Advances in Insect Physiology
Volume 17
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Advances in Insect Physiology edited by
M. J. BERRIDGE J. E. TREHERNE and V. B. WIGGLESWORTH Department of Zoology, The University Cambridge, England
Volume 17
1983
ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers London New York Paris San Diego San Francisco SBo Paulo Sydney Tokyo Toronto
ACADEMIC PRESS INC. (LONDON) LTD 24/28 Oval Road London NW1 7DX United States Edition published by ACADEMIC PRESS INC. 111 Fifth Avenue New York, New York 10003
Copyright 0 1983 by ACADEMIC PRESS INC. (LONDON) LTD
All Rights Reserved No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers
ISBN 0-12-024217-6 ISSN 0065-2806
Printed in Great Britain at The Pitman Press, Bath
Contributors W. Henzel Department of Biology, University of Massachusetts- Boston, Dorchester, Mass, USA
G. J. Goldsworthy Department of Zoology, University of Hull, England
H. Lipke Department of Biology, University of Massachusetts- Boston, Dorchester, Mass, USA
M. Raabe Laboratoire de Neuroendocrinologie des Insectes, P. et M . Curie Universiti, Paris, France
M. Sugumaran Department of Biology, University of Massachusetts- Boston, Dorchester, Mass, USA V. B. Wigglesworth Department of Zoology, University of Cambridge, England
V
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Contents Contributors
V
Mechanisms of Sclerotization in Dipterans H. LIPKE, M. SUGUMARAN and W. HENZEL The Physiology of Insect Tracheoles V. B. WIGGLESWORTH The Endocrine Control of Flight Metabolism in Locusts G. J. GOLDSWORTHY
85 149
The Neurosecretory-Neurohaemal System of Insects; Anatomical, Structural and Physiological Data 205 M. RAABE Subject Index
305
Cumulative List of Authors
314
Cumulative List of Chapter Titles
316
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Mechanisms of Sclerotization in Dipterans Herbert Lipke, Manickam Sugumaran and William Henzel Department of Biology, University of Massachusetts, Boston, Dorchester, Massachusetts, USA
1 Introduction 2 The protomer-matrix transformation 2.1 Criteria for sclerotization 2.2 Kinetics of dimer assembly 2.3 Protein and nucleic acid synthesis 3 Composition and preparation of larval proteins 3.1 The cyclorrhapid integument 3.2 The nematocerid larval cuticle 4 Composition of sclerotized tissue 4.1 The cyclorrhaphid puparial case 4.2 The pupal cuticle of nematocera 4.3 The adult stage 4.4 The egg stage 5 Chemical mechanisms of cross-linking 5.1 Ring substitutions 5.2 p-Sclerotization 5.3 A combined pathway 6 Developments and prospects Acknowledgements References
1 3 3 5 9 14 14 36 38 38 47 49 51 51 53 60 71 73 75 75
1 Introduction
In register with expanding interest in the development of insects, examination of the insect integument currently extends to many processes leading to the synthesis, recycling, deposition and maintenance of skeletal tissue. As a consequence of simultaneous advances in chemistry, microsurgery, microscopy and genetics during the period 1930-70, the influence of the endocrine systems on the control of metamorphosis was established. In the course of these “Advances in Insect Physiology” Volume 17 (edited by M. J. Berridge, J. E. Treherne and Academic Press, London and New York. 1
v. B. Wigglesworth).
2
H. L I P K E e t a / .
early studies, the focus of each investigation was some broad aspect of cuticle development as expressed during pupation, pigmentation or bristle distribution, for example. In the current era, however, and in confirmation of the dictum “everything in the body (cell?) depends on everything else”, students of cuticle biology now assimilate detailed reports on highly specialized systems in the hope that a universal model will obtain. Unfortunately, in the pursuit of this ideal, major inconsistencies in developmental programmes, in enzyme localization, in functional group activity and in protomer composition have become apparent. This search for a generalized mechanism has persisted in spite of the acknowledged heterogeneity of structural components in the cell wall of prokaryotes and plants or in the connective tissue of vertebrates and invertebrates. Indeed, diversity in the biological and chemical aspects of peptidoglycan, lignin, melanin and proteoglycan structure supports a comfortable prosperity, not only among practitioners of these chemical arts, but in the publishing trade as well. The formation and disposal of hardened regions of the exoskeleton require precise integration of virtually all of the major synthetic and catabolic systems of the organism. In species where the trehalose-glucose-glycogen triad provides energy for skeletal development, more than 75% of the carbon can be transferred directly to the integument or consumed in side reactions fuelling deposition of the lamina (Lipke et al., 1965b,c; Ferrus and Kankel, 1981). When mobilization of resources attains this exceptional level, any one of a multiplicity of biochemical systems can be invoked as the key reaction in the formation of the sclerotized entity. Thus peripheral aspects of cuticle development are frequently presented as contributions to the mechanism of sclerotization, per se, when the issue would be better served by greater circumspection. For this reason the present discussion is restricted in two respects, taxonomic and biochemical, With respect to the biochemistry of hardening and bond stabilization, only those reactions contributing to crosslinking and the decline in chemical reactivity will be discussed in depth. Related processes dealing with the characterization of unconjugated phenols, epidermal transformations, gene activations, polymer resorption, endocrine secretion, haemolymph precursors, and wound metabolism will be left to other specialists. In focusing on the Diptera, clear phylogenetic limits are imposed with ample provision for ecological diversity and developmental patterns. Of the 105 species within the chosen taxonomic group, no more than a dozen examples will be taken as representative of the
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N D I P T E R A N S
3
order. Many families of great economic and medical significance have been overlooked by cuticle physiologists on the basis of unavailability or inconvenience. The important families yet to be examined include the Simuliidae, Tabanidae, Gasterophilidae, Tachinidae, Glossinidae, Hippoboscidae and the Tephritidae, to name but a few. At least one species of each of the above families has been reared in the laboratory; lack of material, therefore, cannot be the cause of the neglect. For several reasons, gaps in comparative aspects of cuticle structure should be filled in the near future. Countries formerly dependent on foreign experts have developed indigenous research facilities manned by local personnel. The trend toward integrated pest control requires detailed information on the ecology of each insect-host couple, including the biology of the integument since each life stage is fashioned for a particular environment. On the assumption that safe and efficient pesticides can be developed targeted for those processes without counterparts in benign groups of arthropods or higher forms, unique features of cuticle development are sought as objects for the action of specific inhibitors. The immediate advantages to be derived from these practical exercises are of sufficient consequence to guarantee continued activity in this specialty extending not only to aspects requiring heavy investment in instrumentation but to field practice as well. With these acknowledged limitations concerning the scope of this review, a simplified classification of the Order Diptera is presented in Fig. 1. The phyletics are based solely on the “integumental wisdom” of those investigators choosing to exploit these few groups as experimental subjects. 2 The protomer-matrix transformation
2.1 C R I T E R I A F O R S C L E R O T I Z A T I O N Regardless of the life stage or body region, the changes accompanying crosslinking are easily detected at a superficial level. Although a precise ordering of events has not been realized, the products clearly differ from the reactants in physical and chemical characteristics. A full description of the sequence of reactions requires the delineation of the time-course of each reaction contributing to the completion of the sclerotized matrix. It would appear initially that these objectives would be easily accessible, since crosslinking takes from a few hours to several days to complete in the intact animal, and the process can be accelerated or retarded by manipulation of
4
H. LIPKE e t a / . Order Diptera
Cretaceous)
Suborder
(-108 years)
Vematocera Brachycero
/
+
Tabanomor pha \ A <silo morpha lnfraorder
Lower Oligocene (- 5 x 105 yeors)
\
Cyclorrhapha
' I
Drosophiloidea
Muscdidea
Superfamily
A
'\
Muscidae Family
Subfa mi Iy Callipiorinae
Fig. 1 Simplified phylogeny of the Diptera The Drosophiloidea typify the acalypteratae and the muscoidea represent the calypteratae.
the internal and external environments. In the species examined to date, however, technical difficulties have precluded presentation of a kinetic catalogue using chemical probes. Some of the steps, such as dimerization, for example, call for rapid flow procedure to accommodate time scales in the range of milliseconds or less. Endogenous crosslinkers of natural origin interfere with the monitoring of spin labels by displacement of the probe from the derivatized residues of the primary chain, or by the generation of new signals that interfere with the labelled reactants. When ratios of buried to exposed functional groups are followed during cuticle hardening it has not been possible to distinguish between chemical modification of the side chain of a particular amino acid residue and loss of reactivity due to internalization. The choice is complicated further by the possibility of more than one route to sequestration,
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N DIPTERANS
5
since the transfer of a group to the interior of a globular construct is presently indistinguishable from the compression of fibrous members to form a bundle. A similar ambiguity accompanies the assignment of dehydration indices, since the contributions of each water species can vary with changes in cuticle density, particularly in the categories of non-solvent, immobilized, ice-like and bulk water. Integumental loci subject to crosslinking share properties in common although each manifestation may vary in magnitude and temporal order. Reactions characteristic of puparium formation have counterparts in adult sclerites, the egg shell and in hardened appendages of larval forms. To a degree dependent on the density and hardness of the tissue the criteria for sclerotization include: 1. Decreased solubility of proteins and lipids. 2. Addition of bridges between proteins and between proteins and chitin. 3. Increased molecular weight of the structural polypeptides. 4. Altered packing of protein and polysaccharide. 5. Sequestration of functional groups. 6. Declining response to agents mediating enzymatic or chemical cleavage. 7. Extrusion of water. 8. Reorientation of chitin fibrils. 9. Pigmentation (excluding defensive colorations). 10. Post-translational modification of primary structures. A number of reviews and technical manuals have dealt with various aspects of sclerotization and crosslinking (Horspool, 1969; Rogers, 1978; Andersen, 1979a; Guay and Lamy, 1979; Jungreis, 1979; Silvert and Fristrom, 1980; Miller, 1980; Brunet, 1980; Sherald, 1980; Roberts and Brach, 1981). The breadth of opinions attests to the vigour of this branch of insect physiology and anticipates both lively disagreement and substantial progress in the future.
2.2 K I N E T I C S O F D I M E R A S S E M B L Y The joining of two or more monomeric polypeptides by the introduction of a covalently bound bridging unit calls for chemical definition of the reactants and products, namely the structural proteins, the activated crosslinker, the electron carriers and the fused dimer. Identification of the two proteins participating in the initial stages of matrix formation constitutes the first step, followed
6
H. LIPKE e t a / .
by characterization of the type of residue subject to modification and finally the location of this residue in the primary sequence. If exposure of the susceptible residue is an additional factor in identifying the rate-limiting step, changes in configuration must also be taken into account. The joining of additional protomers to the dimer to form higher n-mers may be very rapid as in an apparent concerted mechanism and may procede by repetition of the dimerization process, although no evidence supports this contention. If indeed the dimer is transient in the route to a multichain complex, the initial bridge would be present in low concentration compared to crosslinks added subsequently and could be overlooked, especially if notably different in chemical structure and stability. In the following discussion it will be shown that sclerotized systems afford a variety of putative crosslinks strongly suggestive of high specificity with respect to participation in different stages of the pathway to oligomers. Although knowledge of the chemical structures is necessary for adequate description of the process, scheduling of the participants is of equal importance to the biosynthetic programme. From the temporal perspective, crosslinking consists of rapid substitution reactions of the order of lo-* to 10-los and slower processes consuming hours or days. The fastest component is associated with the formation of a covalent link between the bridge precursor and the first protein. The interval during which the second structural member is added to the product of the first reaction is also brief, although it may be significantly longer than the initial coupling reaction. The events preceding coupling constitute additional synthetic processes that contribute to sclerotization. In these cases the time scale for synthesis, transport and modification of the polypeptides is much greater than the bridging reaction, per se. Implicit in the elucidation of this slower component is the history of each of the two proteins when viewed in the context of the entire instar. In the simplest case, both proteins are synthesized and transported to the appropriate strata hours or days before crosslinking is initiated. On the other hand dimerization may require monomers of relatively recent origin. These two extremes can be adjusted to accommodate several intermediate conditions to satisfy both the insect and the investigator. The aromatic bridging unit, for example, may be activated by combination with a newly synthesized protein following which an older polypeptide is attacked by the reactive complex. To provide for the large excess of older to newer elements in the Dipteran tanning systems, the initiation step may utilize two pro-
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N D I P T E R A N S
7
teins of recent origin, the resulting dimer serving as the nucleus for subsequent addition of a number of the less reactive older structural polypeptides in concert. In the context of sclerotization, classification of a protein as old or new may be difficult if post-translational modifications immediately precede participation of a preformed polypeptide in dimer formation. The transport of a protein to the reaction site is an additional factor in construction of the time table. An old protein migrating to the reaction locus is modified at least twice, first as a signal for transport to commence, and again on arrival at the site of crosslinking. Proteins such as calliphorin, lucilin, Drosophilin and their counterparts in other families clearly fall within this category (Wolfe et al., 1977; Thomson et al., 1976; Scheller et al., 1980; Roberts and Brach, 1981).
2.2.1 Phylogenetic considerations It is evident that these considerations play a major role in the choice of experimental material. Insects with incomplete metamorphosis synthesize large amounts of cuticle protein during sclerotization, thus an important tool for the study of synthetic mechanisms and reaction kinetics, namely tracer methodology, is compromised. In Dipterans the deposition of structural proteins is by no means uniform either on an anatomical or developmental basis, and tanning procedes with high or low rates of synthesis depending on the tissue and life stage. If the time course and reactants involved in sclerotization are to be described, long lived participants of ready availability are preferred. These requirements speak against the study of crosslinking in the egg, pupa or developing adult and emphasize the advantages derived from the use of the cyclorrhaphan puparium as an experimental subject. In the course of the maggot-puparial transformation, the bulk of the proteins of the ultimate instar are subject to conjugation with companion polypeptides or chitin-protein complexes. In the case of drosophilids, mutants blocked at discrete steps in the pathway to the finished puparial sheath are available. Control of temperature and moisture affords convenient routes to the synchronous development of cultures of the larger Calyptratae. In this group endocrine and nutritional factors are known in sufficient detail to plot the course of ion and precursor flux and a battery of inhibitors are available to block specific steps of matrix formation. In the selection of this model, however, a number of qualifications must be acknowledged. The coloration of the puparium combines two pathways of unknown
8
H . L I P K E eta/.
relationship, first the synthesis of benzenoid chromophor from the arylated amino acid residues of the crosslink and second from the deposition of melanoprotein. The dissociation of these pathways encounters major technical and conceptual difficulties. To some extent, this problem can be circumvented by restricting the search to the early white or amber-pink stages or by organ culture. The assignment of a protein to a given age class, furthermore, constitutes a problem of much greater complexity than originally believed. Whereas gross analysis for chitin and protein suggested that the near constancy of the protein-chitin ratio during pupariation precluded significant turnover, the evidence is no longer convincing with respect to the protein (Fraenkel and Rudall, 1947; Rudall, 1967). That the increase in the dry weight of the puparium is due principally to the dehydration and the deposition of bridging groups and melanin is unquestioned (Hillerton and Vincent, 1979). However, the redistribution of old protein and the synthesis of new polypeptides of unknown function is now well documented. Redistribution increases solubility by loosening non-covalent bonds between two polypeptides. Conditions favourable to dissociation of two chains joined non-covalently would be promoted by participation of one member of the pair in crosslink formation with a third protein (Mitra and Lawton, 1979). The displaced polypeptide may be retained by the integument or may be degraded by epidermal cathepsins (Knowles and Fristrom, 1967; Bautz ef nl., 1973; Deloach and Mayer, 1979; Katsoris et al., 1981). Both de novo synthesis and partial proteolysis, therefore, may be required for sclerotization without causing major changes in protein levels, solvent partition patterns or amino acid composition, and hence may be overlooked by the investigator (Hackman and Goldberg, 1971; Mayer et al., 1979). Inthe Cyclorrhapha the cuticle of the third larval instar is converted to the puparium by modification of both the non-glycosylated proteins and the polypeptide joined to chitin. Some of these proteins are synthesized and deposited in the cuticle early in the last instar and some immediately preceding sclerotization (Snyder et al., 1981). Within the group synthesized at the conclusion of the larval stage, when the maggots have ceased feeding and have migrated from the substrate, additional temporal subdivisions can be detected comprising (a) proteins synthesized during the hours just prior to the retraction of the head and (b) proteins synthesized during formation of the puparium. The destiny of the proteins falling within these two subclasses has not been established. A significant
M E C H A N I S M S O F SCLEROTIZATION I N DIPTERANS
9
portion are added to the puparial case, while others may be directed to the viscera or may initiate particular steps in the deposition of the pupal exoskeleton. In this taxonomic group, completion of the puparium and deposition of the pupal cuticle overlap to the degree that the two processes are not synchronous in head, thorax and abdomen (Roseland and Schneiderman, 1979; Madhaven and Madhaven, 1980; Utsumi and Natori, 1980a). When the entire animal is sacrificed to establish the rate of tissue synthesis, activity of a particular region of the body is confounded at the expense of information on the local economy of protein, nucleic acid and chitin (Pearson, 1974; Kiss et al., 1978; Mayer et al., 1979; White and Lassam, 1979). 2.3
PROTEIN A N D N U C L E I C ACID SYNTHESIS
If deposition of structural proteins was confined solely to that period of the last instar coinciding with food intake, synthesis of protein by the epidermis should undergo a major decline during the post-feeding and white prepuparial stages (Fraenkel and Bhaskran, 1973). By a number of approaches it has been established that such is not the case. Exclusive of fat body, carcass ribosomes originating principally from the epidermis remain more or less constant in number during puparium formation in C. erythrocephala (=C. vicina), thus the potential for polysome formation is retained throughout this interval (Sridhara and Levenbook, 1974). Polysomes prepared from cuticle scrapings purported to consist principally of epidermal cells incorporate [3H]-leucine into ribonucleoprotein when fortified with the pH 5 fraction from rat liver. The synthetic capacity of the polysomes was identical for six-day-old larvae and white (untanned) calliphorid pupae (Fragouli-Fournogeraki et al., 1978). Translation of 5-18s mRNA from 6-day larvae and prepupae on mouse liver ribosomes enabled Fragoulis and Sekeris (1975) to assess gross message synthesis as well as production of dopa decarboxylase, per se. As predicted by differences in ecdysone titres, a four-fold increase in enzyme production was evident in the prepupae judged by titres of specific antibody-precipitable enzyme. In contrast to dopa decarboxylase, total cell-free product including putative structural polypeptides declined only 20% at the commencement of pupation in keeping with the maintenance of appreciable synthetic activity (Table 1). Production of translatable prepuparial mRNA was at the expense of high molecular weight heterogeneous DNAlike RNA. The messenger precursor was recovered from nuclei of
10
H . L I P K E eta/.
TABLE 1 Total cell-free product and immunoprecipitable dopa-decarboxylase from 6-day larval and prepupal 5-18s RNA (from Fragoulis and Sekeris, 1975). The system contained 2.6pg of mRNA from the indicated life stage, mouse liver ribosomal subunits, the pH 5 fraction from rat liver and 3 FCi [14C]leucine. Immunoprecipitation was performed after 30min at 37°C with rabbit anti-dopa decarboxylase
mRNA source
Incorporation of I4C leucine into Total cell-free Enzyme product (A) protein (B)
White prepupae 6-7-day larvae
35000 k 2988 counts/min 250 + 23 48000 k 2111 88f11
B/A 100)
(X
0.70 0.18
epidermal cells at the onset of the maggot wandering stage which falls late in the last instar of C. vicina (Shaaya, 1976a,b). Uniform rates of protein synthesis are expressed by the correspondence of (a) incorporation indices from labelled precursors and (b) the amino acid mole ratios in the polypeptide. With the assumption that precursor pools remain relatively constant, and with normalization of the data to accommodate differences in specific activity of the isotopic building blocks, variations in the time-course of protein synthesis within an instar can be assessed. On the other hand, if the adjusted values for radioactivity are not in accord with amino acid composition, a change in the array of cuticle proteins is indicated during the third instar. When 18 labelled amino acids were administered individually to maggots of Sarcophuga bullata at the commencement of the third instar, major differences in amino acid composition and incorporation rates were evident (Table 2). Discordance of the two parameters was observed within classes of amino acids based on either polarity or nutritional dependence (Sugumaran and Lipke, 1982a). Disparate rates of synthesis could also be inferred from glycopeptide analysis. At least two polypeptides are appended to chitin at the conclusion of pupariation, one present during the larval stage with high levels of glycine in the vicinity of the unions between protein and chitin and a second group added during hardening of the puparium with glutamic acid predominating at these loci (Table 3). The component(s) added late in the process and enriched in glutamic acid could be a protein synthesized and stored prior to polysaccharide modification or could be translated later, at the onset of glycoprotein assembly (Lipke and Strout, 1972; Strout et al., 1976; Kimura et al., 1976).
11
M E C H A N I S M S O F SCLEROTIZATION I N DIPTERANS
TABLE 2 Incorporation of [14C] amino acids into proteins of sarcophagid puparial cases. Each larvae received 1 . s 2 . 0 X lo5dpm of labelled precursor at the commencement of the last larval instar and the puparium harvested following metamorphosis. Portions of the washed and powdered cases were hydrolysed for amino acid analysis and oxidized with HC1O4-H2O2for assessment of total incorporation per puparium (from Sugumaran and Lipke, 1982a) Precursor Tyrosine Lysine Histidine Glutamic acid Proline Aspartic acid Glycine Serine a-Alanine Valine Threonine Isoleucine Leucine Phen ylalanine Arginine Methionine Cysteine Tryptophane
Residue mol percent
percent
3.6 3.6 3.6 12.3 11.8 10.2 10.2 8.6 8.6 8.3 5.9 4.3 3.6 3.1 2.4 nil nil nil
10.0 3.4 8.1 1.2 2.3 1.4 1.4 1.3 3.1 1.9 2.5 1.7 0.9 5.2 8.1 1.4 3.0 5.3
1 4 c
TABLE 3 Mole ratios of amino acids from Sephadex G-10 glycopeptides in formic acid extracts of Sarcophagid cuticle; values expressed as residues00 mg of peptidochitodextrin (from Kimura et a l . , 1976) Fraction Larval LA-I LA-I1 LA-I11 White puparial WA-I WA-I1 WA-I11 Puparial PA-I PA-I1 PA-I11
Glu
Lys
GlY
GlY
0.31 0.23 0.19
0.17 0.28 0.08
0.34 1.47 0.88
0.14 0.44 0.45
1.94 1.77 2.94
0.81 0.62 0.61
12
H. LIPKE e t a / .
2.3.1 Methodology
Without detracting from the value of the maggot-puparial transition as a model system for following crosslink formation, the observations discussed above must be integrated into new proposals on the course of the phenomenon. Regardless of the species under investigation, the appearance of polypeptides with molecular weights in accordance with the dimensions of a dimer or trimer, must be viewed from the position that one or more of the polypeptides are synthesized de novo and may not represent a pre-existing structural member. From the standpoint of methodology, the relevant criteria should include not only classification on the basis of molecular weight, but also (a) the amino acid sequence of the candidates, (b) cross-reactivity with respect to the purported monomer, and (c) the kinetics of assembly of each of the polymeric components of the scheme (Mills et al., 1967; Strout and Lipke, 1974; Willis et al., 1981). 2.3.2 Preparation of larvalproteins
High performance liquid chromatography and two-dimensional polyacrylamide gel electrophoresis are the principal techniques currently favoured for resolution of integument polypeptides. These procedures separate proteins differing very slightly in amino acid composition and molecular weight, but unfortunately problems arise at the preparative rather than the analytical level. With dissociating or non-dissociating gels, thin slabs (0.1 mm) are clearly superior to thick ( l m m ) in resolving power as are analytical scale HPLC supports over larger diameter columns of high capacity. Given the need for unequivocal purification of a polypeptide for sequencing or complement fixation, the current trend is to chemical probes that respond to very low levels of protein recovered from a single narrow band on a thin gel. These new approaches have revealed minor isolates that play important roles in skeletal development. On the other hand, increased sensitivity and resolving power have introduced new uncertainties particularly with respect to microheterogeneity , contamination with non-cuticular protein and spurious variants arising from proteolytic cleavage during sample preparation. The ability of a pure protein to migrate to more than one region of a gel is troublesome on crowded gels and is most frequently caused by substandard ampholytes or protein-protein
MECHANISMS OF SCLEROTIZATION I N DIPTERANS
13
interactions (Cann, 1979). With respect to the presence of contaminating proteins extracted from intersegmental muscles, it may be necessary to establish the cuticular origin of a minor component by immunodiffusion against antibodies to pure muscle proteins (Crossley, 1968). Proteolytic or oxidative changes are minimized by the addition of phenylmethanesulphonyl fluoride and phenylthiourea. Most cuticle proteins exhibit solubility minima below pH 5. In situations calling for minimum exposure to inhibitors, repeated extraction at p H 4-5 under an inert atmosphere in the presence of ascorbic acid can reduce artifacts to acceptable levels. At this acidity cuticle proteins are minimally soluble whereas visceral components pass into solution. A wide choice of equipment and procedures is available for processing both small and large samples. Maggot sheaths can be scraped to release epidermal cells reasonably free of visceral fragments. Providing calliphorin and rel&ed proteins are removed by a preliminary sedimentation, the disrupted epidermal cells are a good source of nuclei, ribosomes and mRNA (Sekeris et al., 1974; Fragouli-Fournogeraki et al., 1978; Sumner-Smith and Phillips, 1979). Fristrom and associates described procedures for the harvest of cuticle proteins applicable to a single Drosophila larva (Fristrom et al., 1978) or for numbers in excess of lo6 larvae (Eugene et al., 1979). Satisfactory preparations have been obtained by judicious operation of conventional blenders and Potter-Elvehjem or Dounce disintegrators, devices capable of considerable flexibility with respect to sample size and solvent volume. The best features of these two apparatuses are combined in the tubular disintegrator designed for Drosophila by Zweidler and Cohen (1971) and modified by Zomer (1978) to accommodate other species. As depicted in Fig. 2, oxygen may be excluded from the system and buffer programmes changed as required. Separation procedures based on attrition of softer organs are suspect with regard to the removal of loosely bound cuticular components along with formed elements from the body cavity. Comparisons of hand versus machine-dissected preparations usually resolve this uncertainty. A genuine need exists for automated methods of separation of individual layers of the cuticle. The practice of retrieval of exuviae for sampling epi- and exocuticular material is difficult in the case of Dipterous larvae inhabiting semi-solid media due to poor visibility, exposure to alkaline pH, and the likelihood of partial autolysis by digestive enzymes secreted into the diet.
14
H. L I P K E e t a / .
I c D E F I Fig. 2 Continuous feed automatic dissection device for dipterous larvae. (From Zweidler and Cohen, 1971; Zomer, 1978) A. Buffer chamber; B. Insect container; C. Mixing chamber on magnetic stirrer; D. Zweidler conical disruptor; E. Second homogenizer; F. Chilled sieving system on magnetic stirrer.
3 Composition and preparation of larval proteins
3.1
THE CYCLORRHAPHID INTEGUMENT
Washed maggot sheaths extracted with 6-8 M urea; pH 8.6; 4-8 M guanidine or 2% NaZB407 containing 0.2-2.0% sodium dodecyl sulphate (SDS) at temperatures ranging from 2°C to 60°C release approximately 50% of the cuticle protein to the solvent. Precise information on the relative solvating ability of the three media has not been forthcoming, usually the proteins can be transferred from one solvent of this group to another without significant loss of individual components. When the soluble proteins are separated by electrophoresis under conditions favouring dissociation it is evident that the bulk of the proteins fall within the molecular mass range between 9 and 30kd with minor components in excess of 50kd. Isoelectric points (PI) range from p H 4 to 6 as assessed with ampholytes poised at these acidities. Unlike the Blattids, the proteins are not conjugated with lipid or carbohydrate and are
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N D I P T E R A N S
Volume eluate ( m i )
'
~
15
1
Fig. 3 Molecular sieve chromatography of proteolytic digests of cuticle from larvae and puparia receiving [7 - I4C] dopamine prior to pupation. Borate extracts of the two cuticles were digested serially with S. aureus V8 protease, subtilisin, Pronase, trypsin carboxypeptidase A and pepsin and chromatographed on Bio-Gel. A, larval; B , amber puparial cuticle. (From Lipke et a / . , 1981)
devoid of pigment if prepared and stored in the presence of reducing agents or copper chelators (Lipke et al., 1965a, 1981; Snyder et al., 1981; Silvert and Fristrom, 1982). Little differences in electrophoretic patterns are manifest when second, early third or first instar sarcophagid proteins are compared. At least one of the genes coding for structural proteins in Drosophila is expressed later than the other four, however (J. W. Fristrom, personal communication). Despite similarities in PI and composition favouring strong noncovalent interactions, shielding of hydrolase-susceptible peptide bonds is not as strongly expressed as in puparial extracts (Lipke and Geoghegan, 1971a). Figure 3 presents the molecular weight distribution of peptides generated from larval and puparial proteins by proteolytic digestion with enzymes specific for linkages of relative abundance in the protein mixture (Lipke et al., 1981). It can be seen from elution pattern A that the bulk of the fragments chromatographed in accord with standards corresponding to amino acids and oligopeptides (0-2-1 kd). When drosophilid proteins were assessed for relatedness by peptide fingerprinting, all the isolates were cleaved by Staphylococcus aureus protease V8 and several afforded fragments of apparent identity (Silvert and Fristrom, 1982). This enzyme cleaves almost exclusively at the C-terminal
16
H. L l P K E
et al.
aspect of aspartyl and glutamyl residues which account for 20-25% of the amino acids in structural proteins of this species (Fristrom et al., 1978). 3.1.1 Unresolved mixtures Amino acid analysis of protein mixtures provided intial stimuli for intraspecific comparisons and selection of reagents for group modification and cleavage, Definitive criteria for homologies, whether mathematical, chemical or immunological are based on properties of purified proteins and can be misleading when applied to unresolved mixtures. Table 4 compares an acalypterate dipteran, D.melanogaster, with three Calypteratae on the basis of amino acid composition. Larval proteins from the drosophilid appear related to the sarcophagid, whereas limited homology has been observed based on N-terminal sequences, total number of proteins or electrophoretic mobility (Mulligan et al., unpublished observations). Conversely, L. cuprina (a calliphorid) and D. melanogaster differ with respect to Asp, Ser, Pro, Ile, Phe and Met content in spite of pronounced immunological crossreactivity (J. W. Fristrom, personal communication). The nematoceran is strikingly similar to C. vicina and L. cuprina, phenylalanine content possibly excepted, whereas habitat and phylogeny are disparate. Comparisons of acidic proteins, furthermore, are suspect without amide titres. This value is 40% for the mixed cuticle proteins from S . bullata. Cuticle extracts comprised of unresolved protein mixtures have provided useful approximations of general properties if interference by companion polypeptides is appreciated. As a group, the proteins soluble in urea are responsible for the 41 8, axial repeat of diffraction patterns (Rudall, 1976). This reflection represents a regularity associated with every four chitobiose residues of a peptidylated chitin chain. Since substantial levels of protein remain attached to the polysaccharide after exposure of the sample to urea, covalently bound protein may be joined to the carbohydrate polymer at a different locus. The patterns could not distinguish between elimination of protein from the 41 8, system or conversion to a less ordered form incapable of generating a reflection at this repeat distance (Rudall and Kenchington, 1973). In this respect, assignment of a given protein to a specified locus in a three-dimensional construct of the cuticle matrix encounters artificial distinctions between structural polypeptides and proteins with enzymic functions. Based on recoveries of soluble enzymes of carbohydrate metabolism as well as
TABLE4 Amino acid composition of extractable larval cuticle proteins of selected Diptera. Values in residues per 1000 ~
Nernatocera" A. aegypti
ASP Thr Ser Glu Pro GIY Ala Val Ile Leu TYr Phe His LYS '4%
Met
80 39 82 117 60 114 132 58 24 46 39 112e 22 42 36
-
Calliphorab vicina
Luciliab cuprina
78.7 48.9 102.8 113.6 80.9 122.3 107.5 76.1 28.1 40.1 37.9 36.8 66.2 42.3 15.0 3.2
80.2 42.6 110.7 109.2 80.6 121.3 115.7 77.1 21.5 43.6 32.4 55.0 52.2 44.2 16.7 2.7
Cyclorrhapha SarcophagaC bullata
140.4 40.2 77.4 141.1 62.1 82.3 87.6 54.1 20.3 49.5 45.8 37.8 63.7 73.4 24.2 -
Drosop hilad rnelanogaster 121 50 66 120 55 99 103 92 51 46 48 28 47 47 26 -
N-termini Ile Val Ala GlY Glu
Asx 51.6% Val 18.4% Gly 14.3% Ala 8.7% Leu 7.0%
Zomer and Lipke (1981). Hackman and Goldberg (1976). Lipke et al. (1981). Fristrom er al. (1978). Includes glucosamine.
18
H. LIPKE eta/.
lysosomal hydrolases, a factor of lo2 probably represents the minimum for the difference in concentration between proteins with a metabolic as opposed to a support function in exoskeleton (Knowles and Fristrom, 1967; Bienz and Diek, 1978; Candy, 1979). However, this classification on the basis of metabolic function is debatable. First, there is virtually no information on the enzymatic activity within the structural proteins as a group, particularly with respect to regulation of ion flux. Second, phenolases, peroxidases and hydroxylases apparently have dual roles in sclerotization, since these enzymes are firmly bound to the matrix, thus constituting structural material while retaining high catalytic activity (Yamazaki, 1969; Locke, 1974; Andersen, 1979b; Barrett and Andersen, 1981; Sugumaron et al., 1982). 3.1.2 Configuration Assessments of secondary and tertiary structure have been attempted in mixtures of cuticle proteins. When prepared from C. vicina by extraction with 7~ urea, eleven components were observed in gels run under dissociating conditions, the polypeptides consisting predominantly of material of M, 14 000-16 000. Perturbation with 20% methyl alcohol or 20% dimethylsulphoxide at p H 6 failed to reveal sequestration of tyrosyl residues by uv difference spectra (Hackman and Goldberg, 1979). The procedure assumes the perturbant did not induce conformational changes in the protein altering the ratio of exposed to internalized groups (Donovan, 1969). Cuticle proteins from cyclorrhaphids are minimally soluble in 20% alcohol at pH5 suggesting this assumption may not obtain for this preparation. The ratio of buried to exposed tyrosyl has also been examined in unresolved samples of proteins from sarcophagid cuticle with the chemical probes, N-acetylimidazole and tetranitromethane (Riordan and Vallee, 1967; Lipke et al., 1981). Borate-SDS extracts containing 21 polypeptides of the same approximate molecular weight distribution as C. vicina were reacted with the acetylating agent for 40min at pH 8-6 and 25°C in the presence and absence of urea. Based on spectrophotometric assessment of the rate of 0-acylation in the two samples, the ratio of buried to exposed functional groups in the native sample was 1: 1. The chitin-bound structural protein on the other hand appeared less ordered, since essentially all phenolic hydroxyl was accessible to modifier (Hennigan and Lipke, unpublished observation). Both chitin-bound and unglycosylated samples were exposed to the borate-SDS extraction medium in the course of sample preparation discounting artifactual
MECHANISMS OF SCLEROTIZATION I N DIPTERANS
19
tertiary structure imposed by previous contact with and removal of detergent. Abundance of helix and p-sheet in urea extracts of Calliphora vomitoria integument varied inversely with the polarity of the solvent selected for measurement of circular dichroism (Hillerton and Vincent, 1979). With C. vicina, addition of less polar solvents was not required for stabilization of either helix or P-sheet (Hackman and Goldberg, 1979). The two investigations differed in selection of solvents and in methods of data processing. Both laboratories agree that cast films show evidence of P-sheet, in confirmation of X-ray analysis of dried material (Fraenkel and Rudall, 1947). The intractability of sclerotized cuticle for the isolation of protomers of larval origin has prevented comparison of primary structures before and after crosslinking. Protein modification as a prerequisite for protomer polymerization remains a strong possibility, particularly when analogies with skeletal material from other phyla are considered. The likelihood of one or more post-translational modifications in larval protomers coincident with pupariation challenges the validity of the assumption that extracts of larval cuticle represent the material ultimately used for puparium fabrication. Without exception, the literature concerning sclerotization accepts post-oxidative coupling of unmodified larval proteins as the rate-limiting step in dimer formation. That this cannot represent the pathway in situ is evident from the absence of dimer and trimer from incubates of sarcophagid larval proteins, dopamine, oxygen and homologous phenolases. Fortification with metal activators, electron carriers, peroxidase, chitin and a wide variety of other stimulants fail to initiate crosslinking in vitro (Strout and Lipke, 1974). In view of widespread success in the resolution of protein mixtures from cuticle and in the development of non-invasive probes of intact organisms, there is little reason to continue experimentation with crude mixtures or to rely too heavily on information culled from these sources. 3.1.3 Drosophilid larval proteins (Superfamily Drosophiloidea) Mass rearing of synchronized cultures coupled with bulk processing enabled Fristrom and associates to prepare litre quantities of third instar larvae from which gram quantities of cuticle were harvested (Fristrom et al., 1978; Silvert and Fristrom, 1982). The urea-soluble proteins were estimated as eight in number, with a later upward revision to include two additional minor components as resolution on the gel was improved (Fig. 4). Five of the proteins coded with a
H. LIPKE e t a / .
20
40
30 20 its
. I I
-
-
-
6 -
I
II
*
+ Fig. 4 Electrophoretic separation and isolation of purified cuticle proteins from Drosophila melanogaster. Upper slab in absence of urea. Lower slab, proteins dissociated with sodium dodecyl sulfate. (From Fristrom, Hill and Watt, 1978)
36 kd DNA segment at chromosomal region 44D, with four of the five falling within 7.9kd of DNA (Snyder e l al., 1981). At least four members of the tetrad cluster were activated for transcription early in the last instar while the fifth component, 8 kd removed from this group was not expressed until later (Chihara et al., 1982).
