased, an effect which could be related to the absolute refractory period of the corresponding neurone. This effect then decreases (Fig. 1 I). It is important to know at what level this spontaneous activity originates. Although this question is not completely settled, it has been established that the threshold of the excitability of a ganglion is rather lower than that of the nerve fibres (Koketsu, 1951; Yamasaki and Narahashi, 1954), while Pichon and Boistel (1966), who have carried out electrical polarizations with microelectrodes inserted in the neuropile, have shown that the most sensitive region is not close

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to the cell bodies but is situated inside the neuropile where the synaptic zones are localized. 2

I

100 mV

I SO msec

3 30

J

1

5

10

15

20

25

30

Intensity of polarization relative to the rheobase

FIG. I I . I . Spikes induced along a giant fibre, intracellularly recorded, by electrical depolarizing shocks of increasing amplitudes applied to the sixth abdominal ganglion of Periplanrm. A : I .Ox rheobase. B: 2.0 x rheobase. C: 2.7 x rheobase. D: 3.3 x rheobase. E: 4.7 x rheobase. F: 6.7 x rheobase. 2. Electrical activity of the same fibre produced by an excessive depolarization of the ganglion. G : 16.7 x rheobase. H: 23.3 x rheobase. 1 : 33 x rheobase. J: 53 x rheobase. 3. Variation of impulses frequency in terms of the intensity of polarization for three different preparations. Current applied with external electrodes. (From Pichon and Boistel, 1966).

C. EFFECTS O F VARIOUS CHEMICAL A N D

P H A R M A C O L O G I C A L S U B STA N C ES

I . Potassium and calcium ions Roeder ( 1948b) has shown that spontaneous electrical activity of the isolated nerve cord of the cockroach is considerably increased by solutions of elevated potassium concentration (35 to 50 mM/1) and is abolished above a critical concentration. Moreover, according to

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Twarog and Roeder (1956), the effects of the increased potassium concentration were enhanced if the ganglion sheath was previously removed. Thus, in a cord-treated with a,solution containing 180 mM of KCI, impulse conduction was blocked after an interval of between 12 and 18 min. The blocking time was, however, reduced to 10 sec if the same preparation had been previously desheathed. Pichon and Boistel (1965a) have, in addition, shown that when the sixth abdominal ganglion of the cockroach was treated by a solution of elevated potassium

FIG. 12. Graphical representation of modifications of synaptic excitability induced by 52 mM of KCI applied to the sixth abdominal ganglion of Periplaneru. Continuous line: level of activity obtained by electronic integration of action potentials (extracellular record). Dotted line: threshold of the synaptic response of a giant fibre. In the upper part of the figure, records, obtained by microelectrodes, either of the response of the same giant fibre to a supraliminary stimulation of cercal nerves at instants marked on the threshold curve by corresponding letters (A, C and D; in A and D, direct and synaptic responses are obtained; in C a direct response only can be induced), or of the activity induced by KCI (B) (point B of the threshold curves). Amplitude scale: 50 mV; time scale: 4 msec for A, C and D. 500 msec for B. (From Pichon and Boistel, 1965b).

concentration, the spontaneous activity recorded between fifth and sixth abdominal ganglia showed a transient decrease before increasing. Moreover, they obtained similar results to those of Roeder with giant fibres impaled by intracellular electrodes (Fig. 12): when the normal physiological solution was replaced by a solution containing 52 mM of KCI, the fibre, which was generally inactive in the beginning, commenced firing, while the rate of its electrical activity, at first slow, then

23 quickly increased to a high value of between 130 and 360/sec. This period of activity was short and generally lasted for only a few seconds. The peak period of activity occurred very late, during the phase of hyperactivity described above (Fig. 12B). These results indicate that the effects of potassium ions are thus similar to those produced by a catelectrotonus and seem to be related to a gradual depolarization either of cell bodies or, as seems more likely, of the synaptic zones (Pichon and Boistel, 1965a). These two effects will be resumed (see p. 30), and discussed in a later section, when the problem of the possible involvement of ACh. in the synaptic transmission will be considered (see p. 36). Although potassium ions exert the most profound influence on the spontaneous activity of the insect nerve cord it is convenient to mention the effects of calcium ions which, as in the vertebrates, seem to contribute to the maintenance of the normal permeability and the excitability of the axon membrane. In this field Roeder (1948b) has shown that repetitive discharges of impulses appear in a cockroach nerve cord exposed to calcium-free saline for 2 or 3 hr. S Y N A P T I C TRANSMISSION-PHENOMENA

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2. Carbon dioxide Boistel and Coraboeuf (1954) have studied the effects of carbon dioxide on the spontaneous activity of the cockroach nerve cord. They have shown that pure CO, induces a very significant increase in activity which then declines. With a gaseous mixture containing 10 or 15% of COz and 90 or 85% of Oz, however, there is an appreciable and sustained increase in activity which appears to originate in the ganglia. In addition the excitability of directly stimulated giant fibres was considerably increased. To explain these effects, Boistel and Coraboeuf (1955) have established that if the above gaseous mixture is applied with a high potassium solution then there is a decrease in excitability. It appears therefore that a rather high membrane potential is necessary in order that the COz may have a facilitating effect. It would be of interest to know whether the effect of 10 or 15% COz is the same at the synaptic than at the axonal level and also whether the same interpretation can be propounded. According to Colhoun (1963) severe treatment of Periplanetu by CO, induces an appreciable loss of ACh. These facts indicate that C 0 2 probably acts by several ways on insect nervous tissue.

3. Acetylcholine (ACh.) We have already seen that a relatively large amount of ACh. exists in the insect nervous tissue. The simultaneous presence of AChE. localized

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in the neuropile, close to the synaptic membranes, suggests that ACh. is indeed the synaptic transmitter. The important point is to know whether ganglia or, more specifically, the postsynaptic membranes are sensitve to this substance. It is relevant in this respect to recall the experiments carried out by Twarog and Roeder (1956). These authors established that it was necessary to use a solution with a concentration of 1.6 x ACh. to increase the spontaneous activity of the cockroach isolated nerve cord, although when the sheath surrounding the ganglion was removed ACh. at this concentration induced appreciable asynchronous electrical activity, which was followed in most cases by a complete block of the synaptic transmission. Under these conditions an was sufficient to induce ACh. concentration of between 4.8 and 8 x ACh. was effective in the case a postsynaptic activity, while 1-6x of eserinized desheathed preparations. Yamasaki and Narahashi (1960) have also found essentially similar results and have observed that lo-* ACh. had some effect on the eserinized desheathed ganglia. Finally Callec and Boistel (1967) have induced rhythmical activity in cell bodies of a cockroach ganglion desheathed but non-eserinized with loW6ACh. The ACh. concentrations necessary to activate the synaptic structures are, therefore, higher than in the case of the vertebrate species which have been investigated and raised the question as to whether ACh. is in fact the transmitter substance in insect nervous tissues. This problem will be discussed in more detail in a later section of this article (see p. 54).

4. Anticliolitiesterases The essential property of these substances is to inactivate AChE. so as to prevent the hydrolysis of ACh. molecules liberated during synaptic transmission. Under these circumstances ACh. action will be appreciably prolonged. The effects of two substances of this type (eserine, DFP) on the electrical activity of insect ganglia will now be discussed. The properties of some of them to induce after-discharges will be further considered in their action on the synaptic activity itself. (a) Eserine. Roeder and Roeder (1939)have shown that application of this substance at a concentration of induces an enormous increase in the electrical activity in the cockroach isolated nerve cord, whereas a concentration of produces a burst of impulses which is then followed by a sudden and complete cessation of activity. According eserine applied to the deto Twarog and Roeder (1957) 3 x sheathed sixth abdominal ganglion of cockroach markedly increases the spontaneous activity and releases an activity in the giant fibres. Similar

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results have been obtained by Suga and Katsuki (1961b) in the T large fibres of the prothoracic ganglia of a locust, Gampsocleis buergeri. These results are in agreement with those obtained in many species of animals. Thus in the abdominal nerve cord of Crustacea (Turner, Hagins and Moore, 1950) and in the central neurones of Molluscs (Tauc and Gerschenfeld, 1960) eserine reinforces the action of ACh. In vertebrates, for example, it is known that eserine prolongs the response of Renshaw cells to synaptic excitation and increases their response to electrophoretic injection of ACh. (Curtis and Eccles, 1958) (see also p. 43). (b) Di-isopropyl fluorophosphate (DFP). Twarog and Roeder (1957) have found that this substance was active at a concentration of with desheathed abdominal ganglia of cockroach. These authors observed after-discharges, together with bursts of action potentials which occurred without electrical stimulation and which were followed by total synaptic block. This substance was also effective on the superior cervical ganglion of vertebrate inducing long-lasting postganglionary discharges (Volle and Koelle, 1961).

5 . Curare and atropine As is well known these substances and related compounds depress and even block the synaptic transmission in vertebrate animals. There is good evidence that this effect is caused by the fact that curare decreases the sensitivity of the synaptic membranes to ACh. (Eccles, 1963). Moreover, Tauc and Gerschenfeld (1961) have shown that a solution of D-tubocurarine applied to a ganglion cell of Aplrysia either decreases or suppresses the action of ACh. The study of the effects of curare on the insect nervous system is therefore critical in the assessment of the involvement of the cholinergic system in synaptic transmission in this group of animals. Roeder’s work (1948~)indicated that the application of a solution of curare to the sixth abdominal ganglion of the cockroach did not seem to have any obvious effect. However atropine, whose effects are similar to those of curare, blocked synaptic transmission in two or three cases (Roeder, 1948~).Suga and Katsuki (1961b) have studied the effects of curare on the auditory synapses of grasshopper. They have shown that if a prothoracic ganglion of this insect D-tubocurarine then the discharges is treated with a solution of 3 x of impulses in the T large fibre markedly decrease after 3 min. Moreover, the same solution abolishes the increased electrical activity produced by prior treatment with ACh. solution. More recently Callec and Boistel (1967) have carried out the following experiment: a cell

26 J . BOISTEL body of the desheathed sixth abdominal ganglion of cockroach was impaled with an intracellular microelectrode and ACh. was then injected in the immediate vicinity of the cell. This treatment produced appreciable spontaneous activity. This effect was not obtained even with 2 x ACh., if the preparation had been previously treated by flaxedil. These results are in agreement with those obtained, for example, by Tauc and Gerschenfeld (1961) and represent evidence for chemical synaptic transmission in insects. The question which now arises is to what extent curare, and also the other substances previously mentioned, are able to reach synaptic structures. It is relevant in this respect to recall that Roder’s experiments were carried out with intact ganglia whereas the sheath was removed in Callec and Boistel’s experiments. 6. Adrenaline arid noradrenaline The effects of these substances, which are the chemical mediators of the sympathetic system in vertebrates, have been studied on the nervous system of insects by Twarog and Roeder (1957). When applied to the desheathed sixth abdominal ganglion of the cockroach at a concentrathey induced asynchronous burst of low voltage spikes, tion of 2 x which were frequently associated with an increase in the synaptic rethe discharges become more masked and are then sponse. At 2 x followed by total block of synaptic activity. 6. 3-Hydroxytyramine (dopamine) This substance which, at least in invertebrates, is a metabolic intermediary of epinephrine has been found in significant amounts in insects by Ostlund (1954). The actual amounts were 5 to 10 g/g tissue, that is to say from 10 to 20 times more than the norepinephrine. Gahery and Boistel(l965) have studied the effects of dopamine on the sixth abdominal ganglion of the cockroach at a concentration of 5.10-5; this substance immediately induces burst of action potentials whose amplitudes were distributed in two groups with mean values of 0.2 and 1 mV, respectively. These responses are similar to those obtained in case of epinephrine. On the other hand dopamine does not appear to have any effect on the synaptic transmission between cercal nerves and giant fibres. This substance has quite a different action on the stretch receptor neurones of crustacea; indeed according to McGeer et al. (1961) it is a more effective inhibitor than y-aminobutyric acid.

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8. DDT The effects of this insecticide, whose toxicity is much higher in arthropods than in vertebrates, are well known. Insects are, in fact, less sensitive to DDT than crustacea (Welsh and Gordon, 1947). A review of the studies concerning the effects of this substance on the insect nervous tissue has been given by Narahashi in the first volume of this series (1963). We shall therefore confine ourselves to mention the following points. According to Yamasaki and Ishii (1957) DDT induces an appreciable electrical activity in the giant neurones of the cockroach nerve cord. This activity seems to be due to a modification of the synaptic processes which must be responsible for the locomotor instability and of the ataxia shown by cockroaches intoxicated by DDT. This effect is more pronounced at a low than that a high temperature. Moreover, these authors have shown that spontaneous discharges produced by a catelectrotonus are increased by DDT. These results suggest that DDT induces directly or indirectly a depolarization of the postsynaptic membranes. In this field Gordon and Welsh (1948) have shown that a decrease in the concentration of calcium ions in the physiological solution increases the repetitive discharges due to DDT while an increase in this cation has the opposite effect. These authors suggest that calcium ions have a stabilizing effect on the fibre membranes. DDT might, therefore, induce changes in the distribution of Ca2+. This hypothesis is interesting but according to Narahashi and Yamasaki (1960) a more complicated relation exists between the distribution of calcium and the action of DDT. An additional point, mentioned by Tobias et al. (1946), could at first sight explain the effects of DDT on the synaptic regions. These authors have pointed out that during the intoxication of the fly and cockroach by DDT, the ACh. content considerably increases to 180 and 210% of the normal level. This increase is particularly pronounced in the head and in the thorax. One might suppose, therefore, that DDT acts primarily by increasing the ACh. content of ganglia. Two facts militate against this hypothesis : first, according to the above-mentioned authors, ACh. accumulates much more in connectives than in ganglia in the cockroach, and secondly this accumulation is seen only during late prostration and not during the earlier hyperactivity.

9. Nicotine According to Roeder and Roeder (1939) this alkaloid markedly increases the electrical activity of the cockroach thoracic ganglia when

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applied at concentrations of loT3.A few minutes later a block of synapses may occur. 1V. PROPERTIES O F S Y N A P T ITRANSMISSION C A. G E N E R A L C H A R A C T E R I S T I C S O F T H E RESPONSES

Most researches in this field have been carried out either on the sixth abdominal ganglion or on the third thoracic ganglion of the cockroach, Periplaneta americana. The anatomy of these two regions is also well known, particularly as a result of the works of Pipa et al. (1959) on the thoracic ganglion, and Smith and Treherne (1963, 1965) on the last abdominal ganglion of the cockroach. Moreover, it is relatively easy to stimulate the different nerves which reach the ganglia and to record the resulting action potentials, either in ganglia themselves, in some nerves or in the connectives. At last one knows that the two ganglia are particularly important because they participate to a fundamental reflex circuit. The electrophysiological study of the sixth abdominal ganglion was first undertaken by Pumphrey and Rawdon-Smith (1937) and was later continued by Roeder (1948) using a similar technique. In these studies the afferent fibres originating in the cercus were electrically stimulated, either by inserting an electrode in the cut end of a cercus and another one in its base or by a pair of electrodes applied directly to the cercal nerves. The electrical activity could then be simultaneously recorded on the cercal nerves and, for example, on the connectives between the fifth and sixth abdominal ganglia. Under these conditions the cercal response occurs for a definite intensity of stimulation. However, the spatial summation is then insufficient to excite the giant fibres synaptically (Fig. 13). Pumphrey and Rawdon-Smith (1937) have shown, however, that in some cases these fibres can be activated by mechanical stimulation of a single cercal receptor. When the intensity of the stimulus was increased it was shown that the amplitude of the cercal potential became larger, as a result of the progressive recruitment of new nervefibres, anda spike occurred in the connectives; a giant fibre was thus stimulated following the convergence of a sufficient number of cercal impulses which had synaptically excited the different branches contributing to this fibre. When the intensity of the stimulus was still further increased several large spikes and also some small ones were recorded. Each large spike, whose amplitude did not change with increasing stimulus strength, corresponded to the activity of a single giant fibre. These

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studies were carried out using external electrodes. Essentially similar results have also been obtained by Pichon and Boistel (1965b) with microelectrodes introduced into the giant fibres themselves (technique perfected by Boistel(l957)). According to the schematic diagrams proposed by Pumphrey and Rawdon-Smith (1937) and Roeder (1948a) it was possible to activate these fibres by the stimulation of either the homolateral cercal nerves or the contralateral ones.

14

m v ~ ~

U

2 msec

I

0.5 mV Ra

FIG.13. Stimulation, in E, of cercal nerves (nX and nXI) in the cockroach induces in these nerves an action potential which is recorded in R1(upper trace of each photograph). Above a certain intensity of stimulation a synaptic response occurs in Rz(B, lower trace). As the stimulus is increased responses of several post synaptic fibres are recorded (C,lower trace) and the synaptic delay decreases. 6: sixth abdominal ganglion. (From Y. Gahery, unpublished observations).

Synaptic responses elicited by the stimulation of the cercal nerves have also been recorded in the region of the metathoracic ganglion (Roeder, 1948a). A complex spike was obtained in the homolateral connective above this ganglion. This latter spike occurred there slightly less than 2msec after the ascending spike complex had entered the ganglion. Some of the ascending fibres entering the ganglion appear to synapse in a 1-to-1 ratio with fibres leaving it. Moreover, under the

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same conditions of stimulation synaptic responses were obtained on thc crural nerves. The response of these motor fibres was not synchronous with the stimulus and the spike complex entering the ganglion. It took the form of a burst of spikes which continued for a variable period after the end of the stimulation (Roeder, 1948a). The synapses between giant and motor fibres seem to be very labile: the number of preganglionic fibres (spatial summation) and the frequency of the afferent impulses (temporal summation) in particular determine the amplitude of the postganglionic response. The excitability of these synapses (motor centres) seems to depend upon the arrival of impulses coming from the supra- and suboesophagian ganglia. Pumphrey and Rawdon-Smith (1937) and Roeder et ul. (1947) have shown that the stimulation threshold of the giant fibres by the cercal nerves is also variable. However, it is difficult in some cases to know whether or not these variations are due to fluctuations in the number of the stimulated cercal fibres. Some factors are able to modify the threshold of synaptic stimulation. The effects of slow temperature variations locally applied to the sixth abdominal ganglion of Bluberu cruniifer have been studied by Bernard et ul. (1965). In these experiments the cercal nerves were electrically stimulated, the presynaptic activity being recorded in these nerves (upper trace) and the postsynaptic activity on the connectives (lower trace) (Fig. 14). The cercal nerves were then given the minimum stimulation necessary to obtain a response on the cord; the amplitude of the cercal potential causing the appearance of the action potential in the cord was then measured at each temperature. The analysis of the records shows that the larger the temperature increases the smaller the cercal potential necessary for the excitation of the giant fibres. Thus the synaptic region seems to be more excitable at a high than at a low temperature. Moreover, the synaptic delay decreases with increasing temperature. These facts suggest that the processes involved in the release of the synaptic response are accelerated at a high temperature. Pichon and Boistel (1965b) have studied the effects of electrical polarization and of high potassium solutions on the sixth abdominal ganglion, treatments which are known to markedly modify the spontaneous activity. In normal conditions when the cerci are not stimulated, the giant fibres are generally electrically inactive. However, a catelectrotonus, which induces an increase in the electrical activity of the ganglion, brings about a decrease in the threshold of synaptic excitability. When the depolarization has lowered the threshold of stimulation by about 50% of the initial value, a rhythmical and persis-

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tent activity occurs in the giant fibres (Fig. 15). The synaptic threshold is, however, increased by an anelectrotonus. Moreover, if the potassium ion’s concentration of the physiological solution bathing the ganglion

FIG.14. Modifications in the excitability threshold of the synapses of the sixth abdominal ganglion of Periplunetu following slow temperature variations. For each photograph: Upper truce: Action potential recorded on the cercal nerve after its electrical stimulation. Lower truce: Action potential of the giant fibres recorded on the cord for the same stimulation. For the different temperatures each photograph was taken when the first action potential appeared on the cord. It can be seen that a smaller cercal potential is necessary to induce a postsynaptic response at a high than at a low temperature. (From Bernard ef ul. 1965).

32

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is increased from 3.1 to 52 mM the synaptic excitability measured in one giant fibre first slightly decreases and then it increases. However, when the depolarizationproduced by K+ ion’s reaches a critical level, synaptic transmission is blocked (Fig. 12C). These changes in synaptic excitability parallel the variations of the total spontaneous activity recorded in the

-----

150--

0

0 0

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T

I I

-4

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t

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0’

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+3 I

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;

Pol” (V)

so

Spontaneous activity 0

FIG.15. Changes in the synaptic excitability (S%) in function of the intensity of electrical polarization (pol” (V)) applied to the sixth abdominal ganglion of Periplunetu. It must be noticed that a catelectrotonus decreases the threshold by about 50% before inducing a long-lasting activity. (From Pichon and Boistel, 196%).

connectives (see p. 20). These phenomena are completely reversible. The above observations confirm Roeder’s opinion (1953) that the spontaneous activity of the isolated nervous system could correspond to the manifestation of an “intrinsic factor” contributing to the state of excitation of the postsynaptic fibres. Such a factor might be related to the

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33

electrical polarization of the synaptic regions. Indeed, Pichon and Boistel (1965b) have established that the electrical depolarization of a postsynaptic fibre successively induces (for increasing intensities): a decrease of the synaptic threshold, together with a rhythmical activity (whose frequency becomes progressively higher) ; and finally the disappearance of both synaptic response and electrical activity. B. S Y N A P T I C DELAY

The first estimates, of about 1 msec, were carried out by Pumphrey and Rawdon-Smith (1937). Using a similar technique Roeder et al. (1947) and Roeder (1953) have measured the time interval between the beginning of the cercal and of the postsynaptic spike. In these experiments the first pair of recording electrodes were placed on the cercal nerve, close to the sixth abdominal ganglion, and the second pair on the ganglion itself. Values of between 1.4 and 1.9 msec were obtained for the synaptic delay. These estimates include the time taken by the presynaptic impulses to pass through the space between the point where the recording electrodes are located and the synaptic zone. Similar values (between 0.97 and 2-08msec) have been obtained by Yamasaki and Narahashi (1960). In the metathoracic ganglion the synaptic delay may be very short, with values of between 0.15 to 03 msec, according to Roeder (1948a). The above values are of the same order of magnitude as those measured in vertebrates (from 0.5 to several milliseconds), in cephalopods (0.6 to 1-2msec in the squid (Bullock, 1948)) and in crustacea (0.35 msec for the crayfish (Takeda and Kennedy, 1964)). These different authors have emphasized the importance of variations in the synaptic delay which occur even when the stimulus intensity is constant during a series of stimulations (Roeder, 1948~).This effect is accentuated when the intensity of stimulation is progressively increased above the threshold, the synaptic delay being decreased by several tenths of a millisecond (Fig. 16). In addition Bernard et al. (1965) have shown that this delay is reduced by increasing temperature. These variations may correspond to fluctuations in the time necessary to reach the threshold of synaptic excitation. C. REFRACTORY PERIOD

The type of responses recorded with microelectrodes in a single giant fibre varies as a function of the applied stimulus intensity at the cercal nerve (Pichon and Boistel, 1965b). If the stimulus intensity is progressively increased above the threshold, a second synaptic response

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frequently occurs after an interval of 2 msec. This action potential has a smaller amplitude than the first, probably because it occurs during the refractery period of the first one. For a stronger intensity of stimulation it appears a direct response without any synaptic delay (Fig. 16). This result has led us to measure the duration of the refractory period at the synaptic level and to attempt to interpret the double response obtained, The refractory period measured in a giant fibre following two close electrical stimuli, applied synaptically to the cercal nerves, does

FIG. 16. Record by microelectrodesof responses of giant fibres to an increased electrical stimulation of cercal nerves of Periplaneta. A: Synaptic response alone; B: “double” synaptic response; C: direct and synaptic responses; D: direct and “double” synaptic responses. Time scale: 4 msec; Amplitude scale: 50 mV; In B and C, same calibration as in A. (From Pichon and Boistel, 1965b).

not seem to be different from this one which corresponds to direct stimulations of the same fibre. One may infer that the refractory period of the synapse is at least equal and perhaps shorter than that of the fibre. The values found for the absolute and relative refractory periods are about 2 and 5 msec respectively, at a temperature of 18°C. The value which has been found for the absolute refractory period (2 msec) is higher than the time interval between two spikes of a double response. The interpretation of this observation presents some diffi-

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culties. It is, however, possible to attribute this effect to the multicellular origin of the giant fibres (Roeder, 1948a), for, at sufficient intensity of stimulation, two synapticjunctions (situated on two different branches of the same giant fibre) react to a supraliminary presynaptic stimulation by a propagated action potential. The combination of these two responses would thus make up the double response. The hypothesis formulated on page 18 might perhaps be applied in this case.

-

D. AFTER D I S C H A R CE

The above experiments suggest that the synaptic region is unstable. This point has been also ascertained by Pumphrey and Rawdon-Smith (1937) in some non-adapted preparation in which single maximal stimuli were applied .to the cercal nerves and induced an appreciable discharge in the giant fibres. As these after-discharges are produced only after a period of synaptic inactivity, the authors supposed that the state of excitation of non-adapted synapses may reach such a level that postsynaptic fibres be re-excited several times, following an afferent volley involving a great number of fibres. Their frequency of discharge was, however, limited by the refractory period produced by the former impulse. An alternative hypothesis might be also proposed, namely that when the frequency of the presynaptic impulses is sufficiently high additional neuronal circuits (involvinga large number of synapses) can be also activated so that the same postsynaptic fibre will be stimulated several times, provided that it is not in a refractory period. One can also imagine the activation of circuits in a closed chain. Several anticholinesterasic substances, mainly diisopropyl-fluorophosphate @FP), hexaethyl tetraphosphate (HETP) considerably increase and prolong the state of excitability of the synapses involving the giant fibres, so that a single presynaptic stimulus induces in these fibres a substantial after-discharge (Roeder et ul., 1947; Roeder, 1948~).Thus a few minutes after application of lo-* DFP, a single presynaptic stimulation induces an after-discharge which may last between 15 and 30 sec. Frequently the after-discharge is followed by a total cessation of electrical activity, for if a fresh afferent stimulation is applied it can be seen that the synapses of the giant fibres are completely blocked. After 30 to 60 sec synaptic activity reappears and new after-discharges can be obtained. The phenomenon is not easily reversible after the washing with normal physiological solution. In no case did DFP induce a permanent block in synaptic transmission. 3 x lo-' tetraethyl pyrophosphate (TEPP) had some action while 3 x lo-' HETP was more effective than DFP (Roeder, 1948~).Physostigmine has a similar

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effect although the after-discharge produced was shown to be very short and the tendency to synaptic block higher (Roeder, 1948~).Phosphine oxides applied to-the sixth abdominal ganglion of the cockroach induce an after-discharge, whose duration can reach 30 sec, and also a marked tendency to spontaneous activity. Following this after-discharge a prolonged block of the synaptic transmission may occur (Roeder and Kennedy, 1955). The analogous effects produced by these different anticholinesterases has caused Roeder and Kennedy to consider that a synapse has a certain safety factor: “This one would be equivalent to the amount of mediator released by the presynaptic terminals, per impulse, over and above that critically necessary to ensure transmission : a fresh, unstimulated ganglion would have a high safety factor, the presynaptic terminals being capable of releasing an excess of mediator from a large actual or potential reserve. A reduction in safety factor would result if the mediator were depleted through previous activity.” Accordingly in a fresh preparation treated by an anticholinesterase an efferent volley might be expected to release a large amount of mediator. This would be accompanied by after-discharge and conduction block if the depolarization of the synapse was excessive. During subsequent stimulation, depolarization block might be of shorter duration, owing to progressive depletion of the mediator reserve and to its dispersion. Under these conditions synaptic excitability could recover. Moreover, if the concentration of anticholinesterase was relatively high, then the development of the synaptic block would be more rapid. The above results agree with those previously described concerning a progressive depolarization of the ganglion by potassium ions or by a catelectrotonus (Pichon and Boistel, 1965a, 1966). Thus, in conclusion, it can be seen that the effects produced by these anticholinesterasic substances indicate that the normal synaptic activity of the cockroach sixth abdominal ganglion requires the presence of a certain quantity of cholinesterase. On the other hand these various substances do not seem to modify the transmission of impulses along the nerve fibres themselves, even when applied at higher concentrations than those mentioned above. E. EFFECTS OF R E P E T I T I V E S T I M U L A T I O N

Pumphrey and Rawdon-Smith (1937) have recorded in the giant fibres of the cockroach the activity induced by repetitive stimulation of the cercal nerves. A response which has disappeared during a repetitive stimulation may reappear if either the frequency or the intensity of the stimulus are increased. These authors suggested that some at least of the giant fibres must be in synaptic connection with two or more pre-

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ganglionic fibres. The latter are distributed along the different branches of each giant fibre and it seems that an increasing recruitment of these branches might induce, by temporal or spatial summation, a progressive increase in the impulse frequency in the giant portion of the fibre. When these cercal fibres are subjected to repeated maximal or supramaximal stimulation, then above a certain frequency of stimulation the number of active postsynaptic fibres is reduced, the remainder becoming intermittent before failing completely. Pumphrey and RawdonSmith considered that this effect resulted from fatigue; in our opinion this could be related to the emptying of ACh. in the presynaptic endings. Different results were obtained if the cercal nerves were stimulated submaximally. Thus for a definite intensity of stimulation there is a critical frequency, beneath which the amplitude of the postganglionic response does not decrease and above which it does. It is possible to obtain a new response by increasing the stimulus intensity, the frequency becoming higher as the intensity of stimulation is increased. The cercal nerve fibres are not concerned in this effect because in the range of frequency which abolishes the response of the giant fibres, a higher frequency of stimulation still induces an increase in the impulse frequency of the cercal nerves without any decrease of the amplitude of the response. Consequently it must be the synaptic zone which is affected. Another relevant observation is that during repetitive submaximal stimulation, to which the postsynaptic fibres failed to respond, an additional stimulus (even though its amplitude is much smaller than those of the series) causes a single response to the stimulus of the regular series immediately following it. Occasionally a response may occur to the interpolated stimulus itself, especially if this should occur immediately after one of the series. Pumphrey and Rawdon-Smith do not refer to fatigue but to adaptation of the synaptic region, and make the following comments. In a non-adapted synapse (i.e. a synapse which has not functioned for a while) the arrival of a single presynaptic impulse produced by stimulation of a single hair on the cercus is sufficient to induce an impulse in the corresponding postganglionic fibre. As soon as a series of presynaptic impulses reach the ganglion the synaptic threshold becomes higher in consequence of adaptation, although this effect will be obtained if the number of stimulated presynaptic fibres is increased. In our opinion, it is likely that the synapses become Iess sensitive to ACh. so that a higher concentration of transmitter is necessary to produce an effect. It is obvious that a synaptic region may reveal very different degrees of excitability. In normal conditions the ganglia of the nerve cord are

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subjected to both excitatory and inhibitory influcnces produced by impulses originating especially in the supra- and suboesophagian ganglia. Several examples have-been given by Roeder (1948a). Thus in the decapitated cockroach a more intense stimulation of the cercal nerves, producing the activation of a higher number of giant fibres, is necessary to obtain a discharge in the motor fibres originating in the metathoracic ganglion. The threshold of stimulation is increased following section of the nerve cord above the metathoracic ganglion. It appears therefore that these different sections eliminate impulses which pass along the connectives and increase the level of excitability of the motor neurones, so as to facilitate the action of the ascending giant fibres. The influence of cephalic centres is complicated. According to Roeder et al. (1960), the cephalic centres of Mantis and Peripplaneta inhibit the last abdominal ganglion, while in Mantis Roeder (1937) has shown that removal of the supraoesophageal ganglia induces a long lasting locomotor activity. It is concluded that these centres have an inhibitory action. This action exerts its influence on the suboesophageal ganglion, which by itself has an excitatory effect on locomotion. Removal of the supraoesophageal ganglion would thus allow the suboesophageal ganglion to manifest its action. On the other hand supraoesophageal ganglia have a facilitating effect on the tonus. Thus the excitability of the insect nervous centre is, as in the case of vertebrates, submitted to antagonist actions of facilitation and inhibition. The problem which remains is the extent to which it is possible to explain such actions at the synaptic level. F. T H E M E C H A N I S M O F S Y N A P T I C T R A N S M I S S I O N

According to the classical conception (see Eccles, 1964) the phenomena which induce synaptic transmission can be summarized as follows: The propagation of an impulse along a presynaptic fibre may occur without decrement, although it seems that the progressive decrease in diameter of this fibre causes a reduction in the amplitude of the action potential as it approaches the terminal. In a synapse of electrical type this presynaptic potential produces currents at the level of the postsynaptic membrane which will depolarize it. In a “chemical synapse” the presynaptic potential induces the release of ACh. molecules from synaptic vesicles. These molecules pass through the presynaptic membrane and come into contact with the postsynaptic membrane where they produce changes of permeability which bring about the membrane depolarization (Fig. 17). In both cases this depolarization will induce a

S Y N A P T I C TRANSMISSION-PHENOMENA

persistent small potential-the

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excitatory postsynaptic potential

(EPSP). If the amplitude of this depolarization is sufficient, or if other

similar potentials summate with it, then phenomena of spatial or temporal facilitation will result in the production of a spike which will rise above the EPSP and will then propagate along the synaptic fibre. In the case of the chemical synaptic transmission the presence of AChE. between pre- and postsynaptic membrane will reduce the time of action of ACh. The synaptic transmission may be blocked by the release of an inhibitory substance at the ends of other fibres. In the case of postsynaptic inhibition in particular, this substance induces an inhibitory potential whose effect is to reduce the EPSP amplitude.

FIG. 17. Diagrams of two categories of synapses. A: One operating by chemical transmission. Two cylinders (pre- and postfibres) are separated by a synaptic cleft in which postsynaptic currents can flow freely; vesicles are shown in the presynaptic fibre. B: One operating by electrical transmission; the synaptic cleft is much narrower than in the first case. (From Eccles, 1964).

The anatomical and histochemical aspects of synaptic transmission have been previously considered and it is now relevant to consider the electrophysiological results in relation to the above scheme. As mentioned previously two groups of synapses have been most intensively studied in the cockroach. The first of these is contained in the sixth abdominal ganglion, and involves transmission between cercal fibres and some giant fibres; the second is the third thoracic ganglion

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(metathoracic) which is concerned with the transmission between giant and leg motor fibres. Some results have also been obtained on the mesothoracic ganglion of the locust, Schistocerca gregaria. Yamasaki and Narahashi (1958, 1960) have studied transmission in the first group of synapses. In this study, the isolated abdominal nerve cord, which included the cercal nerves and the cerci, was bathed in the following physiological solution: 159.6 mM Na+, 31 mM K + , 1.8 mM Ca2+,160-1mM C1-, 0.2 mM H2PO; and 1.8 mM HPOq- (PH of 7.2). The preparation was stimulated by brief shocks applied to the peripheral end of the cercal nerve through a pair of silver wire electrodes. Presynaptic responses were recorded from the cercal nerve just before entering the ganglion. Postsynaptic responses were obtained by another pair of electrodes at the origin of the connectives with the last abdominal ganglion. With the above conditions moderate stimulation resulted in a slow potential change with a rising phase of 2-3 msec and which decays exponentially with a time constant of 5-20 msec. With a higher intensity of stimulation, one or more action potentials will be generated from the summit of the slow potential and will propagate along giant fibres in the connectives. It is assumed that this slow potential, which occurs somewhat later than the cercal potential, is an excitatory postsynaptic potential (EPSP) (Fig. 18). When the second pair of recording electrodes is moved along the connectives far from the last abdominal ganglion the EPSP progressively decreases in height and its time course is slowed. It is electrotonically transmitted from the ganglion. It is, therefore, a local, non-propagated phenomenon. A small monophasic action potential was also observable, immediately before the onset of the EPSP, which progressively increased in height in parallel with presynaptic response as the stimulus intensity was increased. Even though synaptic transmission was blocked by anticholinesterases, this potential was still observable. Furthermore, the onset of this action potential corresponded to the initial peak of the diphasic cercal action potential and coincided with the time of arrival of the latter at the origin of the cercal nerve. It is considered that in these experiments the presynaptic action potential was transmitted electrotonically across the ganglion. Iwasaki and Wilson also (1966) recorded EPSP's in the mesothoracic ganglion of Schistocerca gregaria which, at a critical amplitude, were topped by a spike which could be blocked by eserine at 10-5-10-3. These records were obtained using extracellular glass capillary microelectrodes while electrical stimulation was carried out on the two sensory nerves from the wings.

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The study of the characteristics of the EPSP is complicated, because a relatively small increase in the presynaptic stimulus results in the appearance of an action potential (Fig. 18A). Unfortunately, curare does not easily block insect ganglion as in vertebrates’ synapses (Eccles, 1964); however, Yamasaki and Narahashi (1960) have discovered that

FIG.18. A : a postsynaptic response in the sixth abdominal ganglion of Periplunetu recorded by external electrodes, following a single shock applied to cercal nerves. The EPSP is topped by a series of spikes. B: as in A but 47 min after treatment with 1.3 x eserine. The prolonged ganglionic depolarization is superimposed with discharges. Time intervals between successive records are 15 sec. C: An EPSP set up by a single presynaptic volley under urethane. D: as in C but by repetitive volleys. E: as in C but 17 min after treatment with 2 . 7 ~ eserine; the EPSP is increased and prolonged. F: as in D but 41 min. after treatment with 2.7 x eserine; EPSP’s increased and prolonged. Time markers are 50 c/sec, except for B which is 25 c/sec. Voltage calibration: 0.5 mV. (From Yamasaki and Narahashi, 1958).

phenobarbital and urethane (13 x selectively block synapses. In this way pure EPSP’s were obtained (Fig. 18C) which, when a series of weak repetitive volleys was applied to the preganglionic nerve, could be observed to summate. The amplitude of EPSP’s was increased to several times their initial values; in this way, owing to this potentiation, the critical level of depolarization which was increased by these

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substances, might, however, still be reached so that a propagated response occurred. The stronger the stimulus strength or the higher the stimulus frequency, then the earlier was the onset of the critical amplitude of EPSP. Yamasaki and Narahashi (1958, 1960) have also studied the effects of anticholinesterases, eserine and TEPP, on the EPSP’s. With 1.3 x eserine the EPSP’s increased in size and their declining phase became prolonged. During this action an appreciable after-discharge was superimposed onto the EPSP. When the depolarization due to EPSP exceeded a certain value, the after-discharge stopped (Fig. 18B); it reappeared during the repolarization phase and stopped again close to the end of this phase. A similar type of persistent depolarization associated with prolonged discharges was frequently observed in the absence of presynaptic stimulation. In this case also a restoration was usually achieved after a period of depolarization. These results are in

FIG.19. (a) An EPSP set up by a single presynaptic volley (same preparation as in Fig. 18). (b) As in (a) but by repetitive volleys. (c) As in (a) but 26 min after treatment with eserine and 5 x urethane. Time intervals between successive records: 400 msec except for the last one which is 260 msec. (d) As in (c), but 32 min after and by repetitive stimula-

tions. Time intervals between successive record: 460msec except for the last one which is 100 msec. Voltage calibration: 0.5 mV. Time marker: 50 c/sec. (From Yamasaki and Narahashi, 1960).

agreement with those concerning the effects of ACh. Thus, it was shown that the synapses would function only between two limits of depolarization (Yamasaki and Narahashi, 1960). Moreover, if eserine was applied to a ganglion which had been previously treated by urethane, then the size of EPSP was markedly increased and the repolarization phase extended for as long as 3 sec (Yamasaki and Narahashi, 1960) (Fig. 19). These authors have pointed out that application of ACh. to the intact sixth abdominal ganglion induces a prolonged discharge (5-20 min) of impulses in giant fibres. This discharge is followed by a

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phase of high frequency and by a synaptic block. Application of ACh. is still very effective in a desheathed ganglion while is sufficient to induce impulses in eserinized and desheathed ganglion. This concentration is about 100 times higher than that found to produce equivalent effects in the case of vertebrates. Sympathetic ganglia are, for example, activated by 5 x ACh. in the presence of eserine (see Eccles, 1964). These results emphasize the difficulties involved in elucidating the r61e of ACh. as a transmitter of impulses in insect synapses. This problem will be further discussed in the light of more recent experiments and in connection with possible structural basis for the restricted penetration of substances in the synaptic zones. The fine mechanism of synaptic transmission has been studied in thc third thoracic and sixth abdominal ganglia of the cockroach. On the basis of anatomical works, mainly of Pipa et a!. (1959), Rowe (1963) has carried out an investigation on the metathoracic ganglion of Periplaneta fuliginosa using capillary microelectrodes. In these experiments the nerve cord was exposed but left in position. Particular precautions were taken to keep damage to the tracheal system to a minimum, for it was found that the normal functioning of synapses required a convenient oxygenation. The connective tissue sheath surrounding the ganglion was removed and the physiological solution, which was aerated Hoyle’s saline, was modified by the addition of sucrose to make it isotonic with desheathed cells (cf. Yamasaki and Narahashi, 1959). As is shown in Fig. 20 a pair of excitatory electrodes was put in the femur of each third thoracic leg. In addition it was possible to stimulate one or other of the thoracic and abdominal connectives of the nerve cord. A pair of recording electrodes was placed on the abdominal cord and the electrical activity of the third thoracic ganglion recorded using capillary microelectrode filled with KCl3 M or with KC1-sodium ferrocyanide mixture. The vertical position of the microelectrode was controlled by a micrometer drive. Stained serial sections of the ganglion were used to judge the probable position of the electrode tip. A cathode follower was used to compensate for the electrode capacitance and the indifferent electrode was introduced into the body of the animal. In these experiments the EPSP’s were recorded intracellularly following a single presynaptic stimulation, their amplitudes and durations being about 4 mV and 25 msec, respectively. Some of them were topped by an action potential, the total amplitude of them being 25mV (Fig. 21). For the first time in insects inhibitory postsynaptic potentials (IPSP) were demonstrated following electrical stimulations of the third leg nerve. Figure 22 shows rhythmical activity of one postsynaptic fibre

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FIG.20. Set up used for recording synaptic activities on the third thoracic ganglion of Periplanetafuliginosa. A : Agar-agar electrodes. I: Indifferent electrode. M : Microelectrode.

R: External recording electrode. S: Stimulating electrodes. (From Rowe, 1963).

transiently (about 50 msec) abolished by an IPSP occurring at each stimulation. In some cases EPSP and IPSP were successively recorded following electrical stimulations, thus modulating the electrical activity of the unit. Moreover, some units were stimulated 30 times per second. The stimulation, which was at first ineffective, then induced potentials whose frequency progressively increased. Even after cessation of the

FIG.21. Examples of EPSPs obtained with microelectrodes in the metathoracic ganglion of Periplanetafuliginosa following electrical stimulations. Spikes arise from EPSP's to stimuli 1 and 4. (From Rowe, 1963).

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stimulation, the unit still continued to emit impulses at 30/sec for a period of 1 sec and then progressively decreased during the next several seconds (Fig. 23). This experiment shows again the lability, the tendency of synaptic regions to produce electrical potentials. Different units, whether spontaneously active or not, could be stimulated through up to 3 or even 4 afferent routes, chosen from among the abdominal or thoracic connectives and leg nerves. Moreover, it was noted that the characteristics of responses vary as a function of the afferences which are stimulated. Thus, single unit stimulation through

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FIG. 22. Same preparation as in Fi3 21. Spontaneous activity is shown in the top line of the record. It is temporarily abolished by inhibitory postsynaptic potentials in response to electrical stimulation. Stimulus artefacts are marked by black dots under the record. Spike amplitudes: 6.5 mV. (From Rowe, 1963).

leg nerve induced an all-or-none response in a definite unit whereas stimulation of the right thoracic connective induced in the same unit both postsynaptic responses and action potentials. The above experiments are interesting because they give information about the complicated functional organization of the third thoracic ganglion. Most of the records were obtained in the synaptic zones of the neuropile. It is, however, difficult to know exactly which circuits or neuropilar structures were stimulated. In most cases synaptic stimulation was involved, although it seems reasonable to suppose that in

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other ones, antidromic stimulation might be able to change the rhythm of the units. In the case of the sixth abdoininal ganglion Callec and Boistel ( 1965a, b) continued Yamasaki and Narahashi’s above-mentioned work (1958, 1960) using microelectrodes techniques and preparations similar to the ones described by Rowe (1963). The nerve cord was removed from between the metathoracic and sixth abdominal ganglia, including cercal nerves. The tracheal network of the preparation was left as intact as possible in order to retain a certain amount of air within the tissues.

I

0

I

\

I

2 3 4 5 Time from beginning of stimulation (sec)

r

8

FIG.23. Same preparation as in Fig. 21. Variation of the frequency of postsynaptic spikes during and after an electrical stimulation whose duration is represented by the length of the shaded area while intervals between stimuli are indicated by the height of the shaded area. (From Rowe, 1963).

It was also necessary to oxygenate the physiological solution, which was that of Roeder as modified by Boistel(1957), used in these experiments. This preparation was stimulated through the cercal nerves or connectives (Fig. 24). One pair of recording electrodes was also put on connectives to act as controls. The sixth abdominal ganglion was placed ventral side uppermost and in some experiments the sheath of this ganglion was removed, while in other ones it was left intact. The penetration of the microelectrode was effected by tapping the experimental device. The tips of the microelectrodes used in this were particularly

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fine, being about 0.1 p as measured with the electron microscope (Boisseau and Boistel, 1965). The position of microelectrode tip was located with a devioe similar to the one used by Rowe (1963). A similar marking technique was used, which involved either formation of Prussian blue* or electrophoretic injection of methyl blue.? On histological sections it was possible to localize spots of about 10 p in diameter materializing position of the microelectrode tip. The following results were recorded in the various zones of the ganglion using the experimental technique outlined above. Stimulation

FIG.24. General arrangement of the chamber containing the nerve cord of Periplaiieta ctniericcma. The vertical watertight

partition, situated close to the sixth abdominal ganglion, divides the chamber into two compartments, the right one only being filled with the physiological solution. R1: recording electrodes including one microelectrode and one indifferent electrode. Ra :AgCI/Ag recording electrodes. El :stimulating electrodes including one liquid electrode in which the cercal nerve (N.C.) is sucked up with a syringe and one indifferent electrode. E2:AgCI/Ag stimulating electrodes. Oa: oxygen supply. 3T: third thoracic ganglion. IA, 6A: first and sixth abdominal ganglia. (From C a l k and Boistel, 1965b).

of cercal nerves on one side induced in the corresponding distal half of the sixth abdominal ganglion an action potential which probably originated in the cercus (zone I, Fig. 25). Moreover, in the median part of this zone the potential was replaced by a smaller one, of about 1 mV, whose duration was about 6 msec. This early potential might be either the cercal potential electronically transmitted from the tenuous ends of cercal fibres, or a presynaptic potential. A second region including the neuropile (zone 11, Fig. 25) was mainly * Technique perfected by Bultitude (1958) in vertebrates and adapted to insects by Callec and Boistel (1 965a). t Technique of Thomas and Wilson (1966) for vertebrates and adapted to insects by Callec and Boistel (1966a).

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characterized by potentials occurring after a delay of about 2 msec and with approximately 10 msec duration. These slow potentials are very likely to be EPSP’s, similar to those obtained by Yamasaki and Narahashi (1960). Modifications of these potentials under various conditions of stimulation were studied. It was shown that as the intensity of the presynaptic stimulus was increased the EPSP’s became higher. At a

FIG.25. Longitudinal representation of one-half of the sixth abdominal ganglion of Peripfaneta americanu showing in the upper part the insertion of the cercal nerve and in the lower part the beginning of the connective. Three regions are delimited whose numbers are related to the four graphs on the right side of the figure. In each division the lower record was obtained with a microelectrode from the corresponding region of the diagram (a downward deflexion being negative); the upper record was obtained from connectives. These responses were induced by the electrical stimulation of the homolateral cercal nerve. The vertical scale is related to the lower curve of each photograph, the horizontal one to both curves. (From Callec and Boistel, 1965b).

critical level of depolarization an action potential rose above the EPSP (Fig. 27, Al and A,) (Callec and Boistel, 1966b). Such potentials, but not EPSP’s, were in fact recorded inside the whole of zone 111. In this case the tip of the microelectrode was very likely in a structure of rather large diameter, probably a giant fibre. Temporal summation was observed in zone 11, following stimulation

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49

of the cercal nerves of the same side by two subliminal stimuli separated by a suitable time interval. After the second shock an action potential occurred whereas the first one was ineffective (Fig. 26). A spatial facilitation was also obtained by the application of a first stimulus on the cercal nerves of one side and of a second shock (both ones subliminal) on cercal nerves of the opposite side (Fig. 27, Callec and Boistel, I966b).

10 msec

FIG.26. Synaptic temporal facilitation obtained with two successive electrical shocks of equal intensity applied to one XI cercal nerve (in St) of Peripluneru umericunu; the postsynaptic electrical activity was recorded in R with an intracellular microelectrode introduced by the dorsal side of the sixth abdominal ganglion. Its tip was at a depth of 170 p. From A to D the second shock is progressively brought closer to the first one. In A, B,and C, EPSP's only are obtained. In D the second shock induces a spike. (From C a l k and Boistel, 1966b).

Moreover, injections of ACh. were carried out with micropipettes placed in contact with the surface of the sixth abdominal ganglion which was non-eserinized but desheathed. Under these conditions an EPSP obtained by cercal stimulation and which, before the injection, was unable to induce an action potential, became effective after the injection of a droplet of ACh. (Fig. 28). This action might last about 20 sec (Callec and Boistel, 1967).

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Inhibitory phenomena were also recorded in zone 11 following random microelectrode penetration. For any given electrode position, stimulation of cercal nerves on one side induced an EPSP which at a critical amplitude was topped by an action potential; stimulation of cercal nerves on the opposite side did not induce any action potential,

FIG.27. Same preparation as in Fig. 26. Spatial synaptic inhibition and facilitation obtained with the electrical stimulation of both XI cercal nerves (in Stl and St2) and recorded at a depth of 90 p. Al: A single homolateral stimulation (in Stz) induces an EPSP. A,: A stimulus of a higher intensity induces an EPSP topped by a spike. B: With a single contralateral stimulus applied in Stl one obtains a compound phenomenon: a phase of depolarization followed by a phase of hyperpolarization. C: Successive stimulations applied first in Stl and after in St, (the second stimulus is just supraliminary and progressively brought closer to the first one). A spike is visible in Cl and Cz. For a definite interval between both shocks an inhibition occurs (C3). The spike reappears in Cq.D: Successive stimulations in Stl and St2 (the second shock is now subliminary); for a definite interval between both shocks a spike occurs after the second one (DJ. (From Callec and Boistel, 1966b).

but a compound wave characterized by a depolarization followed by an hyperpolarization. Successive stimulation of nerves of both sides induced, for a definite interval between the two shocks, a facilitation released by the first phase of the wave; for a longer interval, on the other hand, inhibition was induced by the hyperpolarizing phase of the same wave (Fig. 27) (Callec and Boistel, 1966b).

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The results described above concerning modalities of synaptic transmission in third thoracic and sixth abdominal ganglia, although still fragmentary, mainly because of some formidable technical difficulties, are on the whole comparable. In both preparations EPSP’s and IPSP’s have been obtained and in the case of the sixth abdominal ganglion both spatial and temporal summation were demonstrated. These results are in agreement with those described by Eccles e l al. (see Eccles, 1964) in various vertebrate synapses, by Preston and Kennedy (1960) and Takeda and Kennedy (1964) in crayfish neurones and by Tauc (1955,

Q

10 msec Fir;. 28. Same preparation as in Fig. 26. A single shock applied in St only induces an EPSP (A), recorded in R (the tip of the intracellular microelectrode introduced by the dorsal face of the ganglion was at a depth of 200 p). Then a droplet of ACh. is injected in the immediate vicinity of the microelectrode; 7 min later it induces an increase in the amplitude of the EPSP (B as compared with A). 7 min 15 sec after the beginning of the injection a spike tops the EPSP (C). After 13 min of ACh. action the EPSP amplitude is the same as in A. (From Callcc and Boistel, 1967).

1962a, b) in giant neurones of Aplysia. Moreover, as we have already seen, it is very difficult, in opposition to vertebrate synapses (J. C. Eccles, 1946; R. M. Eccles, 1963), to block action potentials with nembutal or curare (Yamasaki and Narahashi, 1960). However, isolated EPSP’s were obtained with urethane, this anaesthetic probably increasing the

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stability of postsynaptic membranes (Yamasaki and Narahashi, 1960). Eccles (1946), in particular, reports a similar action in vertebrates. G . I N H I B I T O R Y PRO CESSES

Although many examples of such phenomena have been described in vertebrates as well as in crustacea and molluscs, these processes are not well understood in insects. In addition to the inhibitory r61e of the suboesophageal ganglia of Periplaneta and Mantis, previously described, some experiments have been carried out by Suga and Katsuki (1961a). These authors have recorded from thoracic connectives of Gampsocleis buergeri impulses which were immediately evoked by the activity of the tympanic nerve at the prothoracic ganglion. These impulses seem to be conducted to the rostra1 and caudal ganglia by one large fibre (T) whose cell body appears to be localized in prothoracic ganglion. Under these conditions the analysis of records shows that impulses of the tympanic nerve on one side have an inhibitory effect on the contralateral T large fibre. A marked increase of impulses occurs in the T large fibre of one side following section of the opposite tympanic nerve (Fig. 29). It is relevant to mention once again results obtained by Callec and Boistel (1966b) using the cockroach sixth abdominal ganglion in which electrical stimulations of, at least, several fibres of the cercal nerve of one side are able to inhibit the synaptic response released by stimulation of the cercal nerve of the other side (Fig. 26C). The diphasic waves described by these authors may be compared to those obtained by Hughes and Tauc (1963, 1965) in their study of the interactions between giant cells of the left pleural ganglion in Aplysia. They show that postsynaptic fibres can be submitted to complicated influences produced by different afferent fibres. The relatively long-lasting blockings of activity rhythms obtained by Rowe (1963) also seem to be comparable to those obtained by Tauc in particular on ganglionic cells of Helix (1955) and Aplysia (1958). The nature of mechanisms which are responsible for inhibition in insects still remains obscure. A great deal of research has been carried out in vertebrates, crustacea and molluscs in an attempt to identify the substance responsible for this phenomenon. Factor I extracted from mammalian brain has a powerful inhibitory effect on different nervous tissues and especially on the discharge from the crustacean stretch receptors (Florey, 1954). It seems to be proved that this extract is a mixture of different substances, among which GABA has the most depressing effect on nerve cells. Other authors, notably Bazemore et al. (1957), have suggested that either this substance or a related amino-acid might

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be the inhibitory transmitter in the vertebrate central nervous system. Doubts still exist in this field, however, because the effects of GABA appear to differ from those of the inhibitory transmitter. On the other

FIG.29. Inhibitory effect of the tympanic nerve on the contralateral T large fibre in the prothoracic ganglion of Gampsocleis buergeri. In A and B the upper and middle beams represent impulse discharges of the right and left large fibres, respectively. A is before and B is after the elimination of the inhibitory effect by cutting the left tympanic nerve. In A and B the sound stimulus (lower beam) is 13 kc/sec. Time signal, 10 msec. (From Suga and Katsuki, 1961a).

hand Kuffler and Edwards (1958) and Edwards and Kuffler (1959) have shown that the synaptic inhibitory action on the crustacean stretch receptor cell could be duplicated in every respect by GABA or its near relatives.

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In contrast to crustacea it seems that molluscs use at least one of the transmitter substances which are widely distributed in vertebrates, and there is now wry good evidence that ACh. is an inhibitory transmitter at many synapses. This is mainly the case for one type of ganglionic cells (the H cells) in ApIysia (Tauc and Gerschenfeld, 1961, 1962). In insects Vereshtchagin ef al. (1961) have shown that the relatively high concentration of l o w 3GABA has a depressing effect on the electrical activity of the nerve chain of the caterpillar of Dendrolinus pini. Suga and Katsuki (1961b) have also studied the effects of GABA on the discharge of T large fibres in the grasshopper Gumpsocleis buergeri. Thus treatment of the prothoracic ganglion with GABA resulted in a decrease of impulse frequency, all activity being abolished within 5 min. This phenomenon was found to be fully reversible. Gahery and Boistel(1965) have also observed that GABA induced a complete inhibition of the synaptic response in the sixth abdominal ganglion of the cockroach elicited in giant fibres by cercal stimulation. Thus GABA was found to be active for very high concentrations; in this field, it is relevant to mention that GABA has no action on spontaneous activity in the crayfish nerve cord (Hichar, 1960b). The evidence outlined above is insufficient to enable GABA to be conclusively identified as the inhibitory substance in insects’ nervous tissues. The possibility also exists that this substance may be the precursor or a by-product of the real inhibitory transmitter. It should be mentioned, however, that Price (1961) has extracted GABA from the head of a fly, Musca domestica, where it seems to be more concentrated than in any other part of the body. Ray (1965) has also shown that this substance exists in the central nervous system of Periplunefa americana, which suggests that GABA must play a significant r61e in metabolism and physiology of insect nervous tissue. V. G E N E R ACONCLUSIONS L The evidence discussed in this chapter has served to emphasize the limits of our knowledge of synaptic transmission in insects. Anatomical studies have shown that synapses, essentially axo-axonic ones, are localized in the neuropilar region of ganglia. These synapses correspond to coupling zones of very fine nerve fibres which lie adjacent to or cross one another. In some cases these zones of contact exhibit a level of organization which is equivalent to that of synaptic knobs. These regions seem to be completely devoid of glial processes, the spaces between the pre- and postsynaptic membranes being about 200 A wide

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for the synapses so far studied. This size of synaptic cleft is usually associated with a chemical mode of transmission of nervous messages. Theclusters of vesicles which occur along these zones seem to be mainly localized in the cytoplasm of one of a pair of fibres, usually the larger one. By analogy with the current concepts of vertebrate synaptic regions, these vesicles might be regarded as containing the ACh. molecules which are released into the synaptic cleft on arrival of presynaptic impulses. It is, perhaps, significant in this respect that appreciable amounts of ACh. have been demonstrated in the neuropile. Moreover, histo-chemical studies have revealed the presence of appreciable amounts of ACh.E. capable of inactivating ACh. The electrophysiological evidence outlined above is in essential agreement with the hypothesis of a cholinergic synaptic transmission in insects : we have seen that several anticholinesterasic substances, such as eserine and DFP (which are known to favour the accumulation of ACh. in synaptic zones and extend postsynaptic membranes depolarization) induce either intense after-discharges following a single presynaptic stimulation or in some cases a spontaneous activity. A total block of synaptic transmission then occurs, most probably as a result of an excessive depolarization of postsynaptic membranes produced by ACh. A similar conduction block also occurs as a result of a catelectrotonus or an elevated potassium ion concentration in the physiological solution. It has also been mentioned that, according to the work of Suga and Katsuki (1961b), rhythmical activity induced by ACh. is decreased by curare in the auditory synapse of the grasshopper.* It would seem to be very desirable that such fundamental observations should be extended to other insect preparations. The most important evidence for a cholinergic synaptic transmission is the demonstration of an action of ACh. on synapses at concentrations equivalent to those which have been found to be effective in other animal species, that is to say at about The first results obtained were rather disappointing, for, according to Twarog and Roeder (1956), 1.6 x ACh. applied to an intact ganglion of cockroach had no effect on synaptic activity; ACh. was, however, found to be effective with ganglia which had been deprived of their connective tissue sheaths (Twarog and Roeder, 1956; Yamasaki and Narahashi, 1960). These results were interpreted as being evidence that the peripheral Similar results have been obtained by Callec and Boistel (1967) on nerve cell bodies of a cockroach ganglion.

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nerve sheath (whose histological structure was studied by Wigglesworth, 1960) functioned as a diffusion barrier restricting the penetration of ACh. into ganglia. More recently, however, Treherne (1962a and b) and Treherne and Smith (1965a and b) have shown that exchanges of different ions and organic substances, including ACh., occur relatively rapidly between haemolymph and central nervous system of Periplaneta americana. It appears likely, according to these latter authors, that the increased sensitivity of desheathed ganglia to ACh. results from drastic secondary modifications caused by the removal of the sheath, particularly to the disruption of the Donnan equilibrium between the extracellular fluid surrounding neurones and the haemolymph. In particular the following modifications have been postulated to result from the desheathing procedure: appreciable changes in the volume of the extracellular fluid, a decrease in osmotic pressure and also of the K +,Na and Ca2+ ions concentrations in this fluid (Treherne, 1962a). Now it is known that transynaptic excitation is dependent upon the chemical environment of the cells and terminations in the synaptic regions. For example, it was shown that in the absence of calcium ions the excitability of postsynaptic cells was increased by the perfusion of ACh. In sympathetic ganglia of vertebrates the excitability was, however, reduced with elevated calcium chloride concentration (Brink et al., 1946). Thus the fall in the calcium concentration in the extracellular fluid (from 17.1 to 4.6 mM/1) resulting from the desheathing procedure could increase the sensitivity of the synaptic membranes to ACh. This is in agreement with experiments of Twarog and Roeder and of Yamasaki and Narahashi. Another possibility advanced by Twarog and Roeder (1956) would be to suppose that the giant fibres, and possibly other neurons, are encased throughout their length by a delicate sheath-like structure. These structures could function to delay the penetration of ACh. into the synaptic regions during these experiments. This hypothesis does not, however, appear to be very likely since the electron microscope studies of Smith and Treherne (1963) have shown that the regions of synaptic activity in the neuropile of insects are very probably confluent with the general extracellular system. Yamasaki and Narahashi (1960) have suggested that the apparent insensitivity of the nervous system to ACh. might be related to the high cholinesterase activity of these tissues and also to a relative insensitivity of the postsynaptic membranes to the transmitter substance. Under these conditions it would not be necessary to postulate the existence of peripheral diffusion barrier. +

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Recent experiments carried out by Callec and Boistel (1967) have removed many of the objections as to identity of the neurally released transmitter substance. ACh. has, for example, been found to be effective on nerve cells situated at the periphery of ganglia where they are, however, encapsulated by extensive concentric glial folds (Smith and Treherne, 1963) which could possibly delay the penetration of ACh. to the synaptic membranes; application of ACh. increases synaptic activity still further. I t must be recalled that these ACh. injections were carried out at the surface of desheathed ganglia. Such a technique is not ideal since ACh. can diffuse away from the point of injection. It is possible therefore that injection of lower concentrations of ACh. could be effective if this was carried out inside the neuropile. Under these circumstances the sensitivity of insect synaptic membranes could be similar to those of vertebrate and mollusc species which have been investigated. Thus, although it is impossible to assert categorically that ACh. is the synaptic transmitter in insects, there are several arguments which are in agreement with this hypothesis. The possibility cannot be excluded, however, that some insect synapses might be electrical. There has even been less progress in the field of synaptic inhibition, for, although there are several examples of this phenomenon in insects, it is impossible to postulate whether GABA or related substances are implicated in these processes.

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Hichar, J. K. (1960b). Etfects of gamma-aminobutyric acid and picrotoxin on spontaneous activity in the central nervous system of the crayfish. Nature, Lond. 188, 1117-1119. Hughes, G. M. and Taw, L. (1963). An electrophysiological study of the anatomical relations of two giant nerve cells in Aplysia depilans. J. exp. Biol. 40, 469486. Hughes, G. M. and Tauc, L. (1965). A unitary biphasic post-synaptic potential (PPSP) in Aplysia “brain”. froc. physiol. Soc. J. Physiol,, Lond. 179, 27-28P. Hunt, C. C . and Kuno, M. (1959). Background discharge and evoked responses of spinal interneurones. J. Physiol., Lond. 147, 363-384. Twasaki, S. and Wilson, D. M. (1966). Neuropile potentials following sensory nerve stimulation in locusts. Comp. Biochem. Physiol. 17, 693-695. Iyatomi, K. and Kanehisa, K. (1958). Localization of cholinesterase in the american cockroach. Jap. J. appl. Ent. Zool. 2, 1-10. Kandel, E. R. and Tauc, L. (1966). Input organization of two symmetrical giant cells in the snail brain. J. Physiol., Lond. 183, 269-286. Kerkut, G. A. and Cottrell, G. A. (1963). Acetylcholine and 5-hydroxytryptamine in the snail brain. Comp. Biochem. Physiol. 8, 53-63. Kerkut, G. A. and Taylor, B. J. R. (1958). The effect of temperature changes on the activity of po‘ikilotherms. Behauiour 13, 261-279. Koketsu, K. (1951). Etude sur la reaction des neurones isoles de chenille Dendrolinus spectabilis Butler a la stimulation par le courant continu. Kyushu Mem. Med. Sci. 2, 113-122. Kuffler, S. W. and Edwards, C. (1958). Mechanism of gamma-aminobutyric acid (GABA) action and its relation to synaptic inhibition. J. Neurophysiol. 21, 589410. Landolt, A. M. and Ris, H. (1966). Electron microscope studies on soma-somatic interneuronal junctions in the corpus pedunculatum of the wood ant (Formica lugubris Zett.). J. biophys. biochem. Cytol. 28, 391-403. Landolt, A. M. and Sandri, C. (1966). Cholinergische synapsen im oberschlundganglion der waldameise (Formica lugubris Zett.). Z . Zellforsch. 69, 246-259. Laverack, M. S. (1961). The effect of temperature changes on the spontaneous nervous activity of the isolated nerve cord of Lumhricus terrestris. Comp. Biochem. Physiol. 3, 136-140. Leghissa, S. (1942). Peculiari connessioni nervose nella catena ganglionare di Carausius morosus. Arch. Zool. Ital. 30, 289-310. McGeer, E. G., McGeer, P. L. and McLennan, H. (1961). The inhibitory action of 3-hydroxytyramine, GABA and some other compounds toward the crayfish stretch receptor neuron. J. Neurochem., Lond. 8, 36-49. Mikalonis, S. J. and Brown, R. H. (1941). Acetylcholine and cholinesterase in the insect central nervous system. J. cell. comp. Physiol., 18, 401403. Mill, P. J. (1963). Neural activity in the abdominal nervous system of aeschnid nymphs. Comp. Biochem. Physiol. 8, 83-98. Narahashi, T. (1963). Properties of insect axons. In “Advances in Insect Physiology” (J. W. L. Beament, J. E. Treherne and V. B. Wigglesworth, eds.), Vol. 1, pp. 175-256, Academic Press, London and New York. Narahashi, T. and Yamasaki, T. (1960). Mechanism of increase in negative afterpotential by dicophanum (DDT) in the giant axons of the cockroach. J. Physiol., Lond. 152, 122-140.

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Osborne, M. P. (1966). The fine structure of synapses and tight junctions in the central nervous system of the blowfly larva. J. Insect. Physiol. 12, 1503-1512. Ostlund, E. (1954). The distribution of catecholamines in lower animals and their effects on the heart. Acta physiol. scand. 31, suppl. 112, 1-65. Palay, S. L. (1958). The morphology of synapses in the central nervous system. Expl Cell. Res., suppl. 5, 275-293. Pichon, Y. and Boistel, J. (1965a). Etude comparative des effets du potassium et de ceux de la polarisation Clectrique sup le sixibme ganglion abdominal de la blatte, Periplaneta americana. J. Physiol., Paris 57, 680-68 I. Pichon, Y. and Boistel, J. (1965b). ComplCments a 1’Ctude electrophysiologique de la transmission synaptique au niveau du sixibme ganglion abdominal de la Blatte, Periplaneta arnericana. Vkme Congrks Internat. U.I.E.I.S., Toulouse, pp. 355-367. Pichon, Y. and Boistel, J. (1966). Effets de la polarisation electrique du &me ganglion abdominal de la blatte, Periplaneta americana, sur I’activitC des fibres geantes. C . r. SOC.Biol., Paris 160, nos. 8-9, 1728-1732. Pipa, R. L., Cook, E. F. and Richards, A. G. (1959). Studies on the hexapod nervous system. 11. The histology of the thoracic ganglion of the adult cockroach, Periplaneta americana. J. comp. Neurol. 113, 401-423. Preston, J. B. and Kennedy, D. K. (1960). Integrative synaptic mechanisms in the caudal ganglion of the crayfish. J. gen. Physiol. 43, 671-681. Price, G. M. (1961). Some aspects of amino-acid metabolism in the adult housefly, Musca dornestica. Biochem. J. 80, 420-428. Prosser, C. L. (1934). Action potentials in the nervous system of the crayfish. 1. Spontaneous impulses. J. cell. comp. Physiol. 4, 185-209. Pumphrey, R. J. and Rawdon-Smith, A. F. (1937). Transmission of nervous impulses through the last abdominal ganglion of the cockroach. Proc. R. SOC.B, 122, 106-118. Ray, J. W. (1965). The free amino-acid pool of cockroach (Periplaneta americana: Dictyoptera) central nervous system. In “The Physiology of the Insect Central Nervous System” (J. E. Treherne and J. W. L. Beament, eds). Academic Press, London and New York. Robertson, J. D. (1961). Ultrastructure of excitable membranes and the crayfish median-giant synapse. Ann. N. Y.Acad. Sci. 94, 339-389. Roeder, K. D. (1937). The control of tonus and locomotor activity in the praying mantis (Mantis religiosa L). J. exp. Zool. 76, 353-354. Roeder, K. D. (1948a). Organization of the ascending giant fiber system in the cockroach (Periplaneta americana). J. exp. 2001. 108, 243-261. Roeder, K. D. (1948b). The effects of potassium and calcium on the nervous system of the cockroach, Periplaneta americana. J. cell. comp. Physiol. 31, 327-338. Roeder, K. D. (1948~).V. The effect of anticholinesterases and related substances on nervous activity in the cockroach. Johns Hopkins Hosp. Bull. 83, 587-599. Roeder, K. D., ed. (1953). “Insect Physiology”, John Wiley and Sons, New York. Roeder, K. D. and Kennedy, N. K. (1955). The effects of certain trisubstituted phosphine oxides on synaptic conduction in the roach. J. Pharm. exp. Ther. 114,211-220. Roeder, K. D. and Roeder, S. (1939). Electrical activity in the isolated nerve cord of the cockroach. I. The action of pilocarpine, nicotine, eserine and acetylcholine. J. cell. romp. Physiol. 14, 1-12.

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Roeder, K. D., Kennedy, N. K. and Samson, E. A. (1947). Synaptic conduction to giant fibers of the cockroach and the action of anticholinesterases. J. Neurophysiol. 10, 1-10. Roeder, K. D., Tozian, L. and Weiant, E. A. (1960). Endogenous nerve activity and behaviour in the mantis and cockroach. J. Insect Physiol. 4, 45-62. Rowe, E. C. (1963). Microelectrode records from an insect thoracic ganglion. Ph.D. Dissertation. Univ. of Michigan. Sherrington, C. S. (1897). The central nervous system. I n “Sir Michael Foster’s: a Text-book of Physiology” (7th ed.). Macmillan and Co., London. Smith, D. S. (1965). Synapses in the insect nervous system. I n “The Physiology of the Insect Central Nervous System” (J. E. Treherne and J. W. L. Beament. eds.), pp. 39-57. Academic Press, London and New York. Smith, D. S. and Treherne, J. E. (1963). Functional aspects of the organization of the insect nervous system. I n “Advances in Insect Physiology” (J. W. L. Beament, J. E. Treherne and V. B. Wigglesworth, eds.), Vol. I, pp. 401484. Academic Press, London and New York. Smith, D. S. and Treherne, J. E. (1965). The electron microscopic localization of cholinesterase activity in the central nervous system of an insect, Peripluneta americana. J. biophys. biochem. Cytol. 26, 445465. Suga, N. and Katsuki, Y.(1961a). Central mechanism of hearing in insects. J. exp. Biol. 38, 545-558. Suga, N. and Katsuki, Y.(I961 b). Pharmacological studies on the auditory synapses in a grasshopper. J. exp. Biol. 38, 759-770. Svidersky, V. L. (1967). Central mechanisms controlling the activity of locust flight muscles. J. Znsect Physiol. 13, 899-91 I . Takeda, K. and Kennedy, D. (1964). Soma potentials and modes of activation of crayfish motoneurons. J. cell. comp. Physiol. 64, 165-182. Tauc, L. (1955). Etude de I’activite elementaires des cellules du ganglion abdominal de I’aplysie. J. Physiol., Lond. 47, 769-792. Tauc, L. (1958). Processus postsynaptiques d‘excitation et d‘inhibition dans le soma neuronique de I’aplysie et de I’escargot. Arch. itul. Biol. 96, 78-1 10. Tauc, L. (1960). Diversite des modes d‘activite des cellules nerveuses du ganglion deconnecte de I’aplysie. C. r. SOC.Biol., Paris 44, 17-2 I . Tauc, L. (1962a). Site of origin and propagation of spike in the giant neuron of Aplysia. J. gen. Physiol. 45, 1077-1097. Tauc, L. (1962b). Identification of active membrane areas in the giant neuron of Aplysia. J. gen. Physiol. 45, 1099-1 115. Tauc, L. and Gerschenfeld, H. M. (1960). L’acetylcholine comme transmetteur possible de I’inhibition synaptique chez I’aplysie. C . r. hebd. Skanc Acad. Sci.. Paris 251, 3076-3078. Tauc, L. and Gerschenfeld, H. M. (1961). Cholinergic transmission mechanisms for both excitation and inhibition in molluscan central synapses. Nature. Lond. 192, 366-367. Tauc, L. and Gerschenfeld, H. M. (1962). A cholinergic mechanism of inhibitory synaptic transmission in a molluscan nervous system. J. Neurophysiol. 25, 236-262. Tauc, L. and Hughes, G. M. (1963). Modes of initiation and propagation of spikes in the branching axons of molluscan central neurons. J. gen. Physiol., 46, 533549.

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Thomas, R. C. and Wilson, V. J. (1966). Marking single neurons by staining with intracellular recording microelectrodes. Science, N. Y . 151, 1538-1 539. Tobias, J. M., Kollros, J. J. and Savit, J. (1946). Acetylcholine and related substances in the cockroach, fly and crayfish and the effect of DDT. J. cell. cornp. Physiol. 28, 159-1 82. Treherne, J. E. (1962a). The distribution and exchange of some ions and molecules in the central nervous system of Periplaneta arnericana. L. J . exp. Biol. 39, 193-2 1 7. Treherne, J. E. (1962b). Some effects of the ionic composition of the extracellular fluid on the electrical activity of the cockroach abdominal nerve cord. J. exp. Bi01. 39, 631-64 1. Treherne, J. E. ( I 966). “The Neurochemistry of Arthropods”, Cambridge University Press. Treherne, J. E. and Smith, D. S.(1965a). The penetration of acetylcholine into the central nervous tissues of an insect (Periplaneta amrricana L.). J . exp. Biol. 43. 13-21. Treherne, J. E. and Smith, D. S. (1965b). The metabolism of acetylcholine in the intact central nervous system of an insect (Periplaneta urnericana L.). J . exp. Biol. 43, 441-454. Tmjillo-Cenoz, 0. (1959). Study on the fine structure of the central nervous system of Pholus labruscoe L. (Lepidoptera). Z . Zellforsrh. 49, 4 3 2 4 6 . Trujillo-Cenoz, 0. ( 1 962). Some aspects of the structural organization of the arthropod ganglia. Z . Zellforsch. 56, 649-682. Turner, R. S., Hagins, W. A. and Moore, A. R. (1950). lnfluence of certain neurotropic substances on central and synaptic transmission in Callianassa. Proc. SOC.exp. Biol. Med. 73, 156-158. Twarog, B. M. and Roeder, K. D. (1956). Properties of the connective tissue sheath of the cockroach abdominal nerve cord. Biol. Bull., Woods Hole 111, 278-286. Twarog, B. M. and Roeder, K. D. (1357). Pharmacological observations on the desheathed last abdominal ganglion of the cockroach. Ann. ent. SOC.Am. 50, 231-237. Vereshtchagin, S. M., Sytinsky, I. A. and Tyshchenko, V. P. (1961). The effect of gamma-aminobutyric acid and beta-alanine on bioelectrical activity of nerve ganglia of the pine moth caterpillar (Dendrofinus p h i ) . J. Insect Physiol. 6, 21-35. Volle, R. L. and Koelle, G. B. (1961). The physiological r6Ie of acetylcholinesterase (AChE.) in sympathetic ganglia. J. Pharmac. exp. Ther. 136, 223-240. Welsh, J. H. and Gordon, H. T. (1947). The mode of action of certain insecticides on the arthropod nerve axon. J. cell. cornp. Physiol. 30, 147-171. Wigglesworth, V. B. (1958). The distribution of esterase in the nervous system and other tissues of the insect Rhodnius prolixus. Q. JI rnirrosr. Sci. 99, 441450. Wigglesworth, V. B. (1960). The nutrition of the central nervous system in the cockroach, Periplaneta arnericana L. The r d e of perineurium and glial cells in the mobilization of reserves. J. exp. Biol. 37, 500-512. Yamasaki, T. and Ishii, T. (1957). Studies on the mechanism of action of insecticides. IX.Repetitive excitation of the insect neurone soma by direct current stimulation and effects of D.D.T. I n “Japanese Contributions of the Study of the Insecticide Resistance Problem”, pp. 163-175. Publ. by the Kyoto University for the W.H.O., Kyoto.

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Yamasaki, T. and Narahashi, T. (1954). Japan. J . appl. Zool. 19, 16. Yamasaki, T. and Narahashi, T. (1958). Synaptic transmission in the cockroach. Nature, Lond. 182, 1805-1806. Yamasaki, T. and NarahaShi, T. (1959). The effects of potassium and sodium ions on the resting and action potentials of the cockroach giant axon. J. Insect Physiol. 3, 146-158. Yamasaki, T. and Narahashi, T. (1960). Synaptic transmission in the last abdominal ganglion of the cockroach. J. Insect Physiol. 4, 1-13.

Spiracular Gills H. E. HINTON Department of Zoology, University of Bristol, England I. Introduction . . I t . The Plastron and the Environment 111. Definition of Stages during Metamorphosis . . I V . Interrelation of Pupal and Adult Respiratory Systems V. Polyphyletic Origin of Spiracular Gills . A. Origin from spiracles . B. Origin from respiratory horns . VI. Isolation of Tissue in Spiracular Gills . A. Origin of isolated tissue . R. Significance of differences in time of isolation of gill tissue C. Attributes of isolated tissue . D. Function of isolated tissue . E. Tissue reservoirs . . VI1. The Plastron . A. Structure . B. Respiratory efficiency . V111. Resistance of Gill to High Pressures . 1X. Spiracular Gills of Pupae . . A. Psephenidae B. Torridincolidae . C. Tanyderidae . D. Tipulidae . E. Simuliidae . F. Blepharoceridae . G. Deuterophlebiidae . H. Empididae . 1. Dolichopodidae . J. Canaceidae . . X. Spiracular Gills of Larvae . A. Torridincolidae . . B. Sphaeriidae and Hydroscaphidae . References .

. . . . .

. . .

. . .

. . . . . . . . . .

. . . . .

.

. ,

.

.

.

65 66 68 71 74 82 84 84 84 90 92 97 101 104 104 105 112 114 114 120 122 123 133 141 144 146 148 152 156 156 158 159

I. I N T R O D U C T I O N The structures that have been called spiracular gills (Hinton, 1953) are either modifications of the spiracle, or of the body wall adjoining the 65

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spiracle, or of both the body wall and the spiracle. Spiracular gills are chiefly confined to the pupal stage of some flies and a few beetles, but they also occur in larvae of beetles of the suborder Myxophaga. All spiracular gills except those of the Chironomidae bear a plastron of one kind or another, and oxygen entering through the water-air interface of the plastron diffuses through air channels directly into the spiracles. In chironomid flies the spiracular gills lack a plastron, and a continuous layer of haemolymph is interposed between the water and the spiracles. Plastron-bearing spiracular gills are known to occur in about 1400 species of pupae. Among these they have been independently evolved on at least 14 occasions. The first good account of the structure of a spiracular gill was that given by Pulikovsky (1927) of Simulium, and it is only in the last decade that enough has been discovered about the different kinds of gills to make possible a comparative account of their evolution and function. It now seems likely that relatively few spiracular gills will be discovered that do not conform to one or another of the types already known or described below. Many of the conclusions about the function of the gills are necessarily based entirely on their structure because often only preserved specimens have been available, but it is hoped that this review will provide a basis for future studies of their physiology. 11. T H E P L A S T R O A NN D T H E E N V I R O N M E N T

The kind of physical gill called a plastron is a gas film of constant volume and an extensive water-air interface. Such films are held in position by a system of hydrofuge structures, and they resist wetting under the hydrostatic pressures to which they are normally subjected in nature. In well-aerated water a plastron enables the animal to remain immersed indefinitely, when it obtains the oxygen it requires from the ambient water. Nearly all aquatic insects with a plastron are found in waters in which the oxygen pressure is maintained at a fairly high level, such as rapidly flowing streams, the littoral of large lakes, and intertidal areas. This ecological distribution is no accident because a plastron also serves as an efficient means of extracting oxygen from the insect should the oxygen pressure of the environment fall below that of its tissues. A few adult beetles-Macroplea (Chrysomelidae) and some weevils-appear to be the only plastron-bearing insects that live in standing water such as marshes and ponds in which the oxygen pressure may drop greatly, especially at night. Presumably these insects move to the upper layers of

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the water or climb out should the oxygen pressure of the water fall sufficiently to cause the plastron to work in reverse. The well-aerated waters in which most plastron-bearing aquatic insects occur are characterized by frequent and often large fluctuations in the level of the water. These environments are thus alternately dry and flooded; and the great selective advantage of the plastron can only be understood in relation to this particular feature of the environment. In water, the plastron provides the insect with a relatively enormous area for the extraction of dissolved oxygen without necessarily involving any reduction in the impermeability of its cuticle. In air, the plastron provides a direct route for the entry of atmospheric oxygen that does not involve water-loss over a large area because the connection between the plastron and the internal tissues may be very restricted. Thus the capacity of the insect to avoid the loss of water when the environment is dry is not necessarily impaired by the provision of a plastron. Plastron-bearing spiracular gills provide a relatively enormous surface area for diffusion (Figs 24, 44),and their structure is such that they do not collapse under the hydrostatic pressures to which they are normally subject. When the pupa or pharate adult is exposed above water, atmospheric oxygen enters through the interstices of the plastron nearest to the spiracle, and the remainder of the plastron ceases to function. Under these conditions the area available for humidity exchanges between the saturated air in the tracheae, and the drier air outside is usually about as limited (Figs 15-16,46, 54) as in terrestrial insects with normally formed spiracles. The ratio between the surface area of the plastron interface and the weight of the pupa differs greatly among species with plastron-bearing spiracular gills (Table I). It may be suspected that, at least in some of the species in which the ratio is poor (e.g. Eutanyderus wilsoni), oxygen uptake through the plastron of the spiracular gills is supplemented by cutaneous respiration. In others, such as the Simuliidae which have a plastron interface of lo5 to lo6 p2/mg fresh body weight, all oxygen requirements may be satisfied by the plastron, and cutaneous respiration through the body wall cuticle may well be of no significance. For instance, using the flagellate Bod0 sulcatus, Fox (1921) found that the pupa of Simulium absorbed oxygen through its spiracular gills but little or none through its body wall cuticle. Once it becomes possible to specify the features of certain aquatic environments that confer a great selective advantage upon the plastron method of respiration, it also becomes possible to distinguish the same features in other environments that at first sight may appear to be very

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different indeed. As we have seen, the essential features of the aquatic environment in which plastron respiration is advantageous are : (1) that it is alternately dry and flooded; and (2) that when flooded the oxygen pressure in the water is maintained at a high level. Both of these essential features of the aquatic environments in which plastrons are evolved are reproduced in the environment of terrestrial insects; the difference is merely that for the terrestrial insects the flooded periods are less frequent and shorter. Whenever it rains heavily, a very large number of terrestrial insects are submerged beneath a layer of water. The immobile stages of these insects, the eggs and pupae, are normally glued or otherwise fastened to the substrate and necessarily remain submerged until it has stopped raining and the water has evaporated or flowed away. Thus in most climates many of the terrestrial insects are alternately dry and flooded. To be submerged in water for several hours or even days is no rare and isolated event but is a normal hazard of their environment. It therefore seems likely on theoretical grounds that many terrestrial insects will be adapted to respiration in water in a manner no less complex than are many aquatic insects. Among terrestrial insects plastron respiration was first reported for the eggs of some flies (Hinton, 1959, 1960d), and it already appears that instances of plastron respiration among terrestrial insects will be much more numerous than among aquatic ones. 111. D E F I N I T I O O FN S T A G E S DURING

M E T A M O R P H O S IS Remarkable differences exist between insects in the time of development of some of the structures of spiracular gills in relation to other events, for instance, the larval-pupal ecdysis. In order to appreciate the existence of such differences, and still more to understand their functional significance, some of the terms normally used to describe events during metamorphosis require more precise definition. The term apolysis (Jenkin and Hinton, 1966) is now used for the freeing of the epidermal cells from the old cuticle, whereas the term ecdysis is used for the act of shedding the cuticle of the previous instar or stage by the new instar or stage. Thus apolysis, together with the secretion of at least some layers of the new cuticle and digestion of part of the old, is an essential preparation for an ecdysis. The term moult is avoided; it has been used indiscriminately to describe the events at both apolysis and ecdysis. Although in recent years some writers have tried LO restrict the use of the term moult for the event here described as

SPJRACULAR GJLLS

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apolysis, this restriction has not been generally accepted because the colloquial meaning of “moult”, like that of la mue and der Haufungsakt, has always been the actual shedding of the old cuticle. It is commonly held that a new instar or stage of an insect begins when the cuticle of the previous instar or stage is shed, that is, at ecdysis. However, after apolysis the old cuticle is only mechanically connected to the living animal, and between the old and new cuticles there is a layer of moulting fluid that may be replaced by air before the emergence of the new instar or stage. To define the instar or stage as beginning at ecdysis is to define out OF existence certain stages of some insects. For instance, in some Chironomidae (Diptera) and some Psychidae (Lepidoptera) the adult female never sheds the pupal cuticle but remains within it and there lays eggs that develop parthenogenetically. In these insects the pupal-adult apolysis is not followed by a pupal-adult ecdysis. Sometimes, as in all Diptera-Cyclorrhapha, the larval-pupal apolysis is not followed by a larval-pupal ecdysis, but at emergence the adult sheds both larval and pupal cuticles simultaneously. After the larvalpupal apolysis, the pupae of most Cyclorrhapha thrust their respiratory horns through preformed areas of weakness in the hardened larval cuticle (puparium). It is sometimes held that this event constitutes the larval-pupal ecdysis and initiates the beginning of the pupal stage. Apart from other difficulties, such a view would deny a pupal stage to those individuals that miss the preformed areas of weakness and so never manage to thrust the respiratory horns through the larval cuticle. In the previous paragraph some of the difficulties created when ecdysis is considered to initiate a new stage are illustrated by examples from insects with atypical life-histories. Much more serious is the fact that unless apolysis is recognized as the event that initiates the new stage, the physiology and activities of the insect between apolysis and ecdysis are inevitably attributed to the previous stage. For instance, in most published studies of the biochemistry of the pupa, the biochemistry of the pharate adult is attributed to the pupa and the early part of the pupal stage, the pharate pupa, is ignored. Frequently activities are attributed to the pupa of which it is not capable, e.g. Imms (1957, p. 582) says of Trichoptera, “ In some species the pupae are able to swim freely about at the surface until they find some suitable objects to crawl out upon.. . .” But it is the fully formed adult that swims and makes use of especially modified pupal legs to do so: the pupa is quite unable to move its legs, and the leg muscles are not striated before the pupaladult apolysis. Each new instar or stage of an arthropod begins enclosed within the

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cuticle of the previous instarorstage.Thatpartof itthatisspentenveloped by the cuticle of the previous instar or stage is known as the pharate phase or stage, or simply as the pharate larva, pupa, or adult, as the case may be (Hinton, 1946, 1958~). Thus the pupal stage of most endopterygotes, for instance, includes both the pharate pupa and the nonlarval-pupal .polyrlr larval-pupal I ecdysis

pupal-adult apolysis

,

pupal-adult ecdyrls

typical endopterygote la

Simullidae upal-adult ecdysis

Dauterophlebildac larval-pupal apolysir

larval-pupalecdyrir pupal-adult apolyrir

ecdysis

/

Tipulidas larval-pupal apolysis

pupal-adult

larval*adult ecdYris pupal-adult ecdyrir

1

.... Cyclorrhrpha

FIG.I . Variations in the duration of the stages during metamorphosis, and the relation between the times of apolyses and ecdyses of different insects.

pharate pupa. However, in a fair number of endopterygotes, particularly in the order Diptera, the pupal-adult apolysis occurs before the larval-pupal ecdysis and in consequence the whole of the pupal stage is passed as a pharate pupa and there is no non-pharate pupal stage.

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71

The time that the new instar spends in the pharate phase varies enormously; it is generally a few hours or a day or so in the larval instars and often several days in the pupal stage. The time that many insects spend as pharate adults may be much greater than the time spent as non-pharate adults. For instance, flies of the family Deuterophlebiidae have a pharate adult stage of about 8 to 9 days (12-18°C) but live only about 2 h r after the pupal cuticle is shed (Kennedy, 1958, 1960). Many, but not all, of the insects that are said to pass the winter as pupae do so as pharate adults; the higher Lepidoptera that diapause in the nonpharate pupal stage are a notable exception. Variations in the duration of the stages during metamorphosis, with special reference to insects with spiracular gills, are shown in Fig. 1. During the pharate stages the insect does not disrupt all relations with the external environment; many of its demands require the maintenance of some relations with the external environment although these have now to be effected through the old cuticle. Because of this necessity, the function of many of the structures of the cuticle cannot be understood by reference to the activities of the stage that produced the cuticle but only by reference to the activities of the succeeding stage. The pupal spiracular gills are a good example of such structures; in many insects they function only during the pharate adult stage and in no way during the pupal stage. O F P U P A LA N D A D U L T R E S P I R A T O RSYSTEMS Y In most of the insects with spiracular gills, the pupal stage is passed entirely within the cuticle of the last larval instar, and the pupal-adult apolysis has already taken place by the time that the larval cuticle is shed and the pupal cuticle is for the first time directly exposed to the environment. It is thus evident that in these insects the functional significance of most of the structures of the pupal cuticle can only be understood in relation to the activities of the pharate adult and not in terms of the activities of the pupa. But even in those insects in which the larval cuticle is shed well before the pupal-adult apolysis, the duration of the pharate adult stage is not necessarily shorter (Fig. 1). Thus the pupal spiracular gills function either exclusively in the pharate adult stage or both in this stage and the pupal stage. The interrelation between the pupal and adult respiratory systems during the pharate adult stage of an insect without spiracular gills is shown in Fig. 2. These interrelations are essentially similar to those of

IV. I N T E R R E L A T I O N

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insects with spiracular gills (Fig. 54). In the insect shown in Fig. 2, as in all of those with pupal spiracular gills, the ecdysial process of the spiracles is of the elatevoid kind (Hinton, 1947b) at the larval-pupal

ecdrrial

tube

.. .-

of adult spiracle

c d u l l CUIICI.

- adull arche. CL

pupal aachea

FIG. 2. Interrelation of pupal and adult respiratory systems during the pharate adult stage of an insect without spiracular gills. Fifth abdominal segment of the beetle SclerOc ~ p h o nfuscus Armstrong (Psephenidae). The relative positions of the pupal and adult cuticles have been slightly altered in order to bring the spiracles of the two stagesinto the same plane. The pupa has the specialized elateroid type of spiracle, and the adult has the Primitive type of spiracle. The non-functional spiracle of the larva is on the ventral side of the pleural extension, and the ecdysial tube of the pupa is formed in the same position. The functional pupal spiracle is on the dorsal surface almost exactly above the external opening of the ecdysial titbe. through which the larval tracheae were withdrawn at the larval-pupal ecdysis. (After Hinton, 1966d).

ecdysis and of the primitive kind at the pupal-adult ecdysis. The functional opening of the pupal spiracle is secreted by a cylinder of epidermis that is formed to one side of the cylinder of epidermis that secretes

K G I 1.I.S

73 the ecdysial tube of the spiracle of the larva. The simultaneous formation of two spiracles at this time becomes a functional necessity because the tracheal linings of the larva cannot be withdrawn at the larval-pupal ecdysis through the newly formed pupal spiracle because cuticular bars that extend across its external opening absolutely prevent this. Instead, the old tracheal lining of the larva is withdrawn through the ecdysial tube and its unoccluded opening. After the larval-pupal ecdysis, the ecdysial tube collapses, and its external opening is closed by centripetal contraction of the peripheral integument. All that then remains of the external opening of the ecdysial tube is a small and usually heavily sclerotized patch of cuticle often called the external or stigmatic scar. At the junction of the collapsed ecdysial tube with the atrial chamber of the functional spiracle there is another scar, the internal scar (Fig. 2). The modification of the pupal spiracle or respiratory horn to form a spiracular gill always prevents its use for the withdrawal of the tracheal lining of the last larval instar and is therefore always accompanied by the formation of an ecdysial tube. In most insects, and in all of those with spiracular gills, the structure of-the adult spiracle does not preclude the withdrawal of the lining of the pupal trachae through it at the pupal-adult ecdysis. A special ecdysial tube is thuspot required and none is formed after the pupal-adult apolysis. The e:dysial process of the spiracles at this stage reverts to the primitive type. In most insects the muscles of the regulatory apparatus of the spiracles are carried over directly or with some modification from the larval stage and are inserted into the regulatory apparatus of the pupa and later the adult. In the pharate stages, the regulatory apparatus appears to function effectivelyto control water loss despite the fact that the tracheae of the previous instar are enclosed within the trachea of the new instar (Fig. 2). However, all beetles and all flies that have spiracular gills in the pupal stage have larvae that lack both a spiracular regulatory apparatus and the appropriate muscles. At the larval-pupal apolysis a regulatory apparatus is not usually formed, e.g. Tipulidae, Deuterophlebiidae, and Dolichopodidae. In the exceptional case of the Simuliidae (see p. 137), a regulatory apparatus is formed at the larval-pupal apolysis but does not become functional until the pupal-adult apolysis, when it is operated by muscles from within the body of the pharate adult (Hinton, 1957b). The problem of water loss through the pupal spiracles should the insect be exposed above water first arises when the larval cuticle is shed. In flies with spiracular gills this event normally occurs after the pupal-adult S PI R A C U L A

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apolysis. Water loss through the functional spiracles is therefore under the effective control of the pharate adult. In a few flies there is a very brief period after the larval cuticle is shed and before the pupaladult apolysis. If the insect should be exposed above water during this time, water loss through the spiracles would be considerably reduced by the dense cuticular meshwork of the spiracular atrium (Hinton, 1947a). which by pressure resistance and frictional drag might be expected to prevent tidal movements of air. In experiments with the larvae of some Diptera (e.g. Tipulu) it was found that the dense cuticular network of the atrial chamber of the post-abdominal spiracles prevented the bulk flow of air through these spiracles even when considerable pressure was applied to the tracheae (Hinton, 1953). Whether or not a significant reduction in the duration of the nonpharate pupal stage preceded the loss of the regulatory apparatus of the spiracles, it is certain that with the loss of this apparatus a further premium is placed upon the completion of the pupal stage before or very shortly after the larval cuticle is shed. This suggestion receives support when we consider the situation of the Diptera as a whole. Dipterous larvae are descended from a common ancestor that had lost the regulatory apparatus of all spiracles. No recent dipterous larvae have re-evolved such an apparatus, although a few Stratiomyidae have a regulatory apparatus that is external to the post-abdominal spiracles (Hinton, 1953). So far as is known, none of the spiracles of any pupa of the order Diptera has a functional regulatory apparatus. The Diptera are the only order of pterygote insects that as a primitive feature lack a regulatory apparatus on both larval and pupal spiracles, and they are the only order characterized by lacking or having only a very brief nonpharate pupal stage: most Diptera (i.e. all Cyclorrhapha) lack a nonpharate pupal stage, and in the remainder such a stage is also absent or, with few exceptions, of very short duration.

V. P O L Y P H Y L E TO I CR I G I NO F S P I R A C U L AGRI L L S Plastron-bearing spiracular gills are a particularly instructive example of the polyphyletic origin of a complex structure that always subserves the same function. As previously noted, they are confined to the pupal stage except in beetles of the family Torridincolidae in which they are present in both larval and pupal stages. They have been independently evolved in at least 5 different groups of beetles and 11 groups of flies, as follows:

SI'I R A < ' U I , A K < i I 1.1,s

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Coleoptera (Myxophaga) 1 . Torridincolidae: larvae 2. Torridincolidae: pupae 3. Sphaeridae and Hydroscaphidae Coleoptera (Polyphaga) 4. Psephenidae: one genus of Eubriinae 5. Psephenidae: all Psephenoidinae 6. 7. 8. 9. 10.

I I. 12. 13.

Diptera (Nematocera) Tanyderidae : Euranyderus Tipulidae (Eriopterini): Lipsothrix Tipulidae (Limoniini): Antocha and Orimargula Tipulidae (Limoniini): Geranomyia Tipulidae (Limoniini): Dicrunomyia and Idioglochinu Simuliidae Blepharoceridae Deuterophlebiidae Diptera ( Brachycera-Orthorrhapha)

14. Empididae : some Hemerodrominae 15. Dolichopodidae : Aphrosylus

Diptera ( Brachycera-Cyclorrhapha) 16. Canaceidae : Canuce The evidence that spiracular ,gills are independently evolved in each of the groups of Diptera listed above has been given recently (Hinton, 1962, 1965, 1967a), as has that for their independent origin on two occasions within the family Psephenidae (Hinton, 1966d). Apart from the Tipulidae, it appears that plastron-bearing spiracular gills have been independently evolved but once in each family of flies. In the Tipulidae, however, such gills have been evolved on no less than four separate occasions. At first sight it seems most improbable that very complex structures that so much resemble each other should have been independently evolved so many times within a single family. However, looking at the matter a little more closely, no other conclusion seems possible. In order to demonstrate the independent origin of a structure in two groups, it is only necessary to show that the common ancestor of the two groups did not possess such a structure. In the family Tipulidae spiracular gills occur only in two of the tribes of the subfamily Limoniinae, the Eriopterini and the Limoniini. In both tribes they occur

76 H. E. HINTON only in specialized freshwater or marine forms and are absent in the vast majority of genera which are terrestrial and have respiratory horns. A brief summary of the evidence for four independent origins of spiracular gills in the Limoniinae is as follows: ( I ) Lipsothrix is a specialized semi-aquatic member of the tribe

FIG.3. Stereoscan electron micrograph of the plastron lines of the tipulid Anrochu vitripennis. The structure of the plastron lines of Orimargula australiensis is similar. A

transmission electron micrograph said by Hinton (1965)to be of the cuticular bridges of a plastron line of the latter species is in fact one of the meshwork of the spiracular atrium. FIG.4. Stereosdan electron micrograph of the plastron lines of Antocho hifdu. (After Hinton, 1966b).

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Eriopterini, all other genera of which have, so far as is known, simple respiratory horns or respiratory horns modified for root-piercing (Hinton, 1953). (2) Geranomyia is a cosmopolitan genus of some 275 described species. So far as is known, the larvae and pupae live in moist terrestrial environments, and a few are semi-aquatic or aquatic. Only two species have invaded the sea, and apparently only these have spiracular gills. (3) Dicranomyia is a cosmopolitan genus with an even larger number of described species. The habits of the larvae and pupae are similar to ,those of Geranomyia. A few species are intertidal, and these have spiracular gills that are sometimes very similar to those of Geranomyia, whereas the terrestrial species of Dicranomyia, like the terrestrial species of Geranomyia, have respiratory horns similar to those of most genera of Limoniini. Although the spiracular gills of the intertidal species of Geranomyia and Dicranomyia are similar, this similarity is due to convergence, a convergence of structure that has not extended to the adults of the two genera. (4) Antocha pupates in freshwater streams as does Orimargula. The latter is considered by some writers to be no more than a subgenus of the former, and in any event there is no good evidence against the view that both are derived from a common ancestor with spiracular gills. The fine structure of the plastron of Antocha (Figs 3-5) and most species of Orimargula is more or less identical. The fine structure of the plastrons evolved on each of the four occasions noted above is rather different. In Lipsothrix (Figs 8 , 38) it consists of grooves in the gill wall covered by a network; in Antocha and Orimargula of tubes that are circular in section (Figs 5-7), the upper part of each tube consisting of interlinked transverse cuticular bridges and the lower part of a groove in the gill wall; in Geranomyia the plastron is composed of more or less separate groups of upright struts that have their distal apices joined together (Figs 10-1 1, 41); and in Dicranomyia (Figs 12, 43) the plastron is a cuticular network supported by vertical struts. Although the structure of the plastron in each of the four groups of Tipulidae is very different, such differences are not nor can be certain evidence of their independent origin any more than identity of structure is certain proof of a common origin. In one species of Orimargula, 0. hintoni Alex., the plastron is not only unlike any other Tipulid but is unlike that of any other insect with spiracular gills. In this species the plastron consists of evenly spaced

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tubercles about 0-3p wide and 0 . 2 5 ~high over nearly all the surface of the gill (Fig. 9), which is otherwise very similar to that of other species of the genus. Jf no mistake has been made in placing this species

FIG.5. Diagram of a typical plastron line of Anrocha bifda. (After Hinton, 1966b). FIG. 6. Anrocha bifda. Transverse section through the fourth gill branch before the apex of the arm of the spiracular atrium. (After Hinton, 1966b). FIG.7. Transverse section near the apex of the fifth gill branch of Antorha bifda. (After Hinton, 1966b).

in Orimurgulu, it is necessary to believe that its plastron is either (a) derived from the kind of plastron lines present in other species of Orimargulu and also in Antochu; or (b) that it represents another independent origin of plastron respiration in the family. The second view is not,

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however, compatible with a common origin for plastron-bearing spiracular gills in Antocha and Orimargula. About 25 species of Idioglochina have been described. The available evidence suggests that all are intertidal and that the pupae of all have plastron-bearing spiracular gills (Hinton, 1967a). Idioglochina is of

FIG.8. Stereoscan electron micrographof the plastron lines near the dorsal rim of the gill of the tipulid Lipsothrix rernota. (After Hinton, 1967a).

FIG.9. Stereoscan electron micrograph of the plastron on one of the gill branches of the tipulid Orimargula hintoni.

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course derived from terrestrial ancestors. If the genus is really distinct from Dicrunomyia, which I doubt, it must represent a fifth instance of the independent evolution of spiracular gills in the Tipulidae. However, even if the genus is not distinct from Dicranomyiu, the possibility remains that two different groups of the genus Dicrunomyia have inde-

FIG. 10. Stereoscan electron micrograph of the plastron of the tipulid Geranoniyitr unicolor.

FIG.1 1 . Stereoscan electron micrograph of the plastron of Gerunoniviu unicolor.

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FIG.12. Stereoscan electron micrograph of the plastron of the tipulid Dicranomyia nionostromia. Compare with the diagram of the plastron of Dicranomyia trifjlatnentosa shown in Fig. 43. FIG. 13. Stereoscan electron micrograph of the plastron of the tipulid Idioglochina marmorata.

pendently invaded the sea from the land or fresh water. At an electron microscope level, there is no difference between the structure of the plastron of Idioglochina marmorata (Osten-Sacken) (Fig. 13) and the species of Dicranomyia. This suggests a common origin but is not certain proof of one.

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Before speculating upon the origin of spiracular gills from spiracles it is necessary to consider briefly plastron respiration by spiracles adapted for atmospheric respiration. The spiracles of both terrestrial and aquatic insects are hydrofuge and resist the entry of water under more or less considerable hydrostatic pressures. When such insects are covered by water, a water-air interface is established across the spiracles. The spiracles thus inevitably function as plastrons. However, even in peripneustic insects the total water-air interface is usually only about 500 to 2000 p2/mg fresh weight, whereas in those insects that have recognizable plastrons the surface area/mg ranges from 1.5 x lo4 (pupa of Eutunyderus (Hinton, 1966~))to 2-5 x lo6 pz/mg. Most terrestrial and aquatic insects readily drown if their spiracles are not exposed to air at relatively frequent intervals. I t may therefore be supposed that the amount of plastron respiration through spiracles that are normally formed, and which are adapted for atmospheric respiration, is insignificant in relation to the oxygen demands of the animals when they are submerged even in well-aerated water. Thus, because of the hydrofuge nature or normally formed spiracles of both terrestrial and aquatic insects there is, paradoxically, plastron respiration in insects without a plastron; that is, without structures especially evolved for plastron respiration. Possible stages in the evolution of spiracular gills from normally formed spiracles in the pupae of the Psephenidae are shown in Fig. 14. Many of the species of this family pupate close to the edges of streams where a slight rise in the water level is likely to submerge them. Many species have their spiracles on the apices of cylindrical tubercles so that the openings of the spiracles are well above the general surface level of the body. The selective advantage of this seems clear enough: the animals are able to utilize atmospheric oxygen even when covered by a thin layer of water. The structure of the pupa of a Japanese Psephenid, Mutueopsephefius japonicus Mats., is particularly significant in this connection. Mutueopsephenus is derived from ancestors with functional spiracles on the first seven abdominal segments. In Mutueopsephenus the spiracles of the first three segments are normally formed, those, of the fourth and fifth segments are non-functional, and those of the sixth and seventh segments are greatly enlarged. The total water-air interface of the spiracles is 1.2 x lo3 p2/mg. If all seven pairs of spiracles were the size of the second and third pairs, the surface area in relation to weight would be 1.0 x lo3 p2/mg. There has thus been no significant gain in surface area as a result

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of the changes undergone in the structure of the spiracles. The selective advantage of the great enlargement of the spiracles of the sixth and seventh abdominal segments must therefore be concerned with some function other than plastron respiration. The lengthened spiracles enable the pupa to utilize atmospheric oxygen even when covered by a thin layer of water. Every increase in length has a selective advantage in that it enables the pupa to utilize atmospheric oxygen when covered by correspondingly thicker layers of water. At some stage in the process of lengthening the spiracles and the consequent increase in their surface

C

D

E

FIG. 14. Origin of different types of spiracular gills from or near a spiracle like that shown in A . (c)Spiracular gill of the simuliid type. ( D ) Spiracular gill of the tipulid type. (E) Presumed stage in the origin of a spiracular gill of the kind found in fsephcnoides from the stage represented in B. (After Hinton, 1966d).

area, plastron respiration becomes significant in satisfying oxygen demands. When this stage is reached, selective pressure for an increase in plastron area as such begins to operate for the first time. It has been shown (Hinton, 1966d) that if all seven pairs of spiracles in Mataeopsephenus were as large as those of the sixth and seventh segments, the

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linear dimensions of the spiracles would only have to be increased by a factor of 2.2 for the animal to have a plastron area per mg equal to that of Eutanyderus, but- plastron respiration would probably be significant long before the linear dimensions of the spiracles were doubled. B. O R I G I N FROM R E S P l R A T O R Y H O R N S

In the Tipulidae and Dolichopodidae sufficient information is available about the terrestrial ancestors of the aquatic species to state that the spiracular gills of the latter have been evolved from the respiratory horns of the former. The typical and apparently also the primitive type of respiratory horn in both families consists of a modification of the body wall adjacent to the spiracle to form a long cylindrical or flattened appendage. The spiracular atrium extends up the appendage. Gas exchanges are effected through aeropyles that extend from the surface of the appendage to the spiracular atrium. When such pupae are submerged, a water-air interface is inevitably established across the aeropyles, but the ratio of the area of interface to the live weight of the animal is probably usually no better than that of terrestrial insects with normally formed spiracles. In order to transform such a respiratory horn into a plastron-bearing spiracular gill, no fundamental change in general structure is required (Fig. 14D). All that is required is an increase in the total number of aeropyles or an alteration of the surface between the existing aeropyles so that a superficial air film can be held against a pressure difference. In the tipulids with spiracular gills the number of aeropyles that drain the plastron is not appreciably greater than the number of aeropyles in the respiratory horns of their terrestrial relatives. I t therefore seems evident enough that in this family the evolution of a plastron from an ordinary respiratory horn adapted for atmospheric respiration did not involve an increase in the number of aeropyles but only a change in the structure of the surface between them. The plastron-bearing gill of Aphrosylus (Dolichopodidae) is also a transformed respiratory horn (Hinton, 1967c), but the intermediate stages in the evolution of the gill from the respiratory horn are not as evident as in the Tipulidae. VI. I S O L A T I O N

OF

T I S S U EI N S P I R A C U L AGRI L L S

A. O R I G I N OF ISOLATED TISSUE

I t has previously been noted (Hinton, 1965) that where one stage with long appendages of one kind or another is succeeded by a stage without

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such appendages, the fate of the epidermis that secreted the cuticle of the appendages of the previous stage may differ as follows : (1) It may be completely retracted out of the old appendages and incorporated into the body of the new stage. This appears to be its normal fate, particularly when the appendages of the new stage are relatively short. The fate of the retracted epidermis is probably similar to that of the body wall epidermis at either the larval-pupal or pupal-adult apolysis. In some of the more specialized endopterygotes there is a total replacement of the epidermis at the larval-pupal apolysis from “imaginal” discs, e.g. Diptera-Cyclorrhapha. At both the larval-pupal and pupal-adult apolyses of the less specialized endopterygotes, and the pupal-adult apolysis of the more specialized endopterygotes, the epidermal cells divide and the new cuticle is secreted by a new generation of epidermal cells (see review by Hinton, 1963). The extent to which the retracted epidermal cells divide and contribute to the new generation of epidermal cells is not known. (2) The epidermis that secreted the cuticle of the appendages of the previous stage may be cytolized in situ and the products of cytolysis utilized by the new stage. (3) The epidermis may be partly retracted, partly cytolized irt situ, and partly discarded. Selman (1960) has shown that at the larval-pupal apolysis of Siulis luturiu L. the epidermis of each gill segment retracts one segment at a time with a pause between each phase of retraction. During each period of retraction some epidermal cells are left behind by the retracting tip. Some of these cells break down but others remain behind just distal to the joints of the gill segments in pockets enclosed by a moulting membrane. In Siulis the epidermal cells that are eventually retracted come to lie within the body of the pupa and are later cytolized. None of them appear to be incorporated into the body wall of the pupa. (4) The epidermis may be completely discarded in the lumen of the appendages of the previous stage, as in the spiracular gills of most insects at the pupal-adult apolysis or even (Simuliidae) before this apolysis. The tissue discarded within the appendages of the previous stage generally ceases to have a function and therefore a selective value for the new stage. The discarded tissue is a loss to the insect. We may suppose that the cost of this loss to the insect is in the long run less than the cost of retrieving it. It may be retrieved, as we have seen, either by retraction into the body of the new stage or it may be cytolized within

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the old appendages and then absorbed into the body of the new stage. To cytolize and absorb the tissue in a long appendage, or to retract all of the epidermis from within it, requires,time: it requires a prolongation of the moulting period, a period when the insect is particularly vulnerable. In short, it seems that in some insects tissue is discarded in certain appendages of the previous stage, because, on balance, the loss it represents as a reserve of food is less than the cost that would be incurred in other ways by its absorption or retraction into the body of the new stage.

FIG.15. Diagram of the base of the Fpiracular gill of the tipulid Liptothrix remora; (B) basal occluding membrane. (After Hinton, 1955).

The habit of discarding epidermis and blood in the lumen of the pupal spiracular gills has been independently evolved on at least 1 1 occasions, as follows : Coleoptera (Myxophaga) I . Torridincolidae Coleoptera (Polyphaga) 2. Psephenidae: one genus of Eubriinae Diptera (Nematocera) 3. Tanyderidae 4. Tipulidae (Eriopterini) : Lipsofhrix 6. Simuliidae 5. Tipulidae (Limoniini) 7. Blepharoceridae

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8. Deuterophlebiidae 9. Chironomidae Diptera (Brachycera-Orthorrhapha) 10. Empididae 1 1. Dolichopodidae

In most insects the isolation of the tissue in the gill occurs at the pupal-adult apolysis. During the whole of the pupal period the lumen of the gill is continuous with the haemocoele, and the epidermal layer of the gill wall is continuous with that of the body wall of the pupa.

FIG. 16. Longitudinal section of the spiracular gill of the tipulid Antocha uitripennis in the very early pharate adult stage when the dissociation of the epidermal cells isolated in the lumen of the gill is beginning. The figure also shows the connection between the spiracular atrium and the trachea; B, basal occluding membrane. (After Hinton, 1957a).

At the pupal-adult apolysis the epidermis of the body wall retracts away from the opening into the gill lumen and in time loses all connection with the epidermis of the gill wall, which is left behind in the lumen of the gill. During the pupal-adult apolysis a thin sheet of cuticle, the basal membrane (Fig. 15) is secreted across the opening into the gill lumen. When the adult cuticle is secreted, the tissue in the gill lumen is isolated not only by the basal membrane but also by a layer of moulting

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C FIG.17. Spiracular gill of Deriterophkbiu inyoensis. The larval cuticle has been shed but the pupal-adult apolysis has not been completed. (A) Section through the base of the gill. (B) Section through the base of the gill at the junction of the plastron and the atrium of the spiracle; b, blood cell. (C) Section through the base of the gill showing the junction of the plastron and the atrial chamber of the spiracle and also the connection still maintained between the epidermis of the gill and that of the body wall through the orifice at the base of the gill; compare with Fig. 18. (After Hinton, 1962).

fluid and the cuticle of the body wall of the pharate adult. The isolation of the tissue in the spiracular gill from the tissues in the body of the insect is both mechanically and physiologically complete : after the

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secretion of the basal membrane the gill may be detached from the insect without in any way affecting the activity or the duration of life of the isolated tissue. It is not known if the basal membrane is secreted by the retracting epidermis of the body wall, or by the epidermis within the gill, or by both. Besides epidermis, a quantity of blood is also isolated in the gill. In most species the blood appears to lack blood cells. Blood cells are

FIG. 18. Section through the base of the gill of Deurerophlebiu inyoensis showing the plastron opening into the atrial chamber of the spiracle. The larval cuticle has been shed but the pupal-adult apolysis is not complete and the epidermis of the gill is still connected to the epidermis of the body wall. (After Hinton, 1962).

certainly absent in the gills of Antocha rjitripennis (Fig. 16), a species that has rather transparent gill walls that make it possible to examine the contained tissue in the live animals. In Orimargula australiensis a few round and spindle-shaped blood cells were found in serial sections of the gill cut before the larval-pupal ecdysis, but such cells were never seen in the lumen after this ecdysis (Hinton, 1965). No blood cells were found in Lipsothrix and others, but none of these were heat-fixed, and it is therefore probable that if any hyaline haemocytes (coagulocytes) were present they would have disintegrated by the time the gills were examined. A few cells presumed to be blood cells were found in the gill

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of Deuterophlebia (Figs 17- 18) on the lumen side of the basement membrane at the time of the larval-pupal ecdysis (Hinton, 1962). However, these cells may well have been epidermal cells that had undergone a precocious dissociation and migration. B. S I G N I F I C A N C E O F D I F F E R E N C E S I N T I M E O F ISOLATION OF G I L L TISSUE

As might be expected, the isolation of tissue in pupal appendages by the secretion of a basal membrane usually occurs at the pupal-adult apolysis whether this be before or after the larval-pupal ecdysis, that is, irrespective of whether or not there is a non-pharate pupal stage. However, the tissue can be isolated in some other ontogenetic stage. For instance, in the Simuliidae the gill tissue is isolated long before the pupal-adult apolysis, whereas in the Tipulidae and Deuterophlebiidae it is isolated at the pupal-adult apolysis. In the Tipulidae (Antocha and Orimargula) the isolation of the gill tissue occurs before the larval-pupal ecdysis, whereas in the Deuterophlebiidae it occurs after the larvalpupal ecdysis. These differences in the time of isolation of the gill tissue in respect to both the pupal-adult apolysis and the larval-pupal ecdysis are of considerable biological significance. The spiracular gills of the Deuterophlebiidae are relatively small in relation to the size of the animal, and there is room enough between the pupal and larval cuticles for them to attain their full size before the larval-pupal ecdysis. There is no noticeable expansion of the gill after the larval-pupal ecdysis when it comes into contact with the ambient water. Before the larval-pupal ecdysis the gill is as large as i t is after the larval-pupal ecdysis, although after this ecdysis there is some slight straightening of the branches. If a spiracular gill is to absorb water in a liquid phase when it comes into contact with the ambient water and expand further, there must be parts of the gill that are ( I ) free of plastron so that there is no layer of gas between the gill wall and the ambient water because of the plastron of spiracular gills becomes gas-filled before the larval-pupal ecdysis, and (2) parts of the plastron-free gill wall have to be readily permeable to water. In the Deuterophlebiidae all parts of the base and branches of the gill support a hydrofuge meshwork that holds the plastron gas, and consequently the tissue in the gill cannot readily absorb water. It thus appears that in the Deuterophlebiidae there is no selective pressure for the secretion of a basal membrane and the isolation of the gill tissue before the larval-pupal ecdysis. If water is to be absorbed by the tissue in a spiracular gill after the

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larval-pupal ecdysis, it would seem necessary to isolate the tissue in the gill from the haemocoele before water is absorbed. And the tissue is indeed isolated in all instances where it is known that water is absorbed after the larval cuticle is shed. The necessity for isolating the gill tissue before the larval-pupal ecdysis if water is to be absorbed may in part account for the absence of a non-pharate pupal stage in many insects with spiracular gills: it may be less difficult to alter the normal time relations between the larval-pupal ecdysis and the pupal-adult apolysis than to secrete a basal membrane before the pupal-adult apolysis. In such Tipulidae as Antocha and Orimargula, the spiracular gills are also relatively small in relation to the size of the animal, and there is room enough between the pupal and larval cuticles for them to attain more or less their full size before the larval-pupal ecdysis. However, in both of these genera, unlike the Deuterophlebiidae, the efficiency of the gill is, as we shall see, dependent upon the maintenance of a high internal pressure. After the larval-pupal ecdysis the high internal pressure is produced and maintained by the gill tissue absorbing water. In order to do this it is not only necessary that there be some plastron-free areas of the gill wall but that at least some of these be permeable to water. If the gill tissue is to absorb water after the larval-pupal ecdysis, it must be sealed off from the haemocoele if the gill is not to provide an uncontrolled route for the entry of water into the living animal. It would therefore appear that spiracular gills of the Antocha and Orimargula kind could only be evolved if the tissue in the lumen of the gill were isolated from the living animal by the secretion of a basal membrane before the larval-pupal ecdysis. The spiracular gills of the Simuliidae vary greatly in size and outward form but their basic structure is identical. Most of the gills are large in relation to the size of the animals, as they are in the primitive Gymnopaidinae. Although a number of groups have gills that in relation to the size of the animal are as small as those of the Tipulidae and Deuterophlebiidae, the balance of evidence suggests that these are specialized and not primitive types. It would seem that a spiracular gill of the basic structure of recent forms is primitively large. This is not, of course, to imply that it originated as a large gill but only to say that by the time it attained the basic structure of recent forms it was a large gill. As in all other insects with spiracular gills, growth of the gill is completed before the larval cuticle is shed, and the plastron is also filled with gas before this event. When the gill comes into contact with the ambient water, water is absorbed through a ventral, plastron-free membrane. In due course the mounting internal pressure ruptures this

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membrane. Rupture of the ventral membrane occurs after the gill is fully stretched but before the concentration of solutes in the isolated tissue and ambient water is equalized. Because water is readily absorbed into the gill through the ventral membrane, the selective pressure for the isolation of the gill tissue before the larval-pupal ecdysis would seem to be the same as that noted for Antocha and Orimargula. But in the Simuliidae the basal membrane is not secreted at the pupal-adult apolysis, as in the Tipulidae. Instead, it is secreted long before the pupal-adult apolysis but after the beginning of the larval-pupal apolysis and after the evagination of the gill into the space between the pupal and larval cuticles (Pulikovsky, 1927). After the evaginated gill has grown to a certain size, the basal membrane, which in the Simuliidae consists of thick cuticle, is secreted. The secretion of the basal membrane prevents any further very appreciable enlargement of the gill, which remains considerably wrinkled and curled in the moulting fluid, between the pupal and larval cuticles. After secretion of the basal membrane, the gill will rapidly expand if it is dissected out and placed in water. It does not expand so long as it is bathed by moulting fluid because the solute concentration of the moulting fluid is greater than that of the tissue isolated in the gill (Hinton, 1957b). At various times diseased pharate pupae of Simulium ornatum Meig. and other species have been found. In some of these diseased individuals the osmotic pressure of the moulting fluid was evidently lowered because the spiracular gills were expanded between the pupal and larval cuticles. The expansion of the gills appeared to inconvenience the pupae greatly, but it was difficult to dissociate the mechanical effects of gill expansion from such other effects as the disease may have had on the activities of the pharate pupae. It would appear that it is the relatively large size of the gills of the Simuliidae that requires the isolation of the gill tissue long before the larval-pupal ecdysis in order to keep the volume of the gill small while it remains between the larval and pupal cuticles. I n this connection it may be noted that it is not length that is critical but volume: in the Empididae (Fig. 70) the gills are long slender filaments that expand to more or less their full length between the pupal and larval cuticles, but each gill does so in a tight coil and so occupies relatively little space. C. A T T R I B U T E S O F I S O L A T E D T I S S U E

The epidermal cells isolated in the spiracular gills of the beetle Torridincola and in the gills of flies of the families Tanyderidae, Tipulidae, Deuterophlebiidae, Chironomidae, and Dolichopodidae show no signs

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of degeneration during the life of the pharate adult. In the Tipulidae and Chironomidae degeneration of the isolated epidermis does not become evident until some time after the adult has shed the pupal cuticle and flown away. It seems probable that the epidermis in the gills of Torridincola and in flies of the families Tanyderidae, Deuterophlebiidae, and Dolichopodidae also remains in good condition for a time after the emergence of the adult. In the tipulid, Lipsothrix remota, it is common to find that the normal appearance of the nuclei is preserved for as long as 14 days (at 20°C) after the isolation of the epidermal cells, that is, for 5 days after the emergence of the adult. In this species, nuclei appreciably more than 10 p wide were never seen in freshly isolated tissue, but from the 11th to 14th days-2 to 5 days after the pupal cuticle is shed-some or most of the nuclei of about a third of the gills examined were 15 to 20 p wide. I . Dissociation and migration of isolated epidermis In some Tipulidae (Antocha, Orimargula, Dicranomyia) and in the Deuterophlebiidae the closely packed and more or less hexagonal epidermal cells become rounded and separated from one another and from the cuticle to which they had previously adhered. In Antocha uitripennis dissociation and migration is completed about 12 hr (at 20°C) after the larval-pupal ecdysis. After dissociation is complete, most of the epidermal cells are present as loose clusters of irregular size and shape, more or less in the middle of the lumen far from the gill walls to which they had previously adhered. In Antocha and Orimargula a similar and contemporaneous dissociation and migration of the epidermal cells lining the spiracular atrium and its branches takes place. Because the outer gill walls are more or less transparent, it has been possible to observe this process of dissociation and migration in whole live mounts of Antocha and Orimargula. There is no dissociation or migration of the isolated epidermal cells in the tipulid Lipsothrix, nor, so far as is now known, in any other families of insects in which tissue is isolated in the gills. 2. Wound repair b-v isolated tissuc The isolated tissue in the gills has been found to be competent to repair damage to the gills whenever it has been put to the test irrespective of whether or not such repairs have a functional significance. It may therefore be supposed that the isolated tissue in the gills is probably also competent to repair wounds in those insects that have so far not been tested. I t would appear that the competence to repair is a general

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property of isolated epidermis and blood so long as the tissue does not degenerate. The competence to repair not only extends throughout the life of the pharate adult but also persists after the emergence of the adult. It has been shown that damage to the gill walls is repaired for long periods (16-20°C) after the emergence of the adult in the following species: 109 hr in Lipsothrix remota Walk.; 7 hr in L . nervosa Edw.; 120 hr in Antocha vitripennis Meig. ; a “few hours” in Orimargula australiensis Alex.; and 10 hr in Geranomyia unicolor Haliday. Cuts or tears in the gill wall are repaired by a plug of dark amber to black material. This material is sclerotin ;it withstands hot concentrated nitric acid for more than 10 min, but it is rapidly dispersed in concentrated nitric acid saturated with potassium chlorate. Furthermore, darkening and hardening of the wound plugs does not occur in the presence of phenylthiourea. The wound plugs can be completely dispersed in a saturated solution of potassium hydroxide held at 180°C for 30 min, from which it appears that if they contain chitin, the chitin does not form a discrete framework. Repair of the spiracular gills by the isolated tissue consists in no more than the formation of a plug of sclerotin that seals the lumen from the ambient water; any part of the plastron that is damaged is never repaired. Damage to the integument of an insect often results in a migration of the surrounding epidermal cells to the site of the wound damage, leaving a peripheral zone where the epidermal cells are very sparse (e.g. Wigglesworth, 1937). I t has been suggested (Wigglesworth, 1965) that the dead or injured cells release substances that provide the stimulus for the migration of the uninjured cells. The possibility of a similar migration of the isolated epidermal cells in a gill towards the site of an injury has been investigated in Lipsothrix remota and Antocha vitripennis (Hinton, 1957a), but no evidence of migration was obtained in either insect. Experiments with Antocha were particularly conclusive because the gill walls are fairly transparent and it is possible to see the cells clearly through them. Gill branches in the pharate adult stage were ligatured near their bases and then cut off and mounted in water in cavity slides. The tip of a gill branch was then cut off and a cover-slip placed over the preparation so that it could be viewed with an oil immersion lens. In this way it was possible to view the cells within 2 min of an operation. At different focal levels, the cells behind the cut edge form different distribution patterns. These were immediately drawn with the aid of a camera lucida, and from time to time afterwards the patterns of cell distribution were compared with those already

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drawn. By determining the position of any group of cells with a point on one of the plastron lines, it was also easy to detect movements of the pattern as a whole. No alteration was observed in any pattern within 7 hr of the operation. In a few instances, the pattern as a whole shifted forwards a few microns, but without any distinguishable change in the relative positions of the cells. Since it is difficult to suppose that a large number of cells can migrate without changing even slightly their relative positions, it was concluded that such slight forward shifts were the result of swelling as water was absorbed through the gill walls. If the cells migrate at all, it seems unlikely that their migration was restricted to the first 2 min or so after an operation when they were not under observation.

3. Cryptobiosis of isolated tissue Insects that pupate in streams have to contend with the fact that to be exposed above water during dry spells is a normal hazard of their environment. In many of the places where such tipulids as Antocha and Orirnargula are found, a drop of a few inches in the water level will suffice to leave them exposed. When so exposed, the spiracular gills shrivel and dry long before the body of the insect is noticeably affected. The great reduction in the surface area of the dry and collapsed gill is of no significance because atmospheric oxygen is taken directly through the numerous aeropyles. Because of the osmotic pressure of the isolated tissue, the gill swells out and its surface area is restored when the water level rises and it is again submerged. But if tears or holes in the gill wall are not now to result in a disproportionate loss of surface area, the isolated tissue that has been dried must when wetted again be capable of once more repairing such damage. Unless breaks in the gill wall are repaired before the concentration of solutes in the lumen of the gill and the ambient water come into equilibrium, the positive internal pressure necessary for maintenance of turgidity and maximum surface area cannot be established. For these reasons it seemed likely that when dry the isolated tissue might enter a state of cryptobiosis, a term that has been restricted (Hinton, 1960b) to describe a state when metabolic activity comes reversibly to a standstill, as it necessarily does in all those plants and animals that, for instance, grow after a sojourn in liquid helium. In preliminary experiments, gills of Antocha vitripennis in the pharate adult stage were dried at 75% R.H. and 24°C for 17 hr, immersed in tap water for 24 hr, and the tips were then cut off some branches of each gill. All cut tips were repaired. Later gills were dried for varying periods up to 70 days over phosphorus pentoxide. Wounds made in such gills

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6 hr after immersion in water were repaired as quickly as if they had not been dehydrated. Drying over phosphorus pentoxide for periods as long as 70 days did not affect the percentage or speed of repair of gills when wounded a few hours after immersion. However, when the period of immersion in water was extended beyond about 12 hr the percentage of gills that repaired fell and the time they required to form a coloured wound plug increased. Gills dried over phosphorus pentoxide for periods of 27 hr to 70 days and then heated in an electric oven for 1 to 2 hr at 103°C before immersion in water successfully repaired, as did gills dried for 70 days and then heated to 130°C for 30 min. These experiments leave little room for doubt that the isolated tissue in the gills is cryptobiotic when dried. Once it is shown that cryptobiosis by dehydration can be induced in the isolated epidermis of the spiracular gills of some insects, the question arises as to whether this is a very special property of the isolated epidermis or whether it is a rather general property of the epidermal cells. At physiological temperatures most insects are killed long before their moisture content falls as low as 20%. However, there is reason to believe that certain tissues may survive conditions that are rapidly fatal to the organism as a whole. When pharate adults of Antocha were dehydrated for periods up to 70 days, it was found that wounds made in the integument of the dead adults after they were immersed in water were repaired by plugs of sclerotin. Wounds were also repaired when the adults were dehydrated and then heated to 103°C for 1 hr. The epidermis of larvae killed by dehydration would also repair wounds after the dead larvae were immersed in water. Thus, cryptobiosis can be induced by dehydration in the epidermis of both larvae and adults of Antochu just as it can in the pupal epidermal cells in the gills. The capacity of epidermal cells to survive total dehydration may be very widespread in insects. When larvae of four families of beetles (Gyrinidae, Haliplidae, Elminthidae, Psephenidae) and a fly (Chironornus) were killed by dehydration the epidermis nevertheless survived (Hinton, 1957a). Evidence of the kind cited above that the epidermis is alive is open to objection. It may, for instance, be claimed that the epidermis is not alive but that damage to it simply destroys a barrier between enzyme and substrate that enables the tanning reaction to proceed. Selman (1961) has produced unexceptionable evidence that in the larva of Sialis lutariu L., an insect that does not tolerate complete dehydration, certain blood cells nevertheless survive the treatment : the coagulocytes clot and form pseudopodia after dehydration as they do before dehy-

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dration. The typical clotting behaviour of blood cells occurs even after dehydration for 17 months and heating when dry to 100°C for 30 min. In this connection it may be recalled that the larva of Polypedilum vanderplanki Hint. (Chironomidae) will tolerate dehydration to a moisture content of 1% for several years. In the dehydrated state it will tolerate immersion in liquid gases, including liquid helium, and it will metamorphose successfully when heated to 68°C for l l hr or to 102-104°C for 1 min (Hinton, 1960a, b). In the larva of this insect the metabolism of all tissues can be brought reversibly to a standstill. There is some evidence that larvae of two other insect species can enter a state of cryptobiosis by dehydration, although a number can do so by freezing in the hydrated state. D. F U N C T I O N O F I S O L A T E D T I S S U E

The immediate terrestrial ancestors are not known of some of those groups of insects that have the habit of isolating tissue in the spiracular gills, e.g. Simuliidae, Blepharoceridae, and Deuterophlebiidae. In some of these, such as the Simuliidae, the attributes of the isolated tissue are always exploited, but not enough is known about the ancestry of these to determine whether the isolation of tissue preceded the exploitation of the isolated tissue or whether both the habit of isolating tissue and the exploitation of its attributes were simultaneously evolved. However, the evolutionary history of the spiracular gills of the Limoniini (Tipulidae) is sufficiently known (see p. 75) to leave no room for doubt that the selective pressures that resulted in the isolation of tissue in the gills were effective long before there was any selective pressure to exploit the attributes of the isolated tissue. Thus in the Limoniini the discarded tissue in ,the gills comes to have a selective value it did not originally possess. It is possible that the evolutionary history of such groups as the Simuliidae was similar in this respect. The tissue isolated in the gills of the Eubriinae, many Tipulidae, Deuterophlebiidae, and Dolichopodidae has no function. Since the spiracular gills of all of these, with the possible exception of those of the Deuterophlebiidae, are of more recent origin than those of the Simuliidae, further but indirect support is had for the view that the isolation of tissue in such groups as the Simuliidae had a long history before the isolated tissue began to be exploited.

I . Contraction ojgill before the larval-pupal ecdysis As we have seen (p. 91) the tissue in the gills of the Simuliidae is isolated by the secretion of a basal membrane long before the larvalpupal ecdysis but after the evagination of the gill into the moulting fluid

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between pupal and larval cuticles. Although the gill grows considerably in size during the pharate pupal stage, it nevertheless remains wrinkled and curled and thus occupies much less space than it otherwise would. Once the basal membrane is secreted, the gill behaves as a simple osniometer. If it is dissected out from the pharate pupa and placed in water, it immediately uncurls and expands. During the time that the gill is bathed by the moulting fluid, it does not swell out because the osmotic pressure of the moulting fluid ( > 3.6 atm) is greater than that of the isolated tissue in the gill lumen (Hinton, 1957b). The importance to the insect of keeping the gill as small as possible during the pharate pupal stage has already been discussed (pp. 91-92).

2. Expansion of gill after the larval-pupa f ecdysis After the larval-pupal ecdysis spiracular gills for the first time come into direct contact with the ambient water. On contact with the water, the gills of the Simuliidae quickly swell and straighten out and assume their normal appearance. The unwrinkling and swelling out of the gill is of course quite independent of any activity of the living animal because the blood and epidermis in the gill lumen are isolated from the living animal (Pulikovsky, 1927; Hinton, 1957b, 1964). In the Simuliidae water absorption occurs through a thin, oval or circular, plastron-free membrane at the ventral base of the gill. Experiments with gills dissected out from the pharate pupa show that the gill can be caused to expand and contract many times by appropriate adjustments of the osmotic preswre of the fluid in which they are immersed, providing only that the turgor pressure is never allowed to rise sufficiently to rupture the ventral membrane. The gills of the tipulids Anrocha and Orimargufa also slightly expand and straighten out on contact with the ambient water. Those of the Empididae, which are in a tight coil during the pharate pupal stage, become quite straight. In all of these expansion and straightening out of the gill is caused by absorption of water by the isolated tissue. 3. Sclerotization of gill wafls If the gills of the Simuliidae are immersed in a fluid of high osmotic pressure immediately after the larval-pupal ecdysis in water, they will contract greatly, almost to their size before the cuticle was shed. They can then be placed in water and will again expand. Alternate contractions and expansions can be caused only a limited number of times-6 to 10 in some experiments. The gradual hardening of the gill walls appears to be the factor that sets a limit to the number of times that the '

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gill can be contracted and expanded. If the gill is allowed to expand, but not enough to rupture the ventral membrane, and then kept like this for a few hours before placing it in a fluid of high osmotic pressure, it wrinkles and flattens but does not again curl anything like as much as it was in the moulting fluid immediately before the larval cuticle was shed. During the process of hardening, the walls of the gills also become somewhat darker. The results of the experiments described above suggest that water entering the gill lumen triggers the epidermal cells further to sclerotize the cuticle. When the process of expansion is not interfered with, mounting turgor pressure quickly ruptures the ventral membrane. In one series of sections of the gill of Simulium ornatum fixed 2 hr after the larval cuticle was shed, few nuclei were intact. After the rupture of the ventral membrane the tissue degenerates and in due course its remnants are washed out of the gill or consumed by micro-organisms; and usually long before the emergence of the adult no tissue can be found in the lumen, which now often contains diatoms or protozoa or both that have entered through the orifice formed by the rupture of the ventral membrane. 4. Maintenance of gill turgidity

In the tipulids Antocha (Hinton, 1957a) and Orimargula (Hinton, 1965) absorption of water by the isolated tissue is responsible for the maintenance of the positive internal pressure necessary to avoid crumpling of the non-rigid gill walls such as would bring the plastron lines close together and so diminish their scanning capacity. When intact gills are fully expanded and there is no net transference of water, the turgor pressure is 4.3 atm in Antocha vitripennis and 4.5 to 4.8 atm in Orimargula australiensis. In the field these pressures will be reduced by the osmotic pressure of the stream water, which was not determined but is probably rarely more than about 0.1 atm. The spiracular gills of all Tipulidae are derived from rigid respiratory horns like those of their terrestrial relatives. The spiracular gills of many aquatic species continue to be rigid, but in Antocha, Orimargula, and possibly a few of the marine species of Dicranomyia, the gill walls have lost their rigidity. In spiracular gills of the tipulid type it would appear that loss of rigidity is only made possible by the presence of the isolated tissue. In their natural habitats in streams the gill branches are moved freely to and fro, a movement brought about by fluctuations of water pressure and the elastic recoil of the gill. Such movements of the gill branches must thin out the diffusion boundary layer, and any thinning

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out of this layer of course increases the efficiency of the plastron. In the tipulids that have retained rigid walls, such as Lipsothrix, Geranomyiu, and many of the marme species of Dicranomyia, the isolated tissue ceases to function very shortly after the larval-pupal ecdysis and may then be destroyed without in any way affecting the activity of the pharate adult: in such species it is not turgor pressure that prevents crumpling but the rigidity of the gill walls. The spiracular gills of Eutanyderus (Tanyderidae) flatten in a few minutes when exposed to room humidities (ca 70% R.H.) despite their rather thick walls (Hinton, 1966~).It thus seems possible that the isolated tissue may play an important part in maintaining turgidity of the gills by absorbing water when they are immersed. 5 . Rupture of gill wall and equalization of internal and ambient pressures In the Simuliidae the shape of the gill is maintained at any hydrostatic pressure by means quite unlike those used by any other insect. As already noted, when the larval cuticle is shed and the gill first comes into contact with the ambient water, water is absorbed by the isolated tissue through an oval or circular, plastron-free membrane at the ventral base of each gill. In due course the mounting internal pressure ruptures the ventral membrane. Rupture occurs after the gill is fully stretched but before the concentration of solutes in the isolated tissue and the ambient water is equalized. With the rupture of the delicate ventral membrane, the ambient water has free access into the lumen of the gill. The shape of the gill then becomes independent of variations in hydrostatic pressure because the pressure inside the gill is always equal to that outside it. 6 . Strengthening of gill by cuticularization In most Blepharoceridae, the gill branches which first develop as tubular structures later flatten to exclude a lumen (Hinton, 1962). During the process of flattening, the isolated tissue in the lumen becomes entirely cuticularized. Each branch of this kind of gill therefore consists of two sheets of heavily sclerotized cuticle pressed closely together. It seems evident that distortion of the general shape of the branches of a gill of this kind would require pressures of quite a different order of magnitude from those of the deepest rivers; and the Blepharoceridae normally pupate only a few inches below the surface of streams and the littoral of large lakes. A number of Blepharoceridae have gills of unusual shapes, e.g. Paulianina (Figs 56, 58-59,62,66-67), but the formation of none of these has been studied. In the gill of Aphrosylus (Dolichopodidae) there is some cuticulariza-

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tion of the isolated tissue. The gill is not distorted when dried in a vacuum of 4 x mm Hg (Hinton, 1967c), but its rigidity is probably chiefly or entirely due to the sclerotization of its walls rather than to the partial cuticularization of the isolated tissue. E. T I S S U E R E S E R V O I R S

1. Function

In some Tipulidae the efficient functioning of the gill depends upon the maintenance of a high positive internal pressure by the isolated tissue. In some of these, e.g. species of Anrocha, the gill may have appendages that are in no way directly concerned in respiration but function simply as tissue reservoirs (Hinton, 1957a, 1966b). In order to understand the function of the tissue reservoirs, it is necessary to consider the sequence of events that follow upon a break in the gill wall. Because of the very great difference between the turgor pressure of the gill ( > 4 atm) and the hydrostatic pressure of its normal environment ( < 0.1 atm), any break in the gill wall results in the rapid outflow of blood or both blood and dissociated epidermal cells, and the internal and external pressures are almost immediately equalized. Blood begins to clot at the site of the break, and the clot is sclerotized in due course. Now, as soon as a clot is formed and the gill is sealed, the osmotic pressure of the tissue that has remained within the gill causes an inflow of water through the semipermeable walls of the gill. Thus a little pressure is almost immediately exerted against the newly formed clot or wound Plug. The rise in the turgor pressure within the gill is slow because the speed of the rise depends upon diffusion rates. Since breaks in the gill wall are usually successfully repaired both in the field and in laboratory experiments, it follows that the gradual increase in the mechanical strength of the wound plug brought about by tanning effectively outpaces the gradual increase in turgor pressure. The turgor pressure that can be obtained after the gill is broken and repaired becomes less after each break, because the total volume of isolated tissue in relation to the volume of the gill is less after each break, owing to the explosive outflow of tissue as the internal and external hydrostatic pressures are equalized. The number of times that a gill can be damaged and again restore a high degree of turgidity depends entirely upon the total amount of the isolated tissue. The amount of tissue lost when the gill is torn, or the end of a branch broken off, is only that necessary to bring about the immediate equalization of the internal and external hydrostatic

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pressures because a clot is formed at the site of the injury before there is time for water to diffuse into the gill and force out more tissue. The amount of blood lost through a hole of any given size therefore depends upon the difference between internal and external pressures and not upon the total volume of tissue. This being so, it follows that the greater the volume of tissue the less it is diluted after an injury, and consequently the greater the volume the greater the ,lumber of injuries that can be sustained before dilution becomes so great that the pressure drops below the level necessary to maintain the turgidity of the gill. The turgor pressure of the intact gill of Antocha vitripennis is 4.3 atm. After the tips of two of its eight branches were cut, the turgor pressure 24 hr later did not exceed 3-5 atm (Hinton, 1957a). The turgor pressure of the gills of Orimargula australiensis is 4.5 to 4.8 atm. When the tip of the second branch was removed the pressure after repair was slightly less than 4.5 atm, which was the lowest pressure recorded for any of the intact gills (Hinton, 1965). The positive internal pressure of the gills of Antocha and Orimargula far exceeds any required in its natural environment in order to resist a change in shape. It is scarcely likely that such species are ever exposed to hydrostatic pressures of the order of 4 atm; in severe floods they might be exposed to pressures of as much as 0.5 atm. But the limiting factor will be wetting of the plastron, which is likely to occur at hydrostatic pressures of about 0.3 atm, judging from experiments on the European .4ntocha ritripennis. A positive internal pressure of 4 to 5 atm is nevertheless not such an unnecessarily wide safety margin as it first appears to be. In nature the gills are often damaged in one way or another, as is evident from the frequency of the dark brown or black wound plugs. The most frequent type of damage found in specimens in the field is loss of the tip of one or more of the gill branches. As we have seen, the concentration of solutes within the gill falls each time that it sustains an injury. It therefore follows that if the turgidity of the gill is to be restored after the loss of some of its tissue, the osmotic pressure of the tissue must exceed that required to maintain the turgidity of the intact gill. In a previous section it was noted that in all Limoniini tissue is isolated in the respiratory horns. The osmotic pressure of this tissue has not been measured, but it may be supposed that it is not very different from that of the other body tissues. In the Tipulidae spiracular gills are always evolved from respiratory horns. When some Tipulidae become aquatic and evolve spiracular gills, they exploit the turgor pressure produced by the isolated tissue, and this property of the isolated tissue is an example of what is meant by preadaptation.

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2. Independeent evolution Tissue reservoirs are present in both known pupae of Antocha and in one of the two known pupae of Orimargula. Antocha vitripennis (Fig. 19) has two hemispherical blood reservoirs on the inner base of the gill, both of which lack a plastron. It is also possible that the dilated apex of the fourth gill branch functions as a tissue reservoir although it supports a plastron (Hinton, 1957a). In Antocha bifida there are two plastron-free tissue reservoirs, a basal appendage on the first gill branch and the 0.20 mm I

FIG. 19. Inner base of right spiracular gill of the tipulid Anrochrt oitripent1i.v showing the two large blood reservoirs, 91 and 92. (After Hinton, 1957a).

whole of the eighth or outermost gill branch (Fig. 20). In Oriniargula hintoni a single tissue reservoir is present at the ventral bases of the first four gill branches. The tissue reservoirs of the two species of Antocha are not homologous structures and are therefore of independent origin. Thus, among the four known species of these two genera, there are no less than three independent origins of blood reservoirs. Perhaps the most interesting of the blood reservoirs is the eighth gill branch of .4ntocha biJ7du. The modification of this branch to form a blood reservoir has involved a great reduction of the arm of the spiracular atrium, which extends only 0.06 mm into the branch, or only about one-seventh as far as it extends into the first seven gill branches. This great reduction of the atrium has resulted in a corresponding increase in the amount of tissue that can be contained in the ventral

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reservoir. It is difficult at first sight to account for the absence of plastron lines on the eighth gill branch. The shortening of the spiracular atrium would of course increase the length of the diffusion path if plastron lines were present, but even if they extended to the tip of the branch, the value of nx, would only be 0.09, and the whole of the plastron would be effectively used. The eighth gill branch lies more or less flat against the front of the prothorax and head. Here both the cocoon and the bases of the other gills hinder circulation of water and therefore the efficiency of any plastron in this position. It seems probable that it is for these reasons that the plastron has been lost on this gill branch. 0.20

mm

FIG.20. Right spiracular gill of the tipulid Anrocha bifida. A small blood reservoir is present near the base of the first branch and the whole of the eighth gill branch has become a blood reservoir and has lost its plastron. (After Hinton, 1966b).