M E C H A N I S M S O F SCLEROTIZATION I N DIPTERANS
21
Cloning was facilitated by the abundance of poly(A) RNA for structural proteins in late third instar epidermal cells compared to embryonic poly(A) RNA which is virtually devoid of this transcription product. The four major proteins were sequenced and the primary structure confirmed by parallel sequencing of the corresponding cloned genes. CP1 and CP2 are homologous except for residues 1-9 and 12 of CP1, and residues 1-5 and 8 of CP2. CP3 and CP4 are 85% homologous; their interior sequences show homology to CP1 and CP2. The proteins were absorbed quantitatively by deproteinized chitin and could be eluted with urea. None were glycosylated to a significant extent although pupal exoskeleton could be fractionated to resolve polypeptides with 1 to 7% neutral carbohydrate appended. Table 5 presents the amino acid composition of the larval isolates calculated for the sequence. It is evident that Asx and Glx constituted about 25% of the total residues, if Ala, Gly and Pro are included, over half the amino N could be accounted for. The low levels of non-polar components together with the abundance of charged residues imply few if any hydrophobic interactions could be accommodated (Fig. 5). The proteins are compact with few pleated sheets and an even distribution of helical and p reverse turns (Fig. 6). Based on electrophoretic mobility, variants of all of the proteins (except CP4) were readily detected, almost invariably associated with heterozygosity. Gel separations could not detect differences between cuticle proteins from the sibling species D.melanogaster and D.virilis, although haemocyte involvement during pupariation may vary within the genus (Rizki and Rizki, 1980). In support of the information on transcriptional differentials discussed previously, proteins of the third instar were separated into early and lately synthesized proteins. This distinction was not manifest in the first and second instar (Chihara et al., 1982). 3.1.4 Larval proteins from the Superfamily Muscoidea Isoelectric focusing of urea extracts of C. vicina separated twelve proteins with PI 44-5.4 and M, 14-16 kd. N-terminal analysis of the mixed proteins by dansylation indicated Ile, Val, Ala, Gly and Glx in this position (Table 4), mole ratios were not specified (Hackman and Goldberg, 1976). Ampholytes poised from pH4-0-6.0 were exploited for resolution of sarcophagid proteins extracted with borate-SDS buffers instead of urea. A minimum of 20 proteins were identified by staining with Coomassie Blue or precipitation with ammonium sulphate in situ on the gel (Lipke et al., 1981).
i.-n
1
CP1 CP2 CP3 CP4 CPX CP2a # 5 64.68
N
CP1 CP2 CP3 CP4 CPX CP2a X 5 64.68
(D
P
P
V
P L
H A
S P
L V
G R S D V S [ R l E l O V I N I A (N IN E)
(N (
H ( A ) D ( V ) L S R H /A) D /V) L S R N V ElIVIIK E L N E&E (A) D (V) V (K1 (S) L
E N
D E
D
A N A N
V) V
20
CPI CP2 CP3 CP4 CPX CP2a # 5 64.68
I
D
D
v)
A
A
R
H G H G
A I L ) K E m (L ~ N ) G (? E ?
CPI CP2 CP3 CP4 CPX
V
A
N
K
V
CP1 CP2 CP3 CP4
A A
R R
A
(D)
N N
40 I I
H
D
E
G
(F)
D
H H
G G
N N
N [ I
W
V
E
w v
s
V D V
D
D]
N (S) N
R K
A E Q S Y S )
A A
A A
30
H H
T T
s
W W
I I
G
E I
S S S S Y
P P P P E I
s ILI
F F
G G
V
S
v
L
S S
K
G
a
S
(N) G (N) G
50 E G E G E G E G Y V
w
(I) (I)
E E V E A
E E
H H H H P
a 0
s (GI S
(G)
V E V K Y l V E V K Y V V N G K T V
D)
V
E E E E Y
N N N N T
G G G G A
Y Y Y Y D
A A
V V
A A
W W Y Y
L L I I
7n 80 ._ Q P S G A W I P T P P P I P E O P S G A W I P T P P P I P E O P Q S D L L P T P P P I P m O P O S D l l P T P P P l P E E T G Y N P K ? V E A
90 E S H E S H W A N O A H P
A I A I A I A I
100 P P P S
P A P E H P A P E H S K NEnd K E E n d
P P
R R
H H
HEnd HEnd
Fig. 5 Primary structure of larval proteins from D . melanogaster and S. bulluta. (From Snyder et al., 1981; Henzel, Mulligan, Mole and Lipke, unpublished results.) []-no residue; inserted to maximize homology.
23
M E C H A N I S M S O F SCLEROTIZATION I N DIPTERANS
TABLE 5 Amino acid composition of Drosophila cuticle proteins (unpublished results of Dr M. Snvder). Values in mole percent
ASP Thr Ser Glu Pro GlY Ala Val Ile Leu TYr Phe His LYS Arg Asn Gln TrP Acidic Basic Diff
CP1
CP2
CP3
CP4
6.1 1.8 9.6 7.9 12.3 8.8 10.5 7.0 5.3 3.5 1.8 1.8 8.8 0.9 4.4 5.3 1.8 2.7 14.0 14.1 0-1
7.3 1.8 1.0 7.3 10.9 8.2 11.8 7.3 5.5 3.6 1.8 1.8 8.2 0.9 4.5 4.5 1.8 2.7 14.6 13.6 1.o
8.3 2.1 8.3 6.3 9.4 7.3 9.4 10.4 7.3 6.3 3.1 2.1 2.1 5.2 1.0 7.3 3.1 1 14.6 8-3 6-3
7.3 2.1 7.3 9.4 9.4 7.3 9.4 10.4 5.2 6.3 3.1 2.1 3.1 5.2 1.o 6.3 4.2 1.0 16.7 9.3 7.4
s
o
Lipke et al., 1981). Heterogeneity of this magnitude was confirmed by isolation and amino acid analysis of the 20 major components (Table 6). That these represented only a portion of the protein complement was reported by Willis et al. (1981) based on staining patterns characteristic of thin gel slabs (Fig. 7). Both laboratories failed to detect appreciable accumulation of bridged intermediates between the monomeric components and the higher molecular species at the onset of pupariation during the period of declining solubility. The paucity of dimers and trimers favoured a concerted rather than a stepwise assembly mechanism for the sclerotized complex with n-mer half lives on the order of seconds or minutes in vivo. In spite of the presence of phenylthiourea and detergent, the extracts deteriorated on prolonged storage, either in solution or as a lyophilized powder. Isolates declined markedly in solubility and recovery of amino N from hydrolysates was low unless stored at low pressure under vapours of mercaptoethanol. These changes suggested autoxidation of tyrosyl residues, however, phenolase did not constitute one of the proteins of the mixture. In concordance with
24
H. L l P K E e t a / . CP I
H2N I
5
10
80
90 95 100
85
I
CP2
eo
85
90 95 100
I05
110
CP3
40
35
30
60
65
70
75
75
70
COOH 50
55
eo
85
so
95
c P4 95
go 85
eo
65
60
55
50
Fig. 6 Secondary structure of larval proteins from D. melanogaster computed by W. Henzel according to Chou and Fasman (1974). (From Snyder et al., 1981)
the findings in Drosophila, no detectable hexose, pentose or hexosamine was observed in 5 mg (0.30 pmole) of pooled proteins. The assays were sensitive to 0.05 pmole of carbohydrate. Extraction with ether and chloroform did not reduce the dry weight of the lyophilized powder. Intact and hydrolysed proteins were transesterified with BF3-methyl alcohol to release presumptive bound lipids. Gas liquid chromatography failed to reveal fatty acid methyl esters. Sarcophagid isolates were subjected to mapping and the Edman degradation to establish relatedness between proteins and for comparison with Drosophila. It can be seen from Fig. 5 that sarcophagid protein 5-64-68 is essentially homologous with the N-terminal regions of Drosophila CP2a, the six terminal residues are identical and the remaining substitutions are conservative. This finding was supported by comparison of termini of four proteins
TABLE 6 Molecular weights, isoelectric points, composition and yields of cuticle proteins from S. bulluta purified by isoelectric focusing and SDS-Dolvacrvlamide gel electrophoresis (from Lipke et ul., 1981). Amino acid values in residues per mole of protein Protein number
PI Molecular weight ( x lO-’Mr) Relative amount (%y Aspartic acid Glutamic acid Threonine Serine Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine p-Aianine OH-Lysine Acidic/Basic Polar/Non-Polar Amino Terminus
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
4.63 4.68 4.73 4.80 4.88 4.90 4.97 5.03 5.08 5.13 5.18 5.23 5.33 5.38 5.50 20 17 21 16 22 21 22 24 21 21 21 21 20 19 17 5 29 34 7
6 8 7 3 29 16 1 1
3 8 23 30 46 32 1 2 1 4 31 27 2 9 31 34 15 16 29 27 6 7 8 9 7 6 7 3 3 11 12 7 16 1 1 0 1
n
o
o
o
o
o
o
o
i
1.2 2.2
2.7 1.2
2.7 1.5
3.5 1.9
1.9 1.9
2.1 1.6
1.9 1.4
1.9 1.4
2.0 1.5
2.2 1.4
1.6 1.3
1.3 2.3
2.8 2.1
-
gly
-
gly
-
-
-
-
-
-
asp
-
-
1 25 30 1 1 28 4 38 15
in
o
10 5
22 12 10
5 6 5 3 2
is
0 1
7 6 13 3 39 35 30 35 48 41 42 48 9 9 8 8 12 12 11 8 9 1 0 1 0 1 0 29 25 22 27 17 18 15 18 30 31 28 32 6 6 8 1 1 8 1 1 1 3 1 0 1 1 9 1 0 2 5 8 9 21 17 16 19 24 22 20 22 0 0 1 2 0 0 0 0
11 30 41 6 10 5 25 16
25 9 1 7
5 11 24 1 0
o
n.d.. not determined. a Soluble protein only. Insoluble protein CB-I represents 46% of the total cuticle protein.
2 9 31 29 38 36 9 7 1 12 14 7 1 0 28 27 15 17 25 2s 1 0 1 2 1 9 1 7 7 5 5 14 23 16 15 1 2 0 0
17
18
19
20
CB-I
5.53 5.75 5.88 5.90 17 17 18 19 63
2 3 7 1 1 3 7 5 20 36 38 19 17 22 25 22 6 31 36 42 38 32 29 30 39 12 3 1 9 1 1 1 2 1 1 8 6 1 1 16 17 21 22 23 22 13 23 33 6 1 2 1 4 1 1 4 6 1 2 1 26 49 57 36 28 25 11 35 24 11 8 10 9 8 9 5 13 36 14 16 16 14 13 21 in 25 9 7 7 6 5 6 8 1 0 8 0 9 0 8 7 7 8 1 0 8 8 7 1 2 5 4 2 3 5 6 6 7 4 4 2 1 4 2 4 3 n.d.* 6 48 9 12 8 5 11 17 LO 3 15 21 18 19 20 10 13 10 3 1 0 0 0 2 1 2 1 0 0 0 0 1 0 0 1 0 o o o o o o o o 0
2.7 2.0 tyr
2.1 2.2 -
1.8 1.8 ~
2.3 1.4
1.7 2.0
2.6
-
leu
-
1.5
-
1.5 asp
H. L l P K E eta/.
26
+
-
L-3d L-4d RS
WP
T-lh
T-2h T-4h
T-8h T-16h T-24h
pH 4 - 6
Fig. 7 Dissociated (guanidine) proteins from larval and puparial cuticle of S. bullata. The mixture was resolved by isoelectric focusing. Larval age in days (L), Puparial age in hours post tanning (T). White pupae (WP) Solubility in guanidine in per cent. (From Willis et al., 1981)
representative of both the pH 4 and pH 6 regions of the gel (Table 7). At neither terminal was homology significant within the species (Lipke et al., 1980; Snyder et al., 1981). On the other hand, when relatedness was assessed mathematically, it was evident that the inner regions of many of the proteins probably bore strong resemblances as revealed by peptide mapping (Fig. 8). The degree of relatedness can be calculated from amino acid composition based on SAQ for all possible combinations of the 19 candidates (Marchalonis and Weltman, 1971; Cornish-Bowden, 1980). Since all were similar in molecular weight an average residue number of 220 was used to establish the “strong test” criterion as SAQ = 38.6 as maximum index of similarity. This approach was first exploited by Willis et al. (1981) for D. m e h o g a s t e r and was subsequently confirmed by
TABLE 7 Partial sequences of selected cuticle proteins from S. bullata (Lipke et al., 1980) Protein
PI
N-term
C-term
1 5 NH2-Gly-His-Asx-Ala-Gly
Ile-Val-Ala-COOH
3A
4.4
9A l l H e
1 5 10 5.28 NH2-Asx-Ser-His-Pro-Asx-Asx-Val-His-Ala-Glu
12A He
1 5.30 NH,-Tyr-Tyr-Tyr-Tyr
14A
1 5 10 5-80 NH,-Leu-Gly-His-?-Gly-Gly-His-?-Glu-Ala
CBI
-
NH,- Asp-Val-Ala-His
Ile-Ala-His-Leu-COOH
His-COOH Blocked
H. L I P K E
28
eta/.
E
c
8
(u
L
I
inj,
15
30 Time (mtn )
45
Fig. 8 Peptide mapping of larval proteins from S. bullutu. The polypeptides were digested with trypsin and chromatographed on ultrasphere ODS by the procedure of Fullmer and Wasserman (1979). (From Henzel, Mulligan, Mole and Lipke, unpublished results)
sequencing. When sarcophagid proteins were ranked according to PI, strong relationships were indicated only among entities with close acid-base reactivities and SAQ 4 38.6 (Table 8). Similarly, when calypterate and acalypterate Diptera were compared on the basis of RNA complexity in the egg, only a distant relationship was manifest (Hough-Evans et ul., 1980). In the case of most cuticle proteins, however, questionable relatedness exists between non-terminal areas of D.melunoguster and S. bullutu when SAQ is calculated for proteins of disparate molecular weight. In view of non-identities for termini, at best the values may reflect internal homologies. An unusual component, 12He differed markedly from the other isolates. When this tyrosine-rich protein was separated by focusing
TABLE 8 SAQ of larval cuticle proteins of Surcophugu bullutu (Marcholonis and Weltman, 1971; Lipke et al., 1981). “Strong test” SAQ 5 38.6 in italics ____
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
____
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
156
149 53
287 261 102
213 190 81 102
245 179 73 104 8
240 180 78 121 23 8
259 208 100 125 25 14 5
244 192 77 80 29 21 18 18
178 126 45 87 23 21 20 20 22
136 136 77 168 45 46 33 34 56 22
141 381 355 544 285 325 303 309 367 297 188
179 229 125 121 171 200 209 212 169 129 181 454
169 225 131 152 207 240 254 259 220 161 208 449 12
145 142 71 148 191 204 211 231 164 149 201 417 103 100
154 124 65 149 193 189 179 207 139 140 184 433 141 161 28
131 45 31 136 96 99 99 118 103 52 71 330 132 148 122 102
138 215 113 140 82 96 79 88 97 71 60 199 185 216 194 166 90
150 36 26 161 132 128 134 147 123 77 111 375 141 138 62 75 47 167
H. L I P K E e t a / .
30
TABLE 9 Composition of tyrosine-rich larval proteins of S.bullutu (from Lipke and Henzel, 1981) 1st Isoelectric point Molecular wt kd 5 0.4 Relative amount (%)" Aspartic acidb Threonine Serine Glutamic acid Proline G1ycine Alanine Valine Isoleucine Leucine Tyrosine (%) Phenylalanine Lysine Histidine Arginine Acidiclbasic Polarmon-polar
12He
4.51 5.30 23 26 1 1 12 25 6 7 21 14 24 20 8 9 31 26 9 15 11 9 2 4 1 6 72 (35) 48 (20) 27 4 15 4 7 1 4.0 2.1 0.5 0.6
Percentage of total protein in extract. Values in molesimole of protein rounded to nearest whole number. Amide N not determined.
on a granulated bed (Bio-Gel P-100) similarity was noted to an aromatic polypeptide recovered from a polyacrylamide disc gel and previously designated 1st (Lipke and Henzel, 1981). The two focusing methods were not strictly comparable due to the different sieving and binding properties of the ampholyte supports. Mobility can also be effected by losses in amide N which are difficult to control at low pH, especially in cuticle proteins with a high degree of amidation of acidic amino acid residues. Proteins 1st and 12He contained exceptionally high titres of tyrosine; in the case of 12He, a oligotyrosyl peptide was located at the N-terminus. The yield of PTH-tyrosine was acceptable for the first four cycles of the Edman degradation, when continued beyond this point, unidentified products were released (Table 9). In view of the ubiquity of aromatic proteins in cyclorrhaphid haemolymph and the sharing of immuno determinants with cuticle proteins, protein 12He may represent a component transferred to the larval integument toward the end of the third instar (Scheller et al., 1980). In the event that protein-
MECHANISMS
OF SCLEROTIZATION I N DIPTERANS
31
TABLE 10 Radioactivity of S. bullata cuticle proteins from larvae administered [7-I4C] dopamine (from Lipke et al., 1981) 14c
Protein number
Per cent of Total durna
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CB-Ib Control 12CProteins 1-20+ [7-W] dovamine ~
Cuticle only.
1.2 1.0 4.8 1.8 3.0 5.5 1.8 8.4 2.8 6.9 1.3 7.5 2.2 2.4 3.7 1.6 1.2 1.8 3.4 3.6 21.1
dumhmole 34 46 34 38 34 51 56 44 62 58 48 72 51 40 41 30 29 37 29 38 79 0.8
~
Polypeptide only
bound tyrosyls are substrates for phenolase or peroxidase in the course of crosslinking, the polytyrosyl sequence would be among the more vulnerable of the sites for modification. In addition to the well-defined aromatic amino acid complement of 12He, a second entity derived from aromatic precursors was associated with the borate-soluble larval proteins. At the conclusion of the post-feeding stage, cuticle proteins acquired radioactivity administered as [714C] dopamine earlier in the third instar (Lipke et al., 1981). The dopamine derivative was probably incorporated during a preparatory stage to crosslinking. The label was distributed more or less uniformly among the integumental polypeptides, in keeping with the concerted mechanism revealed by electrophoretic analysis (Table 10). Non-radioactive larval proteins did not bind dopamine and administration of labelled catecholamine during pupariation did
32
H. LIPKE e t a / .
not generate 14C02 from C7 or C8, thus non-specific binding or recycling did not account for the prevalence of isotope among the polypeptides. Affinity for borylcellulose, a bidentate ligand of o-diphenols, was undetectable (Sugumaran and Lipke, 1982b). These properties indicate that dopamine was converted to an aryl ether or an open chain metabolite in the course of addition to soluble components in anticipation of puparial metamorphosis.
3.1.5 Glycosylated components In contrast to the multiplicity of different proteins in urea or borate extracts of cuticle (Tables 6, 7) the chitin-bound insoluble fraction from S. bullata, fraction CB-I, was composed of a single protein with the N-terminal sequence: HN-Asp-Val-Ala-His-Tyr- and with the C-terminal blocked (Sugumaran et al., 1981, 1982). Brief exposure to chymotrypsin cleaved a 63000 dalton entity from the mucoprotein complex. Fraction CB-I was composed of significantly more proline and serine and less aspartic acid than the boratesoluble components (Table 6). Serial digestion with pronase, subtilisin-BPN and chitinase released a peptidochitodextrin with mole ratios: Gly (5):Glx (4):Asx (4):Thr (1):Val (1):Arg (1):glcNac (20). These values were fully in accord with the excess of glycine over glutamic acid in limited digests of larval glycopeptides previously reported (Kimura et al., 1976). In addition to the predominant protein species, and unlike the soluble protein mixture, phenolase activity characterized the chitin-protein complex. Whereas the cuticular phenolase of C. vicina was released into neutral buffers, the sarcophagid enzyme remained insoluble after prolonged extraction with borate-SDS. The ability of the phenolase to withstand mild proteolysis with thermolysin, pronase, trypsin, or chymotrypsin to affect release from the insoluble matrix was confirmed (Yamazaki, 1977; Barrett and Andersen, 1981). Oxidative activity was expressed with or without solubilization, in either instance thiols, copper chelators and withdrawal of oxygen inhibited quinone production from dopamine. The acellular strata of S . bullata include peroxidase in the enzyme complement and warrant detailed cataloguing of transferases and ion pumps to augment compositional studies (Quesada et al., 1978; Gupta and Hall, 1979; Sugumaran et al., 1981, 1982). The chitin-bound protein was dissimilar to the soluble faction in configuration. Whereas a globular shape was indicated by a 1:l ratio of buried to exposed tyrosyl groups in the
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N D I P T E R A N S
33
borate-soluble fraction, all the phenolic residues were external in the residue (Hennigan and Lipke, unpublished observations). The scheduling of the reactions forming the chitin and the protein chains destined for joining has not been addressed directly. The heavy predominance of older chitin and protein synthesized during the third instar as opposed to new materials synthesized just prior to pupariation makes the kinetics of peptidoglycan deposition difficult to follow by tracer technique. Based on the small amount of protein added to the polysaccharide during tanning, however, relatively few polysaccharide chains would be required to accommodate the new glutamic acid-rich proteoaminoglycan. If the usual pattern of glycoprotein synthesis obtains, protein synthesis precedes saccharide addition. Protein synthesis was unaffected by polyoxin D, an inhibitor of chitin assembly in calliphorid peritrophic membranes, suggesting the polypeptide is supplied to the new chitin-protein complex well before the glycan was synthesized (Ishaaya and Casida, 1974; Peters, 1976; Becker, 1980). On the other hand there is no reason to rule out delayed peptidylation of chitin deposited before sclerotization or the storage and editing of preformed glutamic acid-rich polypeptide until conjugation was initiated. The localization of enzymes oxidizing phenols in cuticle well before visual evidence of pupariation or sclerotization is manifest is coincident with the presence of benzenoid species of low molecular weight (Lipke and Henzel, 1981; Lipke et al., 1981; Sugumaran and Lipke, 1982a). As discussed in Section 3.1.4, at this time labelled dopamine was incorporated into soluble proteins and extensively metabolized. Accordingly, the insoluble chitin-protein complex from larvae also contained label derived from tyrosine or dopamine in a variety of metabolites. Indeed, based on specific radioactivity, the residual Faction CBI exceeded the soluble in isotope uptake (Table 10). Tyrosine was converted to four distinct materials when administered early in the third instar (Sugumaran et al., 1981, 1982). Each metabolite was easily separated from tyrosine and companion substances on a cation exchange resin developed with gradients composed of volatile buffers (Lipke and Henzel, 1981). The best defined products were bi- and tertyrosine, the former present at levels of 2 nmole/mg during the post-feeding stage when peroxidase activity was high and at lower levels thereafter (Fig. 9). The polypeptide including this material in the primary structure was cleaved from the insoluble matrix by 0.1111 NaOH at 100°C for 5 min, yielding the two multi-functional aromatic amino acids on
34
H. L I P K E eta/. I
I
white yellow pink
red
dark brown
PUPARIAL STAGES
Fig. 9 Dityrosine and orthodiphenols during pupariation in S. bulluta. Following removal of soluble components by extraction with borate buffer, p H 9.2, odiphenols were determined in the residue according to Arnow (1937). Dityrosine was assessed by fluorimetry following isolation by chromatography. (From Sugumaran et ul., 1981, 1982.)
acid hydrolysis. The alkali-soluble protein and the original chitinprotein complex released peptides of clearly defined primary structure on extensive treatment with proteolytic enzymes (Fig. 10). Distinction between the two position isomers N-terminal to bityrosine remains to be established. Bi- and tertyrosine was identified in residual larval skins from Tabanus bivattatus but could not be detected in Aedes aegypti. In S. bullata labelled on the schedule described above, the sum of the radioactivity in the two tyrosine conjugates was equal to that of tyrosine. Generally, recovery of bityrosine was five-fold greater than tertyrosine. Bityrosine is refractory to phenolase and is not involved in the pigmentation of insoluble chitin-protein complexes that followed partial proteolytic digestion. In these preparations from S. bulluta, phenolase was active while still bound to the matrix, hence darkening on treatment with proteolytic enzymes was due to release of oxidized products to the buffer or the removal of blocking groups separating the oxidase from substrate(s). The endogenous materials subject to oxidation consisted of two types of substrates. The first group contained monotyrosyl peptides cleaved from the matrix or exteriorized from a sequestered tertiary structure. The peptides were prepared by proteolytic digestion of extracted skins with the phenolase previously inactivated by boiling, and conformed in
M E C H A N I S M S O F SCLEROTIZATION I N DIPTERANS
35
f: Hz H,N -ASP
- NH-CH-CO-P
RO
- S E R-COOH
Fig. 10 Primary structure of Peptide SH-3 from 5’. bullata larval cuticle. Sequence determined by the subtractive Edman procedure and carboxypeptidases A, Y and proline-specific endopeptidase. (From Sugumaran et al., 1981, 1982).
structure and properties to conventional tyrosine polypeptides susceptible to phenolase. The second object of phenolase action was a tightly bound o-diphenol that was alkali-labile unless complexed with molybdate-nitrite, this property also serving as a convenient route to quantitation (Arnow, 1937; White et al., 1979). It can be seen from Fig. 9 that the o-diphenol occurred at levels in excess of bityrosine and that turnover followed a different time course. Following prolonged treatment with chemical or enzymatic oxidizing agents, dark polymeric material accumulated in the reaction medium or was deposited within the chitin-protein complex. This pigment bears an obscure relationship to the pathway of sclerotization as observed in vivo and may be formed by a side reaction of little biological significance from the standpoint of crosslinking. The aromatic precursor of the pigment is of undeniable importance and is probably identical with the benzenoid species liberated by hydrolysis under reducing conditions with mercaptoethanesulphonic acid (Sugumaran and Lipke, 1982a). This reduction product did not chromatograph with dopamine although this catecholamine could serve as a precursor to the aromatic moiety. Two products were generated by the thio acid. In addition to the aromatic species, a non-benzenoid derivative of dopamine formed an acidic mercaptan or thioether clearly distinguishable from the positive charged metabolites discussed above. Whether the disappearance of aromatic character preceeded exposure to mercaptoethanesulphonic acid or is artefactual is not known. There was no question that the insoluble cuticle oxidases and substrates initiated crosslinking first in the matrix and then in the soluble components. During the course of
36
H. LIPKE eta/.
the darkening reaction initiated by the action of thermolysin on the protein-bound diphenol, the aspartyl N-terminus of the structural protein was completely blocked by coupling with quinone and was unreactive to Edman’s phenylthioisocyanate reagent. Masking or non-reactivity was not due to aspartyl removal by thermolysin since titre was unchanged during protease treatment if oxygen was excluded from the system. The attack on the N-terminal aspartyl residue clearly differentiated tanning of the chitin-protein as observed in covalent aggregates of sclerotized puparial proteins. In the latter example, lysyl and histidyl residues located in positions other than the N-termini were modified (Sugumaran and Lipke, 1982a). The enzymic reactions discussed above have an unusual counterpart in a purely chemical transformation produced by the action of 6~ HCI on borate-extracted larval cuticle at room temperature. Under conditions precluding enzymic activity (pH <0.1) and probably oxidative attack as well, the tissue and acid medium acquire a pronounced violet colouration without appreciable change in physical appearance. These conditions are not conducive to cleavage of ~-1-4-N-acety1glucosaminidiclinkages or of the average peptide bond, although P-alanyl and aspartyl links are less resistant than other residues to cold hydrochloric acid. The reaction did not occur in 3 M mercaptoethanesulphonic acid. In HCl, an acid-catalysed condensation reaction in which one or more of bound aromatic species participated was indicated. Mechanistic considerations will be discussed in Section 5.1. 3.2
THE NEMATOCERID LARVAL CUTICLE
3.2.1 Kinetics of deposition
Aedes aegypti metamorphose to the pupal stage following four larval instars. Soluble larval proteins are in the 20-30 kd range in molecular weight (Zomer, 1978). In contradistinction to cyclorrhaphans, the larval cuticle is shed and the pupal stage is encased in an integument formed partly by de novo synthesis. If the mechanism of cuticle synthesis was fully analogous with heterometabolous orders, synthesis of cuticle protein should decline with respect to viscera prior to ecdysis in the interest of economy and to favour catabolism (Lipke et al., 1965b,c). As discussed in Section 2.2, cyclorraphids do not conform absolutely to a pattern at variance with the heterometabola. Although the bulk of protein synthesis is confined to the early period of the last larval instar, some new RNA and protein is
37
M E C H A N I S M S O F S C L E R O T I Z A T I O N I N DIPTERANS
I-
L A B E L D I S T R I B U T I O N IN L A T E L A R V A E (DAY 6) I I
loo%
I00
F
I
-
c
I1
100%
L A B E L DISTRIBUTION IN EARLY PUPAE (DAY 7 )
I
I
100%
C
Fig. ll Time-course of tyrosine metabolism in Aedes aegypti. Isotopic and nonradioactive tyrosine administered during larval stages Days 3, 4 and 5. Values in percent of total counts. F, body fluids; C, cuticle; V, viscera. (From Zomer and Lipke, 1980)
produced just prior to pupariation in cyclorrhaphans. In the ultimate larval stage of A . aegypti, a third pattern intermediate between the two extremes is encountered, namely major synthesis of new protein toward the end of this life stage (Zomer and Lipke, 1980). When labelled tyrosine was administered per 0s via specifically labelled E. coli early in the fourth instar and chased with unlabelled bacterial cells, a three-fold enhancement of incorporation into cuticle protein was recorded at the end of the fourth instar (Fig. 11). The larval cuticle was devoid of bityrosine, hence the fate of the label was probably the tyrosyl of the primary chain. That incorporation was not confined to tyrosine was evident from the ready uptake of labelled lysine and histidine at rates only slightly less than tyrosine. In the presence of MON 0585 and Diflubenzuron a significant portion of the protein and chitin normally synthesized late in the last instar was not deposited in the cuticle, either because synthesis was inhibited or the nascent polypeptides were subject to rapid degradation (Zomer and Lipke, 1981). The two insecticidal
H. L I P K E e t a / .
38
agents inhibited crosslinking and chitin deposition, respectively, but at the doses administered, mortality was confined to the pupal stage. It was clear, therefore, that the inhibition was targeted at a special protein complement characteristic of the end of the fourth instar. These observations introduced the possibility that the Nematocera represent an intermediate developmental level between those orders where (a) the onset of the pupal stage signals the laying down of entirely new polymers and (b) situation in the cyclorrhapha, where new polymer formation is divided into two periods with the majority laid down early and a second smaller increment limited to the end of the last larval instar. The regulation of these distinct pathways of cuticle protein turnover in higher Diptera may be beyond the capacity of the simple ecdysone-juvenile hormone couple to control and may account for the evolution of additional endocrines operating at this stage (Seligman et al., 1977). 4 Composition of sclerotized tissue
4.1
THE CYCLORRHAPHID PUPARIAL CASE
Chemical mechanisms of crosslinking can be examined by tissue analysis or by reconstruction of presumptive pathways in cell-free systems. Only limited success has been reported in the latter instance (Strout and Lipke, 1974; Peter, 1980). Puparial cases exhibit marked variation in chemical stability. After fragmentation with acid, ketocatechols were released from D. melanogaster apparently to the exclusion of other products (Driskell, 1974). On the other hand, non-phenolic benzenoid amino acids were cleaved from the matrix in S. bullata (Lipke and Henzel, 1981; Sugumaran and Lipke, 1982a). The arylated proteins from S. bullata responded sluggishly to enzymatic degradation but were amenable to partial acid hydrolysis or oxidative cleavage with N-bromo-succinimide. The resulting crosslinked peptides were suitable for sequencing and physical probes (Lipke and Geoghegan, 1971b). The peptides ranged from 3 to 25kd and, bore no similarity to conventional tanning models widely accepted since 1940 (Brunet, 1980). 4.1.1 Models of cuticle structure In the course of pupariation, the decline in solubility and titre of hydrolase-susceptible linkages coincides with enhanced uptake of phenolic precursors from the circulation. Two models are favoured
MECHANISMS
OF S C L E R O T I Z A T I O N
IN DIPTERANS
39
for this conversion (Fig. 12). The models differ in: (1) The role of aromatic substituents in crosslinking; (2) the reactions driving the extrusion of water; (3) The degree of polymerization of the bridge; (4)The molecular weight of the modified proteins; (5) The identity of the amino acid residues participating in matrix assembly.
(A) COVALENTLY CROSSLINKED MODEL
(6)NON-COVALENTLY Key:
Protein,
L I N K E D MODEL Aromatic polymer
Amino acid involved in covalent crosslinking Amino acid involved in hydrophobic interaction Amino acid involved in hydrogen bonding
Fig. 12 Stabilization of the insect cuticle by covalent and non-covalent interactions. Model A depicts crosslinking by covalent bonds to the ring and side-chain of an aromatic or quinonoid bridge. Model B emphasizes hydrophobic interaction with a polyaromatic backbone. (From Sugurnaran and Lipke, 1982a)
In Fig. 12A specific residues in the primary chains are substituted by benzenoid groups affording one crosslink per 20-30 amino acid residues. The crosslink is of a relatively low degree of polymerization enabling the modified proteins to compress sufficiently to extrude the bulk of the aqueous domains. Model 12A shows bridging by ring substitution or side chain addition. The latter mechanism obtains in species with lightly sclerotized puparia and the former where a more resistant shield is called for. In either case covalent link to protein is a common feature to both the ring and side chain bridges, clearly distinguishing the crosslinked structure from the alternate non-covalent proposal. In the latter model (Fig.