V11. T H EP L A S T R O N A. S T R U C T U R E

The plastron of most spiracular gills consists of vertical struts which are branched at their apices in a plane normal to their long axes, the

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horizontal branches forming an open hydrofuge network that provides the water-air interface when the gill is immersed. This is the type of plastron of the Tanyderidae (Fig. 35B), some Tipulidae (Figs 11-13,41, 43), Simuliidae (Figs 49-5 I), Blepharoceridae (Figs 60-61), Deuterophlebiidae (Fig. 69), Dolichopodidae (Fig. 77), Canaceidae (Fig. 79), Torridincolidae pupae (Figs 32-34) and larvae (Fig. 84). A few insects have plastrons that differ slightly or considerably from the usual type, as follows: 1. In the tipulid Lipsothrix (Fig. 38) the plastron is a series of grooves in the gill wall covered by a flat network. 2. In the tipulid Antocha it consists of tubes that are circular in section (Fig. 5), the upper part of each tube consisting of interlinked transverse cuticular bridges and the lower part of a groove in the gill wall. The structure is the same in some species of Orimargula (Tipulidae). 3. In the tipulid Orimargula hintoni (Fig. 9) the plastron consists of round tubercles between which the air is held, a plastron rather similar to that of the mite, Platyseius italicus, except that in the latter the tubercles are long and papilla-like. 4. In Psephenoides (Figs 25-28) the plastron consists of tubular outgrowths of the spiracle. 5 . In the pupae of a genus of Eubriinae (Fig. 30A) and in the larvae of the Sphaeriidae and Hydroscaphidae the plastron is the dilated spiracle. B. R E S P I R A T O R Y E F F I C I E N C Y

If the plastron is to serve as an efficient respiratory structure it must (I) resist wetting at the hydrostatic pressures to which it is subjected in nature, (2) resist loss of waterproofing from surface active materials, (3) the ratio between the rate of oxygen consumption and the area of the plastron must be satisfactory, and (4) the drop in oxygen pressure along the plastron should be small enough so that the whole of the plastron is effectively used 1. Resistance to hydrostatic pressure

Figures for the resistance of the plastron to wetting have little meaning unless the time that the pressure was applied is stated. For instance, the plastron of the tipulid Geranomyia unicolor is immediately wetted at 1.4 atm, resists wetting at I atm for about 10 min, and resists wetting at 0-3atm for more than 2 hr. The equations that have been given for calculating the resistance of the plastron to wetting by excess pressures

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(e.g. Crisp, 1964) make no allowance for the time that the pressure is applied and therefore do not allow for creep. Wetting by excess pressures always occurs before theTe is a mechanical breakdown of the plastron meshwork, as is also true of the plastrons of insect eggs (Hinton, 1960~). In most adult insects the plastron consists of a hair-pile. In these too the plastron is wetted long before it is crushed. It has been claimed that in the bug Aphelocheirus the resistance to the entry of water is greater than the mechanical strength of the hair-pile (Thorpe and Crisp, 1947a; Thorpe, 1950), but recent experiments on this bug (H. E. Hinton, unpublished) show that its plastron conforms to the rule and is wetted long before there is a breakdown of the hair-pile. The hydrostatic pressure required to force water through the plastron network and wet the plastron varies with the species. All species that have been tested resist wetting at hydrostatic pressures greater than those to which they are normally exposed in nature. That is to say, all species appear to have a wide safety margin. A wide safety margin to wetting from high pressures has also been found in the plastrons of all eggs and adult insects tested. All insects with spiracular gills live in environments liable to sudden fluctuations of water level. Thus, to be exposed above water is a normal hazard. When the gills are exposed above water they must resist wetting by the pressures exerted by raindrops falling on them. Assuming a raindrop to be spherical, the pressure it exerts on striking the plastron is equivalent to a head of water about 1000 times its diameter. A large raindrop of, say, a diameter of 4 mm will thus be equivalent to an excess pressure of about 31 cm Hg. However, this pressure is only exerted for a minute fraction of a second, and it therefore appears that the least resistant plastrons are more than adequately protected against being struck by raindrops. A few examples of the relation between the resistance to hydrostatic pressures and the ecology of the animals may be given. The larva of the fly Canace riusica Haliday feeds on Enteromorpha in the intertidal zone. I t is usually found in the upper half of this zone. but, where fresh water runs into the sea, it may extend into the lower part of the intertidal zone. The plastron resists wetting at an excess pressure of 1 atm for 9 hr, and more than half of those tested resisted 2 atm for 3 hr (Hinton, 1967b). The plastron thus has a wide safety margin even when the fly breeds in the lower part of the intertidal zone. The plastron of the tipulid Antocha iitripennis resists wetting at an excess pressure of 0.3 atm for 24 hr and sometimes as long as 48 hr (Hinton, 1957a). In the great majority of streams in which the species is found, it would never be

SPIRACULAR GILLS

I07

under more than about 4 ft of water even during exceptionally heavy floods. In the largest streams in which it has been found by me, some of its habitats might be under as much as 6 to 8 ft of water during exceptionally heavy floods, but during the summer months the same places will rarely be covered by as much as 5 ft of water. The degree of resistance to wetting by adult plastron-breathers must be such as to provide not only an adequate safety margin at the depths they normally inhabit but also at those depths into which they are likely to be swept by floods. The latter provision does not appear to be necessary for an easily damaged and relatively immobile pupa: the kind of force required to displace the pupa from its cocoon would probably nearly always result in its death. The resistance to excess pressures of the plastron of a number of species of Simulium has been tested (Hinton, unpublished). S. equinum (L.), S. ornatum Meig., S. reptans (L.), and S. monticola Fried. resist wetting at 48 cm Hg for 24 hr, whereas S . costatum Fried. and S. latipes Meig. do not. Of all these species, the plastron of S. equinum is much the most resistant, and when 66 gills were subjected to I atm for 24 hr, the plastron of 68% remained functional. Some species of Siinulium tend to pupate a greater distance below the surface than others, but such differences are trivial compared to the differences in the resistance of their plastrons. However, there appears to be some correlation between the width o f the kind of streams they inhabit and the resistance of their plastrons. For instance, both S. costatum and S . latipes are generally found only in small upland streams, whereas the other species are commonly found in much larger streams and rivers. Of all the species listed. S. equinum is the one most characteristic of large lowland rivers. The depth to which the site of pupation may be flooded in exceptional circumstances is in a general way related to the width of the stream, and this fact would seem to provide a satisfactory explanation for the great resistance of the plastron of S. equinunt and the relatively low resistance of the plastrons of S . costatum and S. la t ipes. 2. Resistance to loss o f water-prooBng The surface tension of the streams in which Antocha, Simulium, and other species with spiracular gills live is usually about 70 dyn/cm or a little more, and in a few of the smaller upland streams it is as high as 73.5 dyn/cm. The figures for the resistance of the plastron to excess pressures cited in the previous section all relate to experiments in water of a surface tension of 72.8 dyn/cm.

I08

H . E . HINTON

As the surface tension falls, the excess pressures required to wet the plastron diminish. When the surface tension is lowered to about 25 dyn/cm, which corresponds to a reduction in the contact angle to 55-60', the plastron of Antocha and Simulium is wetted at virtually no excess pressure. However, surface tensions as low as this are probably never found in natural waters. For instance, it has been found (Hinton, 1960d) that the surface tension of rain water standing on cow pats is reduced to 50dyn/cm and on decomposing flesh to 39 or 40dyn/cm. The surface tension of water in ponds, marshes, and canals is rarely as little as 68 dyn/cm, and even in an abandoned section of a canal where the water was shallow and stinking from decomposing vegetation, the surface tension was 60 dyn/cm (Hinton, 1961b). For reasons already noted, insects with spiracular gills are confined to waters in which the oxygen pressure is always high, which in nature means that the surface tension is also high : they simply do not live in the kind of environments in which it is at all likely for the surface tension to drop more than a fraction below 70 dyn/cm, and they are therefore probably never subjected to a significant loss of water-proofing from surface active materials. 3 . Ratio bet wreii ox)-get1 consumption mid area of'ylustroii The oxygen consumption of the pharate adult of Similium orncrtum in early, middle, and late stages of development is 2.0, 3.7, and 5.6 yllhr. respectively (Hinton, 1964). The highest rate of oxygen consumption obtained over a period of 1 h r was 6 PI. This included periods when the pharate adult was very actively moving within the pupal cuticle. A t this highest rate the value for q/Ai, is 0.16, where q is the rate of oxygen uptake (ml/sec), A the area of the plastron (cm'). and i, the invasion A value of 0.16 is obtained when coefficient of oxygen ( 4 . 8 ~ A = 2 mm2 and no account is taken of the bulging menisci. Using the total area of the plastron ( A = 4mm2), the value of q/Ai, is 0.08. Clearly the true value lies between these figures. The value of y/Ai, for the early period of pharate adult life lies between 0.06 ( A = 2 mm2) and 0.03 ( A = 4 mm2). The values obtained by Thorpe and Crisp (1949) for the adults of six species of insects range from 0.005 for Aphelochirus to 0.12-0- 17 for Hydrophilus piceus. Since their figures are all calculated for the basal respiration rate, it seems clear that the ratio between oxygen consumption and the area of the plastron is very favourable for Simulium ornatum. The rate of oxygen consumption has not been determined for any other insect with spiracular gills. One of the factors that necessarily

S PI R A ('U L A R Ci I L L S

109

determines the respiratory efficiency of a plastron is its area of water-air interface in relation to the weight of the insect. When this is expressed as units of water-air interface per mg of body weight, a comparison can be made between the efficiency of the plastrons of insects of quite different weights. Also, comparisons can be made between species of which only preserved material is available. The validity of such comparisons rests upon the fact that, in general, the rate of respiration of insects is directly proportional to their total wet (live) weight (see review by Keister and Buck, 1964). In Table I the plastron area/wet weight values are given for 17 species with spiracular gills. TABLE I The ratio between the area of the plastron and the wet body weight. In calculating the water-air interface, the menisci are assumed to be flat. About 25% has to be added to these values to take account of the fact that the menisci bulge. In a few instances (e.g. Geranomyia), the geometry of the plastron is such (Fig. 41) that the water-air interface is probably equal to the total area of the plastron. Species __

-

Interface (!8mg)

_____

~

Simrclirrm hirriteri Simulium eqtiinum Simulium canadense Simulium bequarti Simrrlium ornatum Psephenoides marlier1 Psephenoides gahani Simulium rariegatum Apkrosylirs celtiber Eubriinae (genus ?) Dicranomyia triflamentcw Geranomyia unrcolor Psephenoides colatrlis Antocha tttripennis Dicranomyia monostromia Orimargula arrstraliensis Eutanyderus nilsoni

Total plastron area (F2/mo)

1.9 x 10' 1.8 x lo6 1.1 x 106 9.9 x lo6 8.3 x 105 6.2 x lo5 5.4 x 1 0 5 4.6 x 105 3.9 x 105 3.1 x 105 1.0 x 105

-

7.2 x 104

-

6.7 x 104 -

1.5 x 1 0 4

3.5 x 3.4 x 2.0 x 1.8 x

106 106

1.5 x I .8x 7.2 x 8.4 x 1.6 x 6.2 x 4.5 x

lo6

106 106 106

105 105 lo6 105 105 1 . o 105 ~ 3.6 x 105 4.0 x 105 2.9 x 105 3.4 x 104 2.9 x 104

4. Drop in pressure along the plastron When the plastron can withstand an external pressure Ap sufficiently large to supply the animal with oxygen, it is still necessary to discover if this pressure difference exists throughout the whole of the plastron. If the diffusion of oxygen along the plastron towards the spiracle results

H . E. H I N T O N

110

in significant differences in oxygen pressure within the plastron, there will be a decrease in Ap from the spiracle outwards so that only part of the plastron is fully utilized. The entry of oxygen across the plastron network and its diffusion within the meshwork towards the spiracle is shown in Fig. 21A. The solutions given by Thorpe and Crisp (1947b) and Crisp and Thorpe (1948) relate the average drop in oxygen pressure across the plastron G,obtained from the equation = q/Aio, to the actual drop in pressure (Ap), at distance x from the spiracle, (Ap), = G . n x , cosh n(x, - x)/sinh nx, (1) where x1 is the maximum extent of the plastron and nx, is the function (i,x?/Dh)’ 2. The diffusion constant of oxygen within the plastron will (t)

Y ’ Y

(to)

* _ _ _ _ _ _4-

- -- - - --

X I-------*

* ,-----__

A

FIG.21. (A) Flat plastron showing oxygen diffusing into the plastron across faces XOY and X’O’Y’ (vertical arrows) and within the plastron along XO and X’O‘ (horizontal arrows); x1 is the maximum extent of the plastron. (After Crisp and Thorpe, 1948.) (B) Cylindrical plastron. Radial diffusion of oxygen into the plastron (small arrows) and longitudinal diffusion of oxygen within the plastron towards the spiracles (large arrows) ; x, is the maximum extent of the plastron. (After Crisp, 1964).

be similar to its value in air, 0.18 ml cm-2s-1. The values used for D and i,, when calculating nx, should be reduced appropriately to allow for obstructions caused by very dense plastron meshworks. However, as Crisp (1964) points out. errors caused by imprecise assumptions i n making these adjustments are reduced in importance because of the square root sign. The value (AP),~is maximal at the spiracle where .Y = 0, as indicated by equation ( I ) , (Ap), = , = (&%x,)/tanh nx,

111

SPIRACULAR GILLS

and minimal at the outer edges of the plastron where s

=

s1

( A P ) ~ == ~ ,(&nxl)/sinh n x ,

The extent to which oxygen is drawn uniformly over the whole area of the plastron is therefore dependent only on the function nx,. Values of this function are shown in Fig. 22. When nxl does not exceed the value of 1.0, the curve of ( A p ) , against x deviates little from the average

FIG.22. Values for the drop in oxygen pressure across the plastron interface. The abscissa represents the full extent of the plastron drawn to the scale x I = 1.0. The ordinate gives the drop in oxygen pressure below ambient at any point, ( A p ) x ,relative to the average drop across the whole plastron as a whole,&. Each member of the family of curves shown was obtained by substituting the following values of the function ( i o x ~ / D h ) 1( =~M2I ) into cquation (1); -A, 0.1; B, 0.5: C, 1.0; D.2.0; E, 3.0; F. 5.0; and G, 10.0. Thecurves deviate little from A p = 1.0 if n x , < 1.0 (curve C ) but they deviate considerably when nxl > 3.0. (After Crisp, 1964).

AT,

value of but, as it exceeds the value 1 .O, a progressively smaller proportion of the plastron area is effectively used. The plastrons of the respiratory horns of many insect eggs as well as

I12

H. E . H I N T O N

those of the spiracular gills of some of the pupae of Psephenoides are in the form of a tube (Fig. 25A). The outer surface bears the plastron across which oxygen diffuses from the ambient water and diffuses along the tube towards the spiracular atrium (Fig. 21A). If the tube is assumed to be of uniform radius of cross-section R, h must be replaced by R/2 so that nx, = ( 2 i , ~ f / D R ) "So ~ . far as is known, the value of nx, for all insects with spiracular gills is less than 1 SO,and the whole of the plastron is therefore efficient as a gill. Some typical examples are: ( i,sf / Dh) (2i,~f/DR)"~ Antocha brfida 0.19 Antocha aitripeririis 0.29-0' 3 3 Eutanyderus wilsoni 0.49 Simulium ornatuni 0.64-0.97 Psephenoides gaharii 0.30 Psephenoides marlieri 0.42 0.56 Psephenoides uolatilis In many species, such as the species of Siniulium, the branches of a gill may vary considerably in length without a comparable variation in h. For instance, in Simulium ornaturii n.vl = 0.64 for the shortest branch and 0.97 for the longest gill branch. VIII. R E S I S T A N COEF G I L LT O H I G H P R E S S U R E S The resistance of plastron-bearing gills to high hydrostatic pressures involves two distinct things. Firstly, the plastron must resist wetting at the pressures to which the animal is at all likely to be exposed in nature. Secondly, the surface area of the gill on which the plastron is borne must be maintained and crumpling avoided such as would produce pockets where the water flowed more slowly with a consequent thickening of the diffusion boundary layer. Measurements of the resistance to high pressures of the gill and its plastron show that the resistance of the former is never a limiting factor: as pressure is increased, the plastron is wetted long before the shape of the gill is affected. The diversity of the means by which high pressures are resisted by the gills of different groups is further evidence of their independent origin within each group and witness to the fact that from rather similar basic materials in a similar environment different solutions to the same problem have been evolved. The five chief ways in which spiracular gills resist high pressures are as follows: I . B.1, ri*cidit,i,of'gill wal/.v.In the Deuteroplilebiidae the thickness and

SPIRACULAK GILLS

113

rigidity of the gill walls is such that the pressures required to bend the cuticle are probably never attained in the shallow mountain streams in which the species live. Prolonged drying of the gills scarcely alters their shape (Hinton, 1962). The blood within the lumen would also tend to prevent compression. Because there is everywhere a layer of air-the plastron gas-between the ambient water and the wall of the gill, the turgor pressure of the isolated tissue is probably not high since it cannot absorb water in a liquid phase. In many Tipulidae, such as all of the species of Lipsothrix and Geranorizyia and most or all of the species of Dicranornyia and Idioglochina, the rigidity of the plastron-bearing parts of the gill is quite sufficient to prevent crumpling at the pressures to which they are at all likely to be exposed. In all of these species, parts of the gill wall are free of plastron, and the isolated tissue absorbs water with a consequence that the turgor pressure in the lumen of the gill is high. However, none of the species of these genera appears to have evolved the kind of flexible gill, such as that of the tipulids Antocha and Orimargula, that requires a high turgor pressure for its proper functioning. Aphrosylus (Dolichopodidae) also has a gill that resists high pressures by the rigidity of its walls. When the gill of Aphrosylus is dried in a vacuum of 4 x mm Hg its shape is not affected (Hinton, 1967~). 2. BJIcuticular struts across the lumen. In the beetles Psephenoides (Figs 25A, 28) and Eubriinae (Fig. 30B) the integrity of the lumen and therefore the surface area of the gill is maintained by stout cuticular struts across the lumen. In the fly Canace, in which the gill is also a modified spiracle, the plastron meshwork is very thin and has little rigidity, but the atrial meshwork is stout and rigid. When the gill is subjected to high hydrostatic pressures, the plastron is wetted long before there is any question of distorting the atrial meshwork (see also p. 154). 3. By cuticularizationqj isolated tissue. As explained on p. 100, in the Blepharoceridae the gill branches which first develop as tubular structures later flatten to exclude a lumen. During the process of flattening, the tissue isolated in the lumen becomes entirely cuticularized. Distortion of the general shape of such a gill branch would mean deforming a solid piece of cuticle. Although no experiments have been made on the gills, it is evident that the plastron will be wetted long before there is any question of distorting the surface of the gill. 4. By maintenance of a high internal pressure. In the tipulids Antocha and Orimargula the integrity of the gill is maintained by an internal pressure of a little more than 4 atm, as explained on p. 99. In Anrocha

1 I4

H. E. HINTON

uirripennis the plastron is rapidly wetted when the hydrostatic pressure exceeds about 0.3 atm. The other species of Antocha and the species of Orimargda pupate in similar environments, and it is likely that their plastrons are not appreciably more resistant to wetting from high pressures. Jt should be noted that no plastron is known that will resist an excess pressure of more than 3 atm. So far as damage from high hydrostatic pressures is concerned, it is thus clear that distortion of the gill surface is not a limiting factor, quite apart from the fact that even if the internal pressure of the gill is exceeded the distortion will be negligible because of the relative incompressibility of the internal fluid. It is possible that the surface area of the gill in Eutanyderus (p. 122) is also maintained by a high internal pressure.

5 . By allowing water to mter.freely into the gill lumen. In the Simuliidae the shape of the gill is maintained in a unique manner (p. 100). Absorption of water by the isolated tissue leads to a rapid expansion of the gill after the larval cuticle is shed. Further absorption of water results in the rupture of a delicate ventral membrane. Once the ventral membrane is ruptured and the ambient water has free access into the gill lumen, the shape of the gill becomes independent of variations in hydrostatic pressure because the pressure inside the gill lumen will always be the same as the ambient pressure.

IX. S P I R A C U L G AR I L L SO F P U P A E A. P S E P H E N I D A E

Plastron-bearing spiracular gills have been independently evolved in two groups of the Psephenidae, in the Psephenoidinae and in one genus of Eubriinae (Hinton, 1966d). Although in both groups the gills are formed from the spiracle itself, they are very different in appearance and basic structure. I . Psephenoidinae Psephenoides is the only known genus in this subfamily. It is widely distributed in the Oriental Region, and one species, P. niarlieri (Basilewsky), lives in the littoral of Lake Tanganyika in Africa. All of the species pupate on stones beneath the surface of the water in streams or the littoral of large lakes. Functional spiracles, all of which are branched gills, are present only on the second to seventh abdominal segments.

SPIRACULAR GILLS

115

The general appearance of the pupa is shown in Fig. 23. The spiracles of some species, such as P . volatilis Champ. and P . marlieri, are carried at the end of long cylindrical extensions of the body wall that contain the spiracular atrium (Fig. 24A), whereas in other species, such as P . gahani Champ. (Fig. 24B), the spiracles are not lifted beyond the general level of the body wall. It has been suggested (Hinton, 1947a) that the large spine associated with each gill (Fig. 24B) of such species

FIG.23. Dorsal view of the pupa of the beetle Psephenoides gahani. (After Hinton, 1947a).

as P . gahani may assist in keeping debris from rubbing against the gills. The number of gill branches is variable, e.g. 36-40 in P . gahani, 6-10 in P. volatilis, and usually 14 in P . marlieri. The fine structure of the gill branches of some of the species is shown in the accompanying illustrations. In all species the lumen of each branch opens directly into the spiracular atrium. The spiracular atrium has a very dense meshwork or

,ai A FIG.24. (A) Spiracular gill of right side of third abdominal segment of Psephenoides uoloiilis. (B) Same of Psephetioides gnhoni.

(After Hinton, 1947a).

FIG.25. Spiracular gill of Psephenoides voluiilis. (A) Structure of one of the branches. (B) Plastron network of a branch near its base. (C) Connection of spiracular gill with tracheal system. (After Hinton. 1966d).

S P I K A C U L A R GIL1.S

I17

“felt” of fine cuticular struts. The so-called felt chambers of Psephenoides are proportionally longer than in any other known insect, e.g. the felt chamber of P . ziolutilis is a sixth and that of P.guhani a tenth as

FIG.26. Psephenoides uolatilis. Stereoscan electron micrograph of the inside of a gill branch showing that the thin walls are sometimes supported by transverse struts that arise on the thin longitudinal struts. However, the integrity o f the lumen is chiefly maintained by struts that arise from the thick walls, as shown in Fig. 25A. (After Hinton, 1966d). FIG.27. Psephenoides uolatilis. Stereoscan electron micrograph of the thin longitudinal struts of a gill branch with the plastron network between them. (After Hinton, 1966d).

long as the pupa itself. This extraordinary development of the felt chambers of the spiracles would seem to be related to the fact that the pupae are probably often exposed to very desiccating conditions. They are glued to stones in streams, and when the level of the stream falls they may be

I18

H . E. HINTON

exposed above water. Under these circumstances the very long felt chambers no doubt serve to establish an effective humidity gradient and so reduce water loss from the spiracles. The larvae of Psephenoides are apneustic. The regulatory apparatus of the spiracles is developed at the larval-pupal apolysis but no muscles are inserted in it until the pupaladult apolysis (Hinton, 1958b). I n Psephenoides there is a distinct nonpharate pupal phase when the only control of water loss through the spiracles should the pupa be exposed above water is by the felt chambers.

FIG.28. Structure of a branch of the spiracular gill of Psephenoides gcrhnni. The plastron network on either side is supported by slender struts arising from the outer face of the outer longitudinal strut. (After Hinton. 1966d).

In the succeeding stage, the pharate adult, the felt chambers can scarcely be of the same importance because the spiracles have a functional regulatory apparatus. 2. Eubriinae A species of an unknown genus found in streams in South Africa is the only known member of the subfamily that has spiracular gills. The functional spiracles, all of which are modified to form plastron bearing gills, are restricted to abdominal segments two to seven (Fig. 29). Each spiracle is at the end of a more or less vertical projection of the body

SPIRACULAR GILLS

119

wall. The external form of the spiracle is shown in Fig. 30A. About 30 stout cuticular struts extend around the spiracle. Between the struts there is a fine and open hydrofuge network that provides a large waterair interface when the gill is submerged. Beneath the plastron network there is a dense supporting meshwork of cuticular struts (Fig. 30B). The supporting meshwork is also hydrofuge with the result that if pieces are broken off the spiracle its respiratory function is not affected except that its effective plastron area is reduced. 2.0nitn I

I

FIG.29. Dorsal view of the pupa of an unknown genus of Eubriinae (Psephenidae) from South Africa. The spiracular gill of the right side of the fifth abdominal segment is bent down to show its general shape. (After Hinton, 1966d).

The proximal part of the projection that bears the spiracle is a hollow cylinder through which the atrial chamber extends (Fig. 30A). Except for a large semi-spherical bulb near the apex, the opposed walls of the distal two-fifths of the projection are flattened and cemented together, presumably by a cuticularization of the epidermis between them. The semi-spherical bulb is hollow and contains epidermal cells. The isolation of these cells must occur in the early part of the pupal stage. The isolated cells appear to have no function. The fate of the two layers of epidermal cells in the proximal three-fifths of the projection-the cells that secreted the projection itself and those that secreted the spiracular atrium-was not determined with certainty, but they are probably retracted rather than isolated at the time of the pupal-adult apolysis.

I20

H. E. HINTON

FIG.30. Spiracular gill of an unknown genus of Eubriinae (Psephenidae) from South Africa. (A) Lateral view of spiracular gill of fifth abdominal segment. (B) Meshwork supporting the plastron network. (C) Plastron network between two supporting cuticular ribs; in most areas of the gill the network is less regular than in this drawing. (D) Transverse section near the base of the projection of the body wall that bears the spiracle at its apex. (After Hinton, 1966d). B. T O R R I D I N C O L I D A E

The pupa and pharate adult of the only known species in the family, the African Torridincola rhodesica Steffan, remain within the cuticle of the last instar larva, which serves as a cocoon. The median ecdysial line of the thorax and first few abdominal segments of the larval cuticle are split open sufficiently to permit the extrusion of the four spiracular gills, a pair on each of the first two abdominal segments. The long and slender spiracular gills very much resemble those of the

SPIRACULAR GILLS

121

dipterous family Empididae (cf. Figs 31 and 71). The gills are outgrowths of the body wall. The plastron gas is contained in superficial longitudinal canals. These canals (Figs 32-34) are formed by more or less parallel rows of struts that arise perpendicularly from the surface and are branched at their apices in a plane normal to their long axes.

FIGS. 31-34. Stereoscan electron micrographs of the spiracular gill of Torridincola rhodesica. (31) Base of gill of first abdominal segment. (32) Plastron network of same. (33) Section through middle of gill of second abdominal segment. (34) Plastron network at extreme base of gill of first abdominal segment.

The apical branches are more or less arched and connect each vertical strut to the nearest strut on either side. The arched struts are occasionally connected longitudinally to the arches before or behind by short struts of about the same thickness. When the gill is immersed, a waterair interface is established across the interstices between the arches.

122

H. E. HINTON

In the pharate adult stage tissue is isolated in the lumen of the gill (Fig. 33). It seems probable that the tissue is isolated in the lumen of the gill at the pupal-adult apolysis, but it is not known if this event occurs before or after the larval cuticle is split. Torridincola is the only known insect that has a plastron in both the pupal and larval stages. Because neithef the pupa nor the pharate adult shed the larval cuticle, it seems possible that both of these stages make some use of the larval plastron. However, from an examination of the two rather poorly preserved specimens available, it was not possible to determine to what extent, if any, a functional connection remains between the spiracular gills of the larval and the pupal (and later the pharate adult) tracheal systems. The use by one stage of the plastron of a previous stage is common: in all insects with spiracular gills in the pupal stage, the pupal gills are the chief respiratory organs of the pharate adult. Also in a number of parasitic wasps of the family Encyrtidae, the first three larval instars obtain at least part of their oxygen requirements from the plastron-bearing respiratory horn of the egg shell (Hinton, 1961a). C. T A N Y D E R I D A E

The Tanyderidae are a primitive family of nematocerous Diptera with some 30 described species. The pupae of four genera have been described. Of these only Eutanyderus of Australia has plastron-bearing spiracular gills. The gills of Eutanyderus (Fig. 35A) are outgrowths of the body wall adjoining the first pair of thoracic spiracles. The structure of the plastron is shown in Fig. 35B. Thick vertical pillars are more or less regularly distributed over the whole surface of the gill. The tops of the pillars are connected by horizontal struts that are usually 0.15-0.2 thick but are occasionally two or three times as thick. These horizontal struts form the hydrofuge plastron network, and the interstices between them provide the water-air interface when the gill is immersed. The ratio of effective plastron area to wet weight (1.5 x lo4 pz/mg) is less in Eutanyderus wilsoni Alex. than in any other pupa with structures especially evolved for plastron respiration. Epidermis and blood are isolated in the lumen of the gill, presumably at the pupal-adult apolysis. When the gill is exposed to room humidities (ca 70% R.H.),it is flattened in a few minutes despite its rather thick walls (Hinton, 1966~).It thus seems possible that the isolated tissue in the gills may play an important part in maintaining the turgidity of the gills when they are immersed by absorbing water.

123

SPIRACULAR GILLS

ork

FIG.35. Eutanyderus wilsoni. (A) Lateral view of right spiracular gill. (B) Diagram of the structure of the wall of the spiracular gill showing the plastron. (After Hinton, 1966~). D. T I P U L I D A E

Over 13,000 species of Tipulidae have been described. The larvae of the great majority live in moist habitats-in damp earth, rotten wood, or even fungi-but a fair number are semi-aquatic or aquatic, and Idioglochina and a few species of Dicranomyia and Geranontyia are marine (see pp. 77-80). Most of the larvae are phytophagous but a number are predaceous. In the Tipulidae spiracular gills are always evolved from respiratory horns. Gills are evolved in two tribes of the Limoniinae, the Eriopterini and the Limoniini. Tissue is isolated in the gills of both groups at the pupal-adult apolysis. In the terrestrial Eriopterini, the epidermis that secreted both the walls of the horn and the walls of the spiracular atrium is withdrawn into the body of the adult at the pupal-adult apolysis. In the terrestrial Limoniini, on the other hand, the epidermal cells that secreted the walls of the horn and the spiracular atrium are

I24

H. E

HINTON

FIG. 36. (A) Pupa of Lipsothrix remo/o. (After Hinton, 1955.) (B) Pupa of Atltocho vitripennis. (After Hinton, 1957a).

1OP

FIG.37. Section through the spiracular gill of Lipsothrix remofu about a day before the larval-pupal ecdysis. (After Hinton, 1955).

S P I R A C U L A R GILLS

125

discarded in the gill lumen together with a quantity of blood at the pupal-adult apolysis. Thus the evolutionary history of the spiracular gills of the Eriopterini (Lipsothrix) and the Limoniini is quite different in respect to the isolated tissue: in Lipsothrix the habit of isolating tissue in the spiracular gills is established sometime during the process of evolving a spiracular gill from a respiratory horn, whereas in the Limoniini the habit is well established long before spiracular gills are evolved and persists after they are evolved. As noted on p. 75, plastron-bearing spiracular gills have been independently evolved on at least four occasions within the Tipulidae. A brief summary of the structure of the gills evolved on these four occasions is given below. 1. Lipsothrix. This genus includes some 20 species found in Europe, Asia, and North America. The larvae mine in waterlogged trunks or branches that are either largely submerged in streams or very near the water line and frequently wetted. The larval-pupal ecdysis can occur beneath the surface of the water, but the adults of both the European (Hinton, 1955) and American (Rogers and Byers, 1956) species appear to be unable to shed the pupal cuticle when under water. The shape of the spiracular gill is shown in Fig. 36A and its structure in Figs 15, 37-38. The spiracular atrium completely divides the gill (Fig. 15), but holes through the atrium effect the continuity of the blood spaces on both sides of the atrium. In Lipsorhrix remota Walk. about 1100 epidermal cells are isolated in each gill. After the pupal-adult apolysis, these epidermal cells remain attached to the walls of the gill and spiracular atrium and do not at any time become detached or migrate away from the walls as they do in all other Tipulidae. It has been noted elsewhere that the isolated tissue has no function; it may be destroyed without affecting the duration of the pharate adult stage. The outer surface of the gill except for a ventral area near its base is covered with a reticulate system of plastron lines, each of which is a groove in the cuticle over which there is a network (Fig. 38). In L . remota about 446 aeropyles near the rim of the gill effect the continuity of the air in the plastron lines and that in the spiracular atrium. On the inner side of the gill plastron lines are present only in thick cuticle near the distal margin, but these are short, do not anastomose, and each is drained by an aeropyle close to the row of aeropyles that drain the plastron lines of the outer surface.

2. Geranonzyia. Only two species, G . unicolor Haliday and G . bezzi Alexander and Leonard, of this large genus have invaded the sea. The

I26

H. E. H I N T O N

larvae feed on algae, generally in the upper half of the intertidal zone. They pupate in cocoons. Emergence of the adult apparently only occurs when the tide is out. The general form of the gills and the distribution of the plastron is shown in Figs 39A and 39B. Aeropyles in a row close to the margin of the gill effect the continuity of the air in the plastron and spiracular atrium,

FIG.38. Diagram of a plastron line of Lipsothrix remofa. (After Hinton, 1967a).

as shown in Fig. 40.The structure of the plastron meshwork is shown in Figs 10-1 1,41. Epidermis and blood are isolated in the gill lumen at the pupal-adult apolysis. After this apolysis, the epidermal cells dissociate, become rounded, and separate away from the cuticle and one another. In due course they cluster together in groups mostly confined to the proximal part of the lumen. The isolated tissue appears to have little if

SPIRACULAR GILLS

127

any function. It may possibly assist to a slight extent in the expansion of the gill by absorbing water after the Iarval-pupal ecdysis, but the gill is more or less fully expanded before the larval cuticle is shed. As already noted, the tissue will repair wounds to the gill wall during the entire life of the pharate adult and for some hours after the adult has shed the pupal cuticle. However, because of the rigidity of the gill walls,

FIG.39. Gerunomyia unirolor. (A) Dorsal view of spiracular gill. (B) Ventral view of same. Plastron stippled. ( C ) Aeropyles, surface view.

this capacity seems to be of no advantage to the insect, and the tissue can be destroyed without affecting the life of the pharate adult. 3. Dicrunomyiu. A few species of this large genus are intertidal, and the habits of their larvae and pupae are similar to those of Gerunomyia. There is a remarkable convergence of form and structure between the

128

-

H . E . HINTON

IOP

wropyle

!

spiracular atrium

tion through apex of gill of Geranomyia unicolor showing an aeropyle and FIG. the spiracular atrium. Dissociated epidermal cells are generally absent in the apex of the gill.

FIG.41. Structure of the plastron of Geranomyia unicolor; compare with Figs 10-1 1.

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129

Pupal

Adult

A FIG.42. (A) Spiracular gill of Dicranomyia triflamentosa. (B) Same of Dicranomyia monostromia.

FIG.43. Structure of the plastron of Dicranomyia triflamentosa.

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H. E. HINTON

gills of Dicranomyia and Geranomyia (cf. Figs 42B and 39), and in Dicranomyia the isolated epidermal cells also dissociate and form clusters in the proximal part of the lumen. Some of the species of Dicranomyia, e.g. D . fr~lamentosaAlex., have branched gills (Fig. 42A). The structure of the plastron of Dicranomyia (Figs 12, 43), however, is very different from that of Geranomyia: The 25 described species of Idioglochina are apparently all intertidal. Their gills do not differ in any significant way from those of Dicrano-

FIG.44. Orimargula australiensis. (A) Dorsal view of pupa. (B) Dorsal view of right spiracular gill. (After Hinton, 1965).

myia, and the fine structure of the plastron is identical (Fig. 13). The question of whether or not Idioglochina is generically distinct from Dicranomyia has already been discussed (p. 79): if it proves to be distinct, still another independent origin of plastron-bearing spiracular gills within the family Tipulidae will have to be recognized. 4. Antocha. This genus includes a large number of species from all faunal regions except New Zealand. Orimargula is sometimes considered to be no more than a subgenus of Antocha. It would seem that both genera are derived from a common ancestor possessing spiracular gills.

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131

So far as is known, the larvae of all live in freshwater streams where they feed on algae. They pupate in cocoons attached to stones or other objects below the surface. The gills of both genera are branched (Figs 36B,44A), and the structure of the plastron is similar. The plastron (Figs 3-5) consists of tubes that are circular in section, the upper part of each tube being interlinked transverse cuticular bridges and the lower part a groove in the gill wall (Fig. 5). The density and distribution of the plastron lines or tubes vary greatly: in some species they are transverse (Fig. 45),whereas

FIG.45. The middle of the third branch of the spiracular gill of Orimargula ausfraliensis. (After Hinton, 1965).

in others they are longitudinal or nearly so (Fig. 46). Each plastron line ends in an aeropyle that extends vertically through the cuticle to the spiracular atrium. The plastron of the African Orimargula hintoni Alex. (Fig. 9) is entirely different from that of all other known species of Antocha and Orimargula. The difficulty raised by its very different structure has already been discussed (p. 77). The rBle of the tissue isolated in the lumen of the gills of both Antocha and Orimargula has already been described in considerable detail in an earlier section.

I32

H . E. HINTON

FIG.46. Base of spiracular gill of Anfocha vitripennis showing the plastron lines and the branched spiracular atrium. (After Hinton, 1957a). 0.04

mm

FIG.47. Antocha vifripennis, pharate adult stage. Reconstruction based on sections through the common base of gill branches K and D1 (see Fig. 46) showing the connection between the plastron lines, aeropyles, and spiracular atrium. Dissociation of the epidermis is complete, and there are three broadly oval epidermal cells in the coagulated blood. (After Hinton, 1957a).

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133

E. S I M U L l l D A E

About 1000 species of Simuliidae have been described. The larvae are plankton feeders, usually living in running water but a few species are known to live in the littoral of large lakes. The pupal cocoons are attached to plants or to stones beneath the surface of the water. Nothing is known of the terrestrial ancestors of the family.

FIG.48. (A) Structure of plastron near middle of first gill branch of SirnuIiurn costaturn. (B) Structure of plastron near spiracular atrium of Twinnia hydroides Novak.

In nearly all endopterygotes the pupal cocoon is spun by the larva well before the larval-pupal apolysis. So far as is now known, the Simuliidae are unique among Diptera in that the pupal cocoon is spun by the pupa itself several days after the larval-pupal apolysis (Hinton, 1958a). It is clear from the illustrations given by Sundermeier (1940) that the neuropteran, Myrmeleon europaeus L., also spins a cocoon in the pupal rather than in the larval stage. The Simuliidae appear to be the

134

H. E. HINTON

only insects that have a prolonged feeding phase-up to 4 or more days (1&13"C)-after the larval-pupal apolysis. The pharate pupa is able to move about and feed like the larva because it makes use of many structures, such as the mouth-parts and prolegs, of the old larval cuticle to which it is only mechanically connected. The connections of the pharate

FIG.49. Transmission electron micrograph of the plastron of Simuliuni vuriegatrmi.

FIG.50. Transmission electron micrograph of the plastron of Simuhm

ornutrim.

pupa to the old larval cuticle are apparently all similar: when the epidermis retracts from the old larval cuticle at the larval-pupal apolysis many of the muscle connections to the old cuticle remain. In sections it may be seen that the tonofibrillae associated with many of the skeletal muscles remain attached to the old larval cuticle and extend inwards

SPIRACULAR GILLS

135

through the new pupal cuticle. Many of the tonofibrillar connections to the old cuticle persist until a few hours or minutes before the larval cuticle is shed. The pharate pupa may feed and defecate for four or more days before it begins to spin its cocoon. When it begins to spin its cocoon, much of the pupal cuticle is already heavily sclerotized. For instance, the abdominal hooks of the pupa that will later engage the fabric of the coccon may be as heavily sclerotized before as after the cocoon is spun. The plastron of the spiracular gills normally becomes air-filled before the cocoon is completed. Although the appearance of

FIG.51. Reconstruction of the plastron network of Sinruhnr iiuriegurunr Meig.

air in the plastron sometimes coincides with the appearance of air in the tracheal system, it is independent of the latter process. The gills of 33 species belonging to all of the chief groups in the family have been examined. However unlike their external appearance may be, their basic structure and function are more or less identical. Each gill usually consists of at least two main branches, with or without secondary branching. In many species a large number of very fine secondary branches arise from one or more of the main stems, e.g. in Simulium hunteri Mall., there are 184 fine and long branches. Whatever the outward form of the gill, the fine structure of the plastron of all is very

136

H . E. HINTON

similar. The plastron meshwork consists of cuticular columns or struts normal to the long axes of the branches and base of the gill. The vertical struts are normally not connected to each other below their apices (Fig. 48A) except near the base of the gill (Fig. 48B), where in all species they

FIG.52. Stereoscan electron micrograph of the base of a gill branch of Twinnia tafrensis Novak with part of the plastron meshwork removed to show arrangement of vertical struts. FIG.53. Stereoscan electron micrograph of the middle of two gill branches of Simulium latipes.

are longer than elsewhere. The vertical struts are branched at their apices in a plane normal to their long axes. These horizontal branches or struts form a fine and open hydrofuge network that provides a large water-air interface (Figs 49-53). The plastron network cannot be resolved with the light microscope. When a gill is viewed normal to the

S P I R A C U L A R GILLS

137

surface by transmitted light, total internal reflection in the vertical struts gives the appearance that the surface of the gill is covered by a delicate membrane perforated by fine holes as wide as the vertical struts. This is the view of the structure maintained by Pulikovsky (1927) and more recent writers, e.g. Wigglesworth ( 1965). As may be seen from Fig. 54, during the pharate adult stage the section of the pupal trachea that extends between the pupal and adult cuticles consists of the following parts : (1) a distal chamber, the lumen of which is continuous with the interstices of the thickened part of the

FIG. 54. Diagram of base of left spiracular gill of Sinrdiunr oriiu/uw in the pharate adult stage. (After Hinton, 1957b).

plastron meshwork around the dorsal base of the gill; (2) a “pinchcock” regulatory apparatus immediately behind the distal chamber; (3) a long and very wide tube with helical thickenings; (4) a very short felt chamber near the adult spiracle; and ( 5 ) the collapsed ecdysial tube through which the larval tracheae were withdrawn from within the pupal tracheal system at the larval-pupal ecdysis. A strand of cuticle that extends into the lumen of the felt chamber where in section it is completely choked with “felt” (the “perforated diaphragm” of some writers) anchors this part of the structure to the body wall of the pupa. As is clear from Fig. 54, the muscle that moves the lever of the regulatory apparatus lies within the haemocoele of the adult and is, of course, an adult muscle, not a pupal muscle; the muscle is separated

138

H. E . H I N T O N

from the pupal spiracle by the cuticle of the adult and a layer of moulting fluid. It arises in the thorax not far from the origin of the muscle of the regulatory apparatus of the adult spiracle. The apodeme on which it is inserted very closely ensleeves an apodeme of the pupal cuticle. This pupal apodeme, the tendon of Pulikovsky (1927), is a backward extension of the lever of the regulatory apparatus of the pupal spiracle. Whether the pupal apodeme is held within the adult apodeme by friction or whether the two are cemented together in some way, has not been determined. Before it reaches the adult apodeme, the pupal apodeme extends through a delicate epithelial sleeve which is a modified part of the epidermal layer of the adult (Fig. 55).

FIG.55. Simulium ornatum, pharate adult stage. Epithelial sleeve enclosing apodeme of closing apparatus of pupal spiracle.

SPIRACULAR GILLS

139

During the emergence of the adult from the pupal cuticle, the pupal apodeme is broken off and pulled out of the epithelial sleeve. If a shed pupal cuticle be examined, it may be seen as a delicate thread attached to the lever of the regulatory apparatus. The epithelial sleeve, which looks like a thin papilla projecting out of the side of the thorax of the adult above the first thoracic spiracle is withdrawn into the thorax during the process of emergence of the adult; and, on a recently emerged adult, the point of origin of the epithelial sleeve can be recognized only with difficulty. Judging from the appearance of the broken end of the pupal apodeme, it seems possible that some of it is left enclosed in the adult apodeme and thus retained in the body of the adult (Hinton, 1957b). Among other insects, only flies of the family Psychodidae are known to have a regulatory apparatus on the pupal spiracle that is operated by the pharate adult (Satchell, 1948). Thus, the Simuliidae and Psychodidae are unique among insects in that during the pharate adult stage water loss is controlled not only by the regulatory apparatus of the

FIG.56. (A) Spiracular gill of Paulianina umbra Stuckenberg. (After Stuckenberg. 1958.) (B) Spiracular gill of Edwardsina tosmaniensis Edw. (After Tonnoir, 1924.) ( C ) Spiracular gill of the kind found in such genera as Blepharocera, Liponeura, KeHoRginrC, Hapalothrix, Horuirr, and Peritheates.

FIG.60. Transmission electron micrograph nearly normal to the long axes of the vertical struts of the plastron meshwork ofliponeuru decipiens Beui. FIG.61. Transmission electron micrograph taken at an oblique angle to the surface of the plastron of Liponeuru decipiens. c

FIG.57. Stereoscanelectron micrograph of plastron network MvordsiM gracilis Edw.

FIG.58. Stereoscan electron micrograph of whole gill of Mvardsinn grucilis. FIG. 59. Stereoscan electron micrograph of the lobes on the rim of one of the gill leaflets of Eiwurdsinu dispur Edw.

SPIRACULAR GILLS

141

adult but in addition by a pupal regulatory apparatus also operated by the adult. F. B L E P H A R O C E R I D A E

About 170 species of Blepharoceridae have been described. The larvae live in fast-flowing streams or in waterfalls where they feed on

FIG.62. Stereoscan electron micrograph of whole gill of Paulianinu umbra. FIG.63. Stereoscan electron micrograph of plastron network of gill of Pauliunina umbra.

142

H . E. HINTON

algae. They pupate on the surface of stones, usually beneath the water. The pupae, which lack cocoons, are glued to the surface of the stones by three or four oval areas on either side of the abdomen. The Blepharoceridae have no close relatives among other nematocerous Diptera, and nothing is known of their terrestrial ancestors,

FIG.64. Stereoscan electron micrograph of base of gill of Liponeuru decipiens. FIG.65. Stereoscan electron micrograph of plastron network of gill of Liponeura decipiens.

SPIRACULAR GILLS

143

The spiracular gills are formed entirely by the body wall adjacent to the first pair of thoracic spiracles, and the spiracles do not as a rule extend beyond the general level of the body wall. In the subfamilies Blepharocerinae, Paltostominae, and Apistomyinae each gill almost

FIG.66. Stereoscan electron micrograph of gill of Puuliunina ingens Stuckenberg. FIG.67. Stereoscan electron micrograph of gill of Puuliuninu purnelu Stuckenberg. The plastron network is in the form of more or less round, discrete islands on the dorsal rims of the gill leaflets.

always consists of three or more vertical leaflets that are closely appressed together (Fig. 56C). In the Edwardsininae, which are restricted

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H . E. H I N T O N

to the Southern Hemisphere, the gill structure is extremely variable even in the same genus (Figs 58-59). In some forms, e.g. species of Edwurdsinu (s. h t . ) , the gill may be borne on the end of a projection from the body wall into which the spiracular atrium extends (Fig. 56B), although, as in other Blepharoceridae, the atrium does not extend into the gill leaflets. The surface of the whole gill bears a plastron. The plastron meshwork always consists of vertical struts that are branched at their apices to form a plastron network (Figs 60-61). The fine structure of the plastron network varies greatly. From a view more or less normal to the surface, the network and the supporting struts may be easily distinguished (Fig. 63), or the network may appear to be a flat perforated sheet of cuticle (Fig. 57). Sometimes, as in the species of Liponeuru, the plastron network is very irregular (Fig. 65). The external opening of the spiracular atrium may be simple or branched. The spiracle may open between the median pair of leaflets, as in most species of the family, or it may open on one side. The air held between the vertical struts of the plastron meshwork is always continuous with the air in the atrial chamber. The connection between the plastron meshwork and the atrial meshwork cannot always be resolved with the light microscope, and this fact has sometimes led to the belief that the spiracles are closed (Stuckenberg, 1958). The cuticular projections from the walls of the spiracular atrium vary considerably in size and density, but they are frequently sufficiently dense for the atrial chamber to be described as a “felt chamber”. The isolation of the tissue in the gill lumen and its subsequent cuticularization have already been described (see pp. 100, 113). G . DEUTEROPHLEBIIDAE

Seven species of Deuterophlebiidae have been described, all from mountain streams in Asia and western North America. In two Californian species, which live only about 2 hr after the pupal cuticle is shed (Kennedy, 1958, 1960), the duration of the pharate adult stage is about 100 times that of the non-pharate adult stage. The selective advantage of the very short non-pharate life is not known. The spiracular gills are formed from the body wall adjacent to the spiracles (Fig. 18). They are usually 3-branched (Fig. 68). The plastron meshwork consists of rows of pillars that arise perpendicularly from the surface and meander irregularly over all parts of the base and branches of the gill. The pillars are branched at their apices in a plane normal to their long axes, and each is joined to the nearest pillar of the

145

SPIRACULAR GILLS

row on either side by an arched strut (Fig. 69). The arched struts are joined to those before and behind by more slender struts that are sometimes branched. The atrium of the spiracle opens directly into the spaces between the pillars at the base of the gill (Figs. 17-18). r-

0.40 m m

1

FIG. 68. Deureroph/eh;~rinyot.tr.si.s Kennedy, pharate adult stage. Pupal gill of right side. (After Hinton, 1962).

The larval cuticle is shed shortly before the pupal-adult apolysis, and there is thus a brief non-pharate pupal stage. The duration of the nonpharate stage is not precisely known, but it is probably less than 24 hr. The epidermis and blood in the gill are not isolated until the pupaladult apolysis so that when the gill first comes into direct contact with the ambient water the lumen of the gill is still continuous with the haemocoele.

146

H. E. HINTON

After the isolation of the epidermis in the lumen of the gill by the secretion of a basal membrane, many of the epidermal cells become rounded and separate -from the cuticle and from one another. These cells eventually form loose clusters of irregular size and shape more or less in the middle of the lumen. The process of dissociation of the epidermal cells appears to be very similar to that of the tipulids Antocha

FIG.69. Structure of plastron of gill of Dertrerophlehia inyoensis. (After Hinton, 1962.)

and Orimurguh. The isolated epidermal cells remain in good condition throughout the life of the pharate adult. A few round or broadly oval cells 8-10 p wide were found in the basal part of the gill on the lumen side of the basement membrane (Fig. I7B). These were presumed to be blood cells, but they could have been epidermal cells that had undergone precocious dissociation and migration. H. E M P I D I D A E

The Empididae are related to the Dolichopodidae, but the species with spiracular gills do not pupate in cocoons. Only the species of

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147

Chelyera and Hemerodromia in the subfamily Hemerodromiinae are known to have spiracular gills. In all other dipterous pupae with spiracular gills these are associated only with the first pair of thoracic spiracles, but in the Hemerodromiinae spiracular gills formed by the body wall adjacent to the spiracle are associated with the first thoracic and the first seven abdominal spiracles (Fig. 70). No account exists of the structure of the spiracular gills. The plastron gas is held in superficial canals (Figs 71-72) that extend the length of

FIG.70. Pupa of Henrerodrorniu rrnilineotrr Zett. (After Hinton, 1953.)

the gill and communicate at the base of the gill with the spiracular atrium. The detailed structure of the plastron network has not been established, but there is reason to believe that it resembles that of the beetle Torridincola (Figs 32-34). The gills attain their full length during the pharate pupal phase, but they remain in tight coils in the moulting fluid between the pupal and larval cuticles and so occupy little space. Tissue is isolated in the lumen of the gill at the pupal-adult apolysis, which appears to be completed before the larval cuticle is shed. It would seem that it is water absorbed by the isolated tissue that is responsible for the uncoiling and straightening out of the gills after the larval cuticle is shed.

148

H . E. H I N T O N

FIG.71. Stereoscan electron micrograph of broken tip of a gill of Hemerodromia unilineata. FIG.72. Stereoscan electron micrograph of surface near base of a gill of Hemerodromia unilineata. I. D 0L I C H O POD I D A E

In this family plastron-bearing spiracular gills are known only in the genus Aphrosylus (Fig. 73). All of the species appear to be intertidal, and it is suspected that all genera of the Aphrosylinae will be found to breed only in the intertidal zone.

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149

FIG.73. Pupa of Aphrosvliu celriher Haliday. (After Hinton, 1966a).

B FIG.74. Aphrosylus celtiher. (A) Section through the middle part of the horn below the plastron-bearing $11. (B) Transverse section through the gill near its apex. (After Hinton, 1967~).

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H . E. H I N T O N

The gill of Aphrosylus has been evolved from the kind of respiratory horn present in nearly all terrestrial and freshwater Dolichopodidae, and the structure of the basal sixth of the appendage differs in no significant particular from that of the respiratory horns of other members of

FIG.75. Stereoscan electron micrograph of the spiracular gill of Aphrosylus celriber showing the vertical struts that support the plastron network. (After Hinton, 1967). FIG.76. Stereoscan electron micrograph of the plastron network of the middle side of the gill of Aphrosylus celtiber. The interstices between the bridges of the plastron network appear to be narrower than they are because of the film of gold-palladium alloy evaporated on the specimen.

the family. The spiracular atrium is present oily within the unmodified part of the appendage. It is circular in section, and it contains a dense

SPIRACULAR GILLS

151

meshwork of anastomosing cuticular struts that arise from its walls (Fig. 74A). The appendage beyond the basal sixth, that is the whole of the gill, is a prolongation of the body wall adjacent to the spiracle and does not include any parts that can properly be considered to be parts of the spiracle itself (Fig. 74B). The gill proper is nearly circular in section near its base but becomes progressively flattened towards its apex. The walls of the gill enclose the epithelium that secreted them, but the epidermal cells here, unlike those of the basal sixth of the appendage (the unmodified respiratory horn), are indistinct; in most parts of the

FIG.77. Diagram of the plastron of Aphrosyhrs celtiber. (After Hinton, 1967~).

gill their cytoplasm appears to be largely cuticularized. Except for a median ventral strip that extends close to the apex, the outer wall of the gill consists of a meshwork of cuticular struts that hold the plastron gas (Figs 75, 77). Isolation of tissue in the respiratory horns at the pupal-adult apolysis appears to be universal in the family: such tissue was found in each of the 15 species belonging to five subfamilies examined (Hinton, 1967~). It therefore seems evident that in this family the habit of isolating tissue in the respiratory horns was well established before the modification of these to form spiracular gills.

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H . E. H I N T O N

Aphrosylus is of particular interest because it is the only known pupa with a plastron-bearing spiracular gill that also has functional abdominal spiracles. The presence of normal functional spiracles in Aphrosylus but in no other insect with spiracular gills seems to be related to the fact that when the cocoon is covered by the sea, the air within it is trapped. When this occurs the cocoon itself inevitably functions as a plastron: it has a large water-air interface and the volume of air it contains remains constant so long as the mechanical strength of the cocoon is sufficient to resist the hydrostatic pressures to which it is subjected. Thus, the pupa and pharate adult of Aphrosylus have in effect two different plastrons, that of the spiracular gills and that of the cocoon. A number of other insects with spiracular gills pupate in cocoons, e.g. Simuliidae and Tipulidae. In all of these, however, the cocoon fills with water when it is immersed, and no air is trapped in it. In such insects open abdominal spiracles in the pupa would seem to have no function, and none are formed. Besides functioning as a plastron, the air layer around the pupa must also function as an osmotic barrier: it could account in part for the great success of the Dolichopodidae in invading the sea. There have been no less than ten independent invasions of the sea by members of the family, which is a greater number than is known in any other family of insects with the possible exception of the Chironomidae and Staphylinidae. J. C A N A C E I D A E

The spiracular gills of Cunace nasicu Haliday (Hinton, 1967b) are unlike those of any other known species of Diptera in that they are modifications of the spiracle itself, as are those of beetles of the family Psephenidae. In all other Diptera the spiracular gills are modifications of the body wall adjoining the spiracle or of both the body wall and the spiracle. The general shape of the spiracle is shown in Fig 78. A section through the middle part of the spiracle is shown in Fig. 78C, and one through the middle part of the gill in Fig. 79A. The transition between the structure of the basal part of the spiracle and the plastron-bearing apical part is abrupt: the cuticular wall which is dorsally and laterally 10-20 IJ. thick becomes the plastron network which is only 0.3-0-5p thick. The spiracular atrium extends to the apex of the gill. In the gill part of the spiracle the atrium extends around the dorsal and lateral parts of the thickened ventral wall so that the latter is almost entirely enveloped by the atrium (Fig. 79A). The fine structure of the plastron, as deduced from stereoscan electron micrographs, is shown in Fig. 79B.

S P I R A C U L A R GILLS

153

The function of the rather rigid meshwork that arises from the walls of the spiracular atrium (Figs 80-81) seems evident enough in the gill part of the spiracle. Under high hydrostatic pressures the opposed walls

FIG.78. Canace nusicn. (A) Dorsal view of puparium. The plastron is stippled. (B) Dorsal view of the spiracular gill and part of the base of the spiracle. ( C )Transverse section through the middle of the basal part of the spiracle. (After Hinton, 1967b).

of the atrium would tend to be pressed together because the very thin wall (the plastron network) outside the atrium on the dorsal and lateral sides of the gill has little rigidity. When the opposed walls of the atrium

I54

H . E . HINTON

were forced together, it was found that a large volume of gas was nevertheless retained in the meshwork that lines the atrium. In order to crush the meshwork, and force into solution the air that it normally contains, pressures of another order of magnitude are required. About 35 species of Canaceidae have been described. All appear to

FIG.79. Cunure nctsicu. (A) Transverse section through the middle of the spiracular gill. (B) Diagram of outer wall of plastron network of gill supported by the atrial meshwork. (After Hinton, 1967b).

be intertidal except for two species of Protocanace that have invaded freshwater streams, one in the Hawaiian Islands and the other in Java. All of the Hawaiian Islands originated in the Tertiary, chiefly during and since the Miocene. It has therefore been suggested (Hinton, 1967b) that Protocanace is not a primitive member of its family but a specialized

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form that has secondarily invaded fresh water. A parallel is to be found in the almost .exclusively intertidal Chironomidae of the subfamily Clunioninae, in which some species of the genus Telmatogeton have also secondarily invaded freshwater streams in the Hawaiian Islands (Wirth, 1951). Until now only Canace has been found to have plastron-bearing spiracular gills. Probably many other Canaceidae will be found to have

FIG.80. Stereoscan electron micrograph of inner surface of atrial meshwork of gill of Canace nasica. (After Hinton, 1967b). FIG.81. Transmission electron micrograph of atrial meshwork of gill of Canace nnsica slightly crushed on surface of grid.

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similar structures, but some members of the family have long spiracles without a plastron, e.g. Xanthocanace nigrifrons Malloch.

X. S P I R A C U L AGRI L L SO F L A R V A E Larvae of three of the four known families of the suborder Myxophaga (Coleoptera) are known. All of these (Torridincolidae, Hydroscaphidae, Sphaeriidae) have spiracular gills. The spiracular gills of the Torridincolidae support a typical plastron meshwork, and it appears that the spiracular gills of the Hydroscaphidae and Sphaeriidae also function as plastrons. Although many plastron-breathers are known among adult, pupal, and egg stages of insects, the only larval insects so far known with a plastron are those of the suborder Myxophaga. A. T O R R I D I N C O L I D A E

The spiracles of the first eight abdominal segments of the only known species in the family, the African Torridincola rhodesica Steffan, are

\ 'I

FIG.82. (A) Right spiracular gill of first abdominal segment of second instar larva of Torridincola rhodesica. (B) Right spiracular gill of first abdominal segment of third instar larva of the same species. The extent of the plastron of both instars is indicated by stippling. (C) Right spiracle of first abdominal segment of third instar larva of Sphaerius ovenensis. (D) Right spiracle of eighth abdominal segment of third instar larva of Hydroscapha natans Lec. (Redrawn from Hinton, 1967d).

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modified to form plastron-bearing spiracular gills in both the second and third larval instars. The first larval instar is unknown. The gills are normally 3-segmented, but an occasional gill may be 2-segmented. In the second larval instar (Fig. 82A) the external opening of the

FIG.83. Stereoscan electron micrograph of spiracular gills of right side of young second instar larva of Torridincola rhodesica. FIG.84. Steroscan electron micrograph of junction of first and second segments of spiracular gill of fourth abdominal segment of third instar larva of Torridincola rhodesia.

spiracular atrium is very near the apex of the second segment of each gill. The whole surface of the third segment and all but the extreme base of the second segment supports a fine cuticular meshwork that holds

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H . E. HINTON

the plastron gas. In the third larval instar (Fig. 82B) the external opening of the spiracular atrium is on the apex of the first instead of the second segment of each gill. The whole of the, second and third segments and the distal two-thirds of the first segment support a superficial meshwork (Fig. 84) that functions as a plastron. The fine structure of the plastron of the second and third larval instar appears to be identical. B. S P H A E R I I D A E A N D H Y D R O S C A P H I D A E

The structure and function of the balloon-like spiracles of the Sphaeriidae and Hydroscaphidae has recently been described (Hinton, 1967d). In Sphaerius functional spiracles are present on the first eight abdominal segments, whereas in Hydroscapha functional spiracles are present on the mesothorax and the first and eighth abdominal segment. In both genera the functional spiracles of one segment are like those of any other. Apart from their shape, the spiracles of Sphaerius (Fig. 82C) and Hydroscapha (Fig. 82D) are very similar. In both the peritreme of the spiracle is only about 0.1 p thick. The spiracular atrium extends through the peritreme to open at its distal end. The lumen of the spiracular atrium has a meshwork of very fine cuticular struts that cannot clearly be resolved even with phase contrast. When living larvae of Sphaerius ovenensis (Oke) were immersed in water, the space between the peritreme and the spiracular atrium appeared to be gas filled. There should, of course, be a double layer of epidermal cells here, the layer that secreted the atrium and the layer that secreted the peritreme : clearly more has to be known about the development of the spiracles. The total water-air interface across the openings of the spiracles is about 1.2 x lo4 p2per mg wet body weight in the Sphaeriidae and about 0.9 x lo4 p2/mg in the Hydroscaphidae. Thus, in both families the water-air interface is about an order of magnitude greater per mg wet body weight than that established across the spiracles of terrestrial insects (Hinton, 1966d). I t is almost as great as the water-air interface of the plastron of some insects with plastron-bearing gills, e.g. 1.5 x lo4 pz/ mg in the pupa of Eutanyderus. The total surface area of the six spiracles of Hydroscapha, excluding the area of the openings, is 1.2 x lo6 p2/mg. The shape of the spiracles of Sphaerius is such that it is more difficult to calculate their surface area. Comparable figures for the 16 spiracles of this insect varied from 2 to 4 x lo5 p2/mg, according to the assumptions made in the calculations. The probable error in these figures is not known, but the figures are almost certainly of the right order of magnitude. If the outer wall of

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the spiracles is permeable to oxygen, and there is no reason to believe that it is not, there must be holes in it. However, at a resolution of about 300 A no holes were detected with a stereoscan electron microscope. It was thus not possible to estimate the proportion of the area of the walls that is water-air interface and therefore effective as a plastron. REFERENCES Crisp, D. J. (1964). Plastron respiration. Recent Prog. Surface Sci. 2, 377-425. Crisp, D. J. and Thorpe, W. H. (1948). The water-protecting properties of insect hairs. Discuss. Faraday SOC.3, 210-220. Fox, H. M. (1921). Methods of studying the respiratory exchange in small aquatic organisms, with particular reference to the use of flagellates as an indicator for oxygen consumption. J. gen. Physiol. 3, 565-573. Hinton, H. E. (1946). Concealed phases in the metamorphosis of insects. Nature, Lond. 157, 552. Hinton, H. E. (1947a). The gills of some aquatic beetle pupae (Coleoptera, Psephenidae). Proc. R. ent. SOC.Lond. (A) 22, 52-60. Hinton, H. E. (1947b). On the reduction of functional spiracles in the aquatic larvae of the Holometabola, with notes on the moulting process of spiracles. Trans. R. ent. SOC.Lond. 98,449-473. Hinton, H. E. (1953). Some adaptations of insects to environments that are alternately dry and flooded, with some notes on the habits of the Stratiomyidae. Trans. SOC.Br. Ent. 11, 209-227. Hinton, H. E. (1955). The structure of the spiracular gill of the genus Lipsothrix (Tipulidae), with some observations on the living epithelium isolated in the gill at the pupa-adult moult. Proc. R. ent. SOC.Lond. (A) 30, 1-14. Hinton, H. E. (1957a). The structure and function of the spiracular gill of the fly Taphrophila uitripennis. Proc. R. SOC.B 247, 90-120. Hinton, H. E. (1957b). Some little known respiratory adaptations. Sci. Prog. Lond. 45, 692-700. Hinton, H. E. (1958a). The pupa of the fly Simulium feeds and spins its own cocoon, Ent. mon. Mag. 94, 14-16. Hinton, H. E. (1958b). The spiracular gills of insects. Proc. 10th int. Congr. Ent. (1956) 1, 543-548. Hinton, H. E. (1958~).Concealed phases in the metamorphosis of insects. Sci. Prog. Lond. 46, 260-275. Hinton, H. E. (1959). Plastron respiration in the eggs of Drosophila and other flies. Nature, Lond. 184, 280-281. Hinton, H. E. (1960a). A fly larva that tolerates dehydration and temperatures from -270°C to + 102°C. Nature, Lond. 188, 336-337. Hinton, H. E. (1960b). Cryptobiosis in the larva of Polypedilum uanderplanki Hint. (Chironomidae). J . Insect Physiol. 5, 286-300. Hinton, H. E. (1960~).The chorionic plastron and its role in the eggs of the Muscinae (Diptera). Q. Jl microsc. Sci. 101, 313-332. Hinton, H. E. (1960d). The structure and function of the respiratory horns of the eggs of some flies, Phil. Trans. R . SOC.B 243,45-73.

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Hinton, H. E. (1961a). How some insects, especially the egg stages, avoid drowning when it rains. Proc. S. Lond. ent. nut. Hist. SOC.1960, 138-154. Hinton, H. E. (1961-b). The structure and function of the egg-shell in the Nepidae (Hemiptera). J. Insect Physiol. 7 , 224-257. Hinton, H. E. (1962). The structure and function of the spiracular gill of Deuterophlebia (Deuterophlebiidae) in relation to those of other Diptera. Proc. zoo/. SOC.Lond. 138, 111-122. Hinton, H. E. (1963). Metamorphosis of the epidermis and hormone mimetic substances. Sci. Prog., Lond. 51, 306-322. Hinton, H. E. (1964). The respiratory efficiency of the spiracular gill of Simrrlium. J. Insect Physiol. 10, 73-80. Hinton, H. E. (1965). The spiracular gill of the fly Orimargula australiensis and its relation to those of other flies. Aust. J . 2001. 13, 783-800. Hinton, H. E. (1966a). Plastron respiration in marine insects. Nature, Lond. 209, 220-221. Hinton, H. E. (1966b). The spiracular gill of the fly, Antocha bifida, as seen with the scanning electron microscope. Proc. R . ent. SOC.Lond. (A) 41, 1 07-1 15 . Hinton, H. E. (1966~).The spiracular gill of the fly Eufunydcrus (Tanyderidae). Ausl. J. ZOO^. 14, 365-369. Hinton, H. E. (1966d). Respiratory adaptations of the pupae of beetles of the family Psephenidae. Phil. Trans. R. SOC.B 251, 21 1-245. Hinton, H. E. (1967a). Structure of the plastron in Lipsothrix, and the polyphyletic origin of plastron respiration in the Tipulidae. Proc. R. ent. SOC.Lond. (A) 42, 35-38. Hinton, H. E. (19676). Plastron respiration in the marine fly Canuce. J. mar. biol. ASS. U.K.47, 319-327. Hinton, H. E. (1967~).Spiracular gills in the marine fly Aphrosylus and their relation to the respiratory horns of other Dolichopodidae. J. mar. biol. Ass. U.K. 47,485-497. Hinton, H. E. (1967d). On the spiracles of the larvae of the suborder Myxophaga (Coleoptera). Aust. J. Zool. 15, 955-959. Imms, A. D. (1957). “ A General Textbook of Entomology” (9th Ed. revised by 0. W. Richards and R. G. Davies). Methuen & Co. Ltd., London. Jenkin, P. M. and Hinton, H. E. (1966). Apolysis in arthropod moulting cycles. Nature, Lond. 211, 871. Keister, M. and Buck, J. (1964). Respiration: some exogenous and endogenous effects on rate of respiration. In “The Physiology of the Insecta” (M. Rockstein, ed.), Vol. 3, pp. 617-658. Academic Press, New York and London. Kennedy, H. D. (1958). The biology and life history of a new species of mountain midge, Deuterophlebia nielsoni, from Eastern California (Diptera, Deuterophlebiidae). Trans. Am. microsc. SOC.77, 201-228. Kennedy, H. D. (1 960). Deurerophlebia inyoensis, a new species of mountain midge from the alpine zone of the Sierra Nevada Range, California (Diptera, Deuterophlebiidae). Trans. Am. microsc. SOC.79, 191-210. Pulikovsky, N. (1927). Die Respiratorischen Anpassungserscheinungen bei den Puppen de Simuliiden (und einigen anderen in schnellfliessendenBachen lebenden Dipterenpuppen). 2.Morph. dkol. Tiere 7, 3 8 4 4 4 3 .

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Rogers, J. S. and Byers, G. W. (1956). The ecological distribution, life history, and immature stages of Lipsothrix Sylvia (Diptera: Tipulidae). Occ. Pap. Mus. Zool. Univ. Mich. 572, 1-14. Satchel], G. H. (1948). The respiratory horns of Psychoda pupae. Parasitology 39, 43-52.

Selman, B. J. (1960). On the tissue isolated in some of the larval appendages of Sialir lutaria L. at the larval-pupal moult. .I. Insect Physiol. 4, 235-257. Selman, R. J. (1961). Tolerance to dehydration of the blood of Sialis lutaria L. J. Insect Physiol. 6, 81-83. Stuckenberg, B. R. (1958). Taxonomic and morphological studies on the genus Paulianina Alexander (Diptera: Blepharoceridae). Mem. Inst. scient. Modagascar (E) 10, 97-198. Sundermeier, W. (1940). Der Hautpanzer des Kopfes und der Thorax von MyrmeIeon europaeus und seine Metamorphose. Zool. Jb. (Anat.) 66, 291-348. Thorpe, W. H. (1950). Plastron respiration in aquatic insects. Biol. Rev. 25,344-390. Thorpe, W. H. and Crisp, D. J. (1947a). Studies on plastron respiration. I. The biology of Aphelocheirus (Hemiptera, Aphelocheiridae (Naucoridae)) and the mechanism of plastron retention. J. exp. Biol. 24, 227-269. Thorpe, W. H. and Crisp, D. J. (1947b). Studies on plastron respiration. 11. The respiratory efficiency of the plastron in Aphelocherius. J. exp. Biol. 24, 270-303. Tonnoir, A. (1924). Les Blepharoceridae de la Tasmanie. Ann. biol. lacust. 13, 5-67. Wigglesworth, V. B. (1937). Wound healing in an insect (Rhodnius prolixus Hemiptera). J. exp. Biol. 14, 364-381. Wigglesworth, V. B. (1965). “The Principles of Insect Physiology ” (6th Ed.). Methuen & Co. Ltd., London. Wirth, W. W. (1951). A revision of the dipterous family Canaceidae. Occ. Pap. Bernice P. Bishop Mus. 20, 245-275.

ADDENDUM ADDED IN PROOF In the introduction (p. 66) it was stated that all spiracular gills except those of the Chironomidae bear a plastron. The complex spiracular gills of Chironornus and some related genera of the Chironominae do not bear a plastron. However, it has recently been found that plastron-bearing spiracular gills occur in the subfamilies Tanypodinae and Prodonominae. It therefore appears that plastron-bearing spiracular gills have been independently evolved no less than 15 times among insect pupae. Not enough is known about the relationship of the Tanypodinae to the Prodonominae to determine whether their common ancestor had plastronbearing spiracular gills or whether such structures have been independently evolved in both subfamilies. The fine structure of the plastron of Anafopynia(Tanypodinae) resembles that of the tipulid genus Geranomyia more than it does that of any other known insect (cf. Figs. 10-1 1 and 85-86).

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FIGS85-86. Stereoscanelectron micrographsof the plastron meshwork of the spiracular gill of Anaropynia nebulosa (Meig.).

Comparative Physiology of the Flight Motor J. W. S. PRINGLE Department of Zoology, University of Oxford, England I. Introduction

.

IC. The Generation of Lift and Thrust .

.

General The flight of Coleoptera . Gliding flight of Lepidoptera . The flight of small Diptera . Kinematics of Wing Motion . A. Diptera B. Apis mellifera . IV. Stability in Flight . A. Diptera . B. Other insects . V. The Motor Mechanism of Flight Reflexes . A. List of reflexes . B. Initiation, maintenance and termination of flight C. Control of amplitude, frequency and power . D. Control of velocity E. Control of lift . F. Control of attitude VI. Comparative Studies . A. Axioms B. Differentiation of the flight muscles . References . A.

B. C. D. 111. The

. . . . . . . . .

.

. . .

. . .

. . . . . .

.