H . L l P K E eta/.
40
1
4
8
12
16
20
24
28
32
Fraction Number
Fig. 13 Separation of arylated lysine and histidine metabolites by chromatography on polystyrene sulphuric acid. Puparia from larvae administered labelled tyrosine, lysine or histidine were hydrolyzed in 6 M HCI and acid removed by evaporation. The hydrolysates were loaded on the column and eluted at pH 3.5,7.0 and 12.0 and the fractions assayed for radioactivity and uv-absorbance. Insert describes the absorption spectra; before A, and after B, reduction. (From Sugumaran and Lipke, 1982a)
12B), aromatic precursors are oxidized and converted to a highly polymerized hydrophobic backbone, this reaction favouring the extrusion of water from hydrated protein and chitin. The proteins perturbed by the introduction of the new polymer undergo conformational changes that externalize non-polar amino acid residues sufficiently to induce a major increase in hydrophobic interactions between macromolecules. Little covalent bridging between proteins is invoked. Changes in chemical stability are ascribed to synthesis of carbon-carbon linkages between the monomers of the aromatic polymer and shielding of protein and chitin by the poorly hydrated polyaryl component (Hillerton and Vincent, 1979; Vincent and Hillerton, 1979). Since the principal difference between the two constructs lies in the presence or absence of aryl crosslinks, the models have been tested by examination of total hydrolysates of the
41
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N D I P T E R A N S
TABLE 11 Incorporation of [14C] amino acids and aminodiphenols into puparial cases from S. bullutu (from Sugumaran and Lipke, 1982a). Each larva received 1.02.0 x dpm of labelled precursor at the commencement of the last larval instar. The puparium was harvested 8 days following metamorphosis. Portions of the washed and powdered cases were hydrolysed for amino acid analysis. Basic, acidic and neutral adducts, were identified by cation exchange chromatography
Precursor Tyrosine Lysine Histidine Phenylalanine Arginine Tryptophane Alanine P-Alanine Cysteine Isoleucine Serine Methionine Leucine GIycine Valine Aspartic acid Proline Glutamic acid Threonine
Basic (pH 12.0) 61.9 33.0 39.5 31.6 38.0 50.0 17.2 16.4 15.4 14.8 12.8 12.2 11.6 11.0 9.9 9.7 7.7 5.9 5.7
Percent of radioactivity Acidic Neutral (PH 3.5) (PH 7.0) 5.7 9.7 1.4 3.7 1.1 1.25 27.2 7.1 1.4 15.0 38.2 32.8 18.6 40.1 33.0 19.6 38.3 17.1 50.0
32.4 57.3 53.1 30.5 40.8 45.0 77.5 76.5 61.7 60.7 36.1 24.5 47.4 48.9 45.2 53.8 58.0 52.2 33.0
heavily sclerotized puparium of S. bullutu for arylated amino acids (Sugumaran and Lipke, 1982a). The isolations were facilitated by injection of 18 different labelled amino acids individually prior to chromatographic identification of the presumptive bridge element from puparial hydrolysates. Table 11 shows that tyrosine, phenylalanine, histidine, arginine, alanine, cysteine, lysine, p-alanine and dopamine were incorporated at levels in excess of the other candidates. Ion exchange chromatography of the hydrolysed cases revealed that ring-labelled tyrosine, histidine, and lysine were converted to basic metabolites clearly distinguishable from the respective amino acid precursors (Fig. 13). The figure shows that the basic adducts of lysine and histidine absorbed in the ultraviolet and the spectrum differed markedly from authentic tyrosine or catecholamines in that 254:280nm was 4 rather than 1. The distinction
42
H. L I P K E e t a / .
between the conjugates and their precursors was verified by thinlayer chromatography, high-voltage electrophoresis, hydrophobic and affinity columns. These procedures showed that basic or aromatic adducts were not formed by the other amino acids incorporated at significant rates, namely cysteine, alanine and arginine (Table 11).Phenylalanine was hydroxylated to tyrosine which subsequently was converted to the benzenoid adduct. The puparial proteins could be cleaved with N-bromosuccinimide to afford labelled peptides from which the adducts could be isolated. The isolates did not coincide with dopamine, bi- or tertyrosine, noradrenaline, ketocatechols, quinolinedicarboxylic acid or 1,2-dihydroxylysyl benzenes. A substantial portion of the phenolic groups were masked and were unblocked only by aryl-ether cleavage (Lipke and Henzel, 1981). These observations must be taken into account when considering the models described in Fig. 12. Allowing for generalizations dictated by species variation and for lack of definition of particular features by supporters of each of the two examples, the models clearly differ. Model A links structural proteins via covalent bond to an aromatic bridge of relatively low molecular weight by 1,6nucleophilic addition to the ring or by substitution to the P-carbon of dopamine-like tanning agents. This process leads to the formation of a compact matrix resistant to proteolysis. The alternative model features a polyaromatic backbone of a high degree of polymerization with the protein chains distributed along the polymer by hydrophobic interactions. The stability of the construct is ascribed to enhanced hydrophobic interactions between the benzenoid core and non-polar side chains of the cuticle proteins. The scheme is dependent on configurational changes transferring interior hydrophobic residues to the periphery of the molecule, a process that might also account for the extrusion of water characteristic of puparium formation. For the covalent model to obtain, an interprotein crosslink is required. For the model based on hydrophobic interaction with an aromatic polymer, a melanin or lignin-like framework must be identified. Arguments favouring Model B (Fig. 12) question the significance of the decline in lysine &-aminogroups during pupariation, and the role of the polymeric aromatic pigment deposited as a crosslinker as the cuticle hardens. It is now evident that a portion of the lysyl and histidyl residues were subject to modification by an aromatic component derived from the ring of tyrosine, a situation best explained by the synthesis of covalent crosslinks.
M E C H A N I S M S OF SCLEROTIZATION I N DIPTERANS
43
In this respect coupling of quinones with the &-aminogroup of lysine via 1,4-nucleophilic addition to afford stable conjugates has been described in cockroach ootheca and in plants where phenols and quinones abound (Pau, 1975; Pierpoint et al., 1977; Laird et al., 1979; Eagles et al., 1980). The aromatic component of benzylated lysine and histidine from the sarcophagid puparium differed notably from the catechol-lysine adducts described in plants and cockroaches since most of the o-diphenolic character was lost. Not only did behaviour on an affinity support specific for diols discount a catechol-like structure, but also confirmation was not forthcoming based on spot tests (molybdate) and uv-spectra (Fig. 13). Since models invoking beta substitution on a catecholamine retain a dihydric structure, the above considerations as well as chromatographic evidence speaks against substitution on the sidechain in S. bullata. The ability of NBS to generate peptides which in turn released arylated histidine and lysine on acid hydrolysis was also in accord with a quinonoidal structure with multiple substitutions on the ring. This reagent would preserve benzylated amino acids only if phenolic hydroxyls were blocked or hindered. Model B (Fig. 12) stresses the importance of the resistant polymer remaining after acid or alkaline hydrolysis of puparia. Distinction between the polymer responsible for dehydration and the melanic puparial pigment is not offered by proponents of this scheme. Concerning the abundant puparial pigment, chemical evidence favours a structure with many features resembling typical melanin rather than a lignin-like plasticizer. Long-chain aromatic polymers resembling melanin or lignin require free radical intermediates for assembly; invariably the reactive species persists even after the completion of the chain. It is probable that covalent coupling of proteins to an activated backbone would accompany the configurational changes invoked by Hillerton and Vincent (1979). In addition to enhanced hydrophobic interaction, a free radical mechanism would add protein to the polyaromatic constituent either during polymer formation or after synthesis of the main chain. Free radical-mediated reactions are of low specificity, hence Model B would afford aryl conjugates of many different amino acids. That this was contrary to the findings can be seen from Table 11, where the participation of lysine and histidine in conjugate formation exceeded most of the other amino acids by a factor of three or more. Evidence favouring a generalized free radical mechanism would require a greater proportion of radioactive peptides in the fingerprint record for tyrosine-treated animals, such is not the case.
I /-
I
Fig. 14 Mechanisms of assembly of sclerotized proteins in Sarcophaga bullata. Structural proteins are represented by residue symbols in specific sequences connected by distinguishing lines. From Lipke and Henzel (1981). Stage I: Cleavage of P-alanyl-tyrosine releases (a) tyrosine for conversion to dopamine; (b)
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N DIPTERANS
45
4.1.2 Crosslink heterogeneity Anatomical, kinetic and steric considerations may demand a variety of crosslinks to provide regions of varying flexibility, hardness, or permeability. From the diversity of products obtained from tyrosine and dopamine metabolism in maggot integument and the complexity of the aromatic components in the puparial residue it is likely that the uniform outer appearance of the puparial case is not an expression of a corresponding uniform chemical structure. The modification of the larval structural polypeptides by metabolites of tyrosine and of dopamine just prior to metamorphosis may indicate one pathway for presclerotal activation based on modification of protein 12He and additional pathways expressed following the white puparial stage when the majority of the crosslinks are joined. The later steps would link lysine and histidine with phenols and bityrosine described in Section 3.1.5. In S . bullata and sibling species with heavily sclerotized puparial cases, the two pathways are modelled in Fig. 14. The scheme begins at Step 1 with an ecdysone-dependent dipeptidase providing tyrosine and p-alanine for post-translational modification (Dunn et al., 1977) and is followed by coupling between a dopamine metabolite and soluble cuticle proteins (Step 11) (Lipke et al., 1981). In step 111, oxidation of the oligotyrosyl region of Protein 12He is coupled to synthesis of the first crosslink, depicted in Step IV, ending the presclerotal events (Lipke and Henzel, 1981). Step V represents the major tanning event with the deposition of new protein-protein bonds. These links are bridged with phenols bound to the chitin-protein complex and stored in the circulation.
p-alanine for activation prior to transfer to protein. Stage 11: Non-derivatized larval structural protomers are modified by addition of a fragment in part derived from the side chain of dopamine. Stage 111: Tyrosine-rich initiator protein is activated. Stage IV: Modified structural protomers peptidylate the aryl groups of activated initiator protein. Stage V: (a) Dopamine is converted to a cross-linking benzenoid derivative which then adds to modified structural protomers in concerted polymerizations; (b) p-alanine is added at N-termini of selected components of the complex. Stages I, I1 and I11 are presclerotal, occurring during post-feeding white puparial development; Stages IV and V constitute the major cross-linking and pigmentation reactions. Proteins depicted as linear constructs solely for clarification of the progress of assembly sequence. (From Lipke and Henzel, 1981).
46
H. L I P K E e t a / .
4.1.3 N-terminal P-alanylation N-terminal P-alanylation is arbitrarily assigned to this step based on sound kinetic evidence for the rate of incorporation into puparia and less conclusive chemical information (Bodnaryk, 1978). N-P-alanylation of a dopamine-derived crosslink also satisfies kinetic requirements for acid-catalysed hydrolysis. Evidence for the participation of p-alanine as a crosslink participant remains unconvincing in view of the lability of P-alanyl bonds to acid hydrolysis compared to heavily bridged integumental members. The dipeptide is an effective carrier of aromatic groups in C. vicina which subsequently can be recovered as ketocatechols (Andersen, 1977). In S. bullata a substantial portion of the total body (3-alanine can be recovered as N-P-alanyl-dopamine by extraction of the puparium with N HCl at 25°C. These conditions are too mild for severance of intraprotein bridges but are adequate for removal of a weakly bound aryl storage agent from the matrix (Hopkins et ul., 1982). Pigment deposition has been separated from crosslinking by selection of mutants differing in the degree of melanization. In D. melanogaster the puparium is unusually light in colour in ebony and black, while the adult is darker than the wild type. The transport mechanism for (3-alanine is defective and this lesion results in abnormally diffuse strata in the puparial exoskeleton and disorganization of microfibrillar architecture in the endocuticle (Jacobs, 1978; Sherald, 1980). Until f3-alanine is localized on the modified cuticle polymers, definitive assignment of function is not possible. The sporadic distribution of this precursor in many sclerotized and pigmented dipteran cuticles is particularly difficult to interpret in terms of bridge formation, particularly with respect to minimal participation of p-aln the synthesis of acid-stable basic aromatic adducts (Table 11).
4.1.4 Chitin-protein bonds Preparation CB-I from S. bullutu maggots consists of a single structural protein of known N-terminal sequence (Section 3.1.5). The chemical nature of the carbohydrate-protein linkage is not known, except for the prevalence of glycine in the peptide proximate to the chitin (Kimura et ul., 1976; Sugumaran et al., 1981,1982). With the onset of puparium formation a striking reorientation of polysaccharide chains alters the X-ray reflection pattern from a random distribution with respect to the plane of the puparial case to
M E C H A N I S M S O F S C L E R O T I Z A T I O N I N DIPTERANS
47
an ordered array perpendicular to the main axis of the oblate oval (Fraenkel and Rudall, 1947; Rudall, 1963). Rotation is facilitated by a 50% decline in molecular weight average of the chitin chains as assessed by molecular sieve chromatography and sedimentation equilibrium of the disaggregated fibril (Strout et al., 1976). When chitin-protein bonds were quantified at the commencement and conclusion of reorientation an increase in the peptidylation index of the polysaccharide was recorded ranging from 2 to 6, depending on the method of preparation of the peptidochitodextrin (Kimura et al., 1976). The new polypeptides appended to the chitin had an excess of glutamic over glycine in the region closest to carbohydrate, and contrary to the situation in Drosophila, were devoid of palanine, and phenolic crosslinks (Lipke and Geoghegan, 1971b, Jacobs, 1978; Sherald, 1980). The discordant ratio was manifest in parallel isolates prepared by oxidative, reductive acidic, or alkaline cleaving reagents, and in the case of larval isolates, were almost identical with glycopeptides from crayfish (Herzog et al., 1975). It is evident that compaction of the strata extended not only to the protein complement but also to the environment including polysaccharide and attendant polypeptides. The model shown in Fig. 15 incorporates the information currently available for puparium of S. bullata. The influence of chitin structure on the tensile strength and physical behaviour of cuticle is of major import, hence modification of the polysaccharide by reduction in the degree of polymerization of the chitin and introduction of new protein chains contributes to the dramatic alteration in stability and extensibility. The mobilization of RNA at the time of pupariation, i.e. message production for peptidylation of the glycosaminoglycan, is coincident with the need for limited protein synthesis at this stage (Section 2.3). 4.2 T H E P U P A L C U T I C L E O F N E M A T O C E R A Intermediates in the path to fully sclerotized pupal cuticle were generated by administration of MON 0585 and diflubenzuron to larvae labelled with [ring-14C]tyrosine. The two inhibitors blocked the final steps of pupal stabilization and prevented sequestration of protease-susceptible peptide bonds. Aside from loss of an undetermined fraction of the bridged peptides to the medium, initial steps in the production of bridged precursors and crosslinks were unaffected, including substantial o-alkylation of phenolic hydroxyl with generation of arylethers (Zomer and Lipke, 1981). The labelled arylpeptides released by exhaustive enzymatic digestion were
H. L I P K E e t a / .
48
0 N - Acetylglucosominy l
t Fig. 15 Protein-protein and chitin-protein linkages in dipteran cuticle. Crosshatched regions signify non-covalent interactions. (From Lipke and Geoghegan, 1971b)
purified by ion exchange, molecular sieve and affinity chromatography and analysed by uv- and infrared chromatography spectrophotometry and mass spectrometry. In common with Sarcophaga, more than one crosslink was isolated. The protease limit conjugates from A . aegypti showed M, 500-600. Ratios of unsaturated aromatic moieties to amino acids for two of the benzenoid peptides were 1: 3 and 1:1 with strong yellow fluorescence characterizing the products of 6~ HCl hydrolysis. Complete elucidation of the structure was delayed by decomposition of the peptides during acid hydrolysis necessitating chemical fine-structural delineation by noninvasive probes. With one isolate, the mass spectrometer showed production of 2C fragments, in accord with an open chain structure retaining a conjugated double bond system. Weakness in the ir at 700-900cm-l (aromatic) and strength at 1590cm-l (conjugated double bonds) introduced the possibility that a derivative of cis, cis muconic acid was present with one or both carboxyl groups in amide linkage with an amino acid. No bands corresponding to ketocatechols were in evidence , furnishing additional evidence that composition in acid was at the expense of an open chain unsaturated amino acid
MECHANISMS OF SCLEROTIZATION I N DIPTERANS
49
conjugate. The route to these unusual derivatives probably included at least four oxidative steps in which peroxidase and other copper and iron-dependent enzymes were invoked, since a second group of peptides were conjugated with quinones accumulating as a result of insecticide action. MON 0585 is highly specific for nematocerids as a group and for the sclerotization of the pupal stage within this group (Walton et al., 1979; Zomer and Lipke, 1981). Brachycerans and cyclorrhaphans do not differ markedly in storage forms or crosslink precursors posing the question of the unique target in nematocerans for this remarkable insecticidal agent. At the physiological level the tert-butylphenol mimics anaerobiasis since oxygen bubbled through the larval medium partially restores pigmentation without effecting mortality (Sacher, 1971). At the molecular level, however, the mode of action is uncertain and may include defective transport of bridge precursors and aberrations in post translational editing of structural protein.
4.3 T H E A D U L T S T A G E Net utilization of the tanning precursor [7-I4C] dopamine in cuticle from larva, puparium and adult was assessed at 1:2 :6, in keeping with levels of sclerotized integument in each sarcophagid life stage (Lipke et al., 1981). The direct incorporation of this precursor signifies ready exchange of free dopamine with catecholamine derived from peptide and protein-bound storage depots. The response of imaginal discs to 20-hydroxyecdysone in organ culture or in situ facilitates monitoring of macromolecular synthesis in cyclorrhaphans (Silvert and Fristrom, 1980). In D. melanogaster, about 300 polypeptides were discerned on two-dimensional gels following labelling with [35S]-labelled precursors (O’Farrell, 1975). Similarity between disc types was evidenced by the identity of 90% of the polypeptides recovered. The findings were extended to Sarcophaga peregrina although some decline was observed in heterogeneity when the entire head region was sampled rather than the eye discs alone (Utsumi and Natori, 1980a,b). This approach does not distinguish crosslinked structural proteins from monomeric precursors or cellular from acellular constituents. If it is assumed that adult cuticle resembles earlier stages in amino acid composition, 35S would not reveal proteins with a support function since these are essentially cysteine and methionine-free. Following extraction with urea or borate-SDS, 10% of the disc protein remains bound to the polysaccharide matrix. Additional polypeptide increments are
50
H. LIPKE e t a / .
added as sclerotization proceeds. In accord with separate chitin synthetases for larval, pupal and adult stages, chitin from the imago was predominantly of high molecular weight compared to earlier life stages. The measurements were made with the polysaccharide dissolved in formic acid-HCI, a solvent that extracts non-peptidylated aminoglycans at the expense of chitins with a high titre of bound proteins. In the adult cuticle, the amount of heavily peptidylated chitin chains exceeded counterparts from earlier life stages by a factor of two, the diminished solubility arising either from the high protein content or from M, in excess of 106kd (Strout et ul., 1976). Operationally, adult integument is difficult to free of noncuticular tissue except by prolonged digestion with chemical and enzymatic proteolytic agents. In many Dipterans the product is heavily pigmented invoking the usual reservations concerning distinctions between melanoprotein and structural elements. Assuming both classes of pigmented proteins are abundant in the Muscoidea, determinants common to calliphorin and cogeners are deposited in the adult endocuticle (Scheller et ul., 1980; Levenbook and Bauer, 1980). Antibody raised against calliphorin binds only to regions of closely related primary and tertiary structure in cuticle protein and not to heavily crosslinked regions. These measurements of antigen-antibody complexes were made on the whole cuticle, crosslinked soluble and residual proteins were not separated. Secondary and tertiary structures are subject to major configurational modifications by arylation and glycosylation (Section 3), hence a significant proportion of calliphorin may lose cross reactivity following transfer to the integument. Ultraviolet irradiation of emerging adult sarcophagids decreased pigmentation of thorax and abdomen with low mortality providing dessication was prevented. In S. fulculutu irradiated at 300nm, melanization alone was reduced, whereas both melanization and sclerotization were depressed following exposure to a source emitting at 254nm. The two aspects of aromatic metabolism were assessed visually and by definition of growth zones of the thoracic phragmata (Schlein, 1975). In S. bullutu exposed at 242nm both sclerotization and melanization were affected, providing a facile route to intermediates in the crosslinking of the imaginal exoskeleton (Godbey and Lipke, unpublished data). Following uvexposure a major decline in the recovery of alkali-soluble melanin characterized the irradiated population together with an increase in imperfectly crosslinked polypeptides. Survival and walk proclivities were maintained by the insensitivity of a portion of the crosslink
MECHANISMS O F SCLEROTIZATION I N DIPTERANS
51
mechanism of leg and thorax to ultraviolet, since titres of fully sclerotized basic aryl-lysyl conjugates did not decline during treatment. The results support the contention that heavily sclerotized structures are stabilized by more than one type of crosslink. In the case of adult cyclorrhaphans, catecholamines may supply one requirement and aromatic haemolymph proteins the other via autotanning. Changes in mobility and quantity of individual polypeptides reflect crosslink diversity from eclosion to completion of hardening (Chihara et al., 1982). 4.4
T H E EGG S T A G E
Darkening of the aedine chorion is preceeded by the synthesis of dopa decarboxylase in the Jocyte (Schlaeger and Fuchs, 1974a,b,c). Following a blood meal, the oocyte tans prematurely unless exposed to an inhibitor present in the accessory gland substance from the male. In view of the resistance of aedine eggs to desiccation it is probable pigmentation is an index of crosslinking rather than melanization (Adlakha and Pillai, 1980). The drosophilid chorion consists of six proteins ranging from M, 9.7 to 41 kd, some or all of which are high in glycine, alanine, proline and neutral sugar. Following deposition the insoluble chorion affords bi- and tertyrosine on acid hydrolysis, presumably for crosslinking of the eggshell protein (Petri et al., 1976). Bityrosine is synthesized late in oogenesis at which time peroxidase becomes active in the oocyte. Inhibition of peroxidase with phloroglucinol blocks crosslinking of chorion proteins while exogenous hydrogen peroxide induces premature polymerization of the protein subunits. Crosslinking was assessed by fluorimetry of intact proteins, whether peroxidase activity was in register with the synthesis of bityrosine remains to be determined (Mindrinos et al., 1980).
5 Chemical mechanisms of crosslinking
In the following sections sclerotization will be discussed in terms of probable reaction pathways. The influence of chemical reagents on the structure of cuticle isolates is a major factor in the assignment of putative models, hence proclivity to artefact falls within this subject. The discussion will address the following facts more or less common to Dipteran systems.
52
H . LIPKE e t a / .
(1) Activation of phenolases. (2) Oxygen dependency. (3) Detritiation of ring-tritiated ethanocatechols. (4) Detritiation of side chain-tritiated ethanocatechols. (5) Masking of specific side chains of structural proteins. (6) Polymerization of protein subunits. (7) Formation of coloured products. In previous sections it was shown that more than one type of crosslink can be recovered from Dipteran integument. The biological basis of this versatility is uncertain, however, protein-protein junctions invariably demand attack on ethanocatecholic crosslink precursors via oxygen-dependent peptidylations. The high chemical potentials of the oxidized phenolic-quinonoid intermediates and the amino acid side chains naturally generate multiple products in vivo. It is now evident, however, that heterogeneity also characterizes non-biological reaction systems even though the initial reactants are less diverse. In recent years improved understanding of the loci, sequence and order of peptidylations has emerged, calling for extensive revision of established opinion. New reaction mechanisms of aryl and quinoid reagents must be incorporated into the entomological literature if a full understanding of developmental specialization is to be realized. It is unfortunate that so few contemporary probes of chemical structure, both invasive and non-invasive, have been applied to insect systems, a reflection of the lack of appreciation of the subtleties of acellular tissues among biologists. To date, all reactions in which catechols are used for sclerotization reactions are initiated by oxidations catalysed by one or more phenolases with the reaction sequence depicted in Fig. 16. The semiquinone (2) matches the quinone (3) in affinity for nucleophiles and bears serious consideration as an independent participant in sclerotization process. Further, availability of a variety of nonspecific free radical reactions makes this transient intermediate a R
bo
@lo" - @$lo-- OH
.o
1
2
0
SCLEROTIZEO CUTJCLE
3
Fig. 16 Initial stages of catechol metabolism in sclerotizing cuticle. R = side chain of catecholic precursor. (1) 4-substituted catechol. (2) Semiquinone. (3) Quinone
M E C H A N I S M S O F S C L E R O T I Z A T I O N I N DIPTERANS
53
better candidate for some crosslinks over the more stable quinone (3). The reactions are influenced by cuticular pH and redox potentials. Higher p H leads to more coloured quinoid products and enhanced solubility of oxygen.
5.1 R I N G S U B S T I T U T I O N S As shown in Fig. 17, amino acid and peptide addition to quinones proceed via 1,&nucleophilic addition reactions of the Michael type. Following this stage less defined products accumulate, thus the scheme is highly idealized. Formation of oligomers and polymers
4
5
IF
R
IS
6
PROTEIN D
AUTO
TANNING
Fig. 17 Reactions of o-benzoquinone. R = side chain of catecholic precursor. R, and R, = substituents of secondary amine. R, = side chain of primary amine. R, = side chain of a-amino acid. (3) = quinone. (4) = quinone oligomer. (5) and (6) = secondary amine substituted catechol. (7) = amino catechol. (8) = amino quinone. (9) = amino quinone monoanil. (10) = a-amino acid substituted catechol. (11)= a-amino acid substituted quinone. (12) = amino acid-quinone-monoanil adduct
54
H. LIPKE e t a / .
from quinone and interactions with derivatized amines further complicate this scheme. Additional uncertainties stem from secondary rearrangements initiated by modification of the ring substituents as skeletal development proceeds. During pupariation, for example, rounding of the white puparium is dependent on muscular contractions that enhance the internal pressure several hundredfold (Zdarek et al., 1979). This force plays a significant role in the realignment of chitin chains, the extrusion of water and enzyme-substrate interactions (Strout et al., 1976). In the case of cuticle proteins, pressure dependency initiates local changes in conformation altering the ratio of buried to exposed reactive groups within the polypeptide protomers. Aromatic ring rotation is a function of viscosity and hydrostatic pressure, both properties subject to major increases at the commencement of tanning (Karplus and McCammon, 1981). It follows that molecular environments change in the course of sclerotization, thereby favouring specific reactions and diverting less stable entities to new end products. The chemical fine structure of the reactants provides a clear indication of the course of sclerotization in situ. Since this information is best garnered from cell-free systems, a major redirection of effort toward proton, 15N and 13C NMR can be predicted. 5.1.1 General reactions with amines Quinones do not react with tertiary amines. With secondary amines stable purple products are generated (Fig. 17, structure (6)). Thus proline, hydroxyproline and other cyclic and linear secondary amines yield purple-coloured products which may be responsible for the acid-catalysed production of violet pigments in acidified cuticle (Beavers and James, 1948; Jackson and Kendal, 1949). Primary amines follow a complex course of reactions depending on the nature of amines and the solvent. Generally, aromatic amines yield well-defined products as opposed to aliphatic (Davies and Frahn, 1977). Reaction of aniline with 1,Zbenzoquinone in methanol or water produces 4,5-dianilino-l,2-benzoquinone(Fig. 18; (17)), whereas in ether the product is 2,4-dianilino-l,4-benzoquinone monoanil (Hackman and Todd, 1953; Horspool et al., 1971). This compound (Fig. 18, structure (18)), is justified by the occurrence of two consecutive Michael additions followed by anil formation at the expense of excess aniline. Note stabilization by way of internal hydrogen bonding and the p-quinoid structures. The products are generally dark brown to black in colour in keeping with the hue of
55
M E C H A N I S M S O F SCLEROTIZATION I N DIPTERANS
14
13
15
I
PhNH,
Ph
I
N,\ H \"
I
0"'. .,
c _
+O
PhNH
PhNH
Ph
17
16
Ph
I
phNoo = H
PhNH
PhNH
PhN*
18
"NHPh
H \ N D , l ' H N
I
I
Ph
Ph
Fig. 18 Reaction of o-benzoquinone with aniline. (13) 1,2-Benzoquinone. (14) 4-anilinocatechol. (15) 4-anilino-l,2-benzoquinone. (16) 4,5-dianilinocatechol. (17) 4,5-dianilino-l,2-benzoquinone. (18) 2,4-dianilino-l,4-benzoquinone monoanil.
sclerotized Dipteran integument. The reactions of quinones with aliphatic amines are more complex in biological as well as chemical systems. Chromatograms of lysine-labelled cuticle yielded a family of peaks containing appreciable radioactivity after acid hydrolysis (Sugumaran and Lipke, 1982a). Similarly, in a non-biological reaction system, over 50 products were observed, only two of which corresponded to those presented in Fig. 18 (Davies and Frahn, 1977). An interesting variant characterizes melanin production from dopamine, N-methyl dopamine or dopa (Fig. 19). This pathway is blocked by N-acylation and serves as a branch point for catecholamine utilization in melanized sclerotizing tissues (Blois, 1978; Brunet, 1980). Amino acids resemble amines in their reaction courses with quinones with one notable exception (Fig. 17). When present in excess, non-enzymatic deamination occurs stoichiometrically until all the amino acid is consumed (James et al., 1948; Hess, 1958; Pierpont, 1969a). This reaction accounts for the high titres of ammonia in cuticle extracts and hydrolysates (Fig. 17).
H. LIPKE eta/.
56
o*Dx2c00 ' I,
0'
H o HO
m
c
o
O
H
I, M E L A N I N
20
19
Fig. 19 Self-condensation of dopaquinone (19) during melanin formation. Similar cyclizations also occur with quinones derived from dopamine and N-methyl dopamine. (20) = 2-carboxyl-2,3-dihydroxyindole
5.1.2 Reactions with peptides and proteins The complexity of the reactions of quinones with protein originates in the diversity of the functional groups of the side chains and the influence of secondary and tertiary structure. With cysteinyl and R
21
FH CH
1
POTATO
VIRUS
Hi@NHRl R +
x
H3b,o H"$o
....
I......
I..
'OH OH
22
0
0
23
Fig. 20 Postulated products from the reaction of chlorogenic acid quinone (21) with potato virus X. R' = protein. (22) = Protein-catechol-protein adduct. (23) = Protein-quinone-dimer
lysyl residues the nature of the products are a function of reactant mole ratios (Mason, 1955; Pierpoint, 1969b). N-Terminal aspartyl and &-aminoof lysine in bovine serum albumin couple readily with quinones, both reactions with counterparts in Dipterans (Haider et al., 1965; Stauffer, 1971; Sugumaran et al., 1981, 1982).
57
M E C H A N I S M S O F SCLEROTIZATION IN DIPTERANS
Di- and trimeric proteins accumulate when polyphenol oxidase is added to a mixture of chlorogenic acid and potato virus. Two crosslinks were postulated (Fig. 20), the conventional disubstituted ring (22) and a protein-quinone dimer (23) (Pierpoint et al., 1977; Pierpoint, 1982). 5.1.3 Autotanning-quinones within the primary chain The action of polyphenol oxidase on tyrosyl residues in the presence of oxygen converts the aromatic residue to orthoquinones (Fig. 21).
28
29
4
"
N-(cH,)
PROTE I N
-P
30 p - C H 2 G = 0
*
26 Fig. 21 Mechanism of autotanning (Lissitzky et al., 1962). Tyrosyl residues of protein (24) upon oxidation with polyphenol oxidase (PPO) is converted to protein-bound dopaquinone (26) via protein-bound dopa (25). Schiff's base formation at quinone carbonyl yields crosslink (27). 1,4-Addition of protein to the quinone (26) yields the corresponding quinone (29) and the crosslink (30)
The oxidized products are classified according to the position of the phenolic residue in the primary chain and the uv spectrum. At the C-terminus the dopaquinone-type is produced, at the N-terminus the dopachrome type is formed and internalized tyrosyl affords the protein type (Yasunobu et al., 1959). Oxidation of ribonuclease and tropocollagen resulted in products of all three types (Lissitizky et al., 1962; Dabbous, 1966). These reactions are favoured in
H . L I P K E eta/.
58
tyrosinase per se and in tyrosine-rich proteins where inductive effects are large (Lipke and Henzel, 1981). The storage instability of these proteins is largely a function of this process. The pathway suppresses melanin production and obviates catecholamine transport unless additional tanning agent is called for during dimerization of the oxidized residue with exogenous phenol. The system is common to a wide variety of invertebrates. Thus Knight and Hunt (1974) identified polyphenol oxidase and protein-bound dopa in the egg capsule of leeches. Crosslinking of molluscan periostracum requires secretion of a dopa-rich protein (Waite 1977). Periostracin, the soluble precursor of sclerotized periostracum in Mytilus edulis, contained large amounts of tyrosine and dopa (Waite et al., 1979; Waite and Andersen, 1978, 1980; Waite and Tanzer, 1980, 1981). Among the Insecta the related derivative, bityrosine, constitutes the only recognized example of tyrosyl dimerization. 5.1.4 Reactions with sulphur-containing compounds Quinones react with thiols forming thioethers (It0 and Nicol, 1975; Ito and Prota, 1977) (Fig. 22) .- Tyrosinase-'generated o-benzo-
&) R
RlSH
0 II
R,S
3
'& O H OH
31
I
CH,S CH,R,
OH
33
32 R, = - C H , C H C O O H I NH,
Fig. 22 Reaction of o-benzoquinone with sulphur-containing amino acids. R = side chain of catechol. R,SH = cysteine. CH,S - CH,R, =,methionine. (31) monocysteinyl catechol. (32) dicysteinyl catechol. (33) methionyl catechol.
59
MECHANISMS OF SCLEROTIZATION I N DIPTERANS
quinone and cysteine produced 3-S-cysteinyl catechol (Structure 31; R=H) and 3,6-S-dicysteinylcatechol (Structure 32; R=H). Cysteinyldopas were formed by a similar reaction with dopaquinone and are utilized as initiators of pheomelanin synthesis (Thomson, 1974; Prota and Thomson, 1976). Similar adducts were produced from glutathione, cysteamine and thiohistidine (Bouchilloux and Kodja, 1960; Prota et al., 1970; Ito et al., 1979). Thetins derived from methionine (33) also participate in quinone coupling with retention of positive sulphur (Vithayathil and Murthy, 1972; Vithayathil and Gupta, 1981). The involvement of sulphur amino acids in cuticle hardening and pigmentation remains unclear in view of the absence of information on the total sulphur budget of tanned cuticle as opposed to the three common sulphur amino acids which are only present in trace amounts. 5.1.5 Quinone tanning In Fig. 23, 4-substituted catechol is oxidized by a cuticular polyphenoloxidase to the corresponding quinone which reacts with &-amino or N-terminus to give the catechol adduct (34). Further R
R
R
on
0
on
1
R
n
34
- R1f5
RINl$o c _
0
PRO1E I N
NR;
" n
35
36
Fig. 23 Quinone tanning mechanism for sclerotization of insect cuticle. R = side chain of catechol. R,,R', = cuticular proteins. Sclerotizing catechol (1) upon oxidation with polyphenol oxidase (PPO) is converted to the quinone (3) which couples with cuticular protein to give a protein-catechol adduct (34). Further oxidation t o quinone (35) and coupIing to another protein molecule yields the crosslink (36) which is stabilized by tautomerism.