163 164 164 166 171 173 179 179 186 190 190 195 198 198 199 200 206 209 211 217 217 219

223

1. I N T R O D U C T I O N The study of flight has now thrown light on such diverse aspects of insect physiology that it is impossible to discuss the whole of the subject in a single article. This review will be concerned largely with mechanical phenomena and it has as its objective an understanding of the effector mechanism responsible for motion in the air. Part of the problem is the elucidation of the lines of evolution of the flight system in the various orders. The review will not be concerned with energetics (Weis-Fogh, 1961), with the biochemistry of flight muscle (Maruyama, 1965; Sacktor, 1965) or the control of metabolism (Harvey and Haskell, 1966), nor with central nervous organization responsible for the control 163

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of flight, which is dealt with in a separate article in this volume (Wilson, 1968); these topics have been adequately treated elsewhere. The work to be summarized will be that done recently on the kinematics and aerodynamics of the wing motion, the reflex regulation of that motion and the arrangement and physiology of the power-producing and controlling muscles. The peculiarities of the contractile mechanism of fibrillar flight muscle have recently been considered from a biophysical and biochemical point of view (Pringle, 1967) and will here be treated functionally; that is, in relation to the design of the flight system as a whole. The self-oscillatory property of fibrillar muscle profoundly affects both the nervous and skeletal organization of those insects that possess it and the evolution of this property in several distinct lines within the Insecta must have involved parallel modifications of all parts of the flight machinery. It is at present by no means clear how efficient flight was possible during some of the stages through which these insects must have passed. Since it may be assumed, however, that such functional continuity was achieved in each case, useful clues to the nature of the constituent physiological mechanisms may be obtained from functional arguments. 11. THEG E N E R A T I O OFNL I F TA N D THRUST A. GENERAL

The ultimate requirement for the flight motor is that it should generate sufficient lift to support the weight of the insect and sufficient thrust to pull it through the air. Tn the absence of independently movable tail surfaces, as in birds and aeroplanes, there is a further requirement that the lift and thrust must be controllable in such a way as to enable the insect to balance and to turn and move in the required direction. Since lift and thrust are produced by the interaction between the air and the moving wings, aerodynamics must come first in any discussion of the design of the flight system. In their comprehensive review of earlier theories about the aerodynamics of insect flight, Weis-Fogh and Jensen (1956) emphasized the dangers of simplifying the problem. As they remarked, natural flapping flight is a complicated type of locomotion, and a misleading picture can easily be obtained if the kinematics of the wing motion are not known in detail. Weis-Fogh’s (1956a) analysis of the kinematics of normal locust flight enabled Jensen (1956) to make the first accurate study of the aerodynamics of an insect, and, as a result, it can be stated with some certainty that for an insect of the size and shape of Schistocercu grcguriu,

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beating its wings at about 17 beats per second, classical steady-state aerodynamic theory provides a quantitative explanation of the forces generated by the wing motion (summary in Pringle, 1957). In order to appreciate the researches that have been made since then, it is important to understand why this does not solve the problem for other insects. The complications that arise are matters of scale. Small insects move through the air at low speeds, but if the wing beat frequency is high, the movement involves high accelerations. Vogel(l964) points out that size and speed of the air-flow are complementary factors, since the Reynolds number, the relevant index to the flow rigime, involves a product of length and velocity. [(Re) = (pVd)/q, where p is the density of the air and q is its viscosity.] For the flight of Schistocerca gregaria, (Re) is approximately 2000 (Jensen, 1956). At such values and above, turbulent motion can occur in the air and high values of the lift coefficient C, are obtained from well-designed aerofoils at optimum angles of attack. As (Re) falls to a value of about 100 the maximum lift coefficient gets less and is obtained at higher angles of attack (45-50"; Thom and Swart, 1940; Vogel, 1966, 1967b); the drag coefficient increases and becomes less dependent on the angle of attack. Finally at values of (Re) less than about 20, vortices cannot form and the drag coefficient remains greater than the lift coefficient at all angles of attack; the drag is now almost entirely due to skin friction and is independent of the shape and orientation of the object, being merely proportional to its surface area. Horridge (1956) has argued that very small insects must fly by so changing the surface area of the wings that the drag is different on the down- and upstrokes. Insects with a wing length of less than 0.1 mm tend to have the wing reduced to a central rod with fringes of hairs (Pringle, 1957) and this may facilitate such a change in surface area. Even before the scale of size is reached at which drag rather than lift forces become the dominant feature of the aerodynamics, complications may be introduced by the accelerations implicit in the high frequency of beat in small insects. It is known that higher lift coefficients can occur if the incidence is changing rapidly and if the air is accelerating over the wing surface (Moore, 1956). If an appreciable quantity of air is entrained by the wing motion, the actual angle of attack on the air may not be that deducible from the inclination of the wing and the direction of overall movement. Particularly at low Reynolds numbers, the air mass contained in the boundary layer or an even larger induced mass may be accelerated by the wing motion and contribute to the aerodynamic forces. The reality of this last effect was established by Vogel (1962), who showed that the inertia of the boundary layer could make a signi-

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ficant contribution to the total mass in the mechanically-resonant wingthorax system. In insects where the index P . f-liz.M;’ is large ( I = wing length, f = frequency, M , = wing mass) the inertia of the boundary layer contributes appreciably to the total. Vogel computed this index for those species in which Sotavalta (1952) had shown an effect of air density on the wing-beat frequency and found values between 40 and 190; for those species where beat frequency was independent of air density, the index was between 8 and 20. The extent to which these factors are important can only be decided by detailed examination of the flight of each type of insect. This is now widely appreciated by biologists, but aeronautical engineers are still occasionally tempted into thinking that a n answer can be found by theoretical or practical study of a simplified situation. In the years preceding Weis-Fogh and Jensen’s study of the locust, the most elaborate of these theoretical exercises was that of Osborne (1951), who derived formulae for computing the lift and drag coefficients and the total power of an insect from structural measurements and then applied the formulae to the data of Magnan (1934). The results, in several cases, produced values for the lift coefficient which would be impossible under steadystate conditions with slightly cambered aerofoils, and Osborne concluded that aerodynamic inertial forces due to wing acceleration must play a significant r61e. Weis-Fogh and Jensen (1956) criticized this conclusion on the grounds that the data of Magnan (1934) were not all obtained under the same conditions; they showed that Osborne’s formulae did not give unusually large values of the lift coefficient when used with their own, more reliable measurements of the flight of Schistocerca nor with their reasonably postulated flight data for a “horse-fly ” or a “mosquito”. The Reynolds numbers for these idealized types of “small” insect would be about 5000 and 800, respectively, at which the flow rCgime is nearly normal.

B. T H E F L I G H T O F C O L E O P T E R A

1. Wings

Recently Bennett (1966) has revived the discussion on the basis of an experimental study of a model of the wing of MeIoIontha vulgaris, one of the insects for which Osborne’s analysis of Magnan’s data produced the high value of 2.0 for the mean lift coefficient on the downstroke. The model was made from cellophane stretched over a wire frame and

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was dtiven in such a way that its angle of attack* changed sharply at the top and bottom of the stroke (Fig. 1). The resulting motion was compared with that of the insect in flight, as analysed frame by frame from high-speed motion pictures and was said to differ in three respects: (1) the effect of the elytra was omitted; (2) the rate of change of angle CELLOPHANE TAPE M

TYPICAL WING SECTION

DRIVE PULLEY

BALANCE

WEIGHT

LATERAL SECTION FIG.1. Mechanism simulating wing movement of Melolonthu. (Bennett, 1966).

of attack was much less, requiring 20 degrees of stroke for completion in the model compared with only 5 degrees in the insect; (3) no attempt was made to simulate wing twist or section camber. The interaction between the two wings was simulated by placing a fixed surface as an image plane in the insect’s vertical plane of symmetry; forward motion * “Angle of attack”, in this review means the inclination of the wing surface to an axis in space or the longitudinal axis of the body. The term “incidence” refers to the angle of the wing surface to the true wind direction.

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PHINGLE

was reproduced with a wind-tunnel. With the model in flapping motion, instantaneous induced air velocities were sampled with a hot-wire anemometer of short time constant placed upstream and downstream of the flapping plane around an azimuthal arc at 0.7 of the wing span; the magnitude and direction of the air-flow were thus determined. By analysis of the oscillograms, a measure was obtained of the vertical and horizontal components of the time-averaged induced velocity upstream and downstream. Propeller theory was then used to derive a value of 1.06g for the vertical force; the difference of this from the weight of the insect, 0.96g, is said to represent errors in modelling, in experiment and in calculation. Since the Reynolds number for this model was 3100, the conclusion that the lifting process results from increase in the downward momentum of the air passing through the flapping plane (i.e. from a normal type of lift coefficient), rather than from a high drag coefficient during the downstroke, is hardly unexpected. More controversial is the claim that unsteady effects dominate the simulated performance. The vertical impulse experienced by the air moving through a unit area located at the mid-downstroke azimuth position in the course of a single stroke was computed by integrating the instantaneous vertical force with respect to time; the value obtained was 3.32 x g sec/cm2. Near steady-state conditions were then set up by allowing the model wing to perform a complete rotation (instead of a flapping motion) at the same incidence and with the same tangential velocity; the value was now 1.67 x g sec/cm2. Finally true steady-state conditions were established by setting the wind-tunnel axis normal to the propeller disc g sec/cm2. and adjusting the air speed; the value was now 1-19x Bennett concluded that these experiments do not support Jensen’s (1956) conclusion that insect flight may be treated as a sequence of stationary flow situations. Apart from the possibility that there were undetected differences in the radial component of the air-flow under the three conditions (these would not have been detected by the experimental set-up), it does appear to have been established that unsteady flow effects dominated the performance of the model tested. The author states that it cannot yet be decided whether these were due to vigorous “destalling” (effects due to wing acceleration) or to virtual mass forces (effects due to the inertia of the boundary layer and other air in induced motion). He should also have said that it cannot yet be decided whether the conclusions apply to the actual flight of the insect, since errors in modelling might produce a larger effect than was assumed. The differences between the performance of the model and that of the insect were considerable. Sotavalta (1952) states that the amplitude of the wing

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stroke in Melolontha is 180", not 150" as in the model; he gives the beat frequency as 62/sec, not 46/sec. The interaction between the two wings may not have been correctly simulated by the image plane, and there were certainly important errors in the angles of attack at the top and bottom of the stroke. Changes in camber and twist during the stroke were not simulated. Against Bennett's conclusion is the fact that Melolontha vulgaris is not an insect in which the inertia of the boundary layer would be expected to be significant. Sotavalta (1952) did not study the effect of air density on the wing-beat frequency of this species, but application of Vogel's (1962) index to Sotavalta's structural data gives a value of 11.2; this is well within the range over which the inertia of the boundary layer is negligible. 2. Elytra Bennett (1966) considered that the contribution of the elytra to the total vertical force would be small, owing to their low flapping speed. It has usually been assumed that the elytra, which except in Cetoniidae are held extended at a pronounced dihedral angle, contribute at least to the stability of flight. Stellwaag (1914) found that, although slow flight was possible after unilateral extirpation of an elytron, the insect flew forward in a wide curve. The aerodynamics of the elytra have now been studied experimentally in Oryctes boas by Burton and Sandeman (1961) and in Melolontha by NachtigaIl(l964). Burton and Sandeman (1961) showed by stroboscopic illumination that the elytra were not stationary but moved through an angle of 20" in phase with the wings; their angle of incidence changed from 20" at the top of the stroke to 34" at the bottom. Measurements were made of the lift generated by an insect with its two elytra fixed in their middle position and mounted in a simple wind-tunnel at various angles of attack; similar measurements with elytra removed made it possible to calculate the contribution of the elytra. The results given appear not to be very exact and the lift is only approximately proportional to the square of the velocity above 10" incidence. The data have been plotted in Fig. 2 as L / V 2 ( L = lift produced by the elytra as a percentage of body weight; V = wind speed) and values of the lift coefficient C, have been calculated for a body weight of 5.2 g and a n elytral surface area of 7.2 cm2 (values measured from a large specimen of Oryctes rhinoceros, which is similar to 0. boas). A curve can be drawn through the experimental points which is of the type expected for a highly cambered aerofoil. There is a distinct stall at about 25" incidence and the value of C,,,, of 1.2 is reasonable. Burton and Sandeman

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state that in their wind-tunnel the preferred flying speed at which the net drag was zero was about 4m/sec and that under these conditions the angle of attack of the elytra, at their mid-stroke position, was 26"; they are then producing lift equal to 21% of the body weight. In the more exact study of Melolontha by Nachtigall(1964), measurements of lift and drag were made at a single air speed of 2.25 m/sec at a Reynolds number of lo00 (Fig. 3). The derived curve for the lift of the elytra again shows a pronounced stall at about 30"incidence. Their drag

0

0

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10

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20

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30

35 ANGLE

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FIG.2. Lift produced by the elytra of Oryctes boas at different angles of attack. (Data of Burton and Sandeman, 1961, replotted as described in the text).

is high but the lift exceeds the drag by a small amount over a range of incidences from 8-28'. In the combination of body plus elytra, the drag is always greater than the lift, but the presence of the elytra does improve the lift/drag ratio and under optimum conditions they can carry perhaps 10% of the body weight. They are not held in flight in the position that would give the maximum lifting effect, but at a pronounced dihedral angle; for this reason and because of large errors introduced by small uncertainties in the air velocity, Nachtigall does not consider that it is justifiable to compute values of C, and C , (personal communication). He quotes Demo11 (1918) and his own unpublished observations that Melolontha cannot fly when its elytra have been removed and sug-

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gests that this is due to upset to the resonance of the whole thoracic system rather than to loss of the small contribution that the elytra make to the aerodynamic forces. A further uossible r81e in conferring lateral stability is discussed later.

1

1

FIG.3. Lift L , + , and drag D B f Eof the body with elytra of Melolonrha at different angles of attack. Curves L E and DE are derived curves of lift and drag for the elytra alone. (Redrawn from Nachtigall, 1964, with ordinate values corrected). C. G L I D I N G F L I G H T OF L E P I D O P T E R A

Nachtigall (1967) has made accurate measurements in the windtunnel of the lift and drag of six species of Lepidoptera when the wings were fixed in their gliding attitude. No great differences were found between the different species. Figure 4 shows a polar plot of the lift and drag for a mounted specimen of Agupetes gulutheu. This plot is convenient for showing a number of features of the aerodynamics: (1) For a very slightly cambered wing, the maximum lift occurs at the high angle of attack of 30-40". Jensen (1956) found a maximum at about 15" for the fore-wing of the locust and 25" for the hind-wings. The maximum value of C, works out at about 0.85 (estimated wing area, 8.5 cm2) as compared with 1.3 for the fore-wings and 1.1 for the hindwings of Schistocercu. Both effects may be related to the flexibility of

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the butterfly wings; Jensen points out that flexibility of the locust hindwing can make a difference of 15" in angle of attack between the tip and the base. (2) The stall is very gradual, so that there will be little loss of control if the incidence for maximum lift is exceeded. (3) On this plot, the maximum ratio of lift to drag is given at the point at which a line through the origin touches the curve at a tangent. The angle that this line makes with the lift axis gives the best gliding angle, which is 15-24' in different specimens.

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FIG.4. Aguperes guluthru (Lepidoptera). polar plot of lift and drag of a mounted insect in 2.0 m/sec air-flow. Angle of attack indicated on curve. (Redrawn from Nachtigall, 1967).

(4) Both lift and drag depend on the square of the air speed; the shape of the polar therefore changes with air speed. Without knowing the weight of the live insect (Nachtigall did not have fresh specimens available) it is not possible to determine the minimum flying speed needed to produce lift sufficient for support. There is also insufficient information in this paper to justify the statement that the flatness of the polar near the region of maximum lift results in absolute flight stability. Stability in flight depends on the direction of chord-wise travel of the centre of aerodynamic pressure when the incidence changes. It will be

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helped by the smooth stall and the span-wise twist (“wash-out”) which is stated to be present, but these are secondary influences. The influence of the wing hairs and the wing scales was also studied (see also Nachtigall, 1965). Removing the hairs slightly improved the aerodynamic performance, but removing the scales definitely worsened it, particularly near the region of maximum lift where the reduction varied between 10 and 35%. The scales must act in a manner not fully understood to delay the turbulent break-away of the air-flow on the top surface as the wing approaches stalling incidence. D. T H E F L I G H T O F S M A L L D I P T E R A

Owing to the absence of complications due to the second pair of wings, flies have always been favourite insects for study of the aerodynamics of flight. Apart from some of the larger Nematocera, the wingbeat frequencies are high (Sotavalta, 1947) and at some point in the scale of size covered by the Diptera departures from steady-state aerodynamics are to be expected. Although it cannot yet be said that this point has been determined, several notable advances have been made since 1957. A great gap in knowledge has been filled by Nachtigall’s (1966) detailed account of the kinematics of wing motion in Phormia regina, but since this does not include actual measurements of lift and drag it will be discussed in the next section. A brief note by Baird (1965) reports some strain gauge measurements of lift and drag in Sarcophaga bullara and their correlation with wing position as determined photographically. Unusually high peak values of lift were obtained during brief periods in the cycle, and it is suggested that useful forces are obtained on the upstroke. The full report of this work will be of interest. The first experimental stljciy of a small insect is described by Vogel ( 1966), who established the flight performance of Drosophila uirilis at a Reynolds number of about 100. The lift required to support the body weight (2.0 dynes) was produced at a forward speed of 200 cm/sec with a body angle (upward tilt of the longitudinal axis) of 10”(Fig. 5); during this “standard performance” the beat frequency was 195/sec, the stroke angle 146” and the stroke plane angle +65”. The effect of the low Reynolds number was shown in the slight dependence of the parasitic drag on the body angle; perhaps because of this, there was one notable simplification of the flight control mechanism from that found in the locust. All the stroke parameters measured were found to be independent of the body angle; tilting the fly thus caused the lift and “preferred flying speed” (the speed of air movement at which the net drag was

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zero) to vary with the body angle over the range from - 10 to + 40". I n the locust, experimental change of the body angle brings into play the lift control reaction (see page 209), through the operation of which the lift tends to remain constant; because of this, it is impossible to influence the lift significantly by changing the body angle between 0" and 15-20" (Weis-Fogh, I956a, b). Vogel went to some pains to establish that the performance achieved by his tethered flies was comparable to that in free flight. He found that in free flight the forward velocity was usually less than 200 cm/sec, but that the freely flying animals were climbing along paths 15" to 20" above I

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FIG.5. Flight performance of a specimen of Drosophi/u uirilis. <-), lift; 0 , "preferred flying speed". Dashed lines mark values of body angle and flying speed at 100% lift. (From Vogel, 1966).

horizontal. The speed achieved by tethered flies in the wind-tunnel at 100% lift (equal to the body weight) was near to the maximum possible and occurred with the stroke plane nearly vertical ; under these conditions the forward distance travelled during one full wing cycle was just over twice the total span of both wings, which is a reasonable maximum by conventional standards of propeller performance. The conclusion from this part of the investigation was that the direction of the aerodynamic output force is primarily determined by the body angle and that variations in lift and flying speed can be explained

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in terms of a simple actuator disc with variable inclination, and not by changes in the form of the beat or of the angle of attack. This does not, however, imply that other parameters cannot be changed for short periods; and, in a further paper, Vogel (1967a) describes observations of those flight performances which, though not “successful” in the sense that steady values of lift and preferred flying speed were maintained for the prescribed period, provided useful information about the methods used for “voluntary” control. Body angle was now experimentally fixed at a single value for all measurements on each individual fly and performances were selected in which lift and air speed varied erratically. Flash photographs of 1 psec duration were taken at random times from a position in front of and 40” above the specimen, and from examination of these exposures it was possible to get an indication of changes in the stroke parameters accompanying particular performances. The main results were: (1) Wing-beat frequency changed very little under any conditions. In no case was the frequency more than k 10% of the average value and there was no correlation between frequency and other parameters. A slight decline in frequency was sometimes noted during the course of experiments on a particular fly; lift and thrust were then proportional to the square of the frequency, as in propellers. Since in a flight system powered by fibrillar muscle the frequency is largely determined by mechanical resonance, this parameter is effectively eliminated from those available for control. Vogel points out that, in an insect in which the inertia of the boundary layer has been shown to be a significant fraction of the total wing mass, there should be some influence on frequency of increase in stroke angle or flying speed, since these should reduce the moment of inertia of the boundary layer, but the effect is evidently too small to be detected. (2) Stroke angle (=amplitude of beat) and stroke plane were found to be fully interdependent parameters. The position of the wing at the top of the stroke did not change; increase in stroke angle from 90” to 150” was accompanied by a forward shift in the position of the bottom of the stroke, with only a slight change in its vertical position relative to the body axis (Fig. 6). There was a corresponding change in lift and thrust, and thus of total power. (3) In relation to the insect the angle of attack of the wing surface changed during the beat but was constant over the whole length of the wing, so that the movement was one of rotation from the base, rather than twist along the span. The angle was nearly constant during the whole of the downstroke and was not affected by the flying speed; the

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wing motion thus appeared similar in still air, when the middle of the wing was moving at 200 cm/sec, and in a 200 cm/sec air-flow. Since the incidence to the relative wind must have been very different under these two conditions, this means that there is no regulation of this parameter. Furthermore, since the wing performs an angular movement, an angle of attack which is constant over the span implies a variation in incidence along the length of the wing when there is any appreciable translational movement of the air relative to the fly.

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FIG.6. Mercator projection of the position of the wing-tip of Drosophilu at the top and bottom of the stroke when the stroke amplitude has the value indicated. (Redrawn from Vogel, 1967a).

(4) The wing profile was flat during the upstroke, but a slight camber appeared during the lower two-thirds of the downstroke. Lengthwise bending was observed near the wing base. Both these effects were in the opposite direction from that expected from passive deformation caused by the air-flow and must be actively produced. Alteration of air speed did not change the surface contour of the wing but produced a slight backward shift of the stroke plane.

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The main conclusion from this work was that lift and thrust are increased by Drosophila primarily by increase of the stroke angle, which also changes the stroke plane by moving forward the bottom part of the stroke. No measurements were made of the pitching moment (torque about the transverse axis of the fly) which this might produce, but Vogel points out that there should be a forward movement of the line of action of the total aerodynamic force tending to increase the body angle. Since the earlier study showed that increasing the body angle increases the lift and decreases the preferred flying speed without change in the stroke parameters, the overall result of an increase in stroke angle in free flight will be to make the fly climb more steeply with lower forward velocity. I t is, however, unlikely that this is the only means of control, since there would then be an invariable coupling between power output and the direction of flight. Some control may be exercised by the hind-legs, which are not held tightly against the body as are the proand mesothoracic legs, but control of some other unidentified parameter is also probable. In a third paper Vogel (1967b) has described the aerodynamic characteristics of the Drosophila wing and compared them with those of flat models in the same dimensional range. The most notable features were : (1) the greater L / D ratio at positive angles of incidence of the cambered as compared with the flat wing and the slightly better performance of the flat wing at negative incidence, such as is found during the upstroke. Camber was more effective in the wing than in the models. (2) near constancy of the lift coefficient of the wing over an incidence range from 20-50" (Fig. 7), compared to the distinct stall of the models. Vogel showed by use of the wind-tunnel balance and by means of visualized flow patterns how the stall of the models occurs at higher angles of incidence as (Re) is reduced from 200 to 60; this is the critical range for change of the flow pattern. The stall is further prevented in the natural wing by the microtrichia on the wing surface, which must influence the flow even though they lie within the boundary layer; their function may be to prevent backflow along the top surface, the occurrence of which is known to promote vortices and stalling. It is pointed out that hairs are retained only over the critical parts of the surface (the distal and posterior parts) in partly glabrous wings and that hairiness correlates inversely with aspect ratio (span/chord) in biting midges ; a broad wing is more liable to stall than a narrow one and the hairs may compensate for this.

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The polar curves for a conventional aerofoil, Schistocerco and Drosophilu, are compared in Fig. 7. The constancy of the lift coefficient over a wide range of angles helps to explain the absence of twist in the wing during flight. Due to the angular motion, there must be a very different incidence to the relative wind at tip and base during forward flight, but no part of the wing will stall. Owing to the low Reynolds number, the maximum LID ratio is small, but it does not necessarily follow that flight is energetically inefficient. In this dimensional range, C, is still 1

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FIG.7. Polar diagrams of three different airfoils. A. Conventional profile, NACA 2409, aspect ratio of 6, ( R e ) == 5 x lo8. B. Schistocerca hind-wing, (Re) == 4 x lo3. C. Drosophilu wing, ( R e ) 2 x lo2. On each curve the point of maximum LID is marked, with its value underlined. (From Vogel, 1967b).

greater at high than at low angles of incidence and the fly may obtain some effective lift and thrust forces from the wing drag during the downstoke, which under some circumstances may be inclined downwards and backwards instead of downwards and forwards as in larger insects (Fig. 6). It is not clear whether Vogel took this factor into account in his approximate computation of the expected overall performance from the kinematics of wing motion and the aerodynamic measurements on the

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wings. He found values for the total integrated forces that were only half those needed to sustain flight, but he cautions that the conclusion should not be drawn from this that stall hysteresis and other non-steadystate factors must be important in Drosophila. 111. T H EK I N E M A T I COSF W I N G M O T I O N

It will be apparent that a proper understanding of the aerodynamics of insect flight can only be achieved when full information is available about the precise form of the wing motion. If the beat frequency is sufficiently constant, such information can be obtained stroboscopically; that is, by illumination with brief flashes of light phased to known instants in the stroke cycle. More satisfactory, if the equipment is available, is continuous cinematography at really high speed, since one can then analyse single wing strokes. There remains, however, the problem of describing in a manner which can be appreciated by the reader the vast amount of information so obtained. A. D I P T E R A

Nachtigall (1966) has now used high-speed cinematography to make a detailed study of the form of the wing movements of the large fly Phorniia regina under two well-defined conditions. “Free flight” is defined as the flight of a fly suspended by the tip of the abdomen in an air-flow just sufficient to keep it stationary in space and mounted in a way that permits the fly to take up its preferred orientation; “flight in still air” is the condition when the fly is fixed in space with no air-flow. The wing movements are very different under these two conditions. For a complete description of the results it is necessary to define several reference systems. The system with axes related to the longitudinal axis of the body is defined as the t-system. That with axes related to the direction of the air-flow is the e-system, and that with axes related to the geoclinic vertical is the g-system. In each system, the vertical (or near-vertical) axis is the z-axis, the transverse axis is the y-axis and the longitudinal axis is the x-axis. Suffixes denote the system of reference in use. Thus, since in this investigation bilateral symmetry was always preserved, y, = y e . The body was, however, sometimes tilted up by the body angle, so xt # x, and zt # 2,. Under the conditions studied so far, the e-system and the g-system were coincident. The wing-tip moves on a great circle and various forms of description are possible in two-dimensional graphs. Figure 8 shows the wing-tip path of three different strokes as a projection of the surface of a globe.

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FIG.8. Path of the wing-tip of Phornzio reginn in "free flight", plotted on the surface of a sphere centred on the wing base; ends of strokes shown separately. (From Nachtigall, 1966).

Figure 9 shows the time course of angular movement in the three planes of the t-system. Both these figures related to a fly in fast, forward flight. Figure 10 shows silhouettes from the three axes of the t-system during one complete stroke. The pro-jections of Figs I 1 and 12 are more complicated. The stroke plane is defined as the plane of the great circle which most nearly coincides with the wing-tip path; if this circle is developed

FIG.9. Time course of angular movement of the wing of Phormia regina in fast, free flight, plotted in the three planes defined by the body axes (t-system). Points are individual frames at 1/6400 sec interval. (From Nachtigall, 1966).

FIG.10. Silhouettes of fly from direction of the three body axes during one complete wing beat. (From Nachtigall, 1966).

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into a line by unrolling the left or right half-cylinder on whose surface it lies and whose axis is the longitudinal axis of the fly, the path of the wing-tip is described by the two-dimensional plot of Fig. 11. Unrolling the half-cylinder whose axis is the direction of the air-flow produces the

FIG.1 1 . Path of the wing-tip and angle of attack in relation to the body as plotted on a developed cylindrical surface (see text). The triangles mark the upper leading edge of the wing and are drawn solid for the downstroke. y , is the best great circle defining the stroke plane. (From Nachtigall, 1966).

plot on the right of Fig. 12. On these developed plots, the lines y, and correspond to the stroke plane and the axes 5, and 5, are drawn at the level of the wing-base. It is important to remember that Figs 11 and 12 are obtained by unrolling a cylindrical surface and not by projection of the spherical surface on to a plane; particularly at the top of the stroke there is a large component of wing-tip movement in a lateral direction. The projection of Fig. 12 is the same as that used by Jensen (1956) (his Figs 111.6 and 111.8) to illustrate the motion of the locust wing. yt

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When considering the thoracic mechanisms by means of which the wing movements are produced, the plot of Fig. 11 and the silhouettes of Fig. 10 are the most useful. When considering the probable aerodynamic effect of the movements, Fig. 12 is of greater interest, since a translational movement can be added geometrically to give the path of the wing in space during free flight. The most accurate information about the time course of the movement is given by Fig. 9, on which measurements from individual photographic frames are plotted.

FIG. 12. Path of the wing-tip in relation to the air-flow, together with the path of the wing-tip in space and the incidence of the wing, as plotted on a developed cylindrical surface (see text). Triangles as in Fig. 1 1 . (From Nachtigall, 1966).

Nachtigall derived from the curves of Fig. 9 a plot of the time course of movement and then by successive differentiation could compute the changes of velocity and of acceleration; these have not been reproduced here since they are not directly relevant to the discussion. The other plots are all necessary for a full description of the kinematics. The time course of the stroke will now be described together with some qualitative features of the aerodynamics. It is convenient to distinguish four phases; downstroke, lower reversal movement, upstroke, upper reversal movement. 1. Downstroke (Nos. 3-14 of Fig. 10; Nos. 1-22 of Figs 11 and 12). The wing moves downwards and forwards with a steadily changing

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angle of attack, but Fig. 12 shows that it maintains a positive angle of incidence to the relative wind throughout this phase. During most of the downstroke the velocity of movement is constant and relatively slow. The surface remains flat and shows little passive flexion or bowing between the veins. This is aerodynamically the most important part of the stroke and generates the main lift; only in the middle of the downstroke is there any thrust. 2 . Lower reversal rnooernent. During this phase there is a rapid turnover of the wing, produced actively from the base, so that by the beginning of the upstroke the morphologically upper surface of the wing is the aerodynamic lower surface. The axis of turning lies behind the wing-tip, so that the tip executes a complicated downward and backward path during a period of about I/3 msec while the wing is almost stationary. The surface thus becomes nearly vertical and the reversal finishes with a flick of the wing-tip which is probably passive due to the aerodynamic force acting on it. 3. Upstroke. Much thrust is generated during the first half of the upstroke, when the wing surface is morphologically inverted. Half-way through this phase (No. 45 on Fig. 12) the aerodynamic incidence becomes zero and from then on the upstroke seems to be aerodynamically inefficient. The upstroke is kinematically complicated by the fact that the wing is not a rigid plate but can twist, due to interaction between the torque at its base and the aerodynamic forces. Nachtigall argues, however, that the twist can be almost discounted since it is greatest at those phases of the stroke that are aerodynamically ineffective, namely the reversal movements. During the middle of the downstroke there is almost no twist; during the first part of the upstroke when the twist is maximal (No. 19 on Fig. 10) the angle of attack is constant over the outer two-thirds of the span. The inner third of the fly’s wing has a smaller chord and since it moves at lower velocity owing to the angular nature of the motion, the harmful effect of its twist is minimized. 4. Upper reversal movement. At the end of the upstroke the path of the wing-tip bends round so that it is moving forwards through the air with its lower surface to the front and almost perpendicular to its direction of movement. At the top of the stroke the wing rotates rapidly from this aerodynamically unfavourable orientation, the active torque being helped by passive forces. The downstroke starts at once, at high incidence. These complicated movements are best appreciated by examination of the figures, which display them more adequately than any verbal description. The resulting motion of the wings is not harmonic since

185 the upstroke velocity is higher and its duration shorter than the downstroke. Velocities and accelerations of the wing, computed from the observed motion, are highly irregular; the bending due to aerodynamic forces evidently makes it impossible to deduce anything from these curves about the timing of the muscular contractions. At the start of flight, stroke amplitude builds up steadily, over several cycles at constant frequency, and there is a similar gradual decline in amplitude before motion stops. Figures 13 and 14 illustrate the different form of the wing stroke when the insect is flying in still air. The chief difference is that the planes of the upstroke and downstroke are now the same. The two parts of the stroke have the same duration and the velocities of movement are constant and nearly equal throughout the strokes. It appears that in free flight C O M P A R A T I V E P H Y S I O L O G Y O F T H E F L I G H T MOTOR

FIG.13. Path of the wing-tip in still air, plotted as in Fig. 8. (From Nachtigall, 1966).

the air-flow slows the downstroke and accelerates the upstroke, perhaps partly through a direct influence of the aerodynamic forces acting on the wings. Changes in the angle of attack during the stroke are very similar to those of free flight, showing the active nature of the twisting; at the middle of the downstroke the pronation is slightly greater and at the beginning of the upstroke the supination is greater and occurs earlier. It is impossible from kinematic studies alone to say whether these differences are the passive result of the air-flow or represent active compensatory movements. This detailed study of the wing motion of a fly confirms and extends earlier studies and it raises a large number of interesting questions. Without accompanying dynamic measurements, it does not help to decide whether steady-state aerodynamics can account for the generation of lift and thrust in an insect of this size and wing-beat frequency,

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but it does form an essential preliminary to such a study. It is of immediate relevance to the problem of the mechanics of the thorax and the functional r61e of the various muscles, which will be considered later. It also provides an essential background for discussion of the nature of the compensatory control movements in Diptera.

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FIG.14. Path of the wing-tip and angle of attack in relation to the body during flight in still air. Plot as in Fig. 11. (From Nachtigall, 1966). B. APlS MELLIFERA

The most quoted observations of the wing motion of the bee are those of Stellwaag (1916). Recently, accurate studies of the kinematics have been made by Neuhaus and Wohlgemuth ( I 960), Wohlgemuth (1962) and Herbst and Freund (1962). These authors used high-speed

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cinematography to record the form of the beat during ventilating movements and compared this pattern of behaviour to that observed during tethered flight in an air-flow and in free flight. Unlike the fly, the up- and downstroke of the wing beat in a bee lie close to each other under nearly all circumstances, so that the wing-tip path is never a wide loop or figure eight (Fig. 15). When not turning, the stroke plane remains inclined at about 120" to the longitudinal body axis, but its orientation in space naturally depends on the body angle. Apart from flight, bees use their wings to maintain the aeration of the hive (Fucheln) and also to distribute the secretion from abdominal

FIG.IS. Apis niellifern. Body attitude and path of the wing-tip during A, flight at 3 m/sec airflow; B, "Sterzeh". (Redrawn from Wohlgemuth, 1962).

scent glands (Sterzeht). The insect clings firmly with its legs and raises its abdomen at an angle of 25-30', bringing the stroke plane nearly vertical (Fig. 15B). Wohlgemuth (1962) has shown that there is a smooth gradation of behaviour from that observed in fanning to that characteristic of flight. At one extreme, the wings throughout the stroke remain inclined backward at about 30" to the transverse axis of the body; the beat frequency in this position averages 120/sec and the angle of attack changes during the stroke as shown in Fig. 16B. At the other extreme is the flight pattern in a 3 m/sec air-flow, when the whole stroke is rotated forwards; the beat frequency is now 200-225/sec, and the angle of attack changes as shown in Fig. 16A. The stroke angle (amplitude) is not correlated with fore-and-aft positioning of the stroke, nor with beat frequency; in either the fanning or flight attitudes, or in

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the intermediate attitudes adopted during Sterzeln, it is 65-120”, depending on the intensity of the behaviour. Addition of the air-flow vector to the wing motion shows that the angle of incidence to the relative wind at the widest part of the wing is about 15” during flight for almost the whole of the downstroke. For nearly two-thirds of the upstroke, the wing incidence to the relative wind is approximately zero; in contrast to the fly, the upstroke during flight therefore generates no useful aerodynamic forces. This conclusion is reinforced by examination of the form of the wing-tip path and by the changes in wing-tip velocity during the stroke (Fig. 17A). During

FIG.16. Apis mellifera. Path of the wing-tip and angle of attack of the widest part of the wing during A, flight at 3 m/sec airflow; B, fanning. (From Neuhaus and Wohlgemuth, 1960.)

the downstroke, the path of the tip is variable, suggesting a high aerodynamic loading and some wing flexibility (Figs 15, 16); during the upstroke it is straight and constant. Fig. 17A shows that the downstroke lasts longer than the upstroke and that for 30% of the downstroke the velocity is constant at a moderate value, indicating that the muscles are heavily loaded; the upstroke shows no such pause but sometimes a slight acceleration. The curvature of the wing-tip path (Fig. 16B) and the changes in tip velocity (Fig. 17B) suggest that during fanning the loading is high during both strokes, and this is borne out by calculation that during much of both strokes the wings are at a high incidence to the relative wind. The whole body of the insect actually oscillates up and down during strong fanning movements. The “Sterzeln” pattern spans this range of behaviour. Low-frequency movements with the wings

C O M P A R A T I V E P H Y S I O L O G Y O F T H E F L I G H T MOTOR

189

drawn back are similar to fanning except that the incidence to the relative wind is lower and the motion more nearly sinusoidal; this pattern is also seen at the start of ventilating activity. High-frequency “Sterzeln” movements with the wings drawn forwards are similar to those of flight. Neuhaus and Wohlgemuth (1960) state that the stroke pattern seen in tethered flight at 3 m/sec air-flow closely resembles that found in freeflight cinematographs of bees leaving the hive, but that when a returning bee nears the hive the body angle increases and the stroke plane is tilted backwards. They did not investigate other air-flow velocities or body attitudes. Herbst and Freund report similar observations of the range of ventilating behaviour, but include some accurate graphs of the angle of beat and angle of attack during the stroke cycle. They show that the

A

8

B

4 0

Downstroke

Upstroke t

FIG.17. Apis rnellifru. Velocity of the wing-tip in lateral projection during A, flight at 3 m/sec airflow; B, fanning. (From Neuhaus and Wohlgemuth, 1960).

rates of the reversal movements at the top and bottom of the stroke are variable, that the full range of beat frequencies is 85 to 240/sec and that the beat interval may vary by 10% over the space of a few cycles. Since the bee’s flight mechanism is powered by self-oscillating, fibrillar muscle, the last two observations suggest that the aerodynamic damping of the mechanical resonance must be very high and that there must also be some control of the elastic compliance of the thorax and muscles. One may conclude from these studies that the flight system of Apis differs from that of Phormiu in that the wing is more flexible and that,

190

J . W . S. PRINGLE

at any rate under the flight condition studied (3 m/sec air-flow), the upstroke contributes little to the aerodynamic force. The following parameters are under active control : (1) the fore-and-aft position of the stroke, which appears also to influence beat frequency. (2) the stroke amplitude. (3) the angle of attack during the downstroke, possibly with independent control of the angle of attack in different parts of the stroke. (4) the angle of attack during the upstroke (evidence so far only from ventilating behaviour).

To these must be added the old observations by Stellwaag (1916) of control of ( 5 ) the stroke planc, which will be discussed in the section dealing with flight reflexes.

IV. ST A BI L I T IYN FLIGHT A. D I P T E R A

As was first pointed out in the context of insect flight by Hollick (1940), control is required both of the magnitude of the lift and thrust (in order to vary the vertical and horizontal motion) and also of the line of action of the aerodynamic resultant in relation to the centre of gravity. In an animal which does not possess secondary stabilizing surfaces such as the tail of a bird or aeroplane, turning couples (torques) in one or more of the three planes of space will be generated if the resultant of the total aerodynamic forces does not pass through the centre of gravity. Furthermore, stability (that is, a tendency to return to the original attitude when displaced) will only be present if the line of action of this resultant moves, on angular displacement of the insect, in such a sense as to produce a restoring torque. No further experimental measurements of the torque acting on a flying Dipteran have been made since Hollick (1940), but some deductions are possible from the more accurate kinematic studies that have been made since then. Stability in a flying machine may be passive or active; that is, it may be inherent in the aerodynamic properties of the structure or it may involve active movement of the control surfaces. In a flying insect, the question is whether or not reflexes are always involved. Stability can be studied in flies in the probable absence of reflexes by using the phe-

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nomenon of “anaesthetic flight” (RuuschJlug), the activity which occurs during recovery from certain anaesthetics due to maintained motor activation of the self-oscillatory indirect flight muscles (Pringle, 1949). Hollick showed that free flight in this condition is stable if the wings are undamaged, but that there is not the usual compensation for bilateral asymmetry which is provided by reflexes from the halteres (Schneider, 1953; Faust, 1952; discussion in Pringle, 1957). Anaesthetic flight in calm air is, in fact, just stable in all three planes of space and we may consider for each plane in turn how this stability is achieved through the design of the flight system. Factors which make for inherent lateral (i.e. rolling) stability in a flying machine are location of the wings above the centre of gravity, mounting the wings at a dihedral angle and having the angle of incidence greater at the base than at the wing-tip (“wash-out”). In Phormia the angle of attack of the wing giving the maximum lift occurs in the middle of the stroke (points 12-15 on Fig. 12), when the wing is still above the horizontal. This suggests that the first two of the above factors may be operative. A difference in angle of incidence along the span is said by Nachtigall to be absent during the parts of the stroke that are most effective aerodynamically and this factor could only operate, if at all, during the lower reversal movement. Inherent stability in yaw (i.e. turning about a vertical axis) demands that the thrust be delivered by the wings at a point in front of the centre of drag of the body. The position of the latter has not been determined, but it may be assumed to be about one-third of the way along the body which, in a fly, is very near to the wing base. Nachtigall (1966) shows that the main thrust comes from the beginning of the upstroke. The thrust is, therefore, delivered well forward of the wing base and some inherent stability in yaw may be expected. Stability is most important in the pitching plane, and this was the plane considered by Hollick (1940), who first described both the inherent and the reflex mechanisms in Muscina stabuluns. His observations may be summarized as follows : (1) In still air, the magnitude, direction and line of action of the aerodynamic resultant are independent of the body angle. There is no inherent or reflex regulation by gravity. (2) In still air, spontaneous variability during different periods of flight showed that there is a close correlation between the amplitude of beat (stroke angle) and the line of action of the resultant (Fig. 18). A large majority of the flies tested in still air flew in the condition in

J . W . S. P R I N G L E

192

Fig. 18B.Although the magnitude of the aerodynamic force was sufficient or more than sufficient to support their weight, when released they immediately plunged forward due to the large pitching torque. (3) Exposure to an air-flow reduced the amplitude of beat and increase in body angle now produced a further considerable reduction

120

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FIG. 18. Muscina stabulans. A. Relationship between stroke amplitude and the horizontal distance from the centre of gravity to the line of action of the aerodynamic resultant. The outlines show stroke amplitude as viewed at right angles to the stroke plane. B. Diagram showing the modal magnitude and line of action of the resultant in still air. (From Pringle, 1957, after Hollick, 1940).

(Fig. 19). An effect of body angle on stroke angle was also seen after amputation of the antennae, (4) Increasing air flow also changed the path of the wing-tip as shown in Fig. 20A. In intact flies the change was primarily a moving forward of the path of the downstroke. After removal of the antennae, air-flow

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193

merely reduced the forward travel of the wing at the bottom of the stroke and moved backward the last part of the upstroke (Fig. 20B). Hollick concluded that these effects conferred two types of longitudinal stability : (a) Inherent stability dug to the fact that, in an air-flow, increasing body angle decreased the stroke amplitude and thus moved the line of 140' c

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FIG.19. Muscina stabulans. Average angle between the wings at the bottom of the stroke at different body angles in still air and in an air-flow of different velocities. (From Hollick, 1940).

action of the resultant backward. This must produce a restoring torque. Since this is a direct influence of the aerodynamic forces on the wing motion, the restoring torque will be greater at higher forward speeds, exactly as in a conventional flying machine with auxiliary tail surfaces. (b) Reflex stability due to the change in the path of the wing-tip (longitudinal position of the downstroke) when the air-flow stimulates

194

J . W . S . PRINGLE

the antennae. This reflex will produce a zero pitching moment only at a certain air speed; the direction of the torque at lower or higher speeds is such as to make the insect dive or climb until the optimum speed is reached. These suggestions are entirely consistent with the accurate kinematic studies of Nachtigall (1966), in spite of the different species of muscid Dipteran used. Caution is needed in comparing Fig. 20 with Figs 1 1 , 12 and 14, since the former is a projection of the spherical surface on which the wing-tip moves and Figs 11, 12 and 14 are developments of a cylindrical surface. The following features of the wing motion of Phormia are significant in the comparison : (1) In an air-flow the downstroke path moves forward from its position in still air (Figs 1 1 and 14). This is evidently the reflex effect described by Hollick (1940).

A

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FIG.20. Musca dontcsticn. Lateral view of the path of the wing-tip at different air speeds in intact insect and after removal of the antennae. (From Hollick, 1940.)

(2) The wing surface is at right angles to its direction of motion at the top and bottom of the stroke. It is thus at these instants that air-flow may be expected to produce the greatest longitudinal displacement of the wing-tip path due to the direct influence of the aerodynamic forces (Fig. 20B). (3) Hollick found that the passive reduction in stroke amplitude in an air-flow was greatest at large body angles and that it arises from a curtailment of the beat at the bottom of the stroke (Fig. 19). Figures 1 1 and 12 show that increase in body angle will cause the wing to be moving at above-optimum incidence earlier in the downstroke; its

C O M P A R A T I V E PHYSIOLOGY O F T H E F L I G H T MOTOR

195

movement will thus be more resisted by the increased drag and the stroke angle will decrease. There are thus good reasons for thinking that the form of the wing motion shown by a fly in an air-flow automatically confers inherent stability in the pitching plane, in spite of the absence of fixed auxiliary surfaces. The stable movement of the line of action of the resultant arises from two features of the mechanism, (a) the plane of the wing-beat, such that at the bottom of the stroke the wing is in front of the centre of gravity: (b) the gradual supination at the end of the downstroke, so that, when increase of body angle increases the incidence above the optimum, this part of the stroke is curtailed and its contribution to the lift reduced. Decrease of body angle will have the opposite effect, increasing the stroke amplitude at the bottom and increasing the component of lift acting in front of the centre of gravity. We have already seen how inherent stability in yaw may be produced by the fact that the main thrust comes early in the upstroke when the wing is in front of the centre of gravity, and how inherent stability in roll may result from the twist at the lower reversal movement and from the fact that the lift is mainly produced when the wing is above the centre of gravity. If further studies show this to be correct the remarkable conclusion will be reached that by virtue solely of the kinematics of the wing motion, a fly has slight inherent stability in all three planes of space. R . OTHER INSECTS

In the course of his study of the reflex control of velocity of flight in the honey-bee, Heran (1959) made some observations of this insect in tethered, anaesthetic flight and was able to show that, as in the fly, reflexes are absent. He found a small but significant influence of the air-flow on the amplitude of beat, but he did not study how this may be influenced by body angle nor did he consider the question of longitudinal stability. Anaesthetic flight in the honey-bee is not so stable as in the fly, and it is possible that this species relies more completely on reflex stabilization. Faust (1952) performed experiments on a variety of insects in free flight in total darkness to determine stability in the absence of an orienting light stimulus. He found that Diptera and Odonata flew normally and that slow-flying Lepidoptera preserved a normal flight attitude, though they did not move far in flight. The other insects showed varying degrees of disorientation. Coleoptera often showed long periods

196

J . W. S. PRINGLE

of rather unsteady flight, usually in a wide spiral; the moths Plusia and Agrotis and the honey-bee, though maintaining normal attitude, flew down in a steep spiral; most of the Hymenoptera, Sphinx (Lepidoptera) and Cetonia (Coleoptera) were completely disoriented. These experiments did not, of course, eliminate the influence of proprioceptive reflexes, but they suggest that, in all except the last group, some measure of inherent stability must be present, especially in the pitching plane; stability in roll appears to be better in most Coleoptera than in the other Orders. Haskell (1960) added the locust to the species tested. Completely blinded insects were sometimes able to fly as far as loom, but the

FIG.21. A. Movement of the wing-tips of Schistocerca gregaria in relation to the air during standard flight conditions. B. Lift generated in different parts of the stroke. (Replotted from Jensen, 1956).

majority were unstable in roll and eventually went into a spin. Goodman (1965) made a laboratory study of visual stabilization in Schistocerca by mounting the insect in front of a wind-tunnel in a framework that was free to rotate about the longitudinal axis, but this arrangement would not have detected inherent lateral stability; it was intended to demonstrate rolling reflexes. Gettrup (1966) found instability around the three main body axes in Schistocerca after destruction of all companiform sensilla on the wings and stressed the importance of reflex control. The locust is the only one of these insects for which sufficiently accurate data are available to permit an analysis of the expected stability

COMPARATIVE PHYSIOLOGY OF THE FLIGHT MOTOR

197

of the aerodynamic mechanism. Figure 21 shows the movement in space of the fore- and hind-wing-tips during steady flight, together with the instantaneous values of the lift generated. An approximate estimate of the lateral stability to be expected from the contribution of each pair of wings can be obtained by summing over the whole cycle the product of the instantaneous lift and the vertical distance of the wing-tip above or below the wing base. For the curves of Fig. 21 this gives values (arbitrary units) of + 2240 for the fore-wings and - 940 for the hind-wings. The hind-wings contribute 71% of the total lift (Jensen, 1956), but by themselves would create a system which is unstable in the rolling plane owing to the fact that the lift is mainly delivered when they are below the horizontal. The smaller lift contribution from the fore-wings greatly increases lateral stability. Overall, these data suggest that the insect should just be stable, but no account has been taken of wing interaction, wing twisting and several other factors which may complicate the situation. An interesting feature of this analysis for the locust is that it suggests a possible evolutionary origin for the state of affairs found in the Coleoptera. Here the hind-wings nearly meet in the mid-ventral line at the bottom of the stroke (Magnan, 1934), and it seems likely that their contribution to the lift is an even more unstable one than in the locust; the elytra, on the other hand, are held in flight at a pronounced dihedral angle and the appreciable lift that they are now known to produce should compensate for this instability, particularly during fast, forward motion. Faust’s (1952) demonstration that Cetonia is noticeably less stable in roll in the absence of visual stimuli is significant, since this family, alone in the Coleoptera, flies with its elytra folded. Stability in yaw will not be a problem in insects with a long abdomen, since this will act like a rudder to maintain the direction of movement in the air. In the locust the main thrust is delivered by both wings in the middle of the downstroke and by the hind-wings only rather late in the upstroke; for this reason and because the whole of the beat of the hindwings takes place with the wing behind the transverse (y,z,) plane, stability in yaw might be expected to be poor if the centre of drag of the body were located at the wing base. A long abdomen will also contribute to stability in pitch, and it may be for this reason that the Odonata are able to fly stably in complete darkness. The extreme development of this mechanism is found in the genus Petalura, where the anal appendages are expanded into flat surfaces which must function like a conventional tailplane (Tillyard, 1908). The passive mechanism found in flies could hardly operate in an insect

198

J . W . S. P R l N G L E

like a locust where there is not the same close correlation between the stroke plane and the stroke amplitude. It may be that the shortening of the abdomen, found in flies and bees and necessary if the insect is to perform rapid turning movements, is only acceptable aerodynamically in insects with a high-frequency wing-beat, because of its inevitable effect on stability in the pitching plane. It also demands a more elaborate and accurate system of reflex control of flight, which is superimposed on the necessary minimum of inherent stability.

v. T H E MOTORMECHANISM OF

FLIGHT REFLEXES

A . LIST O F R E F L E X E S

In this section we shall be concerned only with those reflexes that arc known to produce a rapid change in the spatial or temporal pattern of wing movements. Many other types of afferent stimulus affect features of insect behaviour that involve flight, but these will not be discussed unless it is known how their efferent effect is produced. The following reflexes have been described : (1) A reflex from a stretch receptor at the base of both pairs of wings, affecting the frequency of the neurogenic rhythm (Schistocerca : Wilson, 1961; Gettrup, 1962, 1963; Wilson and Gettrup, 1963). Study of this reflex gives no information about the effector mechanism of the flight motor and it will not be further discussed. (2) Reflexes from campaniform sensilla on the ventral surface of the costa and subcosta controlling aerodynamic lift and various features of the wing movement (Scltistocercu: Gettrup, 1965a, b, 1966). (3) A reflex from Johnston’s organ in the antennae, affecting (a) the path of the wing-tip during the downstroke (muscid Diptera: Hollick, 1940; Nachtigall, 1966). (b) the stroke amplitude and the velocity of forward flight (Calkphora: Burkhardt and Schneider, 1957; Apis: Heran, 1959). (4) Reflexes from the halteres, counteracting rotation in each of the three planes of space (Diptera: Fraenkel and Pringle, 1938; Fraenkel, 1939; Pringle, 1948; Faust, 1952). (5) A reflex from neck receptors (probably hair plates) controlling stability in the rolling plane by differential wing twisting (Odonata: Mittelstaedt, 1950). (6) A reflex from hairs on the front of the head, regulating the yawing torque in relation to the direction of the air-flow (Schistocerca and Locusta: Weis-Fogh, 1949, 1950; Guthrie, 1966).

COMPARATIVE PHYSIOLOGY OF THE FLIGHT MOTOR

199

(7) A reflex initiating the motor discharge to the flight muscles on loss of contact by the legs (nearly all insects except Coleoptera: Fraenkel, 1932). Further stimuli may be needed to maintain flight (Calliphora: Hollick, 1940; Schistocerca : Weis-Fogh, 1956b). (8) Visual control of stability in roll (Schistocerca: Goodman, 1965; Odonata: Mittelstaedt, 1950) and yaw (Schistocerca: Dugard, 1967). A dorsal light reaction and a reaction to the position of the horizon in the visual field are distinct sensory influences. These reflexes are integrated with the neck proprioceptive reflexes (5, above). (9) Dynamic visual (optomotor) reflexes, regulating the orientation and velocity of flight in relation to movement of the visual field (Apis: Schaller, 1960; Kunze, 1961; Heran, 1955; Heran and Lindauer, 1963. Muscid Diptera: Smyth and Yurkiewicz, 1966; Nachtigall and Wilson, 1967. Oryctes (Coleoptera) : Burton, 1964). B.

INITIATION,

M A I N T E N A N C E A N D T E R M I N A T I O N OF FLIGHT

Both in insects with synchronous and with asynchronous motor control, flight is normally initiated by starting the discharge of impulses in the nerves to the indirect flight muscles. In Schistocerca (Wilson, 1961) the motor discharge is synchronous with the frequency of beat from the first cycle of activity; at the beginning and end of flight, elevator muscles activity predominates over depressor activity. In muscid Diptera (Pringle, 1949; Nachtigall and Wilson, 1967) and in the beetle Oryctes (Darwin and Pringle, 1959) there is a high-frequency burst of motor nerve impulses to the indirect muscles at the start of flight and the frequency then falls to the low value characteristic of steady activity. However, in the water-bug Lethoceros, where flight is always preceded by a period of warming up, no change may be observable in the motor discharge to the indirect flight muscles at the instant that wing movements commence (Barber and Pringle, 1966). Motor excitation of the indirect muscles is also involved in the warming-up process before flight in the water-beetle Acilius, though mechanical activity during this process is limited to movements of minute amplitude at the wing base (Leston et al., 1965). Nervous excitation and minute thoracic oscillations at about twice the normal wing-beat frequency occur in muscid Diptera shortly before the wings are drawn forward into the flight position (Nachtigall and Wilson, 1967). Spontaneous termination of flight in Vespa and in flies is usually signalled by cessation of motor nerve impulses followed by a gradual

200

J . W . S . PRINGLE

decline in the amplitude of wing beat (Roeder, 1951), but wing movement in flies may be stopped abruptly on one or both sides of the body without change in motor activity to the indirect muscles (Boettiger, 1957; Nachtigall and Wilson, 1967). It is thus clear that activity in muscles other than the power-producing indirect muscles may be involved in initiation and termination of flight in insects with fibrillar muscles. Nachtigall and Wilson (1967) have confirmed by electrical recording that the non-fibrillar tergotrochanteral muscles of muscid Diptera are excited by a volley of 1 , 2 or 3 impulses (and that there is a strong jumping thrust from the mesothoracic legs) about 12 msec before the start of wing movements. This special adaptation for rapid take-off by initial elevation of the wings is not, however, present in all Diptera (Smart, 1958, 1959) nor in bees or beetles. Flight can start even in muscids after the lower insertion has been cut by removal of the legs; the tergotrochanteral muscle is therefore not necessary for the start of the auto-rhythmic activity. The main requirement if power is to be drawn from the activated fibrillar muscles is that the wing position and thoracic elasticity should be such that a mechanical resonance is created in a frequency range that is matched to the properties of the muscles. The mechanism by which this is achieved and the means of control of the power take-off involves other sets of muscles. On the sensory side, flight is initiated in most insects (not Coleoptera and only under some conditions in aquatic Hemiptera; Dingle, 1961) by loss of contact by the legs (reflex 7). In Drosophila, Muscina and some other insects it may then continue for long periods in still air, but in Schistocercu, Apis and most muscid Dipteru further stimuli are needed to maintain activity. These include wind-sensitive receptors on the head (Schistocercu: Weis-Fogh, 1956b) or antennae (Hollick, 1940), moving visual field (Schaller, 1960) or “wind on the moving wings” (WeisFogh, 1956b), which Gettrup (1966) has shown to mean the campaniform sensilla on the lower surface of the fore-wings. The central nervous organization by which these stimuli maintain excitation to the flight muscles is unknown. C . CONTROL O F AMPLITUDE, FREQUENCY A N D POWER

Insects require to control the mechanical power output in flight in order to move forwards or upwards at different velocities and to lift a variable body weight. This cannot be done, as with a rotating propeller, simply by increasing the angular velocity. Weis-Fogh (1965) has emphasized that energetic efficiency in a flapping-wing system like an

COMPARATIVE PHYSIOLOGY OF THE FLIGHT MOTOR

201

insect is only achievable if there is considerable storage of kinetic energy in elastic structures. This implies that the thorax, muscles and wings must together form a mechanically resonant system, and it imposes limitations on the parameters which are available for the control of power output. They are, essentially, three: (a) The amplitude of beat, implying a change in the distance through which the muscles shorten. (b) The frequency of beat, implying a change in the elastic properties of the thorax (since the inertia cannot change) and of the velocity of shortening of the muscles. (c) The aerodynamic incidence of the wings, implying a change in the loading of the muscles. It is now possible to outline how these parameters are used by various types of insect for the control of power output. 1. Indirect muscles

In the locust with synchronous power-producing muscles, neither the stroke angle (amplitude of beat) nor the beat frequency vary enough to account for the variation of power (Weis-Fogh, 1956a); the main controlled variable is the aerodynamic incidence of the fore-wings, and therefore the loading of the muscles. In order that the same velocity of shortening be achieved, the excitation must therefore increase. Wilson and Weis-Fogh (1962) found by electrical recording that there was an increase both in the number of active motor units and in the number of impulses in the volleys in the indirect muscles when power output was higher. The total number of nerve impulses reaching the powerproducing musculature per second thus increased, though the volleys still occurred at the frequency of wing beat. Changes in the timing of the volleys as the number of active motor units increased were correlated with a slight increase in beat frequency, assisting the increase in power. In insects with asynchronous power-producing muscles, the wing-beat frequency is directly determined by and not merely related to the mechanical resonant frequency of the thorax/wing system. Apart from a small change in the effective elasticity of the fibrillar muscles produced by increase of their excitation, beat frequency can therefore only be changed through auxiliary mechanisms. Increased excitation of the fibrillar muscles will produce an increase in stroke angle if the incidence and loading do not change, and increased excitation is required in order to maintain the same amplitude of beat under conditions of increased loading.

202

J . W. S. PRlNGLE

Nachtigall and Wilson (1967) measured not the power output but the aerodynamic lift at constant thrust of several species of large fly and found, during spontaneous variations in performance, a high correlation between lift and the frequency of motor nerve impulses to all the indirect flight muscles. This is an indication that power is controlled by adjustment of the power generator. Smyth and Yurkiewicz (1966) recorded a reduction in impulse frequency in dorsoventral and dorsal longitudinal muscles of a blow-fly when a pattern of stripes was moved from front to rear in the visual field (reflex 9), indicating that there is reflex regulation of this parameter. All authors agree, however, that there is not a large change in stroke amplitude (amplitude of beat) in flies when power output changes, except at the very beginning and end of a period of flying. Correlated increase in aerodynamic loading due to changes in angle of attack and also an increase in wing-beat frequency due to a change in thoracic elasticity must absorb most of the additional mechanical power generated. Evidence for a mechanism of this sort was provided by Chadwick (1951), who showed in Drosophila that change of air density did not produce the change in stroke angle expected in the absence of other compensatory effects (see also Chadwick, 1953). Large changes in stroke angle and of the amplitude of muscular contractions occur only at large body angles (Fig. 19), which would be unlikely to occur in life except as transient attitudes. Increases in motor nerve impulse frequency to the indirect fibrillar muscles have also been reported by Burton (1964) in the beetle Oryctes boas and correlated with increase in the amplitude of beat. Since these increased excitation frequencies were only observed unilaterally in the experiments described, one cannot conclude with certainty that stroke angle is a variable parameter in changes of output power in beetles, but the possibility is clearly indicated. No measurements appear to have been made of the frequency of excitation of the indirect muscles in the honey-bee, where large changes of stroke angle are involved in control of the velocity of flight (Heran, 1956; see later). 2. Direct inuscles

Changes in the angle of attack of the wings during the stroke are produced in the locust by the timing and force of contraction of the direct (basalar and subalar) muscles (Wilson and Weis-Fogh, 1962; Wilson, 1962). It is mainly the supination-producing subalar muscle whose degree of excitation is under reflex control and, since supination increases the aerodynamic loading during the downstroke, there is an

COMPARATIVE PHYSIOLOGY O F T H E F L I G H T MOTOR

203

automatic correlation between loading and total power production during the operation of this reflex. A similar situation is found in Odonata, where Neville (1960) showed that the angle of attack during the middle of the downstroke (Fig. 22) is controlled by the balance of excitation to the large second basalar and second subalar muscles, both of which contract at wing-beat frequency and are synergic so far as depression of the wing is concerned.

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FIG.22. Diagrams showing the angle of attack of the fore-wings of Aeshizu at different instants in the stroke and the effect of cutting various muscles. The leading edge is shown by a dot. Series (i) are downstroke and series (ii) upstroke. (a) normal stroke, (b) without anterior coxoalar, (c) without anterior coxoalar and third subalar, (d) without third subalar, (e) without first and second basalars and third subalars, (f) without second subalar. (From Neville, 1960).

He found that supination for short periods at the bottom of the downstroke and the latter part of the upstroke was also produced by contraction, respectively, of the anterior coxoalar and third subalar muscles, the latter of which operates tonically and transmits its force through a long elastic ligament. The size of these muscles shows, however, that they can contribute little to the control of power and they must be involved in attitude or directional control.

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Among well-studied types of insect with asynchronous flight mechanisms, Coleoptera and Hemiptera have several fibrillar muscles other than the indirect dorsal longitudinal afid dorsoventral. In both these Orders, the main basalar inuscle is fibrillar and also the indirect oblique dorsal which tends to supinate the wing. In beetles but not in Belostomatid bugs, the subalar is also of this type (Pringle, 1957; Barber and Pringle, 1966). Simultaneous increase in frequency of excitation to all the power producing muscles, direct and indirect, as found by Burton (1964), might therefore be expected to produce an increase in stroke angle, since there is no obvious provision for change in relative phasing of the cycles of mechanical activity of different muscles such as is found in the synchronous locust and therefore no simple way of achieving simultaneously a change in angle of attack during the downstroke and an increase in muscle loading. Such a mechanism would require that the basic time constants of “delayed tension” in the direct fibrillar muscles be different from that of the indirect muscles (Pringle, 1967), and would be sensitive to small changes in beat frequency, making it unreliable as a means of control. It is interesting that direct fibrillar muscles are found in some of the lower Hymenoptera (Daly, 1963) and in Diptera Nematocera (Smart, 1957, 1959). The highly simplified condition of the fibrillar musculature found in the Apoidea, Vespoidea and the muscid Diptera is evidently a late evolutionary development, but its independent evolution in these two lines may indicate that there is some inherent disadvantage, such as that suggested above, in the control of angle of attack by an autorhythmic mechanism. As explained earlier, power can only be drawn from the fibrillar muscles if the wing is coupled to them so as to generate the correct mechanical resonance, and a possible mechanism for the control of power exists in flies and other insects through the folding of the wings, which decouples them from their normal basal articulations. Folding is generally produced by the “adductor” muscle (terminology of Ritter, 1911 ; 3rd axillary muscle in many insects, Pringle, 1957), unfolding either by tonic contraction of the indirect muscles (honey-bee, Pringle, 1961) or, in muscid Diptera, by the “abductor” muscle (“anterior episternal basalar”, Smart, 1959). Nachtigall and Wilson (1967) show that the abductor muscles of flies are involved in drawing forward the wings at the start of flight and that full amplitude and power are not reached until this movement has occurred. When the wings are folded, minute vibrations of the pleural wall occur at approximately twice the normal beat frequency. They find, however, that neither the abductor nor the

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adductor muscles are excited during steady, straight flight, and this seems to exclude the promotion and remotion of the wing as a parameter that is used in flies for the control of total output power. Differential effects in this system during turning will be discussed later. There is no other evidence that a wing opening and folding mechanism is used for control during normal flight. 3. Accessory indirect muscles Muscles of this tonically contracting group change the structural characteristics of the thoracic box (Bonhag, 1949; Pringle, 1957). The mode of action of only two of them is known. It was first shown by Boettiger and Furshpan (1952) that tonic contraction of the pleurosternal muscle in Sarcophaga under the influence of CCl, moves inwards the mesopleural wing articulation and introduces a click action into the mechanical coupling between thorax and wings. During flight, this means that the velocity of wing movement on the up- and downstrokes is faster than it would be in a harmonic motion; with the wings folded, it provides a mechanical load with the right characteristics to permit oscillations to occur when the wing inertia is not coupled into the resonant system. Nachtigall and Wilson (1967) proved by electrical recording from the pleurosternal muscle in flies that it is indeed brought into action at the start of flight before any oscillations begin, and also before the movement forward of the wings couples them to the source of power in the indirect muscles. Non-linearity in the wing-thorax coupling appears to be widespread in the Pterygota, but in other Orders it has not been proved to be controlled by the pleurosternal muscle. A click action of the articulation has been demonstrated in Coleoptera (Pringle, 1957; Leston, Pringle and White, 1965), and in the metathorax of Schistocerca (T. Weis-Fogh, unpublished, quoted by Pringle, 1957), but is apparently absent in bees (J. W. S. Pringle, unpublished). The pleurosternal muscle tends to be replaced by skeletal or ligamentous internal bracing in many Lepidoptera (Chadwick, 1959); in scarabeid Coleoptera and in the Apoidea, its location is such that it is hard to see how it could greatly influence the thoracic elasticity, and its function may have been taken over by small tergopleural 01 other muscles. The great development of the click mechanism in Diptera may perhaps be correlated with the peculiar ability of these insects to start into flight without the preliminary warming up that is characteristic of many of the higher Orders. The oscillation frequency at which fibrillar muscles deliver their maximum power is greatly influenced by temperature (Machin, Pringle and Tamasige,

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1962), whereas the mechanical resonance of the wing-thorax system is almost temperature-invariant ; a proper matching of the two will therefore normally be achieved only at a certain temperature. In flies, however, the click mechanism ensures that shortening does not occur until tension has been developed; power output is thus much less sensitive to proper matching of the properties of the thorax and the muscles and therefore to the temperature. The second accessory indirect muscle whose action can be partially understood is the muscle of the axillary lever in bees. This slender tonic muscle operates the mechanism of the axillary lever (Fig. 23) and its contraction moves backward the 4th axillary and alters the relative positions of notum and pleuron through the axillary arm. Pringle (1961, 1965) has suggested that this gives a control of power through increase in the maintained stretch imposed on the indirect muscles. If this is proved to be correct, it will represent a distinct means of control from that found in other insects. An additional function for the axillary lever system is outlined in Section E.2. D. CONTROL OF VELOCITY

I . Apis inellifera It has long been known from field observations that a foraging worker-bee regulates its speed of flight according to the velocity of the wind with or against which it is flying. Heran (1955, 1956) showed that this involves sensory indication both of the velocity of movement through the air and of the velocity of movement over the ground. The former is obtained by means of the antennae (reflex 3b) and the latter using the eyes (reflex 9). The effector coordination is apparently similar in the two cases, though Heran (1959) made measurements of only one parameter, the amplitude of beat as viewed from a fixed direction in front of the insect; this may not give reliable values of the stroke angle if the stroke plane alters, He states that, in fixed bees, the normal stroke angle of about 120" was reduced to about 80" in an air-flow of 6-7 m/sec, with some change in the path of the wing-tip. This reflex was lost after amputation or fixation of the antennae. It is evidently a natural response, since bees fly to a good food source at about 8 m/sec and can exceed 10 m/sec through the air in a head-wind. The optomotor control was studied by Heran (1955), using a striped drum rotating about the transverse axis and placed below the bee so that it subtended an angle of 70". At a visual speed of 2.5 m/sec, the stroke amplitude was reduced by 12", and at 4*6m/sec by 23"; the

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magnitude of the effect was not changed when an air-flow was present. The reduction in stroke angle occurred mainly in the lower part of the beat. At a visual speed of 10.6 m/sec the reduction in stroke angle was less, and at about 20 m/sec it was hardly apparent; at these speeds of drum rotation the stripes are probably passing a point in the visual field above the flicker fusion frequency of the eye. Rotation of the drum also produced a change in the stroke plane, but this was not measured. A different response to optomotor stimulation was reported by Schaller (1960), who used two moving bands of vertical stripes, one on each side of the suspended bee; stroboscopic illumination was used to observe the wing positions. Schaller states that when the stripes moved from head to tail at the same speed of 35 cm/sec on each side, the stroke angle was increased from an initial value of less than 90" to a higher value, with an increase in the inclination of the stroke plane. As viewed from the front the pronation during the downstroke was reduced from 30" to 10" and the supination during the upstroke increased from an average of 40" to about 85". Both lift and thrust forces were increased and the duration of flight greatly prolonged. It seems probable that in these experiments the level of illumination was too low to produce the full amplitude of wing-beat in the absence of further excitation and that the stimulus of stripes moving at slow speed supplied this. The largeamplitude condition observed by Schaller was therefore the initial condition in Heran's experiments. In still air with a stationary and dimly lit visual field, the wing motion of a suspended bee is more similar to that used for fanning than for flight. Heran's observations are therefore indicative of the free-flight situation and they suggest that in bees the stroke angle is reduced as air speed and ground speed increase, thus regulating the velocity of flight in the way observed in the field. If other stroke parameters do not change much, this will be automatically correlated with a reduction of output power, and it is presumably achieved on the motor side by reduction in excitation to the indirect flight muscles. 2. Diptera Mention has already been made of the experiments of Hollick (1940) in which it was shown that the chief effect of increased air-flow in Muscina was a change in the path of the downstroke (Fig. 18). This, by, itself, will not regulate the aerodynamic thrust and the velocity of flight; the regulation is of the pitching moment, which leads to a change in velocity only when the fly has gained speed through a short dive or

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lost it through a short climb. The small influence of air-flow on stroke angle at small body angles was probably a direct effect and indicated the absence rather than the presence of a velocity regulating reflex. The antennal reflex was further studied by Burkhardt and Schneider (1957), who found in free-flight experiments that under strong lighting conditions removal of the antennae had little effect on forward velocity. In dim light, however, when an intact CuZliphoru normally either hovers or moves forward very slowly, an insect without antennae flew fast round the room and frequently collided with the walls. This does not, however, prove that the control of velocity by a visual reflex in bright light and by the antennae in dim light is done directly as in the bee; the control could be indirect through a change in pitching attitude. Further proof that the intensity of illumination has an important influence on the behaviour of a fly was given by Goodman (1960), who described the leg and wing movements involved in the landing response of LuciIiu sericutu and showed that they could be evoked merely by a reduction in light intensity. At the same time as the legs were lowered, the beat frequency was reduced and the stroke plane changed from its normal inclined position into a mere vertical orientation. From what is known from other studies, this was probably simply a sign of reduced power output from the flight motor as flight terminates, since it is the effect to be expected from relaxation of the pleurosternal muscle and reduction of excitation of the indirect muscles. The evidence, though inconclusive, suggests that although antennal and optomotor stimuli produce qualitatively similar effects on the speed of flight in large flies and in bees, the effector mechanisms may be different. A fly appears not to have (or to need) such an accurate control of velocity and it may achieve control largely by change in body angle. Vogel(l966) states that this is the chief means of control in Drosophilu and that no change in the angle of attack of the wings during the downstroke occurs when the air-flow is increased from 0 to 2m/sec. The automatic coupling between stroke angle and stroke plane and the movement of the line of action of the aerodynamic resultant in muscids will tend, in free flight, to produce an automatic relation between power output and lift, rather than between power output and forward velocity. The bee, on the other hand, appears not to vary the path of the wing on the downstroke to the extent seen in muscids and seems able to change the direction of the aerodynamic resultant without change of body angle. This gives it a wider control of forward velocity and enables it to maintain a constancy of the orientation of the body in space that may be needed in an animal that relies so much on accurate visual stimuli.