60
H. L I P K E eta/.
oxidation of this product to quinone (35) as well as Schiff’s base formation joins the two proteins to give the crosslink (36) which is stabilized by tautomerism. Although an unsubstituted catechol can couple at three sites (Fig. 18), 4-substituted catechols add at two sites only. Structures (37), (38), (39) from recent reviews are chemically improbable since model reactions do not afford substituents at these loci (Fig. 24). When the pathway depicted in Fig. 23 is augmented with the products of (1) autotanning, (2)
“‘“-6”’. pJo H
R
II +N R, 0
37
NH R,
Hy’
II
Rz
0
38
R R2N@,N;R2 H
OH
39
Fig. 24 Structure of improbable cross-links. R = side chain of catechol. R, = protein.
quinonoid-protein dimers and (3) bityrosine-linked couples the rationale for multiple hydrolysis products becomes self evident. When the influence of strong acid and high temperature are also considered, the analytical problems are of serious consequence. Quinone adducts similar to (36) behave as vinylous amides and regenerate the parent amine upon hydrolysis (Cranwell and Haworth, 1971). To some extent prior reduction of the sclerotized material at pH 9-12 decreases this artifactual influence but the resulting phenylpropanoid adduct may itself be suspect due to the influence of the alkaline reducing agent (Davies et al., 1975; Laird et al., 1979; Eagles et al., 1980). 5.2 p-Sclerotization
Two proteins can be joined via a bridge contributed by the P-carbon of ethanocatechols such as N-acetyldopamine to give the adduct (Structure 40) (Andersen, 1970; Andersen and Barrett, 1971). OH
PROTEIN -C-PROTEIN
I
CH,R
40
MECHANISMS O F SCLEROTIZATION IN DIPTERANS
61
The evidence favouring this arrangement can be summarized as follows:
1. Mild acid hydrolysis of sclerotized cuticle releases (a) 2-hydroxy-3’,4‘-dihydroxyacetophenone and (b) 2-amino-3’ ,4’-dihydroxyacetophenone. 2. Enzymatic digestion with proteases generates soluble material with speGtral characteristics in disagreement with the above two compounds. However, acid hydrolysis of the digests affords the postulated 2-amino-3’,4’-dihydroxyacetophenone. 3. Sclerotization is accompanied by loss of 3H from [p-3H]N-acetyldopamine but not from [ R ~ I I ~ - ~ H and ] [a-3H]-acetyldopamine. Two crosslinks have been proposed with the structure of (40) R=NHCOCH3 produces 2-amino-3’,4’-dihydroxyacetophenone; R=OH, produces 2-hydroxy-3’ ,4’-dihydroxyacetophenone; on acid hydrolysis. In the course of comprehensive inquiries into the products of acid digestion of exoskeleton, four classes of catechols were revealed together with the probable parent material (Table 12). In this discussion, mechanistic aspect considerations must be in accord with these isolates when the strengths and weaknesses of P-sclerotization are assessed. In sensu strictu only those products retaining Ca warrant serious consideration as bridge participants in the p-sclerotization model. In view of the many routes by which crosslinked polypeptides are accessible, the significance of side reactions must be weighed when distinction between mechanisms is called for. Of particular import is the fate of catechols, not only as bridging elements but also as participants in self conjugations and dismutations. From the latter pathways several valuable insights have obtained that and warrant further discussion. 5.2.1 Mechanism of catechol dimerzzation Intact cuticle incubated with N-acetyldopamine yielded not only polymerized insoluble components but also low molecular weight materials (Andersen, 1972). The latter products were isolated and characterized as dimers of the parent compound. Hydrolysis with 6~ HCl at 100°C for 30min yielded N-acetyldopamine (41), 2-amino-3’,4’-dihydroxyacetophenone(49) and its N-acetyl derivative (48). Hydrolysis at 100°C with 1M HCl for 3 h yielded dopamine (42) and 2-hydroxy-3’,4’-dihydroxyacetophenone (50). On the
TABLE 12 Catechols recovered from sclerotized insect cuticle following mild acid hydrolysis (Andersen, 1971, 1975; Andersen and Roepstorff, 1978;Roepstorff and Andersen, 1980;Barrett, 1977, 1980)
Structure
No.
Name
R=
(41) (42) (43) (44)
Phenethylamine derivatives N-acetyldopamine dopamine 3,4-dihydroxyphenethylalcohol 3,4-dihydroxyphenylaceticacid
-CH2CH,NHCOCH, -CHZCHZNHz -CHzCH,OH --CH,COOH
(45) (46) (47)
P-hydroxyphenethylamine derivatives N-acetylnorepinephrine Norepinephrine 3,4-dihydroxyphenylglycol
-CHOHCHZNHCOCH, -CHOHCH,NH, -CHOHCHZOH
ketocatechols N-acetyl-2-amino-3’,4’-dihydroxyacetophenone 2-amino-3’,4’-dihydroxyacetophenone 2-hydroxy-3‘,4’-dihydroxyacetophenone 3,4-dihydroxyphenylglyoxal 3,4-dihydroxyphenylglyoxylicacid 2-(3’,4’-dihydroxyphenyl)-2-oxo-ethyl-acetate
4OCHzNHCOCH3 -COCHzNH2 -COCH,OH -COCHO -COCOOH -COCHZOCOCH3
3,I-dihydroxybenzoate derivatives 3,4-dihydroxybenzaldehyde 3,4-dihydroxybenzoic acid
-CHO -COOH
(48) (49) (50) (51) (52) (53)
(54) (55)
.
Origin
(41) (43)
(45)
(48) (50)
(51) (52)
(54)
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N D I P T E R A N S
63
other hand, hydrolysis of the methylated dimer produced unmodified N-acetyldopamine and 2-hydroxy-3‘,4’-dimethoxyacetophenone. Based on these observations structure (56) was proposed.
CH NHCOCH,
This structure accounted for the tritium released from [p-3H] N-acetyldopamine during enzymatic oxidation of Cp and the release of ketocatechols from sclerotized cuticle. However, it did not explain the near quantitative release of 2-hydroxy-3’ ,4’-dihydroxyacetophenone (50) on acid hydrolysis of the dimer (Andersen et al., 1980). Following additional spectroscopic investigation the structure of (56) was modified to (57) (Andersen, 1979a; Andersen et al., 1980; Andersen and Roepstorff, 1981; Roepstorff and Andersen, 1981).
CH,NHCOCH,
Unfortunately, this compound does not account for the formation of 2-amino-3’ ,4’-dihydroxyacetophenone and its N-acetyl derivative. Furthermore, formation of (45), (47), (48), (50) and (53) is not justified because positions other than Cp are attached. This structure also invalidates the unique release of tritium from the Cp position and retention at Ca as first reported (Andersen, 1976; 1977). On reinvestigation of the release of 3H from a and p-3H substrates a new model involving both (Y and p carbon atoms was proposed (Fig. 25) (Andersen et al., 1980; Waite and Andersen, 1980). The dimer fraction was separated and heterogeneity of a major order revealed (Table 13). The significance of these compounds to sclerotization is not clear. However, their presence can
H. L l P K E et al.
64
FH2 CH2
CH* CHI
FH- PROTEIN
NHCOCH,
NHCOCH,
NHCOCH,
41
FH-PROTEIN
58
59
Fig. 25 Mechanism of a,p-sclerotization. (Andersen et a l . , 1980; Waite and Andersen, 1980). N-Acetyldopamine (41) is activated at Ca and Cp (structure 58) and coupled to proteins to give the crosslink (59)
explain the acid-catalysed release of many of the catechol derivatives listed in Table 12. A scheme has been proposed in which (57) arises from the oxidative coupling of N-acetyldopamine with a sidechain desaturated product (66) (Fig. 26) (Andersen and Roepstorff, 1981).
*
@$OH
@OH
FH 2
CH I1 CH
NncocH,
NHCOCH,
y
2
41
-
a : : all
FH2
NHCOCH,
CH2 hHCOCH,
I
66
57
Fig. 26 Mechanism of dimerization (Andersen and Roepstorff, 1981). N-Acetyldopamine (41) is desaturated in the side chain to give (66). Coupling of (41) and (66) yields the dimer (57)
Formation of (66) implies the presence in the cuticle of a dehydrogenase similar to fatty acyl CoA dehydrogenase or succinate dehydrogenase for which no evidence exists. Secondly, (66)is unstable and would undergo rapid hydrolysis to acetamide and 3,4-dihydroxyphenylacetaldehyde(Fig. 27) (Stamhuis, 1969). Formation of various dimers at the expense of N-acetyldopamine and cogeners further complicates the designation of any one compound as the bridge element. Presumably the failure of 3,4-dihydroxybenzaldehyde, 3,4-dihydroxyphenethylalcohol, 3,4-dihydroxy2-hydroxy-3’,4’-dihydroxyacetophenone and phenylglycol, N-acetylnorepinephrine to participate in crosslinking is ascribed to high specificity of a key enzyme in the pathway to Cp bridged
TABLE 13 Structure and hydrolytic products of dimers isolated from the sclerotized cuticle (Roepstorff and Andersen, 1981; Andersen and Roepstorff, 1981)
Structure
No.
Compound
Hydrolysis product(s)
R, (57) CH,CH,NHCOCH,
NHCOCH,
(60) CHOHCH,NHCOCH,
NHCOCH,
(61) CHOHCH,OH
NHCOCH,
(62) COCH20H (63) CHO
NHCOCH, NHCOCH,
(64) CH2CH2OH
NHCOCH,
(65) CH,CH20H
OH
R2
N-acetyldopamine (41) and 2-hydroxy-3',4'-dihydroxyacetophenone (50) N-acetylnorepinephrine (45) and 2-hydroxy-3' ,4'-dihydroxyacetophenone (50) 3,4-dihydroxyphenylglycol(47) and 2-hydroxy-3' ,4'-dihydroxyacetophenone (50) 2-hydroxy-3',4'-dihydroxyacetophenone(50) 3,4-dihydroxybenzaldehyde(52) and 2-hydroxy-3',4'-dihydroxyacetophenone (50) 3,4-dihydroxyphenethylalcohol (43) and 2-hydroxy-3' ,4'-dihydroxyacetophenone (50) 3,4-dihydroxyphenethylalcohol (43) and 2-hydroxy-3' ,4'-dihydroxyacetophenone (50)
H. L I P K E e t a / .
66
+
C"
H CH
hll
CH
CH
p H COCH,
N I
1
II
$"
cn,cow,
2
cno
COW,
67
66
68
Fig. 27 Reactions of desaturated N-acetyldopamine (66). Desaturated N-acetyldopamine (66) after facile isomerization to (67) decomposes to acetamide and 3,4-dihydroxyphenylacetaldehyde (68). (Stamhuis, 1969)
polypeptides. Two other questions remain unanswered, namely, the distinction of intermediates formed spontaneously from those formed enzymatically, and the basis for the unusual lability of the bonds between proteins and the catecholamine side chain carbons. An alternative mechanism justifies formation of catechol derivatives, tritium release from @-position and catechol dimerization. The proposed pathway is based on reactive quinone methides as intermediates and accommodates both isolates from biological systems and the established chemical behaviour of aromatic and quinonoid reagents.
5.2.2
Quinone methides-general reactions
Quinone methides are analogues of quinones with one oxygen atom of the quinone replaced by a methylene group (Wagner and Grompper, 1971) (Fig. 28). In addition to the usual electrophilic reactivity of the carbonyl group, these compounds undergo Michael additions similar to 0
0
13
69
0
70
71
Fig. 28 Structure of simple quinones and quinone methides. (13) = 1,2-benzoquinone. (69) = 1,Cbenzoquinone. (70) = 1,2-quinone methide. (71) = 1,4quinone methide
MECHANISMS OF SCLEROTIZATION I N DIPTERANS
67
quinones. Moreover, methylene group participates in nucleophilic addition reaction with both strong and weak bases (Fig. 29). Thus the addition of various mild bases follow the order: PhOH > Carbohydrate hydroxyl > HzO > CH30H > C ~ H S O H . These reactions transform the quinonoidal structure (72) to the benzenoid, (73), hence 1,6 additions are highly favoured (Wagner and Grompper , 1974). CHCHBr CH, II
?iORCHBrCH,
73
72
Fig. 29 Reaction of a quinone methide (72) with weak bases. ROH = Phenol, sugar, water, methanol, or ethanol
Quinone methides dimerize, polymerize and participate in DielsAlder condensations. The addition product of 5-methoxy-4methyl-o-benzoquinone (74) and butadiene is ascribed to the tautomeric quinone methide intermediate (75) (Mazza et al., 1974) (Fig. 30).
S
0’JC fH =’ 0
JP
0
0
OH
74
7s
OH
76
Fig. 30 Reaction of 5-methoxy-4-methyl-o-benzoquinone (74) with butadiene. o-Quinone (74) tautomerizes to the corresponding quinone methide (75) before yielding the Diels-Alder addition product (76) with butadiene.
The above reaction, apart from illustrating the novel reactions of quinone methides also shows the facile generation of quinonemethide tautomers from substituted quinones. In fact, this tautomerism is commonly observed in 4-methylene o-benzoquinones and accounts for the abnormal reactions of 4-alkylnaphothquinones (77) (Fieser and Fieser, 1939; Fieser and Bradsher, 1939; Cassebaum, 1958) (Fig. 31).
H. L I P K E e t a / .
68 CH,R
I
77
78
Fig. 31 Tautomerism of 4-alkylnaphthoquinones (77). (77) Quinone form. (78) Quinone methide form
Naphthoquinones of this type (77) decompose readily upon exposure to oxygen and heat and have a number of reactions through their tautomeric quinone methides. 5.2.3 Quinone methide sclerotization N-Acetyldopamine is generally accepted as the unique sclerotizing agent, hence, it is proper to envisage the corresponding quinone and its tautomeric quinone-methide as intermediates during tanning. Cuticular polyphenol oxidases convert N-acetyldopamine to the corresponding quinone (80) (pathway a (Fig. 32)). Thus formation of tautomeric quinone methide is probable. Note exchange of P-hydrogen with water is obligatory (Andersen, 1976, 1977). What is more intriguing is the feasibility of direct synthesis of (81) from (41) as depicted by the pathway b (Fig. 32). In this respect, cuticular polyphenol oxidases strongly favour catechols bearing a methylene group at 4-position (Yamazaki, 1969; Andersen, 1978, 1979b). Regardless of the origin of quinonemethide, either by the direct or by the tautomeric route, its presence accounts for the chemical behaviour of certain cuticles (Fig. 33). Water addition generates N-acetylnorepinephrine (45). Self condensation leads to dimers and oligomers. Addition to N-acetyldopamine and its quinone yields other dimers with complex structures. Above all, reaction with protein or chitin generates cuticle bound catechol (82). Regeneration of quinone-methide (83) by oxidation of (82) and further reaction with protein and/or chitin accounts for crosslink (84). This reaction would proceed with amino acid side chains of proteins, presumably imidazolyl (His), amino (Lys and N-terminal), phenolic (Tyr) and hydroxyl (Ser, Thr) and with hydroxyls at the 3 and 6 position of N-acetylglucosaminyl units. This scheme would account for protein-protein, protein+hitin and chitin-chitin crosslinking in the cuticle.
MECHANISMS OF SCLEROTIZATION I N DIPTERANS
R
69
= CH,NHCOCH,
OH
R I
CH,R
I
Y*O
0
80
H
- C-H kl
Y.0
I
0
81
Fig. 32 Mechanism of quinone and quinone methide formation from N-acetyldopamine. N-Acetyldopamine (41) after initial oxidation to the semiquinone (79) yields the quinone (80) by Pathway a and the quinone methide (81) by Pathway b
Peter (1980) observed the release of 11.5% of tritium from [fb3H] N-acetyldopamine to water and stoichiometric formation of N-acetylnorepinephrine. This reaction probably includes quinonemethide intermediates and is distinctly different from the alternate possibility via dopamine-P-hydroxylase. The final product formed was optically inactive whereas dopamine-P-hydroxylase generates optically active norepinephrine (Taylor, 1974). Quinone methides are relatively long-lived intermediates and in nonaqueous solvents have a half life in the order of seconds (Creed, 1976). However, in aqueous systems their decay is markedly accelerated, one of the principle reactions being addition of water to give sidechain hydroxylated products (Fig. 33). Upon incubation of 4-methylcatechol with cuticle preparation from S . buttutu we obtained not only 3,4-dihydroxybenzylalcohol but also 3,4-dihydroxybenzaldehyde. Similar conversions also occurred in
70
H. LlPKE e t a / .
-
R:
R ‘H
81
@on OH
82
It
6
,R
-0 C
OH
0
83
3
R‘H
R’
I CHOH R
R-C-R
0
0
80
@on OH OH
45
84
Fig. 33 Quinone-methide sclerotization R = CH,NHCOCH,, R’H = Protein or chitin. N-Acetyldopamine (41) is oxidized in the cuticle to both quinone (80) and quinone methide (81). Water addition to (81) leads to N-acetylnorepinephrine (45). Four different modes of dimer formation is also possible. Reaction of quinone methide (81) with protein or chitin ( = R ’ ) gives cuticle bound catechol (82). Further conversion of (82) to the quinone methide (83) ensures the formation of cross-link (84)
the case of 3,4-dihydroxyphenylaceticacid and 3,4-dihydroxyphenethylalcohol confirming the intermediate formation of quinonemethides (Sugumaran and Lipke, 1983a,b). The release of tritium from only the P-position of labelled N-acetyldopamine (Andersen, 1976, 1977) and the recovery of ketocatechols and P-hydroxyphenethylamines from sclerotized cuticle are also in accord with this intermediate. The reason why structure (82) yields (45) and (47) and structure (84) yields (48) and (50) on hydrolysis becomes self-evident. Several modes of dimerizations are shown in Fig. 33. Since the dimers isolated by the Danish group represent only a few percent of the N-acetyldopamine incorporated, their relevance to sclerotization seems doubtful. The course of reactions shown is clearly a function of oxygen concentration in the cuticle. This imposes strong reservations on investigations where this variable was not controlled. In addition, existence of short-lived intermediates between sclerotizing agents and end product falls within the purview of rapid reaction chemistry and renders the static approach somewhat obsolete.
M E C H A N I S M S O F S C L E R O T I Z A T I O N I N DIPTERANS
71
5.2.4 p-Sclerotization in dipterans Table 14 summarizes the occurrence of ketocatechol in puparial hydrolysates. In Drosophila melanogaster 66% of the radioactive tyrosine injected could be recovered as 2-hydroxy-3’,4’-dihydroxyacetophone; the remainder included tyrosine and an unidentified diphenol (Driskell, 1974). Sarcophaga bullata, on the other hand, does not exploit p-sclerotization to an appreciable degree, since the major portion of the injected tyrosine is converted to basic, non-catecholic adducts (Section 4.1). Indirect evidence is available in C . vicina for the occurrence of both quinone tanning and p-sclerotization based on detritiation of ring and side chain labelled N-acetyldopamine (Andersen, 1976, 1977; Barrett and Andersen, 1981). TABLE 14 Occurrence of ketocatechols in dipteran species SDecies
Lucilia cuprina Calliphora erythrocephala Sarcophaga barbata Sarcophaga bullata Musca dornestica Nephrotorna suturalis Drosophita viritis Drosophila rnelanogaster
Compound (50) (46) or (49) Reference
+ + + n.d. + + n.d. +
n.d.
+
n.d.
+ + + +
n.d.
Atkinson et al., 1973 Andersen, 1970; Andersen and Barrett, 1971 Andersen and Barrett, 1971 Sugumaran and Lipke, 1982b Andersen and Barrett, 1971 Andersen and Barrett, 1971 Andersen, 1972 Driskell, 1974
+ = present. n.d. = not determined. (50) = 2-hydroxy-3‘,4’-dihydroxyacetophenone. (49) = 2-amino-3’,4’-dihydroxyacetophenone. (46) = norepinephrine.
5.3
A COMBINED PATHWAY
The mechanisms underlying quinone tanning and quinone methide tanning have been examined separately. These data also accommodate a combination of both mechanisms. Radioactive experiments have failed to verify all or none systems establishing quinone or quinone methide tanning uniquely in Dipterans (Sugumaran and Lipke, 1983~).Release of tritium from side chain of N-acetyldopamine is accompanied by liberation from the ring and vice versa (Andersen, 1974; Andersen et al., 1981). These results can be explained by assuming either both mechanisms occur simultaneous-
72 H
I
R'-C
-R
H. L I P K E e t a / .
R'H
OH
82 R'H H
R'
I
C-R
R'-
R'-
h-
CH.R
R
H
R2Nl$LNR* 0
+OH
a4
a7
1
R'H
RM,~
H
R' R'-C-
I
R
'.:d-
OH O H
I
R'-C-R
'zN@N
on
R,
aa Fig. 34 Combined quinone and quinone methide tanning. R = CH,NHCOCH3. R'H = Protein or chitin. R2NH2= Protein. (41) N-acetyldopamine. (82) side chain substituted catechol adduct. (85) ring substituted catechol adduct. (84) crosslink from quinone methide tanning. (87) crosslink formed from quinone tanning. (86, 88, 89 and 90) mixed crosslinks from combined quinone and quinone methide tanning
ly and independently or the two processes are interdependent. An interdependence of the two mechanisms demands formation of additional novel cross-links. Figure 34 describes the postulated adducts and crosslinks formed by the operation of both mechanisms with the same sclerotizing agent. This scheme yields novel crosslinks with catechol to protein ratio of 1:4 as well as 1:3 while conventional crosslinks have only 1:2. Some of these attachments could be via chitin chains. Hydrolysis of these structures yield the amino acids in near quantitative yield, thus speculations based on similar recoveries of amino acids before and after tanning should not be interpreted as evidence for the absence of crosslinks.
M E C H A N I S M S OF SCLEROTIZATION I N DIPTERANS
73
6 Developments and prospects
The systems presently described call for an unusual combination of interests on the part of the practitioner. In this respect the authors have presented information treating two sclerotal intervals, first the events preceeding release of structural protein and associated enzymes from ribosomes (translation and transcription) and second, crosslinking and reordering of bridged polymers. The technology of these specialized aspects of cuticle biology is so restrictive in terms of bench time and financial commitments that no single laboratory has bridged the conceptual barrier between the two areas of expertise. If one could predict the course of the next major advance, the formulation of cell-free systems capable of translating and crosslinking the appropriate macromolecules in their nuturulproportions would come first on the agenda. It is unlikely that this accomplishment will yield to a single investigator, anticipating much cooperation between laboratories hitherto viewed as independent. Judging by the mutual respect and data sharing proclivities of the younger contributors to the two areas delineated above, the prospects are encouraging, both for insect physiology and for travel agencies. To this end, the authors submit that a substantial catalogue of new developments are in order. Investigators with a broader acquaintance with cell biology will no doubt add many items to the list. From the information discussed in the present review, the following methodological hiatuses obtain: (1) Group cleaving reagents for amino acid and glycosamino glycan side chains of higher specificity than is presently the case. (2) Sequencing procedures for multichain complexes. (3) Chemical or enzymatic tools for localization of Gly, Ala and Leu. (4) Non-invasive indices of configurational changes. (5) Blocking agents for each step in the path to matrix deposition, either chemical or genetic. (6) Microtomy for the reliable separation of integument strata. (7) Microprobe and microelectric analysis of ion flux in specific cuticle loci. (8) Ultrastructural visualization of polymers associated by chemical bridges and non-covalent forces. (9) Synthesis of arylated amino acid models for structural comparison with natural products.
74
H. L I P K E e t a / .
(10) Microchemical criteria for positioning of substituents on conjugated ring systems. (11) Procedures for separation of peptidylated from unconjugated chitins. These aids to the elucidation of aryl and glycosyl bridges have applications transcending the acquisition of stability by the exocuticle. As noted previously, a wide array of biological systems pose identical problems in polymer function, thus the advances listed above benefit plant and microbial physiologists as well as pathologists addressing connective tissue malfunctions. There is no question that the integument presents many advantages for inquiry into particular aspects of the life process, witness the ready cooperation of specialists from other disciplines following a serious effort on the part of the entomologist to outline a problem of mutual interest. Of immediate concern are a number of issues of wide applicability to all living systems, the list including: (1) Control of the position of each protein and chitin entity following exocytotic extrusion from the Schmidt layer. (2) Reading of neutral sugar insertions appended to protein and chitin. (3) Interlaminal pH and the extracellular pumping of protons. (4) Regulation of flow through pore canals in the course of strata deposition. ( 5 ) Ring current adjustment during arylation. (6) Free radical half lives in situ. (7) Localization of amino residues destined for modification. (8) Juxtaposition of phenolases and peroxidases to structural proteins. (9) Resistance of the ambient to fibril rotation. (10) Environmental and evolutionary influences on the direction of tanning mechanisms. Given this partial list of projects worthy of attention by interested parties, one might anticipate an air of optimism within the exoskeletal brotherhood/sisterhood. In truth, however, a bleak prospect confronts the investigator who has the good fortune to devise a model satisfying all the idiosyncracies of sclerotizing systems. On that happy (and preposterous) occasion, the attention of cuticle biologists will be redirected to the biochemistry of a system of even greater complexity, the epicuticle. In a region accounting for no more than a few percent of the total exoskeleton thickness, all the
MECHANISMS O F SCLEROTIZATION I N DIPTERANS
75
problems of aryl and glycosyl bonding are encountered together with formidable aspects of hydrocarbon metabolism. The interactions between these components are even more rapid and sequential than those of the underlying strata, at this writing no means of uncoupling of the individual processes have been described. The authors join with their colleagues in the profound wish that the granting agencies continue to overestimate the ingenuity of applicants vis-u-vis Phylum Arthropoda.
Acknowledgements
The skill and devotion of K . Mulligan and G. DeMatteo in sequence assignments and configurational analysis are greatly appreciated. We acknowledge the valuable assistance of B. Henzel, R. Rutchick, and M. Burke in the preparation of text and figures. Dr M. Snyder of the California Institute of Technology, Dr J . Fristrom of the University of California-Berkeley and Dr John Mole of the University of Massachusetts were most generous in providing access to unpublished data. Fast Atom Bombardment Mass Spectroscopy was performed by Dr Catherine Costello of the Massachusetts Institute of Technology. Funds for this review were furnished in part by the United States Public Health Service, National Institute of Health Grant NIH-2-RO l - A 1- 14753-04.
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The Physiology of Insect Tracheoles V. B. Wigglesworth Department of Zoology, University of Cambridge, UK
1 Definition and formation of tracheoles 1.1 Definitions 1.2 Formation of tracheoles 1.3 The tracheoles during ecdysis 2 Histology and histochemistry of tracheoles 2.1 The intima and its variations 2.2 The cytoplasmic wall 3 Visualization of tracheoles 3.1 Staining methods and injection of dyes 3.2 Injection with metallic sulphides 3.3 Injection of osmiophilic oils 4 Respiratory functions of the tracheoles 4.1 Site of respiratory exchange 4.2 The diffusion theory of respiration 4.3 Safety margins of oxygen supply 4.4 Tracheoles and mitochondria 4.5 Tracheal gills and ion absorbing epithelia in aquatic insects 4.6 Limiting size of tracheoles 5 “Intracellular” tracheoles 5.1 Plasma membrane invagination 5.2 The sarcoplasmic reticulum of the flight muscles 5.3 Tracheoles in flight muscles 5.4 Distribution of injected tracheoles in flight muscles 6 Adaptive responses of tracheoles during growth and moulting 6.1 Tracheoles in the epidermis of Rhodnius 6.2 Tracheole entry into the flight muscles 6.3 Formation of plasma membrane invaginations 6.4 Maturation of flight muscles: tracheoles and mitochondria 6.5 Mitochondria and tracheoles in extreme exhaustion 7 Restoration of tracheation without moulting: tracheole capture 8 Appearance of air in the tracheal system 8.1 Absorption of fluid contents 8.2 Site of fluid absorption
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8.3 The nature of the absorption process 8.4 The role of oxygen in absorption 8.5 Liberation of gas from solution 9 Movements of fluid in the tracheole endings 9.1 Visible movements in the living insect 9.2 Possible control of movement by osmotic pressure 9.3 The tracheoles during normal respiration in the flea 9.4 Measurements of osmotic pressure and reassessment 9.5 Osmotic forces from ionized proteins 9.6 Elastic forces of protein structure 9.7 Site of action of metabolic products 9.8 Metabolic products in flight muscles 9.9 Role of surface tension in the tracheole endings 9.10 Role of active transport 10 Tracheole supply to the photogenic organs of fireflies: a new hypothesis 11 Permeability and tracheole function: a new theory 11.1 Variability in fluid content of tracheoles 11.2 Permeability differences in the tracheole walls 11.3 Permeability and oxygen supply References
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1 Definition and formation of tracheoles
1.1 D E F I N I T I O N S According to the description by Meyer (1849) the tracheal tubes, with a cellular sheath and with a spiral filament in their walls, terminate in “tracheal end cells” of stellate form, where they divide to give fine tubes which, as first noticed by Leuckart, are without a visible spiral thread. These fine tubes were long referred to as “tracheal capillaries” but are now commonly called “tracheoles” (Pantel, 1898) and the stellate tracheal end cell which Holmgren (1895) preferred to call the “transition cell” became recognized as their matrix cell or “tracheoblast”. It was shown by Weismann (1863) in the young larva of Culliphora, and by PCrez (1910) in the pupa of the same insect, that the single stellate end cell may be replaced by a variable number of nucleated cells each of which serves as the tracheoblast for one tracheole, single or branched. That is the common arrangement in Sciaru (Keister, 1948) and Rhodnius (Wigglesworth, 1954); whereas large stellate tracheal cells serve as tracheoblasts for a large number of tracheoles in Lepidoptera (Beaulaton, 1964) and Hymenoptera: Nusonia (Tiegs, 1922) and Apis (Dreher, 1936). The term “tracheal cell” can involve misunderstanding. The
PHYSIOLOGY OF INSECT TRACHEOLES
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tracheal end cells described by Schultze (1865) in the luminous organs of Lampyridae are not tracheoblasts but specialized cells to be considered in connection with the luminous organs (see Section 10). The haemoglobin containing cells of Oestridae (Enderlein, 1899; Keilin, 1944) which may be richly tracheated fat body cells, have been called “tracheal cells”; and the glandular cells on the tracheae of larval Arctiidae (Vieweger, 1912) are tracheal cells which have acquired a special function. It is therefore preferable to adopt the term “tracheolar cells” for those end cells of the tracheae which are in fact the true matrix cells or tracheoblasts of the tracheoles. In the epidermis of Rhodnius, in which it is possible to trace individual tracheoles over their entire course, they were mostly 200-250pm in length (often with several branches) and each with the nucleus at about one third of the distance from the point of origin on the terminal tracheae. They have a diameter of 0.7-1.0 pm at their point of origin and a usual diameter of 0.2-0.3 pm at their ending, with some fine branches going down to 0.1 p or less. When examined in the electron microscope (Richards and Anderson, 1942) the tracheoles in the body cavity of the honey-bee no longer appeared smooth but had spiral or more often annular folds right up to the point where they ended bluntly at 0.2pm diameter. In the ovary of Periplaneta the abundant tracheoles lie in the outer ovarial sheath, which is a delicate cellular meshwork consisting of modified adipose cells well supplied with mitochondria, glycogen and small lipid droplets. The tracheoles have an average length of 175 pm (maximum 200 pm) and all end blindly (Bonhag and Arnold, 1961). In the fat body of Drosophila the tracheoles are applied to the cells, bound down to their surface by the amorphous basal lamina (Rizki and Rizki, 1979) which lines the entire body cavity of the insect. In this way the tracheoles provide delicate strands between the cells. In early descriptions of the tracheal system the tracheoles were commonly described as forming an anastomosing network. Anastomosis is frequent in the tracheae; there seems to be no obvious reason why it should not occur in the tracheoles. But it was soon discovered that anastomosis of tracheoles did not occur in the general body cavity (Koeppen, 1921; Richards and Korda, 1950); close examination showed that it did not occur in the rectal gills of Odonata (Koch, 1936); the apparently well established anastomosis in the luminous organs of Lampyridae has been disproved by electron microscope studies (Smith, 1963; Ghirandella, 1977); and it
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is not certain that it occurs even in the fibres of flight muscles (Wigglesworth and Lee, 1982) in which it has frequently been described. 1.2
FORMATION OF TRACHEOLES
The formation of tracheoles was well described by Weismann (1863) in the living embryo of Culliphoru, in which clusters of formative cells (tracheoblasts) form elongations extending beyond the terminal column of tracheal cells. Some of these filamentous matrix cells form single unbranched tracheoles, others become stellate and form tracheoles with branches in some of their processes but not in others. Pantel (1898) made similar observations in the living larva of the Tachinid Thrixion: he observed the tracheole lumen to appear first as a “filiform vacuole”, produced apparently by secretion within the cell, the walls of this vacuole being gradually transformed into a definite membrane. A good description of a tracheole arising from the side of a trachea was given by Keister (1948) from observations on the living larva of the Mycetophilid Sciuru. One cell, the tracheoblast, enlarged beyond its neighbours in the tracheal epithelium. At first it was triangular, then spindle shaped, and later stellate. The lumen first appeared as a pale streak in the cytoplasm; at first indistinct and incomplete but gradually acquiring a regular outline along the whole course of the future tracheole. Many of the processes of the tracheoblast did not form a lumen. The lumen when formed does not grow outward from the developing tracheal trunk but grows inwards to meet the trachea, starting its development in the more distal processes and body of the tracheoblast, and finally laying down the intima throughout its length. Distally the tracheoles end more or less abruptly without tapering to a point, and a slender wisp of cytoplasm extends a little beyond the termination of the intima. Tiegs (1922) studying the larva of the parasitic Hymenopteron Nusoniu, suggested that the tracheole lumen was formed by a longitudinal fold in the plasma membrane invaginated in the tracheoblast; and Beaulaton (1968) favours this idea, although he admits that there is no direct evidence for it (cf. Noirot and Noirot-TimothCe, 1982). The same applies to a possible terminal invagination from one end of the tracheoblast. Most observers of the process in living insects seem to have favoured the coalition of cytoplasmic vacuoles (Prenant, 1900). An invagination of the plasma membrane might be expected to persist as a “mestracheon”
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(Smith, 1968) but this could be difficult to see in the tenuous cytoplasm of the mature tracheoblast.
1.3 T H E T R A C H E O L E S D U R I N G E C D Y S I S It is commonly asserted that whereas the intima of the tracheae is always cast off at ecdysis, the lining of the tracheoles is not; and that this is another definitive character of tracheoles. But it was shown by Keister (1948) that throughout the four larval stages of Sciuru the intima is moulted entirely, to the finest extremities. At the moult to the pupal stage, however, there is a difference: at definite points the new intima, instead of investing the entire length of the old tracheal tube, stops abruptly and closes round the wall. The old tube breaks at this point and here the lumina of the old larval tracheole and the new pupal trachea become continuous. The new trachea is always wider than the old tracheole that is retained. In the pupa the slender tracheoles appear to sprout from short stumpy tracheal endings. Likewise at the next ecdysis, the adult tracheal system is formed by piecing together the new tracheae and the slender existing tracheoles. How widespread this difference between larval and later moults may be, in other Nematocera, or in other Diptera, or in the early stages of other insects is not known. It was shown by Noirot and Noirot-Timothee (1982) that in the rectal pads of Kulotermes and of the cockroach Bluberus the intima of the larval tracheoles is shed at the time of moulting. It was pointed out by Whitten (1957, 1968, 1972) that in the Cyclorrhapha large sections of the existing tracheal system are discarded and not replaced at pupation and at adult development; but that is something different: it is a part of metamorphosis. In Rhodnius the tracheoles fail to shed the intima throughout all the moulting stages; so that the tracheoles (at least of the epidermis) which were formed at the time of hatching from the egg are still functioning in the adult (Wigglesworth, 1954). In each instar a new segment of trachea is added to the end of each existing trachea and at its termination new tracheoles are developed (in the same manner as in Sciuru). Thus the tracheal system grows like a plant with “nodes”, where the tracheoles are attached, marking the points where moulting occurred in each successive instar (Wigglesworth, 1954). This process of tracheole retention raises the question of how the continuity between the new trachea and the old tracheole is secured. In Rhodnius the new trachea closes down on the old tracheole at a
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Fig. 1 A. Diagram of tracheal ending, shortly before moulting; a , old tracheal cuticle; b , old terminal tracheoles; c, new tracheal cuticle; d, newly-formed terminal trachea; e, new terminal tracheoles. B. The same at the moment of moulting; the old tracheal cuticle (a) with short segments of tracheole attached is being withdrawn. C. The same showing the tracheal epithelium and the rings of cement ( f ) securing the old tracheoles to the new tracheal cuticle. D. Actual tracheae and tracheoles in Rhodnius; g, tracheole attachments where moulting has occurred; h, attachments of recently formed terminal tracheoles
point about 1-3 pm from its attachment to the old trachea (Wigglesworth, 1954) (Fig. 1A). A ring of cement is here secreted by the tracheal cells (or by the tracheolar cell) which fastens the new tracheal wall to the old tracheole cuticle (Fig. 1C). At ecdysis the old tracheole ruptures at this point, so that a short length (1-3 pm) at the base of the old tracheole is drawn out and shed along with the entire lining of the tracheae (Wigglesworth, 1954) (Fig. 1B). The cement ring can be visualized by osmium/ethyi gallate staining, and the presence of this black ring (along with the disproportionate diameter of the tracheal ending, as described by Keister (1948), can be used to distinguish tracheoles which are survivors from earlier instars from newly formed tracheoles, which of course show no cement rings (Wigglesworth, 1959) (Fig. 1D). Cement rings of this kind are seen likewise in the tracheal supply to the cryptonephridial system in the larval stages of Tenebrio (unpublished observations).
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There are no cement rings in the 4th stage larva of the mosquito Aedes (Wigglesworth, unpublished): presumably, as in Scaria (Keistor, 1948), the tracheoles are moulted to their extremities during the larval stages. 2 Histology and histochemistry of tracheoles
2.1
T H E INTIMA A N D ITS VARIATIONS
It was shown by Locke (1966) that the first step in the deposition of the insect cuticle is the appearance of minute curved plaques of “outer epicuticle” (termed by him the “cuticulin layer”) at the apex of the microvilli of the epidermal cells. At the outset these plaques are separated from one another, but they grow at their margins by accretion and thus unite to form the continuous outer epicuticle, a trilaminate structure with a total thickness of about 150A. This layer, formed in the same way by closely spaced microvilli, is to be seen in the deposition of the new tracheal cuticle during moulting in Rhodnius (Wigglesworth, 1973), and by Beaulaton (1968) both in the moulting tracheae of Bombyx mori, and in the tracheoles. Here again the plaques gradually fuse to form a continuous layer over the microvilli, and during this process a progressive folding of the plasma membrane leads to the spiral folds. This taenidial rudiment is reinforced before moulting by the addition below the folds of a very thin layer of “inner” or dense epicuticle. There is usually no taenidial filament (Beaulaton, 1968). The structure of the tracheolar intima differs from that of the tracheae only in the tenuous nature of the inner epicuticle. As in the smaller tracheae, there is no chitin demonstrable in the intima of the tracheoles by the chitosan test (Campbell, 1929). The outer epicuticle (cuticulin layer) seems to be indistinguishable from that of the outer epicuticle of the body surface. Its chemical nature is unknown. It appears to be protein-free; at least it reacts negatively to Millon’s test (Beaulaton, 1964). It is not sclerotized, and is certainly not sclerotized lipoprotein (“cuticulin” as defined by Wigglesworth, 1975). The apical contents of the microvilli by which it is secreted, and the fused material after deposition, stain a deep blue black with Sudan B after osmium fixation and mild treatment with hypochlorite (Wigglesworth, 1973) and a uniform black after partition in myrcene or farnesol in solution followed by osmium tetroxide. It thus stains as lipid (Wigglesworth, 1975, 1981). Perhaps it is some novel lipid-staining polymer (Wigglesworth, 1976). It has
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been noticed that the intima of the new tracheoles in the epidermis of Rhodnius is blackened by osmium tetroxide fixation shortly before moulting; but after moulting it no longer reacts in this way (unpublished observations). It would appear that some form of polymerization may be occurring which occludes the unsaturated bonds in the lipid present. When the flight muscles of insects are examined by freezefracture, and the underlying tissue is dissolved away with hypochlorite after the surface has been shadowed with tungsten and tantalum, the extensions of the tracheoles, even the narrow terminations of about 0.05km diameter, resist solution and appear as electron dense tracks each leading to the fractured margin of a tracheole (Wigglesworth and Lee, 1982). It is uncertain whether it is the outer or the inner epicuticle or both which is resisting solution (Fig. 2, B and C). In the finer terminations of the tracheoles in the flight muscles of Musca and Tenebrio, with a lumen of 0.05 p,m or less, the annular or spiral folding is absent; the intima is either weakly beaded or quite smooth (Wigglesworth and Lee, 1982). (Helical folds occur invariably in the tracheoles of many groups, such as Lepidoptera; in others such as Musca and its relatives and Rhodnius the folds are always annular.) (Richards and Korda, 1950.) There are occasional specializations in the structure of the tracheoles such as the tracheoles of the luminous organs of fireflies (see Section 10) in which the spirally folded intima is strongly reinforced with transverse bars across the folds, and a taenidial thread is present (Smith, 1963; Ghirandella, 1977, 1978); and the thickened walls of the tracheoles in the physogastric queens of higher termites in which 1p,m tracheoles (as well as trachaea) have extensive irregular deposits of dense material (procuticle) below the Fig. 2 A. Flight muscle fibre of Pieris seen in optical section of a whole mount after injection of tracheal system. Larger tracheoles seen out of focus running transversely and longitudinally. Smaller tracheoles run transversely and form an apparent rectangular network with black points at the corners. (The slightly darker shade between alternate pairs of tracheoles is due to the out of focus Z lines; further details in the text.) ~ 1 2 0 0B. . Freeze-fracture preparation of Musca flight muscle showing four mitochondria (m); with five plasma membrane sheaths, each enclosing one or more terminal tracheoles (long arrows). The irregular shadows (short arrows) represent the undissolved residues of the cuticular lining of the tracheoles. X13 000 (by W. M. Lee). C. Freeze-fracture of Pieris flight muscle showing surface view of the muscle plasma membrane with some of the sites of invagination of the transverse tubules and the contained tracheoles (long arrows). The residues of tracheole linings are also visible (short arrows). ~ 1 000 3 (by W. M. Lee)
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epicuticle. In these tracheoles a definite taenidium is present (Bordereau, 1975). The presence of taenidia is described also in the pupal tracheoles of Cyclorrhapha and is attributed by Whitten (1968) to the greater stress to which these tracheoles are exposed.
2.2
THE CYTOPLASMIC WALL
The cytoplasm of the tracheoblast, as described by Beaulaton (1968) shows the same activities during growth and moulting as are seen in the epidermis of the general cuticle (Wigglesworth, 1957, 1963): enlargement of the nucleolus, formation of endoplasmic reticulum with abundant ribosomes and activation of the mitochondria. This is the typical response to ecdysone, and Beaulaton observed that in Bornbyx it occurs during the peak of the ecdysone titre in the haemolymph. The cytoplasmic wall has of course a plasma membrane like that of other cells. The thickness of the cytoplasmic wall is highly variable (see Section 7 ) .In the tracheoles of the fat body in Drosophila (Rizki and Rizki, 1979), with an internal diameter of 0.3-0.5 pm, the volume of the cytoplasmic sheath appears to be 4-5 times that of the lumen of the tube. During development the tracheole lumen is filled with a gel-like substance with a high concentration of microfibrillar material, with fibres of 30-35A diameter oriented in the long axis of the tube (Beaulaton, 1968). This material is comparable with the moulting fluid gel as seen in Hyalophora (Passonneau and Williams, 1953). The contents of the tracheoles are presumably digested and absorbed, like the general moulting fluid as described by Wigglesworth (1933) and Passonneau and Williams (1953). The cytoplasm of the tracheoblast always contains microtubules lying in the long axis of the tracheole (Locke, 1966). These are greatly increased during the time of growth of the tracheoles (see Section 6) when they are considered by Hasskarl et al. (1973) to provide movement by causing the tracheoles to uncoil. In the Cyclorrhapha the pupal tracheal system is fully developed 24 h after the formation of the puparium. Within a few hours the pupal head is everted, the thoracic spiracles pierce the puparial wall, and the pupal tracheae become air-filled. Many tufts of fine tracheae come off from various points on the tracheal system, and each terminates in a 0.7 pm tracheole which is tightly coiled and hangs freely in the body cavity. Then these tracheoles proceed to uncoil: between the 3rd and 6th day all are spread out over the surface of the muscles and other tissues; they come to lie close to the muscle fibres, but
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unlike the adult tracheoles (see Section 5.3) they do not penetrate the tissues. This process as described by Whitten (1968) in Sarcophaga and Drosophila, and by Houlihan and Newton (1979) in Calliphora, appears to show active movement by the tracheoles (see Section 6.2). Microtubules in the tracheoles of Rhodnius epidermis become extremely numerous when the tracheoles are subject to mechanical tension-as when they are being drawn along by contractile strands from the epidermal cells (see Section 7). In this situation the microtubules would appear to be resisting tension (like the microtubules in the “tendon cells” of the epidermis) rather than inducing movement (Wigglesworth, 1977). 3 Visualization of tracheoles
3.1
STAINING METHODS A N D INJECTION OF DYES
The standard method for visualizing the tracheal system is to immerse the insect or the required organ in glycerol or glycerol jelly (Kielich, 1918; Landa, 1948) but this is unsuited for studying the finest tracheoles which rapidly fill with fluid. Injection methods using Sudan dyes dissolved in oil are unsatisfactory because even with strong solutions of black Sudan B the fine tracheoles are invisibly pale (Wigglesworth, 1950). For his classic measurements of the tracheal system, for testing the diffusion theory of insect respiration, Krogh (1917) used a turpentine extract of alkanna root mixed with paraffin, beeswax and colophony to melt at 40°C. This gave a solid injection which was used only for measuring the tracheae and not the tracheoles. Hagmann (1940) used vacuum injection of Periplaneta with trypan blue in 10% acetic acid with a detergent (Santomerse No. 3 of Monsanto Chemical Co.) followed by fixation in formol/acetic acid with barium chloride and sectioning in celloidin or paraffin. This gave visible injections extending to many of the tracheoles. An alternative procedure was to take advantage of the reaction of osmium tetroxide with the intima of the tracheoles by exposing the living insect to osmium tetroxide vapour, as used by Heinemann (1872) to demonstrate the tracheoles of the luminous organ of Pyrophorus; and Bongardt (1903) obtained staining of tracheoles by exposure of the living insect in 0.15% osmium tetroxide for 3-4h. The silver impregnation methods of Golgi and Cajal were found to stain the finest branches and were used by Cajal (1890), Holmgren
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(1907), Prenant (1911), Morison (1927). These silver methods often gave beautiful preparations when applied to the flight muscles, revealing intracellular networks which were, however, only doubtfully connected with the tracheal system (Athanasiu and Dragoiu, 1914; Montalenti, 1926; Smith, 1961a). We shall return to this question in discussing the intracellular tracheoles of muscle (see Section 5.3). 3.2
INJECTION WITH METALLIC SULPHIDES
A new approach was to seek some substance for injection which would subsequently react with another reagent to form a dark precipitate (Wigglesworth, 1950). The most promising reagents were metals such as iron, lead and cobalt, which form black precipitates with hydrogen sulphide. The tests were made on small insects such as Drosophila, Cimex larvae, Xenopsylla or other fleas. Injections were made by evacuation in an atmosphere of hydrogen, immersion in the solution, and injection by admission of air at atmospheric pressure. But it proved impossible to obtain any injection at all of aqueous solutions of copper sulphate, cobalt nitrate etc. Addition of a wide range of wetting agents to aqueous solutions was equally ineffective. The use of intermediate organic solvents such as alcohols or acids led to rapid escape of the injected solutions into the tissues. The metals were therefore tested in the form of oil soluble naphthenates (which are widely used as oxidation catalysts in paints). Of these, cobalt naphthenate proved satisfactory as a 30% solution in “white spirit” (petroleum in the boiling point range 15O-19OoC). This solution readily entered the spiracles and filled the system; then sulphide formation was produced by exposure to H2S gas. This method can provide good preparations for whole mounts of the tracheal system, often with good injection of the finer tracheoles; and it was possible to fix specimens in Carnoy’s fixative and to prepare sections for the light microscope (Wigglesworth, 1950). Christophers (1960) used ammonium sulphide solution for gassing the injected material and obtained excellent preparations of the tracheal system in mosquitoes. Burrows (1980) used injection into the major tracheal trunks of Schistocerca to obtain good preparations of the tracheal distribution in the central nervous system with intensification of the staining by silver impregnation. But for the study of tracheoles the method has serious shortcomings. Even in the best preparations of Drosophila, for example, the injection of
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intracellular tracheoles is very incomplete. It is necessary to expose the tissues very rapidly to the hydrogen sulphide in order to get a smooth coloration of the tracheoles. If the insect is injected after hardening of the cuticle the gas enters too slowly and the cobalt sulphide forms an irregular precipitate. This resulted in some misleading results when the method was used on the subchorionic respiratory system of insect eggs (Wigglesworth and Beament, 1950; Hinton, 1961; Wigglesworth and Salpeter, 1962).
3.3
INJECTION OF OSMIOPHILIC OILS
In recent years an improved method has been used, which consists in injecting the tracheal system with lipid material containing abundant ethylenic double bonds and which are then exposed to osmium tetroxide, and the bound osmium visualized by blackening on exposure, after sectioning, to ethyl gallate in Farrants medium. This method also involves snags and difficulties-some of which have proved illuminating when studying the properties of insect tracheoles. Thus the presence of fatty acids (oleic and linoleic acid) leads to the slow penetration of the oil through the tracheole walls. If the injected oil contains too many double bonds it takes up so much osmium that the volume of the tracheal contents increases (especially after treatment with ethyl gallate) and the tubules become far more convoluted than is normal. That often happens with linseed oil, and with myrcene alone (which has three double bonds in a molecule with 14 carbon atoms). The most satisfactory mixture found was myrcene with an equal volume of Shell odourless kerosine (a petroleum of B.P. 166-184°C) followed by fixation in formol and glutaraldehyde and then by osmium tetroxide. After embedding in Spurr’s medium (1961). sections for the electron microscope show the tracheole contents deeply blackened. Sections are cut at 1-2 pm for the light microscope and mounted in Farrants’ medium containing ethyl gallate (2%). Whole mounts of injected organs can be mounted directly in Spurr’s medium. In order to avoid any general darkening of the tissues, they should not be treated with ethyl gallate or warmed above room temperature. It is interesting to note that injections of pure paraffins (odourless kerosine or medicinal paraffin) followed by osmium fixation, gave dark coloration of the contents of the tracheoles (though not of the larger tracheae). These paraffins do not react with osmium tetroxide; so what is being blackened is presumably unsaturated
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lipid dissolved from the intima of the tracheoles (Wigglesworth and Lee, 1982). The myrcene/paraffin mixture has given very good results for the complete injection of the tracheoles, without any escape of the injection fluid through their walls in most tissues, and good results also for injection of the aeroscopic chorion. But, as will be discussed later (see Section 11.2) in the tracheoles of the flight muscles of some insects the mixture escapes through the walls of certain of the terminal tracheoles and was therefore replaced by a mixture of medicinal paraffin and 30% myrcene, or by arachis oil alone. In this way escape through the tracheole walls has been eliminated; but with these heavier oils the total injection of the system is less certain and the failure of some branches to fill is not uncommon. 4 Respiratory functions of the tracheoles
4.1 S I T E O F R E S P I R A T O R Y E X C H A N G E The respiratory functions of the tracheoles are twofold: mainly they are concerned in the respiratory exchanges in the active tissues of the body; but in some aquatic insects they function in gills. Respiratory exchange involves the uptake of oxygen and the discharge of carbon dioxide. There is no great difference between the rates of gaseous diffusion of 0 2 and CO2 but there is a great difference in solubility and in the consequent diffusibility through the tissues; in these respects carbon dioxide is more readily diffusible and it is generally agreed that the limiting factor in respiratory exchange is usually the diffusion of oxygen. It is also generally agreed that respiratory exchanges occur chiefly through the walls of the tracheoles. Remy (1925) quoted Joanny Martin (1893) as having injected indigo white into the body cavity of insects and shown that crystals of indigo blue were deposited only around the tracheal capillaries; from which Martin concluded that oxygen diffusion occurs only through the tracheoles beyond the disappearance of the spiral thread. Remy himself who made extensive use of this procedure obtained a blue coloration around the tracheal tubes also, but the deposit around the tracheoles was far more intense. Likewise von Frankenberg (1915) in Corethru and Kreuscher (1922) in Sphingid larvae, found some passage of oxygen through the large tracheal tubes. These observations and the tenuous nature of the walls of the tracheoles support the belief that the tracheoles are indeed more permeable than the tracheae.
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But not only is the intrinsic permeability of the tracheoles greater but they offer a greatly increased surface area per unit length for the gaseous exchange to take place. It has generally been found that when tracheae divide the combined area of cross-section in the branches is equal to that of the parent tracheae (Krogh (1917) in Tenebrio larva; Thorpe and Crisp (1947) in Aphe~oc~eirus). The cross-section of the diffusion path remains approximately constant at each level of branching in Rhodnius (Locke, 1958). There have been no precise measurements concerning the change from tracheae to tracheoles; but if a 5 pm trachea divides to give rise eventually to 50 0.5 pm tracheoles, which is not an unreasonable figure, they will provide more than a sixfold increase in surface per unit length. 4.2
THE DIFFUSION THEORY OF RESPIRATION
The theory advanced by Thomas Graham (1833) and Dutrochet (1837) that the oxygen supply to the tissues of insects is dependent on gaseous diffusion in the tracheal system, was substantiated by the measurements and calculations of Krogh (1920a,b). Taking into consideration the average cross-section of the tracheal diffusion path, the mean length of this path, the oxygen consumption of the insect and the diffusion constant of oxygen, Krogh showed by calculation that diffusion alone would provide an adequate supply of oxygen to the tissues and yet maintain at the tracheal endings a partial pressure of oxygen not more than 2 or 3% below that in the atmosphere. In the case of Myriopoda, larvae of Tenebrio, and the caterpillar of Cossus, which show no respiratory movements, it was assumed that diffusion took place from the spiracles; in the Dytiscus larva, from the periphery of the great elliptical tracheae which are ventilated mechanically. These calculations took into consideration the diffusion to the endings of the tracheae proper; it was taken for granted that diffusion would also account for exchanges in the finest tracheoles. Weis-Fogh (1964a,b) extended such calculations to the oxygen supply for the flight muscles of insects, in which the rate of metabolism far exceeds that in the insects studied by Krogh-but which are provided with elaborate systems of ventilation which greatly shorten the diffusion path. Indeed in the most active flying insects ventilation is effective up to a point very close to the commencement of the tracheoles. Weis-Fogh made various assumptions about the tracheole supply in order to make possible the calculations; and he reached the conclusion that the highest
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demands could be met by simple diffusion. Weis-Fogh made use of a parameter termed the “hole factor”, that is the percentage of the total area as seen in cross section which is represented by the lumina of tracheae and tracheoles. But the richness of the distribution of fine tracheoles has since been shown to be very much greater than appeared in the preparations available at the time (Wigglesworth and Lee, 1982). In fact the richness of the tracheal supply is such that Weis-Fogh’s general conclusions can undoubtedly be accepted. Throughout his calculations Krogh assumed that the spiracles were kept constantly open. But it was discovered by Hazelhoff (1927) that in most insects the spiracles are held tightly closed most of the time and are only opened enough to meet their needs for taking in oxygen or releasing carbon dioxide. This does not affect Krogh’s conclusions; it merely means that during periods of rest the insect probably tolerates lower levels of oxygen partial pressure in the tissues than would be expected if the spiracles were always open.
4.3
SAFETY MARGINS O F OXYGEN SUPPLY
Insects are adapted to breathe atmospheric air, and some indication of the safety-margin of their respiratory system can be got by exposing them to mixtures with a reduced content of oxygen. In the case of the growth process, Gaarder (1918) found that in pupae of Tenebrio oxygen uptake remains constant down to 5% 0 2 in nitrogen; below this level oxygen consumption becomes proportional to oxygen tension: the rate of diffusion becomes a limiting factor. Kalmus (1937) found in the pupa of Drosophilu that the interval between pupation and emergence is a functon of oxygen tension: as oxygen fell from 20% to 3% this interval increased from 4-8 days to 7.2 days. And Davis and Fraenkel (1940) found that the blowfly Lucilia (flying for periods of 2-20min) consumed oxygen at an average rate of 187ml 02/g wet weight/h in 21% oxygen; in pure oxygen there was a slight increase in consumption; in 10% oxygen in nitrogen, uptake was reduced to half or less; and in 5% oxygen in nitrogen, few insects would fly and those which did showed a still greater reduction in uptake. In the most actively flying insects the respiratory system cannot function with complete efficiency at much below the normal oxygen content of the atmosphere.
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4.4 T R A C H E O L E S A N D M I T O C H O N D R I A The tracheole supply to the tissues varies with their oxygen requirement. This is highest in the muscles, above all in the flight muscles in which the energy utilized for mechanical movement in the most active fliers, such as the blowfly or the honey-bee, may amount to some 2200 kcal/kg muscle/h, equivalent to about 4 ml Oz/g muscle/ min; and this is achieved without the accumulation of any detectable oxygen debt. There is no prolongation of heightened oxygen consumption after flight is arrested (Sacktor, 1970). The enzymes of the oxidizing system (dehydrogenases, cytochromes, cytochrome oxidase) are confined to the mitochondria (Levenbook and Williams, 1956; Walker and Birt, 1969) and direct evidence has been given for the presence of all the main tricarboxylic-cycle and acetate activating enzymes in the mitochondria of the flight muscles in the honey-bee (Hoskins et al., 1956). The mitochondria constitute perhaps 3 0 4 0 % of the mass of these flight muscles; so that the consumption of oxygen by the mitochondria will be some 6ml 02/ml/min or 0.1 ml/ml/s. In other words, a single mitochondrion will consume about one-tenth of its volume of oxygen per second. In these muscles the tracheole supply is concentrated upon the rows of mitochondria that lie between the contractile fibrils (Edwards and Ruska, 1955). The flight muscles are a special case so far as intensity of metabolism is concerned. But with regard to the distribution of tracheoles the principle applies throughout the body: mitochondria are most plentiful where there is an increased rate of metabolism and these mitochondria are serviced by a corresponding supply of tracheoles. This is notable where protein synthesis is active, as in the fat body and in the epidermis responsible for the formation of the cuticle; and around the ovaries and testes, and the silk glands; also around the cells of the gut epithelium which synthesize enzymes and absorb the products of digestion; around the Malpighian tubules engaged in active secretion and reabsorption; and very conspicuously in the cryptonephridial system of Tenebrio and related Coleoptera and the enlarged rectal cells (forming the rectal papillae etc.) which are concerned in the active resorption of ions and water from the excretory residue which may be completely dessicated. The prothoracic glands which secrete ecdysone and the corpus allatum and corpus cardiacum, also engaged in hormone synthesis, are richly supplied. The central nervous system, viewed as a whole,
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receives a conspicuous tracheal supply; the tracheoles are concentrated upon the glial cells which are rich in mitochondria (Wigglesworth, 1959; Longley and Edwards, 1979) and around the synaptic junctions, notably in the optic lobes-suggesting that synaptic transmission is an oxygen demanding process (Burrows, 1980).
4.5
TRACHEAL GILLS A N D ION ABSORBING EPITHELIA I N AQUATIC INSECTS
In some larvae of Nematocera such as Chironomus and Simulium and the Lepidopteron Acentropus, the spiracular branches of the tracheae are obliterated and respiration is effected through the general surface of the skin, which is supplied with a rich network of tracheoles. The same is true of various parasitic larvae such as Microcentrus (Ichneumonidae) and Nemeritis developing in caterpillars of Ephestia. The spiracles become open and functional only when the larva leaves its host. The survival of Nemeritis larvae may be limited by the supply of oxygen; if parasites are too numerous some of them are killed by asphyxiation (Fisher, 1963). In the rectum, or upon the exposed surface of aquatic insect larvae, there are often epithelia covered by thin cuticle, which are concerned in the uptake of ions from fresh water. The general character of these cells resembles that of the resorptive epithelia of the rectum of terrestrial insects: there are deep infoldings of the plasma membrane associated with large numbers of greatly elongated mitochondria well supplied with tracheoles. Alongside these epithelia there are often well defined “tracheal gills” which are very richly supplied with tracheoles running closely below a tenuous epithelium covered by very thin cuticle and with few mitochondria. It was established by Wallengen (1915) and confirmed with improved techniques by Koch (1936) that in the larvae of Aeschna the uptake of oxygen by these gills is dependent solely on diffusion. Examined in the electron microscope the rectal gills of Aeschna, Libellula and Odonata-Zygoptera are characterized by a lack of the elongated mitochondria and the infolded membrane of the salt absorbing cells, and of course by the plentiful tracheoles closely applied below the thin cuticle (Greven and Rudolph, 1973; Saini, 1977). Both gills and salt absorbing cells may take the form of anal papillae. In the beetle larvae Elodes minuta and Cyphon palustris the essential structural features suggest their involvement in osmotic regulation and ion absorption; whereas in Elodes miniata there is a
PHYSIOLOGY
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1Of
moderate tracheal supply suggesting some respiratory function, but this supply is very small when compared with that in Elmis bangei in which the abundant tracheoles, going down to 0 . 2 p m diameter. indicate a predominant respiratory function (Wichard and Komnich, 1974a). (Salt uptake was demonstrated by Treherne (1954) in Helodes. ) Likewise in the anal papillae of Trichoptera larvae: in the Glossosomatidae the anal papillae show the combined characteristics of ion transporting and respiratory epithelia; infoldings of the apical plasma membranes and abundance of mitochondria, combined with numerous tracheoles (0-2-1 pm lumen), which are enclosed in plasma membrane sheaths invaginated from the base of the cells and coming to lie close beneath the thin (0.3 pm) cuticle, with the tracheoles enclosed in tracheoblast cytoplasm and all orientated in the long axis of the papilla. Whereas in other families of Trichoptera there are purely respiratory papillae with few mitochondria and devoid of apical folds. And in yet other families there are purely ion transporting papillae with exaggerated plasma folding and many mitochondria but comparatively little tracheation (Nuske and Wichard, 1972). In the thread-like tracheal gills of Trichoptera-Limniphilini the arrangement of tracheoles is highly organized; invaginated from the base of the epithelial cells they come to lie close beneath the cuticle at uniform distances apart. The arrangement ensures the maximum uptake of oxygen with the minimum of tracheoles. At each moult additional tracheoles are added, the distance between tracheoles decreasing regularly in correlation with the decreasing diameter of the tracheoles in successive larval stages (Wichard, 1973). Similar arrangements are present in the tracheal gills of stonefly larvae (Wichard and Komnick, 1974b). In certain parasitic larvae tracheated tail filaments function as tracheal gills. Such filaments are enormously developed in two species of the Agromyzid Cryptochaetum, parasites of the giant scale Aspidoproctus. In the third stage larva they are ten times the length of the body and are packed with a great mass of fine tracheal branches which extend at least two-thirds of the way to the tip and are commonly entangled among the tracheae of the host. They are of obvious importance in respiration and the same sort of calculation as that applied by Krogh to the diffusion of oxygen to the tracheal endings (see Section 4.2) shows that the filaments have reached the maximum effective dimensions (Thorpe, 1941). In mosquito larvae such as Aedes aenypti or Culex pipiens the anal
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papillae are only moderately tracheated and they seem to be of little importance in respiration though highly important in ion absorption (Koch, 1938; Wigglesworth, 1938a; Copeland, 1964). But in Aedes argentiopunctalis the use of the respiratory siphon is becoming reduced, the anal papillae are exceedingly richly supplied with tracheoles and are apparently respiratory; in addition these larvae have flattened ventro-lateral papillae on the head, which likewise are rich in tracheoles, and the larva drives a current of water over the surface of all these papillae by the use of the mouth brushes (Lewis, 1949). 4.6
LIMITING SIZE OF TRACHEOLES
The question was raised by Weis-Fogh (1964b) whether the rate of gaseous diffusion in the tracheoles might be impeded if the diameter of the lumen should fall below the length of the mean free path of gaseous oxygen molecules. In a mathematical analysis of this problem Pickard (1974) showed that diffusion will begin to be impeded only when the effective radius of the tracheole (a) falls to a point where the value of 2alA, where A is the mean free path of the oxygen molecule, roughly 0.072p,m, is no longer greater than 1. Since the average minimum diameter of the tracheole lumen, as deduced from published electron micrographs, ranged from 0.160.28 pm, the minimum value of 2alA will be about 38. As pointed out later (see Section 5.4) there are extensive terminal regions in the tracheoles of many insect flight muscles which have a lumen diameter in the range 0.05-0.08pm and some even less (Wigglesworth and Lee, 1982) which brings the value of 2alX into the critical region. But for the vast extent of the tracheole system the figures quoted by Pickard apply and it is evident that the insect has kept clear of this problem.
5 "lntracellular" tracheoles
It was early recognized that cells of many types are entered by tracheoles. Kupffer (1873) observed fine tracheoles entering cells of the salivary glands of Muscids and penetrating as far as the nucleus. Van Lidth de Jeule (1878) saw the same in the silk glands of Bornbyx. Leydig (1885) observed tracheole endings entering muscles and Anglas (1904) described tracheal cells invading the flight muscle fibres to give rise to intracellular tracheoles. They have been
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described also by Faur6-Fremiet (1910) in the labial glands of aquatic Hemiptera; by Vieweger (1912) in the tracheal gland cells of Arctiid larvae; in the rectal papillae of Calliphora by Graham-Smith (1934) and Gupta and Berridge (1966); and in the prothoracic gland cells of Antheraea by Beaulaton (1964).
5.1 P L A S M A M E M B R A N E I N V A G I N A T I O N Tracheoles of about 0.3 pm diameter were seen by Tahmisian and Devine (1957) in electron microscope preparations of the thecal cells of the testis in Melanoplus; and they recognized that there was “no actual intracellular penetration”, for besides the cytoplasmic sheath of the tracheole with its plasma membrane, the tracheole was enclosed in the invaginated plasma membrane of the thecal cell. They concluded that the apparent penetration was effected “by causing or following an invagination of the thecal cell membrane to the inner portion of the cell”. This process was recognized by Edwards et al. (1958) in the flight muscles of Dytiscus and Hydrophilus. They concluded that extensions of the tracheoblasts indent the surface of the muscle cell, thus providing “intracellular” tracheolization “like a finger pushed into the surface of a balloon”. They were never able to observe invasion of this kind in the lemnoblast cytoplasm of the nerve axons. 5.2
THE SARCOPLASMIC RETICULUM O F THE FLIGHT MUSCLES
It has been recognized in the muscle fibres of vertebrates, that membranes of the sarcoplasmic reticulum, a modification of the endoplasmic reticulum of other cells, were concerned in the conduction of nerve impulses (waves of depolarization) from the surface membrane of the muscle to the contractile fibres. In a detailed study of the fibrillar flight muscles of Tenebrio, Smith (1961b) observed that the invading tracheoles draw with them a sheath of plasma membrane from the surface and extending to all depths in the fibre; and that this invaginated sheath gives rise to subsidiary tubules which branch and spread over the surface of mitochondria and contractile fibrils. In these muscles the sarcoplasmic reticulum is reduced to small vesicles bounded by a simple 5081 membrane with which the plasma membrane tubules (made up of two layers, with an intervening gap, at a total thickness of about 75A) became associated to form “dyads”. In Aeschna both components of the reticulum are present: the
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tubular invaginations of the fibre plasma membrane lie in indentations of the sarcosomes and cross the fibrils midway between the Z and H levels, two tubes to each sarcomere (Smith, 1961~).Smith (1962) concluded that the sarcoplasmic reticulum had two components: (i) the classical reticulum, more or less vesicular and (ii) an intermediary component derived from the plasma membrane. In the flight muscles the cisternal element is reduced and the tubular membrane system is an invagination drawn into the fibre by the tracheoles. Both types of membrane were considered to conduct excitations to the contractile fibrils. In Lepidoptera the tubular plasma membrane system is arranged like that in Aeschna, In Hemiptera there are extensive irregularly dispersed spaces derived from the T-system tubules coming both from the surface plasma membrane and from the tracheole sheaths (Smith, 1965). Similar invaginated plasma membranes and a Tsystem derived from them, with a greatly reduced sarcoplasmic reticulum was described by Smith and Sacktor (1970) in Phormia.
5.3
T R A C H E O L E S IN F L I G H T M U S C L E S
The relation between the tubular membrane system in the flight muscles and the tracheoles is a long and complicated story (Smith, 1961a). Cajal (1890) found that staining insect flight muscles by the Golgi method revealed a network which he believed to be a system of intracellular tracheoles. Fusari (1894), however, observed such networks in all classes of vertebrates as well as in insects; he therefore regarded them as being independent of the tracheae. Veratti (1902) came to the same conclusion-believing that the tracheae ended on the sarcolemma (as they do, of course, in skeletal muscles apart from the flight muscles). Sanchez (1907) observed similar reticular formations after silver staining in the sarcoplasm of vertebrates, crustaceans and insects. A full review of the literature of these “reticular” organizations within the striated muscle cell, as seen with the light microscope, is given by Smith (1961a). Tiegs (1955) supported the opinion of those authors who claimed that the silver staining network in the flight muscles did represent tracheoles. But it would probably be correct to say that the general belief in recent years has been that these fine networks represent not tracheoles but the widespread tubular systems which have been revealed by electron microscopy. However, in 1907 Holmgren had turned to a study of insect flight muscles to see whether the supposed intracellular tracheoles bore
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any relation to the “trophospongium” which he believed to be almost universal in the cells of all animals. The trophospongium was pictured as a system of intracellular channels formed by the invagination of the plasma membrane. This conception comes very close to the present day conception of the plasma membrane tubular system. The chief difference between the two is that the modern tubular system has been shown by Smith (1966) and Smith and Sacktor (1970) to be occupied by fluid continuous with the haemolymph: particles of ferritin, introduced into the haemolymph, diffuse into the system and can be seen within the channels of the tubular system at all levels within the cell. On the other hand Holmgren believed that the trophospongium contained strands of cytoplasm and that the channels were formed and occupied by the inward growth of cytoplasmic processes from certain interstitial cells. In the case of insect flight muscles he claimed that the tracheal end cells were in fact the interstitial cells concerned; that their cytoplasmic processes led the way into the muscle fibres; and that the tracheoles formed within these processes were thus continuous with the trophospongium. Prenant (1911) accepted Holmgren’s interpretation which, as he pointed out, involved the assumption that a trosphongium could exist equally with or without containing tracheoles.