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E . CONTROL OF LIFT

It is convenient to discuss the locust first under this heading since only here has the mechanism of a lift-control reaction been fully elucidated; in this insect it appears to be the regulated parameter, while the forward velocity was found by Weis-Fogh (1956a) to vary from a maximum of 5.5 m/sec at the start of flight to a minimum of 3.5 m/sec, below which the insect cannot maintain itself in the air. The lift-control reaction (Weis-Fogh, 1956b; Gettrup and Wilson, 1964) was first identified when it was discovered that the lift produced by a locust flying in front of the wind-tunnel remained almost constant when the body angle was increased from 0 to 15". Since neither the beat frequency, the stroke angle nor the stroke plane changed enough to account for this, it was concluded that the controlled parameter was the angle of attack during the downstroke. Gettrup and Wilson (1964) established that the angle of attack of the hind-wings did not change and that therefore there is a compensatory change of pitching moment as well as a regulation of the total aerodynamic lift. Wilson and Weis-Fogh (1962) showed by electrophysiological recording that the effector action is chiefly mediated through variable excitation to the subalar muscle, and Gettrup (1965a, b; 1966) showed that it is chiefly the campaniform sensilla on the lower surface of the hind-wings that monitor the lift during their downstroke and form the sensory element in the reflex. Of all the reflexes involved in the control of insect flight, this is probably the most fully worked out. 2. Other insects In other insects, measurements of the lift have not been dissociated from measurements of total aerodynamic power, and it is therefore not possible to conclude that this parameter is under specific control. Thus Vogel's (1 967a) demonstration of a correlation in Drosophila between lift and stroke angle may merely show spontaneous variability in the excitation of the indirect flight muscles. In this species the lift control reaction is definitely absent (Vogel, 1966). Hollick (1940) does not state how lift varies with body angle in Muscina and one cannot therefore decide on present evidence whether the absence of the reflex in Drosophila is characteristic of flies in general or of small insects. The former would be consistent with the conclusion that velocity of flight in muscid Diptera is controlled indirectly by change of attitude. The control of lift presents special problems in insects that can hover or fly backwards, that is, that can remain airborne without forward

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velocity. The wing kinematics required for hovering have not been fully elucidated, but in some insects it seems to be achieved (as in humming birds, Greenewalt, 1960) by bringing the stroke plane nearly horizontal. The simplest method of doing this is by an increase in body angle. Vogel (1966) showed for tethered Drosophila that lift equal to 100% body weight was produced at a body angle of +65" at zero forward velocity. Demo11 (1918) showed in MeZoZontha in free flight that reducing the area of the wings led to flight at a higher body angle and at slower forward speed, but that the insect could remain airborne; this

FIG. 23. Semi-diagrammatic internal view of some lateral mesothoracic muscles of Xylophaga (Apidae). (Original).

suggests that lift is the controlled parameter and that regulation, as in muscids, is by change of body angle. In contrast to this, the honey-bee hovers with little change of the attitude of the body and must either change the stroke plane in relation to the body (Stellwaag, 1916; Heran, 1959) or change the kinematics in a way not understood. Syrphid Diptera hover with the stroke plane vertical (Magnan, 1934). Mention has already been made of the mechanism of the axillary lever in the Apoidea (Fig. 23). This peculiar development of the posterior

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notal wing articulation must be correlated with some special control mechanism in bees. Contraction of the muscle acts through a large leverage to change the relative position of notum and pleuron, the force being transmitted through the axillary arm and the 4th axillary sclerite. In addition to increasing the mean length of both dorsoventral and dorsal longitudinal muscles, so that more power will be drawn from them, it also appears to rotate the stroke plane into a more horizontal position. Unfortunately the muscle is very inaccessible in the flying insect, and this anatomical deduction cannot be checked experimentally. There has been no modern account of the flight muscles of Syrphidae, but Bonhag (1949) gives a detailed description of Tabanus sulcifrons, which can also hover. There is no movable portion of the postphragma in any way resembling the axillary lever. F. CONTROL OF ATTITUDE

1. Pitch

We have now to consider in turn the reflexes that enable a flying insect to maintain its orientation in space. The question of inherent stability, due to a direct influence of the aerodynamic forces on the wing motion, was discussed in Section IV; here we are concerned with the active contraction of direct flight muscles. Reflex stabilizing mechanisms in the pitching plane have already been described in the antenna1 reflex of Muscina (Hollick, 1940) and in the lift control reaction of Sclzistocerca (Gettrup and Wilson, 1964). In Schistocerca a reduction in the angle of attack of the fore-wings during the downstroke, and the absence of a similar change in the hind-wing, leads to a forward pitching moment when the body angle is increased; in Muscina, a forward pitching moment is produced by backward displacement of the path of the downstroke when the air-flow on the antennae is reduced. In Schistocerca, the compensation for change of attitude is accompanied by a regulation of total lift; in Muscina, with only a single pair of wings, the magnitude and the line of action of the aerodynamic resultant are independently controlled. The change in wing motion in Calliphora during operation of the dynamic reflexes from the halteres (reflex 4) was described by Faust (1952). Figure 24C shows that forward rotation (when one would expect there to be a compensatory backward pitching moment) leads to an increase in angle of attack during the downstroke, especially at the beginning and end of the stroke. If one compares these outline tracings with the silhouettes of Fig. 10 and with the angles of attack shown on

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A

FIG. 24. Tracings from high-speed photographs of the downstroke of Calliphora. A, during backward pitching rotation at 2.5 rotations/sec; B, during normal flight in still air; C,during forward pitching rotation at 2.5 rotations/sec. (From Faust, 1952).

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the developed cylindrical plot of Fig. 11, it is clear that the drag at the top of the stroke and the lift at the bottom of the stroke should both be greater with the motion shown in Fig. 24C; both these will produce a torque tending to resist the forward rotation. Faust did not observe his flies from the side and so would not have detected any change in the path of the wing-tip; it is therefore possible that the path of the downstroke moved forward in Fig. 24C and that is a similar effector response as that observed by Hollick in Muscina. 2. Roll Reflex stabilization in roll has been studied in Schistocerca by Goodman (1965) and Gettrup (1966), in Anax by Mittelstaedt (1950) and in Calliphoru by Faust (1952) and Schneider (1956). The sense organs involved include the eyes, the neck proprioceptors, the campaniform sensilla at the base of the fore-wings and the halteres. In all cases where it has been investigated, the effector movement is a small differential change in the angle of attack in the middle of the downstroke. The movement is particularly clear in Odonata, where it can be elicited when the wings are not beating; in this Order, where the mechanisms of the fore- and hind-wings are entirely independent, all four wings twist (Mittelstaedt, 1950). Mittelstaedt did not determine which muscles are used to produce this motion, but from the work of Neville (1960) it seems probable that it is due to antagonistic action of one or more muscles of the basalar and subalar complexes. Control through a subalar muscle is probable since this will lead to a more powerful downstroke of the wing with the greater angle of attack. There is no reason to doubt that this is the general mechanism of control in roll but a complication arises in those insects in which the fore- and hind-wings are coupled together; here the angle of attack of the forewings cannot be changed without change in the azimuthal position of the hind-wings. The difficultyis resolved in the case of the Apidae by the fact that the direct basalar and subalar muscles of the metathorax are the only muscles producing downward torque on the hind-wings ; both are tonic, non-fibrillar muscles acting through elastic apodemes, and if control is exercised mainly through the larger subalar muscle, then increased angle of attack and relative downward displacement of the hind-wing will be produced simultaneously.

3. Yaw Turning need not be linked to banking in a flying machine that lacks a rudder and tail surfaces. Insects therefore require reflex control in both

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roll and yaw, though the two may be linked; if there is forward motion through the air, a banked turn is aerodynamically more efficient, but in hovering flight banking is unnecessary. Control of directional change has been studied in Schistocercu by Weis-Fogh (1956b) and Dugard (1967), in muscid Diptera by Faust (1952), Smyth and Yurkevicz (1966), Nachtigall (1967) and Nachtigall and Wilson (1967), in Apis by Stellwaag (1916), Schaller (1960), Kunze (1961), Heran and Lindauer (1963) and in Oryctes by Burton (1964). There seems to be a real difference between different insects in the way turning movements are brought about. In Sclzisfocercu flying in front of a wind-tunnel and induced to turn by greater illumination of one-half of the visual field, the chief effector action was a greater and earlier pronation of the left fore-wing during a left turn ; the left wing also moved more rapidly than the right wing in its downstroke (Dugard, 1967). The change was produced by earlier and double firing of the first basalar muscle and by bringing into action one or both of the motor units in the second basalar muscle; the motor discharge to the subalar muscle was constant. The basalars on the outer side of the turn had some activity, even during rapid turns. There was no change in the pattern of movement of the hind-wings or of the motor impulses to metathoracic flight muscles, but legs and abdomen were extended towards the inside of the turn, the former through excitation of the second coxal abductors and anterior rotators at flight frequency and synchronized, respectively, with the up- and downstrokes. In the locust, control in yaw is therefore exercised by variation in angle of attack during the downstroke, but different muscles are used from those involved in the lift-control reaction and in adjustment of pitch. No difference in the frequency of motor excitation to the indirect muscles on the two sides during a turn has been reported in Schistocercu, but in Oryctes Burton (1964) states that the motor nerve impulse frequency to all six pairs of fibrillar flight muscles is increased unilaterally on the side away from which the insect is turning during asymmetrical visual stimulation. In muscid Diptera, Smyth and Yurkiewicz (1966) state that there is no difference between the left and right sides, and Nachtigall and Wilson (1967) confirm this in the extreme case when one wing is folded and not beating. This feature may be peculiar to Coleoptera. In Oryctes the amplitude of beat (stroke angle) is increased on the outer side during a turn, producing both a yawing and a rolling moment owing to the fact that the aerodynamic resultant is directed downwards and backwards in forward flight; the result must be a banked turn. In Diptera the power does not appear to be a controlled variable in

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regulation of yaw, which must be done by change of other parameters under the control of tonic muscles; yaw and roll can thus be dissociated in the way that would be expected in insects that can hover or fly forward very slowly. There is disagreement in the literature about the nature of the control movement. Nachtigall and Wilson (1967) observed spontaneous asymmetries of wing movement in tethered flies in still air. They found that asymmetrical beating was accompanied by excitation of the direct abductor or adductor muscles. When only the left wing was beating, the right adductor and the left abductor muscles were active; when only the right wing was beating, the reverse pattern was observed. During symmetrical wing movements, both muscles were usually silent, but were sometimes observed to fire at lower frequency when there was not a visually obvious turn in progress. That this is a normal mechanism of producing yawing torques is inferred from an observation by Magnan (1934) of similar asymmetries in free flight and small fore-and-aft displacements seen in high-speed photographs. Compensatory movements quite different from this were described by Faust (1952) during yawing rotations of Culliphora with intact halteres (Fig. 25). During neither direction of rotation was there any change of stroke angle, nor any modification of the upstroke, but during the downstroke the angles of attack were extremely unequal on the two sides. Examination of these tracings together with the wing-tip trajectory of Fig. 14 shows that, for example, the wing motion of Fig. 25A must produce greatly increased drag from the right wing and some increase in lift from the left. These are exactly the torques required to produce the most rapid compensation for a yaw to the left in free flight. It must therefore be doubted whether the large differences in foreand-aft wing position and in stroke angle seen in the experiments of Nachtigall and Wilson (1967) represent a normal effector pattern for regulation in the yawing plane. Their experiments were done in still air in which the flight of many muscid Diptera is intermittent. The drawing back of the wing by the muscles of the third axillary sclerite normally accompanies the termination of flight in all insects except the Odonata and is evidently used by Diptera for quick stops (Boettiger, 1957). The large size of the tergal muscle of the basalar (abductor of Ritter, 1911) is also a peculiar feature of the Diptera and could be correlated with their ability to start flight very rapidly; it is small or absent in Apidae and Oryctes, where the wings are brought forward and held forward in flight largely through the tonic contraction of both sets of indirect muscles (Pringle, I961 and unpublished observations). The observation by Nachtigall and Wilson (1967) that neither abductor nor adductor

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muscles were excited during steady flight is also consistent with the view that this antagonistic pair of muscles is concerned with starting and stopping and not with control in yaw. In the honey-bee, Stellwaag (1916) figured and described briefly a quite distinct mechanism of control, a differential change in stroke plane

B

C

FIG. 25. Tracings from high-speed photographs of the downstroke of Cafliphora. A, during yawing to the left at 2.2 rotations/sec; B, during normal flight in still air; C, during yawing to the right at 1.5 rotations/sec. (From Faust, 1952).

on the two sides. We have already seen that the ability to change the plane of the wing beat is a peculiar feature of the wing mechanism of the bee, and it has been suggested that it is achieved by the axillary lever and used for the control of lift. Anatomically, the axillary lever

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mechanisms of the left and right sides are entirely distinct and could be used differentially. The result would be a banked turn. Schaller (1960), on the basis of some not very convincing flash photographs, states that there is yet another mechanism of control in yaw and one which, unlike all other cases described in insects, involves a change in the form of the upstroke. Schaller induced turning by moving a pattern of vertical stripes past a tethered bee in opposite fore-and-aft directions on the two sides. His photographs show asymmetrical positions of legs and abdomen and large differences in the angle of attack of the wings on the two sides in the same way as was described by Faust for flies (Fig. 25), but he interprets them as showing changes in the angle of attack during the upstroke. Since an increased angle of attack on the side to which the bee is trying to turn correlates with a slight reduction in stroke amplitude, this does not make sense, since a large angle of attack during the upstroke would produce a smaller aerodynamic loading. Kunze (1961) measured torques of up to g cm with the bee in a very similar attitude, and we may conclude that Schaller’s interpretation of his results was wrong and that the same compensatory movements are made by the honey-bee during optomotor yawing reflexes as by the fly during yaw detected by the halteres. V I . COMPARATIVE STUDIES A . AXIOMS

A physiologist is interested in comparative studies primarily because a knowledge of variety may make it easier to understand basic mechanisms. It is axiomatic in the study of insect flight that the basic organization arose only once and that the anatomical and physiological patterns that are now to be found in the different Orders have evolved by differentiation in different ways from a common plan. It follows that homologies can be traced in the flight muscles and in the nerves and sense organs that are involved in flight reflexes. More important, these reflexes themselves should have a common pattern, so that it would be surprising (though never, of course, impossible) that an entirely different control movement should be used in one insect from that common to the others. At the extremes of evolutionary specialization, these homologies may be difficult to unravel, but the assumption that they are there to be unravelled provides the stimulus for this sort of essay. Kinematic and aerodynamic principles provide a further unifying

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influence. The insect wing moves essentially about a pivot, formed by complex folding of the cuticle at its base and local thickening with elastic or hard pads to form the axillary sclerites. It has three degrees of freedom in space and one in time. Thus, it can show elevation and depression, promotion and remotion, pronation and supination, and each of these motions can occur at different velocities. Furthermore, the wing is not a rigid structure, but can sometimes twist about its long axis, change section or even fold. There are, however, only certain combinations of these movements which generate lift and thrust, and once these are understood in a few insects in different size ranges, it begins to be possible to appreciate the common themes. Differences in shape between different insects arise mainly because of differential growth of the epidermal cells which lay down the cuticle. Since the muscle fibres insert directly into the cuticle, such differential growth can give rise to apparent migration of the insertions of muscles on to adjacent sclerites. By differentiation of the amount and type of cuticle laid down in different places, apparently new cuticular structures can form or old ones can change their shape. In extreme cases of this process, the nature of the reflex patterns of which certain muscles form a part may be the best clue to homology. There are few flight muscles in any insect that cannot reasonably be derived from one of the ten basic muscles shown in Fig. 26. It is the

A

B

FIG.26. Diagrammatic views of the ten muscles in the right half of a pterothoracic segment. A, more medial muscles; B, more lateral muscles. dlm, dorsal longitudinal; odm, oblique dorsal; dvm, dorsoventral; fin, wing-folding muscle of 3rd axillary;pm, pleurosternal; hi,basalar; sm, subalar; tpnil, anterior tergo-pleural; isw, intersegmental ; @ma, posterior tergo-pleural. (From Pringle, 1957).

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thesis of this review that not only was the anatomical pattern laid down early in insect evolution, but that the pattern of nervous organization was also defined at an early stage, so that it is, in principle, possible to trace continuity of function for each of these ten muscles and for the reflexes of which they form the effector machinery. This means that though the muscle may change its size and the insertions of different parts of it may move owing to differential cuticular growth, the contribution the muscle makes to the movements of the whole insect in the air either remains unchanged or changes by slow degrees so that at all stages of the process it is of advantage to the insect. It is argued that this continuity of function is preserved for each individual muscle even as it changes its physiological properties in a way that would make it seem, at first sight, inevitable that it must play a different r81e in the flight machinery. B. D I F F E R E N T I A T I O N O F T H E F L I G H T MUSCLES

Histologically and physiologically it is possible to distinguish three types of muscle in the flight system: tubular, comprising all the tonic muscles and the twitching, power-producing muscles of Odonata and cockroaches; close-packed, forming the power-producing muscles of Orthoptera, Lepidoptera and the other “synchronous” insects ;fibrillur, forming the self-oscillatory muscles of Hymenoptera, Diptera, Coleoptera and Hemiptera (Pringle, 1957). It used to be thought that within any one Order the distribution of these types of muscle was constant, but recent work has revealed considerable diversity and generalization is now dangerous. As a final summary an outline will be given of the functions and patterns of differentiation shown by the ten muscles, emphasizing newly discovered facts. The information about Hymenoptera comes from Daly (1963) unless otherwise stated. 1. Dorsal longitudinal (dlm) Probably always an important power-producing downstroke muscle. Reduced in Odonata. Close-packed in Orthoptera, mesothorax of Lepidoptera, Cicadidae, Sirex and Cephus (Hymenoptera). Fibrillar in metathorax of Coleoptera and Xyelu (Hymenoptera), mesothorax of Diptera and all other Hymenoptera, Hemiptera and Homoptera. The rnetathoracic dlwi of Apis and nearly all Hymenoptera is tubular and serves to control the transmission of energy from the mesothorax to the hind-wings, thereby preserving its original function (Pringle, 1961).

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2. Oblique dorsal (odm) This indirect muscle appears to act, where it is present, as an upstroke supinatar, thus resembling the direct basalar and subalar muscles of some insects in providing both power and control of angle of attack. Absent or tubular and reduced in Orthoptera, Odonata and Lepidoptera. Close-packed in mesothorax of Cicadidae. Fibrillar in metathorax of Coleoptera, both segments of Xyela, mesothorax of Diptera (large except in Ptychoptera, Smart, 1959), other Hemiptera and Homoptera (Barber and Pringle, 1966), and some Hymenoptera but not Apis or Vespa. One may speculate that possession of this muscle, in either closepacked or fibrillar form, enables insects to achieve a greater degree of supination of the wing during the upstroke. All species known to possess it can hover; the honey-bee, which can hover, does not possess it, but can rotate the stroke plane by means of the axillary lever. 3. Dorsoveiztral (dvm) A complex of muscles in most insects, inserting dorsally on the tergum but ventrally on sternum, coxa or trochanter. The main powerproducing upstroke muscle, following the durn closely in its histological type. Part of it differentiates into the close-packed tergo-trochanteral muscle in many Diptera, where the rest of the muscle is fibrillar (Smart, 1959). Absent in Leptomastix (Chalcidae), which thus has no muscular antagonist to the fibrillar dlm, but instead an apodemal ligament. Absent in metathorax of Apis and Vespa but fibrillar in the metathorax of some Hymenoptera Apocrita, where this portion attaches to a detached notal sclerite. Neville (1960) describes a similar situation in Odonata, where the close-packed anterior coxoalar muscle produces supination at the end of the downstroke and throughout the upstroke by separate movement of the lateroprescutum. 4. Third axillary muscle

Present and tubular in all insects that can fold their wings. One of the most constant functional r8les of all the flight muscles.

5. Pleurosternal Tubular in all insects. Its location in Oryctes and Apidae makes it hard to see how it can influence the lateral stiffness of the thoracic box, but elsewhere, especially in Diptera, it evidently has this function. Replaced by a skeletal bridge in some Lepidoptera (Chadwick, 1959).

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6. Basalar Dorsal insertion on basafar sclerite of pleuron, if this is differentiated; ventral insertion on sternum, coxa, or pleuron. Produces downstroke pronation. Close-packed in both segments in Schistocerca, Odonata, Sirex and Cephus. Fibrillar in metathorax of Coleoptera and some Hymenoptera (pleural portion), in mesothorax of Belostomatidae (Barber and Pringle, 1966) and some Hymenoptera including Ichneumonidae. Tubular in Apis, Vespa and Diptera. The basalar complex may contain up to three distinct muscles. In Schistocerca contraction of the first basalar is required for proper pronation, and the excitation of its single motor unit is normally a pair of impulses; the second basalar has more variable excitation but produces less supination (Wilson and Weis-Fogh, 1962). The second basalar is the larger muscle in Odonata (Clark, 1940). The first basalar becomes a tubular tonic muscle in Ichneumonidae and some other Hymenoptera in which the second basalar is fibrillar, but the whole muscle is tubular and tonic in Apis and Vespa, where pronation is produced primarily by closure of the scutal cleft under the influence of the indirect muscles and only small adjustments are required by the direct muscles (Pringle, 1965). The tubular pleural musculature of the 1st axillary sclerite in Diptera may represent part of the basalar complex.

7 . Subalar Dorsal insertion on the subalar sclerite, if differentiated; ventral insertion on sternum, coxa or pleuron. Close-packed in both segments in Schistocerca. Fibrillar in metathorax of Coleoptera. Coxal portion fibrillar in mesothorax of Xyela (Hymenoptera) and Diptera Nematocera (Smart, 1959); tubular in other Hymenoptera. First and second subalars close-packed in Odonata, third subalar tubular with a long resilin ligament (Weis-Fogh, 1960); Neville (1960) shows how the third subalar of dragon-flies comes into action near the top of the upstroke, producing a phased supination in spite of the fact that it can only contract tonically (Fig. 22a, d). In Apidae the subalar sclerite is connected to the posterior part of the 2nd axillary sclerite by a ligament, so that tonic contraction of the subalar muscle will partially oppose the natural pronation movement of the wing. In the mesothorax of higher Diptera, where there is no subalar sclerite, the tubular pleural muscles of the 4th axillary probably represent the subalar complex. It is reasonable to suppose that the lift-control reaction of insects other than Schistocerca is also mediated through the subalar muscles.

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Extra power can be added when these muscles are close-packed or fibrillar and extra power can be drawn from the indirect muscles by tonically-controlled supination in other cases. This is a good example of the way reflex functioning may be preserved by a particular muscle even though its physiological type changes in the course of evolution.

8. Anterior tergopleural Dorsal insertion on the prealar arm of the tergum; ventral insertion on or near pleural ridge or basalar sclerite. Probably always tubular and usually small, but large in Diptera, where it forms the main abductor muscle of the wing (Bonhag, 1949; Nachtigall and Wilson, 1967). Part forms the remotor muscle in the metathorax of Coleoptera, where it could exercise an opposing tension to the oscillatory basalar (Darwin and Pringle, 1959). 9. Intersegtnental muscle Ventral insertion on sternal apophysis ;posterior, dorsal insertion on lateral part of post-phragma. The involvement of this muscle in the flight machinery has been doubtful and Neville (1960) has now shown that in Odonata it produces elevation of the abdomen. It is important in this review only because the axillary lever muscle of the Apidae has been homologized with it on morphological grounds (Daly, 1964); this seems improbable in view of Neville’s discovery, an‘d the axillary lever muscle will here be assumed to be a differentiation of part of the posterior tergopleural complex. 10. Posterior tergopleural Dorsal insertion on postero-lateral part of tergum near posterior notal wing process; ventral insertion on pleuron. Always tubular. Small in Orthoptera and Odonata, but one of the largest controlling systems in Hymenoptera and Diptera, where it becomes complex. Pringle (1961) suggested from an examination of the anatomy of bees that one of these muscles (which he called the scutellar muscle, no. 75 of Snodgrass, 1942) might be responsible for control of the pitching moment. Pronation at the start of the downstroke in a bee is produced by closure of the scutal cleft under the pull of the large, fibrillar dorsal longitudinal muscle acting through the lateral arm of the postphragma (Fig. 23) and does not require the phasic contraction of the direct muscles, as in Schistocerca and Odonata; all of these, being tubular in nature, would be unable to follow the frequency of wing beat. Tonic contraction of the “scutellar” muscle resists closure of the scutal cleft and might delay

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pronation at the start of the stroke. Some of the pleural muscles of the posterior notal process in Diptera (Bonhag, 1949) are so placed as to have a similar action. As stated above, the muscle of the axillary lever in bees must now be assumed to be part of the posterior tergopleural complex, so that control of power and of the stroke plane must be added to the possible functions of the tergo-pleural complex. It will require much difficult experimental work to sort out the separate controlling actions of these small tubular muscles and deductions from comparative studies are thus permissible in the absence of better evidence. REFERENCES Baird, J. L. (1965). Aerodynamic behavior of the flesh fly Sarcoplraga bullata (Diptera). Am. 2001.5, 706. Barber, S. B. and Pringle, J. W. S. (1966). Functional aspects of flight in Belostomatid bugs (Heteroptera). Proc. R. SOC.B. 164, 21. Bennett, L. (1966). Insect aerodynamics : vertical sustaining force in near-hovering flight. Science, N . Y. 152, 1263-1266. Boettiger, E. G. (1957). The machinery of insect flight. In “Recent Advances in Invertebrate Physiology” (B. T. Scheer, ed.), pp. 117-142. University of Oregon Publications. Boettiger, E. G. and Furshpan, E. (1952). The mechanics of flight movements in Diptera. Biol. Bull. Woods Hole 102, 200-21 1. Bonhag, P. F. (1949). The thoracic mechanism of the adult horsefly (Diptera: Tabanidae). Mem. Cornell agric. Exp. Sta. no. 285. Burkhardt, D. and Schneider, G. (1957). Die Antennen von Calliphora als Anzeiger der Fliiggeswindigkeit. Z. Naturf. 126, 139-143. Burton, A. J. (1964). Nervous control of flight orientation in a beetle. Nature, Lond. 204, 1333.

Burton, A. J. and Sandeman, D. C. (1961). The lift provided by the elytra of the rhinoceros beetle, Oryctes boas. S. Afr. J. Sci. 57, 107-109. Chadwick, L. E. (1951). Stroke amplitude as a function of air density in the flight of Drosophila. Biol. Bull. Woo& Hole 100, 15-27. Chadwick, L. E. (1953). The motion of the wings. In “Insect Physiology” (K. D. Roeder, ed.). John Wiley and Sons, New York and Chichester. Chadwick, L. E. (1959). The furcopleural muscles of Lepidoptera. Ann. m t . SOC. Am. 52, 665-668. Clark, H. W. (1940). The adult musculature of the Anisopterous dragonfly thorax (Odonata, Anisoptera). J. Morph. 67, 523-565. Daly, H. V. (1963). Close-packed and fibrillar muscles in the Hymenoptera. Ann. ent. SOC.Am. 56,295-306. Daly, H. V. (1964). Skeleto-muscular morphogenesis of the thorax and wings of the honey bee Apis mellifea (Hymenoptera: Apidae). U.Cal. Publ. Ent. 39, 1-77. Darwin, F. W. and Pringle, J. W. S. (1959). The physiology of insect fibrillar muscle. I. Anatomy and innervation of the basalar muscle of lamellicorn beetles. Proc. R. SOC.B, 151, 194-203.

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Demoll, R. (1918). “Der Flug der Insekten und der Vogel.” Gustav Fischer, Jena. Dingle, H. (1961). Flight and swimming reflexes in giant water bugs. Biol. Bull. Woods Hole 121, 117-128. Dugard, J. J. (1967). Directional change in flying locusts. J. Insect Physiol. 13, 1055-1 063. Faust, R. (1952). Untersuchungen zum Halterenproblem. 2001.Jb., Allg. 2001. Physiol. 63, 325-366. Fraenkel, G. (1932). Untersuchungen uber die Koordination von Reflexen und automatisch-nervosen Rhythmen bei Insekten. I. Die Flugreflexe der Insekten und ihre Koordination. Z. vergl. Physiol. 16, 371-393. Fraenkel, G. (1939). The function of the halteres of flies (Diptera). Proc. zool. Sor. Lond. A, 109, 69-78. Fraenkel, G. and Pringle, J. W. S. (1938). Halteres of flies as gyroscopic organs of equilibrium. Nature, Lond. 141, 919-921. Gettmp, E. (1962). Thoracic proprioceptors in the flight system of locusts. Nature, Lond. 193,498499. Gettrup, E. (1963). Phasic stimulation of a thoracic stretch receptor in locusts. J. exp. Biol. 40,323-333. Gettrup, E. (1965a). Control of fore-wing twisting by hindwing receptors in flying locusts. Proc. XIIth int. Congr. Ent., London 1964, 190-192. Gettrup, E. (1965b). Sensory mechanisms in locomotion : the campaniform sensilla of the insect wing and their function during flight. Cold Spr. Harb. Symp. quant. Biol. 30,615-622. Gettrup, E. (1966). Sensory regulation of wing twisting in locusts. J. exp. Biol. 44, 1-16. Gettrup, E. and Wilson, D. M. (1964). The lift-control reaction of flying locusts. J. exp. Biol. 41, 183-190. Goodman, L. J. (1960). The landing responses of insects. I. The landing response of the fly, Lucilia sericata, and other Calliphorinae. J. exp. Biol. 37, 854-878. Goodman, L. J. (1965). The role of certain optomotor reactions in regulating stability in the rolling plane during flight in the desert locust, Schistocerca gregaria. J. exp. Biol. 42, 385-407. Greenewalt, C. H. (1960). “Hummingbirds.” Doubleday & Co., New York. Guthrie, D. M. (1966). The function and line structure of the cephalic airflow receptor in Schistocerca gregaria. J. Cell Sci. 1, 463-470. Harvey, W. R. and Haskell, J. A. (1966). Metabolic control mechanisms in insects. Adv. Insect Physiol. 3, 133-205. Haskell, P. T. (1960). Sensory specialization in response to environmental demands. Symp. zool. SOC.Lond. 3, 1-23. Heran, H. (1955). Versuche uber die Windkompensation der Bienen. Naturwissenschaften 42, 132. Heran, H. (1956). Ein Beitrag zur Frage nach der Wahrnehmungsgrundlage der Entfernungsweise der Bienen. Z. vergl. Physiol. 38, 168-21 8. Heran, H. (1959). Wahrnehmung und Regelung der Flugeigengeschwindigkeit bei Apis mellijica L. 2.vergl. Physiol. 42, 103-163. Heran, H. and Lindauer, M. (1963). Windkompensation und Seitenwindkorrectur der Bienen beim Flug uber Wasser. Z. vergl. Physiol. 47, 39-55. Herbst, H. G. and Freund, K. (1962). Kinematik der Flugel bei ventilierenden Honigbienen. Deufsch. ent. Z. N.F. 9, Hft. I/II, 1-29.

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Hollick, F. S. J. (1940). The flight of the dipterous fly Muscina stabulans Fallbn. Phil. Trans. B. 230, 357-390. Horridge, A. (1956). The flight of very small insects. Nature, Lond. 178, 1334-1335. Jensen, Martin (1956). Biology and physics of locust flight. 111. The aerodynamics of locust flight. Phil. Trans. B. 239, 511-552. Kunze P. (1961). Untersuchung des Bewegungssehens fixiert fliegender Bienen. 2. vergl. Physiol. 44 656-684. Leston, D., Pringle, J. W. S. and White, D. C. S. (1965). Muscular activity during preparation for flight in a beetle. J. exp. Biol. 42, 409414. Machin, K. E., Pringle, J. W. S. and Tamasige, M. (1962). The physiology of insect fibrillar muscle. IV. The effect of temperature on a beetle flight muscle. Proc. R. SOC.B. 155,493499. Magnan, A. (1934). “Le Vol des Insectes.” Hermann & Cie, Paris. Maruyama, K. (1965). The biochemistry of the contractile elements of insect muscle. In “The Physiology of Insecta” (M. Rockstein, ed.), vol. 2. Academic Press, New York and London. Mittelstaedt, H. (1950). Physiologie des Gleichgewichtsinnes bei fliegenden Libellen. 2. uergl. Physiol. 32, 422-463. Moore, F. K. (1956). Lift hysteresis at stall as an unsteady boundary layer phenomenon. N.A.C.A. Report, No. 1291. Nachtigall, W. (1964). Zur Aerodynamik des Coleopterenflugs: Wirken die Elytren als Tragfliigel? Verhdl. dtsch. 2001. Ges., Kiel319-326. Nachtigall, W. (1965). Die aerodynamische Funktion der Schmetterlingsschuppen. Naturwissenschaften 52, 216-217. Nachtigall, W. (1966). Die kinematik der Schlagiliigelbewegungen von Dipteren. Methodische und analytische Grundlagen zur Biophysik des Insektenflugs. Z. vergl. Physiol. 52, 155-21 1. Nachtigall, W. (1967). Aerodynamische Messungen am Traglliigelsystem segelnder Schmetterhge. Z. vergl. Physiol. 54,210-231. Nachtigall, W . and Wilson, D. M. (1967). Neuromuscular control of dipteran fight. J. exp. Biol. 47, 77-97. Neuhaus, W. and Wohlgemuth, R. (1960). uber das Facheln die Bienen und dessen Verhaltnis zum Fliegen. Z. vergl. Physiol. 43, 615-641. Neville, A. C. (1960). Aspects of flight mechanics in anisopterous dragonflies. J. exp. Biol. 37, 631-656. Osborne, M. F. M. (1951). Aerodynamics of flapping flight with application to insects. J. exp. Biol. 28, 221-245. Pringle, J. W. S. (1948). The gyroscopic mechanism of the halteres of Diptera. Phil. Trans. B. 233, 347-384. Pringle, J. W. S. (1949). The excitation and contraction of the flight muscles of insects. J. Physiol. 108, 226-232. Pringle, J. W. S. (1957). “Insect Flight.” Cambridge University Press, London. Pringle, J. W. S.(1961). The function of the direct muscles in the bee. Proc. XZth int. Congr. Ent., Vienna 1960, 1, 660. Pringle, J. W. S. (1965). Locomotion: fight. Zn “The Physiology of Insecta” (M. Rockstein, ed.), pp. 283-329. Academic Press, New York and London. Pringle, J. W. S. (1967). The contractile mechanism of insect fibrillar muscle. Prog. Biophys. molec. Biol. 17, 1-60. Ritter, W. (191 1). The flying apparatus of the blow-fly. Smithson misc. Coll. 56.

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The Consumption and Utilization of Food by Insects G. P. WALDBAUER Department of Entomology, Uriiilersity of Illinois, Urbana, Illinois, US.A . I. Introduction [I. Consumption, Growth and Utilization Indices . A. Consumption and growth . B. Digestibility and efficiency of conversion . “1. Measuring Consumption and Utilization by Weight . A. General considerations . B. The gravimetric method . C. Indirect methods using markers . IV. Food Consumption . V. Digestion and Conversion of Fresh and Dry Matter . A. Limitations of the data . B. Comparison of species . C. Comparison of foods . D. Effects of environmental factors E. Variations with age and sex . VI. Utilization of Dietary Constituents . W. Utilization of Energy . Acknowledgement . References

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I. INTRODUCTION A great deal is known concerning the qualitative nutritional requirements of insects. The quantitative aspects of insect nutrition have, however, received less attention, and there have been few studies on the rates of intake and the efficiency of food utilization. In particular, relatively little is known concerning the intake, digestibility and efficiency of conversion of defined diets. Quantitative work with artificial diets has usually involved only measurements of the amount of a particular nutrient required per unit of diet (House, 1959, 1962, 1965a). This defines the relationship between the requirements for particular nutrients (Sang, 1956, 1959), but says nothing of food intake, absolute requirements or the efficiency of food utilization. Most measurements 229

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of intake and utilization have been made with insects feeding on natural foods. The information available in this area is useful, but some of it is difficult to interpret because measurements have not been standardized, methods have varied in accuracy and confusion has resulted from the application of indices of utilization derived from vertebrate nutrition. I have, therefore, included an extensive discussion of these points. Lafon (1951) reviewed some of the earlier studies of insects other than the silkworm Bombyx mori. Most of the numerous studies of food utilization by Bornbyx are summarized by Legay (1957, 1958) and Yokoyama (1963). I have omitted discussion of most of this work since it would be repetitious in the context of this review. Recent papers on Bombyx which are not cited by the above reviewers are as follows: Hassanein and El Shaarawy (1962a, b), Mukaiyama and Ito (1962a, b), Shyamala et al. (1956), Takeuchi and Kosaka (1961). Trouvelot’s (1867) measurements of food efficiency in Antheraea polyphemus are of historical interest but too crude to be useful. Insects as a group feed upon a remarkably diverse list of organic substances. At the same time most species show a high degree of specificity in their choice of food. Gordon (1959) hypothesized: “Competition and natural selection gradually drive and bind each species to a specialized food supply that it can utilize more efficiently than any of its competitors.” Nevertheless, the qualitative nutritional requirements of growing insects seem to be relatively uniform (Fraenkel, 1953, 1959; House, 1962). It seems apparent that adaptive nutritional differences must be sought on a quantitative level and that a meaningful comparative nutrition of insects will not emerge until quantitative studies are emphasized. The determination of absolute requirements for dietary constituents depends upon the measurement of intake. Differences in food efficiency can be demonstrated only by measuring intake and growth. Digestibility should also be measured since it can be expected to vary widely with different foods. The efficiency with which digested food is used for growth will vary not only with the maintenance requirement for energy but also with the balance of nutrients (Gordon, 1959). Measures of intake and utilization have played little part in the classical studies of insect nutrition although their usefulness in this connection is obvious. For example, instances of poor growth may not be due to the nutritional inadequacy of the diet but to a low rate of intake due to the absence of a non-nutrient phagostimulant. Conversely, the addition of a nutrient with phagostimulatory activity might lead to increased growth although the nutrient is neither required nor utilized (Dadd, 1960). Sang (1959) suggested that there may be more than one optimal

231 diet for a given species. Measures of intake and utilization can give an indication of this, since patterns of utilization may be different although diets are similar in their ability to support growth. For instance, low intake might be offset by high digestibility or a high utilization of digested food for growth or vice versa. Poor digestibility might be offset by the efficient utilization of digested food or vice versa. One of the major concerns of modern ecology is the elucidation of the energetic relationships within and between communities. A knowledge of the food efficiency of insects is thus of particular importance to ecology since, in terms of either numbers or biomass, insects are a major component of almost all terrestrial or fresh water communities. Engelmann (1966) reviewed the field of terrestrial energetics and discussed its ecological significance. He also discussed the importance of food efficiency data in ecological energetics. CONSUMPTION A N D UTILIZATION O F FOOD

11. CONSUMPTION, GROWTH A N D U T I L I Z A T I O N INDICES An overall understanding of the utilization of a food requires answers to the following questions: At what rate is the food eaten? How much of the eaten food is actually digested? What part of the food is incorporated as body substance? These questions cannot be answered without a reliable and accurate method for determining the weight of the food ingested, the weight of the feces which correspond to the ingested food and the weight gained by the insect during the experiment. These three measurements are basic and necessary for the calculation of the rate of feeding, the digestibility and the efficiency of conversion of food to body substance. This is true whether the question is the utilization of whole food (fresh weight), dry matter, energy, carbohydrates, nitrogen or other nutrient constituents of the food. Utilization data are most useful if they are reduced to terms which allow one to compare, for example, the utilization of different foods, utilization from instar to instar, or the effects of environmental factors on utilization. Various indices of consumption, digestibility and efficiency of conversion have been devised by the students of vertebrate nutrition (Kleiber, 1961;Tyler, 1964). The use of these indices in insect nutrition has been discussed by Trager (1953), Gordon (1959) and Waldbauer (1962, 1964). The indices mentioned below can be calculated on the basis of fresh or dry weight, but it should be noted that fresh and dry weight indices are not comparable since the percentage dry matter of food, feces and insect are likely to differ.

G. P. W A L D B A U E R

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A . CONSUMPTION A N D G R O W T H

I . Consumption index In his classic work on accessory growth factors Hopkins (1912) pointed out that absolute quantities cannot be used to compare the intake of rats growing at different rates-that valid comparisons can be made only on the basis of the rate of intake relative to the mean weight of the animal during the feeding period. The consumption index (C.I.) is, therefore, calculated as :

C.I. F T A

r =

_f_

TA

fresh or dry weight of food eaten duration of feeding period (days) = mean fresh or dry weight of animal during feeding period.

= =

The mean weight of the animal is most accurately calculated from the area under its growth curve as determined by integration or direct measurement. A weighted average of daily weights will give an almost identical value if the growth curve is smooth (Waldbauer, 1964). Other indices of consumption have been proposed. Lafon (1951) used mg of food eaten per g of insect (final weight) in thirty days. The usefulness of this index is difficult to discern since it neglects the initial weight of the insect. For instance, an insect which eats 500 mg as it grows from 10 to 100 mg is obviously eating at a greater rate relative to its weight than an insect which eats the same quantity in the same time as it grows from 50 to 100 mg. Legay (1957) proposed mg of food eaten per g of insect (initial weight) per day. In this case two insects of the same initial weight which eat the same quantity of food in the same time will have identical indices even if one grows more than the other. Smith (1959) used weight of food eaten divided by weight gained. This yields an infinitely large index if the insect eats but does not grow, a dramatic expression of the failure to grow but not a useful measure of feeding rate. See Section IV for further discussion of feeding rates. 2. Growth rate The reZatiue growth rate (G.R.) is calculated as: G.R.

=

G TA

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233

G = fresh or dry weight gain of animal during feeding period

T A

= =

duration of feeding period (days) mean fresh or dry weight of animal during the feeding period.

Workers who measured efficiency of utilization in insects usually did not compute true growth rates (see, however, Waldbauer, 1964). B . DIGESTIBILITY A N D EFFICIENCY OF CONVERSION

1. Conversion of ingested food The eficiency of conversion of ingested food to body substance (E.C.I.) is calculated as :

E.C.I. =

wt gained x 100 wt food ingested

or

G.R. E.C.I. = C.I. The E.C.I. is an over-all measure of an insect’s ability to utilize for growth the food which it ingests. The E.C.I. will vary with both the digestibility of a food and the proportional amounts of the digestible portion of that food which are, on the one hand, converted to body substance and, on the other hand, metabolized for energy to maintain life. Thus the E.C.I. will rise and fall with the A.D. (approximate digestibility) and the E.C.D. (efficiency of conversion of digested food to body substance). 2. Digestibility The approximate digestibility (A.D.) is calculated as :

A.D. =

wt of food ingested-wt of feces wt of food ingested

Many authors including House (1965a), Trager (1953) and Waldbauer (1964) referred to this measure as the “coefficient of digestibility”. However, this is misleading since, as will be discussed below, the difference between the weight of ingested food and the weight of the feces does not represent the amount actually digested. It should be noted that approximate digestibility as here defined for insects differs from apparent digestibility as defined for mammals. Although mammals pass urine and feces separately, their feces do not, strictly speaking, consist only of undigested food nor do they contain all

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of the undigested food. The gases produced in digestion, mainly C 0 2 and CH4, are lost. Mammals on a nitrogen-free diet continue to pass nitrogen in the feces. This metabolic fecal nitrogen presumably comes from intestinal secretions and the debris of gut cells. Thus, with mammals the true digestibility is not obtained by subtracting the weight of the feces from the weight of the ingested food. The value obtained is called the apparent digestibility and is considered to be a reasonable approximation of the true digestibility (Kleiber, 1961; Mitchell, 1964; Tyler, 1964). The feces of insects are certainly not free of fecal metabolic products; the peritrophic membrane is an obvious example. The presence of the urine in the feces further complicates the measurement of digestibility in insects. The difference between the weight of food ingested and the weight of the feces actually represents the food which is stored or metabolized less metabolic wastes discharged in the urine or as fecal metabolic products. Thus, the above formula yields what I have called approximate digestibility, a value which is always lower than the corresponding apparent digestibility. The magnitude of the difference depends upon the relative amount of urine in the feces. Birds mix the urine and feces in the cloaca and thus present a similar problem (Kendeigh, 1949; Mitchell, 1964). Kendeigh refers to the energy in the ingested food less the energy in the excrement as the metabolized energy. It can be argued that the use of “digestibility”-even in the sense of “ approximate digestibility”-is inappropriate unless one accounts for at least the urine content of the feces. However, the available alternatives, “retention” (Evans, 1939b) and “utilization” (Kasting and McGinnis, 1959; Hirano and Ishii, 1962) are also inexact. Furthermore, “utilization” should be reserved as a general term which includes digestion, metabolism and conversion to body substance. An approximation of apparent digestibility can be obtained if the estimated urine content is subtracted from the weight of the feces. I will refer to the value thus obtained as the coeficient ofapparent digestibility (C.A.D.). . Determination of the C.A.D. may be an unnecessary refinement with most insects which eat foods with a moderate protein content. Their feces will probably contain relatively little urine, and thus the difference between the A.D. and the C.A.D. is likely to be very small. The major component of the urine of terrestrial insects is uric acid (cf. Patton, 1953; Wigglesworth, 1965). Analyses of the feces of phytophagous insects indicate a low uric acid content. It ranges from 051% for the 1st instar down to 0.24% for the 5th instar in the dried feces of Bombyx

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235

mori (Hiratsuka, 1920). Subtracting the uric acid content from the feces results in a negligible increase of the A.D. It is raised from 46.0% to 46.4% for the 1st instar and from 36.5% to 36.7% for the fifth instar (calculated from data of Hiratsuka, 1920). The dried feces of other leaffeeding insects are also low in uric acid, 0.62% for the 4th instar and 0.52% for the 5th instar of Neodiprion sertifer (Janda, 1961), and about 4% for Melanoplus bivittatus (Brown, 1937a). A similar correction for the 4% uric acid content of the feces of Tenebrio molitor larvae raises the A.D. from 46.3% to 485% (Evans and Goodliffe, 1939). Gupta and Sinha (1960) found a higher concentration of uric acid in the feces of the adults of stored grain beetles, about 12% of the dry weight for Sitophilus granarius, 18% for Tribolium confusum and almost 20% for Cryptolestes ferrugineus. The feces of insects which eat foods high in protein are likely to contain a relatively greater percentage of urine. The approximate digestibility may, therefore, differ greatly from the coefficient of apparent digestibility. The larvae of the webbing clothes moth, Tineola bisselliella, eat hair and other substances which consist largely of keratin. Their dried feces contain about 28% uric acid plus far smaller amounts of ammonia and urea (Hollande and Cordebard, 1926). Using this figure to correct Titschack’s (1925) data for Tineola (Table V) raises the A.D. of 29% to a C.A.D. of approximately 56%.

3. Conversion of digested food The eficiency with which digested food is converted to body substance (E.C.D.) is calculated as: E.C.D.

=

wt gained x 100 wt of food ingested - wt of feces

E.C.D. will decrease as the proportion of digested food metabolized for energy increases. Thus the E.C.D. is affected by factors which influence the amount of energy devoted to the maintenance of physiological functions or the support of activity. E.C.D. is not directly dependent upon digestibility, but it does vary with the level of nutrient intake. Since the maintenance requirement remains more or less constant, the proportion of food available for growth will decrease as intake decreases. E.C.D. also varies with the nutritional value of the food. For example, Hopkins (1912) demonstrated that a vitamin deficiency caused rats to waste an enormous amount of absorbed food (see Gordon’s (1959) lucid discussion of the significance of food efficiency).

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111. MEASURING CONSUMPTION A N D UTILIZATION B Y WEIGHT A . GENERAL CONSIDERATIONS

The selection of the period during which utilization is to be measured is critical. An arbitrarily defined period, a specified length of time not marked by physiological events, offers the advantage of convenience. However, data obtained on this basis are of limited value since, as will be shown below, utilization varies quantitatively from instar to instar and within instars. Physiologically defined periods are less convenient, but are more likely to yield reproducible results which can be validly compared with the results of other experiments. A physiologically defined period may include the entire life cycle, an entire stage or one or more instars. In any case its beginning and end are marked by physiologically significant events. It may begin with hatching or the completion of a molt. Its end may be marked by the premolt condition, completion of a molt or an event in the life of the adult. The use of physiologically defined periods presents some inconveniences. It may be difficult to obtain enough newly hatched or molted insects to begin an experiment. Growth rates may vary, necessitating frequent inspections to fix the end of the period for each individual. Physiologically defined periods were used by Crowell (1 941), Hiratsuka (1920), So0 Hoo and Fraenkel (1966), Waldbauer (1964) and others. Some workers used mixtures of instars or failed to specify instar or the length of the period. Others, for instance Evans (1939b) and Chauvin (1946), used arbitrary periods of time within specified instars. Evans (1939b) stated that he used larvae of different physiological ages within the same instar. Accuracy demands the collection of all feces derived from the food eaten during an experiment. Residual food which may be in the gut at the beginning or the end of an experiment can cause an error in both weight gain and the weight of feces. The gut contents of feeding early 4th instar Protoparce sexta is about 9.3% of the fresh body weight (Waldbauer, 1962). McCay (1938) found that residual food in the gut of feeding Blattella germanica amounts to less than 5% of the dry body weight. The use of physiologically defined periods simplifies quantitative collection of the feces since the gut will be more or less empty just before or after a molt. It seems that most insects empty the gut before each molt. Indeed food could be retained only in the midgut since the linings of both fore- and hindgut are molted. Protoparce larvae do not retain a significant residue of food through the molt (Waldbauer, 1964). How-

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237

ever, So0 Hoo (1962) states that considerable food may be retained in the midgut of Prodenia eridania after a molt. The larvae of Protoparce empty the gut during the first few hours of the premolt period (Waldbauer, 1964). The time required to empty the gut after cessation of feeding may be related to the speed with which food passes through the gut during feeding. House (1965a) summarized passage times which range from 25min for adults of the cucujid Oryzaephilus surinamensis to 2-73 hr for Prodenia larvae and 28-32 hr for adult females of Aedes aegypti. With farm animals the feces derived from the experimental food are often identified by feeding an indigestible marker at the beginning and end of the experiment. Only those feces which appear between the two markers are collected (Kleiber, 1961). Foods which produce feces of different colors were used to the same end by Phillipson (1960) in his study of the food consumption of a phalangid. Similar methods have apparently not been used with insects although Crowell (1943) and Husain et al. (1946) used indigestible markers to measure the rate at which food passes through the gut. Chauvin (1946) tried to eliminate residual food by starving adult Orthoptera for 24 hr before the experiment and until they stopped passing feces after the experiment. This procedure may not injure grasshoppers, but insects which feed almost continuously (i.e. aphids or many lepidopterouslarvae) may be seriously stressed by a short period of starvation. Furthermore, Waldbauer (1964) found that starved larvae retained considerably more residual food than newly molted ones. Evans (1939b) devised an elaborate indirect method using four groups of larvae. At the beginning of the experiment the first group was sacrificed and dried to a constant weight. The second group was starved and their feces dried and weighed to provide an “excreta equivalent”. The two remgiling groups were provided with food. At the end of the feeding period the experimental group was dried and weighed. The fourth group was then starved and another “excreta equivalent’’ obtained. The difference between the two “excreta equivalents” was added to the weight of the feces of the experimental group and subtracted from their dry weight gain. Evans’ (1939b) method avoids long and possibly deleterious starvation, but is certainly tedious and probably less accurate than the use of a physiologically defined experimental period. The molting of insects presents a special problem in the determination of the E.C.I. and the E.C.D. since the insect reaches a maximum weight in each instar and loses weight during the molt. Both the molted cuticle and the energy used during the molt contribute to this loss. Fifth instar

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G . P. WALDBAUER

Protoparce which attained a maximum fresh weight of 7.14 g (after emptying the gut) weighed only 3.94 g after molting to the pupa, a loss of about 45% (Waldbaucr, 1962). Thus a net weight gain lower than the gross or total weight gain is obtained if the experimental period includes one or more molts and if gain is taken as the difference between the weight of the insect at the end and the beginning of the experiment. Use of the net weight gain yields a net E.C.I. or E.C.D., a considerably lower figure than the gross E.C.I. or E.C.D. which include the weight lost at each molt. Some workers used groups of insects rather than individuals. Hiratsuka (1920) began his experiments with four groups of lo00 Bombyx mori larvae. Evans and Goodliffe (1939) used a single group of over 2000 Tenebrio molitor larvae. However, the use of groups may be complicated by mortality, cannibalism or the eating of dead individuals. With many insects, especially those which feed on powdered diets, the likelihood that they will eat feces is increased. Hiratsuka (1920) minimized the problem of mortality by removing all sick larvae each day and replacing them with healthy larvae of similar size. Evans and Goodliffe (1939) did not mention the problem of mortality. Crowell (1941), Evans (1939a, b), Nagy (1953), Balogh and Gere (1953) and Davey (1954) used smaller groups. Crowding is likely to affect the rates of feeding and growth (see Section VD). The size of the container in which the experiment is carried out may be of importance. Fewkes (1960) found that the 1st and 2nd instars of the hemipterous predator Stalia major had a lower E.C.I. than the latter instars. He felt that this may have been due to the use of similar sized cages for all instars. The ratio of search area to size of predator was higher for the early instars. Thus they required proportionally more energy to obtain each meal. B . THE GRAVIMETRIC METHOD

Most workers have used the classical gravimetric method to measure the utilization of food. Other methods will be discussed in Section IIIc. In essence the gravimetric method involves the following procedure: 1. The insect’s weight gain is found by subtracting its weight at the beginning of a feeding period from its weight at the end. 2. Food eaten is determined by subtracting the weight of uneaten food from the weight of the food provided. 3. All feces are separated from the uneaten food and weighed. If the experimental period includes a molt the difference between initial and final weights yields the net weight gain of the insect. Gross

239 gain is then obtained by adding to net gain the weight lost during each molt. This loss is determined by subtracting the weight of the newly molted insect from the maximum weight attained in the previous instar. Some of the major difficulties encountered in the determination of weight gain should be mentioned. The presence of residual food in the gut can cause a considerable error as discussed above. The best solution is to begin the experiment with newly molted larvae whose guts are naturally empty, and to end with either premolt or newly molted larvae whose guts are again naturally empty. An additional error is avoided by weighing individuals as soon as possible after they have molted or entered the premolt stage. Premolt Protoparce which are about to undergo a larval molt are inactive and lose only about 0.25% of their fresh weight per hour. Newly molted larvae are more active and can be expected to lose more weight. Larvae preparing for the pupal molt are extremely active and lose about 1.3% of their fresh weight per hour (Waldbauer, 1960). If an experiment need include only one instar the penultimate is the most convenient choice since it eats fairly large quantities and the weight lost during the premolt is at a minimum. The weight of silk or other products must be considered if gross efficiency is to be calculated. It is, of course, impossible to measure dry weight directly at the beginning of an experiment. An estimate can be calculated from the mean per cent dry matter of an aliquot of similar larvae dried to constant weight at 100°C. It is convenient to kill the insects first by placing them in a freezer for a short time. At the end of the experiment dry weight may be determined either directly or by means of another aliquot. A d libitum feeding is convenient and natural, and has generally been used with insects in preference to some form of controlled feeding. Taking the weight of the food eaten as the difference between weight of the food provided and weight of the uneaten food poses some problems. If fresh weights are used it is necessary to control for a loss or gain of water by the food. Water loss can be minimized by maintaining a humid atmosphere. Evans (1939a, b) estimated the loss from leaves provided as food from the loss of similar leaves kept without insects in containers similar to those used in his experiments. He calculated the “natural loss” of the food as: % wt loss of aliquot x wt food introduced2 + wt uneaten food A more precise estimate is obtained if the correction factor is calculated CONSUMPTION A N D UTILIZATION OF FOOD

240

G. P. WALDBAUER

both as the ratio of loss to the initial weight of the aliquot (a) and as the ratio of loss to the final weight of the aliquot (6). Then: corrected wt food eaten

=

[1 - -4 [W - ( L + bL)]

W = wt of food introduced L = wt of uneaten food. In this calculation the full correction is applied to the uneaten food because it lost weight during the entire experimental period. One-half the correction is applied to the eaten food since it is assumed that it was being eaten at a constant rate. This calculation differs slightly from one suggested by Waldbauer (1962). The use of an aliquot to estimate “natural loss” in this way involves at least two additional assumptions: I . The % weight loss of the food is independent of the amount of food. 2. Feeding itself does not increase “natural loss” by constantly providing a freshly cut surface. The absolute amount of “natural loss ” and, thus, the relative size of the error will increase with the amount of uneaten food which remains at the end of the experiment. If excess food is held to a minimum “natural loss” will often be negligible (Carne, 1966 and Waldbauer, 1962). However, Waldbauer (1962) found that excised leaves of some plants lose as much as 4% of their initial weight in 24 hr. The dry weight of foods which cannot be dried before feeding must be estimated from the % dry matter of an aliquot. Some foods, particularly leaves, demand judicious selection of the aliquot. Chauvin (1946) found that variations in the dry matter content of leaves necessitated the cutting of an aliquot from each leaf used. Brennikre et al. (1949), Waldbauer (1964) and So0 Hoo and Fraenkel(l966) found that greater precision is obtained by cutting the leaves into two symmetrical portions along the midrib, using one portion as food and the other as the aliquot (Table I). Hiratsuka (1920), Evans (1939a, b), Smith (1959) and others used whole leaves as aliquots. This probably had little effect on the accuracy of Hiratsuka’s (1920) results since he used very large quantities of leaves. The dry weight loss of excised leaves by metabolism is probably negligible. It can be minimized by frequent changes of food and the immediate drying of uneaten food. The potential error from this source could be further minimized by estimating the dry weight of eaten food from the mean of the % dry matter of the aliquot and the % dry matter of the uneaten food. The small but inevitable discrepancy between the % dry matter

241

CONSUMPTION A N D UTILIZATION OF FOOD

TABLE I A comparison of the use of whole leaves and asymmetrically and symmetrically cut leaf pieces as aliquots for the estimation of % dry matter of whole leavesa

Plant

Range of % dry matter of whole leaves

Lycopersicon esculentum Solanum fuberosum Taraxacum oficinafe Arctium minus

12’7-16.6 11.5-17.2 13.6-1 7.0 17.4-245

Mean difference in % dry matter of: apical and basal right and left halves of leaves portions of leaves 1.02 1-32 2-01 2.04

0.16 0.58 0.14

0.26

* From Waldbauer (1964).

determined from the aliquot and the actual % dry matter of the food causes an error in the estimated dry weight of the food provided. The dry weight of uneaten food is not subject to this error since it is measured directly. The error is passed on when food eaten is calculated by subtracting weight of uneaten food from the weight of food provided. The absolute size of the resulting error in weight of food eaten will increase with the quantity of food introduced, while its relative size will increase with the proportion of uneaten food. Adding an excess equal to the quantity eaten will thus double the error. The food intake of blood-sucking insects can be conveniently determined by weighing the insect immediately before and after a meal. Buxton (1930) and Friend et al. (1965) used this method with Rhodnius prolixus. Titschack (1930) and Johnson (1937 and 1960) used it with Cimex lectulurius. Titschack (1930) pointed out that Cimex passes a drop of excrement immediately after feeding. Fewkes (1960) and Evans (1962) measured the food intake of predaceous Hemiptera by supplying weighed prey which were reweighed after the predators had fed. The attempts to measure the rate of sap intake by aphids were reviewed by Auclair (1963). Robinson (1961) found that weighing previously starved aphids before and after feeding did not give reliable results. This was probably because they began excreting honeydew as soon as they resumed feeding. Experiments with reproducing adults were further complicated by the birth of nymphs. Mittler (1957) tried to estimate sap intake from the amount of sap which exudes from embedded stylets severed from feeding aphids. However, the rate of

242

G . P. W A L D B A U E R

exudation was sometimes somewhat less than the rate of honeydew excretion by comparable aphids. Apparently the turgor pressure of the plant is not solely responsible for the flow of sap into the aphid’s alimentary canal. The dry weight of the feces can be measured directly. Feces should be collected frequently and dried as soon as possible to avoid decomposition. Brown (1930) found that after 24 hr there had been a marked change in the carbohydrate composition of the moist feces of Automeris io. He thought that the change was due largely to the growth of mold. The fresh weight of feces may be difficult to determine because of a loss or gain of water. The feces of leaf-feeding insects are likely to lose water rapidly. Those of the larvae of Automeris lose about 26% of their fresh weight in 24 hr (Brown, 1930). Evans (1939a, b) minimized this loss by conducting experiments in air-tight cans and collecting the feces each hour. Legay (1953, 1957) devised an ingenious but tedious gravimetric method of measuring food intake and excretion in fresh weight. He placed a Bornbyx mori larva on the pan of a beam balance, noted its initial weight and gave it a leaf to eat. Every 15 min he removed the leaf and noted the larva’s weight gain. The weight of freshly passed feces was taken by noting total weight before and after brushing the pellets from the pan. The weight of food eaten was taken as the weight gained by the larva between successive weighings plus the weight of feces passed. Legay (1957) pointed out that his method does not account for the weight of water vapor or carbon dioxide lost by the larvae. Legay felt that this error is small but significant, and suggested that it could be estimated by measuring the weight loss of starving larvae. The error is not small. Recalculating the data of So0 Hoo and Fraenkel(l966) for Bornbyx on the basis of fresh weights (using Legay’s (1957) figures for water content of feces) shows that of the food eaten by the 4th instar 29% is excreted as feces, 48% is used for growth and 23% is metabolized and thus lost in the form of water and carbon dioxide. Furthermore, frequent disturbance of the larvae may seriously affect their feeding rate. Legay’s (1957) method is certainly useful for determining the fresh weight of feces, but is of doubtful value for measuring intake. C . I N D I R E C T M E T H O D S USING M A R K E R S

Indigestible markers have been used to measure the utilization of food by vertebrates, especially farm animals, for the last forty years (see Elam et al., 1962; Kleiber, 1961;Maynard, 1951;Smart et al., 1954). The most frequently used method involves the addition of a known concentration

CONSUMPTION A N D UTILIZATION O F FOOD

243

of chromic oxide to the diet and the determination of the concentration of chromic oxide in the corresponding feces. The C.A.D. or A.D. can then be determined without quantitative collection of the feces or ineasurement of the food eaten:

M F = concentration of marker in food M E = concentration of marker in excreta. If either the weight bf feces or the weight of food eaten are known it is possible to calculate: wt food eaten = or

5 x MF

wt feces

MF x wt food eaten ME McGinnis and Kasting (1964a) developed a sensitive colorimetric analysis for chromic oxide which made it possible to use the marker technique with individual insects. The technique was first used to measure the A.D. of insects, larvae of Agrotis orthogonia, by McGinnis and Kasting (1964b). Later they compared the A.D.3 and dry weight food consumption of nymphs of Melanoplus bivittatus and larvae of Agrotis as measured by the chromic oxide and gravimetric methods (McGinnis and Kasting, 1964~).They claimed that the two methods gave more or less comparable results. However, in three of six tests the chromic oxide method gave A.D.’s considerably lower than corresponding A.D.’s obtained gravimetrically. They concluded that the gravimetric method had overestimated the A.D., arguing as follows: 1. One of their diets and the resulting feces were crumbly, making it difficult to separate uneaten food and feces for gravimetric determination. Complete separation is not required for determination of the A.D. with the chromic oxide method. 2. Variations in the % dry matter of aliquots led to an error in the calculated dry weight of the food introduced. They had, however, introduced almost four times as much food as the insects ate, greatly increasing the relative size of this error (see discussion above). They also found that the chromic oxide method gave less variable A.D.’s than the gravimetric method. However, with the gravimetric method Waldbauer (1964) obtained A.D.’s with standard deviations of about the same size as those obtained by McGinnis and Kasting (1964b, c) with the chromic oxide method. wt feces

=

244

G . P . WALDBAUER

The chromic oxide technique was subsequently used by McMillian et al. (1966) to compare the digestion by Heliothis zea and Spodoptera frugiperdu of various lyophilized plant parts incorporated in an agar diet fortified with vitamins. Larvae on these diets gained less weight than larvae on the usual laboratory diet. The authors suggest that the chromic oxide may have deterred feeding. McGinnis and Kasting (1964~)stated that feces need not be collected quantitatively in order to determine the A.D. In fact they did collect feces quantitatively and analyzed all of the feces of each insect for chromic oxide. It should be noted that a sampling error may result if only a part of the feces are used for analysis. Elam et al. (1962) reported that they and others observed that the concentration of chromic oxide in the feces of sheep, goats and cattle appears to vary with time. As yet we have no idea of whether or not sampling errors will be a serious problem in using the chromic oxide method with insects. As pointed out by McGinnis and Kasting (1964b), a marker used to measure food utilization should meet the following criteria: 1. It should not affect feeding or be toxic at the concentrations used. 2. It should not be altered by or absorbed from the gut. 3. It should be possible to distribute the marker uniformly in the food. 4. An accurate and relatively rapid analysis for the marker should be available. According to McGinnis and Kasting (1964~)the chromic oxide method is more rapid and practical and avoids some of the error which may result from use of the gravimetric method. The gravimetric method is laborious but, as shown above (Section IIIB), error can be minimized or reduced to insignificance. McGinnis and Kasting (1964~)pointed out that for determination of food consumption both methods require the quantitative separation of food and feces, often a difficult procedure. With the chromic oxide method uneaten food need not be quantitatively collected, thus allowing greater flexibility in the choice of methods of separation. The chromic oxide method is probably usable only with foods which can be ground and mixed to assure even distribution of the marker. The utilization of whole leaves could be measured if it were possible to distribute the marker evenly on the surface of a leaf. However, the amount of chromic oxide ingested would depend upon the area rather than upon the weight of leaf eaten. Thus an accurate determination is possible only if the relationship between leaf area and weight is known and constant. A naturally occurring constituent of the food would be the ideal marker. The constituent chosen should be evenly distributed in the food, not vary greatly in concentration, and be amenable to rapid and accurate

CONSUMPTION A N D UTILIZATION O F FOOD

245

analysis. Ideally it should not be absorbed from the insect’s gut. Elam that using lignin as a marker with sheep gave a low C.A.D. because all of the lignin in the food was not recovered in the feces. They state that while some workers found varying degrees of lignin digestibility others found lignin to be a satisfactory marker. Brown (1 930) used the starch in leaves as a marker for the determination of A.D. in Automeris larvae. The results were similar to those he obtained with a gravimetric method which was probably inaccurate. Chromogens, naturally occurring plant pigments, have been used as markers to measure the digestibility of dried, ground plant material by grasshoppers (Heinrichs and Pruess, 1966). Their method is an adaptation of one devised by Reid et al. (1950) for use with dairy cows. Heinrichs and Pruess (1966) claimed a general correlation between the results of the chromogen-ratio and gravimetric methods, although the results differed widely in four of twenty-one tests with different plants or plant parts. They did not describe their gravimetric method. Further work on the development of methods using naturally occurring markers would certainly be worthwhile. Such methods should allow one to measure utilization by phytophagous insects living on their food plants in the field. Perhaps similar studies would be possible with predators or bloodfeeders. Radioactive tracers have also been used to estimate food consumption. Crossley (1963a) stated that when an insect reaches a steady-state concentration of a radioisotope through feeding on tagged food, then the rate of intake is equal to the rate of “biological elimination” of the radioisotope (see also Crossley, 1963b, 1966). Crossley (1963a) used cesium-137 to estimate the intake of Chrysomelu knabi feeding on SaZix nigra growing in a former disposal area contaminated with the radioisotope. Cesium-137 estimates of the daily food consumption of 3rd instar larvae in the field agreed closely with the results of direct laboratory measurements. S ~ c r o s e - U - ~or ~ Ccell~lose-U-~~C were used as markers to estimate food consumption by 1st and 5th instar Agrotis (Kasting and McGinnis, 1965). At the end of the feeding period the insects, their feces and the CO, they had expired were measured for radioactivity. The use of s ~ c r o s e - U - ~with ~ C 5th instar larvae gave results which were generally higher than those obtained with either the gravimetric or the chromic oxide method. Kasting and McGinnis (1965) suggested that the larvae may have extracted moisture from the diet independently of dry matter, thus increasing their ingestion of the soluble s~crose-U-~~C. The use of insoluble cell~lose-U-~~C gave results which agreed closely with those e l aZ. (1962) found

246

G . P. WALDBAUER

obtained by the chromic oxide method. Larvae fed a diet containing ~ellulose-U-~~C produced an amount of 14C02equivalent to less than 1% of the 14Cingested. No measurable radioactivity remained in their bodies. The 14C02 produced by larvae fed a diet containing sucroseUJ4C was equivalent to about 10% of total consumption. A large amount of radioactivity remained in their bodies. Krishna and Saxena (1962) found that the gravimetric method could not be used to estimate food consumption by Tribolium custuneum or Trogoderma grunarium because the sticky feces and the flour diet could not be completely separated. They then devised an indirect method based upon their assertion that ethanolic extracts of larval feces do not contain sugars which react with anthrone. Thus, they used colorimetric determinations with anthrone to estimate the weight of uneaten flour in an inseparable mixture of flour and feces by the following calculation : wt uneaten flour

=

S2 s 1

x

wt of flour introduced

S1= total sugars in ethanolic extract of a sample of flour S, = total sugars in ethanolic extract of a sample of the mixture of feces and uneaten flour. Any food constituent which is completely absorbed or completely altered might be similarly used to determine the proportions of feces and food in an inseparable mixture.

I V . FOODCONSUMPTION The dry weight-fresh weight consumption index (C.I.) is calculated from the dry weight of the food eaten and the fresh weight of the insect. It is of nutritional interest since it measures the rate at which nutrients enter the digestive system. The fresh weight C.I. (calculated from fresh weight of food and insect) is probably the better measure of the behavioral response to the food. Physiological and behavioral responses are not independent of each other and are certainly not totally separable. However, it seems reasonable to assume that the feeding rate is limited by the response to the bulk, water content and other physical and chemical properties of whole fresh food, and that the rate of dry matter intake is largely a function of this response. So0 Hoo and Fraenkel(l966) and Waldbauer (1964) found that dry weight C.I.’s (calculated from dry weight of food and insect) were

247

C O N S U M P T I O N A N D U T I L I Z A T I O N O F FOOD

always higher than the corresponding fresh weight C.I.'s because the insects in question contained a lower percentage of dry matter than their food. The rank order of either dry weight-fresh weight or dry weight C.I.'s is not necessarily the same as the rank order of corresponding fresh weight C.I.'s. This is, of course, due to differences in the % dry matter of the food. Thus dry weight intake may be higher on one food than another although fresh weight intake is lower (Table 11). TABLE I1 Summary of Consumption Indices (C.I.) Food plant

Ly copersicon escuIentum Lycopersicon esculentum Lycopersicon esculentum Lycopersicon esculentum Solanum tuberosiim S. dulcamara Taraxacum oficinalec Arctium minusC Verbascum thapsus".

% dry matter of leaves

C.I. calculated from : Fresh wt food Dry wt food Dry wt food Fresh wt Dry wt Fresh wt larva" larvab larva"

17.28

1*87

0.33

2.92

15439

2.22

0.35

3.30

15.87

2.13

0.34

3.08

15.93 13.60 19.75

1 a87 1.96 1.84

0.30 0.26 0.37

2.67 2.40 3.22

21.12 19.79

1 *66

I 44

0.34 0.28

3.14 2.74

25.81

1 *28

0-33

3.14

* From Waldbauer (1964). Unpublished data of Waldbauer. accepted only after maxillectomy. Supports only slow growth.

Not normally eaten;

So0 Hoo and Fraenkel(l966) presented a number of dry weight C.I.'s for 5th instar Prodenia. The daily dry matter intake on plants which supported good growth was from 1.2 to 2.4 times the dry body weight of the feeder. On plants which did not support growth or supported little growth the daily dry matter intake ranged from 1.0 to 2.2 times the dry body weight. Thus in some cases the rate of intake may be greater on a nutritionally inadequate diet than on an adequate diet (see also Table 11). The rate of food intake by Celerio euphorbiae larvae appears to increase with decreases in the nutrient level of the diet. House (1965b) used four

248

C. P. W A L D B A U E R

diets which contained the same amounts of cellulose and agar, decreasing amounts of the same balance of nutrients (85%, 70% and 50% of the highest nutrient content), and correspondingly increasing amounts of water, approximately 87%’ 89%’ 90% and 92% of the respective diets (the latter calculated from House’s data). Total fresh weight consumption increased with the dilution of the nutrients. Thus the larvae partly compensated for a low nutrient concentration. Approximate mean C.I.’s calculated from House’s (1965b) data show this nicely. Fresh weight C.I.’s were 1.4, 1.5, 1-6 and 1.8 in order of decreasing nutrient concentration. The corresponding dry-fresh weight C.I.’s were 0.20, 0.20, 0.18 and 0.16. McGinnis and Kasting (1966) found that grasshoppers more than doubled their intake when the nutrient content of the diet was halved by replacing half of the lyophilized wheat sprouts with cellulose. In this case the % H 2 0 in the diet remained the same. House’s (1965b) comparison of a balanced diet with a nutritionally imbalanced diet is not so clear. Total fresh and dry weight intake were lower on the imbalanced diet, but the differences cannot be shown to be statistically significant. Approximate mean C.I.’s calculated from House’s data suggest that the rate of fresh weight intake of the imbalanced diet (0.86) was lower than the rate of fresh weight intake of the balanced diet (0.95). The rates of dry matter intake are identical (0.14)’ presumably because of the greater dry matter content of the imbalanced diet, 15.8% versus 12.7% in the balanced diet (calculated from House’s data). These observations raise interesting questions concerning the behavioral and physiological control of the feeding rate. Is there a direct behavioral response to increased succulence or dilute flavor? How do the physiological effects of a diet influence the rate of feeding? Can balanced and imbalanced diets taste noticeably different to insects? If so, can an imbalanced diet be avoided because its taste comes to be associated with physiological disturbances by conditioning? The reader is referred to the papers of Garcia et al. (1967) and Revusky and Bedarf (1967) for related discussions. There appears to be a negative correlation between the rate of dry matter intake and the efficiency of conversion of ingested or digested food by Prodeniu larvae (So0 Hoo and Fraenkel, 1966). House’s (1965b) data seem to suggest a similar relationship for the larvae fed diets with varying nutrient concentrations. As the mean dry-fresh weight C.I.’s decreased from 0.20 to 0.16 the corresponding mean E.C.T.’s increased from 18 to 22 (calculated from House’s data). Table 111 shows the proportional food intake of the larval or nymphal

249

C O N S U M P T I O N A N D U T I L I Z A T I O N OF FOOD

TABLE 111 Per cent eaten by each instar of the total quantity of food eaten during the entire larval or nymphal stage Insect, food and authority

O;

It

I ~-

_.

-

Agrotis orthogonia 0.2 I Triticum aestivum sprouts Triticuni durum sprouts 0.15 Kasting and McGinnis (1959)" Agrotis ypsilon Corn foliage 0.06 Satterthwait ( 1 933)' 0.04 Bonibyx mori Morus leavesh 0.08 Hiratsuka (1920) Caraicsius morosils Hedera helix leaves" 1.9 Titschack (1924) Paropsis atomaria Eucalyptus shoots" 1.8 Came (1966) Pieris brassicae Cabbage leaves 0.12 David and Gardiner (1962)" Protoparce sexta Tobacco leaves" 0.08 Wolcott (1 937) Rhodnius prolixirs Rabbit blood" I *2 Buxton (1930) Schistocerca gregariu Ficus leavesa I .9 Husain et al. (1946) Stalia major Carpophilus larvae' 3.8 Fewkes (1960) ~~

.' Measured in fresh weight.

instars.

of total consumption by instars I11 IV v VI .. -.