5.4
DISTRIBUTION OF INJECTED TRACHEOLES IN FLIGHT MUSCLES
The tracheoles in flight muscles can be most reliably distinguished histologically from the tubular system by injection methods. The tracheole system has been reinvestigated (Wigglesworth and Lee, 1982) in a series of flying insects by the injection of lipids reactive to osmium tetroxide (see Section 3.3) examined in whole mounts and in electron microscope sections, combined with electron microscope examination of freeze-fracture preparations of uninjected specimens. The general conclusion has been that the tracheoles follow the tubular system much further and into much finer branches than previously supposed. And that some large tracts of the transverse tubular system, not usually considered to be concerned in respiration may carry thin-walled tracheole tubes within them. The general observations on the insect species concerned will be set out here but the full implications of the results will be discussed later in this review (see Section 11).
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Weis-Fogh (1964a,b) divided the tracheal supply to the flight muscles into a primary system consisting of the large tracheae and associated air sacs which are largely ventilated by moving air and extend up to the muscle surface; a secondary system consisting of tracheoles which run mainly radially into the muscle fibres; and a tertiary system which consists of branches or continuations of the radial tracheoles which run longitudinally between the contractile fibrils and so among the mitochondria. In Muscu the secondary system consists of 1-1.5pm radial tracheoles, a dozen of which may arise fan-like from a terminal trachea. The tertiary system consists of vast numbers of small annulated branches around 0.3 pm maximal internal diameter. These run in all directions, dividing into smaller branches in the 0-05-0.1 pm range, passing between and encircling the mitochondria. As already pointed out these finer branches may have smooth walls or be only weakly beaded. Virtually every mitochondrion is encircled by one or more fine tracheoles (Fig. 2B). All these tracheoles are invested in a very thin cytoplasmic sheath and plasma membrane, and outside this the plasma membrane sheath invaginated from the muscle surface (Wigglesworth and Lee, 1982). In Tenebrio the flight muscles show large tracheae at intervals along the muscle fibres, breaking up into smaller branches which enter the fibres at many points and then run inwards giving off branches (0.15-0-4 pm diameter) which run longitudinally (cf. Smith, 1961b) separated by some 6-8pm from one another, and which have been regarded as the ultimate source of the diffusing oxygen. But these longitudinal tracheoles give off numerous branches going down to 0.05-0.06pm internal diameter and always closely applied to the mitochondria (Wigglesworth and Lee, 1982). In Apis the tracheoles of the secondary system, derived from large thin-walled tracheae wrapped around the muscle fibres, run transversely across the fibres, branching repeatedly. They show no tendency to run longitudinally. As they branch they dip between the mitochondria. There are abundant tracheoles in the 0.1-0.2 pm range and in the 0.05-0-08 km range, applied to the mitochondria. In Schistocercu the tracheae between the muscle fibres send small branches into the fibres, which break up into numerous tracheoles (0-18-0.24 pm diameter) that run longitudinally and are usually separated by no more than two or three contractile fibres. These longitudinal tracheoles are far more abundant than appeared in Weis-Fogh’s preparations of the same material. They have commonly been regarded as the terminal system supplying oxygen to the
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muscle. But the richness of the supply is far below that shown in the insects described above; and injection of mature locusts after exposure to exhausting flight shows abundant tracheoles in the 0.05-0.10pm range, everywhere applied to the surface of the mitochondria; and these observations can be confirmed in freezefracture preparations (Wigglesworth and Lee, 1982). In Pieris again it is usually necessary to subject the insect to exhausting flight before complete injection of the tracheole system can be obtained. It then appears that the system consists of two parts: (i) there are rows of tracheae dividing into tracheoles which enter the muscle fibre and run transversely across it, giving off a rather small number of tracheole branches running in the long axis of the muscle between the fibrils; (ii) there is a transverse system, which is less readily injected, that consists of regular tubules (0-1-0.2 pm diameter) which run transversely at every level in the fibre; they are precisely positioned to cross the fibrils, two to each sarcosome, one on each side of the H-zone. As seen in whole mounts in the light microscope, they run chiefly between the mitochondria and are deflected up or down as they reach a contractile fibril, so that an optical section of the fibre shows a rectangular network with black points at the corners, where the tracheoles are seen in transverse optical section (Fig. 2A). The appearance of a network is an optical illusion: the longitudinal sides of the rectangles, represent the margins of the contractile fibrils; but it remains possible that there are anastomosing networks, in the transverse planes of the fibre. This appearance closely resembles the network figured by Holmgren (1907). In the electron microscope it can be seen, of course, that these transverse tracheoles are running within the familiar transverse tubule system. It is everywhere in close contact with the mitochondria. The transverse tracheoles arise directly from tracheoles applied to the surface of the fibre, which enter invaginations of the surface plasma membrane (Wigglesworth and Lee, 1982) (Fig. 2C). Rhodnius prolixus is a flying insect in its homeland in Venezuela, but it will not fly under the conditions of culture in the laboratory. Consequently, complete injection of the tracheal supply to the flight muscle has not been obtained. But as in Pieris there are two components: (i) a system of abundant tracheoles, less than 0.5 pm diameter, running longitudinally between the contractile fibrils, which do not seem to give off any fine branches; (ii) a transverse system of thin-walled T-tubules which cross the fibrils at the middle of the H-zone, one across each sarcomere, in which there are
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thin-walled tracheole extensions-which have been only partially filled with the injection mixture. These transverse tracheoles probably arise directly from tracheoles applied to the surface of the fibres, as in Pieris (Wigglesworth and Lee, 1982). The Odonata are unique in having flight muscle fibres of some 20 p,m cross-section which have a rich tracheal supply on the surface but show no “intracellular” tracheoles (Smith, 1966). In view of the active flight of dragonflies, it is surprising that this should be so. In a few preliminary observations on the damsel fly Ischnura it was observed that some of the transverse tubules of the plasma membrane system have shown injected tracheoles within them (Wigglesworth and Lee, 1982). But these observations will require confirmation in the large and powerful Anisoptera. We shall return to the physiology of the tracheole system of the flight muscles in later sections of this review (see Sections 6.4 and 11). 6 Adaptive responses of tracheoles during growth and moulting
6.1
TRACHEOLES IN THE EPIDERMIS OF RHODNIUS
The responses of the tracheoles to the demands of growth are seen at their simplest in the epidermis of Rhodnius (Wigglesworth, 1954). As the column of new tracheal cells (formed by mitosis) extends from the termination of a trachea, early in the moulting process, the individual cells show filamentous and often branching outgrowths by means of which they presumably draw themselves along. At the leading point of this column, one or two cells separate from the main body and form solitary elongated processes, each of which will provide a new tracheole. At their terminations these tracheoblasts likewise show numerous branched filaments which are presumably in the nature of pseudopodia concerned with the outward migration of the growing tracheoie. As was shown by Keister (1948) only a few of these filaments become canalized to form the tracheole lumen (see Section 1.1). At moulting, the insect increases in size; the tracheae grow outward, and at their new terminations a sufficient number of new tracheoles are developed to make good the oxygen requirements. If the main trachea, supplying the epidermis over one half of a given tergite, is cut when moulting is beginning, so that the entire half tergite is deprived of its oxygen supply, new tracheae and tracheoles are produced from the tracheae of adjacent segments; and these
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grow inward throughout the anoxic area to provide the requisite supply of tracheoles at the normal density. If, in addition to cutting the main trachea to the half tergite, a corpus allatum (as a convenient organ with a high oxygen requirement, as evidenced by its normal rich tracheal supply) is implanted below the epidermis in the centre of the anoxic area, there is the same inward growth of tracheae and tracheoles; but this is exaggerated, and in the immediate vicinity of the implanted gland the tracheoles pursue a highly convoluted course. The gland becomes invested by a dense tangle of tracheoles. Some of these tracheoles penetrate deeply into the organ, insinuating themselves between the cells just as in the normal gland. The intense convolution of tracheoles is presumably a product of active growth rather than migration, but the result is reminiscent of the “klinokinesis” of an organism, responding to a diffuse stimulus (Fraenkel and Gunn, 1940; Wigglesworth, 1941). It must be supposed that the lack of oxygen, or the acid metabolites which result from the lack of oxygen, act as an attraction or as a stimulus to tracheole growth (Wigglesworth, 1954). The response of the tracheoblasts in Cyclorrhapha to implants was studied by Pihan (1971). He showed that the attractive factor produced by implanted organs was linked to metabolic activities: dead organs or artificial bodies were unattractive. He concluded that a chemical substance produced during partial anaerobiosis was probably responsible. There are examples of respiratory developments in the tracheal system of host insects in response to the presence of endoparasitic insects. Simmonds (1947) observed the presence of numerous tracheoles spreading over the eggs of Bracon in the body cavity of Loxostege-but this was not a constant occurrence. Thorpe (1936) described a remarkable relationship between the host tracheal system of the scale insect Saissetia (Lecanium) and the Chalcid parasite Encyrtus-but this relation is of a totally different character and is not concerned with the responses of tracheoles. 6.2
TRACHEOLE ENTRY INTO THE FLIGHT MUSCLES
Particular interest attaches to the invasion of the flight muscles by the tracheoles at the time of metamorphosis. This process was studied by Brosemer et al. (1963) in Locustu. During the period of anaerobic metabolism of the developing flight muscle, at an early stage of the adult moult, lactic acid dehydrogenase increases to a
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very high level. This is only a temporary increase; the level becomes extremely low again after the moult to the adult when aerobic metabolism has been established. The invasion (or invagination) of the muscle by the tracheoblasts begins only in the last day or so before the moult to the adult. It may well be that lactic acid or other metabolites are attractive (Brosemer et ul. 1963). This interpretation is supported by the observations of Pihan (1972) on the entry of tracheoles in Cyclorrhapha in which the tips of the tracheoblasts are activated and attracted by products of the anaerobic metabolism of carbohydrates, of which lactate is considered the most important. Experimentally, lactate at a pH of 7.5 was most effective. Another factor in the growth of the tracheoblasts is the presence of the moulting hormone ecdysone, which induces growth changes in the cytoplasm of the tracheole cells as in the general epidermis (Beaulaton, 1968) (see Section 2.2). Ecdysone stimulates growth and hence the entry into invaginations of the flight muscle fibres. Implantation of an active corpus allatum into Locustu at its final moult inhibits the ingrowth of tracheoblasts-that is doubtless because it inhibits secretion of ecdysone (van den Handel-Franken and Flight, 1981) or because it sustains the larval state in which flight muscle development is suppressed. Bautz and Pihan (1971) found that in the full grown larva of Culliphoru, 24-48 h after injection of ecdysone, there is a rapid enlargement of the clusters of tracheoblasts. They proliferate by mitosis, and increase in volume, and proceed to form tracheae. Indeed the tracheoblasts proved even more reactive to ecdysone than the histoblasts (cf. Pihan, 1972). 6.3
FORMATION OF PLASMA MEMBRANE INVAGINATIONS
As we have seen, it is commonly supposed that the growing tracheoles are responsible for the invagination of the muscle plasma membrane and the creation of the tubular system (see Section 5.1). But since this system exists in the cells of animals without tracheae, it seems reasonable to regard the tracheae merely as making use of preformed invaginations. This suggestion was made by Beinbrech (1969) who found that, in Phorrniu, tracheolation does not begin in the pupal stage until the muscle is well formed and the T-tubule system is entering the surface of the fibres. The tracheoblasts do indeed make use of this preformed route into the muscle fibres and by following the highly branched T-system they secure a good distribution of tracheoles. In Antherueu pernyi the T-transverse system begins its formation by invagination of the plasma mem-
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brane in the 9-day-old pupa. The tracheoblasts do not begin to enter these prepared invaginations until 13 days (Bienz-Isler, 1968). Haskarl et al. (1973) examined the behaviour of tracheoles towards the wing discs of Galleria in tissue culture. In a medium containing ecdysone the tracheoles migrate into the lacunae of the imaginal discs. The authors attribute this movement to the microtubules in the tracheoles: addition of colchicine and vinblastin prevented the tracheole migration without affecting the viability of the discs. The tracheoles will not migrate in the absence of a tissue to migrate into; they are presumably attracted by some product of anaerobic metabolism. The uncoiling of the tracheoles in the pupa of Cyclorrhapha, effected perhaps by the action of the microtubules, has already been discussed (see Section 2.2). These tracheoles are merely applied to the surface of the developing muscles. Entry into the muscles is accomplished by the newly growing tracheoblasts of the adult tracheal system; this is clearly a process of growth (Houlihan and Newton, 1979). 6.4
MATURATION OF FLIGHT MUSCLES: TRACHEOLES AND MITOCHONDRIA
The tracheoles which make their way into the flight muscles are filled with fluid. Metabolism is largely dependent upon glycolysis, as evidenced by the high content of lactate dehydrogenase in the developing muscles of Locustu (Brosemer et al., 1963). In Calliphora the tracheae of the pharate adult are filled with fluid until a few hours before adult emergence (Houlihan and Newton, 1979). They fill with air at the same time as the resorption of the general pupal moulting fluid occurs (Wolfe, 1954b). When the tracheae fill with air there is a large increase in oxygen consumption, and of course a much greater increase when movement begins (Houlihan and Newton, 1979). After emergence there is a further period of development during which the efficiency of the flight mechanism undergoes extensive changes. Changes which involve the interaction of tracheoles and mitochondria. It is generally assumed that by the time of ecdysis to the adult the development of the tracheoles is complete and that the maturation process concerns primarily the mitochondria. The course of this process varies in different insects. In the blowfly Luciliu cuprina the period of maximum change in the mitochondria extends from 13 days before emergence to 1 day after emergence. During this period the number of cristae per mitochondrion, the
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estimated mitochondrial volume and the total mitochondrial protein all increase two- or three-fold (Lennie et al., 1967) but the enzymic activity is virtually complete by the time of emergence (Walker and Birt, 1969). In Phormia the sarcosomes of the freshly emerged fly had an average diameter of about 1km; whereas by 7 days of continuous growth the average diameter was 2.5p.m; and there had been a 3-fold increase in the content of cytochrome C. Cytochromes (present exclusively in the sarcosomes) continued to increase in titre for 7 days after emergence (Levenbook and Williams, 1956). Locusts are unable to fly immediately after moulting. Within three or four days they will vibrate their wings and make short flights. Full flight powers come later. By 8 days after moulting the mitochondria and contractile fibrils in the adult Locusta appear fully formed (Brosemer et al., 1963), but the enzymes of the citric acid cycle are still rising steeply in the mitochondria of the adult 8 days after moulting (Beenakers et al., 1975). The honey-bee is capable of sustained flight only after some 20 days of adult life. The defect is correlated with progressive changes in the respiratory metabolism in the mitochondria. Myofibril ATPase is at a maximum at emergence and falls gradually to a constant level by 10 days, whereas mitochondrial ATPase gradually increases during the first week of adult life (Maruyama and Sakagami, 1958). The pyruvate metabolizing system of the mitochondria is not complete until 16-20 days after emergence: sustained flight is not possible until the Krebs cycle is fully functional (Balboni, 1967). Whether there are maturation changes in the flight muscle tracheoles after adult emergence is not known. The finest terminations of the tracheoles were more readily demonstrated by injection in the fully mature locust (Schistocerca) (Wigglesworth and Lee, 1982) but this may well have been due to indirect causes (see Section 9.8). 6.5
MITOCHONDRIA A N D TRACHEOLES I N EXTREME EXHAUSTION
We may include here some observations which illustrate the intimate relations between mitochondria and tracheoles in mature adult insects. The wasp Vespa germanica flown in a current of air to complete exhaustion shows a fall in activity of the citric acid cycle enzymes in the flight muscle mitochondria (Hoffmeister, 1961). In these muscles the mitochondria exhibit swelling and disruption with
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extrusion of the contents (brought about by the accumulation of acid metabolites). After recovery during i-1 h, about one-third of the mitochondria have regained their original form and structure; the muscle is functional again and the dispersed internal membranes are once more enclosed in an outer membrane (Hoffmeister, 1961, 1962). In the mosquito Culex tarsalis flown to exhaustion, discrete mitochondria cannot be discerned; membranes resembling cristae are present throughout the sarcoplasm, but there are no limiting outer mitochondrial membranes, only a continuous mitochondrial mass (Johnson and Rowley, 1972). Afzelius and Gonnert (1972) found that in the hornet Vespa crabro about 1% of the mitochondria in the flight muscles are penetrated by tracheoles (around which no plasma membrane is visible). Such a condition could well arise from the changes observed by Hoffmeister (above) after mitochondrial disruption and fusion.
7 Restoration of tracheation without moulting: tracheole capture
There have been many claims that after injuries to insects the tracheal supply may be restored in the absence of moulting. This possibility was investigated in 4th-instar larvae of Rhodnius (Wigglesworth, 1954). At one day after feeding, the tracheal supply to one half of the fourth tergite was cut off by making a minute incision through the cuticle near the margin of the segment, where the main trachea arises from the longitudinal trunk. A fine entomological pin with a hooked point was inserted through the incision and the trachea torn through close to its origin. There is no anastomosis with the dorsal tracheae of the opposite side, so that the entire half tergite, as far as the dorsal vessel, is deprived of oxygen in its tracheal supply. After varnishing the cuticle with shellac the tracheae and larger tracheoles can be observed in the living insect and their movements followed from day to day. They soon begin to migrate inwards from the segments behind and in front, and from the opposite side of the fourth segment. By 10 days the tracheoles have moved to the middle of the fourth segment, some 700pm from their starting point, and there is an adequate new tracheal supply for the affected area. The implantation of a corpus allatum in the middle of the deoxygenated area intensifies and accelerates the response. But there is no convolution of tracheoles around the gland, as was seen after
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moulting (see Section 6.1) when new growing tracheoles were concerned and there is no penetration into the gland. These migrating tracheae and tracheoles have been taken from adjacent segments. The tracheae supplying these regions resist this movement. There is a “tug of war” and the tension in opposite directions leads to tracheae and tracheoles being drawn out straight and taut. Considerable forces are clearly involved. It was at first assumed that the active agents were probably amoebic extensions of the tracheolar cells reactivated by oxygen want. At the most active period of movement the tracheal system was injected and the cells deeply stained, but no visible changes could be seen in the tips of the tracheoles; and the position of the nucleus, about one-third of the distance along the tracheoles, remained unchanged. The newly growing tracheoles formed during moulting are filled with fluid, and it is not unreasonable to expect that they should migrate towards anoxic sites. But this is unlikely to be the case with the fully developed tracheoles in Rhodnius which are filled with air to their extremities. This thought suggested the possibility that the force of traction might be exerted, not by the cytoplasm of the tracheole, but by the epidermal cells which are in need of oxygen; and that the tracheoles play only a passive role (Wigglesworth, 1959b). Preparations of whole mounts of the integument of Rhodnius larvae, stained by the osmium/ethyl gallate method (Wigglesworth, 1957), after the tracheal system had been injected with equal parts of olive oil/kerosine, showed completed filling of the tracheoles right up to their bluntly rounded endings. In preparations made a day or so after section of a main trachea (as already described) there were no visible filaments arising from the epidermal cells. But the preparations did show that many of the “migrating” tracheoles were led not by the tip of the tracheole but often by a hair-pin loop; and that in the neighbourhood of such a loop the mitochondria in the epidermal cells were mostly on the side of the nucleus nearest to the loop and were orientated towards it-as though this were the site of formation of the conical base of a filament. It was then found that if the epithelium was fixed in picro-formol (Bouin) before exposure to osmium tetroxide, the filaments became visible and could be seen running from virtually every epidermal cell and converging upon the loops of the advancing tracheoles. Indeed when the preparation is closely examined scarcely a cell is to be found, within a radius of 100-125pm from the tracheole, which is not connected to it by a strand. Most of the strands are exceedingly
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Fig. 3 Epidermal cells engaged in “tracheole capture” in Rhodnius. A . Two epidermal cells forming a filament attached to a tracheole at 3 days after tracheal section. B. Similar preparation, 4 days after tracheal section, showing attachment to a tracheole loop. C. Two epidermal cells sending out darkly staining processes in the direction of a tracheole loop about 90pm distant. Bouin, osmium tetroxide, ethyl gallate
fine and only just discernible in the light microscope; each arises from a deeply staining cone (Fig. 3A, B). Darkly staining outgrowths that have not yet made contact with a tracheole may sometimes be seen (Fig. 3C). Sometimes the filaments from a number of epidermal cells may fuse to form composite strandssometimes as much as l p m in thickness; they are then quite conspicuous. The total distance from the attachment of the tracheole to the most distant reacting cells may be about 150pm. The cells of the neighbouring segments, from which tracheoles are being withdrawn, also form filaments pulling in the opposite direction. It is probable that epidermal strands are utilized in normal life to secure an equitable distribution of the available tracheoles among the epidermal cells. For if a Rhodnius 4th-stage larva was transferred to an atmosphere of 4% oxygen in nitrogen at 5 days after feeding, and the tracheal system was then injected one day later and the integument mounted, most of the epidermal cells were seen to be sending out cytoplasmic strands that were attached to the tracheoles and these were being pulled in all directions. The
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distances are so short that the strands in question are relatively thick and quite easy to see. In insects kept in air, only an occasional strand of cytoplasm from an epidermal cell can be seen attached to a tracheole (Wigglesworth, 1959b). The existence of cytoplasmic bridges or epidermal feet is well known in insects (Locke and Huie, 1981; Wigglesworth, 1982) but whether the same response is given by other tissues is not known. And the possibility that in some tissues or in some insects, new air-filled tracheae or tracheoles can be regenerated in the absence of moulting, has not been excluded; but it has not yet been demonstrated. The tracheae of the imaginal discs in the larvae of Lepidoptera grow extensively during the interecdysial period (Pate1 and Madhavan, 1969), but no clear indication is given that they become functional and contain air, continuous with the existing system, before ecdysis occurs. Examination of epidermal cells of Rhodnius in the electron microscope confirm the observations on “tracheole capture”. In the normal epidermis there are cytoplasmic processes of varied thickness running in every direction and most of them contain numerous microtubules. The filaments responsible for tracheole capture represent an enhancement of this normal activity; they are merely sent out over greater distances and become very slender, often no more than 50-60nm in diameter; but some fused strands are stout processes 1-2 pm in thickness. They contain mitochondria, rough ER, free ribosomes, occasional microtubules and often plentiful microfilaments. Where they become attached to a tracheole they commonly expand to form a conical attachment (Wigglesworth, 1977). The tracheoles which are being transported may commonly be around 0.75 pm in diameter, but their cytoplasmic sheath is often greatly enlarged to give an overall diameter of 3 p m or even more-as compared with a cytoplasmic coat which is normally quite thin, less than 1pm. The cytoplasm of the “migrating” tracheoles contains vast numbers of microtubules, perhaps induced by the tension to which they are exposed. In the early stages of the process, when the leading tracheoles are probably being transported at a speed of 1pm per minute, the epidermal cells must be in a highly dynamic state and their attachments to the tracheoles must be in a continuous state of flux. It is in the late stages when equilibrium is becoming established between filaments pulling in opposite directions that the extreme changes in the tracheole sheaths become apparent (Wigglesworth, 1977).
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In response to injury, such as excision of the integument, the epidermal cells around the wound leave their original stations and may migrate long distances over the gap, while new cells are produced by mitosis in the deserted outer zones (Wigglesworth, 1937). In tracheole capture the cells are never seen to migrate; they merely send out processes to draw the tracheoles towards themselves.
8 Appearance of air in the tracheal system
Throughout development in the egg (or until very shortly before hatching) the tracheal system contains fluid. The same is true of the newly formed system developed before each moult; the existing tracheae and tracheoles retain their gaseous content but the space between these tubes and the walls of the newly formed tracheae is filled with fluid. About the time of hatching from the egg, or at the time of ecdysis, the fluid is replaced by air-with the exception of some aquatic larvae (Chironomidae; Acentropus (Lep.) etc.) and in some larvae of parasitic Hymenoptera, in which the earlier instars have their tracheal system completely or partially filled with fluid, and replacement by air first takes place after later larval moults. 8.1
ABSORPTION OF FLUID CONTENTS
It was shown by Weismann (1863) that in the newly hatched larva of CaZZiphoru the fluid is absorbed into the tissues. Keilin (1924) gave a full critical review of previous work on the appearance of air in the tracheal system of aquatic insects without functional spiracles, and showed convincingly that in Dasyhelea (Chironomidae), as in Calliphora, the fluid is absorbed, the column of fluid is ruptured, and the system is filled with gas diffusing from the tissues, and thus from dissolved gas in the surrounding water. There is in fact little difference between the filling with air in the open and closed systems. In larvae of the grain moth Sitotroga the system normally fills with air while the larva is still bathed in fluid in the egg. The spiracles do not open until the larva has emerged and the skin dried (Sikes and Wigglesworth, 1931). The same is true of Lucilia in which the tracheae fill while the larva is still in the egg; and Weismann (1863) showed that in Calliphora the tracheal system will fill even if the egg is submerged in water. (In Lucilia this only happens if the egg is near the surface of the water.) In Tenebrio the
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tracheal system normally fills from the outside air after hatching; but if the egg is allowed to hatch under water the system will fill with air from gases in solution as in aquatic larvae. The flea Ceratophyllus normally fills, like Tenebrio, as soon as the spiracles are exposed to the air and was never seen to fill under water. And in such insects as Cirnex and the sucking louse Polyplux filling does not take place until the larva has escaped from the egg, cast the embryonic cuticle, and is exposed to the atmosphere (Sikes and Wigglesworth, 1931). There seem to be all grades in the ability to achieve filling without exposure of the spiracles to the air. During the filling of the tracheae with air in the moulting larva of Agrionid dragonflies, there is a negative pressure in the tracheal system which will aspirate water into the cut ends of the tracheae (Koch, 1936). 8.2
SITE OF FLUID ABSORPTION
During the moulting process a copious layer of fluid, the exuvial fluid, accumulates between the newly forming cuticle and the old cuticle that is to be discarded. This fluid contains enzymes, chitinase and protease, which dissolve the inner layers of the old cuticle (Wigglesworth, 1933c; Passonneau and WiIliams, 1953). Shortly before moulting the exuvial fluid is removed; in certain insects such as caterpillars some of it is swallowed, but for the most part it is absorbed by the epidermal cells through the substance of the new cuticle, the surface of which becomes dry, and the space between old and new cuticle becomes filled with air. The exuvial fluid is continuous with the contents of the newly formed tracheae, in which there is a similar process of digestion of the main substance of the tracheal wall. In the fully developed pupa of Calliphora the drying up of the remains of the exuvial fluid only just precedes or is simultaneous with the filling of the tracheal system with air (Wolfe, 1954b). The filling of the tracheal system is essentially the same process as the absorption by the epidermis of the moulting fluid. It must therefore be inferred that in the tracheae this absorption is a general process distributed throughout the system and is not confined to the terminal tracheoles-although in all probability they also are taking part. The idea of a general absorption is supported by the observation that during the entry of air into the tracheal system of the mosquito larva Aedes (Wigglesworth, 1938b) and the larva of Sciara (Keister and Buck, 1949) columns of air may approach and unite from
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opposite directions in one unbranched trachea, showing that absorption must be in progress at that site. There seems no reason for distinguishing the air filling at hatching from the egg from that after moulting. 8.3
THE NATURE OF THE ABSORPTION PROCESS
In studying the filling of the tracheal system with air in the larva of Dasyhelea, Keilin (1924) concluded that the absorption was probably by “protoplasmic imbibition”. The term “imbibition” is often used for the physical uptake of fluid by colloidal material, such as the swelling of gelatin. But Keilin presumably intended “secretion” or “active transport” by the living tissues, such as we have been implying in the uptake of the moulting fluid. In discussing the absorption of fluid after hatching, in the series of insects mentioned above (Sikes and Wigglesworth, 1931) an unconvincing attempt (based in part upon ideas put forward by Weismann in 1863) was made to attribute the absorption of fluid to the production of a salt-free liquid within the tracheal system, which could then be absorbed by a rise in osmotic pressure, exerted by the metabolic products of excessive muscular exertion associated with the hatching process. This idea was tested on the newly moulted larva of Aedes aegypti. These larvae are unable to liberate gas in the tracheal sytem when totally submerged, even in well oxygenated water. They can only fill the system with atmospheric air taken in through the spiracles of the respiratory siphon. If the eggs hatch under water the tracheae do not fill. When the larvae are allowed to reach the water surface the valves of the siphon are drawn open by surface tension and air enters the spiracles, the fluid is absorbed from the lumen of the tracheae and the system fills with air in 15-30min. If the larvae are kept submerged for 24 h, filling, on making contact with the surface, is more rapid and is complete within 12min. More prolonged submersion after hatching leads to slower absorption and after 4 days most larvae fill the system only partially and some not at all. In second instar larvae the ability to absorb the contents of the tracheal system is lost if they are kept for 8 h before coming to the surface (Wigglesworth, 1938b). The loss of the capacity for absorbing fluid through the tracheal walls agrees with the progressive impermeability of the surface of the general cuticle of the abdomen in the later stages of moulting (Wigglesworth, 1933~). If the newly hatched larvae are lightly narcotized with chloroform
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before being put in contact with the surface, the spiracles open at the surface as usual (they are controlled by surface forces) but no air enters while the larva is motionless. Movement is restored in 20-30min. There is then a further delay of 10min or so before absorption starts; it is completed at the usual rate. The inhibition of absorption by narcosis suggests that absorption is a secretory phenomenon controlled by the nervous system (Wigglesworth, 1938b). This nervous control of resorption followed by impermeability is reminiscent of the changes in the cuticle induced by neurosecretion (bursicon) soon after moulting (Cottrell, 1962; Fraenkel and Hsiao, 1965). Indeed the whole procedure recalls the ability of the newly emerged adult Calliphora to defer the hardening of the cuticle so long as it is burrowing through the soil (Fraenkel, 1935). 8.4
THE ROLE O F OXYGEN IN ABSORPTION
The absorption of the tracheal fluid after hatching and moulting is an oxygen demanding process. It does not occur in Corethra in oxygen free water (von Frankenberg, 1915). Filling in Sciara larvae is likewise inhibited in the absence of oxygen; but as little as 0.3% of oxygen in nitrogen is sufficient to permit gas to appear in the liquid filled tracheae (Keister and Buck, 1949). Filling could also be arrested indefinitely in Sciara by low temperature, 0°C (Keister and Buck, 1949). Some strange results were obtained on the filling and reversal of filling in premoult larvae of Sciara, but these are best studied in the original (Buck and Keister, 1955). The importance of a supply of oxygen and an adequate temperature support the conception of an active absorption of fluid. It has been noted that if the entry of air by way of the spiracles is prevented by allowing the insect to hatch in water and keeping it submerged for some hours, and it is then exposed to the water surface, the rate of filling is accelerated: the whole system may fill in less than thirty seconds (Sikes and Wigglesworth, 1931). This suggests that as the result of the delay the absorbing force has increased in strength.
8.5 L I B E R A T I O N O F G A S F R O M S O L U T I O N From the foregoing discussion it is clear that there is no essential difference between the entry of gas through the spiracles and filling by the liberation of dissolved gas by suction. At one time it was
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thought that a very high negative pressure would be necessary to break the column of water and initiate the process. It is now well known that that is true only when the fluid is in contact with a completely wettable surface: any greasy or waxy point in the surface will permit the formation of a bubble at very little negative pressure, and once a bubble is formed its enlargement from gas in solution again requires only minimal suction (Wigglesworth, 1953).
9 Movements of fluid in the tracheole endings
The question of whether the tracheole endings during life contain liquid or air occupied many authors in the last century. It was well known that after death they soon fill with fluid (Lubbock, 1860; Wielowiejski, 1882; Emery, 1884) and become to all intents invisible. There is no doubt, therefore, that in many cases the upper limit of fluid has been mistaken for the abrupt termination of the tracheole, and this was one cause for the controversy which raged around this subject. The earlier authors (Schultze, 1865; Tozetti, 1870) believed that the tracheoles contained fluid and von Wistinghausen (1890) believed that this fluid flowed in and out of the cells, so constituting a form of inspiration and expiration. Pantel (1898) and Wahl (1899), however, asserted that during life the tracheoles always contain air, and this view became generally accepted (Keilin, 1924; Remy, 1925). But Lund (1911) still maintained that the tracheoles to the photogenetic organs of Lampyridae contain fluid, Koeppen (1921) that this was so in Dytiscus and Davies (1927) in Sminthurus. 9.1 V I S I B L E M O V E M E N T S I N T H E L I V I N G I N S E C T In reflecting on this problem around 1930 I conceived the idea that if the fluid containing endings of the tracheoles supplying a muscle were bounded by a suitable semipermeable membrane, water might well seep through the membrane during rest and be drawn along the fine tube by capillarity until its progress was arrested by the osmotic pressure of the tissue fluids. During muscular activity the liberation of metabolites would increase the osmotic pressure, which would bring about the absorption of water from the tracheoles and the extension of the column of air into finer and finer regions of the tubes until a new balance between osmotic pressure and capillarity was established.
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I tried in vain to devise experiments to test this idea, until the chance observation of a living larva of Aedes aegypti by transmitted light under the 4mm objective revealed the tracheoles in the head coming from a small trachea and running towards the antenna1 muscle. The trachea and the tracheoles were air-filled, appearing black by total deflection of the transmitted light as they divided up within a typical stellate end cell and ran toward the muscle. At a certain point the air in each tracheole ended abruptly and beyond this point the tube was invisible. But when the larva began to struggle (particularly when the respiratory siphon was separated from the water surface) the air began to extend along the tracheoles into finer and finer regions until they could no longer be resolved. On admission of air, and renewed repose of the larva, the fluid slowly rose again in the tracheoles. 9.2
POSSIBLE CONTROL OF MOVEMENT B Y OSMOTIC PRESSURE
This was a gratifying discovery but, as stated in the published report (Wigglesworth, 1930) “the observations recorded create more problems than they solve”. It was shown that if the tracheoles were exposed to the haemolymph from another resting larva (by puncturing the head and applying the foreign haemolymph to it) the columns of air in the tracheoles did not move. But if the haemolymph came from a larva which had been partially asphyxiated (by struggling, out of contact with the air) the tracheoles quickly filled with gas. Thus the circulating fluid was effective. Dilute saline solutions were ineffective until the concentration exceeded about 310p,M/1 and then the tracheoles filled with air as before. The same effect was given by potassium lactate, again when the same osmolarity was exceeded. When lactic acid was used, filling was rapid but almost at once the fluid rose again: permeability had been increased. As was pointed out “a membrane impermeable to a lactate is not a structure with which biochemists are familiar”. Two alternative possibilities were suggested: (i) that the osmotic pressure changes might be due to the acidity of metabolites altering the dispersion of proteins and so inducing the inhibition of water; (ii) in view of the great changes in tension at oil-water interfaces which follow slight changes in pH (Hartridge and Peters, 1922), surface tension changes might play an important part. But in view of the effectiveness of potassium lactate at neutral p H these mechanisms did not seem called for.
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My inclination was to leave the matter there until more information was available. But the referee of the paper urged that it ought to be shown by calculation that the mechanism could work. At that date the parameters needed were unknown. The model used was the standard experiment from the physics books for measuring the surface tension by capillarity measurements in a grease-free glass tube; but instead of setting capillarity against gravity it was set against osmotic pressure-and by making appropriate assumptions for the unknown parameters a tolerable balance was demonstrated. The author’s comment reads: “these are serious assumptions, and too much weight must not be attached to the results”. The movement of fluid in response to asphyxiation was confirmed in a range of terrestrial insects: the flea Cerutophyllus, the larva of Tenebrio, Bluttellu, the adult mosquito Aedes. In Cirnex, fluid could be seen in the tracheole endings but no movement occurred on flooding with water-doubtless because the insect showed no muscular activity. There were small changes in level from day to day in resting insects but no consistent change during extreme desiccation until after death, when the air extended to the tracheal endings as the tissues dried (Wigglesworth, 1931). In the unfed larva of Aeschnu the tracheoles on the surface of the gut were largely filled with fluid; after feeding, they filled with air and air-filled branches dipped down to form basket-like clusters around the epithelial cells (Wigglesworth, 1930). 9.3
THE TRACHEOLES D U R I N G NORMAL RESPIRATION I N THE FLEA
In the course of observations on the flea XenopsylZu, confirming and extending the conclusions of Hazelhoff (1927) on the control of the opening and closing of the spiracles in Periplunetu, in response to oxygen and carbon dioxide, the opportunity was taken for observing changes in the tracheole endings at the same time (Wigglesworth, 1935). The flea was held in a small gas chamber under the microscope. The tracheoles of the abdominal wall showed the air columns ending abruptly as in the mosquito larva. Slight movement downward followed muscular activity, with the fluid rising again during rest. At a raised temperature of 35°C these movements were exaggerated: within 5 s of the commencement of struggling, the air columns had shot down into the finer branches of the tubes. In many tracheoles the meniscus was moving rapidly up and down almost all the time.
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Meanwhile the spiracles were opening and closing more rapidly; it was shown that a major factor in the opening of the spiracles is the “oxygen debt” due to the accumulation of unoxidized metabolites. If the proportion of oxygen in the air was changed, there was a corresponding change in the equilibrium position in the tracheoles during rest: the column of gas extends further in 10% 0 2 and still further in 5% 0 2 , and in cylinder nitrogen containing 0.8% 0 2 it extends virtually to the extremities. In pure 0 2 the column of gas retreats into even wider parts of the tube than it does in air: it would appear that at equilibrium in air there is a small oxygen debt with a small normal accumulation of metabolites. In air at 22°C the spiracles open and close at about 6-7 s intervals. This looks like a respiratory rhythm; but that is not so; each act of opening or closing depends upon the chemical state of the tissue fluids. The duration of the closed period is determined (i) by the accumulation of unoxidized acid metabolites due to want of oxygen, and (ii) by the accumulation of carbon dioxide resulting from complete oxidation. The duration of the open period is determined mainly by the time taken for carbon dioxide to diffuse out and therefore by the duration of the closed period which precedes it. In 5% or 10% 0 2 , acid metabolites accumulate sooner and much less carbon dioxide is formed in these brief periods. In 100% 0 2 it takes a long time for acid metabolites to accumulate-indeed the opening of the spiracles may well be due mainly to accumulated COz, and there is always a long open period to get rid of it. In other words, the average level of unoxidized metabolites will be progressively greater as the oxygen content of the air is reduced. In 1.8% 0 2 the movement of the spiracles is almost too rapid to record accurately: C 0 2 accumulation must be playing virtually no part in control. In all these observations the inferred accumulation of metabolites is associated with the removal of fluid from the tracheole endings.
9.4
M E A S U R E M E N T S O F OSMOTIC P R E S S U R E , A N D REASSESSMENT
In 1937 it became possible for the first time to measure directly the osmotic pressure in the haemolymph of single mosquito larvae (about 0.3 mm3) by the vapour pressure method of Hill and Baldes (Baldes, 1934). It was readily shown that in asphyxiated larvae of Culex the osmotic pressure in the haemolymph increased by 25%
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TABLE 1 Osmotic pressure of haemolymph in mosquito larvae, expressed as % NaCl
Culex Resting larvae: 0.88, 0.85, 0.89, 0.85 Asphyxiated larvae Air extending to finest tracheoles: 1.08, 1.06, 1.16 Less struggling, air not in finest tracheoles: 0.94 Aedes Resting larvae in tap water: 0.81, 0.86, 0.89, 0.81 Resting larvae in 1.01% salt water: 1.05, 1.08, 1.06, 1.05 Asphyxiated larvae from salt water: 1.27, 1.31
in those larvae which had shown the greatest muscular activity (Table 1). In earlier work (Wigglesworth, 1933b) it had been found that in larvae of Aedes acclimatized to water containing 1.2% NaCl, the level of fluid in the tracheoles was unchanged-from which it was concluded that the larva was homoiosmotic. But it was now found that in a medium of l-Ol%NaCl the osmotic pressure in the haernolymph had risen to the equivalent of about 1.05-1*08% NaCl, while the position of the air column in the tracheoles was at the same sort of level as in fresh water; and on asphyxiation there was a rise in haemolymph osmotic pressure by a further 25% and the tracheoles filled with air (Table 1). These results demanded a new assessment of the osmotic hypothesis as formulated in 1930. It was obvious that the direct osmotic pressure exerted by inorganic ions and small organic molecules acting on the tracheole wall, which served as a semipermeable membrane, could not by itself explain the fluid movement observed. It was concluded that the products of metabolism under conditions of anoxia, acted upon the proteins in the cytoplasmic walls of the tracheoles, or in the cells by which the tracheole endings are invested; and that it is the osmotic forces, or “imbibition”, by these proteins which is responsible (Wigglesworth, 1938a). 9.5
OSMOTIC FORCES FROM IONIZED PROTEINS
These are the forces which were turned aside in the 1930 paper because the movement of fluid had been induced by neutral electrolytes. But they were subsequently exhibited very strikingly in the anal papillae of mosquito larvae (Wigglesworth, 1933a). The thin cuticle of the anal papillae is freely permeable to sodium
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a
Fig. 4 Cytoplasmic swelling and tracheole filling in anal papillae of intact mosquito larva, Aedes aegypti. a. Normal papilla of living larva in fresh water: tracheole endings largely filled with fluid. b. The same, after 2 min in 2% NaCI: the cells greatly swollen and tracheole endings filled with air. c. The same, 15 min after return of larva to fresh water: the cells contracted down again and tracheole endings filling with fluid
chloride; the underlying cells actively absorb chloride and other ions (see Section 4.5). If the intact larva is exposed to an increasing concentration of NaCl, or other salts with monovalent ions, these have no visible effect on the cells of the anal papillae, until the solution reaches an osmolar concentration of about 300 mM/1, which is slightly hypertonic to the haemolymph. The cells then-swell to an enormous size and at the same time fluid is extracted from the “intracellular” tracheoles which they contain and the tracheoles fill with air (Fig. 4a,b). This swelling was compared with the action of NaCl on gelatin as interpreted by Thimann (1930): from the Donnan relationship the Na+ and C1- ions enter the gel, increase the ionization of the protein by the formation of complex ions (as was to be expected from the Zwitterion conception) and the protein swells. In the case of the anal papillae, non-electrolytes such as sucrose, glycerol and urea fail to cause swelling in hypertonic solutions; they induce only a general shrinkage of the body of the larva (Wigglesworth, 1933a). These colloid osmotic forces do not have the magnitude of the total osmotic pressure of the ion containing tissue fluids. But as Bult (1939) pointed out it is highly improbable that the tracheole intima should have an angle of contact approaching that of grease-free glass. In 1930 we knew nothing of the nature of the outer epicuticle;
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we do not know yet its chemical composition (see Section 2.1); but in staining properties it behaves like a lipid. Although its fine structure always appears the same, its wetting properties may well vary at different levels and in different insects. In any case its angle of contact is probably quite large and the resulting capillarity could readily be balanced by the osmotic forces exerted by ionized protein. That was the conclusion reached in the paper of Wigglesworth (1938a). The same conclusion was reached by Bult (1939) who made an extensive experimental study of the movements of fluid in the tracheoles of the isolated gut of Blattella (Phyllodromia). (Bult includes a reference to the above paper but it is evident that his conclusions were arrived at quite independently.) Bult’s main conclusion was that extension of the air columns “is caused by the influence of anaerobic metabolites on the swelling of the cell proteins”. But besides the products of anerobic glycolysis he also suggests that the breaking of -S-S- bridges with the formation of -SH groups may be a major factor in enhancing the uptake of water molecules by protein. 9.6
ELASTIC FORCES OF PROTEIN STRUCTURE
Another force which may be involved in the movements of the tracheole fluid derives from the elastic properties of the protein structure in the cell. This was recognized by Bult (1939) and by Beadle (1939). It could be seen operating in both directions in the anal papillae of Aedes. (1) After exposure of the intact larva to hypertonic NaCl as described above (see Section 9.5), (Fig. 4a,b), if the larva is quickly returned to fresh water the excess salt is removed, the elasticity of the cell structure reasserts itself, the cells contract down to their normal size and fluid again extends up the tracheoles (Wigglesworth, 1933a) (Fig. 4c). (2) The converse effect is seen if the anal papillae are amputated and immersed in hypertonic NaCl, so that the cells are exposed to the same solution on both sides, and the cells shrink by osmosis. The protein structure will now be acting to restore the normal cell size and this elastic force draws out the tracheole fluid with its solutes and the tracheoles fill with air (Wigglesworth, 1933a). When the salt content of the haemolymph rises as a result of increased salinity of the
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medium, the protein structure of the cells must become adapted (see Section 9.4). This was shown in more extreme form by Beadle (1939) in Aedes detritus which can live in highly saline waters. 9.7
S I T E OF A C T I O N O F METABOLIC P R O D U C T S
The mode of ending of the tracheoles has already been described (see Section 1.1 and 5): they may be freely exposed on the surface of the tissues; they may form basket-like arrays between closely packed cells, as in the intestinal epithelium and in certain muscles; or they may be invaginated deeply into the cells, notably in the flight muscles and various gland cells. Any action of the products of anaerobiosis must be exerted on the cuticular wall of the tracheoles or on their cytoplasmic sheaths. Von Frankenberg (1915) observed the swelling and expansion of the cuticular wall of the tracheal air sacs of Corethra on asphyxiation. The membrane contracts on drying, swells instantly again on moistening; and this swelling is greatly increased in NaC1. Nothing is known about the swelling properties of the tracheole intima. But it seems likely that the cytoplasmic sheaths (see Section 2.2) of the tracheole may be concerned in absorbing the fluid contents when exposed to metabolites in the general body fluid. The products of anoxia are often set free into the haemolymph and quantitative estimates of them have frequently been made. We saw that the osmotic pressure in the mosquito larva subjected to asphyxia under water for +-1h showed an increase of about 28%, from about 294 to about 382mM/1 (Wigglesworth 1938a). In the beetle Sitophilus under anaerobic conditions, glycerophosphate, pyruvate and particularly lactate accumulate; but the total accumulation amounted to less than half the glycogen utilized; presumably there are other unidentified products (Bond, 1965). In the diapausing caterpillar of Laspeyresia anoxia produced increases in alanine, and particularly in lactate which rose from llmM/l to 58 mM/1 in 6 h and to 98 mM/1 in 24 h (Sgmme, 1967). In the leaping muscle of grasshoppers after exhaustion there is an accumulation of lactate up to 10 times the resting value; but this rise in metabolites is confined to the muscle tissue; there are no significant changes in the circulating haemolymph (Bishai and Zebe, 1959). In the case of glandular cells the invaginated tracheoles are invested not only by their own cytoplasmic sheath but also by the active cytoplasm of the gland cells, and will be subject to the ionized
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protein produced in these cells by anoxia. Indeed that was the main site of the metabolites envisaged by Bult (1939) who makes no reference to the tracheole cytoplasm. 9.8
METABOLIC PRODUCTS I N FLIGHT MUSCLE
In the flight muscles, as we have seen (see Section 5.3) the tracheoles are deeply buried within the fibres and closely associated with the mitochondria. In the thorax of Musca subjected to anoxia there was an accumulation of 8-10 times the normal level of a-glycerophosphate; but comparatively little appeared in the general haemolymph (Heslop and Ray, 1964). In the flight muscles the abundant tracheales lie in the path between the contractile fibrils and the mitochondria. They are right at the centre of metabolic activity. Any failure by the mitochondria to achieve complete oxidation of the carbohydrate or lipid fuels will lead to the accumulation of acid metabolites, notably pyruvic acid, and thus the swelling and dispersion of ionized proteins which will bring about removal of fluid from the tracheole endings and relieve any oxygen deficiency. In Musca and Culliphoru there is such a rich supply of aircontaining tracheoles that it is difficult to observe the presence of fluid in the terminations. When a fly is kept immobile at rest for some hours at 4"C, there is probably an increase in liquid containing endings (Wigglesworth and Lee, 1982)-but it is difficult to be sure of this; if only the extreme endings are filled with fluid they would be impossible to observe. However, Sacktor and Wormser-Shavit (1966) observed a very striking increase in pyruvic acid at the initiation of flight in the blowfly Phormia; the concentration in the thorax increases four-fold in the first few seconds, shortly followed by increases in alanine and acetyl carnitine. But very rapidly the level of these metabolites falls to their initial level and remains so during flight. Clearly on the commencement of flight pyruvate is not metabolized in the Krebs cycle as fast as it is formed by glycolysis. Sacktor (1970) has suggested various biochemical factors which may bring about the required adaptation; but it could well be that improved oxygen supply to the mitochondria as the result of removal of fluid from the tracheole endings could be responsible. In Schistocerca injection of the tracheal supply to the flight muscles in the resting insect fills the longitudinal tracheoles that run parallel with the contractile fibrils, but it does not fill the rich system of fine tracheoles in the 0.05-0.1 size range which lie beyond them
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and which invest the mitochondria. But if the locust if flown to exhaustion in a falling oxygen tension induced by evacuation, the entire system is readily injected (Wigglesworth and Lee, 1982). This again strongly suggests, though it does not prove that during rest the finer tracheoles fill with fluid. In the flight muscles of the cabbage butterfly Pieris the main tracheole supply is the system of transverse tracheoles already decribed (see Section 5.4). These are packed in between the mitochondria. During rest (in insects that have been kept at 4°C) they are filled with fluid. They are not visible in freshly dissected muscles viewed in the light microscope and they often fail to be injected by the usual procedure. But if the muscles of butterflies flown to exhaustion during evacuation in air are examined, they now have a silvery appearance; the air filled tracheoles are readily seen by transmitted light; and they fill completely when injected after evacuation in hydrogen. Here there is no doubt that the most important part of the terminal tracheoles is filled with fluid during rest (Wigglesworth and Lee, 1982). It was established by Zebe (1954) that in Lepidoptera lipid is used as the immediate fuel for flight, and carbohydrate i s converted to fat before it can be used by the flight muscle. Zebe then found that Lepidoptera build up a considerable oxygen debt at the beginning of flight, which he attributed to the slower metabolism of lipid-but which may depend in part upon the presence of fluid in the tracheoles in the early stages of flight. 9.9 R O L E O F S U R F A C E T E N S I O N I N T H E T R A C H E O L E E N D I N G S The problem offered by the tracheal system, of excluding fluid from the narrow channels, is seen also in the alveoli of the vertebrate lung. Pattle (1958) pointed out that the alveoli have a curved diameter of 40pm, and if the fluid on their walls had the surface tension of blood serum (y = 55 dyneskm) the alveoli would fill with fluid and the lung would collapse. But this is prevented by a highly surface active layer some 50A thick, believed by him to be an insoluble protein, which reduces the surface tension to a very low level. On squeezing a fragment of fresh lung a highly stable foam is produced. When Pattle cut through the head, thorax and abdomen of a Calliphora adult and squeezed out bubbles and fluid, these contained no surface active factor. He suggested that control of the entry of fluid into the tracheoles of an insect is probably by active transport of water through the cellular membranes at their termina-
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tions. With the surface properties of the tracheole intima as set out above (see Section 9.5), that would seem to be in no way necessary. 9.10
ROLE OF ACTIVE TRANSPORT
We know little about the composition of the fluid in the tracheole endings. In studying the distribution of ions in the salivary gland cells of Periplunetu by electron probe X-ray microanalysis on frozen-hydrated sections, B. L. Gupta (in unpublished work to which I am very kindly permitted to refer (Gupta and Hall, 1982)) came across the transverse section of a fluid filled vessel, about 0.5 pm in diameter, between the glandular cells. The Ringer bathing the preparation had the ionic composition in mM/1: Na: 120 Mg:2 C1: 135 K: 10 with mannitol 60 and trehalose 5.
Ca: 2
Pod: 2
The fluid in the presumed tracheole:
K: 14 Ca: 6 P: 7 S: 7 . The content of organic solutes in the “tracheole” could not be measured, so that the total molarity was not known. The figures merely indicate that, as we have long supposed, it is a fluid containing a quantity of solutes. We have no indication whether the composition is constant. (The ion content of the fluid in the secretory ducts was quite different.) There is good evidence that the fluid present in the tracheal system at hatching and at moulting is actively absorbed throughout the tracheal system (see Section 8.3) but the physical mechanism of this absorption is unknown. In mosquito larvae it is controlled by the nervous system (perhaps by a hormone) and can take place only shortly after moulting. This initial absorption seems to be a quite different process from the absorption of fluid in the tracheole endings, which can continue throughout life. There is no experimental evidence to suggest that this is a controlled secretory process. In a theoretical discussion of this continued movement of fluid in the tracheoles, Beament (1964) postulates that the lining of the tracheole is completely wettable with a contact angle with water approximating to zero. He leaves open the question whether the water in the tracheoles contains solutes or not, but he contends that in either case, with a fully wettable tracheole wall, to empty the tubules will require active transport of water or ions or both. Na: 28
Mg: 3
C1: 91
V. 6.WIGGLESWORTH
134
With present knowledge of ion and water transfer in cells by electrical coupling and other modes, there is no reason why these movements of fluid could not be under “vital” control. But there is no experimental evidence that “active transport” is in fact being used. The movements with which we are concerned are those which appear to be (i) the adventitious consequences of muscular contraction, or other forms of metabolic activity by the animal, or (ii) the result of the application of reagents by the experimenter. These phenomena can be explained, without recourse to “active transport” by the physical processes already described: osmotic forces by ionized proteins and elastic forces by protoplasmic structures. The requirements are that the intima of the tracheole must have a large angle of contact (to reduce the force of capillarity) and it must be porous. These are characters with which we are familiar, among the diversified properties of the epicuticle of the body surface. Furthermore, the inner (cuticular) wall of the tracheole must be more permeable than the outer (plasma membrane) wall bounding the cytoplasmic layer (Wigglesworth, 1953). These properties are characteristic of the walls of the anal papillae of Aedes larvae, where the outer (cuticular) wall is more permeable than the inner (basement membrane) wall separating the cytoplasm of the cells from the haemolymph (Wigglesworth, 1933a). So they are not unreasonable demands.
10 Tracheole supply to the photogenic organs of fireflies: a new
hypothesis
The luminescence of fireflies is characterized by the emission of light in flashes. Two main hypotheses have been advanced to describe the control of flashing: (i) that the nerve impulse itself evokes the necessary enzymic process in the photogenic cells, (ii) that the flash is evoked by the sudden admission of oxygen by way of the tracheoles which supply the photogenic cells. Once the intermediate complex of luciferin-luciferase is formed, the availability of molecular oxygen is the final requisite for its oxidation and the consequent emission of light with 100% efficiency, one light quantum for each luciferin molecule (McElroy , 1965). Concerning the second hypothesis, two suggestions have been put forward as to the nature of this oxygen control: (a) that the large tracheal end cell provides a mechanical valve capable of suddenly admitting oxygen by a propulsive mechanism (Dahlgren, 1917) or
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by the alternating activity of a circular muscle, which closes a sphincter around the tracheoles in the tracheal end cell, and a set of radial muscles in the tracheal end cell which draw the sphincter open (Alexander, 1943); (b) that the tracheoles supplying the photogenic cells are normally filled with fluid, but that nervous activation of the photogenic cells leads to metabolic changes (like those in contracting muscles) which induce an osmotic extraction of fluid from the tracheole lumen and thus admit oxygen (Maloeuf, 1938). These mechanisms were discussed at length by Buck (1948). He dismisses the Maloeuf hypothesis on the ground of the osmotic mechanism being too slow to explain the rapid flashes. That may well be true; but Buck had in mind the relatively slow movements in the tracheoles of mosquito larvae (Wigglesworth, 1930). Under different conditions, for example in the abdomen of the rat flea at a temperature of 37”C, the meniscus will move up or down the tracheole over many wm in a fraction of a second and this in a system not specially adapted for the present need (Wigglesworth, 1935). But another strong objection to the osmotic extraction idea lies in the complexity of the tracheal end cell system. This specialized structure is present only in the adult fireflies which emit flashes; it is absent in the glow-worm (Lampyris) and the larvae of fireflies, which give out only a steady glow. It seems almost self-evident that this elaborate system must be concerned in flash control. The idea of circular and radial muscles controlling a sphincter at the level of the tracheal end cell has been excluded by study of the fine structure (Beams and Anderson, 1955; Smith, 1963): no such muscles exist. But the tracheal end cell is packed with radial mitochondria and invaginations of the plasma membrane (like the water and ion absorbing cells in the rectum of insects (see Section 4.5): the tracheal end cell almost completely envelopes the tracheolar cell (Kluss, 1958) which is responsible for the intima of the entire length of the tracheoles in question; on its outer surface the tracheal end cell is conspicuously separated from the photogenic cells by a continuous gap bounded by membranes and containing a basal lamina reinforced by plaques; the nerves end in pad-like terminations between the tracheal end cell and the tracheolar cell (Smith, 1963) not on the surface of the photocytes. The tracheal end cell and the tracheolar cell which it encloses appear to form a selfcontained “organelle”, and it seems that it is the nerve ending on this organelle which controls the rate of flashing in the photogenic cells.
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In all the flashing fireflies the tracheoles of the luminous organ have stiffened walls with supporting bars which prevent their collapse (Ghirandella, 1978). As a variant on former hypotheses I would suggest that during the time of functional activity the tracheoles of the light organs are filled with air to their extremities, but that the basal organelle does operate as a localized valve by alternately liberating fluid, which occludes the lumen, and reabsorbing it to re-open the lumen, at a rate dependent on the frequency of the nerve impulses. The nerve wraps around the tracheolar cell and appears to synapse on it (Ghirandella, 1977). As shown by Buck and Case (1961) the flash response is closely similar in all respects to the response of muscles to neural excitation, with a normal flashing rhythm of about 0.5s; but the rate of flashing is much higher in Pteroptyx and other Asiatic fireflies (Peterson and Buck, 1969). The operation suggested is the same process with which we have long been familiar in the tracheoles supplying muscles. I suggest that in the fireflies it may have been adapted and refined to form a new type of valve. All that would be needed is a succession of nerve impulses, each of which will give a momentary increase in hydrogen ion concentration, and thus induce ionization and swelling of the cytoplasmic proteins, with resorption of tracheole fluid at the site of the valve-so releasing a pulse of oxygen to cause the flashinstantly followed by oxidative restitution of pH and release of fluid to block the valve again. (It should be pointed out that Ghirandella (1977), who described in detail the structural characters of the tracheolar cell and its reinforced tracheoles, hinted at an osmotically controlled mechanism for regulating the flash. The mechanism here suggested was inspired by his observations and by his idea.) Would it be possible to obtain evidence in support of such a mechanism? It is unlikely that visual confirmation could be obtained. But one requirement would be a highly permeable tracheole wall at the site of the valve. Perhaps this was already demonstrated by Schultze (1865) who found that in intact Lampyrids exposed to osmic acid vapour the entire tracheal end cells are blackened by the entry of osmium at this point. By injection of myrcene or other similar materials, followed by osmium treatment after fixation, the permeability of this area, and indeed of the whole tracheole tract to the photogenic cells could be tested, as in the tracheole supply to the flight muscles (Wigglesworth and Lee, 1982) (see Section 11.2).
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11 Permeability and tracheole function: a new theory
In a closing comment in the paper describing the removal of fluid from the tracheole endings during muscular activity (Wigglesworth, 1930), it was claimed that the ideas put forward “provide for the adjustment of local respiration to meet the demands of individual organs; just as variations in the capillary bed subserve the same purpose in the internal respiration of vertebrates” in which the capillaries are mostly closed during rest, and are caused to dilate by the metabolites produced during activity. But it was pointed out in a later paper (Wigglesworth, 1931) that this analogy was only superficial, because whereas in vertebrates a restricted capillary circulation during rest is necessary to relieve the work of the heart, there is no apparent reason why all the tracheoles of insects should not be kept always filled with air. Since that time the physiological significance of the movement of fluid has remained a mystery. 11.1
VARIABILITY I N F L U I D CONTENT O F TRACHEOLES
It soon became apparent that fluid was not always present in the tracheoles in some insects. In Pediculus all tracheoles appeared to contain air to their extremities (Wigglesworth, unpublished). In Sciara, once the tracheal fluid had been absorbed the whole system was always filled with air (Keister, 1948). In Rhodniw, as in Sciara, the tracheole endings in the epidermis once filled with air remained so permanently: the air-filled tubes on reaching about 0-2p,m diameter ended in a blunt rounded tip. On the other hand, the numerous longitudinal tracheoles between the contractile fibrils of the flight muscles of Rhodnius could be seen on dissectjon ending abruptly at a diameter of 0.1-0.2 p,m with the terminal section filled with fluid (Wigglesworth and Lee, 1982). It was established that the level of fluid in the tracheoles of Aedes larvae is not determined by a simple relation between capillarity and the osmotic pressure of the blood (Wigglesworth, 1938a); and it was noted that whereas larvae of all the Culicine mosquitoes examined: Aedes aegypti, A . albopictus and other species, Armigeres, Culex (several species), Lutzia, and Rachionotomyia had fluid in the terminal parts of the tracheoles during rest, in all Anopheline larvae examined: Anopheles maculipennis, A. hyrcanus, A . barbirostris, A. maculatus, A. sundaicus, A. subpictus and A . vagus the tracheoles always contain air as far as they can be traced in the head, and in the anal papillae they contain fluid only in the finest
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branches. There was no difference in the osmotic pressure (276288mM/1) in members of the two groups that were tested. Presumably there are differences in the permeability and capillarity of the tracheole walls. 11.2
PERMEABILITY DIFFERENCES I N THE TRACHEOLE WALLS
Differences in the permeability of the walls of the tracheoles in different parts of the system and in different insects were demonstrated by chance. As already described (see Section 3.3) the injection of a mixture of equal parts of myrcene and odourless kerosine, followed by osmium treatment, proved an excellent method for the visualization of tracheoles. It was extensively used for the supply to the integument (see Section 7) and for the nervous system in Rhodnius. When applied to the flight muscles of Cyclorrhapha it was equally successful with tracheoles going down to about 0.2 km and some as small as 0.1 pm; but many of the finer terminal tubules of 0.07-0.05 pm were allowing the injection fluid to escape into the mitochondria which they encircle. In the flight muscles of the honey-bee the escape of the mixture was so extensive that the method was quite unsuitable for use on the terminal tracheoles, although there was no obvious escape from tracheoles of 0-3-0-5pm diameter. In Pieris and in Tenebrio the mixture oozed out through the walls of quite large tracheoles, up to 0.45pm and extensively of course from the finer tubules. And in Rhodnius the longitudinal tracheoles as well as the tubules of the transverse system were extremely permeable (Wigglesworth and Lee, 1982). As already described, in order to avoid escape of the injection fluid, heavier oils such as medicinal paraffin and arachis oil were used for studying the distribution of the tracheoles. No systematic study of the permeation of different oils in different insects has been made. But the one conclusion is quite clear: that the permeability of the tracheoles in the flight muscles is much greater than that in the tracheoles elsewhere. 11.3
PERMEABILITY A N D OXYGEN SUPPLY
This increased permeability may be of great physiological importance. With the enormously high rate of consumption of oxygen in the flight muscles (see Section 4.4) the increased permeability of the tracheole walls may well be a significant factor in oxygen supply. It
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can be argued that a tissue like the integument of Rhodnius can be adequately supplied with oxygen by tracheoles which are sufficiently impermeable to water to remain empty to their extremities. Whereas it will be more efficient for tissues like flight muscle, with very high oxygen requirements during activity but very low requirements during rest, to have highly permeable tracheole endings, which will fill with liquid during periods of rest. For, as we have seen, the liberation of unoxidized metabolites during activity will increase the uptake of fluid, and its extraction from the tracheole endings, and thus allow air to extend to the highly permeable extremities. There is good evidence of this happening in Pieris flight muscles and highly suggestive evidence in Muscid flies (see Section 9.8). In discussing the metabolism of insect flight muscles, Weis-Fogh (1961) wrote: “it is to be expected that evolution has proceeded so that every possible mechanism has been tested for intensifying work production and metabolism of wing muscle”. The theory of tracheole function here proposed suggests a new “possible mechanism” that will contribute to this aim.
Acknowledgements
This work was supported by a grant from the Agricultural Research Council. I should like to take this opportunity of thanking Mrs J. E. Short for her excellent service as secretary and typist over many years.
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The Endocrine Control of Flight Metabolism in Locusts G. J. Goldsworthy Department ofZoology, University of Hull, UK
1 Introduction 1.1 Nature of the transport system 2 Basic features of flight metabolism in locusts 2.1 Utilization of carbohydrate 2.2 Utilization of lipid 2.3 Utilization of other fuels 3 Hormones and flight 3.1 Site of synthesis and release of adipokinetic hormone 3.2 Dynamics of release of adipokinetic hormone 3.3 Control of the release of adipokinetic hormone 3.4 Chemical nature of adipokinetic hormone 3.5 Actions of adipokinetic hormone 3.6 Octopamine 3.7 Neurosecretion and other factors 4 A comparative overview of the hormonal control of flight metabolism in locusts 4.1 Metabolic changes 4.2 Lipid transport 4.3 The role of hormones in energy metabolism Acknowledgements References
149 150 152 152 153 154 155 156 157 158 160 162 181 184 184 184 186 192 194 194
1 Introduction
Locusts are morphologically indistinguishable from grasshoppers. The distinction between them is based on gregarious and migratory habits of the former, which has led to their notoriety as pests. Not surprisingly, locust flight has been studied extensively. To a “Advances in Insect Physiology” Volume 17 (edited by M. J. Berridge, J. E. Treherne and V. B. Wigglesworth). Academic Press, London and New York. 149
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physiologist, the attractions are many: locusts are bred easily in large numbers and are sufficiently large to facilitate detailed analysis of all aspects of flight, from behaviour to neurophysiology, biochemistry and endocrinology. Perhaps more importantly, locusts fly for long periods in the wild and will often do so readily in the laboratory. This raises a number of questions relating to how fuels are supplied to the flight muscles in sufficient quantity, because increases in metabolic rate during transition from rest to flight may be 100-fold (see Sacktor, 1975). It is the control of substrate supplies to locust flight muscle which will form a major theme of this review.
1.1
NATURE O F THE TRANSPORT SYSTEM
Insect blood plays little or no part in the transport of respiratory gases. Instead, oxygen is carried directly to tissues, and carbon dioxide escapes from them, by a network of internal tubes, the tracheal system. This ensures that gaseous oxygen is carried to within a few microns of the mitochondria and is so efficient that insect flight muscle metabolism is entirely aerobic. Indeed, contractions of wing muscles themselves aid ventilation of the tracheal system during flight and flight activity is not limited in any way by requirements of oxygen supply to muscles. The reserves of respiratory fuels in insect flight muscles are, however, usually limited to amounts sufficient to meet immediate energy requirements at the initiation of flight, but are too small to sustain prolonged flight (see Bailey, 1975). Thereafter, muscles utilize fuels made available to them in the haemolymph, which itself is an important pool of respiratory fuels. In locusts, for example, the amount of carbohydrate in the haemolymph is greater than that stored in other tissues (Goldsworthy, 1969), but the opposite is true for fat reserves (Jutsum et al., 1975). Nevertheless, for prolonged flight, stored fuel must be mobilized. The fat body, which is the main site of storage, is a heterogeneous tissue (see Keeley, 1978) which is considered to fulfill many of the equivalent metabolic functions carried out by mammalian liver and adipose tissue (Kilby, 1965); the degree of functional differentiation between cell types is, however, not understood. Glycogen and triacylglycerol are the main energy reserves (see Bailey, 1975), and the role that hormones play in coordinating the supply of suitable fuels to the flight muscles, by initiating or maintaining mobilization of reserves from the fat body, will be a second major theme of this review.
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The circulation of haemolymph is effected largely without blood vessels and major redistribution is almost certainly achieved by muscular activity of the insect itself; the haemolymph moves about in a tidal rather than circulatory manner. This occurs to some extent during walking but will be intense during flight. However, because there is no capillary system, an adequate supply of substrates to the tissues requires that the diffusion distances are as small as possible and that steep gradients of concentration of metabolites are maintained between haemolymph and tissue. However, since the flight muscles are impregnated by the T-tubules (Smith and Saktor, 1970), the presence of metabolites in extracellular fluid will help to reduce diffusion distances. In addition, concentrations of fuels in insect haemolymph are often characteristically much higher than in vertebrates, to ensure saturation of enzyme systems involved in the first steps of metabolism when inadequacies of circulation allow fluctuations in fuel concentrations to occur (Crabtree and Newsholme, 1975). A consequence of the use of high concentrations of fuels in insect haemolymph appears to have been the adoption of transport metabolites which are different from those used in vertebrates. For example, trehalose, a disaccharide of glucose, is the major haemolymph carbohydrate of most insects and in laboratory locusts is present in concentrations ranging from c. 17 kg/kl (Loughton and Orchard, 1981a) to c. 30 pg/pl (Goldsworthy, 1969). Free glucose is present also, but only at concentrations of c. 1-3pg/pl (Goldsworthy, 1969). Presumably trehalose offers the advantages of being non-reducing and contributing less to osmotic pressure than an equivalence of free glucose. Most insects mobilize lipids in the form of diacylglycerols (see Bailey, 1975), but high levels of these neutral lipids, although avoiding the necessity for high harmful concentrations of fatty acids in the haemolymph, impose a special requirement for their transport. Diacylglycerols are insoluble in aqueous media and are therefore transported in haemolymph as part of macro-molecular complexes with proteins, called lipoproteins. Locusts have developed a special mechanism involving formation during flight of a new lipoprotein carrier complex (Mwangi and Goldsworthy, 1976, 1977b, 1978, 1980,1981). The role of hormones in formation of this new complex, lipoprotein A + , and in controlling substrate oxidation in flight muscle, will provide a third major theme of this review.
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2 Basic features of flight metabolism i n locusts
At the initiation of flight, glycogen in the thoracic muscles can provide sufficient energy for 2-3 min. Although endogenous fuel will support flight activity for only this relatively short period, its oxidation enables flight to be undertaken readily and is adequate to support short (trivial) flights from one plant to another. For longer (migratory) flights, other sources of fuel are used. It is now established that locusts utilize two major fuels during prolonged flight; haemolymph trehalose is the predominant source of energy on the initiation of flight, but there is subsequently a switchover towards oxidation of lipid (Mayer and Candy, 1969a; Goldsworthy et al., 1975; Jutsum and Goldsworthy, 1976; Houben, 1976; Van der Horst et al., 1978a,b). Such a change in oxidative metabolism during flight was suggested by pioneering work of Krogh and Weis-Fogh (1951) concerning changes in respiratory rate and quotient during locust flight; recent major advances concern dynamics of carbohydrate and lipid utilization, and their control.