VII

~

0.42 0.48

2.3 3.1

31.6 32.9

56.8

9.1

0.18

0.77 0.52

2.6 1.6

10.4 4.4

86.0''

-

0.1 I

19.6

73.7"

046

2.6

12-0

84-8

-

3.3

6.7

11.6

24.9

51.5

2.2

19.4

75.5

-

-

0.56

2-1

11.5

85.7

-

0.53

1.9

10.5

86.4

-

3.3

10.3

25.0

60.1

-

4.4

11.1

20.0

62.7

-

7.2

12.2

26.8

50.1

-

~

~~~~

Measured in dry weight. Mature in seven instars.

8.7

54.2

~

Measured in area.

Mature in six

instars of several species. The percentages are based on total food consumption during the entire larval or nymphal stage. The great mass of food is eaten during the last two or three instars. Bombyx and Protoparce, both lepidopterous leaf-feeders, eat about 97% of their total

250

G . P . WALDBAUER

intake during the last two instars and about 99% during the last three i nstars. The ideal utilization study will, of course, encompass the entire life cycle. The efficiency of food utilization by the early instars is certainly of interest. The nutritional adequacy of a food can be judged only by its ability to support the growth of successive generations (Gordon, 1959). However, studies which include only one or more of the later instars are often useful. For instance, from an ecological point of view the rates of digestion and conversion during the last two or three instars might serve as a reasonably precise estimate of digestion and conversion during the whole larval stage. V . DIGESTIO AN N D CONVERSIO ON F F R E S HA N D D R Y

MATTER A . LIMITATIONS OF THE DATA

Tables IV to VIII summarize much of the available informatioii on the approximate digestibility and the efficiency of conversion of dry matter and fresh matter to body substance by insects. The data in these tables should be compared with discretion. First, apart from questions of accuracy, data may not be strictly comparable for various reasons which will be discussed below. Second, the data were obtained by methods of varying accuracy. I have, however, omitted from the tables data which appear to be grossly inaccurate or were not accompanied by a statement of methods. I have also omitted data which are in no way comparable with the main body of available information. For example, Schwerdtfeger’s (1930) data are practically useless for comparative purposes since he took only the fresh weight of the food, only the dry weight of the feces, and used the increase in the length of his caterpillars as the only measure of growth. McCay (1938) grew successive groups of roaches on the same batch of diet until all of it had been consumed and nothing but feces remained. He calculated the E.C.I. from the total weight of roaches produced and the initial weight of the diet. The significance of this figure is doubtful because of the high probability that the roaches ate feces. Thus McCay’s (1938) E.C.I. probably falls somewhere between a normal efficiency on a nutritious intake and a very low efficiency on an intake consisting mostly of feces. The further digestion of the feces adds additional complications. Dry weight and fresh weight indices are not comparable, as can be seen by examining corresponding indices derived from the same set of

C O N S U M P T I O N AND U T I L I Z A T I O N O F F O O D

25 1

insects in the same experiment. Corresponding dry weight (Table IV) and fresh weight indices (Table V) are available for Bombyx, Malacosoma, Phalera, Aglais, Pieris, Dermestes, Tribolium and Anagasta. Corresponding fresh and dry weight indices may differ to varying degrees, the difference depending upon the water content of the food and the insect and the % utilization of water by the insect. Fresh weight E.C.D.’s for Dermestes, Anagasta and Tribolium are two to three times higher than corresponding dry weight E.C.D.3. This probably reflects a high rate of retention of the small amount of water taken in with the food, and the storage of metabolic water obtained by the oxidation of carbohydrates. The data in Tables IV and V were taken over different lengths of time and during different portions of the life cycle. It should be kept in mind that the efficiency of utilization is likely to differ from stage to stage, from instar to instar or even within an instar. Thus measures made over a portion of an instar or stage are not necessarily representative of that entire instar or stage (see Section VE). The data of Brennikre et al. (1949), Teissier (1931) and Wolcott (1924) were further complicated by their use of groups of insects of mixed ages. Some of the efficiencies of conversion were calculated with gross weight gain while others were calculated with net weight gain (see footnotes of Table IV). An efficiency calculated from gross gain (includes weight lost during molts) will, of course, be higher than a corresponding efficiencycalculated from net gain. For example, all of the efficiencies of conversion of ingested food (E.C.I.) available for piercing-sucking predators or blood-feeders are based on net gain (Table V). In each case gain was determined by subtracting the weight of an individual before feeding from its weight after feeding and molting to the following instar. The efficiency of utilization will vary with temperature, relative humidity and other physical factors. Teissier’s (1931) efficiencies of conversion of digested food (E.C.D.) represent Tenebrio larvae kept at temperatures well below the optimum, and eating either wheat flour or powdered milk with suboptimum moisture contents, 12-5 and 23%, respectively. These conditions were reflected in an extremely slow growth rate and are probably also reflected in the E.C.D. (Table IV). An insect may utilize a food which it does not normally eat far less efficiently than its natural food. This has been demonstrated with Prodenia (So0 Hoo and Fraenkel, 1966) and Protoparce (Waldbauer, 1964) (Table IV). Edwards (1964) found an extraordinarily low E.C.I. of carrot root by the larvae of Hepia/us (Table V). Carne (1966) suggested that carrots may not be one of the natural foods of Hepialus.

t 4

TABLE 1V The utilization of dry matter by insects

Insect and authority

Temp. (C) and r.h.

Food

Sex and Instar or Stage

VI

h,

A.D.

Leaf Feeders Locusta migratoria (Orthoptera: Acrididae) Dadd (1 960) Melanoplus bilituratus (Orthoptera : Acrididae) Smith (1959)

Schistocerca gregaria (Orthoptera : Acrididae) Brennihre et al. (1949)

As above Chauvin (1946) A s above Dadd (1960)

As above Norris (1961)

32" 55%

Grass

30"

Renown wheat Triticuni nestivutn Agropyron smithii western wheat grass Ajax oats Acena satica Scarole (escarole) Cichorirrni endiuia ? (Compositae)

-

30' -

30"

-

32' 55% 28-35" -

3 Days early in instar V 39

Whole nymphal stage

32

As above

32

As above Instar IV (7 days) gregarious phase isolated gregarious phase in groups solitary phase As above Adult 6 (24 hr) Adult 9 (24 hr) 3 Days early Bran in instar V Grass As above Mixed grasses, mostly Poa sp. Adult 6 and Loliiirii sp. 1st to 10th day crowded isolated

32 55

54 61 33 33

40 39 42" 3 6"

E.C.I.

E.C.D.

1 I th to 20th day

Gryilus domesticus 30' (Orthoptera: Gryllidae) Chauvin (1946) Cararisius morosus 22" (Orthoptera: Phasmidae) Brenniitre et al. (1949) As above 20" Lafon (1951) Phaneropterafalcata 30" (Orthoptera : Tettigoniidae) Chauvin (1946) Hyphantria cunea (Lepidoptera : Arctiidae) Balogh and Gere (1953)b As above Nagy (1953) Bontbyx mori 22" (Lepidoptera : Bom bycidae) Hiratsuka (1920) As above Shyamala et a / . (1960) As above 27" So0 Hoo and Fraenkel(l966) Malacosoma neustria (Lepidoptera: Lasiocampidae) Evans (1939b)b Lynrantria monacha 20-22"

Scarole (escarole) Cichoriunr endivia ? (Compositae) Hedera helix (Araliaceae)

crowded isolated Adult 8 (24 hr)

41" 38" 42

Adult p (24 hr) Instar IV (30 days)

37 55"

Hedera helix (Araliaceae) Lettuce Lactuca satica (Compositae) Acer negundo (Aceraceae)

Whole nymphal stage+?)

51

Adult d (24 hr) Adult F (24 hr) Whole larval stage

41 41 22

Malus punrila (Rosaceae) Morus alba Goshoerami variety (Moraceae) Morris alba

Whole larval stage

31"

-

-

Whole larval stage

37"

23"

62c

Instar IV-V

39

19""

48"

Morus alba

Instar IV

44

21

47

Salix uiminalis (Salicaceae)

Part of (24 hr?) last instar

34

14"

41

Fagirs sp.

Instar IV

29

C

i

rN

2 0

z

0

n 0 0

tl

TABLE Iv-continued

Insect and authority (Lepidoptera: Lymantriidae) Sattler (1939)b

Agrotis orthogonia

(Lepidoptera: Noctuidae) Kasting and McGinnis (1 959)

Temp. ((3 and r.h. ca 75%

-

-

Sex and Instar

Food

or Stage

A.D.

E.C.I.

E.C.D.

-

-

-

(Fagaceae) Alnus sp. (Betulaceae) Picea sp. (Pinaceae) Sprouts of Thatcher wheat

Instars V and VI Instar IV

21 35

Instars V and VI

25

-

-

(Triticum aestivum)

Instar Iv-VI

5oC

28"

56"

Instar IV-VI Early instar V to pupa

55" 49

26c 14'."

46c 29"

As above

24

3%e

14'

Instar V

50

21

42

As above As above As above

66" 66 70

29" 33 38

43" 49 54

As above

60

31

52

As above

65

10

16

As above

40

18

44

Sprouts of golden ball wheat (Triticum durum)

Mamestra brassicae

25"

(Lepidoptera: Noctuidae) Hirano and Noguchi (1963)

-

Beet, Beta sp. (Chenopodiaceae) sweet potato Ipomoea batatas

(Convolvulaceae) Prodenia eridania

(Lepidoptera: Noctuidae) So0 Hoo and Fraenkel (1966) (For data of So0 Hoo and Fraenkel, plants belong to last family mentioned if family is not otherwise indicated)

27"

-

Morus alba Phaseolus lunatus

(Leguminosae) Mean of data on simple leaves trifoliate leaves P. vulgaris Taraxacum oficinale

(Compositae) Antirrhinum majus

(Scrophulariaceae) Ipomoea batatas

(Convolvulaceae)

As above Crowell (1941)b Phalera bucephala (Lepidoptera: Notodontidae) Evans (1939b)b As above Evans (1939a) Aglais urticae (Lepidoptera: Nymphalidae) Evans (1939b)b Pieris brassicae

Nicandra physalodes" (Solanaceae) Solanum tuberosum Lycopcrsicon esculentirni Abutilon theoplirasti" (Malvaceae) Chenopodium album (Chenopodiaceae) Spinacia olcracea Phytolacca americana (Phytolaccaceae) Viola ~ p . ~ (Violaceae) Malus floribunda" (Rosaceae) Prunus serotinaa UImus pumila (Ulmaceae) Acer saccharinuni (Aceraceae) Phaseolus vulgaris London Hort. variety (Leguminosae) Carpinus betula (Betulaceae) Corylus avellana (Betulaceae) Urtica dioica (Urticaceae)

Cabbage

As above As above As above

66

60

64

20 34 22

30 57 35

As above

50

14

29

As above As above

44 60

23 26

51 42

As above

58

30

52

As above

53

10

19

As above As above

36 47

11 8

29 18

As above

44

11

25

As above Instars V and V1

46 48

12 34"

26 69"

Part of (24 hr?) last instar

35

-

-

24 hr? instar?

35

-

-

Part of (24 hr?) last instar

26

17c

63

TABLE IV-continued

h,

wl

o\

Insect and authority (Lepidoptera: Pieridae) Evans (1939b)b Chilo suppressalis (Lepidoptera: Pyralidae) Hirano and Ishii (1962) Protoparce sexta (Lepidoptera : Sphingidae) Wolcott (1937)b As above Waldbauer (1 964)

Temp. (0 and r.h.

-

28"

-

26-30'

-

(For data of Waldbauer, plants belong to last family mentioned if family is not otherwise indicated)

BIatta orientalis (Orthoptera: Blattidae) Lafon (1951) As above Chauvin (1 946)

Food Brassica oleracea (Cruciferae) young leaves old leaves Steam sterilized rice stems

Tobacco Nicotiana sp. (Solanaceae) Lycopersicon esculentuni (Solanaceae) Mean of all data Solanum tuberosum S. dulcamara Taraxacum oficinaled (Compositae) Arctirim minusd Verbascuni thapsusa* (Scrophulariaceae)

Sex and Instar or Stage

A.D.

E.C.I.

36 39 57

-

Instar IV

50"

25"

Instar I V

49"

27"

As above As above As above

56 42 39

36 27 21

As above

41

20

As above

32

12

95

-

63 59

-

Part of (24 hr?) last instar As above First 20 days of larval stage, ca instar I-V

20"

-

Non-Leaf Feeders Last 2 nymphal instars Powdered milk, yeast and cholesterol

25'

Wheat flour

-

Adult d (24 hr) Adult 0 (24 hr)

13c*"

E.C.D.

Gryllus domesticus (Orthoptera : Gryllidae) Chauvin (1946) As above Brennitre et al. (1949) Dermestes maculatus (Coleoptera : Dermestidae) Fraenkel and Blewett (1944)b Tenebrio molitor (Coleoptera : Tenebrionidae) Evans and Goodliffe (1939) As above Teissier (1931)b

Tribolium confusuni (Coleoptera : Tenebrionidae) Fraenkel and Blewett (1944)b Anagasta kuehniella [ =Ephestia] (Lepidoptera: Phycitidae) Fraenkel and Blewett (1944)b

-

30"

Wheat flour

Adult d (24 hr) Adult 0 (24 hr)

-

30"

Wheat flour

Nymphs of 118-272 mg (1 5 days)

1 9"

25" 70% 50% 30%

Brewer's yeast, fructose and cholesterol As above As above

Instar I to pupa As above As above

19" 16" 11'

27"

Wheat bran

Larvae (4 days)

-

17"

Wheat flour

1 8"

20" 25" 70% 20%

Powdered milk White flour and yeast

Larvae of mixed ages (18-45 days) As above (14-1 6 days)

23"

Instar I to pupa As above

19" 14"

25" 70%

Whole wheat flour Instar I to pupa As above As above

16" 11" 8"

-

-

-

20%

> 0%

As above

As above As above

A.D. = approximate digestibility PA),E.C.I. = efficiency of conversion of ingested food to body substance, E.C.D. = efficiency of conversion of digested food to body substance. The E.C.I. and E.C.D.are based on gross gain unless otherwise indicated. The A.D. and E.C.D. have not been corrected for uric acid content of the feces. Unless otherwise indicated experiments ran from the beginning to the end of the instar or stage mentioned or from the beginning of the first to the end of the last instar mentioned. See text for discussion of author's methods or other comments. Calculated from 8 Plants which do not or only rarely support growth. author's data. Plants not usually eateniaccepied only after maxillectomy. Based on net weight gain. Exuviae included in weight of feces.

TABLE V

h,

ch 00

The utilization of fresh weight of food by insects

Insect and authority

Carausius morosiis (Orthoptera: Phasmidae) Titschack (1924)b Paropsis atomaria (Coleoptera: Chrysomelidae) Carne (1 966) Bombyx mori (Lepidoptera: Bombycidae) Hiratsuka (1920) Hepialus humuli (Lepidoptera: Hepialidae) Edwards (1964)b Malacosoma neustria (Lepidoptera: Lasiocampidae) Evans (1939b)b Dendrolimuspini (Lepidoptera: Lasiocampidae) Lebedev and Savenkov (1932)" Phalera bucephala (Lepidoptera: Notodontidae) Evans (1939b)b As above Evans (1939a)"

Temp. (C) and r.h.

-

22"

-

20" 98%

-

Sex and Instar Food

or Stage

Leaf Feeders

A.D.

E.C.I.

Hedera helix (Araliaceae)

Whole nymphal stage ( 0 ) Whole adult stage (0)

-

Ei4calyptus blakelyi young shoots (Eucalyptaceae) Morus alba Goshoerami variety (Moraceae) Slices of carrot root

Instar I to prepupa

20"

Whole larval stage

25"

Instar I to pupa

-

2"

Salix uiminalis (Salicaceae)

Part of (24 hr?) last instar

22'

-

-

Pinirs sp. (Pinaceae)

Whole larval stage

13'

-

Carpinus betula (Betulaceae)

Part of (24 hr?) last instar

28"

Corylus avellana (Betulaceae)

24 hr? instar?

34

-

E.C.D.

Aglais urticae (Lepidoptera: Nymphalidae) Evans (1939b)" Pieris brassicae (Lepidoptera: Pieridae) Evans (1939bIb

As above David and Gardiner (1962)b Snterinthus populi (Lepidoptera: Sphingidae) Evans (1939b)b

Cryptotermes brevis (Isoptera: Kalotermitidae) Wolcott (1924)b

Cimex lectularius (Hemiptera: Cimicidae) Johnson (1960)"

Urtica dioica (Urt icaceae)

Cabbage Brassica oferacea (Cruciferae) Young leaves Old leaves Cabbage Brassica oleracea Salix sp. (Salicaceae)

Part of (24 hr?) last instar

24

14

Part of (24 hr?) last instar As above

20 24

15c

Whole larval stage

-

13"s"

First instar

64"

32c

12

-

41

-

51

-

58

-

-

3oc.e

Non-leaf Feeders Seasoned, air-dry wood Mixed stages Asubo Sideroxylon foetidissimrni Pomarrosa Eugenia jambos Sitka spruce Picea sitchensis Flamboyan Poinciana renia Instar 11 (unfed) to Blood of hum& 1 instar V (unfed) AS above Blood of human 2 As above As above Blood of mouse As above As above Blood of fowl Instar 11(unfed) to instar IV (unfed)

16"

3oc.e

-

-

3oc.e

-

-

3W"

-

0 0

t3

N

E

TABLE V-continued

Insect and authority

Stalia major (Hemiptera: Nabidae) Fewkes (1960)

Rhodnius prolixus (Hemiptera : Reduviidae) Friend et al. (1965)b Phonoctonus nigrofasciatus (Hemiptera : Reduviidae) Evans (1 962) Sitophilus granarius (Coleoptera: Curculionidae) Richards (1947) Dertnestes maculatus (Coleoptera : Dermestidae) Fraenkel and Blewett (1944)b Tribolium confusutii (Coleoptera : Tenebrionidae) Fraenkel and Blewett (1 944)b

Temp. (C) and r.h.

Food

30" Blood of man 92% 20" Asabove 86% 13.5-1 9" Larvae of Carpopidus and dimidiatus 20-22" (Coleoptera: Nitidulidae) (pooled results of two experiments) 26-27" Rabbit blood -

Sex and Instar or Stage Instar I (unfed) to instar V (unfed) Instar I (unfed) to adult (unfed) Jnstar I (unfed) to instar V (unfed)

A.D.

E.C.I.

E.C.D.

0 cd

Instar I (unfed) to instar V (unfed)

E

9

-

r tl W

9

27"

75:4

Nymphs of Dysdercus fasciatits Instar I (unfed) to (Hemiptera: Pyrrhocoridae) instar V (unfed)

25"

Whole grains of wheat

Instar I to adult

14e

Brewer's yeast, fructose and cholesterol Asabove Asabove White flour and yeast

Instar I to pupa As above As above

38" 35" 36"

Instar I to pupa As above

39" 30"

-

70% 25" 70% 50%

30% 25" 70% 20%

As above

sP

c)

0

z

(A

C

3 %!

Anagasta kuehniella [ = E'hestia] (Lepidoptera: Phycitidaz)

Fraenkel and Blewett (1944)b Tineola bisselliella

(Lepidoptera: Tineidae) Titschack (1925)"

See Table IV for footnotes.

25"

70% 20% iO%

Whole wheat flour As above Asabove

Instar I to pupa As above As above

-

-

-

-

42' 30" 22"

r!

2 ; t,

30" 30" 20" 20"

Wool dipped in brew of horse feces As above As above As above

Instar I to adult ( 6 ) As above ( 0 ) As above (d) As above ( 0 )

39" 41"

45' 45"

16".' 18C.e 22C.'

41C*e 38's" w.e

49c.e

C

2

' N

>

2

0

z

C

7 ?I

0 0

0

262

G.

P. W A L D B A U E R

Beck (1956) pointed out some of the difficulties which may be involved in the use of excised plant tissues in nutritional studies. Excised tissue may change rapidly because of biochemical degradation, changes in water and hydrogen ion relationships and microbial attack. Some of the data quoted in the tables were obtained by methods which may have led to relatively minor inaccuracies. Fraenkel and Blewett (1944) were unable to separate the silk spun by Anagasta kuehniella larvae from the mixture of uneaten food and feces. This makes their figure for food digested somewhat too low and thus inflates the E.C.D. Titschack (1924), Crowell (1941), Husain et al. (1946) and David and Gardiner (1962) calculated the weight of food eaten from the area eaten. This was probably reasonably accurate if there is little variation in the relationship of area to weight. Sattler (1939) used woody twigs with attached leaves both as food and aliquots for dry weight determinations. He selected twigs which appeared to have woody portions of the same length and thickness, and trimmed enough leaf material from each so that all had the same fresh weight. Variations in the proportion of woody material may have made his dry weight determinations somewhat inaccurate. Lebedev and Savenkov (1932) determined the number of pine needles which had been eaten and calculated their weight from the average weight of a needle which had been previously determined from an aliquot of needles taken from the same twig. Davey (1954) used the % dry matter of the grass left uneaten after 24 hr to estimate the dry weight of the grass initially provided. This probably inflated his figure for the dry weight of grass provided, thus deflating the A.D. Balogh and Gere (1953) took the air-dry weight of food and feces. Their figures for consumption and excretion are, therefore, probably somewhat higher than those of workers who oven-dried this material. A.D.’s determined on a fresh weight basis will be somewhat low if there was a loss of moisture from the feces. Data for small species or early instars may be less accurate than data for larger forms because of the small quantities involved. Hiratsuka (1920) states that the “average digestibility” of dry matter during the whole larval stage of Bornbyx is 40.9%. An examination of his data reveals that this figure was indeed arrived at by taking the mean of the A.D.’s for the five instars. This average figure is not the same as the % digestibility of the total amount of food consumed during the larval stage. A.D. is at its highest in the first instar, but the food intake of this instar is only 0.08% of the total. The food intake of the 5th instar is 85% of the total, but A.D. is at its lowest in this instar (Table VII). Thus the mean is biased by a high A.D. at a time of low intake.

CONSUMPTION A N D UTILIZATION O F FOOD

263

Calculating from the total weights of food eaten and feces passed during the entire larval stage gives a considerably lower A.D. of 37%. Several authors calculated similar “average values”. In each case I have calculated the actual overall value for presentation in this paper. B . COMPARISON OF SPECIES

A glance at Tables IV and V shows that approximate digestibility and efficiency of conversion differ widely from species to species. On the whole it is probably a safe assumption that these differences are largely the result of differences inherent in the normal insect-food relationship. However, as pointed out above, comparison is complicated by variations in accuracy, experimental design, environmental conditions and other factors. The data of Table IV suggest that leaf-feeding Lepidoptera may in general utilize food for growth more efficiently than the nymphs of leaffeeding Orthoptera. Unfortunately, all three indices are available for only eight of the Lepidoptera and three of the Orthoptera. Considering only these species and omitting the nutritionally inadequate foods, it can be seen that the Lepidoptera on the whole convert ingested food to body substance (E.C.I.) more than twice as efficiently as the Orthoptera. The difference must be attributed largely to the greater efficiency of conversion of digested food (E.C.D.) by the Lepidoptera, since approximate digestibility does not differ much in the two groups. Of the Lepidoptera under consideration only Mamestra and Prodenia on a few of its foods show low E.C.D.’s. So0 Hoo and Fraenkel (1966) compared the utilization of mulberry (Morus) leaves by Bombyx and Prodenia under nearly identical experimental conditions. They used the penultimate larval instar of each species, 4th instar Bombyx and 5th instar Prodenia. The E.C.I.’s were the same. However, the higher A.D. in Prodenia compensated for its lower E.C.D. (Table IV). The data of So0 Hoo and Fraenkel(l966) and Waldbauer (1964) for Protoparce permit similar but somewhat less precise comparisons (Table IV). On tomato (Lycopersicon) Prodenia has a somewhat lower E.C.I. than Protoparce. However, the considerably higher A.D. in Prodenia again compensates for its far lower E.C.D. A similar situation pertained when potato (Solanum tuberosum) leaves were fed. On Taraxacum leaves Prodenia had the higher E.C.I., but this was clearly the result of a higher A.D. Taraxacum is not a natural food for Protoparce and usually is accepted only by maxillectomized larvae. These observations are of considerable interest. Both Bombyx and Protoparce are oligophagous. Bombyx will normally eat only mulberry

264

G . P . WALDBAUER

or a few other species of Moraceae. Protoparce in nature eats tobacco or tomato, but will accept other Solanaceae in the laboratory. Prodenia, on the other hand, is polyphagous and readily accepts a wide variety of plants. One would expect Bombyx and Protoparce to be specifically adapted to digest and convert their limited diets. On the other hand, one would expect Prodenia to be more generalized, adapted for the reasonably efficient utilization of a variety of foods. Thus the comparatively high E.C.D.’s in Bombyx and Protoparce suggest specific adaptations to the nutrient balances offered by their natural foods. The low E.C.D. in Prodenia suggests a less precise correspondence between its requirements and the nutrient balance of its diet. Perhaps the high A.D. in Prodenia is an adaptation which compensates for a decrease in the efficiency of conversion resulting from moderate nutritional imbalances. C . C O M P A R I S O N O F FOODS

Several comparisons of the utilization of different foods by the same species are available. I refer the reader to the data for Melanoplus, Gryllus, Lymantria, Mamestra, Prodenia (So0 Hoo and Fraenkel, 1966) and Protoparce (Waldbauer, 1964) in Table IV and Cryptotermes and Cimex in Table V. It is clear that at least among the leaf-feeding species digestibility and efficiency of conversion vary widely with the species of food plant. Maxillectomized Protoparce larvae will feed on some normally rejected, non-solanaceous plants (Waldbauer and Fraenkel, 1961). Some nonsolanaceous plants were efficiently utilized while others did not support normal growth although they were eaten at the same or higher rates than plants which supported good growth (Waldbauer, 1964) (Tables I1 and IV). So0 Hoo and Fraenkel(l966) obtained similar results with the intact polyphagous larvae of Prodenia (Table IV). These findings are contrary to Fraenkel’s (1959) earlier contention that the leaves of all plants are of similar nutritional value. They do not, however, conflict in any way with Fraenkel’s (1959) theory of host plant selection, that secondary plant substances serve as “ token feeding stimuli” (sign stimuli). Gryllus adults digested wheat flour at a much higher rate than escarole leaves (Chauvin, 1946) (Table 1V). This is not surprising since the crude fiber content as a percentage of dry matter is lower in flour than escarole. Wolcott (1924) found that the approximate digestibility of various woods by Cryptotermes ranges from 12 to 58% (Table V). Cimex converts the blood of humans, mice or fowl with equal efficiency.

C O N S U M P T I O N A N D U T I L I Z A T I O N O F FOOD

265

In each case the E.C.T. was 30% (Johnson, 1960) (Table V). Unfortunately the other indices are not available for any of these insects. It is also clear that leaves of the same species of plant may vary in nutritional value. So0 Hoo and Fraenkel (1966) measured the utilization of Phaseolus lunatus leaves by Prodenia eleven times. The means of these measurements are given in Table IV. In these tests, made at different times of the year, A.D. ranged from 56 to 72%, E.C.I. from 19 to 38% and E.C.D. from 33 to 52%. The utilization of tomato leaves by Protoparce seems to be less variable. The means of data obtained from four experiments conducted in July are given in Table IV. A.D. ranged from 43 to 52%, E.C.T. from 24 to 30% and E.C.D. from 55 to 59%. Waldbauer (1964) thought that some of the variation may have been due to differences in the water content of the field-grown leaves. In both Prodenia and Protoparce the variation in the utilization of different species of plants is greater than the variation in the utilization of different samples of the same plant species. Evans (1939b) found that for Pieris old cabbage leaves are more digestible than young leaves, and that both are converted to body substance with almost equal efficiency (Tables IV and V). D . EFFECTS O F ENVIRONMENTAL FACTORS

A large body of literature shows that mortality, growth, fecundity and other of the vital statistics of insects vary widely with environmental factors. However, almost nothing is known of the effects of the environment on the rate of consumption, digestibility, or the efficiency of food conversion. Existing data suggest that relatively small changes in the physical or biotic environment may have significant effects. The discovery and elucidation of these effects should prove to be an interesting and fertile field of research. The amount of food consumed and the efficiency of its utilization may vary with the degree of crowding. Crowded Schistocerca gregaria nymphs consumed more food than nymphs reared in groups of three (Davey, 1954). Norris (1961) found that A.D. is higher in crowded Schistocerca adults than in isolated adults. However, Brennittre et a/. (1949) found little difference between the A.D.’s, E.C.T.’s and E.C.D.’s of 4th instar gregarious phase Schistocerca raised in crowds or in isolation (Table IV). Hyphantria cunea larvae reared individually or in small groups grew somewhat more slowly than larvae reared in large groups (Gere, 1956). Long (1953) found that crowded Plusia gamma completed the larval stage more quickly than isolated individuals, although isolated individuals appeared to gain more weight. Furthermore, the relative

266

G . P . WALDBAUER

effect of crowding was the greatest on the nutritionally best plants. Crowding led to the largest proportional increase in the rate of development on those plants which supported the most rapid growth of isolated larvae. The effect of temperature on the efficiency of Tineolu and Hyphuntria is discussed below (Section V E ~ )The . amount of food consumed by Bombyx larvae varies widely with temperature. Taking the amount consumed in a given time at 22" as loo%, consumption fell to 46% at 14' and rose to 203% at 32-33'. The A.D. did not vary greatly from 18 to 32', but it was 44% lower at 14" (Legay, 1957). Sattler (1939) found that the A.D. of Lymuntria fell with increasing temperatures above 14', but that the magnitude of the fall varied with the food plant and the instar (Table VI). TABLE VI The effect of temperature on the approximate digestibility (%) by Lymanfria monacha of the leaves of Alnus incana, Fagus sylvaticus, and Picea excelsas

Temperature "C 12-14 20-22 24-26 a

4th instar Alnus Fagus 60 36 30

32 29 30

5th and 6th instars Picea

Fagirs

28 21 22

44 25 18

From Sattler (1939).

Little is known concerning the effecton food intake and utilization of relative humidity and other factors which influence water loss from the body. Many insects are known to drink (Barton-Browne, 1964), but others, particularly stored-product pests, and perhaps many leaf-feeders, probably get all or most of their water in the food. Water loss from the body would presumably be compensated for by increasing food intake -either for its water content or as a source of metabolic water. In either case increased intake would probably lead to a decrease in the efficiency of utilization. Fraenkel and Blewett (1944) found that Anugasta, Dermestes and Tribolium, pests of stored dry products, convert less of the digested food to body substance as the relative humidity of the air and the water content of the food are decreased (Tables IV and V). A larger amount of food was digested at the lower relative humidities, but much of it was used as a source of metabolic water. The decrease in efficiency probably

CONSUMPTION A N D UTILIZATION OF FOOD

267

involved not only the loss of the carbohydrates metabolized for water, but also a corresponding waste of digested nitrogen. Larvae of Diataraxia oleracea grew normally and did not drink free water when they were fed turgid cabbage leaves which contained about 85% water. However, when fed wilted cabbage leaves (70% water or more) they became dehydrated and drank large quantities of water (Mellanby and French, 1958). Legay (1957) found that Bombyx larvae fed mulberry leaves containing at least 70% water retained much of the ingested water. About 30% was passed out with the feces, 10% was lost as vapor and 60% was stored. The data of Sharada and Bhat (1957) suggest that Bonibyx larvae may digest and convert both dry matter and nitrogen more efficiently at 1000/,r.h. than at a lower but unspecified ambient r.h. Evans (1939b) stated that lepidopterous larvae which ate leaves with a high water content ( > 60-90%) absorbed relatively little of the water ingested with the food (20-30%), while larvae which ate leaves with a low water content ( < 60- > 50%) absorbed much more water (6040%). Legay (1957) commented that a study of utilization based entirely on dry weights ignores the important question of water balance. However, we have little quantitative information on the intake and utilization of water by insects. The data will be difficult to obtain. Perhaps the most difficult problem will be the accurate determination of the fresh weight of feces. E . V A R I A T I O N S W I T H A G E A N D SEX

1. Variation with age Approximate digestibility and efficiency of conversion apparently do not remain constant, but rather vary with age during the growth period of an insect. Table VII summarizes the data which permit comparison of A.D. from instar to instar. In each case A.D. declines with age. Evans’ (1939b) data for Phalera bucephala do not show this tendency as clearly as do the rest, probably because he did not measure A.D. over the entire instar, but sampled it for 24 hr periods at intervals of from one to nine days. This could yield figures not representative of entire instars since, as will be shown below, A.D. varies within instars. Calculations made from the data of Kasting and McGinnis (1959) show the A.D. for Agrotis remaining more or less constant in the last three instars. They stated that their data for the first three instars are not reliable. This does appear to be the case. In two instances calculations from their data yield impossible percentages of digested food retained

268

G . P. WALDBAUER

TABLE W The A.D. from instar to instar I

Insect, food, and authority Bombyx mori Leaves of Morus alba

Hiratsuka (1920) Plialera bucephala Leaves of Carpinits hefula Dry weightR Fresh weight” Evans ( 1939b) Scliltocerca gregariu Mixed grasses Davey ( 1954)” S. gregaria Leaves of Ficits sp. Husain et a / . (1946)”

I

I1

47

44

24 70

18

Instar III

IV

V

38

38

37

43

22 42

24 46

19 45

78

52

45

34

35

48

42

45

38

31

All figures are OR a dry weight basis unless otherwise indicated. a Means of A.D.’s measured over 24 hr at intervals of several days, probably not representative of the A.D. for the entire instar. See text for discussion of author’s methods or other comments.

as body substance, 133% for 1st instar larvae feeding on Golden Ball wheat and 122% for 2nd instars feeding on Thatcher wheat. Other workers who did not give data for individual instars also found that A.D. declines during the larval or nymphal stage. Dry weight A.D.’s for sixteen of the twenty days of the larval stage of Hyphantria can be calculated from Nagy’s (1953) data. The A.D. of the leaves of Malus pumila is 49 on the first day, fluctuates about a mean of 34 for the next fourteen days, and falls to 12 during the last five days. However, calculations from the data of Balogh and Gere (1953) show dry weight A.D.’s of Acer negundo leaves for Hyphantria larvae of 39 for the first fourteen days, 19 for the next ten days, and 23 for the last eight days. Smith (1959) found that the dry weight A.D.’s of Agropyron smithii (western wheat grass), Triticum aestiuurn (wheat) or Auena satiua (oats) for nymphs of Melanoplus bilituratus decreased during the first twenty days, tended to level off during the next ten days, and decreased again during the last ten days of the nymphal stage. The initial A.D.’s of the grass, wheat and oats were 81, 65 and 54, respectively. They fell to between 20 and 30. Sylven (1947) found that the larvae of Phyfometra

CONSUMPTION A N D UTILIZATION O F FOOD

269

gamma digest less of soya bean leaves with increasing age. He measured consumption in square millimeters and feces in dry weight, but his findings are probably valid for this comparison. Calculations from the data of Schwerdtfeger (1930) show that the digestibility of pine needles for the larvae of Bupalus piniarius decreased gradually as they grew from a length of 2.8 to 29-3mm during the larval stage of seventy-two days. He measured consumption in fresh weight and feces in dry weight, but his figures are also probably valid for this comparison. The reasons for the decline of A.D.are not entirely clear. One would expect digestive efficiency to decline with growth since an animal which doubles its weight and volume will increase the surface area of the digestive tract by a factor of only 1.8 (Gordon, 1959). With chewing insects it could be argued that small individuals chew off smaller pieces of food and thus present a greater surface area for digestion. Small leaffeeders might also ingest a greater proportion of easily digested broken cells (Biedermann, 1919). Evans (1939b) pointed out that the composition of the food selected by leaf-feeders changes as they grow older. First instar larvae eat from between the small veins of the leaf. Somewhat older larvae also eat the small veins, while larger larvae eat almost the whole leaf. Thus it is likely that the older larvae ingest a larger proportion of indigestible crude fiber. A.D. also varies within instars. The dry weight A.D. for Bombyx varies as follows in the 5th instar: days 1 and 2,40%; day 3,47%; day 4,42%; day 5,39%; and on days 6 and 7 27% (Hiratsuka, 1920). Calculations from the data of Evans (1939b) show that the fresh weight A.D. of Salix leaves for a group of Smerinthus populi larvae varied as follows from the first to the tenth day of the 1st instar: 92,74,61,56,56, 56, 59, 52, 50 and 53%. Many workers have observed that leaf-feeding insects pass more and wetter and greener feces near the end of an instar, especially the last instar. This is probably a sign of a decrease in A.D. Table VIII is a summary comparison of the variation of the efficiency of conversion of ingested food (E.C.I.) from instar to instar. With the exception of Fewkes’ (1960) data for Stalia major there is an obvious tendency for the E.C.I. to decrease with age. The E.C.I. of Stalia nymphs, however, increased with age, but Fewkes (1960) thought that this may have been because all instars were kept in cages of the same size. The ratio of search area to the size of the predator was, therefore, higher for the early than for the lateinstars. Thus the early instars probably expended more energy to obtain each meal. According to Edwards (1964) the fresh weight E.C.J. of Hepialus hzrmzrli rises during the early instars, reaches more or less of a plateau

270

G . 1'. W A L D B A U E R

TABLE VILI The E.C.I. from instar to instar Insect, food and authority Agrotis orthogonia* Triticum aestiuunz sprouts Triticum durum sprouts McGinnis and Kasting (1959) Bombyx morid Morus alba leaves Hiratsuka (1920) Ciniex 1ectulariu.PBlood of man Blood of mouse Johnson (1960) Phonoctonus nigrofasciatuP. Dysdercus fasciatus nymphs Evans (1 962) Rliodnius prolixusc* Blood of rabbit Friend e f al. (1965) Stalia majorc* Carpophilus dimidiatus larvae Fewkes (1960)b ~~

~~

~~

rr

I _-

-

Instar [TI IV

v

VI

-

~

-

35" 37"

33" 26"

27" 21"

28" 22"

17" 16"

31

27

25

25

23

-

-

41"

33" 33"

31" 34"

32" 28"

24'

-

-

-

53"

54

43

42

33

-

22

24

24

19

-

-

43

41

51

52

54

-

~~

Indices are based on gross weight gain unless otherwise indicated. See text for discussion of author's methods or other * Calculated from author's data. comments. Fresh weight basis. Dry weight basis. Based on net weight gain.

and then drops to its lowest in the 12th instar. The significance of his data is doubtful since slices of carrot root are apparently an unnatural and a very poor food for this insect. The E.C.I.3 are exceptionally low, ranging from 0.58 to 4-13. Calculations from the data of Evans (1939b) suggest that the fresh weight E.C.I. of Phuleru increased from the 1st to the 4th instar and decreased in the 5th. However, as pointed out above, his data are probably not representative of entire instars since they are based on eleven 24 hr measurements scattered over a larval stage of forty-five days. Recalculation of the data of Carne (1966) shows that the fresh weight E.C.I.'s of Puropsis utomuriu feeding on shoots of Eucalyptus blukelyi are 18, 34, 30 and 18 from the 1st to the 4th instar. There is little information on variation of the E.C.D. from instar to instar. Hiratsuka (1920) found the E.C.D. of Bombyx to be 66, 61, 66, 65 and 61 from the 1st to the 5th instar, respectively. Calculations from

C O N S U M P T I O N A N D U T I L I Z A T I O N O F FOOD

271

Evans' (1939b) data show the fresh weight E.C.D. of Phalera rising from the 1st to the 4th instar and dropping sharply in the last: 21, 48, 68, 72 and 43, respectively. His data are, however, of doubtful significance as stated above. Calculations from Smith's (1959) data show the E.C.D. rising early in the nymphal stage and falling near the end of the stage. The decline of the E.C.I. with age is at least partly the result of the concomitant decline of the A.D. Comparison of Hiratsuka's (1920) indices for the five instars of Bombyx shows that the E.C.I.'s and the A.D.'s decline together and at about the same rate. The E.C.D.'s, however, do not show a tendency to decline with age. Thus, at least with Bombyx, the decline of the E.C.I. seems to be due to the decline in digestibility and not to a decline in the efficiency of conversion of digested food. The E.C.I. can also vary within an instar. Carne (1966) reported a rise and fall of the fresh weight E.C.I. from the beginning to the end of the 4th and last instar of Paroysis, 19, 22, 18 and 4 on the 13th, 14th, 15th and 18th days of the larval stage, respectively. He suggested that decreases in the E.C.I. are associated with energy-consuming physiological activities associated with recent molts and the approach of maturity. Calculations of Evans' (1939b) data for the 1st instar larvae of Smerinthus populi show the E.C.I. varying from the 1st to the 10th day of the instar as follows: 17, 28, 39, 39, 37, 36, 32, 36, 32 and 28. 2. Variation with sex The sexes may differ in the efficiency with which they utilize food, but whether or not sexual differences are of general occurrence is not clear. Adult Gryllus males apparently digest more of either escarole leaves or wheat flour than the females. The A.D. of wheat flour also seems to be greater in adult males of Blattella (Table IV). In Tineola raised from hatching to the adult stage at 30" approximate digestibility and net efficiency of conversion differ only slightly from male to female. At 20" both sexes digest and convert more efficiently, but although A.D. is the same in both sexes, the E.C.I. of the female is higher because of an increase in E.C.D. (Table V). Macko and Jasic (1959) measured A.D. in the last two larval instars of Hyphantria reared at different temperatures and feeding on the leaves of different species of trees. Approximate digestibility of the more digestible leaves was consistently higher in the female than the male. However, the sexes differed far less in their ability to digest the less digestible leaves. The A.D.'s of both sexes varied with temperature, but the % difference between corresponding A.D.'s of

212

G . P. W A L D B A U E R

female and male remained almost constant. In other species efficiency does not seem to differ with sex. Chauvin (1946) found A.D. to be the same in adult males and females of Schistocerca and Phaneropteru .falcata (Table IV). Davey (1954) reported that total food intake during the nymphal stage is greater in the female than the male of Schistocerca. In the 5th instar the A.D., E.C.I. and E.C.D. of male and female Bombyx larvae are similar, although the females seem to convert a little more efficiently than the males (Hiratsuka, 1920). Legay’s (1957) findings with Bombyx are similar. V I . U T I L I Z A T I OONF D I E T A R YC O N S T I T U E N T S The literature offers but few data on the utilization by insects of thc nutrient constituents of either natural foods or chemically defined diets. House (1959, 1962 and 1965a) pointed out that quantitative work with defined diets has generally consisted of determining the amount of a nutrient required per weight or volume of diet. Minimal or optimum requirements expressed in these terms say nothing about the absolute quantities required, but define only the relationships between the requirements for particular nutrients (Sang, 1956, 1959). This sort of information facilitates the formulation of diets, but conveys a minimum of biological meaning pertinent to questions of metabolism, ecological relationships or the adaptation of insects to their natural foods. House (1962) felt that absolute requirements have been neglected because their determination requires the laborious task of measuring food intake. The coeficient of apparent digestibility for the constituent X of the food is usually expressed as a percentage and is calculated as:

C.A.D.(X) =

amount of X in food ingested - amount of X in feces amount of X in food ingested

The eficiency with which the digestible portion of the constituent X is converted to body substance is calculated as : E.C.D.(X)

=

amount of X in body amount of X in food ingested - amount of X in feces

x 100

The eflciency of coizversiori of ingested constituent X to body substance is calculated as: E.C.I.(X)

=

amount of X in body amount of X in food ingested

C O N S U M P T I O N A N D U T I L I Z A T I O N OF FOOD

273

Measurements of the utilization of nitrogen (N) by insects are complicated by the presence of the urine in their feces. The feces, thus, contain all of the absorbed N which was metabolized and excreted as waste. Thus, if the amount of urine N is not determined and subtracted from total fecal N, calculation by difference (N in food eaten less N in feces) yields total N retained in the body rather than the amount of N digested. The C.A.D. (N) and the E.C.D. (N) are, therefore, practically meaningless if they are calculated from data which have not been corrected for urine N. The uncorrected C.A.D. (N) and the E.C.I. (N) should, of course, be identical since ingested N less fecal and urine N should be the same as the amount of N retained in the body. The uncorrected E.C.D. (N) should always be 100% for the same reason. Both uncorrected calculations are meaningless truisms. The last column of Table JX shows that most of the E.C.D.’s (N) actually deviate considerably from the expected 100%. It can be reasonably assumed that these deviations are due to the incomplete recovery of N during analysis. The uncorrected E.C.D. (N) thus serves as a measure of the accuracy of the N determination. Balogh and Gere (1953), however, ascribed an apparent surplus in N retention to the fixation of atmospheric N. An approximate correction for urine N can be made for only three of the species listed in Table IX. Evans and Goodliffe (1939) and Hiratsuka (1920) gave the uric acid content of the feces and Evans (1939a) gave both the uric acid and ammonia content. Other nitrogenous wastes are not accounted for, but with the insects in question correction for uric acid alone probably closely approximates correction for total urine N. Available analyses show that, except in blowfly larvae and some aquatic larvae, uric acid generally accounts for 80% or more of the excreted N (Stobbart and Shaw, 1964). The resulting difference between the corrected and uncorrected C.A.D.3 (N) varies. With Bonibyx the corrected C.A.D. (N) is about 7”/, higher, with Phalera about 15% and with Tenebrio about 38%. Calculations from the data of Hiratsuka (1920) give a corrected E.C.D. (N) of 92% for Bornbyx as opposed to the uncorrected value of 100.4% (Table IX). It should be noted that even if all of the urine N is accounted for the apparent rather than the true digestibility of N is obtained because of the fecal metabolic N (is. peritrophic membrane, see Section 11B2). The accuracy of E.C.J. (N) is not, of course, affected by the presence of urine or other metabolic N in the feces. Whether an efficiency of conversion is based on the gross retention or the net retention of N may make a considerable difference. The former

Insect, food and authority Agrotis orthogonia Thatcher wheat sprouts Golden ball wheat sprouts Kasting and McGinnis (1959) Bombyx mori Morus alba leaves Hiratsuka (1920) Chilo suppressalis Autoclaved rice stems Hirano and Ishii (1962) Hyphantria cunea Acer ncgicndo leaves Balogh and Gere (1953) Mamestra brassicac Beet leaves Sweet potato leaves Hirano and Noguchi (1963) Phalera bucephala Corylus avellana leaves Evans (1939a) Tenebrio molitor Wheat bran Evans and Goodliffe (1939)

TABUIX The utilization of nitrogen C.A.D. % (Total N) C.A.D. % corrected for E.C.I. (Protein N) uric acid (Total N)

r4 E.C.D. (Total N) corrected for uric acid

E.C.D. (Total N) not corrected for uric acid

4 P

1158*c 95".

78"

63"

608

70

-

62"

-

44"

-

31" 12"

54

-

92".

1 14" 97"

94"

56

The experiments in which these data were obtained are summarized in Table IV. E.C.I.(N) and E.C.D.(N) are based on net retention of N unless otherwise noted. a Calculated from author's data. Corrected for ammonia in feces in addition to uric acid. Based on gross retention of N; thus N lost in the exuviae at the molt is included.

u0 P

crn w

C O N S U M P T I O N A N D U T I L I Z A T I O N O F FOOD

275

includes the N content of the exuviae and products such as silk while the latter does not. The N content of the exuviae of Bombyx is small. According to calculations made from Hiratsuka’s (1920) data the exuviae of 5th instar larvae contain an average of 0.56 mg N, or about 0.54% of the N eaten and 0.91% of the N digested during the instar. However, the average N content of the silk in one cocoon is about 41 mg, or about 35% of the N eaten and 58% of the N digested during larval life (calculated from data of Hiratsuka, 1920). Balogh and Gere (1953) confused thei: data by lumping the exuviae and silk with the feces. Table IX shows that the E.C.I. (N) ranges from 12% (on a plant which supported only poor growth) to 62%. Unfortunately, without more data there is no way to tell just how much of this variation is due to differences in the apparent digestibility of N and how much to differences in the amount of digested N which is metabolized rather than stored. Only Hiratsuka (1920) supplied sufficient data to permit calculation of the corresponding corrected C.A.D. (N) and E.C.D. (N). His data show that Bombyx incorporates almost all of the digested N (92%) in its body (Table IX). The generally high C.A.D.’s (protein N) would lead one to expect the C.A.D. (N) to be generally high. The following C.A.D.’s (protein N) are available in addition to those listed in Table IX: Agluis, 63% ;Malacosoma, 72% ;Pieris, 83% (calculated from data of Evans, 1939b) and Prodeniu, 91% (Crowell, 1941). However, it should be noted that the amount of protein N “digested” and the amount absorbed are not necessarily the same. A difference could result if, as seems likely, some of the peptides and amino acids resulting from the breakdown of protein pass out in the feces. If these breakdown products of protein are not all accounted for, the “digestibility” of protein N will be greater than its absorbability, and the digestibility of nonprotein N will appear to be less than it actually is. With most leaffeeding insects the amount of non-protein N utilized is likely to be small since protein N has been found to make up generally from 80% to over 90% of the total N in leaves (Evans, 1939a, b; Fraenkel, 1953; Hiratsuka, 1920). Recently McGinnis and Kasting (1966) used the chromic oxide method to measure both dry matter and nitrogen utilization by 5th instar nymphs of MeIanoplus bivittatus. Table X shows that the digestibility of sugars is very high, the C.A.D.’s for total sugars ranging from 59% to 94%. The non-reducing sugars, essentially sucrose in leaves, appear to be highly digestible, but it does not follow that their breakdown products have been completely absorbed. It is quite probable that a part of the hydrolysates of sucrose,

TABLE X The coefficients of apparent digestibility of carbohydrates and lipids Insect, food and authority Aglais urticae Urtica dioica leaves Evans (1939b) Bombyx rnori Morus alba leaves Hiratsuka (1920) Cliilo suppressalis Autoclaved rice stems Hirano and Ishii (1962) Malacosorna neustria Salix viniinalis leaves Evans (1939b) Phalera bucephala Corylus aoellana leaves Evans (1939a) Pieris brassicae Young cabbage leaves Evans (1939b) Prodenia eridania Phaseolus vulgaris leaves Crowell (1941) Tenebrio molitor Wheat bran Evans and Goodliffe (1939)

Reducing sugars

Non-red uc i ng sugars

Total sugars

Starch

50"

13'

65'

9'

38"

55"

-

-

-

-

4?

60"

63

96

87

54

61

-

64"

88"

76"

35"

68"

27"

71"

93

78"

0

46'

63"

58"

62"

59"

0

29"

-

56

99

85"

0

65"

-

53"

95"

94"

59

54"

73"

The experiments in which these data were obtained are summarized in Table IV.

Total sugars and starch

Calculated from author's data.

Lipids

CONSUMPTION A N D U T I L I Z A T I O N O F FOOD

277

the reducing sugars fructose and glucose, are passed out with the feces. The presence of these hydrolysates in the feces would make it appear that the reducing sugars are less digestible than they actually are. The non-reducing sugars would presumably be hydrolysed very rapidly by digestive enzymes. The fact that some of the non-reducing sugars pass through the gut unhydrolysed suggests that a corresponding part of the food was simply not reached by the digestive enzymes. The digestion of starch varies widely (Table X). Among the leaffeeding species investigated only Malacosoma digested a large percentage of the starch. Aglais digested very little starch, and the three remaining leaf-feeders digested no starch at all. Another leaf-feeder, Automeris, has also been found not to digest starch(Brown, 1930).Brown (1937b) concluded from an analysis of its feces that Melanoplus bivittatus had not digested starch. He had not, however, measured the amount of starch intake. Both Tenebrio and Chilo normally eat foods with a high content of starch, and both digested over 50% of the starch they ingested (Table X). Ullman (1932) found that amylase from several species of insects, including Tenebrio and several leaf-feeding Lepidoptera, did not hydrolyse whole unbroken grains of starch. However, starch was hydrolysed after the outer layer of amylopectin had been broken by cooking the starch or grinding it with sand. It would be interesting to know if Chilo was able to digest a large percentage of starch in Hirano and Ishii's (1962) experiment because the grains had been ruptured by autoclaving. Tsutsui and Sato (1954) had previously reported that Chilo excretes starch unaltered. They found that the starch is, however, gradually hydrolysed to simple carbohydrates in the feces. They suggested the hypothesis that Chilo may be coprophagous and thus utilize these starch hydrolysates. Hirano and Ishii (1962) felt that their findings are in accord with this hypothesis. Hiratsuka (1920) reported that Bombyx larvae do not digest the crude fiber fraction of mulberry leaves. Phaleru larvae do not digest cellulose (Evans, 1939a). Available data suggest that on the whole leaf-feeding insects do not digest cellulose (Trager, 1953; Wigglesworth, 1965). Tenebrio, however, digests about 36% of the hemicellulose which it ingests (Evans and Goodliffe, 1939). Some wood-feeding insects do, of course, digest cellulose either with or without the aid of intestinal symbionts. With the exception of Malacosoma the apparent digestibility of lipids is fairly high (Table X). Hiratsuka (1920) found the average E.C.T. of lipids for the five instars of Bombyx to be about 213%. Thus Bombyx

G. P. W A L D B A U E R

278

larvae store over twice as much fat as they digest, indicating the transformation of carbohydrates into fats. The nutritive ratio,

% digestible carbohydrate + (% digestible fat % digestible crude protein

x

2-25)

has been used as an expression of nutrient balance for domestic animals. Digestible fat is multiplied by 2.25 since its heat value is that much greater than the heat value of carbohydrate. The nutritive ratio for Bombyx rose from the 1st to the 5th instar, 1.16, 1.25, 1.21, 1.26 and 1.50, respectively. The comparatively wide ratio obtained for the 5th instar, according to Hiratsuka (1920), reflects the greatly increased rate of fat deposition in that instar. Evans (1939b) gave nutritive ratios of 0.65 for Malacosoma, 0.32 for Aglais, 0.44 for Pieris and 1-18 for Phalera. He pointed out that these ratios differ widely from ratios ranging from 4-7 to 7-0 determined for growing cattle, sheep and pigs. The nutritive ratio for Tenebrio molitor, 4.7, appears to be exceptionally wide for an insect, and probably reflects the oxidation of large quantities of carbohydrate as a source of metabolic water (Evans and Goodliffe, 1939).

V I I . UTILIZATION OF E N E R G Y The coeficient of metabolizable energy (C.M.E.), usually expressed as a percentage, is calculated as : C.M.E.

=

gross energy in food eaten - gross energy in feces gross energy in food eaten

The gross energy (or heat of combustion) of a substance is usually measured with a bomb calorimeter and is defined as the energy liberated as heat when the substance is completely oxidized to COz and H20 (Kleiber, 1961; Tyler, 1964). Metabolizable energy has been defined for mammals as the gross energy in the food eaten less the gross energy in the feces, urine and the methane produced in digestion. This is the energy available for the production of heat, body substance or work (Kleiber, 1961). The above formula does not take into account the possibility that insects produce methane or other digestive gases. Metabolizable energy for insects as here defined is thus comparable to metabolizable energy for birds as defined by Kendeigh (1967, personal communication).Hiratsuka (1920) subtracted the gross energy in the feces (urine included) from the

CONSUMPTION A N D UTILIZATION OF FOOD

279

gross energy of the food eaten by Bornbyx and called this value the physiologically available energy. He made no mention of the possibility that insects may produce digestive gases. I have found neither qualitative nor quantitative data on the production of digestive gases in insects. Krogh (1916) stated that carnivorous animals usually produce small quantities of methane, but that herbivores may produce large quantities. The eficiency of storage of ingested energy is calculated as: E.S.I. (E)

=

gross energy stored in body gross energy in food eaten

The eficiency of storage of metabolizable energy is calculated as : E.S.M. (E)

=

gross energy stored in body x 100 gross energy in food eaten - gross energy in feces

Hiratsuka’s (1920) study of Bombyx remains to this day the most complete study of the intake and expenditure of energy by an insect. TABLE XI Comparison of the utilization of dry matter and energy by Bombyx niori To end of 5th instar Dry matter Energy

To newly emerged adult Dry matter Energy _____ A.D. 37 C.M.E. 4 2 A.D. 37 C.M.E. 42 E.C.I. 23 E.S.I.(E) 28 E.C.I. 8 E.S.I.(E) 12 E.C.D. 62 E.S.M.(E) 67 E.C.D. 22 E.S.M.(E) 28 Conversion indices are based either on gross gain from hatching to the 5th instar just before spinning or on net gain from hatching to the newly emerged adult. Calculated from data of Hiratsuka (1920).

Table XI shows that the utilization of energy is higher than the utilization of dry matter, but that the two are roughly comparable. Hiratsuka (1920) also found that the consumed portion of the leaves had a somewhat greater heat value than the total of leaves supplied to the larvae. Table XI1 summarizes the utilization of energy by the five larval instars of Bombyx. The C.M.E. and the E.S.I.(E) vary with age much as do the corresponding A.D. and E.C.I. of dry matter (Tables VII and VIII), declining in the early instars and then tending to remain more or less constant. The E.S.M.(E), however, increases with age, reaching its peak in the 5th instar, while the corresponding E.C.D. of dry matter tends to remain relatively constant from instar to instar (see Section

.L lnm70

FULLY FED LARVA 3 5 3 kcol. 652.1.

280

A

d

HEAT AND WORK

037 kcol

047 k c d

69%

0'12 kcol

8 6%

PUPA 2.00 hcol. 370%

ADULT

k c d , 27.47.

-DEATH

, \

PUP01

Feces a Urine 7 7 2 hcd

Exwoe 005kcal.09%

Silk

1.13 k c o 1 . 2 0 9 %

ENERGY IN

Q

A

PRODUCTS

HEAT AND WORK 061 hcol

ADULT 3 hcal. 28.5%

Feces B urine

8.76 kcol

First 4 Exuvme 0'03 h c d , 0 4 %

h Spermotozoo

ENERGY

IN PRODUCTS

Fro. 1. The energy economy of male and female Bombyx mori (based on data of Hiratsuka, 1920).

0l5kcol

-DEATH

G. P. W A L D B A U E R

Y

CONSUMPTION A N D UTILIZATION O F FOOD

28 1

TABLE XI1 The utilization of energy by the larval instars of Bombyx mori Instar I I1

m N V

C.M.E.

E.S.1.Q

E.S.M.(E)

52 49 42 44 42

32 29 28 29 28

61 60 66 66 68

Conversion indices are based on gross gain in each instar. Calculated from data of Hiratsuka (1920).

VEl). The large amount of energy stored during the 5th instar is, of course, needed to support the non-feeding pupa and adult. Figure 1 is a graphic representation of Hiratsuka’s (1920) elucidation of the energy economy of Bombyx. The female ingests about 16% more energy and retains about 21% more metabolizable energy than the male. Very little of the metabolizable energy, about 2%, is lost in the exuviae, although about 20% is expended in the silk. From hatching to the completion of reproductive activity about 52% of the metabolizable energy is respired by either sex. A large share of the metabolizable energy of the female, over 12%, goes into the eggs. This is not much less than the extra 17% (1.12 kcal) of metabolizable energy available to the female in comparison with the male. After reproductive activity is completed the female goes to her death retaining less than 14% (0-89kcal) of the total metabolizable energy while the male goes to his death retaining over 25% (1.37 kcal). Erhan et al. (1966) looked for circadian rhythms in the utilization of energy by Bombyx. They measured the ingestion, excretion and metabolism of energy for 24 hr in the 4th and 5th instars, but found no evidence of circadian rhythms. Kitzawa (1959) quoted Nakamura’s unpublished data on the utilization of dry matter and energy by the larvae of Pieris rupae. The methods used to obtain the data were not described. The accuracy of the data is doubtful since the A.D. and C.M.E. for the 2nd instar, 95% and 97%, respectively, are unbelievably high. Teissier’s (1931) figures are stated to represent the amount of energy or dry matter consumed by the larvae of Tenebrio. However, his method actually yields the metabolizable energy or the approximate digestibility of dry matter. He provided groups of larvae with a known dry weight of

282

G . P. W A L D B A U E R

food, and at the end of the experiment determined the dry weight of the mixture of left over food and feces. His figures for “consumption” represent the difference between these weights. The mean E.S.M.(E) was 26% for larvae fed wheat flour and 32% for larvae fed powdered milk (see also Table IV). However, the larvae were kept at conditions of temperature and moisture far below the optimum and grew very slowly. The wheat flour and the powdered milk contained only 12.5% and 2.5% moisture, respectively. The experiments were run at ambient temperatures of about 17°C and 20°C, respectively. ACKNOWLEDGEMENT

I wish to thank my colleague, Dr. Gottfried Fraenkel, for his critical reading of the manuscript and numerous helpful suggestions. Errors and omissions, however, remain solely my responsibility. REFERENCES Auclair, J. L. (1963). Aphid feeding and nutrition. Ann. Rec. Ent. 8, 439-490. Balogh, J. and Gere, G. (1953). uber die Ernahrungsbiologie und Luftstickstoffbindung der Hyphantria-Raupen. Acta biol. Hung. 4, 431-452. Barton-Browne, L. B. (1964). Water regulation in insects. Ann. Rev. Ent. 9, 63-82. Beck, S. D. (1956). The European corn borer, Pyrausta nubilalis (Hubn.), and its principal host plant. 11. The influence of nutritional factors on larval establishment and development on the corn plant. Ann. ent. SOC.Am. 49,582-588. Biedermann, W. (1919). Beitrage zur vergleichenden Physiologie der Verdauung. VIII. Dringen Verdauungsfermente in geschlossene Pflanzenzellen ein ? Archs ges. Physiol. 174, 358-425. Brennikre, J., Jover, H. and de Malmann, R. (1949). Sur la nutrition de quelques Orthoptkres. Rev. Path. VPg. 28, 134-140. Brown, A. W. A. (1937a). Studies on the excreta of a grasshopper (Melanoplus bivittatus Say). J. exp. Biol. 14, 87-94. Brown, A. W. A. (1937b). A note on the utilization of polysaccharides by a grasshopper. Will. ent. Res. 28, 333-336. Brown, F. M. (1930). The utilization of hexose carbohydrates by lepidopterous larvae. Ann. N. Y. Acad. Sci. 32, 221-234. Buxton, P.A. (1930). The biology of a blood-sucking bug, Rhodniusprolixus. Trans. R. ent. SOC.Lond. 78, 227-236. Carne, P. B. (1966). Growth and food consumption during the larval stages of Paropsis atomaria (Coleoptera: Chrysomelidae). Ent. exp. appl. 9, 105-1 12. Chauvin, R. (1946). Notes sur la physiologie comparee des Orthopttres. IV. Le coefficient &utilization digestive, le rythme &excretion et le transit intestinal. Will. SOC.ent. Fr. 51, 24-29. Crossley, D. A., Jr. (1963a). Use of radioactive tracers in the study of insect-plant relationships. In “Radiation and Radioisotopes Applied to Insects of Agricultural Importance.” Proc. Symp. intern. Atomic Energy Agency, Vienna, 1%3,43-54.

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Crossley, D. A., Jr. (1963b). Consumption of vegetation by insects. In “Radioecology” (V. Schultz and A. W. Klement, Jr., eds.), pp. 427-430. Reinhold Pub. Corp., New York. Crossley, D. A., Jr. (1966). Radioisotope measurement of food consumption by a leaf beetle species, Chrysomela knabi Brown. Ecology 47, 1-8. Crowell, H. H. (1941). The utilization of certain nitrogenous and carbohydrate substances by the southern armyworm, Prodenia eridania Cram. Ann. ent. SOC. Am. 34, 503-512. Crowell, H. H. (1943). Feeding habits of the southern armyworm and rate of passage of food through its gut. Ann. ent. SOC.Am. 36, 243-249. Dadd, R. H. (1960). Observations on the palatability and utilization of food by locusts, with particular reference to the interpretation of performances in growth trials using synthetic diets. Ent. exp. appl. 3, 283-304. Davey, P. M. (1954). Quantities of food eaten by the desert locust, Schistocerca gregaria (Forsk.) in relation to growth. Bull. ent. Res. 45, 539-551. David, W. A. L. and Gardiner, B. 0. C. (1962). Observations on the larvae and pupae of Pieris brassicae (L.) in a laboratory culture. Bull. ent. Res. 53,417-436. Edwards, C . A. (1964). The bionomics of swift moths. I. The ghost swift moth, Hepialus humuli (L.). Bull. ent. Res. 55, 147-160. Elam, C. J., Reynolds, P. J., Davis, R. E. and Everson, D. 0. (1962). Digestibility studies by means of chromic oxide, lignin and total collection techniques with sheep. J. Anim. Sci. 21, 189-192. Engelmann, M. D. (1966). Energetics, terrestrial field studies, and animal productivity. In “Advances in Ecological Research” (J. B. Cragg, ed.), vol. 3, pp. 73115. Academic Press, London and New York. Erhan, E., Burlacu, G., Petre, Z. and Nersesian-Vasiliu, C. (1966). Metabolismul energetic nictemeral la viermele de matase (Bombyx mori L.). Stud. Cerc. Biol., Ser. Zool. (Bucharest) 18, 271-280. Evans, A. C. (1939a). The utilization of food by the larvae of the buff-tip, Phalera bucephala (Linn.) (Lepidopt.). Proc. R. ent. SOC.Lond. A 14, 25-30. Evans, A. C. (1939b). The utilization of food by certain lepidopterous larvae. Trans. R. ent. SOC.Lond. 89, 13-22. Evans, A. C. and Goodliffe, E. R. (1939). The utilization of food by the larva of the mealworm, Tenebrio molitor L. (Coleop.). Proc. R. ent. SOC.Lond. A 14, 57-62. Evans, D. E. (1962). The food requirements of Phonoctonus nigrofasciatus Stil. (Hemiptera, Reduviidae). Ent. exp. appl. 5, 33-39. Fewkes, D. W. (1960). The food requirements by weight of some British Nabidae (Heteroptera). Ent. exp. appl. 3,231-237. Fraenkel, G. (1953). The nutritional value of green plants for insects. Trans. 9th intern. Congr. Ent. Amsterdam, 1951 2, 90-100. Fraenkel, G. (1959). The raison d’Ctre of secondary plant substances. Science, N.Y. 129, 1466-1470.

Fraenkel, G. and Blewett, M. (1944). The utilization of metabolic water in insects. Bull. ent. Res. 35, 127-139. Friend, W. G., Choy, C. T. H. and Cartwright, E. (1965). The effect of nutrient intake on the development and the egg production of Rhodniusprolixus StBhl. (Hemiptera: Reduviidae). Can. J. 2001. 43, 891-904. Garcia, J., Ervin, F. R., Yorke, C. H. and Koelling, R. A. (1967). Conditioning with delayed vitamin injections. Science, N. Y.155, 716-718.

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Gere, G. (1956). Investigations into the laws governing the growth of Hyphantria cunea Drury caterpillars. Acra biol. Hung. 7,43-72. Gordon, H. T. (1959). Minimal nutritional requirements of the German roach, Blattella germanica L. Ann. N. Y. Acad. Sci. 77, 290-35 1. Gupta, P. D. and Sinha, R. N. (1960). Excretion and its products in some storedgrain infesting beetles. Ann. ent. Sac. Am. 53, 632-638. Hassanein, M. H. and El Shaarawy, F. (1962a). The nutritional value of certain mulberry varieties. Revue Ver Soie 14, 21 1-259. Hassanein, M. H. and El Shaarawy, F. (1962b). The physiological effect of feeding with different mulberry varieties on certain races of the silkworm (Bombyx mori L.). Revue Ver Soie 14, 261-317. Heinrichs, E. A. and Pruess, K. P. (1966). Chromogen-ratio method for determining digestibility of plants by grasshoppers. J. econ. Enr. 59, 550-552. Hirano, C. and Ishii, S. (1962). Utilization of dietary carbohydrates and nitrogen by rice stem borer larvae, under axenic conditions. Ent. exp. a&. 5, 53-59. Hirano, C. and Noguchi, H. (1963). Food utilization by the last instar larvae of the cabbage armyworm, Mamestra brassicae L., fed on plant leaves of different food values. Jap. J. appl. Enr. Zool. 7, 311-315. Hiratsuka, E. (1920). Researches on the nutrition of the silk worm. Bull. ser. Exp. Sta. Japan 1, 257-315. Hollande, A. C. and Cordebard, H. (1926). Notes chimiques et physiologiques se rapportant aux excrkments de la teigne du crin (Tinella biselliella Hummel ; syn. crinella Treitsche-Duponchel). Bull. SOC.Cliim. Biol., Paris 8, 631-635. Hopkins, F. G. (1912). Feeding experiments illustrating the importance of accessory factors in normal dietaries. J. Physiol., Lond. 44,425-460. House, H. L. (1959). Nutrition of the parasitoid Pseudosarcophaga afinis (Fall.) and of other insects. Ann. N. Y. Acad. Sci. 77, 394-405. House, H. L. (1962). Insect nutrition. A, Rev. Biochem. 31, 653-672. House, H. L. (1965a). Digestion. In “The Physiology of Insecta” (M. Rockstein, ed.), Vol. 2, pp. 815-858. Academic Press, New York and London. House, H. L. (1965b). Effects of low levels of the nutrient content of a food and of nutrient imbalance on the feeding and the nutrition of a phytophagous larva, Celerio euphorbiae (Linnaeus) (Lepidoptera : Sphingidae). Can. Enr. 97,62-68. Husain, M. A., Mathur, C. B. and Roonwal, M. L. (1946). Studies on Schistocerca gregaria (ForskH1). XIII. Food and feeding habits of the desert locust. Indian J. Ettt. 8, 141-163. Janda, V. (1961). The total metabolism of insects. 12. Metabolism during the intermoulting period of Neodiprion sertifer Geoffr. larvae (Hym. Tenthredinoidea). VPst. c‘sl. Spol. 1001. 25, 306-317. Johnson, C. G. (1937). The relative values of man, mouse and domestic fowl as experimental hosts for the bed-bug, Cimex lectularius L. Proc. zool. SOC.Lond. A 107, 107-126. Johnson, C. G. (1960). The relation of weight of food ingested to increase in bodyweight during growth in the bed-bug, Cimex lectularius L. (Hemiptera). Ent. exp. appl. 3, 238-240. Kasting, R. and McGinnis, A. J. (1959). Nutrition of the pale western cutworm, Agrotis orthogonia Morr. (Lepidoptera: Noctuidae). 11. Dry matter and nitrogen economy of larvae fed on sprouts of a hard red spring and a durum wheat. Can. J. Zool. 37, 713-720.

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Kasting, R. and McGinnis, A. J. (1965). Measuring consumption of food by an insect with carbon-14 labelled compounds. J. Insect Physiol. 11, 1253-1260. Kendeigh, S.C. (1949). Effect of temperature and season on energy resources of the English sparrow. Auk 66, 113-127. Kitzawa, Y.(1959). Bio-economic study of natural populations of animals. Jap. J. 2001. 12,401448. Kleiber, M. (1961). “The Fire of Life, an Introduction to Animal Energetics”, 454 pp. John Wiley & Sons, Inc., New York. Krishna, S. S. and Saxena, K. N. (1962). Measurement of the quantity of food ingested by insects infesting stored food material. Naturwissenschafen 49, 309. Krogh, A. (1916). “The Respiratory Exchange of Animals and Man”, 173 pp. Longmans, Green & Co., London. Lafon, M. (1951). Quelques documents sur I’appetit et la consommation alimentaire chez les insectes. Ann. Niitr., Paris 5 , 485-504. Lebedev, A. G. and Savenkov, A. N. (1932). Die Nahrungsnormen des Kieferspinners (Dendrolimus pini L.). Z . angew. Ent. 19, 85-103. Legay, J. M. (1953). Sur une methode d’etude quantitative de la prise de nourriture chez le ver a soie. C.r. hebd. SPanc. Acad. Sci. Paris 236, 326-328. Legay, J. M. (1957). La prise de nourriture chez le ver i soie. Ann. Znst. Natl. Recherche Agron., Skries C, NumPro hors skrie, 1-169. Legay, J. M. (1958). Recent advances in silkworm nutrition. Atin. Reo. Ent. 3, 75-86. Long, D. B. (1953). Effects of population density on larvae of Lepidoptera. Trans. R. ent. SOC.Lond. 104,543-584. McCay, C. M. (1938). The nutritional requirements of Blattella germanica. Physiol. Z O O / . 11, 89-103. McGinnis, A. J. and Kasting, R. (1959). Nutrition of the pale western cutworm, Agrotis orthogonia Morr. (Lepidoptera: Noctuidae). I. Effects of underfeeding and artificial diets on growth and development, and a comparison of wheat sprouts of Thatcher, Triticum aestiuunt L., and Golden Ball, T.durum Desf., as food. Can. J. Zool. 37, 259-266. McGinnis, A. J. and Kasting, R. (1964a). Colorimetric analysis of chromic oxide used to study food utilization by phytophagous insects. J. agric. Fd Chem. 12, 259-262. McGinnis, A. J. and Kasting, R. (1964b). Chromic oxide indicator method for measuring food utilization in a plant-feeding insect. Science, N . Y. 144, 14641465. McGinnis, A. J. and Kasting, R. (1964~).Comparison of gravimetric and chromic oxide methods for measuring percentage utilization and consumption of food by phytophagous insects. J. Insect Physiol. 10, 989-995. McGinnis, A. J. and Kasting, R. (1966). Comparison of tissues from solid- and hollow-stemmed spring wheats during growth. IV. Apparent dry matter utilization and nitrogen balance in the two-striped grasshopper, Melanoplus bivittatus (Say). J. Insect Physiol. 12,671-678. Macko, V. and Jasic, J. (1959). Zur Frage der Ernahrung phytophager Lepidopteren. In “The Ontogeny of Insects”, Acta Syrnposii de Evolutione Itwectorum, Praha 1959, 317-320, Czechoslovak Acad. Sci., Prague.

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McMillian, W. W., Starks, K. J. and Bowman, M. C. (1966). Use of plant parts as food by larvae of the corn earworm and fall armyworm. Ann. ent. SOC.Am. 59, 863-864. Mathur, L. M. L. (1967). On the food utilisation and digestion of major food nutrients in the lepidopterous larvae feeding on cauliflowers. Zoo1 Anz. 178,82-89. Maynard, L. A. (1951). “Animal Nutrition”, 3rd Ed., 474 pp. McGraw-Hill Book Co., Inc., New York. Mellanby, K. and French, R. A. (1958). The importance of drinking water to larval insects. Ent. exp. appl. 1, 116-124. Mitchell, H. H. (1964). “Comparative Nutrition of Man and Domestic Animals”, vol. 11, 840 pp. Academic Press, New York and London. Mittler, T. E. (1957). Studies on the feeding and nutrition of Tubcrolachnussalignrrs (Gmelin). J. exp. Biol. 34, 334-341. Mukaiyama, F. and Ito, T. (1962a). Digestion experiments in the silkworm, Bombyx mori, by means of artificial diets (I). Amount of food consumed, amount of food digested, and coefficient of digestibility (in Japanese with English summary). J. Ser. Sci. Japan 31, 317-322. Mukaiyama, F. and Ito, T. (1962b). Digestion experiments in the silkworm, Bombyx mori, by means of artificial diets. (11). Effect of cellulose powder added in diets (in Japanese with English summary). J. Ser. Sci. Japan 31, 398406. Nagy, B. (1953). Der Nahrungsverbrauch der Raupe des amerikanischen weissen Barenspinners (Hyphantriu cunecz Drury) unter Konstanten Verhaltnissen. Acta agron. Hung. 3, 215-223. Norris, M. J. (1961). Group effects on feeding in adult males of the desert locust, Schistocerca gregaria (Forsk.), in relation to sexual maturation. Bull. ent. Res. 51,731-753. Patton, R. L. (1953). Excretion, In “Insect Physiology” (K. D. Roeder, ed.), pp. 387-403. John Wiley & Sons, Inc., New York. Phillipson, J. (1960). The food consumption of different instars of Mitopus morio (F.) (Phalangida) under natural conditions. J. Anim. Ecol. 29, 299-307. Reid, J. T., Woolfolk, P. G., Richards, C. R., Kaufman, R. W., Loosli, J. K., Turk, K. L., Molner, J. I. and Blaser, R. E. (1950). A new indicator method for the determination of digestibility and consumption of forages by ruminants. J. Dairy Sci. 33, 60-71. Revusky, S. H. and Bedarf, E. W. (1967). Association of illness with prior ingestion of novel foods. Science, N. Y. 155, 219-220. Richards, 0. W. (1947). Observations on grain-weevils, Calandra (Col., Curculionidae). I. General biology and oviposition. Proc. zool. SOC.London 117, 143. Robinson, A. G. (1961). Some problems associated with weighing aphids that have fed after starving, as a method of measuring intake of plant sap. Can. Ent. 93, 156-160. Sang, J. H. (1956). The quantitative nutritional requirements of Drosophila melanogaster. J. exp. Biol. 33, 45-72. Sang, J. H. (1959). Circumstances affecting the nutritional requirements of Drosophila melanogaster. Ann. N. Y. Acad. Sci. 77, 352-365. Satterthwait, A. F. (1933). Larval instars and feeding of the black cutworm, Agrotis ypsilon Rott. J. Agric. Res. 46, 517-530.

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Sattler, H. (1939). Die Entwicklung der Nonne, Lymantria monacha, in ihrer Abhangigkeit von der Nahrungsqualitat. 2. angew. Ent. 25, 543-587. Schwerdtfeger, F. (1930). Untersuchungen uber Dauer des Eistadiums, Wachstum und Stoffwechsel des Kiefernspanners (Bupaluspiniarius L.). Z. angew. Ent. 16, 513-526. Sharada, K. and Bhat, J. V. (1957). Influence of humidity on feed utilization by the silkworm Bombyx mori L. J . Indian Inst. Sci. 39, 220-223. Shyamala, M. B., Sharada, K., Bhat, M. G. and Bhat, J. V. (1960). Chloromycetin in the nutrition of the silkworm Bombyx mori L. 11. Influence on digestion and utilization of protein, fat and minerals. J. Insect Physiol. 4, 229-234. Shyamala, h4. B., Venkatachala Murthy, M. R. and Bhat, J. V. (1956). Effect of chloromycetin on feed utilization by the silkworm, Bombyx mori L. J. Indian Inst. Sci. 38, 177-185. Smart, W. W. G., Jr., Matrone, G. and Smart, V. W. (1954). Use of copper derivatives of chlorophylls in ratio method for estimating digestibility of forages. J. agric. Fd Chem. 2, 1331-1332. Smith, D. S. (1959). Utilization of food plants by the migratory grasshopper, Melanoplus bilituratus (Walker) (Orthoptera : Acrididae) with some observations on the nutritional value of the plants. Ann. ent. SOC.Am. 52, 674-680. So0 Hoo, C. F. (1962). Selection and Utilization of Food Plants by the Larvae of Prodenia eridania (Cramer) with Additional Utilization Studies on the Slikworm, Bombyx mori (Linnaeus), Ph.D. Thesis, 202 pp. University of Illinois, Urbana, Illinois. So0 Hoo, C. F. and Fraenkel, G. (1966). The consumption, digestion, and utilization of food plants by a polyphagous insect, Prodenia eridania (Cramer). J. Insect Physiol. 12, 71 1-730. Stobbart, R. H. and Shaw, J. (1964). Salt and water balance: excretion. In “The Physiology of Insecta” (M. Rockstein, ed.), Vol. 3, pp. 189-258. Academic Press, New York and London. Sylven, E. (1947). Undersokningar over gammaflyet, Phytometra gamma L. St. Vaxtskyddanstalt Med. 48,42 pp. Takeuchi, Y. and Kosaka, T. (1961). On the amount of food ingested and digested during the first instar in the silkworm, Bombyx mori L. (in Japanese with English summary). Acta Ser. 37, 65-72. Teissier, G. (193 1). Recherches morphologiques et physiologiques sur la croissance des insectes. Trau. Stn. biol. Roscof9, 25-238. Titschack, E. (1924). Untersuchungen uber das Wachstum, den Nahrungsverbrauch und die Eierzeugung. 1. Carausius (Dixippus) morosus. Z . wiss. Zool. 123,431487. Titschack, E. (1925). Untersuchungen uber den Temperatureinfluss auf die Kleidermotte (Tineola biselliella Hum.). Z. wiss. 2001. 124, 213-251. Titschack, E. (1930). Untersuchungen uber das Wachstum, den Nahrungsverbrauch und die Eierlegung. III. Cimex lectularius. 2.Morph. dkol. Tiere. 17,471-551. Trager, W. (1953). Nutrition. In “Insect Physiology” (K. D. Roeder, ed.), pp. 350386. John Wiley & Sons, Inc., New York. Trouvelot, L. (1867). The American silkworm. Am. Nut. 1, 30-38, 85-94, 145-149. Tsutsui, K. and Sato, A. (1954). Studies on nutritional physiology of rice stem borer (in Japanese). Kontyli 20, 99-100. Cited by Hirano, C. and Ishii, S. (1962), Ent. exp. appl. 5 , 53-59.

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Tyler, C. (1964). “Animal Nutrition”, 253 pp. Chapman & Hall, London. Ullman, T. (1932). Uber die Einwirkung der Fermente einiger Wirbellosen auf polymere Kohlenhydrate. 2. uergl. Physiol. 17, 520-536. Waldbauer, G. P. (1960). Feeding and Growth on Solanaceous and Non-Solanaceoirs Plants by Normal and Maxillectomized Larvae of the Tobacco Hornworm, Protoparce sexta (Johan.), (Lepidoptera: Sphingidae), Ph.D. Thesis, 134 pp. University of Illinois, Urbana, Illinois. Waldbauer, G. P. (1962). The growth and reproduction of maxillectomized tobacco hornworms feeding on normally rejected non-solanaceous plants. Ent. exp. appl. 5, 147-158. Waldbauer, G. P. (1964). The consumption, digestion and utilization of solanaceous and non-solanaceous plants by larvae of the tobacco hornworm, Protoparce sexta (Johan.) (Lepidoptera : Sphingidae). Ent. exp. appl. 7 , 253-269. Waldbauer, G. P. and Fraenkel, G. (1961). Feeding on normally rejected plants by maxillectomized larvae of the tobacco hornworm, Protoparce sexta (Lepidoptera, Sphingidae). Ann. ent. SOC.Am. 54, 477485. Wigglesworth, V. B. (1965). “The Principles of Insect Physiology”, 6th Ed., 741 pp. Methuen & Co., Ltd., London. Wolcott, G. N. (1924). The comparative resistance of woods to the attack of the termite, Cryptotermes brevis Walker. Dept. Agr. Labor, Insular Expt. Sta., Rio Piedras, Puerto Rico, Bull. 33, 3-15. Wolcott, G. N. (1937). An animal census of two pastures and a meadow in northern New York. Ecol. Monogr. 7 , 1-90. Yokoyama, T. (1963). Sericulture. Ann. Rev. Ent. 8, 287-306.

The Nervous Control of Insect Flight and Related Behavior‘ DONALD M. WILSON Department of Biological Sciences, Stmford University, Stanford, California, US.A. I. Introduction

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TI. Kinematics and Aerodynamics

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A. Wingbeat frequency and animal size . 111. Neurogenic Flyers . A. Locusts . B. Dragonflies . C. Jipidopterans . IV. Myogenic Flyers . A. Motor patterns . B. Significance of multiphasic and multistable patterns C. Hypothesis on coordination in flies . V. General Model for Flight Control . VI. Related Behavior A. Temperature and flight . B. Sound produdion using the wings . VII. General Discussion . References .

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1. ~ N T R O D U C T I O N Before writing a “recent advances” type review, one should ask what service can be provided by another article at this time? The general subject of insect flight has been well covered (Chadwiek, 1953a, b, c; Pringle, 1957, 1965) and is also the subject of an article in this volume (Pringle, 1967b). In addition, Pringle (1965, 1967a) gives some especially useful accounts of the current state of our knowledge concerning the physiology of the flight muscles. These papers, and also that of Wilson (1964a), review briefly most aspects of nervous control of flight, and although this is probably the area of most rapid advance a new literature survey is hardly warranted. Thus I feel free to take this opportunity not to make an exhaustive survey of the subject, but rather to I wish to acknowledge the assistance of Mrs. E. Reid in preparing many illustrations. Thanks are due to W.Kutoch, N. Wsner, A. Kammer, and R. Kiester, for permission to produce previously unpublished figures. The original work reported here was supported by NIH grant NB 03927 and NSF grant GB 2116. 289

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provide a synthesis of a special but significant aspect, namely the neuromotor coordination underlying the flight movements. The aim of the entire discussion will be to suggest how the ganglionic output pattern arises. The emphasis will be on the output pattern itself, with reference to inputs only when such reference contributes directly to understanding the intraganglionic coordinating mechanisms. The organization of the paper will be as follows: (I) A brief description of wing movements and aerodynamics, especially recent results. (2) An extensive survey of motor output patterns in a variety of flying insects, together with descriptions of patterns of use of the same output elements in other behaviors such as singing, walking, and behavioral temperature regulation. (3) Discussions of some related special topics; sound production, effects of temperature on flight motor patterns, temperature regulation. (4) Theoretical discussions of coordinating mechanisms. Much information is included only in the figure legends. Insect flight has fascinated many biologists and even attracted the serious attention of two of the greatest comparative physiologists, Krogh and Von Holst. Part of this fascination is due to the aesthetic qualities of the animals and their movements. In addition, even though insect flight and the mechanisms producing it are highly evolved and specialized processes, their study is not merely a branch of entomology, but may make significant contributions to general biology. For example, the recent work of Pringle on the myogenic muscles is yielding knowledge on the molecular mechanisms of contractility which could not be easily obtained on other systems. It is my hope that these studies on motor coordination eventually will contribute significantly to a general theory of neural control of behavior. 11. KINEMATICS A N D AERODYNAMICS

There is no need for a full review in this section, for this subject is fully covered in another article in this volume (Pringle, 1967b), but some general comments will be useful for later sections. For other earlier reviews see Pringle (1 957) and Weis-Fogh and Jensen (1956). A brief summary of notions of aerodynamic behavior in the locust Schistocerca will be useful. Much of this summary is condensed from Weis-Fogh (1956a). The wings beat at about 17 c/sec in the tilted stroke plane (see Fig. 1). Frequency varies with power but the range of frequency variation is small, and although it is correlated with other parameters of wingstroke variation it is probably not the most significant parameter in itself. Parasite drag (drag on the body and legs) increases

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sharply with body angle, and the norinally encountered body angle varies only from about 0” to 20”. Over this range of body angle, often lift does not change. This unexpected result is due to the fact that twisting of the wing is actively controlled and compensates both for variation in wing velocity from the wing base to tip and for changes in body angle. In the “constant lift” reaction (relative to body angle) the

FIG.1 . A. The main axes and rotations of a flying insect. B. The body angle @) and the stroke plane angle (b).

principal controlling parameter is twisting of the forewings. For general control of power, frequency and amplitude can be varied. For turning, asymmetrical variation in angle of attack and wingbeat amplitude probably suffice. Most insects use four wings during flight, but in many cases they are moved nearly as a single pair. True bugs and bees hook the fore- and hindwings together. In flies the hind pair of wings are lost as aerodynamic power organs, but serve as pure sense organs for flight stabilization. Lepidoptera mostly move the two pairs of wings synchronously although they are not mechanically hooked. The smaller hindwings are

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partly covered by the hind edge of the forewing and appear to be pressed down by it during downstroke. Some beetles do not flap the forewings (elytra), or at least not through a large range, and the hindwings must produce most of the power. The elytra, however, may be used as gliding planes and thus contribute to lift; and if they are capable of even slow adjustment of angle of attack, could be used in control of direction. They could also help to regulate the airflow over the hindwings and thus to some extent control power. Other insects move the fore- and hindwings relatively separately, but always at the same frequency. It has been suggested that the difference in the phase of the wing movements helps to keep the hindwings out of the turbulent wake of the forewings (Chadwick, 1953a). In dragonflies the phase difference is rather large (Neville, 1960), but highly variable (personal observations). The variation is probably correlated with changes in wingbeat frequency and amplitude, but no quantitative study of this has been made. In locusts the phase difference is steadier at about 30" with the hindwings leading (Weis-Fogh, 1956a). Steven Crow (personal communication) has calculated that this phase difference does not make the two wing pairs aerodynamically independent, but on the contrary places the hindwing in the upward wake of the forewing (at least during the powerful downstroke). If true, this means that the lift of the hindwing will be influenced by the behavior of the forewing. The pair of wings might be thought of as a unit with an anterior control surface which regulates airflow over the larger posterior plane. This is consistent with Gettrup's finding (see later) that in the lift-control reaction the hindwings, which produce most of the lift, also measure the lift, but do not themselves vary in motor power in order to regulate aerodynamic power. Instead sensory input from the hindwing lift sensors influences discharge rate in muscles which set the angle of attack of the forewings. Notice that this reflex, in which sensory information from the hindwing influences mainly the forewings, provides a beginning basis for the evolution of the fly halteres which can only regulate the forewings. Both reflexly and aerodynamically the two wing pairs are integrated in a complex fashion. This should lead one to accept aerodynamic studies which are based on the study of one wing at a time and summation of the individual results (Jensen, 1956) as first approximations only. Indeed, Jensen also recognized that the fore- and hindwings form an integrated aerodynamic system. Several kinematic and aerodynamic studies have been made recently on flies. Baird (1965) has measured forces during the wingstroke in Surcophugu and finds that peak lift varies between positive and negative

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values several lo's of times greater than average lift. This large oscillation of lift force could be expected to cause the animal to bob up and down, but, in fact, at the particular frequency of occurrence of this force and with the actual mass, the expected oscillatory motion is only a few microns (Nachtigall, 1966). (In large saturniid moths this problem must be worse. The frequency may be less than 10 c/sec, and the forces should be quite large. Bobbing of the body is apparently compensated at least in part by a motion of the heavy abdomen which is in antiphase with the wings. At least in tethered flight the abdominal-thoracic junction flexes about 30" on every wingbeat, the abdomen going down relative to the body as the wings go up. The abdomen is more or less stiff. In this fashion more of the energy of the moving wings is imparted to the air, and the body should remain relatively level.) Nachtigall(l966) has completed a very careful kinematic study of flight in Phornzia which should become the basis of future aerodynamic and motor control studies on flies. He finds that angle of attack is optimal at all points in the cycle, and concludes that it is actively controlled by muscles as well as being a passive result of the special construction of the joint. Most lift is generated during downstroke. Thrust is generated during middownstroke and the first half of the upstroke. The time course of wing movement is not harmonic but contains much high frequency energy. Aerodynamic studies on very small insects have just begun. Vogel (1965, 1966, 1967) presents the first extensive study. Generally he finds a reduction in the number of controlled parameters of the wingbeat. For example the stroke plane and stroke amplitude co-vary; there is not an adjustment of stroke plane as in bees and large flies. Also the wing pitch is not adjusted with velocity changes, nor does the wing twist along its length; thus angles of attack vary widely. The lack of control of angle of attack is allowable, since at low Reynolds number performance of the airfoil is relatively little dependent upon angle of attack. Vogel finds little variation in wingbeat frequency and feels this parameter is not very significant in control. On the other hand he found that changes in stroke angle (together with changes of amplitude of wingbeat) were associated with changes in lift and thrust. Furthermore, the body angle, at any given stroke amplitude, determines the direction of the output force and therefore the relative values of lift and thrust. The Drosophila points himself in the direction he wants to go! Since Drosophila has a small, relatively short body, changes in body angle do not much change drag associated with the body, and control by this parameter is not as costly as it would be in larger insects. Body angle control is partly associated with the stroke amplitude, but may also be effected

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by using the hindlegs as elevators. As far as the wings themselves are concerned, perhaps all control is achieved by simple variation in power of the wingstroke, with banked turns resulting from power asymmetry. A. WI NGB E AT FREQUENCY A N D S I Z E

It has long been recognized that high wingbeat frequency is associated with smaller size. Greenewalt (1960) has collected data on many species of insects and birds. Most species fall along a more or less straight line on a log-log plot. The large winged butterflies deviate noticeably toward lower frequencies, and the smallest insects fall short of the predicted values. The expectation of such a frequency-length relationship is based

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FIG.2. Wingbeat frequencies in a series of differently sized but similarly shaped (except RomaIea and Agymnatus) acridid grasshoppers. The large-bodied, short-winged Romalea does not fit with the others. Each point is an average of several or many individuals. Since there is species variation in the tendency to maintain long tethered flights, measurements were made a few seconds after starts, before the more saltatory species had slowed down.

on arguments which require geometric similarity. The neatest results are thus obtained when individuals of a single species or small taxonomic group are compared. Figure 2 give measurements from a series of nearly similar acridids. The smallest species has a wingbeat frequency of up to 60 c/sec, but this is not as large as one would expect by extrapolation of a power function based on the other species. Perhaps this species approaches some limiting condition with respect to frequency. In the

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whole range of sizes of acridids the nervous patterns during flight are likely to be similar. Even the very small Dissosteira pictipennis fires single motor units in pulse couplets during energetic flight, but the action potential durations and intrapair intervals are smaller than in large species (Fig. 3). When geometries are really dissimilar, wingbeat frequencies may vary unpredictably. In the very large acridid Romalea microptera the wings are only 25 to 30 mm in length whereas in the smaller Schistocerca gregaria they average about 55 mm in females. The Schistocerca females have a wingbeat frequency of about 17 c/sec (Weis-Fogh, 1956a).

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FIG.3. Recording from the metathoracic subalar muscle of three very different-sized acridids during flight. (a) Schistocercu greguria. (b) Agymnustus ingens. (c) Dissosteira pictipennis. Wing lengths for the species are, respectively, about 55 mm, 30 mm, 20 mm. Both the wingbeat and intrapair firing intervals and the duration of the action potentials vary.

Romalea flaps its wings in a stridulatory display at frequencies around 15 clsec. The Romalea weigh twice as much as Schistocerca and cannot fly at all, but the wing flapping is controlled by a motor output like that in flight (see section on sound production). The “wind-on-head” flightinitiating reflex is absent. Possibly Romalea was already a large species when it lost the ability to fly and began using the wings for display only, and the nervous system did not evolve to compensate for the new wing size. This seems to be true not only of the nervous activity pattern but also of some unneeded anatomical features. The motor axons of the dorsal longitudinal muscles are among the largest in the body (excluding

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the fast fiber of the jumping muscle) as in other acridids, but the muscles themselves are vestigial and capable of only very weak contractions. Another lubber grasshopper, Agymnastus ingens, has short-winged females, but the muscles are normally developed and flight reflexes are operative. A suspended animal begins flapping when wind blows on the head and the wing movements are normal in appearance. The frequency is high for the body size, but exactly that expected for wings of this length. When thrown in the air Agymnastus females can maintain stability, but fly only in a steep descent (about 45" is the best which I have seen). Males of Agymnastus are much smaller than the females, but have wings of about equal length. They can fly quite normally. The female Agymnastus appears to have evolved large size of all the gross body features except the wings, and to have retained the physiological flight control system appropriate to the smaller males. 111. N E U R O G E N IFCL Y E R S I will begin with the description of motor patterns in the more primitive or neurogenic flying insects, or those in which the muscular rhythm is under direct control of the nervous output. Since flight is a fairly highspeed oscillatory phenomenon, most of the flight muscles in these animals are fast or twitch-type muscles. Each nerve impulse is followed after a brief delay by a large muscle action potential, and this is followed by a conspicuous mechanical twitch. Repeated nerve impulses give rise to decreasing amplitude muscle action potentials, but the mechanical action summates. The tetanic fussion frequencies are quite high. Data on locust muscle is presented in Fig. 4 (Neville and Weis-Fogh, 1963).

Two methods have been used to determine the timing of individual muscles in flight. One was to cut the muscle and observe the deficit in wing-movement behavior. Sandeman (1961) has done this for Locusta and Neville (1960) has done so with dragonflies. A second method was direct observation of the timing of the muscle activations by means of electronic recordings of the muscle-action potentials. This method was first extensively applied during flight to Schistocerca by Wilson and Weis-Fogh (1962) and has now been used more widely. Data on flightmuscle function during flight (and other behavior) are summarized in Table I. Muscle identifications are provided in Fig. 5, which is from Sandeman (1961). Some of the muscles attach both to wing and leg parts. Examination of the table will reveal that functional combinations of muscles in different behaviors are different. Figure 6 illustrates the

FIG.4. Muscle action potentials and twitch activity in locust dorsal longitudinal flight muscle. (a) A single muscle potential is followed by a large twitch (solid lines). Widely spaced impulses give separate, non-facilitating twitches. At shorter interval, paired impulses give rise to summated contractions. (b) Very close pairs of nerve impulses (not shown) cause strong antifacilitation of the muscle impulses (but transmission is still one-to-one) and the summated mechanical result is decreased in relation to the size of the second muscle potential. (c) There is practically no tetanic fusion of twitches even at 4Oc/sec at muscle temperature of 34°C. (Redrawn from NevilleIand WeisFogh, 1963).

TABLE I Numbers, names, and functions of flight muscles in grasshoppers and homologous cricket singing muscles Functions Walking*

Mesothorax

Metathorax

81

I 12

Dorsal longitudinal

Depresses wing

-

83 84 85

1 I3

First tergosternal Second tergosternal Pleuro-alar

-

89 90 91 97

1 18 119

127

Anterior tergocoxal First posterior tergocoxal Second posterior tergocoxal First basalar

98

128

Second basalar

99

129

Subalar

103

133

Tergotrochanteral

Elevates wing Elevates wing Folds wing (at end of flight) Elevates wing Elevates wing Elevates wing Depresses and pronates wing Depresses and pronates wing Depresses wing (99 supinates also) Elevates wing

114

120

Muscle name

Flight

Cricket song (mesothorax only)

Slat closes wing 81bt opens wing

-

Promotes coxa Remotes coxa Remotes coxa

Closes wing Closes wing

Promotes coxa

Opens wing

Remotes coxa

Opens wing

Depresses trochanter

Closes wing$

-

~~

Numbers are from Snodgrass (1929). Functions for Locusfa (Sandeman, 1961) and Schistocercu and Melanoplus (Wilson, 1962) agree. Snodgrass' common names are not used since they are misleading as to function. Cricket data are from Bentley and Kutsch (1966). Cricket flight is probably functionally similar to that of grasshoppers. Information on the cricket is incomplete (- indicates no function). * During stridulation in Gomphocerippus rufus the metathoracic wing-to-coxa muscles are used in the same functional relationships as in walking (Elmer, 1967) and raise and lower the hindlegs. t In crickets a functional subdivision of the dorsal longitudinal muscle is apparent. Perhaps the parts are homologous to those of grasshoppers which receive innervation from different ganglia (Neville, 1963) but which nevertheless have similar function in grasshoppers. 2 The main parts of muscle 103, the proper tergotrochanteral muscle, have this function. Another branch which is really the 3rd basalar muscle (?) may differ in timing (Bentley and Kutsch, 1966).

r 0 2: v,

FIG.5. The thoracic Hight muscles of a locust. (a) View from median aspect. (b) Similar, but with longitudinal muscles and some other structures removed. Most of the wing elevators are shown. (c) The most lateral row of flight muscles, the controller depressors. N a m e , numbers, and functions are listed in Table I. (From Sandeman, 1961).

300

D. M . W I L S O N

rearrangements which must occur in the motor patterns when a locust changes from walking to flight.

I

Muscles

FIG. 6. Relationship between the main groups of bifunctional leg-wing muscles in the thorax of grasshoppers. Synergists for leg movements are antagonists for wing movements and vice-versa. (From Wilson, 1962). A . LOCUSTS

1 . Motor pattern The basic flight motor pattern in grasshoppers consists of two reciprocally active small populations of motor units in each winged segment, the segments being slightly out of phase. Only 70 to 80 “fast” units are involved, and not all need to be active in weak flight. The general pattern is summarized in Fig. 7. During weak flight the wingbeat frequency is low and some units are silent. As flight becomes more energetic more units are recruited, the wingbeat interval decreases, and single units may be activated multiply during each half wingbeat cycle (Fig. 8). When the animal is very excited, individual units may fire three or four times per wingbeat. When this is so there is a slight acceleration in the burst. The second and third intervals are shorter than the first (Fig. 9) (recently measured by Waldron (1 967a) in intact flying animals and by Wilson on deafferented preparations). This acceleration is considered important in relation to our current hypothesis regarding the coordination of the locust motor output.

30 1

NERVOUS CONTROL OF INSECT FLIGHT

I )nits per

i

/ I 15

'

I

30

45

1

I

60

~

75 msec

1

1

I

longitudinal depressors

I vertical

depressors

rl

I

6-t) -I

1

Y Y

I

-4-44-

L-

I

elevotoi s -

I I

h l a longitudinol 5

6-8 -

i Elil

1

1

vertical

depressors I

-k&-lj I

depressors

~

!

& elevators -

-

FIG.7. The pattern of activity in the fast motor unils of the locust flight motor system in relation to the wing position time course. The larger spikes represent the several somewhat out-of-phase units in each group and are followed by smaller repeat discharges. Basically there are only two firing times, and elevator and depressor spikes are clustered at these two times, although the sets for the two wing pairs are slightly out-of-phase. Each active unit fires once or twice per cycle during average flight. (FromWilson, 1964a).

2. Deafleeren tat ion If the sensory inputs to the locust thoracic ganglia are cut and wind is blown on the head or electrical stimuli are applied to the nerve cord,

302

D . M . WILSON

0.1 sec

FIG.8. One motor unit during flight at different wingbeat frequencies in a locust (male Schistocerca gregaria) shows increasing burst length with increasing frequency. Decrease of muscle action potential amplitude is due to antifacilitation or response amplitude refractoriness at the neuromuscular junction. In the bursts of three or four spikes a slight acceleration of frequency can be seen.

b

100 micc

FIG.9. (a) Records from flight muscles of a largely deafferented Melanoplus differentialis. The pattern is nearly normal except that frequency is too low owing to the prolonged interval from elevator to depressur firing times. (b) A similar preparation of a disco-ordinated Schistocerca greguria. Elevator spikes are upward and depressors downward in this pushpull recording. Elevator bursts accelerate for the first three or four spikes.

303 the flight motor output can still be produced (Fig. 9), although somewhat slowly (Wilson, 1961). The electrical input may even be a random one (Wilson and Wyman, 1965). Apparently the information for the production of the flight motor command is built into the central nervous system of the locust. It is probably genetically coded, but this is not tested . NERVOUS CONTROL OF INSECT FLIGHT

3. Effects of input Sensory input from the wings plays two sorts of role. One role is a general excitation of the flight control system. The other is specific

FIG. 10. The thoracic ganglia and wing nerves of grasshoppers. The numbering of the nerves is according to Ewer (1953,1954). Nerve IBb contains the wing hinge stretch receptor. IA innervates the tegula and the campaniform sensillae of the wings. Nerves IBa and I1 contain the motor innervation to the flight muscles. Wind sensitive hairs on the head send flight initiating and maintaining impulses via the tegmentary nerves (black) and main nerve cords to the thoracic ganglia. (From Wilson and Gettrup, 1963).

control of individual parts to produce controlling manoeuvres. There are two main sensory wing nerve branches both entering the ganglion together in nerve 1 (Fig. 10). The posterior branch innervates the posterior wing-hinge region and contains the axon of a large proprioceptor (Wilson, 1961; Gettrup, 1962; Pabst and Schwartzkopf, 1962;

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D. M . WILSON

Pabst, 1965), and other sense organs not thought to have important function in flight. The anterior branch innervates the wing itself as well as hair sensillae on the tegula, at least. Input from the tegula can be excitatory for flight, but this input seems not to be important either (Wilson and Gettrup, 1963). Input from campaniform sensillae on the wing veins is involved in the control of angle of attack of the forewings (Gettrup and Wilson, 1964; Gettrup, 1965, 1966). Both the frequency controlling stretch reflex and the lift controlling campaniform sensilla reflex operate slowly relative to the duration of the wingbeat itself. If artificial stimulation is applied to the proximal stump of the cut stretch receptor nerves, the motor output frequency increases gradually over several seconds (Wilson and Gettrup, 1963), and there can be virtually complete phase independence between the input and motor output (Wilson and Wyman, 1965). Thus if the thoracic ganglia receive normally or abnormally patterned input from the nerves, steady wind on the head, or random stimulation via the ventral nerve cord, they can produce a well-coordinated flight motor output. Another well-known reflex, the tarsal contact, inhibition-of-flight reflex (Fraenkel, 1932; Weis-Fogh, 1956b), can be electrically activated by means of paired stimulating wires inserted into the femora. This causes local twitching which will provide proprioceptive inflow and probably also directly stimulates sensory nerve fibers. The stimulation causes a drop in fight intensity. Wingbeat frequency may decline dramatically and flight may even stop. The wingbeat frequency can be controlled by adjusting stimulus parameters. However, the wingbeat does not usually become phase locked to the stimulus timing even if the two frequencies are similar (Waldron, 1967b, has made the only careful analysis of this phenomenon), and long bouts of inhibitory stimulation are needed in order to drive wingbeat frequency to low values. The only input which has been found which can affect wingbeat phase is flashing light. Waldron (1967~)has shown that over narrow ranges of frequency (about 3% variation) the wingbeat will synchronize with a stroboscope. This effect would not seem to be particularly relevant in nature. Nor does it as yet suggest much about the organization of the flight control system. 4. Elementary features of the ganglionic coordination Not all deafferented animals give normally coordinated responses. Such preparations are moribund, and the nervous tissues may lack

N E R V O U S CONTROL OF INSECT F L I G H T

305

oxygen after the more radical operations. When relatively discoordinated preparations are stimulated rhythmically or randomly on the ventral nerve cord, varying amounts of the flight control system may be brought into operation. Some results of this technique have been reported before (Wilson, 1961; Wilson and Wyman, 1965), and a few new ones can also be presented now. In the extreme case one can find that only a single flight motor unit responds to stimulation. This unit may require a large number of facilitating shocks and then fire in a strongly fixed latency relationship with respect to the stimulus, and then exhibit a long, slowly decaying after-discharge when the stimulus ceases (Wilson, 1961). It may fire on every input shock to the cord or divide the input frequency. The division may give rise to somewhat complex patterns due to interplay of stimulus timing and summating excitation or decay of refractoriness (Wilson, 1964b). Often two motor units of a single muscle or several muscles respond. Two units, even in the same muscle, may behave entirely independently except for common frequency trends which may follow their common input (Wilson and Wyman, 1965). Often two related units fire in synchrony more frequently than expected by chance. Units in the same muscle or in the lateral homolog seem to show this tendency most strongly. This effect could be due to weak electrical interaction since the behavior is similar to electrically coupled motorneurons in crayfishes (Evoy, Kennedy, and Wilson, 1967). Another degree of complexity of pattern may be added between two units in the same muscle, namely a tendency toward a fixed latency following (see Fig. 11). This suggests an excitatory synaptic interaction (Wilson, 1966). Thus the latency histogram of Fig. 1 1 seems to be made up of three modes, a near synchronous mode (due to electrical interaction or common following on input?), a sharp second mode at about 7 msec (due to excitatory synaptic interconnection?), and a third broad mode (resulting from no interaction at all or from totally unknown factors). The histogram looks about the same when latency is measured from either unit to the other. In other words the interactions are approximately symmetrical, implying, therefore, a closed, positive feedback loop. Most often elevator motor units respond when the anterior nerve cord is stimulated. The tergosternal muscles generally have the lowest threshold. It is frequently found that if depressor units are activated at all, flight-type coordination appears between all elevators and depressors. Stimulating the posterior nerve cord may give similar effects, but often there is no response to such stimulation or even an inhibition of ongoing activity in the flight system. In either case when the stimulation

D . M. W I L S O N

306

is terminated the preparation often begins a long “flight”, even if it had been previously refractory to normally excitatory inputs.

0

10’

Latency

30

40

of spike 1 ofter I I r n m 1

FIG.11. Muscle potentials in two units of a mesothoracic tergosternal muscle (a wing elevator) of Schistocercu during random stimulation of the anterior nerve cord of a largely d d e r e n t e d preparation which no longer produced flight coordination. The top record is the stimulus monitor. The smallest potentials reflect current spread from the other unit. The histogram plots the latency (cross-interval) from each upper line spike to the next lower line spike. Non-random latency is manifest in too many near-synchronous firings and too many latencies of about 7 msec. A spike in the upper line seems to elicit a later one in the lower line which in turn excites a new one in the upper line. When the lower line does not “answer”, only single spikes occur in the upper. When the two fire in perfect synchrony there is no “answer” either, possibly because of blockage due to refractoriness.

5. Model of locust flight coordination mechanism At present we have information on the behavior of the motor neurons only and we have tried to make models of the flight system based on only one layer of neurons. At first sight a likely neural network to produce the alternating upstroke and downstroke motor activity would seem to be a reciprocal inhibition network between populations of antagonistic neural units. The simplest form of reciprocal inhibition network does not work, however (Wilson, 1964b). In the locust output pattern the cessation of the burst of activity in one set of synergists considerably precedes the beginning of activity in the other. Thus one set stops by itself and is not turned off by inhibition from the other set. Groups of neurons can produce self-terminating bursts if they are mutually exciting (Wilson, 1966a). Positive feedback via synaptic path-

NERVOUS CONTROL O F INSECT FLIGHT

307

ways may result in runaway activity until fatigue or refractoriness overtakes excitation, at which time the burst decelerates to quiescence. If the external (or spontaneous) excitation is continued, the network will recover and burst again. Thus reciprocal inhibition is not needed for one of the basic aspects of the locust pattern, namely the repetitive production of short bursts of impulses in individualunits, with different units in the same population firing approximatelytogether. This suggestion of positive synaptic feedback between synergists was uncovered by digital computer and electronic analog modeling. Since then four lines of evidence have appeared to substantiate it. 1. Bursts of motor unit discharges in the locust show acceleration, as do the analog models. 2. Statistical analysis of partially discoordinated preparations shows a tendency for fixed short latency, but not synchronous, coupling between closely related motor units. 3. J. Kendig (1967) has found excitatory synaptic potentials in branches of flight motor-neurons while antidromically stimulating others. 4. Under special reflex conditions Waldron (1967a) has found that the depressor muscles may continue to burst normally in the apparent absence of elevator activity. It seems necessary to invoke cross-inhibition between antagonistic population in order to produce the phase settings between elevators and depressors in the whole flight pattern, but not to terminate the bursts. If cross-inhibition is present, it will likely affect the bursts also. It could delay the beginning of the next antagonist firings, or it could accelerate them by inhibitory rebound. So far there is no direct evidence for inhibition between motor-neurons even via internuncials. Models employing interneuronalpacemakers will be discussed later in this paper. B . DRAGONFLIES

Compared to locusts, power in dragonfly flight is supplied by relatively fewer and larger muscles. The dorsal longitudinal muscles are vestigial and several of the other depressor muscles are small, probably slow, muscles involved in control but not contributing much power directly (see Neville, 1960). Essentially power is supplied by four muscles per wing, the large second basalar, the first subalar depressor, and the two corresponding tergal muscles which elevate the wing. Each muscle appears to contain several units. Electrical stimulation of the muscle surface produces twitches in several step increases of amplitude as voltage is increased. Based on measurements at low temperatures, Heidermanns (1931) had suggested that there would be fusion of contractions at flight frequencies. However, the tetanus frequency is high at flight temperatures and individual twitches are possible at flight

308

D. M. WILSON

frequency. The electrical record from a single muscle during flight is complex. At low wingbeat frequency and amplitude there are several muscle action potentials per cycle, but these seem to be from the separate units. As frequency increases the dispersion in time of the several units decreases and the recorded potential is the sum of several simultaneously firing units. At the highest frequencies this may look like a unitary potential. I have seen no evidence for multiple firing, but cannot exclude it either. However, it does appear that power is graded mainly by recruitment and that synchronization increases with frequency. The temporal spread at lower frequency should even out the power production (see Fig. 12a). Phase relationships between the wings of dragonflies are quite variable relative to those of locusts. Variations occur both between segments and sides. The large degree of independence of con-

FIG.12. (a) Typical record from one of the large flight muscles of a dragonfly. Each large muscle contains several motor units which seem to fire at most once per wingbeat and arc temporally somewhat dispersed at low wingbeat frequency but synchronize as frequency increases. (b) and (c) Records from a laterally homologous pair of large flight muscles which may be nearly in-phase or may be quite out-of-phase as the animal manoeuvres. Time mark 60 clsec.

trol of the four wings is not surprising in animals having such well developed manoeuvring ability. Recordings from the powerful flight muscles show that they also vary strongly in phase (Fig. 12b, c). The phase shifts are not necessarily associated with changes in frequency. Often the muscles of one wing shift phase together, but at other times the subalar muscle units may shift with respect to the basalars. It appears that the motor neurons of each power muscle form small, fairly tightly coupled populations that fire synchronously only when highly excited, and that the different populations controlling separate muscles are more loosely coupled so that differential excitation can result in conspicuous phase shifts. A thorough study of the motor patterns of all the flight muscles of dragonflies in connection with observations on mechanics and aerodynamics should be very interesting.

N E R V O U S C O N T R O L OF I N S E C T F L I G H T

309

Only the first results of what we may hope will be a complete analysis of flight control in dragonflies have been presented by Tamasige’s laboratory (Hisada, Tamasige, and Suzuki, 1965). They have demonstrated a dorsal light response during flight and partially elucidated the neural responses involved. Interestingly, they suggest that tonic discharge in one connective modifies the phasic output of the pterothoracic ganglia so that the wings on the side away from the light increase their stroke amplitude, and hence lift, thus producing a roll toward the light. They also find that the wings do not synchronize with a flashing strobe lamp. C . LEPIDOPTERANS

Some swift-flying lepidopterans such as hawk moths and skippers have a neuro-motor control closely resembling that of locusts (Kammer, 1967b). Each motor unit fires once or twice per cycle. In other species (Saturniids, monarchs) with relatively large wing surfaces each motor unit fires throughout one half-cycle of wingbeat. Wingbeat frequencies are rather low. As frequency changes, burst length does also, but in opposite fashion compared to locusts (Kammer, 1967a). Since the burst fills the half-cycle its length increases with decreasing frequency. Changes in the number of impulses to each motor unit during one-half wing cycle are also positively correlated with changes in wingbeat amplitude. Both of the latter two features are different than in locusts. The motor system coordinating flight of lepidopterans seems not to be quite so strongly coupled as the one in locusts. During the warmup behavior, for example, related but quite incompatible patterns occur (see section on temperature and flight). Furthermore in an apparently fully developed flight in the lime hawkmoth, Kammer (1967b) found that one pair at antagonistic motor units ran at a frequency slightly different from other parts of the flight system (see Fig. 13). Thus decoupling of a small subset of the whole flight system is possible. This subset behaved normally within itself, but its phase of activity relative to the wings and remainder of the flight system drifted rather irregularly. It seems possible that this pair of antagonistic motor units represents some minimum element of the flight control system. IV. MYOGENIC FLYERS A . MOTOR P A T T E R N S

The apparently more advanced insects, flies, bees, etc., which are generally small in size compared to roaches and grasshoppers, have

D. M. WILSON

310

c

*- m

y

i

I

FIG.13. Motor patterns during warmup and flight behavior in lepidopterans. (a) Saniiu cynrhiu, a large satumiid. Upper line, elevator and lower line, depressor muscle potentials.

Flight is characterized by long alternating bursts of potentials. The flight stops and lowamplitude wing vibrating ensues during which time the two muscles fire in very brief bursts or singlets, but in near synchrony and at about three times the frequency of flight. (b) Mimas tiliue, the lime hawkmoth, in transition from warmup to flight. Upper trace, an elevator muscle; lower trace, the subalar muscle, a depressor. Time mark, lO/sec. In the top records flight antagonists are nearly synchronous. They shift gradually to the typical flight pattern of the bottom record. (c) Relative coordination in Mimas Mae. In the upper trace the upper spike is an elevator unit and the downward one is a dorsal longitudinal muscle unit. In the lower trace another elevator and the subalar muscle are shown. Within either pair the antagonistic relationship is approximately constant, but the two sets drift relative to one another. However, analysis of long records shows some preference for certain phase relationships. (After Kammer, 1966b).

relatively simple motor output patterns to the flight muscles. This is presumably a derived simplicity dependent upon the changed neuromuscular control necessitated by small size, namely the acquisition of myogenicity. [For a discussion of this kind of motor control see Pringle (1957, 1965). Briefly it can be said that in the myogenic flight muscles a nerve impulse activates the muscle for a certain time course but the

NERVOUS CONTROL O F INSECT F L I G H T

31 1

actual contractions are initiated by stretching. There is a delay between the stretch and the onset of contraction. Mechanical click mechanisms or inertial loading conditions give rise to oscillatory contractions during the activation following a single nerve impulse. During a train of input nerve impulses the state of muscle excitation may remain at a high value and the many contractions bear no particular temporal relationship to the arriving impulses.] Since in myogenic muscles there is phase independence between motor impulses and contractions during maintained excitation, there is nothing compelling the impulses in different motor units to be patterned with respect to each other. Early reports suggested both unpatterned (Roeder, 1951) and patterned (Pringle, 1949) conditions. As will be shown shortly both seem to be true in different cases; however, even in the patterned cases there would seem to be no effect of the pattern on the behavior of the muscles as long as the average nervous frequency is high. Wilson and Wyman (1963) make an argument that in flies it is frequency of nerve impulses, averaged over several cycles, and frequency alone which can convey information to the flight muscles. This system approaches the engineer’s former idea about nervous systems, namely that they are systems operating with a pulse frequency modulated code. Burton (1964) has added to this suggestion by showing that in the beetle Oryctes yawing is effected by increasing the frequency of nerve impulses to the fibrillar muscles of one side. To the extent that sequential patterns of activity in several motor units in flies do occur, most often they are expressed only by units in the same muscle or related muscle grocp (Wyman, 1966). In the calliphorids he studied the two dorsal longitudinal muscles and the sets of vertical elevator muscles all showed independent firing times, but units within any one muscle or elevator group showed repeated sequences like those figured by Pringle (1949). The sequences often did not repeat indefinitely, however, but after a time altered abruptly to form a new pattern which in turn might be stable for some time (see Fig. 14). These multistable patterns in calliphorids seem to be based on some sort of exclusion principle. The several motorneurons involved in the pattern seem to fire as separately as possible and are very rarely synchronous even when there is much noisy jitter. This is surprising since they are synergisticin motor action. We will examine later what significance these out-of-phase patterns may have for understanding the ganglionic coordination. In other flies other patterns have been found. Wyman, Kiester, Waldron, and Nachtigall (personal communications) have examined numerous species. Some show more strongly out-of-phase patterns than the calliphorids already reported. Others show patterns in which there is a tendency for

3 12

D . M. WILSON

b

"

"

"

"

'

FIG.14. Multistable phase patterns in the indirect flight muscles of a calliphorid fly. Units 1, 2, 3, and 4 are in the same muscle. Unit 5 is in a different muscle. The arrows indicate the abrupt change in phase position of unit 2 relative to unit one intervals. The histograms plot the total occurrences of given phase relationships in a long sample. The single-moded histogram (left) is of the phases of unit 1 spikes in unit 3 intervals. The bimodal one is for unit 2 in unit 1, the quadrimodal one is for 2 in 4, and the flat or random one is for unit 1 in unit 5 intervals. Phase of a spike is measured as the fraction of another unit's interval which has elapsed when the spike occurs. (Redrawn from Wyman, 1966).

NERVOUS CONTROL OF INSECT FLIGHT

313

synchrony between synergistic units (e.g. Eristalis). R. Kiester (unpublished) has demonstrated relatively coordinated activity between motor units in Muscina (Fig. 15). This seems to represent the weakest form of motor unit phase coupling. Two units hold a stable, but jittery, phase relationship for a while, and then begin to drift only to stabilize again in the same phase at a latter time. Nachtigall and Wilson (1967) have recently investigated the roles of the different muscles in flight control. The myogenic muscles vary in

Phw

FIG.15. Firing pattern between two relatively coordinated motor units in a fly, Muscina. Each point stands for the phase of one unit's single spike in the interval between two firings of the other. The two units are independent at first, the linear drift in phase resulting from a small frequency difference. They then stabilize at one phasic pattern for many cycles after which phase drift begins again, this time the frequency difference is opposite. The phase histogram shows a strong preference for certain patterns, but cannot indicate the trends visible in the temporal record. (Data presented by permission of Ross Kiester).

impulse frequency as flight speed and lift change, but the two sides do not vary differentially, even during turns. The pleurosternal muscle is more highly activated at high wingbeat frequencies, as expected. The abductor and adductor muscles of the two sides are differentially active during strong turns, and they may be agents in controlling turns by controlling degree to which the wing is held forward or back relative

3 14

D . M. W I L S O N

to the body. These authors conclude that the myogenic system of muscles varies only in total power and that special muscles regulate the distribution of power to the two wings. Little is known about motor control of flight in bees, beetles, and bugs. Mention has been made already of Burton’s (1964) study on the beetle Orycfes.Barber and Pringle (1966) had limited success at getting belostomatid bugs to fly in the laboratory. Their results on motor control suggest that in these bugs phasing of the motor units is nearly random. Similar results have been found in bees except that motor units in antagonistic muscles may show trends many cycles long of negatively correlated activity (Mulloney, unpublished). B . SIGNIFICANCE OF THE MULTIPHASIC A N D MULTISTABLE PATTERNS

Wilson (1965) and Wyman (1966) have suggested that the significance of the complex motor patterns in calliphorids and other flies may be to smooth the production of muscular power in single muscles by activating the several units in a staggered sequence. The active state of each unit, and therefore the power of the oscillatory contraction, wanes over a time span which might cause considerable variation in power in a unit excited rhythmically at only a few times per second. Random sequencing of the units would tend to smooth the summed power of all units in a muscle. The special antagonistic sequencing would be even better in this regard. On the other hand, some flies, such as Eristalis, show synchrony between intramuscular units and these flies do not seem to be poorer at flight. It is possible that the several observed pattern types in flies have no relationship to the flight behavior and are in that respect irrelevant. Be this as it may, the patterns are relevant to a knowledge of how the ganglion works, and in fact may represent natural experiments on ganglionic function. The motor output of the ganglion gives a window into the ganglion just as does the sensory inflow. In some respects the former is a clearer window, for all commands which issue from the ganglion have to be a function of the ganglion whereas much of the information content of the sensory inflow may be unused. I have suggested previously (Wilson, 1964a) that in the locust stretch reflex which controls wingbeat frequency, much information is filtered or ignored, not simply lost in noise, in the ganglionic transfer, and that one learns most about the functioning of the reflex by studying its effects on output, not by cataloging the information content of the input. In other words the efficient approach to the study of ganglionic function is to work backwards from output to input, and not vice-versa. The same may be

NERVOUS CONTROL OF INSECT FLIGHT

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true with regard to the motor output in flies. Working forward in the causal chain one may find only that much of the pattern has no importance. Working backwards this cannot be so. Each aspect of the pattern must have a cause, and finding these causes is equivalent to unravelling the problems of structure and function of the ganglion. C . H Y P O T H E S I S O N C O O R D I N A T I O N I N FLIES

Since the myogenic animals are derived from neurogenic ones with accompanying loss of the need for phasic motor unit coordination, it is reasonable that they may represent some simplification of the general insect flight control system without the introduction of new parameters. The main features of the fly motor outputs can be summarized as follows. (a) All of the motor units to the indirect (main power) muscles fire at about the same frequency and undergo simultaneous frequency trends. (b) Units in the same muscle have near identical frequencies. (c) In some species units in same muscle tend to fire synchronously; in other species units in the same muscle tend to be locked out-of-phase except at the start of flight when they are synchronous; in still others phasing is irregular. In species in which phase preferences are exhibited the preferred phase can be independent of the overall frequency (Wyman, 1966). A system which can produce these features is the following. Mutual interactions between the motor neurons (or their drivers) of either synchronizing or antiphasing character can produce the common frequency setting. For example, mutual inhibition between several active neurons may set them into firing in a regularly repeated sequence with the whole cycle time divided more or less equally between the several members (Wilson, 1966a). The phase settings can be independent of frequency over wide ranges because each neuron fires on the gradual recovery slope from inhibition rather than on the sudden rise of an excitatory synaptic input (see Fig. 16). Mutual inhibition can also give rise to synchronous firing when the units are evenly matched and the several fall by chance into the same phase once. Firing in synchrony is not excluded because there is a small delay in transmitting the inhibition between neighbors. Each unit is then inhibited synchronously and all fire together again on recovery. However, if there is much jitter in the system it will not remain synchronized since the out-of-phase condition is more stable. If there is any additional synchronizing force the system can become a stable inphase one. Either electrotonic junctions or field affects could play such a role. Tight junctions are now being found in insect neuropile (Osborn, 1966) and bundles of close-packed naked neuronal processes are well

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,

FIG.16. Results of mutual inhibition or mutual excitation in a two-membered artificial neuron network under conditions in which there is one-to-one alternation of firing between the pair. The upper set of drawings (a) illustrates behavior of the reciprocally inhibiting pair. Each unit fires after recovery of threshold and during asymptotic recovery from an inhibitory synaptic potential. If the network is symmetrical, it is possible for the pair to fire synchronously (not illustrated), but noise will likely result in some asynchrony and then the antagonism establishes a stable alternating pattern. Increasing the state of excitation of the network decreases the time taken to recover from inhibition, but the phase relationships remain the same. All intervals are decreased in proportion because of the symmetrical relationship and the fact that firing occurs on the gradual recovery slope. In the cross-excitation case (b), although the network is constructed approximately symmetrically one unit tends to drive the other and not vice-versa. Firing of the second occurs more or less on the peak of an excitatory synaptic potential. The first unit is refractory when it receives an input and does not fire until it recovers. Thus firing time is not similarly determined in the two units. (If the first were not refractory, the pair would begin a positive feedback runaway until fatigue turned them off. During the runaway burst symmetrical relationships could lead to phase constancy.) If the state of excitation of the pair is increased, the latency from first unit to second unit spikes (arrow) is not much changed since the EPSP has more or Iess constant delay. Both units fire higher on the threshold recovery curve. The near constant delay between units one and two gives rise to a changing phase with changing frequency.

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known (Trujillo-Cenbz, 1959; Smith and Treherne, 1963). The latter can be expected to give rise to significant synchronizing field effects. Mutual excitatory synaptic interconnections do not seem likely to produce frequency independent "out-of-phaseness ", nor exact synchrony (see Fig. 16). The " out-of-phaseness" needs gradually rising synaptic excitation which could be produced by large numbers of interacting neurons, and then the exact phasing between certain individuals would be lost, or it could be produced by unusually long duration synaptic potentials such as we have never seen in the thoracic neuropile. An even more remote possibility is that electronic interaction between two neurons can (unusually) produce antiphase locking when the transmission time-constant is such that the high frequency spike is mostly lost but the after-hyperpolarization is transmitted. The latter can have an inhibitory effect (Lewis, 1966). This effect is stable over only very narrow ranges of conditions, however. On the basis of present knowledge the most likely model to explain the flight motor coordination in flies is one in which highly related motor neurons or their pacers are connected in a mutual inhibition network and in which some electrical or synaptic synchronization also occurs. Then, in different species one or the other factor may dominate, or the coupling may be so loose that short-term patterning is lost, as it always is between distant motor units anyway. This model seems to fit available evidence best, but it is not tested by direct electrophysiologicalobservations within the ganglion. V . G E N E R A MO L D E LFORFLIGHT CONTROL We may suppose that the fly motor patterns represent a simplified or degenerate system evolved from a more primitive but complexly patterned one by the loss of very special phasic coupling between motor neurons. Since in myogenic insects the motor nerve impulses no longer set the phase of the muscle contractions, phasic patterns may be fortuitous products of an evolutionary background. Still they may indicate properties of the more primitive system from which they are derived. The several kinds of fly motor patterns which have been seen, seem to suggest either inhibitory coupling between motor neurons controlling the same muscle or some synchronizing coupling, or both. The synchronization might be due to electronic junctions or field effects only. A combination of mutual inhibition and synchronizing coupling can give rise to all the patterns seen so far between motor neurons in myogenic insects.

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Locusts and other neurogenic insects require some kind of positive interconnections between synergistic motor units, or common interneuronal excitatory drive. As indicated earlier, there is evidence for excitatory interactions between synergistic motorneurons in locusts. Recently D. Bentley (personal communication) has found evidence for interneuronal pacemakers in the flight and singing system in crickets, and it seems likely that these will play a significant role in locusts too. In addition to coupling between synergists there needs to be some inhibitory relationship between antagonistic populations. Considering the great anatomical similarity between lodust and fly ganglia (at the level of fiber tract topology) it seems reasonable to try to make a common model for their flight control systems. This would consist in the most general form of a system of mutual inhibition between motor neurons (or their immediate antecedents) with superimposed excitatory coupling between some groups at least. In flies the excitatory coupling might be weak and nonspecific. Whether there is a maskedinhibitory network linking synergistic motor neurons in locusts is entirely unknown, but such a network extending to all motor neurons in the flight system could give rise to the alternation between antagonistic populations while excitatory effects dominated within synergistic pools. Whatever kind of neuron network controls insect flight it must in some cases be a loosely coupled one. Relative, or drifting, coordination in both intact flies and butterflies show this. Furthermore, as discussed in the next sections, the same motor neurons are used in other behaviors and are then coordinated in different patterns. V I . RELATEDBEHAVIOR A. TEMPERATURE A N D FLIGHT

Body temperature influences nervous and muscular activities during flight, and muscular activity affects body temperature. This reciprocity has interesting homeostatic effects. The most commonly measured flight parameter, wingbeat frequency, shows different temperature dependence in different species. In Periplaneta frequency is proportional to environmental temperature between 17°C and 30°C (Richards, 1963). Numerous flies also show a strong dependence of wingbeat frequency on ambient temperature over a wide range (Sotavalta, 1963). In several bees wingbeat frequency is independent of ambient temperature over the normal behavioral range (Sotavalta, 1963). Since the muscular activity associated with flight itself produces heat in the thorax, the measurement of

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ambient temperatures does not indicate the temperature functions of the flight machinery itself. As a first approximation one could guess that small insects might be more dependent upon environmental temperature than large ones since the latter will lose heat less rapidly relative to the volume of heat producing muscle (Sotavalta, 1963). However, there seems to be no such clear generality. Rather variations in temperature independence seem to be correlated with behavioral differences (Sotavalta, 1954). 1. Locusts

A good example to discuss first is that of the locust Schistocercu. Between ambient temperatures of 25" and 35°C flight speed varies insignificantly (Weis-Fogh, 1952), and, after the first 2 min of flight, wingbeat frequency is also nearly independent of temperature (WeisFogh, 1956a). In this range thoracic temperature is about 6°C greater than environmental temperature during steady flight. This temperature excess is independent of position in the temperature range (Church, 1960). These relationships can be partly understood by considering the properties of the flight muscles (Neville and Weis-Fogh, 1963). The twitch duration is highly dependent on temperature. At 25°C it is about 50 msec; at 40"C, about 25 msec. Locusts can begin flight at 25" ambient temperature and within a few minutes the thoracic temperature rises to about 31". Since the wingbeat period is about 50 to 60 msec, at the lower temperatures each muscle twitch lasts nearly the whole cycle and antagonists are active at the same time. The overlapping of antagonistic twitches results in wastage of energy as far as aerodynamic output is concerned, but it results in a rapid rise of thoracic temperature until the twitch duration has decreased. This provides a sort of passive regulatory mechanism up to thoracic temperatures at which there is little overlap of antagonistic twitches (about 30°C). Over the normal flight temperature range, the nervous system must operate in a nearly constant manner. At first I believed this might be due to a reflex effect. The stretch receptors which are significantly involved in frequency control measure physical aspects of the wingbeat such as amplitude and frequency. It is easy to imagine a feedback arrangement of this reflex which would compensate for temperature. However, eliminating the reflex does not change the temperature function of the flight control system. Ten males of Schistocercu were flown at several ambient temperatures and wingbeat frequency measured after t min in each case. Between 25" and 35" the frequency increased only from 21 c/sec to 23 c/sec. The animals were then operated upon to remove the

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stretch receptors and, as expected, the frequencies for all decreased, but the slope of frequency over temperature was not changed sigdicantly. Finally, several groups of animals were more highly deafferented by cutting away the dorsal half of the thorax. Each group was tested at a different temperature since survival time was too short to carry individuals through a sequence of tests. Between 25" and 35" these preparations produced flight commands for frequencies averaging 11.5 and 13 c/sec, respectively. Apparently the relative temperature independence resides within the CNS.

s

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FIG. 17. Intrapair firing intervals of Schislocercu flight muscles vary with temperature, even though wingbeat interval is relatively temperature independent. (a) and (b) Similar wingbeat interval at two temperatures. (c) and (d) Two wingbeat intervals at the same temperature. The intrapair interval is probably strongly influenced by relative refractoriness.

Some of the nervous parameters of the fight controlling motor output are more strongly dependent on temperature than is the flight rhythm itself. The duration of the muscle action potential and its recovery time course from refractoriness after a test impulse are shorter at higher temperatures (Neville and Weis-Fogh, 1963). Also the interval between multiple firing of single units in flight decreases with increased temperature while it is nearly independent of all other parameters (Wilson, 1965) (see Fig. 17). This factor should also result in some automatic temperature regulation since more widely spaced muscle impulses

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at the low temperatures will result in longer contraction duration and thus greater overlap between antagonists.

2. Bees Several hymenopterans including Apis can have a thoracic temperature excess of up to 15°C and the degree of heating is related to wingbeat frequency (Sotavalta, 1954). Esch (1964) has shown that thoracic heating may occur without visible wing movements, but even then action potentials can be recorded in the flight muscles. Under these conditions heating is greater than during flight, either because less mechanical work is done or because cooling by transpiration is less.

3. Lepidoptera Thoracic heating during flight and during a warmup period prior to fight has been observed in numerous lepidoptera (for review see Sotavalta, 1954). Interest in these animals has recently revived. In some species, unlike the locusts, there is not a constant temperature excess, but instead during flight the thoracic temperature is regulated to a nearly constant value (Adams and Heath, 1964). Kammer (1967b) has undertaken a detailed examination of the neuromuscular control mechanisms in flight and warmup in Lepidoptera. During normal flight the control patterns to the flight muscles resemble generally those of locusts. There are bursts of activity in all units in one set of synergistic muscles alternating with bursts in the antagonists. During warmup behavior, which may consist of small or medium amplitude wing vibrations at either flight frequencies or much higher frequencies in different species, the nervous control patterns are highly variable (for details see earlier section and Fig. 13). In Samia Cynthia elevator and depressor muscles may fire at the same time rather than alternating, and the fundamental frequency is much higher than during flight. In CeZerio Zineata, depressors fire synchronously in flight but alternate during warmup. Elevators and depressors may shift gradually in phase from synchrony to antiphase during the transition from warmup to flight in Mimas tiliae. Also in Mimas some normally alternating sets of antagonists decouple from other sets or even oppose them. In the skipper Hylephila the dorsal longitudinal muscles and the subalar are alternately active, while some elevators fire with each of the depressor groups during warmup. In all these examples the mechanical result is that activity in antagonistic muscles is overlapping so that less aerodynamic work is possible and more heat is produced. The pattern of muscular activation is not itself simply a function of

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temperature, although temperature effects on the ganglion may have some influence on pattern in addition to influencing frequency of wing movement. At low temperature a moth may ready itself by warmup behavior, but extreme stimulation can cause normally patterned, if weak, flight, so the warmup pattern must represent a really different mode of activity of the flight control machinery. B . S OUND PRODUCTION USIN G THE WINGS

Many insects communicate with sounds (see Haskell, 1961, 1964). In some cases the sounds are produced by the thoracic wing-moving mechanisms. The simplest example is that of the flight tone itself. As the wings and thorax vibrate energy is imparted to the air, and in the case of smaller high-frequency insects the airborne sound may be used for communication. Mosquito sounds provide a well-known example (Kahn and Offenhauser, 1949a). The fundamental frequency in the flight sound is the wingbeat frequency itself, but the energy content of overtones may be large. Sotavalta (1947) listened to the sounds in order to estimate wingbeat frequency. The overtone energy content varies under different flight conditions in flies, at least. These variations are under active control and therefore could provide the basis for a repertoire of different communicatory signals. The utility to insects of signals of different frequency content is questionable, however, given OUT current knowledge of the function of insect hearing organs.

I. Neurogenic insects In larger winged insects the low-frequency wingbeat vibration is hardly audible but stridulatory mechanisms involving the wings are common, at least in orthopterans. The wings may simply hit the hindlegs or each other during flight. In Romalea microptera, in which the wings are much reduced relative to the adult body size, the wings may be elevated and flapped weakly, rubbing over each other and producing a dry grass rustling sound. The flapping frequency is about 10-15 c/sec, the frequency expected if this large species had normally developed wings and could fly, and not the frequency expected for wings of such small length. The special flight muscles are so reduced as to be certainly ineffective (the dorsal longitudinals, tergosternals, and first basalars), while the wing-to-leg muscles are somewhat better developed (the 2nd basalars and subalars, tergo-coxalb and tergo-trochanterals). The latter muscles are activated in the flight pattern characteristic of acridids during the flapping. In Romalea the wings serve only as display and

323 communicatory organs, but the neuromuscular control mechanism is clearly only a slightly reduced flight control system. In crickets the sound-producing mechanism is more specialized but the animals retain the ability to fly. Sound is produced by moving one elytrum over the other, special stridulatory structures producing a tone burst or pulse during each wing cycle. The muscular control of these movements has been studied recently by Ewing and Hoyle (1965) and Bentley and Kutsch (1966). The muscles are activated in approximately N E R V O U S CONTROL O F INSECT FLIGHT

L

FIG.18. Motor unit activity and sound patterns in chirping crickets. (a) A tergocoxal muscle (90) and the calling song. (b) the subalar muscle (99) and the calling song. (c) The tergocoxal (90) and subalar (99) muscles of the right side during song. These alternate as during flight. (Courtesy of Wolfram Kutsch).

the same pattern as that of flying acridids (Fig. 18). Each muscle unit receives one motor impulse or a very short burst per wing cycle, arid there is alternation between the major sets of muscles. The energy of the tone pulse is influenced by the number of units active and the number of impulses in the burst. The pulses are repeated at approximately the the same frequency as the wingbeat during flight. The main differences from flight are that the wings hold quite a different posture, perhaps due

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to the activity of accessory muscles, and in some song types the movements are turned abruptly on and off with only 3 to 4 pulses in a single bout. Huber (1963) has found that electrical stimulation of certain sites in the neuropile of the second thoracic ganglion can elicit elytral stridulation independent of the stimulus frequency. This kind of stimulation produced continuous stridulation but did not call forth the specific temporally patterned song rhythms, whereas stimulation in the brain could elicit specific songs. As in locusts it appears that the wing movement control system is inherent in the thorax, but the system may be turned on and off by commands from the brain. Once the thoracic

FIG.19. (a) Activity of the hindwing subalar muscle (129) during one syllable of the courtship song of the grasshopper Gomphocerippiis rufus. (b) Activity in both the right and left subalar muscles (129). Activity begins synchronously but gradually shifts to an exactly alternating pattern, although these two muscles are always synchronous during flight. Time mark, 50 c/sec. (Courtesy of Norbert Elsner).

mechanisms themselves are understood their control by higher centers might be better studied in the crickets. Other sound production methods are found in orthopterans which do not involve the flight coordination itself, but which are nevertheless of interest here. In the very small acridid Gomphocerippus rufus the legs are alternately rubbed across the elytra. Elsner (1967) has found that this movement involves those flight muscles which attach to both wings and

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a

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FIG.20. (a) Activity in the right tergocoxal muscle (119) and right subalar muscle (129) of the hindwing of Gomphocerippus during flight. (b) The same muscles during a syllable of song. Time mark, 50 c/sec. These muscles are antagonists for flight and synergists for leg movement (see Fig. 6). Time mark, 50 c/sec. (Courtesy of Norbert Elsner).

legs (Fig. 19). The leg movements take place at the same frequency as the flight wingbeat frequency (Fig. 20). However, the muscles are coordinated according to the synergistic-antagonistic relationships found in walking in both acridids (Wilson, 1962) and gryllids (see Fig. 21). In Gomphocerippus, flight and stridulation use the same muscles at the

a

2oornrec

FIG.21. Bifunctional muscles in crickets. (a) The subalar muscles (99) of right and left side during song. (b) The same muscles during walking. (Courtesy of Wolfram Kutsch).

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same frequency, but there is reversed synergist-antagonist relationship between the muscle sets on one side (see Fig. 6) and reversed synergism between the two sides. 2. Myogenic insects Communicatory sounds produced by the wings of insects with myogenic flight rhythm have received considerable attention, but are still not well understood. In the simpler cases perhaps the unmodified flight tone is involved. However, bees and flies produce a variety of sounds which are different from the usual flight tone (Esch and Wilson, 1967). In the past these special sounds have been attributed to other causes, such as spiracular vibration during expiration, but the wing mechanism is probably involved in all those with which I am familiar. (For a review of facts and theories see Sotavalta, 1947.)

H 20 msec

FIG.22. Wingbeat associated courtship sounds in two sympatic species of Drosophilu. Upper D. pseudo-obscuru. Lower D . persiniilis. (From Waldron, 1964).

The flight tone attracts male mosquitos to females (Kahn and Offenhauser, 1949a). In Drosophila males court females with a wing-vibrating display which could be communicated either (or both) visually or auditorily, in the latter case probably by substrate borne vibration. Waldron (1964) has shown that the courtship sounds, which consist of short repetitive tone bursts are not identical in frequency to the flight tone. In fact in two sympatic species which have identical flight sounds the courtship sounds are different from each other, as well as from the flight sound, thus accomplishing some specific reproductive isolation by

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behavioral means between the two hybridizable species (see Fig. 22). D . persimilis has a courtship tone of about 480 c/sec, D . pseudo-obscura one of about 290c/sec, and both have a flight frequency of about 200 c/sec. The difference in mechanism of the several tones is not known. They are not harmonically related to each other. A good guess is that the frequency of vibration of the thorax is controllable over a wide range by special settings of accessory muscles or wing and wing-hinge sclerite postures. Beekeepers have long known that bees make a variety of sounds associated with special behaviors or individuals. Recently Esch (1961)

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FIG.23. Sounds produced by bees during the waggle dance. The lower record shows the wingbeat sound (bracketed) superimposed on the waggle movement at the abdomen. They are not related in phase. (Redrawn from Esch, 1961).

and Wenner (1962a) have shown that the sounds produced during the waggle dance can convey information about the distance to a food source. The insect emits short sound pulses of 5 to 7 cycles of about 250 c/sec sound. The pulses do not correspond in phase to the body waggles (see Fig. 23). The wings are neither completely folded nor fully extended while the sound is produced. The pulses resemble those of the courting Drosophila. It would seem likely that each pulse was due to a single synchronous firing of many of the flight muscles, but Esch (1964) provides evidence that this is not so. He has recorded from some flight muscles during the pulse sounds and finds no special phase relationships between the muscle action potentials and the sounds. At present

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one can conclude that the flight muscles are kept in an active state and that an accessory muscle, perhaps a twitch type one, modulates the vibrations. Wenner (1962b, 1964) has recorded other bee sounds such as piping and quacking, and has analysed these with the Kay sonagraph. He concludes that these other sounds are also produced by the wingvibrating mechanism, but that the harmonic content is so different that in some cases nearly all the energy appears in higher harmonics.

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FIG.24. Oscillograms of thoracic vibrations of a calliphorid fly during flight. A diversity of waveforms from that associated with quiet flight to that of the dissonant buzz are shown. In the lowest record the wings were cut quite short.

Some insight as to the mechanisms of communicatory sound production in bees may be provided by observations of my own on flies. Everyone should be familiar with the dissonant buzz or squeal made by flies which are restrained. Sotavalta (1947) and others call this the “whining tone”. During studies on motor mechanisms in flight, I happened to record the thoracic vibrations during Such sounds and subsequently collected records of as many sorts of sounds as the flies would produce. Oscillograms of some of these are shown in Fig. 24. These are rather similar to the wingbeat sounds which function in courtship in mosquitos (Kahn and Offenhauser, 1949b; Offenhauser and Kahn, 1949). The

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upper records are associated with more or less normal flight. Spectral analysis shows that most of the energy is in the first, second or third harmonic. The lower records clearly contain higher frequencies as well. They occur especially when the wings are not fully unfolded or when the wings are cut short. In the bottom record the wings were cut off and the fundamental frequency is increased, but in addition a much higher frequency is conspicuous. This record is characteristic of those taken during the squeal or noisy buzz of restrained flies. It does not sound like a nice tone, even one with a rich harmonic content. Sonagrams made with the Kay sonagraph often show the whole vibration analysed into a series of harmonics of the wingbeat frequency. However, one can show that this does not imply a single vibratory mechanism, for the same result can be obtained from a record produced by one oscillator which is periodically reset by a lower frequency one (Esch and Wilson, 1967). A mechanical model could be a tuning fork which is struck by a metronome. The results of a spectral analysis will depend upon the properties of the analyser. If the sampling time is long relative to the lower frequency (or fundamental) driving mechanism, then the whole record will be analysed into harmonics of that frequency. If the frequencies of the two oscillators are irrationally related, then the series of energy containing harmonics will be infinite. If the frequencies differ by, say, nearly exactly 5 to 1 then most of the energy will appear in the fifth harmonic of the fundamental. Such a two oscillator system seems to be working in the fly. A magnified bit of record is shown in Fig. 25. The wingbeat frequency can change without affecting the higher frequency vibration, but the latter is reset twice per wingbeat. The higher frequency vibration seems to be independent of temperature, whereas the wingbeat frequency itself has a strong temperature dependence. Since the higher frequency vibration appears when the wings are folded or cut off, the following hypothesis seems warranted. Reducing the loading on the wing mechanisms allows increased acceleration of the thoracic parts by the myogenic muscles. This might in a simple way allow the system to hit stops at each end of its possible excursion range and thus send some parts into a resonant vibration as in a tymbal. Alternatively the relatively unlimited acceleration of the thorax past its click point may result in a slackening of the active muscles. The antagonistic, stretched muscle does not become actively contractile for a few milliseconds, so there is a brief period in which muscular elasticity and damping forces may fall to insignificant values, leaving the skeletal parts to vibrate at their independent resonant frequency. This is consistent with the temperature independent nature of the higher frequency oscillation.

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Whether the above analysis is exactly correct or not needs further test. It is reasonably certain, however, that some double oscillatory mechanism is operative in producing the high-frequency sounds. This is very likely so in bees also. If so, the different sounds produced by bees may all be due to wing vibrating, but under control of accessory wing muscles which adjust the wing posture and therefore strongly influence the inertial and aerodynamic loading of the wings. Relatively pure highfrequency tones could be obtained by an active control which adjusted the wingbeat frequency to some sub-multiple of the higher frequency, but what information the bee could use in making this adjustment is unclear. Leston et ul. (1965) have recorded sounds during pre-flight warmup

10 msec FIG.25. Amplified record like that of the lowest trace of Fig. 24. The wingbeat period increases between upper and lower record, but the smaller oscillation which gives the high frequency energy has not changed.

in a beetle. The wings were folded and moved not at all, yet thoracic vibrations were produced by the flight muscles. They conclude that the thorax was clicked by the myogenic muscles in a fashion similar to the tymbal action in cicadas (Pringle, 1954), except that both directions of click were muscularly induced. They suggest also that the wings were disarticulated in their folded position so as not to be coupled to the thoracic movements. Each direction of click produced a single pulse of sound. In the buzzing or “whining” tone of flies the same explanation probably holds with the additional factor that each clicking movement

331 results in a damped oscillation which lasts until the opposing motion begins. N E R V O U S C O N T R O L OF I N S E C T F L I G H T

VII. G E N E R AD L I SC US S ION In the models discussed earlier it was assumed that interactions between nervous elements at a single level could explain the output patterns of flying locusts and flies. It was further assumed that these elements were the motorneurons (or perhaps a set of driver neurons nearly equal in number) with the added possibility of internuncials operating between these elements (comparable to some inhibitory internuncials in vertebrates). There is no strong argument favoring or disproving this notion of one-layeredness. An alternative view that the organization consists of a hierarchical many-layering of pacemakers and followers with feedbacks from lower levels to higher ones is also possible (Hoyle, 1964; Ewing and Hoyle, 1965). This view is especially appealing when one considers the fact that the same set of motor neurons can be used in two or more behaviors, and have strikingly different patterns of activity in each case. It is easy to suggest that different pacemaking centers establish the patterns and the motorneurons act only as final common paths for commands originating from higher decision making centers. However, in part this begs the question of how the pattern originates. Until there is demanding evidence in favor of many-layered models we should continue to develop simpler ones as well. Even in several layered systems these models may also be useful at each level. Consider the examples of leg movement and wing movement in grasshoppers or flight and warmup behavior in lepidopterans. A set of motorneurons is involved in each and it is divided into different subsets according to the behavior involved. It is hard to believe even in figurative terms that the motorneuron sets are connected by two sets of wiring which can be switched to produce the two modes of behavior. On the other hand, it is conceivable that each set has more than one interaction mode (remember the multistable phase patterns in flies) and that the source and kind of input, temperature, or other factors might bias the system to a particular output mode. Even more plausible is the idea that the whole set of neurons engaging in flight is not the same as the whole set involved in walking, and that the common or overlap set behaves differently depending upon which ensemble it belongs to at the moment. These last notions are consistent with our accumulating knowledge of command fiber functions in crayfishes (Wiersma and Ikeda, 1964; Evoy and Kennedy, 1967). The latter are long interneurons which

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control activity in groups of lower center neurons. Command fibers can be individually identified as to which specific sets of motorneurons they activate or inhibit. They may produce fairly complexly coordinated outputs, but the detailed pattern of their own discharge is apparently irrelevant. That is, the temporal pattern of output activity depends on what set of neurons they drive, and not on a temporally coded message in their own activity. Insects likely have comparable command fibers and these might bias the activity of sets of thoracic neurons toward the different kinds of behavior outlined above. In the difference between flying and walking in grasshoppers one could imagine fundamentally different mechanisms operating. For example one activity, flight, might be primarily driven by a genetically built-in motor score which is coded in the structure of the central nervous system, while the other, walking, might be dominated by proprioceptive reflexes which incorporate relatively more current environmental information into the short-term aspects of the motor pattern. Both, of course, could be dominantly instinctive, or genetically controlled behaviors, but they could differ in the way suggested by Hoyle (1964). Namely, flight might be under the control of a motor “tape” while walking is patterned by a sensory “tape” system which adjusts motor output until the sensory feedback matches a predetermined template. The motor output which achieves this feedback-template match may not always be the same. This latter system could adapt to varying external (to the CNS) conditions and thus show plasticity. Until recently I had sought in vain to demonstrate plasticity in the flight system, but modifications in the walking patterns are, of course, well known (Wilson, 1966b). Now plasticity in the locust flight control system is also demonstrated (as yet unpublished personal observations). Therefore this best known case of a central motor score command system also shows features of Hoyle’s sensory tape systems. Even though some of the same muscles are used in walking and flight the proprioceptive feedbacks are almost exclusively different and their effects are different also. The flight reflexes are all slowly acting relative to the wingbeat interval as far as is known at present. The walking reflexes on the other hand are very fast and can affect the motorneurons within a single legstep cycle even at high speeds (Wilson, 1966~).These differences in reflex speed may account for much of the apparent difference in modifiability of the motor outputs. Difficult to explain by the possible motor tape-sensory template differentiation are the differences in pattern in flying grasshoppers and stridulating ones. Here the motor patterns, while differing importantly

333 in details of muscle synergisms, are essentially the same in type of pattern and frequency. Both patterns seem equally likely to be built-in motor scores, and it seems that only a switching mechanism need be sought. In the differences between flight and warmup in butterflies equally great variations in motor pattern occur, but these may be continuously graded between extreme types and one certainly ought to postulate a single modifiable mechanism in this case. Less dramatic than changes from one behavior to another are control modulations of the flight itself. Little is known yet about steering or other control mechanisms which are reactions to visual input, for example, although study of visual reflexes affecting flight has begun. Burton (1964) has shown asymmetrical changes in motor unit discharge frequency in the myogenic beetle Oryctes flying in a horizontally moving visual environment. Smyth and Yurkiewicz (1 966) induced changes in discharge frequency in the indirect flight muscles of blowflies by moving the visual field fore and aft under the animals, suggesting a kind of ground speed control. Changes in wind velocity were ineffective. In yawing locusts there are differences in the number of impulses to laterally homologous muscles (Waldron, 1967b; Dugard, 1967). These results indicate that descending inputs to the flight system can differentially bias parts of the system to increased or decreased activity without changing the main pattern, but much needs to be done yet to find how these commands are coded and how their effects are achieved. There seems to be an almost smooth gradation of types emerging in the above series of behavioral motor pattern differences between those which can be named as distinct behavioral acts and those which are merely control variations on one basic act. If motor tapes and sensory templates exist in any real sense at all, they probably also merge into one another in most cases. (The existence of a sensory template is clearly demonstrated probably only in a particular case of bird song development. Nottebohn, 1967.) It may be worth ending by pointing out the most important work that lies ahead. Insects have proven especially tractable material for the aqalysis of behavior in terms of their neuromuscular physiology and motorneuron activity and further comparative studies will likely be useful. However, we must begin to move in other directions as well. Much can be gained by copying those who work on crustaceans. Command fibers in insects should be investigated. Intraganglionic studies with both extra- and intracellular electrodes should be pursued, especially in relationship to insect behavior, which is better known and probably more interesting than that of crayfishes. If we can, as appears NERVOUS CONTROL OF INSECT FLIGHT

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probable, describe the physiological operation of the neuron networks which pattern insect behavior, and if these networks are indeed largely genetically constructed, then two interrelated problems loom large. One is the analysis of the development of the networks by methods, such as tissue culture, which are so far poorly worked out for insects. The other is the description of the anatomy of the nervous system in sufficient detail to provide a basis for physiological and developmental hypotheses. At present insect neurophysiology has moved considerably beyond a secure anatomical basis. REFERENCES Adams, P. A. and Hcath, J. E. (1964). Tempcraturc regulation in the sphinx moth, Cclcrio lineata. Nature, b n d . 20, 20-22. Baird, J. L. (1965). Aerodynamic behavior of the flesh fly Sarcopliga hullata (Diptera). Am. Zool. 5, 706. Barber, S. B. and Pringle, J. W. S. (1966). Functional aspects of flight i n belostomatid bugs (Heteroptera). Proc. R. SOC.B, 164, 21-39. Bentley, D. R. and Kutsch, W. (1966). The neuromuscular mechanism at stridulation in crickets (Orthoptera: Gryllidae). J. cxp. Biol. 45, 151-164. Burton, A. J. (1964). Nervous control of flight orientation in a beetle. Narure, Lo/id. 204, 1333. Chadwick, L. E. (1953a). The motion of the wings. Itz “Insect Physiology” (K. D. Roeder, ed.), pp. 577-614. John Wiley & Sons, New York. Chadwick, L. E. (1953b). Aerodynamics and flight metabolism. In “Insect Physiology” (K.D. Roeder, ed.), pp. 615-636. John Wiley and Sons, New York. Chadwick, L. E. (1953~).The flight muscles and their control. In “Insect Physiology” (K. D. Roeder, ed.), pp. 637-655. John Wiley and Sons, New York. Church, N. S. (1960). Heat loss and the body temperatures of flying insects. I. Heat loss by evaporation of water from the body. J . exp. Biol. 37, 171-185. Dugard, J. J. (1967). Directional change in flying locusts. J. Insect Physiol. 13, 1055-1064. Elsner, N. (1967). Muskelaktivitat und Lauterzeugung bei einer Feldheuschrecke. Verh. dtsch. Zoof. Ges. Heidelberg. (In press). Esch, Harald (1961). Uber die Schallerzeugung beim Werbetunz der Honigbiene. Z. vergl. Physiol. 45, 1-1 1. Esch, H. (1964). Uber den Zusammenhang zwischen Temperatur, Aktionspotentialen und Thorax bewegungen bei der Honigbiene (Apis mellifca L.). Z . vergl Physiol. 48, 547-551. Esch, H. and Wilson, D. M. (1967), The wingbeat sounds of flies and bees. Z. vergl. Physiol. 54, 256-267. Evoy, W. and Kennedy, D. (1967). The central nervous organization underlying control of antagonistic muscles in the crayfish. J. exp. Zool. 165, 223-238. Evoy, W.,Kennedy, D. and Wilson, D. M. (1967). Discharge patterns of neurons supplying tonic abdominal plexol muscles in the crayfish. J. exp. Biol. 46, 393411. Ewer, D. W. (1953). The anatomy of the nervous system of the tree locust, Acorrthacris ruficomis. I. The adult inetathorax. Ann. Natal Mus. 12, 367-381.

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Ewer, D. W. (1954). The anatomy of nervous system of the tree locust, IT. The adult mesothorax. J . ent. SOC.S. Afr. 17,27-37. Ewing, A. and Hoyle, G. (1965). Neuronal mechanisms underlying control of sound production in a cricket, Acheta domesticus. J. exp. Biol. 43, 139-153. Fraenkel, G. (1932). Untersuchungen iiber die Koordination von Reflexen und automatisch-nervosen Rhythmen bei Insekten. I. Die Flugreflexe der Insekten und ihre Koodination. Z . vergl. Physiof. 16,371-393. Gettrup, E. (1962). Thoracic proprioceptors in the flight system of locusts. Nature, Lond. 193,498-499. Gettrup, E.(1965).Sensory mechanisms in locomotion: the campaniform sensilla of the insect wing and their function during flight. Cold Spr. Harb. Symp. quanl.

Biol. 30, 615-622. Gettrup,E.(1966).Sensoryregulation ofwing twistingin1ocusts.J. exp. Biol.44.1-16. Gettrup, E. and Wilson, D. M. (1964).The lift control reaction of flying locusts. J . exp. Biof. 41, 183-190. Greenewalt, C. H. (1960). The wings of insects and birds as mechanical oscillators. Proc. Am. Phil. SOC.104,605-611. Haskell, P. T. (1961).“Insect Sounds.” 189 pp. Witherby Ltd., London. Haskell, P. T. (1964). Sound production. In “The Physiology of lnsecta” (M. Rockstein, ed.), vol. I, pp. 563-608. Academic Press, New York and London. Heidermanns, C. (1931). Reizphysiologische Untersuchungen der Flugmuskulatur von Aeschna coerulea. Zool. Jb. 50, 1-31. Hisada, M., Tamasige, M. and Suzuki, N. (1965).Control of the flight of the dragonfly Sympetrum darwinianum Selys I. Dorsophotic response. J. Fac. Sci., Hokkaido Univ., Ser. VI, Zool. 15, 568-577. Hoyle, G. (1 964). Exploration of neuronal mechanisms underlying behavior in insects. In “Neural Theory and Modeling” (R. Reiss, ed.), pp. 346-376. Stanford University Press. Huber, F. (1963).The role of the central nervous system in orthoptera during the coordination and control of stridulation. In “Acoustic Behavior of Animals” (R. G. Busnel, ed.), Chapter 17,pp. 440-488. Elsevier, Amsterdam. Jensen, M. (1956). Biology and physics of locust flight. 111. The aerodynamics of locust flight. Phil. Trans. R. SOC.B 239, 511-552. Kahn, M. C. and Offenhauser, W. (1949a).The first field tests of recorded mosquito sounds used for mosquito destruction. Am. J. trop. Med. 29, 811-825. Kahn, M. C.and Offenhauser, W. (1949b).The identification of certain west African mosquitos by sound. Am. J. trop. Men. 29, 827-836. Kammer, A. (1967a). Muscle activity during flight in some large Lepidoptera J. exp. Biof. 47,277-295. Kammer, A. (1967b). Motor patterns during flight and warmup in Lepidoptera. J. exp. Biol., (In press.) Kendig, J. J. (1967). Coupling between locust flight motorneurons. (Submitted.) Leston, D., Pringle, J. W. S. and White, D. C. S. (1965). Muscular activity during preparation for flight in a beetle. J. exp. Biol. 42, 409-414. Lewis, E. R. (1966). Synchronization in small groups of neurons: a study with electronic models. (Paper presented at 1966 Bionics Symposium.) Nachtigall, W. (1966). Die Kinematik der Schlagfliigelbewegungen von Dipteren. Methodische und Analytische Grundlagen zur Biophysik des lnsekten flugs. 2.vergl. Physiol. 52, 155-21 1.

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Nachtigall, W. and Wilson, D. M. (1967). Neuro-muscular control of dipteran flight. J. exp. 47,77-97. Neville, A. C. (1960). Aspects of flight mechanics in anisopterous dragon flies J. exp. Biol. 37, 631-656. Neville, A. C . (1963). Motor unit distribution of the dorsal longitudinal flight muscles in locusts. J. exp. Biol. 40, 123-136. Neville, A. C. and Weis-Fogh, T. (1963). The effect of temperature on locust flight muscle. J. exp. Biol. 40, 111-121. Nottebohm, F. (1967). The role of sensory feedback in the development of avian vocalizations. Proc. XIV Int. Ornith. Congr. (D. W. Snow, ed.), pp. 265-280, Blackwells, Oxford. Offenhauser, W. H. and Kahn, M. C. (1949). The sounds of disease-carrying mosquitoes. J. Acont. SOC.Am. 21, 259-263. Osborn, M. P. (1966). The fine structure of synapses and tight junctions in the central nervous system of the blowfly larva. J. Insect Physiol. 12, 1503-1512. Pabst, H. (1965). Elektrophysiologische Untersuchung des Streckrezeptors am Fliigelgelenk der Wanderheuschrecke Locusta migratoria. Z. vergl. Physiol. 50. 498-541. Pabst, H. and Schwartzkopf, J. (1962). Zur Leitung der Flugelgelenkrezeptoren von Locusta migratoria. Z. vergl. Physiol. 45, 39-04. Pringle, J. W. S. (1949). The excitation and contraction of the flight muscles of insects. J. Physiol., Lond. 108, 226-232. Pringle, J. W. S. (1954). A physiological analysis of cicada song. J. exp. Biol. 31, 525-560. Pringle, J. W. S. (1957). “Insect Flight.” 132 pp. Cambridge University Press. Pringle, J. W. S. (1965). Locomotion: Flight. In “The Physiology of Insecta” (M. Rockstein, ed.), Vol. 11, pp. 283-329. Pringle, J. W. S. (1967a). The contractile mechanism of insect fibrillar muscle. Prog. Biophys. molec. Biol., 17, 1-60. Pringle, J. W. S. (1967b). Comparative physiology of the flight motor. In “Advances in Insect Physiology” (J. W. L. Beament, J. E. Treherne and W. B. Wigglesworth, eds.), vol. 5, pp. 163-227. Academic Press, London and New York. Richards, A. G. (1963). The effect of temperature on wing-beat frequency in the male of the cockroach, Periplaneta americana. Ent. News 74, 91-94. Roeder, K. D. (1951). Movements of the thorax and potential changes in the thoracic muscles of insects during flight. Biol. Bull., Woods Hole 100, 95-106. Sandeman, D. C. (1961). Some aspects of the flight mechanism of the migratory locust, Locusta migratoria migratoroides R. & F. Master’s thesis, Dept. of Zoology, University of Natal, Pietermaritzburg. Smith, D. S. and Treherne, J. E. (1963). Functional aspects of the organization of the insect nervous system. Ado. Insect Physiol. 1, 401-484. Smyth, T. and Yurkiewicz, W. J. (1966). Visual reflex control of indirect flight muscles in the sheep blowfly. Comp. Biochem. Physiol. 17, 1175-1180. Snodgrass, R. E. (1929). The thoracic mechanism of a grasshopper, and its antecedents. Smithson Misc. Coll. 82 (2), 1-1 11. Sotavalta, 0. (1947). The flight-tone of insects. Acta ent. fenn. 4, 1-117. Sotavalta, 0. (1954). On the thoracic temperature of insects in flight. Ann. 2001. SOC. ‘‘ Vanamo”, 16, 1-21.

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Sotavalta, 0.(1963). The flight sounds of insects. In “Acoustic Behavior of Animals” (R. G. Busnel, ed.), Chapter 13, pp. 374-390. Elsevier, Amsterdam. Trujillo-Cen6z, 0. (1959). Study on the fibre structure of the central nervous system of Phallus labruscoe, L. (Lepidoptera). Z . Zellforsch. mikrosk. Anat. 49, 43246. Vogel, S. (1965). Aspects of flight at low Reynolds number. Proc. XII Int. Congr. Enr. London, (1964), 188-189. Vogel, S. (1966). Flight in Drosophila. I. Flight performance in tethered flies. J. exp. Biol. 44, 567-578. Vogel, S. (1967). Flight in Drosophila. 11. Variations in stroke parameters and wing contour. J. exp. Biol. 46, 383-392. Waldron, I. (1964). Courtship sound production in two sympatic sibling Drosophila species. Science, N . Y. 144, 191-193. Waldron, I. (1967a). Mechanisms for the production of the motor output pattern in flying locusts. J. exp. Biol. 47, 201-212. Waldron, I. (1967b). Neural mechanism by which controlling inputs influence motor output in the flying locust. J. exp. Biol. 47,213-228. Waldron, I. (1967~).The mechanism of coupling of the locust flight oscillator to oscillatory inputs. Z . Vergl. Physiol. (In press). Weis-Fogh, T. (1952). Fat combustion and metabolic rate of flying locusts (Schistocerca gregaria Forskal). Phil. Trans. R. SOC.B. 237, 1-36. Weis-Fogh, T. (1956a). Biology and physics of locust flight. 11. Flight performance B. 239,459-510. of the desert locust (Schistocerca gregaria). Phil. Trans. R. SOC. Weis-Fogh, T. (1956b). Biology and physics of locust flight. IV. Notes on sensory mechanisms in locust flight. Phil. Trans. R. SOC.B. 239, 553-584. Weis-Fogh, T. and Jensen, M. (1956). Biology and physics of locust flight. I. Basic principles of insect flight. A critical review. Phil. Trans. R. SOC.B. 239,415-458. Wenner, A. (1962a). Sound production during the waggle dance of the honey bee. Anim. Behau. 10, 79-95. Wenner, A. (1962b). Communication with Queen honey bees by substrate sound. Science, N. Y. 138, 446-448. Wenner, A, (1964). Sound communication in honeybees. Scient. Am. 210, 4, 116. Wiersma, C. A. G. and Ikeda, K. (1964). Interneurons commanding swimmeret movements in the crayfish, Procambarus clarki (Girard). Comp. Biochem. Physiol. 12, 509-525. Wilson, D. M. (1961). The central nervous control of flight in a locust. J. exp. Biol. 38,471-490. Wilson, D. M. (1962). Bifunctional muscles in the thorax of grasshoppers. J. exp. Biol. 39, 669-677. Wilson, D. M. (19%). The origin of the flight motor command in grasshoppers. In “Neural Theory and Modeling” (R. Reiss, ed.), pp. 331-345. Wilson, D. M. (1964b). Relative refractoriness and patterned discharge of locust flight motor neurons. J. exp. Biol. 41, 191-205. Wilson, D. M. (1965). The nervous coordination of insect locomotion. In “The Physiology of the Insect Central Nervous System” (J. E. Treherne and J. W. L. Beament, eds.), pp. 125-140. Academic Press, London and New York. Wilson, D. M. (1966a). Central nervous mechanisms for the generation of rhythmic behavior in arthropods. Symp. SOC.exp. Biol. XX, 199-228. Wilson, D. M. (1966b). Insect walking. Ann. Rev. Enr. 11, 103-122.

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Author Index Niunbers in italics are the pages on which the references are listed

A Adrian, E. D., 11, 12, 57 Arvanitaki, A., 16, 57 Auclair, J. L., 241, 282 Augustinsson, K. B.. 7, 57

B Baird, J. L., 173, 223, 292, 334 Balogh, J., 238, 253, 262, 268, 273, 274, 275,282 Barber, S. B., 199, 204, 219, 223, 314, 334 Barton-Browne, L. B., 266, 282 Bazemore, A. W., 52, 57 Beccari, A., 5, 57 Beck, S. D., 262, 282 Bedarf, E. W., 248, 286 Bennett, H. S., 7, 59 Bennett, L., 166, 167, 169, 223 Bentley, D. R., 298, 323, 334 Bernard, J., 30, 31, 33,59 Bhat, J. V.,230, 253, 267, 287 Bhat, M. G., 253,287 Biedermann, W., 269, 282 Blaser, R. E., 245, 286 Blewett, M., 257,260,261,262,266,283 Boettiger, E. G., 200, 205, 215, 223 Boisseau, J., 15, 47, 58 Boistel, J., 10, 14, 15, 16, 17, 20, 21', 22, 23, 24, 25, 26, 29, 30, 31, 32, 33, 34, 36, 46, 47, 48, 49, 50, 51, 52, 54, 55, 57, 57, 58, 61 Bonhag, P. F., 205, 211, 221, 223, 223 Bowman, M. C., 224,286 Brennitke, J., 240, 251, 252, 253, 257, 265,282 Brink. F.. 56.58 Bronk, D. W., 56, 58 Brown, A. W. A., 235,277,282 Brown, F. M., 242,245,217,282 Brown, R. H., 8, 60

Buck, J., 109,160 Bullock, T. H., 33, 58 Bultitude, K. H., 47, 58 Burkhardt, D., 198,208, 223 Burlacu, G., 28 1,283 Burton, A. J., 169, 170, 199, 202, 204, 214,223, 311, 314,333,334 Buxton, P. A., 241,249, 282

339

c. w-* 1259

C Cajal, S. R., 1, 2, 5, 58 Callec, J. J., 10, 14, 15, 16, 17, 24, 25, 46,47,48,49, 50, 51, 52, 55, 57,58 Carne, P. B., 240, 249, 251, 258, 270, 271,282 Cartwright, E., 241, 260,270,283 Chadwick, L. E., 8, 58, 202, 205, 220, 223,289,292,334 Chalazonitus, N., 16, 57, 58 Chane, S. C., 8, 58 Chauvin, R., 236, 237, 240, 252, 253, 256,257,264,272,282 Choy, C. T. H., 241,260, 270,283 Church, N. S., 319, 334 Clark, 220 Colhoun, E. H., 8,20, 23, 58, 59 Cook, E. F., 28, 43,61 Coraboeuf, E., 23,58 Cordebard, H., 235,284 Cottrell, G. A., 8, 60 Crisp, D. J., 106,108, 110, 111,159,161 Crossley, D. J. Jr., 245, 282,283 Crowell, H. H., 236, 237, 238, 256, 262, 275,276,283 Curtis, D. R., 25, 59

D Dadd, R. H., 230,252,283 Daly, H. V., 204, 219, 222, 223

340

AUTHOR INDEX

Darwin, F. W., 199, 221, 223 Davey, P. M., 238, 262, 265, 268, 272, 283 David, W. A. L., 249, 259, 262, 283 Davis, R. E., 242, 244, 245, 283 De Lorenzo, A. J., 7, 59 Demoll, R., 170, 210, 224 De Robertis, E., 7, 59 Dingle, H., 200, 224 Dugard, J. J., 199,214,224, 333, 3-74

E Eccles, J. C., 7, 38, 39,41,43, 51, 52, 59 Eccles, R. M., 25, 51, 59 Edwards, C., 53, 59, 60 Edwards, C. A., 251,258,269,283 Elam, C. J., 242, 244, 245,283 Elliott, K. A. C., 52, 57 El Shaarawy, F., 230, 284 Elsner, N., 298, 324, 334 Engelmann, M. D., 231, 283 Erhan, E., 281, 283 Ervin, F. R., 248,283 Esch, H., 321,326,327, 329,334 Evans, A. C., 234, 235, 236, 237, 238, 239,240,242,253,255,256,257,258, 259. 265. 267. 268, 269. 270,271,273, 274; 275; 276; 277,278; 283 Evans, D. E., 241, 260,270,283 Everson, D. O., 242, 244, 245, 283 Evoy, W., 305, 331, 334 Ewer, D. W., 303, 334 Ewing, A., 323, 331, 335

Friend, W. G., 241, 260, 270, 283 Furshpan, E., 205,223

G Gahery, Y., 26, 30, 31, 33, 54, 57, 59 Garcia, J., 248, 283 Gardiner, n. 0.C., 249, 259, 262,283 Gere, G., 238, 253, 262, 265, 268, 273, 274, 275,284 Gerschenfeld, H. M., 16, 25, 26, 54, 62 Gettrup, E., 196, 198,200,209,211,213, 224,227, 303, 304,335,338 Goodliffe, E. R., 235,238,257,273,274, 276, 277, 278,283 Goodman, L. J., 196, 199,208, 213,224 Gordon, H. T., 27, 63, 230, 231, 235, 250, 269,284 Gordon, I. M., 27, 59 Graham, M., 7, 57 Greenewalt, C. H., 210, 224, 294, 335 Gupta, P. D., 235, 284 Guthrie, D. M., 198, 224

H

Hagins, W. A., 25, 63 Hagiwara, S., 18, 59 Harvey, W. R., 163, 224 Haskell, J. A., 163, 224 Haskell, P. T., 196, 224, 322, 335 Hassanein, M. H., 230, 284 Heath, J. E., 321, 334 Heidermanns, C., 307, 335 Heinrichs, E. A., 245, 284 Heran, H., 195, 198, 199, 202, 206, 210, F 214,224 Faust, R., 191, 195, 197, 198, 211, 212, Herbert, H. G., 186, 224 Hess, A., 3, 4, 5, 6, 11, 59 213,214, 215, 216,224 Fewkes, D. W., 238, 241, 249,260,269, Hichar, J. K., 14, 54, 59, 60 Hinton, H. E., 65,68,70, 71, 72,73,74, 270,283 75, 76, 78, 79, 82, 83, 84, 85, 86, 87, Fielden, A., 13, 59 88, 89, 92, 94, 95, 96, 97, 98, 99, 100, Florey, E., 52, 57, 59 101, 102, 103, 104, 106, 108, 113,114, Fox, H. M., 67,159 115, 116, 117, 118, 119, 120, 122, 123, Fraenkel, G., 198, 199, 224, 230, 236, 124, 125, 126 130, 131, 132, 133,137, 240, 242, 246, 247, 248, 251,253, 254, 139, 145, 146, 147, 149, 150, 151, 152, 257,260,261,262,263,264,265,266, 153, 154, 155, 156, 158,159,160 275,283,287,288, 304,335 Hirano, C., 234, 254,255,274, 276,277, French, R. A., 267, 286 284 Freund, K., 186,224

34 1

AUTHOR INDEX

Hiratsuka, E., 235, 236, 238, 240, 249, 253, 258, 262, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 28 1,284 Hisada, M., 309,335 Hollande, A. C., 235,284 Hollick, F. S. J., 190, 191, 192, 193, 194, 198, 199, 200, 207, 209, 211, 225 Hopkins, F. G., 232,235,284 Horridge, A., 165, 225 House, H. L., 229, 230, 233, 237, 247, 248, 272,284 Hoyle, G., 323, 331, 332, 335 Huber, F., 324,335 Hughes, G. M., 13, 18, 52,59,60, 62 Hunt, C. C., 13, 60 Husain, M. A., 237, 249, 262, 268, 284

I Ikcda, K., 331,337 Imms, A. D., 69, 160 Ishii, S., 234, 255, 274, 276, 277, 284 Ishii, T., 27, 63 Ito, T., 230, 286 Iwasaki, S., 40, 60 Iyatomi, K., 8, 60

J Janda, V., 235, 284 Jasic, J., 271, 285 Jenkins, P. M., 68, I60 Jensen, M., 290,292,335,337 Jensen, Martin, 164, 165, 166, 168, 171, 182, 196, 197, 225,227 Johnson, C. G., 241, 259, 265, 270, 284 Jover, H., 240, 251, 252, 253, 257, 265, 282

K Kahn, M. C., 322, 326, 328, 335, 336 Kammer, A., 309, 310, 321, 335 Kandel, E. R.,16,60 Kanehisa, K., 8, 60 Kasting, R.,234,243,244,245,248,249, 254,267,270,274, 275,284,285 Katsuki, Y., 25, 52, 53, 54, 55, 62 Kaufman, R. W., 245, 286 Kearns, C. W., 8, 58 Keister, M., 109, 160

Kendeigh, S. C., 234, 278, 285 Kendig, J. J., 307, 335 Kennedy, D., 33, 51,62, 305, 331,334 Kennedy, D. K., 16, 51, 61 Kennedy, H. D., 70, 144, 160 Kennedy, N. K., 10, 30, 33, 35, 36, 61 Kerkut, G. A., 8, 18, 19, 60 Kitzawa, Y., 281,285 Kleiber, M., 231,234, 237, 242,278,285 Koelle, G. B., 25,63 Koelling, R. A., 248, 283 Koketsu, K., 20, 60 Koll Ros, J. J., 8, 27, 63 Kosaka, T., 230,287 Krishna, S. S., 246, 285 Krosh, A., 279, 285 Kuffler, S. W., 53, 59, 60 Kuno, M., 13, 60 Kunze, P., 199, 214, 217, 225 Kutsch, W., 298, 323, 325, 334, 335

L Lafon, M., 230, 232, 253, 255, 285 Landolt, A. M., 3, 6, 7, 8, 60 Larrabee, M. G., 56, 58 Laverack, M. S., 19, 60 Lebeden, A. G., 258, 262, 285 Legay, J. M., 230, 232, 242, 266, 276, 272, 285 Leghissa, S., 3, 4, 60 Leston, D., 199, 205, 225, 330, 335 Lewis, E. R., 317, 335 Lindauer, M., 199, 214, 224 Long, D. B., 265,285 Loosli, J. K., 245, 286

M McCay, C. M., 236, 250,285 McGeer, E. G., 26, 60 McGeer, P. L., 26, 60 McGinnis, A. J., 234, 243,244,245,248, 249, 254, 267, 270, 274, 275, 284, 285 Machin, K. E., 205, 225 Macko, V., 271,285 McLennan, H., 26,60 McMillian, W. W., 244, 286 Maenan, A., 166, 197, 210,215,225 Malmann, R., de, 240, 251, 252, 253, 257, 265, 282

342

AUTHOR INDEX

Maruyama, K., 163, 225 Mathur, C. B., 237, 249, 262, 268,284 Matrone, G., 242, 287 Maynard, L. A., 242, 285 Mellanby, K., 276, 286 Mikalonis, S.J., 8, 60 Mill, P. J., 12, 60 Mitchell, H. H., 234, 286 Mittelstaedt, H., 198, 199, 213, 225 Mittler, T. E., 241, 286 Molner, J. I., 245, 286 Moore, A. R., 25, 63 Moore, F. K.. 165, 225 Morita, H., 18, 59 Mukaiyama, F., 230, 286

N Nachtigall, W., 169, 170, 171, 172, 173, 179, 180, 181, 182, 183, 185, 186, 191, 194, 198, 199,200,202,204,205,214, 215, 221, 225, 293, 313, 335,336 Nagy, B., 238,253,268,286 Narahashi, T., 2, 20, 24, 27, 33, 40, 41, 42,43, 46,48, 51, 52, 55, 56,60,64 Nersesian-Vasiliu, C., 281, 283 Neuhaus, W., 186, 188,189,225 Neville, A. C., 203, 213, 220, 221, 225, 292,296, 297, 298, 307, 319, 320,336 Noguchi, H., 254,274,284 Norris, M. J., 252, 265, 286 Nottebohn, F., 333, 336

0 Offenhauser, W., 322,326,328,335,336 Osborne, M. F. M., 166,225 Osborne, M. P., 5, 7, 61, 315, 336 Ostlund, E., 26, 61

P Pabst, H., 303, 304,336 Palay, S. L., 7, 61 Patton, R. L., 234, 286 Petre, Z.,281, 283 Phillipson, J., 237, 286 Pichon, Y.,20,21, 22,23,29, 30,32, 33, 34, 36, 61 Pipa, R. L., 28, 43, 61

Preston, J. B., 16, 51, 61 Price, G. M., 54,61 Pringle, J. W. S., 164, 165, 191, 192, 198, 199,204,205,206,215,219,220,221, 222,223,224,225,289,290,310,311, 314, 330,334, 335, 336 Prosser, C. L., 14, 61 Pruess, K. P.,245, 284 Pulikovsky, N., 66, 92,98, 137, 138,160 Pumphrey, R. J., 28, 29, 30, 33, 35, 36, 61

R Rawdon-Smith, A. F., 28, 29, 30, 33, 35, 36, 61 Ray, J. W., 54, 61 Reid, J. T., 245, 286 Revusky, S. H., 148, 286 Reynolds, P. J., 242, 244, 245, 283 Richards, A. G., 28,43,61, 318,336 Richards, C. R., 245,286 Richards, 0. W., 260, 286 Ris, H., 3, 7, 60 Ritter, W., 204, 215, 225 Robertson, J. D., 7,61 Robinson, A. G., 241, 286 Roeder, K. D., 8, 10, 11, 12, 13, 14, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35, 36, 38, 55, 56, 61, 62, 63, 200, 226, 311,336 Roeder, S., 12, 24, 27, 62 Rogers, J. S.,125, 160 Roonwal, M. L., 237,249,262,268,284 Rowe, E. C., 10, 14, 43, 44,45, 46, 47, 52,62, 307, 336

S Sacktor, B., 163, 226 Samson, E. A., 10,30,33,35,61 Sanchez, D., 2,58 Sandeman, D. C., 169, 170, 223, 296, 298,299,336 Sandri, C., 6, 7, 8, 60 Sang, J. H., 229,230,272,286 Satchell, G. H., 139, I61 Sato, A., 277, 187 Satterthwait, A. F., 249, 286 Sattler, H., 254, 262, 266, 286 Savenkov, A. N., 258, 262, 285

343

AUTHOR INDEX

Savit, J., 8, 27, 63 Saxena, K. N., 246,285 Schaller, F., 199,200,207,214,217,226 Schneider, G., 191, 198, 208, 213, 223, 226 Schwartzkopf, J., 303, 336 Schwerdtfeger, F., 250, 269, 286 Selman, B. J., 85, 96, 161 Sharada, K., 253, 267, 287 Shaw, J., 273,287 Sherrington, C. S.,1, 62 Shyamala, M. B., 230, 253, 287 Sinha, R. N., 235, 284 Smart, J., 200, 204, 219, 220, 221, 226 Smart, V. W., 242, 287 Smart, W. W. G., Jr.. 242, 287 Smith, D. S.,2, 3, 4, 5, 6, 7, 8, 9,28, 56, 57,62,63,232,240,252,268, 271,287, 317,336 Smyth, T., 199, 202, 214, 226, 333, 336 Snodgrass, R. E., 298, 336 So0 Hoo, C. F., 236,237,240,242,246, 247, 248,251, 253,254,263, 264, 265, 287 Sotavalta, O., 166, 168, 169, 173, 226, 318, 319, 321, 322, 326, 328, 336,337 Spencer, E. Y.,8, 59 Starks, K. J., 244, 286 Stellwaag, F., 169, 186, 190, 210, 214, 216,226 Stobbart, R. H., 273, 287 Stuckenberg, B. R., 139, 144, 161 Suga, N., 25, 52, 53, 54, 55,62 Sundermeier, W., 133, 161 Suzuki, N., 309,335 Svidersky, V. L., 13, 62 Swart, P.,165, 226 Sylven, E., 628, 287 Sytinsky, I. A., 63

T Takeda, K., 33, 51, 62 Takeuchi, Y.,230,287 Tamasige, M., 205, 225, 309, 335 Tauc, L., 16, 17, 18, 25, 26, 51, 52, 54, 60, 62 Taylor, B. J. R., 18, 19, 60 Teissier, G., 251, 257, 281, 287

Thorn, A., 165,226 Thomas, R. C., 47,63 Thorpe, W. H., 106, 108, ll0,159,16Z Tillyard, R. J., 197, 226 Titschack, E., 235, 241, 249, 258, 261, 262,287 Tobias, J. M., 8, 27, 63 Tonnoir, A., 139, 161 Tozian, L., 12, 13, 38, 62 Trager, W., 231, 233,277,287 Treherne, J. E., 2, 3, 4, 5, 6, 7, 8, 9, 28, 56, 57, 62, 63, 317, 336 Trouvelot, L., 230, 287 Trujillo-Cen6z, O., 3, 4, 5, 63, 317, 337 Tsutsui, K., 277, 287 Turk, K. L., 245,286 Turner, R. S., 25, 63 Twarog. B. M., 22, 24, 25, 26, 55, 56,63 Tyler, C., 23 1, 234, 278, 287 Tyshchenko, V. P., 63

U Ullman, T., 277, 287

v Venkatachala, Murthy, M. R., 230, 287 Vereshtchagin, S. M., 63 Vogel, S., 165, 173, 174, 175, 176, 177, 178,208,209,210,226,293,337 Volle, R. L., 25, 63

W Waldbauer, G. P., 231, 232, 233, 236, 237,238,239,240,241,243, 46,247, 251,255,263,264,265,287, 88 Waldron, I., 300,304,307,326,333,337 Weiant, E. A., 12, 13, 38, 62 Weis-Fogh, T., 163, 164, 166, 174, 198, 199,200,201,202,209,314,220,221, 226,227,290,292,295,296,297,304, 319, 320, 336,337, 338 Welsh, J. H., 27, 59, 63 Wenner, A., 327, 328,337 White, D. C. S., 199, 205, 225, 330, 335 Wiersma, C. A. G., 331, 337 Wigglesworth, V. B., 2, 8, 56, 63, 94, 137, 161, 234, 277,288

5

344

AUTHOR INDEX

Wilson, D. M., 40, 60, 164, 198, 199, 200,201,202,205,209,211,214,215, 220,221,224,225,227,289,296,298, 300,303, 304,305,306, 311,313,314, 315, 320, 325, 326, 329, 332, 334, 335, 336,337, 338 Wilson, V. J., 47, 63 Wirth, W. W., 155, 16I Wohlgemuth, R., 186, 187, 188, 189, 225, 227 Wolcott, G. N., 249, 251, 255, 259, 264, 288

Woolfolk, P. G., 245, 286 Wyman, R. J., 303, 304, 305, 311, 312, 314, 315,338

'Y Yamasaki, T., 20, 24, 27, 33,40,41, 42, 43,46,48, 51, 52, 55, 56, 60,63,64 Yokoyama, T., 230, 288 Yorke, C. H., 248, 283 Yurkiewicz, W. J., 199, 202, 214, 226, 333,336

Subject Index A Acetylcholine and acetylcholinesterase, 8-9 and carbon dioxide, 23 and curare and atropine, 25-26 and DDT, 27 and eserine. 25 in ganglia, 7-8 and mechanism of transmission, 3839,42,43,49, 55-57 and rhythm modification, 23-24 and temperature, 20 Acetylcholinesterase and acetylcholine, 8-9 in ganglia, 8-9 and mechanism of transmission, 39, 40, 42, 55 and rhythm modification, 23-25 and temperature, 20 Acilius, flight reflexes, 199 Action potentials in fast flight muscle, 296 in synaptic transmission (see Synaptic transmission) Adrenaline and noradrenaline in synaptic transmission, 26 Aedes aegypti, passage time of food in gut, 237 Aeshna abdominal ganglion, 12 flight reflexes, 203 After-discharge, 35-36 Agapetes galathea, lift and drag when gliding, 171-173 Age and variation in food utilization, 267-271 Aglais urticae, feeding dry matter, 255 fresh matter, 259 indices, dry and fresh weight, 25 I nitrogen, 275, 276, 277 starch, 278 34s

Agrotis, flight stability, 196 Agrotis orthogonia, feeding and age, 249, 267, 270 consumption, 245 dry matter, 243, 254 nitrogen, 274 Agrotis ypsilon, food intake, 249 Agyrnriastils ingens, wingbeat frequency, 294, 295, 296 Anaesthetics, use in study of flight, 191 Anagasta kuehniella, feeding dry matter, 257 fresh matter 261, 262 indices, dry and fresh weight, 251 and water, 266 Anax, flight reflexes, 213 Animals other than insects Aplysia, nervous system, 16, 17, 18, 25, 5 I, 52, 54 birds food utilization, 234, 278 song, 333 cephalopods, 33 crayfish, nervous system, 16, 33, 52, 305,331,333 Crustacea, nervous system, 2, 33, 52, 333 farm animals, food utilization, 237, 242, 244, 245 Helix, synaptic transmission, 16, 52 Lumbricus, nervous system, 19 molluscs, nervous system, 2 rat, food utilization, 232, 235 snail, acetylcholine, 8 vertebrates nervous system, 2, 8, 51, 331 nutrition, 231, 242 Antennae, effect on flight, 192-193, 194, 206,208 Antheraea polyphemus, food efficiency, 230 Anticholinesterases and after-discharge, 35-36

346

SUBJECT INDEX

Antich olinesterases-cont. di-iso propyl fluorophosphate (DFP), 25, 35, 55 eserine, 24-25,42,55 hexaethyl tetraphosphate, 35 phosphine oxides, 36 physostigmine, 35 tetraethyl pyrophosphate, 35, 42 Antocha spp., spiracular gills, 75, 77, 78, 90,91,93,95,96,98,99, 100, 102, 103, 105, 107, 108, 113, 114, 130-133 Antocha bifida, spiracular gills, 76, 78, 102, 103, 104, 112 Antocha vitripennis, spiracular gills, 76, 87, 88, 93, 94, 95, 99, 102, 103, 106, 109, 112, 114, 124, 132 Aphelocheirus entry of water, 106 oxygen consumption, 108 Aphid, feeding rate of sap intake, 241 starvation, 237 Aphrosylus spp, spiracular gills, 75, 84, 100, 113, 148-152 Aphrosylus celtiber, spiracular gills, 109, 149, 150, 151 Apidae differentiation of flight muscle, 220, 221, 222 flight reflexes, 213, 215 Apis rnellifera abdominal scent glands, 187 fanning, 187-1 88 flight differentiation of muscles, 219, 220 reflexes and direct muscles, 204 reflexes and indirect muscles, 202 reflexes and flight initiation, 200 reflexes and velocity control,206-207 reflexes and vision, 199 reflexes and yaw control, 214 hive aeration, 187 Apoidea, flight reflexes, 204, 205, 210 Apolysis definition, 68-71 and tissue isolation in spiracular gills, 85 et seq. Atropine and synaptic transmission, 25-26

Automeris io, feeding starch digestion, 277 starch as marker, 245 Water loss from faeces, 242

B Be acetylcholine, 7 flight aerodynamics, 198, 291 differentiation of flight muscle, 222, 223 motor control, 314 reflexes and accessory indirect muscles, 205 reflexes and flight initiation and termination, 200 reflexes and notal wing articulation, 21 1 reflexes and velocity control, 206207, 208 reflexes and yaw control, 217 and sound, 326, 327-328, 330 and temperature, 318, 321 Beetle dehydration, 96 flight aerodynamics, 292 nervous control, 314 reflexes and accessory indirect muscles, 202 reflexes and direct muscles, 204 reflexes and flight initiation and termination, 200 and sound, 330 spiracular gills (see Spiracular gills) Belostomatid bugs, flight aerodynamics, 291 muscle, 220 nervous control, 314 reflexes, 204 Blabera craniifer, abdominal ganglion and temperature, 30 Blatta orientalis, utilization of dry matter, 256 Blattela germanica, feeding residual food in gut, 236 and sex, 271 Blepharoceru, spiracular gills, 139

SUBJECT INDEX

Blepharoceridae, spiracular gills, 75, 87, 97, 100, 105, 113, 139, 140, 141-144 Blocking agents of synapses, 41 Blood, isolation in spiracular gills, 86, 88-89, 122

Blood-sucking insects, food intake, 241, 25 1

Blowfly indirect flight muscles, 202, 333 nitrogen utilization, 273 Bombyx mori, feeding carbohydrate and lipid, 276 digestion and conversion of fresh and dry matter, 251, 253, 258, 262-272 energy utilization, 279,280,281 fibre, 277 intake, 242,249 nitrogen, 273, 274, 275 nutritive ratio, 278 recent papers, 230 and sex, 272 uric acid in faeces, 235 use of groups, 238 Bupalup piniarius, digestibility and age, 269

Butterfly, wingbeat frequency, 294

C Ca2+ and DDT, 27 and synaptic transmission, 23, 56 Calliphora, flight reflexes and antennae, 208 and Johnston’s organ, 198 and maintenance of flight, 199 and pitch, 211, 212 and roll, 213 and yaw, 215,216 Campaniform sensillae, role in flight, 198, 304 Canace nasica, spiracular gills, 75, 106, 113, 152, 153, 154, 155 Canaceidae, spiracular gills, 75, 105, 152-156 Carausius, synapses, 3,4 Carausius morosup, feeding and age, 249 dry matter, 253 fresh matter, 258

347

Carbohydrates, 231, 251, 276, 278 Carbon dioxide and acetylcholine, 8 and electrical activity, 23 Celerio euphorbia, food intake, 247 Celeriolineata,flightand temperature,321 Cellulose, digestion, 277 Cephup, flight muscle differentiation, 219,220 Cetonia, flight stability, 196, 197 Cetoniidae, elytra, 169 Chalcidae, flight muscle differentiation, 220 Chelifera, spiracular gills, 147 Chilo suppressalis, feeding carbohydrate and lipid, 276 dry matter, 256 nitrogen, 274 starch, 277 Chironomidae, spiracular gills, 66, 69, 87, 92, 96, 152, 155 Chironomus, dehydration, 96 Chromic oxide, as marker in food utilization, 243 Chrysomela knabi, food intake, 245 Chrysomelidae, spiracular gills, 66 Cicada, tymbal action, 330 Cicadidae, flight muscle differentiation, 219 Cimex lectularius, feeding and age, 270 different bloods, 264 intake, 241 fresh food, utilization, 259 Cockroach flight muscle differentiation, 219 nerve cord, electrophysiological properties, 10-57 (see Synaptic transmission) Coleoptera flight differentiation of flight muscles, 219, 220, 221 lift and thrust generation, 166-171 reflexes and click action of articulation, 205 reflexes and fibrillar muscles, 204 reflexes and flight initiation, 200 reflexes and yawing, 214

348

SUBJECT INDEX

Coleoptera-cont. flight stability, 195, 196, 197 food utilization dry matter, 257 fresh matter, 258, 260 spiracular gills Hydroscaphidae, 105, 156 158-159 Psephenidae, 72, 74, 75, 82, 83, 86,

Diptera and DDT, 27 flight aerodynamics, 292, 293 control, 318 coordination, hypothesis, 3 15-317 differentiation of flight muscles,

96, 114-120 Sphaeriidae, 74, 105, 156, 158-159 Torridincolidae, 74, 86, 105, 120122,156-158

lift and thrust, 173-179 motor patterns, 309-314, 331 multiphasic and metastable patterns, 314-315 and sound, 326, 328-330 stability, 190-195 and temperature, 318 wing motion kinematics, 179-186 spiracular gills Blepharoceridae, 75, 87, 97, 100,

Cricket singing muscles, 298 sound production, 323, 325 Cryptobiosis, 95-97 Cryptolestcs fcrrugincirs, uric acid in faeces, 235 Cryptoternies brcuis, utilization of fresh matter, 259, 264 Curare and synaptic transmission, 25-

2 19-223

105, 113, 139, 140, 141-144

Canaceidae, 75, 105, 152-1 56 Deuterophlebiidae, 70,73,75,87,90, 91,92,93,97,105,112,144-146

26, 41, 51, 55

D

Dolichopodidae, 73, 75, 84, 87, 92,

DDT and synaptic transmission, 27 Dehydration, 95-97 Dendrolimus pini and GABA, 54 and utilization of fresh matter, 258 Dermestes niaculatus, feeding dry matter, 257 fresh matter, 260 intake, 251 and water, 266 Deuterophlebiainyoensis, spiraculargills,

Empididae, 75,87,92,98, 121, 146-

70, 73, 75, 87, 90, 91, 92, 93, 97, 105, 112, 144-146 Deuterophlebiidae, spiracular gills, 70, 73, 75, 87, 90,91,92, 93, 97, 105, 112, 144-146 Diataraxia oleracea, water content of food, 267 Dicranomyia nionostromia, spiracular gills, 81, 109, 129 Dicranomyia trifilanicntosa, spiracular gills, 81, 109, 129

Digestion (see Food) DiGsopropyl fluorophosphate (DFP) as ant ichol inesterase, 25

97, 100, 105, 113, 148-152 148

Simuliidae,73,75,85,87,90,91,92, 97, 98, 100, 105, 114, 133-141, 152. Tanyderidae, 75, 86, 92, 99, 105, 122-1 23 Tipulidae, 73, 75, 77, 79, 84, 90, 91, 92,93,97,99, 100, 105, 113, 123133,152 Dissosteira Carolina, wingbeat frequency, 294 Dissosteira pictipennis, wingbeat frequency, 294, 295 Dolichopodidae, spiracular gills, 73, 75, 84, 87,92, 97, 100, 105, 113, 148-152

Dopamine and synaptic transmission, 26

Dragonfly electrical activity, 13 flight aerodynamics, 292 differentiation of flight muscle, 221 motor patterns, 296 nervous control, 307-309

SUBJECT INDEX

Drosophila spp., flight aerodynamics, 293 initiation of flight, 200 lift and reflexes, 209, 210 lift and thrust generation, 173-179 and sound, 326, 327 stroke angle, 202 velocity control, 208 Dytiscirs, action potentials, 12

E Ecdysis definition, 68-71 and isolation of tissue in spiracular gills, 90 et seq. Edwursina spp., spiracular gills, 139,140, 144 Elateroid, definition of term, 71 Elytra in flight, 169-171, 197, 292 in sound production, 323-324 Empididae, spiracular gills, 75, 87, 92, 98,121,146-148 Energy, utilization, 231, 278-282 Environment and insects with plastron, 66-68, 106108 and utilization of food, 265-267 crowding, 265 humidity, 266-267 temperature, 266 water content of food, 267 Eristalis, flight motor patterns, 313, 314 Eserine as anticholinesterase, 24-25 Eirtanyderus, spiracular gills, 75, 82, 84, 99, 114 Eutunyderus wilsoni, spiracular gills, 67, 109, 112, 122, 123, 158 Evolution of spiracular gills blood reservoirs, 102-104 from respiratory horns, 84 from spiracles, 81-84

F Flight motor, comparative physiology, 163-227 comparative studies, 217-223 axioms, 2 17-21 8

349

Flight motor, comparative physiologycont. comparative studies-cont. flight muscle differentiation, 219-223 lift and thrust generation, 164-179 in Coleoptera, 166-171 in Diptera, 173-179 in Lepidoptera, 171-173 reflexes, motor mechanisms, 198-217 amplitude, frequency and power control, 200-206 initiation, maintenance and termination, 199-200 list of reflexes, 198-199 velocity, lift and attitude control, 211-217 stability, 190-198 in Diptera, 190-195 in others, 195-198 wing motion, kinematics, 179-190 in Apis mellgera, 186-190 in Diptera, 179-186 Flight and related khaviour, control, 289-338 kinematics and aerodynamics, 290-296 model for flight control, 317-318 ,,,yogenic flyers, 309-317 coordination in flies, 3 15-31 7 motor patterns, 309-314 multiphasic and metastable patterns, 314-315 neurogenic flyers, 296-309 dragonflies, 307-309 Lepidoptera, 309 locusts, 300-307 related behaviour sound production using wings, 32233 1 temperature and flight, 3 18-322 Faeces and digestibility, 233-239, 242, 244, 250, 262, 272-281, 278 Feedback, positive, and flight coordination, 306 Food, consumption and utilization, 229288 digestion and conversion, 250-272 and age and sex, 267-272 comparison of foods, 264-265 comparison of species, 263-264

SUBJECT INDEX

Food, consumption and utilizationdigestion and conversion-cont. and environmental factors, 265-267 limitations of data, 250-263 indices, 231-235 of consumption, 232 of conversion of digested food, 235 of conversion of ingested food, 233 of digestibility, 233-235 of growth rate, 232-233 intake, 246-250 measuring by weight, 236-246 gravimetrically, 238-242 using markers, 242-246 utilization of constituents, 272-278 utilization of energy, 278-282 Formica lugubris acetylcholinesterase,9 soma-somatic junctions, 3, 7 synaptic vesicles, 6

G GABA, inhibiting effect, 26, 52-54, 57

Ganglion electrical activity, 11-28 organization, 2-1 1 (see also Synaptic transmission) Gampsocleis buergeri, synaptic transmission and eserine, 25 inhibitory processes, 52, 53, 54 Geranomyia spp., spiracular gills, 75,76, 77,99, 113, 123, 125-127, 130 Geranomyia bezzi, spiracular gills, 125 Geranomyia unicolor, spiracular gills, 80,93, 105, 109, 125, 127, 128 Gliding flight of Lepidoptera, 171-173 Gomphocerippus rufus, sound production, 298, 324, 325 Grasshoppers flight motor patterns, 298, 300, 303 nervous control, 331, 332, 333 wingbeat frequency, 294 food utilization, 245 synaptic transmission and curare, 25 Growth rate index, 232-233 Gryllus domesticus, digestion and conversion, 253, 257, 264, 271

H Hairs on wing, and flight, 165, 177, 198 Haliplidae, epidermal cells, 96 Halteres and flight, 198, 292 Hapalothrix, spiracular gills, 139 Heliofhis zea, food utilization, 244 Helminthidae, epidermal cells, 96 Hemerodrontia imilineata, spiracular gills, 147, 148 Hemiptera flight muscle differentiation, 219 reflexes, 200,204 food intake, 241 utilization, 259-260 Hepialus humufi, digestion and conversion, 251, 258, 269 Homoptera, differentiation of flight muscles, 219 Honeydew excretion in aphids, 241-242 Horaia. spiracular gills, 139 Hydrophilus piceus, oxygen consumption, 108 Hydroscapha natans, spiracular gills, 156 Hydroscaphidae, spiracular gills, 105, 156,158-159 Hylephila, flight and temperature, 32 I Hymenoptera, flight muscle differentiation: 219 reflexes, 204 stability, 196 Hyphantria cwea, feeding and age, 268 and crowding, 265 and sex, 271 and temperature, 266 and utilization of nitrogen, 274

I Idioglochina, spiracular gills, 75, 78, 80, 113, 123, 130 Idioglochina marmorata, spiracular gills, 80, 81 Ions and synaptic transmission Ca2+,23,27, 56 K + , 21-23, 30-31, 55, 56 Na+, 56

351

SUBJECT INDEX

lnhibition in synaptic transmission, 43, 52-54, 57 Isoptera, food utilization, 259

J Jumping and flight initiation, 200

K K and synaptic transmission and blocking, 55 after desheathing, 56 and ganglia, 21-23 and spontaneous activity, 30-3 I Kelloggina, spiracular gills, 139 Kinematics, use in study of wing motion, 179-190,290-294 Apis mellifera, 186-1 90 Diptera, 179-1 86 +

L Leech, electrical activity of ganglion, 18 Lepidoptera flight aerodynamics, 291 differentiation of muscles, 219, 220 gliding, 171-173 nervous control, 309, 310, 331 re@xes, 205 stability, 195, 196 and temperature, 321-322 food starvation, 237 utilization of dry matter, 253-256, 263 utilization of fresh matter, 258-259, 261 water content of food, 267 metamorphosis, 69, 71 Leptomastrix, differentiation of flight muscle, 220 Lezhoceros, flight reflexes, 199 Lift and thrust in flight, 164-179 Coleoptera, 166-171 general considerations, 164-166 Diptera, 173-1 79 Lepidoptera, 171-173 Lipids, digestibility, 276, 277, 278 Liponeura spp., spiracular gills, 139,140, 142, 144

Lipsothrix spp, spiracular gills, 75, 77, 89, 93, 99, 125 Lipsothrix neruosa, spiracular gills, 93 Lipsothrix remota, spiracular gills, 79, 86, 92, 93, 94, 124, 126 Locust, flight coordination mechanism, 306-307, 309,318 deafFerentation, 301-303 effects of input, 303-304 ganglioniccoordination, 304-306 impulses during yawing, 333 lift, 292 motor patterns, 297, 300-301 plasticity in control system, 332 reflexes, 201,202, 204, 209, 3 I4 single level interactions, 33 I and temperature, 319-321 thoracic flight muscles, 299 wing movements, 182 Lociista, flight, 198, 296, 298 Locusta migratoria electrical activity, 14 utilization of dry matter, 252 Lucilia sericata, flight reflexes, 208 Lyniantria monacha, feeding and temperature, 266 utilization of dry matter, 253, 264

M Macroplea, spiracular gills, 66 Malacosotna neustria, feeding nutrititive ratio, 278 utilization of carbohydrate, 276 dry matter, 251,253 fresh matter, 258 lipid, 276, 277 nitrogen, 275 Mamestra brassicae, feeding utilization of dry matter, 254,263,264 utilization of nitrogen, 274 Mantis religiosa, synaptic transmission elimination of afferent impulses, 12, 13 inhibition, 38, 52 Marker techniques in food utilization, 242-246 anthrone, 246

352

SUBJECT INDEX

Marker techniques in food utilization -cont. chromic oxide, 243-244, 275 lignin, 245 radioactive tracers, 245-246 Metaesphenus japonicus, spiracles, 8284 Melanoplus bifituratus, utilization of dry food, 252, 264, 268 Melanoplus bivittatus flight muscles, 298 wingbeat frequency, 294 feeding uric acid, 235 utilization of dry matter, 243, 275 utilization of nitrogen, 275, 277 Melanoplus devastator, wingbeat frequency, 294 Melanoplus diJerentialis, deafferentation and flight, 302 Meloloritha vulgaris, flight elytra, 170-171 model of wing, 166169 regulation of lift, 210 Metamorphosis, definition of stages, 6871 Microtrichia on wing and stalling, 177 Mimas tiliae, flight, 310, 321 Mosquito, sound using wings, 322, 326, 328 Moths, saturniid, aerodynamics, 293 Moult, usage of term, 68-71 Moulting and digestibility, 237-238 Musca domestica GABA in head, 54 stability in flight and antennae, 194 Musrina stabulans, flight nervous control, 3 13 reflexes, 200, 207, 209, 21 1, 213 stability, 191-194 Muscles flight differentiation, 21 8-223 in dragonflies, 307-309 in grasshoppers, 298, 300 in myogenic insects, 310-317 in neurogenic insects, 296309 properties, 3 19

Muscles, flight-cont. and temperature, 3 18-322 thoracic, of locust, 299 singing, in cricket, 298 Myogenic insects, flight coordination in flies, hypothesis, 315317 motor patterns, 309-314 multiphasic and metastable patterns, 314-315 sound production by wings, 326-33 1 Myrnieleon europaeus, cocoon, 133 N Na+ and synaptic transmission, 56 Neniatocera, wingbeat frequency, I73 Nembutal, and blocking of action potentials, 51 Neodiprion sertiffr, uric acid, 235 Neurogenic insects, flight dragonflies, 307-309 Lepidoptera, 309 locusts, 300-307 sound production by wings, 322-326 Nervous system (see Synaptic transmission, Flight) Nicotine and synaptic transmission, 2728 Nitrogen utilization, 231, 234, 273-5 Nutrition (see Food) Nutrition balance, 278

0 Odonata, flight differentiation of flight muscles, 218222 reflexes, 198, 199, 203, 213, 215 stability, 195, 197 Orimargula spp., spiracular gills, 75, 77, 78,90,91,93,95,98,99, 102, 105, 113, 114 Orimargicla australiensis, spiracular gills, 76,93,99, 102, 109, 130, 131 Oriniargula hintoni. spiracular gills, 77, 79, 103, 105, 131 Orthoptera flight differentiation of muscles, 219, 222 sound using wings, 322

S U B J E C T INDEX

Ort hoptera-con/. food starvation, 237 utilization of dry matter, 252, 253, 256,257,263 utilization of fresh matter, 258 Oryctes spp., flight initiation, 199 and vision, 333 yawing, 3 11 Oryctes boas, flight elytra, 169, 170 muscle differentiation, 220 stroke angle, 202, 214, 215 Oryctes rhinoceros, elytral surface area, 169 Oryzaephilus surinaniensis, speed of food through gut, 237 Oxygen and spiracular giils, 108-1 I2

353

Pharate, definition, 70-71 Phenobarbital and synapse blocking, 41 Pholus labruscoe, ganglia, 3,4, 5 Phonoctonus nigrofasciatus, food utilization, 260, 270 Phormia regina, flight kinematics, 293 stability, 191, 194-195 wing movement, 173, 179-186, 189190 Phormia terrae-novae, synapses, 5, 7 Phytometra gamma, feeding and age, 269 Pieris brassicae, feeding carbohydrate and lipid, 276 dry matter, 255 fresh matter, 259 indices, fresh and dry weight, 25 I intake, 249 nitrogen, 275 nutritive ratio, 278 Pieris rapae, energy utilization, 281 P Plastron (see Spiracular gills) Paropsis atoniaria, feeding Platyseius italicus, spiracular gills, 105 Plusia spp. intake, 249 utilization of fresh food, 258,270,271 crowding and growth, 265 Paulianina spp, spiracular gills, 100, 139, flight stability, 196 141, 143 Polypedilum vanderplanki, dehydration, Periplaneta americana 96 electrophysiology, 1-57 (see Synaptic Polarization level and ganglionic transmission) rhythm, 20-21 wing beat frequency and temperature, Pressure and spiracular gills 318 drop along plastron, 109-1 12 Periplaneta fuliginosa, metathoracic high, and resistance, 112-1 14 ganglion, 43-46 hydrostatic, and resistance, 105-107, PeritheateJ; spiracular gills, 13P 153 Petalura, flight stability, 197 Prodenia eridania, feeding Phagostimulants and nutrition, 230 carbohydrate and lipid, 276 Phalera bucephala, feeding dry matter, 247, 248, 254, 263, 264, and age, 267, 268, 270,271 265 indices, dry and fresh weight, 251 nitrogen, 275 utilization of passage time of food in gut, 237 carbohydrate and lipid, 276 unnatural foods, 251 cellulose, 277 Protein and nutrition, 234235,274-275, dry matter, 255 278 fresh matter, 258 Protocanace, spiracular gills, I54 nitrogen, 273, 274 Protoparce sexta, feeding Phaneroptera falcata, feeding different foods, 265 utilization of dry matter, 253 dry matter, 256 variation with sex, 272 gut contents, 236, 237

354

SUBJECT INDEX

Protoparce sexta, feeding-cont. intake, 249 and moulting, 238, 239 unnatural foods, 263, 264 Psephenidae, spiracular gills, 72, 74, 75, 82, 83, 86, 96, 114-120, 152 Eubriinae, 97, 105, 109, 113, 118-120 Psephenoidinae, 114-1 18 Psephenoides spp., spiracular gills, 83, 105, 112, 113, 114-118 Psephenoides gahani, spiracular gills, 109, 112, 115, 116, 117 Psephenoides marlieri, spiracular gills, 109, 112, 114, 115 Psephenoidrs volatilis, spiracular gills, 109, 112, 115, 116, 117 Psychodidae, spiracle, 139 Pterygota, flight reflexes, 205 Ptychoptera, flight muscle differentiation, 219

Rhodnius prolixus, feeding-coiit. oonversion of ingested food, 260 intake, 241 Romalea microptera sound production, 322 wingbeat frequency, 294, 29s

S

Sarnia Cynthia, flight motor patterns, 310 and temperature, 321 Sarcophaga spp.. flight peak lift, 292-293 reflexes, 205 wing position and lift, 173 Schistocerca gregaria flight aerodynamics and kinematics, 290291, 294,295 differentiationof flight muscle, 220, 221,222 R lift and thrust generation, 164, 165, Radioactive tracers in food utilization, 166, 171, 174, 178 245-246 motor patterns, 296, 298, 302, 306 Reflexes reflexes, 198,199,200,205,211,213, evasion in cockroach, 11 214 flight stability, 196-197 amptitude, frequency and power and temperature, 319-321 control, 200-206 wingbeat frequency, 294, 295 initiation, maintenance and terfood mination, 199-200 and age, 249, 268 list, 198-199 and crowding, 265 and size, 296 dry matter, 252, 272 stretch reflex, 304, 314 metathoracic ganglion, 40 and the two wing pairs, 292 Schistocerca shoslione, wingbeat freand temperature, 3 19 quency, 294 Refractory period, 33-35 Sclerocyphonfnscus, respiratory system, Repetitive stimulation, 36-38 72 Respiration Sex and variation in food utilization, action potentials in nerve cord, 12 271-272 and spiracular gills (see Spiracular Sialis Maria gills) apolysis, 85 Reynolds number and flight, 165-166, dehydration, 96 170,173,178,293 Simuliidae, spiracular gills, 73, 75, 85, Rhodniusprolixus 87, 90, 91, 92, 97, 98, 100, 105, 114, acetylcholinesterase, 8 133-141, 152 feeding Sinrulium spp, spiracular gills, 66, 67, and age, 249, 270 107, 108

SUBJECT I N D E X

Siniuliuni bequarti, 109 Simulium canadense, 109 Simulium costatum, 107, 133 Simulium equinum, 107, 109 Simulium hunteri, 109, 135 Simuliwn latipes, 107, 136 Simulium monticola, 107 Simulium ornatum, 92, 99, 107, 108, 109, 134, 137, 138 Simulium reptans, 107 Simulium variegatum, 109, 134, 135 Sirex, differentiation of flight muscles, 2 19,220 Sitophilrrr granarius, feeding uric acid in faeces, 235 utilization of fresh matter, 260 Size of insect and wingbeat frequency, 294-296 Sinerinthus populi, feeding and age, 271 fresh matter, 259, 269 Soma-somatic junctions, 3 Sound production using wings, 322-33 I myogenic insects, 326-331 neurogenic insects, 322-326 Sphaeriidae, spiracular gills, 74, 105, 156,158-1 59 Sphaerius ovenensis, spiracular gills, 156 Sphinx, stability in flight, 196 Spiracle regulatory apparatus, 139 in Sphaeriidae and Hydroscaphidac, 158-159 Spiracular gills, 65-161 high pressure resistance, 112-1 14 isolation of tissue, 84-104 attributes, 92-97 function, 97-100 origin, 84-89 time differences in, 89-92 tissue reservoirs, 100-104 in larvae, 156-159 Sphaeriidae and Hydroscaphidac, 158-159 Torridincolidae, 156158 metamorphosis, definition of stages, 68-71 plastron and environment, 66-68

355

Spiracular gills, plastron-cont. respiratory efficiency, 105-1 12 structure, 104-105 respiratory systems, pupal and adult interrelationships, 71-74 in pupae, 114-156 Blepharoceridae, 141-1 44 Canaceidae, 152-156 Deuterophlebiidae, 144-146 Dolichopodidae, 148-1 52 Empididae, 146148 Psephenidae, 114-120 Simuliidae, 133-141 Tanyderidae. 122-123 Tipulidae, 123-133 Torridincolidae. 120-122 Spodoptera frugiperda, feeding, cliromic oxide as marker, 244 Stability in flight, 190-198 Diptera, 190-195 others, 195-198 Stalia major, feeding and age, 238,249, 269, 270 fresh matter, 260 Staphylinidae, invasion of sea, 152 Starch, digestion, 277 Stratiomyidae, spiracle, 74 Sugars, digestibility, 275-277 Synapse criteria, 10 definition, 1 types, 2-7 (see also Synaptic transmission) Synaptic transmission, 1-64 ganglia acetylcholine and acetylcholinesterase content, 7-9 acetylcholine and activity, 23-24 anticholinesterases, 24-25 adrenaline and noradrenaline, 26 carbon dioxide, 23 curare and atropine, 25-26 DDT, 27 dopamine, 26 electrical phenomena, description, 11-18

general considerations of organisation, 9-1 1 K + and Ca2+,21-23

356

SUBJECT INDEX

Synaptic transmission, ganglia-cont. nicotine, 27-28 rhythm modification, 18-21 structure of synaptic regions, 2-7 properties after-discharge, 35-36 general characteristics, 28-33 inhibitory processes, 52-54 mechanism, 38-52 refractory period, 33-35 repetitive stimulation, 36-38 synaptic delay, 33 Syrphidae, flight reflexes, 21 1

T Tabanus sulcifrons, flight reflexes, 21 1 Tanyderidae, spiracular gills, 75, 86, 92, 99, 105, 122-123 Telmatogeton, secondary invasion of freshwater, 155 Temperature and flight, 318-322 and rhythm in ganglia, 18-20 Tenebrio molitor, feeding carbohydrate and lipid, 276 conversion of digested food, 251 dry matter, 257 nitrogen, 273, 274 nutritive ratio, 278 uric acid in faeces, 235 use of groups, 238 utilization of energy, 281-282 Tineola bisselliella, feeding fresh matter, 261 and sex, 271 and temperature, 266 uric acid in faeces, 235 Tbula, spiracular gills, 74 Tipulidae, spiracular gills, 73,75,77,79, 84, 90, 91, 92, 93, 97, 99, 100, 105, 113, 123-133, 152 Torridincola rhodesica, spiracular gills, 92, 120-122, 147, 156, 157 Torridincolidae, spiracular gills, 74, 86, 105, 120-122, 156-158 Triboliumcastaneum, food consumption, 246 Tribolium confusuni, feeding dry matter, 257

Triboliurn confusum, feeding-cont. fresh matter, 260 indices, dry and fresh weight, 251 uric acid in faeces, 235 and water, 266 Trichoptera, pupa, 69 Trogoderma granarium, food consumption, 246 Twinnia hydroides, spiracular gills, 133 Twinnia tatrensis, spiracular gills, 136 Tymbal action in cicadas, 330

U Urea and digestibility, 235 Urethane and synapse blocking, 41, 5 I Uric acid and digestibility, 234-235,273, 274 Urine and digestibility nicasurcnient, 233-235, 273, 278

V Vespa, flight differentiation of flight muscles, 219, 220 reflexes, 199 Vespoidea, flight reflexes, 204 Vision and flight in dragonflies, 309 and reflexes, 199 and velocity control, 206-208 and wingbeat phase, 304 and yawing, 214,217 Vitamin deficiency, 235

W Waggle dance in bees, sounds, 327-328 Weevil, spiracular gills, 66 Wings and Coleopteran flight, 166-169 folding, 204-205 kinematics of motion, 179-90 Apis mellifera, 186-190 Diptera, 179-186 sensory input from, 303 and sound production, 322-33 1 wingbeat frequency inhibition, 304

SUBJECT INDEX Wings-cont. wingbeat frequency-cont. in locust, 302 and size, 294-296 in small Diptera, 175 and sound production, 322-331 and temperature, 3 18-322

357

Wound repair in spiracular gills, 93-95, 101-1 02

X Xanthocanace nigrifrons, spiracle, 156 Xyelu, differentiation of flight muscles, 219, 221

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Cumulative List of Authors Nitnibers in bold face indicate the oolunie number of the series

Aidley, D. J., 4, 1 Andersen, Sven Olav. 2, 1 Beament, J. W. L., 2, 67 Boistel, J., 5, 1 Burkhardt, Dietrich, 2, 13I Bursell, E., 4, 33 Burtt, E. T., 3, I Catton. W. T., 3. I Chen, P. S., 3, 53 Colhoun, E. H., 1 , I Cotti-ell, C. B.. 2. 175 Dadd, R. H., 1, 47 Davey, K. G., 2, 219 Gilbert, Lawrence I., 4, 69 Harvey, W. R., 3. 133 Haskell, J. A.. 3, 133 Hinton, H. E.. 5, 65 Kilby, B. A., 1, 1 1 I Lees. A. D.. 3, 207 Miller, P. L.. 3, 279 Narahashi, Toshio, 1, 175 Neville, A. C., 4, 213 Pringle, J. W. S., 5. 163 Rudall, K . M., 1, 257 Shaw, J., 1, 315 Smith, D. S., 1, 401 Stobbart, R. H., 1, 315 Treherne, J. E., 1, 401 Waldbauer, G. P., 5, 229 Weis-Fogh, Torkel, 2, I Wigglesworth, V. B., 2, 247 Wilson, Donald M., 5, 289 Wyatt, G. R., 4, 287

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Cumulative List of Chapter Titles Numbers in bold face indicate the volume number of the series

Active Transport and Passive Movement of Water in Insects, 2, 67 Amino Acid and Protein Metabolism in Insect Development, 3, 53 Biochemistry of Sugars and Polysaccharides in Insects, 4, 287 Biochemistry of the Insect Fat Body, 1, 11 1 Chitin Orientation in Cuticle and its Control, 4, 213 Chitin/Protein Complexes of Insect Cuticles, 1, 257 Colour Discrimination in Insects, 2, 131 Comparative Physiology of the Flight Motor, 5, 163 Consumption and Utilization of Food by Insects, 5, 229 Control of Polymorphism in Aphids, 3, 207 Control of Visceral Muscles in Insects, 2, 219 Excitatioli of Insect Skeletal Muscles, 4, 1 Excretion of Nitrogen in Insects, 4, 33 Feeding Behaviour and Nutrition in Grasshoppers and Locusts, 1,47 Functional Aspects of the Organization of the Insect Nervous System, 1,401 Hormonal Regulation of Growth and Reproduction in Insects, 2, 247 Image Formation and Sensory Transmission in the Compound Eye, 3, 1 Insect Ecdysis with Particular Emphasis on Cuticular Hardening and Darkening, 2, 175 Lipid Metabolism and Function in Insects, 4, 69 Metabolic Control Mechanisms in Insects, 3, 133 Nervous Control of Insect Flight and Related Behaviour, 5, 289 Osmotic and Ionic Regulation in Insects, 1, 315 Physiological Significance of Acetylcholine in Insects and Observations upon other Pharmacologically Active Substances, 1, 1 Properties of Insect Axons, 1, 175 Regulation of Breathing in Insects, 3, 279 Resilin. A Rubberlike Protein in Arthropod Cuticle, 2, 1 Spiracular Gills, 5, 65 Synaptic Transmission and Related Phenomena in Insects, 5, 1

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