2.1
UTILIZATION OF CARBOHYDRATE
Although there is variation in published values for the concentration of trehalose in locust haemolymph, both before and after flight, there is general agreement that its concentration falls rapidly during the first 30min of flight in Schistocerca (Mayer and Candy, 1969a; Robinson and Goldsworthy, 1976; Strang and Clement, 1980) and Locusta (Goldsworthy et al., 1972b, 1973b; Wajc, 1973; Jutsum and Goldsworthy, 1976; Lee and Goldsworthy, 1975, 1976; Houben, 1976; Van der Horst ,et al., 1978b, 1980). The concentration of trehalose then stabilizes rapidly at a steady state of c. 50% the pre-flight value (Jutsum and Goldsworthy, 1976; Van der Horst et al., 197%). The haemolymph volume does not change during flight (Beenakkers, 1973), and therefore the initially rapid decrease in trehalose concentration represents its utilization by the flight muscles; Jutsum and Goldsworthy (1976) showed that little or no trehalose is released from storage tissues into the haemolymph during the early stages of flight. From changes in its concentration, Jutsum and Goldsworthy (1976) conclude that utilization of trehalose during the first 10min of flight is c. 270pg/min, but this decreases rapidly to c. 10 pg/min from 30 min onwards. Van der Horst and his colleagues (197814 have estimated turnover of haemo-
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lymph trehalose during flight by pulse labelling of the haemolymph pool with ITC-trehalose.They confirmed the observations of Jutsum and Goldsworthy (1976) concerning trehalose oxidation during the first 30min of flight, but concluded that it is still used at c. 40 pgimin during the period 0.5 to 3 h flight. Although absolute values for rates of trehalose utilization at different times of flight are important, a most interesting observation is that the utilization of haemolymph trehalose is curtailed dramatically at a time when only half has been used (see Jutsum and Goldsworthy, 1976). Why should this be so? There are a number of levels at which this question may be approached, as we shall see later, but at a first attempt we can correlate decreased utilization of trehalose with provision of an alternative fuel for the flight muscles, made available by mobilization of lipid reserves from the fat body.
2.2
UTILIZATION O F LIPID
Concomittant with rapid decreases in amounts of haemolymph trehalose during the initial stages of flight, there is a steady increase in the concentration of diacylglycerol (Tietz, 1967; Beenakkers, 1969; Mayer and Candy, 196%) until a relatively constant level is obtained after about an hour which is considered to be a steady state where mobilization matches oxidation (Mayer and Candy, 1969a; Spencer and Candy, 1974; Jutsum and Goldsworthy, 1976; Van der Horst ef a!. , 1978a, 1980). Correspondingly, when locusts are rested after flight, the haemolymph diacylglycerol concentration increases, at least for a short time, because lipid mobilization continues in the absence of utilization. The difference in rates of change of diacylglycerol concentration between rest and flight, approximates to rates of utilization. Thus, diacylglycerol oxidation is thought to increase gradually during flight from about 38 pgimin initially (Jutsum and Goldsworthy, 1976) to reach c. 80 pgimin (Spencer and Candy, 1974) in Schistocerca, and c. 8Spgimin in Locusta (Jutsum and Goldsworthy, 1976). when its concentration in the haemolymph is at steady state. Van der Horst and his co-workers (1978a) have studied turnover of diacylglycerol in Locusta using pulse labelling with IT-oleic acid and their estimates for the first 30min of flight are similar to those above, while those at steady state are somewhat lower (58 pgimin). Such rates of diacylglycerol oxidation are well within the capacity of fat body to mobilize lipid (Mwangi and Goldsworthy, 1977a).
G . J . GOLDSWORTHY
154
2.3
UTILIZATION OF OTHER FUELS
Other metabolites change in concentration in haemolymph during flight. It is largely unknown to what extent these contribute to the energy requirements during flight, but it is most likely very minor compared with trehalose and diacylglycerol. 2.3.1 Glucose The concentration of haemolymph glucose in Schistocercu shows a small transient increase during flight (Mayer and Candy, 1969a). No significant changes in glucose concentrations are found in Locusta, however (Jutsum and Goldsworthy, 1976; Houben, 1976), and in both species it is present throughout flight in such low concentrations that it is unlikely to contribute appreciably towards flight muscle metabolism. 2.3.2 Amino acids Mayer and Candy (1969a) and Houben (1976) have described changes in concentrations of amino acids during flight in Schistocerca and Locustu respectively. While the two studies agree to some extent (glycine and glutamine increase steadily during flight), there are also some contradictory findings and they allow no definite conclusions about the participation of amino acids in substrate provision. 2.3.3 Ketone bodies
In Schistocercu, the haemolymph concentration of ketone bodies increases rapidly to reach a steady level within 5min of flight; acetoacetate is the major component and shows the greatest change in concentration, from 0-1mM to 0.3 mM, while D-3-hydroxybutyrate is present at about one-tenth this amount (Hill et pl., 1972). These concentrations (c. 30 ng/p,l during flight) are too low compared with other fuels to represent a major source of energy (see Bailey, 1975). 2.3.4 Glycerol
In Schistocerca, the haemolymph glycerol content increases gradually during a 1h flight to a level 10 times that at rest (Candy et al.,
THE ENDOCRINE C O N T R O L O F F L I G H T M E T A B O L I S M
155
1976). Glycerol is not released from fat body but from flight muscle; immediately flight is stopped, the glycerol concentration drops by c. 40 ngiplimin and is almost at pre-flight levels within 5 min. Candy and his colleagues (1976) argue that this represents uptake by the fat body and approximates to the rate of glycerol release from the flight muscles. Why should flight muscles release glycerol into the haemolymph? It seems likely that the glycerol is derived from hydrolysis of diacylglycerol in active muscle because the pattern of haemolymph glycerol accumulation during flight, both in its time course and proportional increase, is similar to that of haemolymph diacylglycerol. Furthermore, Candy and his colleagues (1976) calculate that glycerol release of c. 40ngiplimin requires hydrolysis of c. 6 mg diacylglycerolilocustih, which is comparable with estimates of diacylglycerol oxidation. But why d o flight muscles release glycerol arising in this way? Is it not a useful source of energy? Locust flight muscles oxidize 14C-glycerol to I4CO2 in vitro (Candy, 1970; Robinson and Goldsworthy, 1977a) but the glycerol kinase activity is inadequate to convert all the glycerol produced by hydrolysis of diacylglycerol to glycerophosphate (Newsholme and Taylor, 1969). The liberated glycerol returns instead to the fat body and provides an important source of glycerophosphate for re-esterification of fatty acids produced during conversion of triacylglycerol to diacylglycerol. Thus, during flight, glycerol carries fatty acids as diacylglycerol from fat body to flight muscle, and then returns as free glycerol for re-esterification in the fat body to give more diacylglycerol (Candy et al., 1976; see also Van der Horst et a l . , 1983).
3 Hormones and flight
The possibility that juvenile hormone controls flight muscle development in larval (Poels and Beenakkers, 1969) and teneral stages (Van der Hondel-Franken et al., 1980), and flight behaviour in adults (Wajc and Pener, 1971; Wajc, 1973; Goldsworthy et al., 1972b, 1977; Michel, 1973; Lee and Goldsworthy, 1975, 1976) has been discussed previously (Goldsworthy, 1976; Rankin, 1977; Beenakkers et al., 1981) and will not be dealt with further. We shall be concerned only with direct effects of hormones on locust flight metabolism.
156
3.1
G . J. GOLDSWORTHY
SITE OF SYNTHESIS A N D RELEASE OF ADIPOKINETIC HORMONE
Realization that locusts possess a hormone which controls mobilization of lipid during flight arose from two independent investigations: in Schistocerca, Mayer and Candy (1969b) described a small peptide hormone, which they named “adipokinetic hormone”, which is present in the corpora cardiaca and stimulates release of diacylglycerol from the fat body when released shortly after the onset of flight: Beenakkers (1969) reported simultaneously that a similar factor existed in corpora cardiaca of Locusta. Locust corpora cardiaca are composite organs containing storage and glandular components. The glandular part consists of paired lobes, containing some axons from cerebral neurosecretory cells, but made up largely of phloxinophillic “parenchyma” cells (Highnam, 1961). These secretory cells possess characteristically large electron-dense secretory granules (200-600 nm in diameter) and appear to be of only one cell type (Cassier and Fain-Maurel, 1970; Cazal et al., 1971; Mordue and Goldsworthy, 1974; Rademakers and Beenakkers, 1977; Krogh and Normann, 1977). I t is argued from ultrastructural evidence, and by analogy with intrinsic secretory cells of other insects, that locust glandular lobe cells are modified neurosecretory cells (Goldsworthy and Mordue, 1974; Krogh and Normann, 1977). The secretory granules are synthesized in the Golgi zones but granules occur in only small numbers in the perinuclear cytoplasm, whereas they are densely packed in storage processes radiating from the cells (Rademakers and Beenakkers, 1977; Krogh and Normann, 1977). The glandular lobes possess most of the adipokinetic activity of whole corpora cardiaca (Goldsworthy et al., 1972a) and electrondense secretory granules isolated from glandular lobes of Schistncerca contain adipokinetic hormone (Stone and Mordue, 1979, 1980a). The glandular lobe cells are therefore, the source of hormone. Rademakers (1977a,b) and Rademakers and Beenakkers (1977) have studied the ultrastructure of these cells in Locusta. They conclude that the secretory granules, which have a diameter of 200-300nm, are of one type only. Other workers have indicated a slightly larger size range for these granules (Cassier and FainMaurel, 1970; Cazal et a / . , 1971), but described also the common occurrence of larger dense granules with diameters up to 5 p m ; Lafon-Cazal (1 974) calls them “ergastoplasmic granules”. LafonCazal and Michel (1977) show that in their strains of Locusta and
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Schistocerca ergastoplasmic granules decrease in number during a 3 h flight, and they disappear completely after flights of 20 h. Age and environmental conditions such as temperature and humidity also influence the number of these granules. Surprisingly, although Rademakers (1977a) did not find ergastoplasmic granules in glandular lobes from unoperated animals, large dense granules with a diameter from 2-3 b m were present in glandular lobes from individuals implanted with an extra pair of corpora cardiaca. Apparently, they did not change in number during flight. It is possible that different rearing conditions are responsible for the usual absence of ergastoplasmic granules in the Locusta used by Rademakers. In Locusta implanted with extra corpora cardiaca, host glandular lobes show signs of reduced synthetic activity (Rademakers, 1977a). Ergastoplasmic granules may therefore represent a stage in inactivation of the synthetic apparatus, or in eventual breakdown of old granules. The failure of Rademakers (1977a) to observe a decrease in these granules during flight may be explained by the short flight periods he employed compared with those in the study of Lafon-Cazal and Michel (1977); perhaps destruction of ergastoplasmic granules is initiated relatively late in flight, after synthesis and release are activated. 3.2
DYNAMICS OF RELEASE OF ADIPOKINETIC HORMONE
Adipokinetic hormone can be detected in the haemolymph during flight by bioassay (Mayer and Candy, 1969b; Houben and Beenakkers, 1973; Houben, 1976; Cheeseman et al., 1976; Cheeseman and Goldsworthy, 1979). Attempts at quantifying release are based either on the magnitude of lipid mobilization during flight (Goldsworthy et al., 1972b), or during a rest period following a short flight (Jutsum and Goldsworthy, 1975, 1976; Cheeseman and Goldsworthy, 1979), or on measurements of lipid-mobilizing activity extracted from haemolymph of flown locusts (Mayer and Candy, 1969b; Houben, 1976; Cheeseman et at., 1976; Cheeseman and Goldsworthy, 1979). All methods show that only a small fraction of total activity from a pair of corpora cardiaca is released during 30min of flight. The adipokinetic activity of locust corpora cardiaca varies with adult age (Goldsworthy et al., 1973b), but glandular lobes in mature male Locusta contain between 100-200pmol of adipokinetic hormone (Stone et al., 1976; Cheeseman et al., 1977; Stone and Mordue, 1980a), whereas rather more (200-250pmol) is present in those from Schistocerca (Stone and Mordue, 1980a). In
158
G .J. GOLDSWORTHY
male Locustu, the peak of haemolymph adipokinetic activity is reached after about 30min flight and corresponds to 4fmoVp.l or, assuming a volume of 250 p1, c. 1 pmol of hormone circulating in the haemocoel (Houben, 1976; Cheeseman and Goldsworthy, 1979). The half-life of the hormone is approximately 20 min (Cheeseman et al., 1976) so the total amount released will be somewhat greater than 1pmol. Nevertheless, this represents between only 1 and 2% of the activity within the glands. Why should corpora cardiaca contain such an apparent excess of hormone? It seems likely that measurements of total extractable activity give a false impression concerning the amount of hormone in a state to be released readily. Assuming that secretory granules are synthesized continuously, they will be broken down after some time unless they are released. Such a system operates in vertebrate neurosecretory cells, and material synthesized most recently is released preferentially (Heap et ul., 1975). There is evidence to suggest that a similar system operates in locust neurosecretory cells (Girardie and Girardie, 1979). Perhaps the ergastoplasmic granules (Lafon-Cazal, 1974; Lafon-Cazal and Michel, 1977; Rademakers, 1977a), if they do represent a stage in the destruction of older granules, represent one pool of adipokinetic activity in whole glands which is not in a readily releasable form.
3.3 C O N T R O L O F T H E R E L E A S E O F A D I P O K I N E T I C H O R M O N E The precise stimuli responsible for initiating release of adipokinetic hormone during flight are unknown. Receptors associated with wing movements or wind receptors, or changes in metabolite levels, could be involved but none has been established (see Goldsworthy , 1976). Nevertheless, a flight of only 2min is sufficient to cause prolonged mobilization of diacylglycerol ( Jutsum and Goldsworthy, 1976), and Rademakers and Beenakkers (1977) measured a significant increase in frequency of exocytotic profiles in glandular lobe cells after only Smin flight. A search for those particular events which initiate release of adipokinetic hormone must centre therefore on the very first minutes of flight. It seems unlikely that hormone release is initiated as a direct response of glandular lobe cells to changes in haemolymph content, whether metabolites or hormones, because in implanted corpora cardiaca these cells do not show ultrastructural signs of enhanced release when host locusts are flown (Rademakers, 1977a). Secretory activity appears to be under direct control of axons containing electron-dense granules of c . 100 nm diameter, which make synaptic contact with the glandular
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159
lobe cells (Rademakers, 1977a). Using a cobalt diffusion technique, the cell bodies of the axons were located as a small group on either side of the protocerebrum; their axons run to the glandular lobes via the NCCII (nervi corporis cardiaci 11) exclusively (Rademakers, 1977b). Indeed, Orchard and Loughton (1981a) have shown that electrical stimulation of this nerve releases adipokinetic activity from corpora cardiaca in vitro. These authors found stimulation of the NCCI alone does not cause release, but when the NCCI and I1 are stimulated simultaneously there is potentiated release of activity. This suggests that axons of the NCCI play no part in initiation of hormone release, but could play a modulatory role (Rademakers, 1977b; Orchard and Loughton, 1981a). However, when release of adipokinetic hormone is assessed in vivo, severance of both the NCCI and NCCII are required to prevent lipid mobilization during flight (Goldsworthy et al., 1972b). I t has been suggested (David and Lafon-Cazal, 1979; Orchard and Loughton, 1981b) that control of the glandular cells is octopaminergic, but a firm conclusion concerning this must await further studies on the pharmacology of the secretomotor system. Factors in the haemolymph may modulate hormone release. If the concentration of haemolymph carbohydrate in Locusta is increased by injection of trehalose, the characteristic increase in haemolymph lipid during flight is prevented (Houben and Beenakkers, 1973; Van der Horst et al., 1979), or delayed for a period depending on the amount of trehalose injected (Cheeseman et al., 1976). Trehalose-injected locusts do not show an increase in haemolymph lipid when rested after a short flight. It seems reasonable, therefore, to conclude that release of adipokinetic hormone is delayed by injection of trehalose prior to flight because hormone cannot be detected in the haemolymph (Cheeseman et al., 1976). Intriguingly, diacylglycerol turnover increases in comparison with resting locusts when trehalose-injected Locusta are flown (Van der Horst et al., 1979), although it is not clear whether this is a response to flight, or trehalose injection itself, since this was not tested; Mwangi and Goldsworthy (1977~)have shown that trehalose injection into starved Locusta induces a rapid decrease in haemolymph lipid concentration, presumably by stimulating fat body uptake. Although experimentally produced high concentrations of haemolymph trehalose prevent lipid mobilization, it is unlikely that decreases in carbohydrate concentration during flight act as a trigger for hormone release (Cheeseman et al., 1976; Houben, 1976). Freshly caught (solitary) field Locusta from Mali, and their first and
160
G . J. G O L D S W O R T H Y
second generation progeny, have high levels of haemolymph carbohydrate compared with stock laboratory Locusta (Wajc, 1973) so that the trehalose-injection, phenomenon could have some significance in terms of release in field populations. On the other hand, starved laboratory Locusta (Jutsum et al., 1975) and freshly caught field Locusta from Jedda have high levels of haemolymph lipid (Wajc, 1973), and Cheeseman and Goldsworthy (1979) suggest that high levels of diacylglycerol exert a negative feedback influence on release of adipokinetic hormone. Finally, it is possible that the hormone exerts a negative feedback influence on its own release; implantation of corpora cardiaca appears to suppress activity in host glandular lobe cells, possibly by an action on the secretomotor axons (Rademakers, 1977a). 3.4
CHEMICAL NATURE OF ADIPOKINETIC HORMONE
The peptide nature of adipokinetic hormone was first suggested by Mayer and Candy (1969b) when they found that lipid mobilizing activity in extracts of corpora cardiaca was destroyed by incubation with chymotrypsin, pronase, and pepsin, but not by boiling. Methanolic extracts of glandular lobes were the starting points for two methods of isolation (Stone et al., 1976; Cheeseman et a l . , 1977) resulting in pure preparations of a blocked decapeptide Glu-LeuAsn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 (Stone et a [ . , 1976). This structure has been confirmed by synthesis of the peptide (Broomfield and Hardy, 1977) which we shall call AKH (adipokinetic hormone). A detailed account of the isolation and chemical sequencing of A K H has been given by Stone and Mordue (1980a,b). Subsequently, a second lipid mobilizing peptide, compound 11, has been found in corpora cardiaca of Schisrocerca (Carlsen et al. , 1979) and Locusta (Gade, 1981b). On the basis of a similar, but not identical, amino acid composition (Asp, Thr, Ser, Glu, Gly, Leu, Phe, Trp in equimolar amounts), Carlsen and his colleagues (1979) speculate that compound I1 is a blocked octapeptide which resembles the prawn-red pigment concentrating hormone which has the structure Glu-Leu-Asn-Phe-Ser-Pro-Gly-TrpNH2 (Fernlund and Joseffson, 1972). Compound 11, like A K H , is found predominantly in extracts of glandular lobes, but is present in smaller amounts compared with A K H , although it is claimed that it has about the same molar activity; it could account for 20% of the total adipokinetic or hyperglycaemic activity (Jones et al., 1977; Van Norstrand et al., 1980) of locust corpora cardiaca (Carlsen et al., 1979).
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161
The physiological significance of compound I1 remains unknown, but it is intriguing that a single cell type in the glandular lobes (Rademakers and Beenakkers, 1977) produces two peptides with similar biological activities. Are they chemically related in terms of their synthesis? Do they have a common precursor molecule? Stone and her colleagues (1978) have undertaken a detailed study of structure-activity relationships for the lipid-mobilizing activity of AKH (see also Mordue and Stone, 1981). They conclude that the first eight residues starting from the N-terminus (the L-enantiomer of pyroglutamic acid), the C-terminal threonine amide residue, and perhaps the sequence Pro-Asn-Trp in positions 6, 7 and 8 (where there may be a p-turn of the peptide) are important in determining full activity (Table 1). A tentative configuration for AKH is shown in Fig. 1. Interestingly, prawn red pigment concentrating hormone TABLE 1 Adipokinetic activities of adipokinetic hormone and structurally-related compounds (from Stone et al., 1978)
Compound 1 H-Asn-Trp-Gly-Thr-NH2 2 Ac- Asn-Trp-Gly-Thr-NH2 3 H-Thr-Pro-Asn-Trp-Gly-Thr-NH? 4 H-Phe-Thr-Pro-Am-Trp-Gly-Thr-NH2 5 Glu-OH 6 Glu-Leu-Asn-OH 7 Glu-Leu-Asn-Phe-OH 8 Glu-Leu-Asn-Phe-Thr-Pro-OH 9 Glu-Leu-Asn-Phe-Thr-Pro-NH2 10 Glu-Leu-Asn-Phe-Thr-Pro-Trp-NH2 11 D-Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-NH2 12 Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-NH2 130 Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NHz 14 Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-NH, 15 Glu-Leu-Asn-Phe-Thr-Pro-Gly-Trp-Gly-Thr-NH2 16 Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Thr-Gly-NH2 17 D-Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 18 Glu-Leu-Am-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH? (AKH) (Residue 1 2 3 4 5 6 7 8 9 10)
Relative agonist activity
<0.3 <0.3 <0.3 <0.3 <0.3 <0.3 <0.3 <0.3 4.3 <0.3 8 20 20 7 30 3 5 100
Prawn red pigment concentrating hormone (Fernlund and Josefsson. 1972). The activity of adipokinetic hormone is defined as 100. Other preparations were compared with hormone standard on a molar basis. ED,,, of adipokinetic hormone is 3 4 M . S pmolilocust. Activities shown as <0.3 indicate that the maximum dose tested (200pmol/locust) did not produce a significant adipokinetic response. a
162
G . J. GOLDSWORTHY
has only 20% of the activity of AKH. From the study of Stone et al. (1978) it is difficult to see how Compound 11, if it is as Carlsen et al. (1979) suggest similar chemically to the prawn hormone, can have a similar lipid mobilizing potency to A K H (see Table 1). 1 2 3 4 5 6 %lu-Leu-Asn-Phe - T h r - Pro
I H2N-Thr
1
- Gty 0
- Trp - Asn
9
8
7
Fig. 1 A possible configuration for AKH (from Mordue and Stone, lY78). Hydrogen bonding between residues 3 and 10, and 5 and 8, would provide some stability. Indirect evidence for such a conformation involving a P-bend associated with residues 6 and 7 is provided by substitution of glycine for asparagine at position 7 (compound 15 in Table l), which reduces the decapeptide activity and also decreases the probability of the P-bend occurring
Little is known of the metabolism of AKH once it is released. The mechanisms of its inactivation or excretion are not understood, but the Malpighian tubules excrete it in an inactive form (Mordue and Stone, 1978). Whether other inactivating mechanisms exist, in fat body or other tissues, remains to be determined.
3.5
A C T I O N S OF A D I P O K I N E T I C H O R M O N E
3.5.1 Lipid mobilization It is thought that during increased fat mobilization, triacylglycerol is degraded by a non-stereospecific lipase to 2-monoacylglycerol (Tietz and Weintraub, 1978) which is subsequently acylated by a stereospecific microsomal monoacylglycerol-acyltransferase (Tietz et a/. , 1975) which synthesizes preferentially sn-l,2-diacylglycerol (Tietz and Weintraub, 1980). Diacylglycerols in haemolymph have almost exclusively the sn- 1,2 configuration (Tietz and Weintraub, 1980; Lok and Van der Horst, 1980). By analogy with vertebrates (Khoo and Steinberg, 1974), it was anticipated that adipokinetic hormone activated triacylglycerol lipase but this proved difficult to demonstrate (see Spencer and Candy, 1976). Nevertheless, exogenous dibutyryl cyclic AMP mimics adipokinetic hormone action in vivo (Gade and Holwerda, 1976) and in vitro (Spencer and Candy, 1976), and endogenous levels of cyclic AMP increase in the fat body after hormone injection (Spencer and Candy, 1976; Gade and Beenakkers, 1977;
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163
Gade, 1979) and during flight (Gade and Holwerda, 1976). Recently Tietz and her colleagues succeeded in demonstrating that adipokinetic hormone activates cyclic AMP dependent protein kinase, triacylglycerol lipase and monoacylglycerol transferase (Tietz and Weintraub, 1978; Pines et al., 1981). The precise mechanism of activation and involvement of cyclic AMP-dependent protein kinase has yet to be determined, but an interesting feature is the lack of stimulation of monoacylglycerol transferase activity by cyclic nucleotides in vitro, despite the fact that prawn red pigment concentrating hormone produces a marked activation in vivo (Pines et al., 1981). It is possible that activation of monoacylglycerol transferase occurs in response to a second messenger other than cyclic AMP; Spencer and Candy (1976) showed that the action of AKH on the fat body is Ca2+-dependent. Perhaps intracellular Ca2+ effect some of the actions of adipokinetic hormone, as has been proposed for the cockroach hyperglycaemic factor (McClure and Steele, 1981). It should be mentioned that the magnitude of the fat body response to A K H in vitro, at least in terms of diacylglycerol release, is very poor compared with that in vivo. Hence, in Locusta. maximal diacylglycerol release in vivo after hormone injection is c. 6 mgih, but release of diacylglycerol from fat body in the presence of adipokinetic hormone in vitro is only c. 0.3 mg/h (Mwangi, 1977). Similarly, in Schistocerca, mobilization of diacylglycerol during flight is c. 5mg/h (Spencer and Candy, 1974) but release of diacylglycerol from hormone-treated fat body in vitro is only 0.4mgih (Spencer and Candy, 1976). There is one report, however, which suggests that fat body of Locusta will release diacylglycerol in v i m at rates of c. 2mg/h after prolonged exposure to large amounts of prawn red pigment concentrating hormone (Pines et al., 1981); such rates of release are still low but approach more closely those found in vivo. The reasons for the poor response of fat body to physiological amounts of A K H in vitro are not known, but diacylglycerol release is energy-dependent in normal (Tietz, 1967) and hormonetreated fat body (Spencer and Candy, 1976) in vitro. Obviously, the provision of a suitable incubation medium containing all the necessary components of the acceptor lipoprotein system is important, but the disruption of the tracheal system in explanted fat body tissue could be a crucial factor. Indeed, the lipid mobilizing response to adipokinetic hormone is completely abolished during chronic carbon dioxide or nitrogen anaesthesia (Goldsworthy, unpublished observations).
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G . J . GOLDSWORTHY
Haemolymph lipoproteins from resting locusts have been studied in detail. In Locusta, diacylglycerol transporting lipoprotein from resting haemolymph has an estimated molecular weight of 340 000 (Peled and Tietz, 1975), 450000-550000 (Mwangi, 1977), 550000 (Chen et al., 1978), 700000-850000 (Gellissen and Emmerich, 1980), 450000 (Van der Horst et al., 1979), or 580000 (Chino and Kitazawa, 1981). It appears to be a single protein in polyacrylamide electrophoresis gels (Peled and Tietz, 1975; Gellissen and Emmerich, 1980; Chino and Kitazawa, 1981; Wheeler, 1981). In SDS-polyacrylamide electrophoresis, however, two apoprotein subunits of c . 85 000 and 250 000 molecular weight separate (Gellissen and Emmerich, 1980; Chino and Kitazawa, 1981). The smaller apoprotein contains between 1-3% mannose (Gellissen and Emmerich, 1981; Chino and Kitazawa, 1981). The amino acid composition of locust apoproteins has been studied by a number of workers (Table 2) and, although the methods of preparation are often very different, there is reasonable agreement concerning Locusta apoproteins, but also about similarities with apoproteins of other insects. Nevertheless, antibodies raised against locust diacylglycerol-transporting lipoproteins do not cross-react with lipoproteins of cockroaches or silkmoths (Chino and Kitazawa, 1981). Diacylglycerol-transportinglipoprotein in resting haemolymph is yellow due to the presence of carotenoids (Peled and Tietz, 1975), and will be referred to as Ayellow (see Mwangi and Goldsworthy, 1977b); other lipids are carried also, but diacylglycerol is the major component (see Peled and Tietz, 1975; Chino and Kitazawa, 1981). Gellissen and Emmerich (1978, 1980) have shown that Ayellow is distinct from vitellogenin despite earlier reports that they had common antigenic determinants (Gellissen et al., 1976; Chen et al., 1976). We shall be concerned only with the Ayellow lipoprotein because, as in silkmoths (Chino et al., 1969, 1976; Thomas and Gilbert, 1968), locust vitellogenin plays only a minor role in diacylglycerol transport. There is little doubt that in fed resting locusts, when the rate of lipid mobilization is low, Ayellow is the acceptor lipoprotein for fat body diacylglycerol (Peled and Tietz, 1975; Chino and Kitazawa, 1981). However, Mwangi and Goldsworthy (1976, 1977b, 1978) demonstrated that at times of increased lipid mobilization (after adipokinetic hormone injection, or during flight or starvation), a new lipoprotein complex appears in the haemolymph (see also Mayer and Candy, 1967). This new lipoprotein is called A+ because
TABLE 2 Amino acid composition (mole %) of total apoprotein of diacylglycerol-carrying lipoproteins from some insect species Silkworm LP- 1 (a)
Amino acid
Cockroach (b) Resting 11.0 6.6 6.9 10.8 3.8 6.4 6.8 8.4 0.6 4.1 10.7 3.0 4.7 9.3 3.9 2.7
12.6 4.9 6.9 10.4 4.7 6.7 6.3 7.4
Asp Thr Ser Glu Pro GlY Ala Val cys Ile Leu Tyr Phe LYS His
+
-
5.8 9.0 2.8 4.8 10.7 2.8 3.7
Arg
Met Tro
~
0.5
0.3
-
-
Tobacco hornworm (larvae) (c) 12.5 3.') 7.7 9.7 5.0 6.6 7.5 7.1 -
54 8.7 3.8 5.2 8.5 2.9 4.1 1.3 -
Locust C-peak proteins
Locust Ayellow (d) (el ffi (g) Resting
Locust A + Elevated*
Resting
Elevated*
12.2 11.9 11.4 12.2 6.0 5.5 6.0 5.8 8.8 7.6 7.1 6.Y 12.1 12.0 11.2 11.9 3.4 5.1 3.8 4.Y 6.6 6.2 6.3 6.2 8.4 7.0 7.2 7.4 7.9 7.0 8.4 8.0 _ 0.7 5.6 5.1 4.2 5.6 11.2 9.6 10.6 1O.Y 2.4 3.9 2.7 1.3 4.4 4.7 4.7 5.0 8.2 7.6 9.2 8.1 2.6 1.9 3.7 2.2 3.2 3.9 2.6 3 4 _ 0.2 1.0 - -
12.2 6.2 6.1
12.7 5.7 7.1 15.8
(g )
(g)
( 'Y)
12.1 6.5
5.2 15.9 5.5
11.5 5.4 10.5 11.2 6.5 9.5 10.7 7.0
-
-
-
15.4 34 4.5
4.9 11.0 1.4 3.2 7.4
3.0 2.2 ~
44
4.5 10.7 0.8 2.1
3.3 4.6 1.7
5.4 7.4 0.6 3.8 2.7 4.0 3.8
-
-
-
-
~
* Extracted from haemolymph of AKH-injected locusts. From
(a) Gilhert and Chino (1973): ( h ) Chino el a / . (I981 : (c) Pattnaik et al. (197Y): (d) Peled and Tictz (1975; (e) Gellissen and Emmerich (1980); I f ) Chino and Kitazawa (1981: (g) Van dcr Horst Ct c d . (personal communication). Van der Horst and his colleagues (scc Van der Horst. 1982) suggest that the greater amounts of glutamate and alanine. a n d lesser amounts of proline, glycine, valine, phenylalaninc. lysine and arginine in A + compared with AjeUow. correlate with some of the changes in amino acid composition of the C-peak proteins. and could be accounted lor by the participation of C,-proteins in A + formation.
:;I tE2ao
I'
Diac y lgI y c er 01
Fraction nu%
Fig. 2 A model for lipoprotein A + formation in locust haemolymph. The UV-absorbance elution profiles from resting and elevated ammonium sulphatetreated haemolymph separated on Ultrogel AcA22 are shown with the distribution of diacylglycerol indicated by the histograms below (from Mwangi and Goldsworthy, 1977b). Note the absence of UV-absorbance or diacylglycerol at the void volume (Vo) of the column. The UV-absorbance of the A and C peaks decreases concomitantly with the appearance of A + , and the diacylglycerol distribution changes correspondingly. It is envisaged that during increased triacylglycerol (TGL) breakdown (after A K H injection, or during flight or starvation), Ayellow and C,-proteins from the C peak combine with diacylglycerol (DGL) from the fat body to form A + . The fat body is represented in this and other figures as a combined adipose/liver type of tissue (see Section 1.1)
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it elutes on Sepharose 6B or Ultrogel AcA 22 just ahead of a group of proteins, including Ayellow, called the A peak. Two other protein peaks (B and C) are identified (Fig. 2) in resting haemolymph, but neither contains diacylglycerol (Mwangi and Goldsworthy, 1977b). Treatment of haemolymph with ammonium sulphate stabilizes lipoproteins in haemolymph from resting and hormone-injected or flown locusts, and can be used in such a way that the precipitate obtained is 6 6 % of the total protein, and contains less than 6% of the total lipid (Mwangi and Goldsworthy, 1977b; Wheeler, 1981). From a comparison of the elution profiles of resting and elevated (from adipokinetic hormone-injected locusts) haemolymph separated by gel chromatography, Mwangi and Goldsworthy (1976, 1977b) propose a unique mechanism whereby a new haemolymph lipoprotein forms by a combination of resting lipoprotein, Ayellow, with diacylglycerol from the fat body and non-lipid-carrying proteins from the C peak (Fig. 2). Lipid loading of Ayellow does not require protein synthesis (Peled and Tietz, 1973) and neither the total haemolymph protein content (Mwangi and Goldsworthy, 1977b) nor haemolymph volume (Jutsum and Goldsworthy, 1976) change during flight or after hormone injection; A + formation must therefore involve an interaction of existing haemolymph proteins and lipoproteins (see Fig. 2). Further evidence for this comes from measurement of changes in protein concentration of individual chromatographic peaks (Mwangi, 1977), and of those fractions of total haemolymph diacylglycerol and protein which can be precipitated by heparin treatment (Mwangi and Goldsworthy, 1977b,c, 1978, 1980, 1981 ; Wheeler and Goldsworthy, 1983a). Formation of insoluble lipoprotein-polyanion metal complexes is well documented as a technique for study of plasma lipoproteins in vertebrates (Cornwell and Kruger, 1961; Burstein et al., 1970; Burstein and Scholnick, 1973) and can be applied to insect lipoproteins. Ayellow lipoprotein precipitates when haemolymph is treated with heparin in the presence of Ca2+ (Robinson, 1975; Mwangi and Goldsworthy, 1977b, 1978; Wheeler and Goldsworthy, 1983a). A + lipoprotein, when it is present, remains in the supernatant as a soluble heparin complex, but precipitates on addition of EDTA (Mwangi and Goldsworthy, 1978; Wheeler, 1981). When used with haemolymph from male !ocusts, where there is no complication with vitellogenin, the heparin-EDTA technique allows a quicker and more easily quantified study of lipoprotein A + formation than procedures based on chromatographic separations. Using the heparin-EDTA technique, changes in amounts of Ayellow and A +
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251
7 Ii f
10
V
J:::
5
0
I
30 60 Time after AKH injection min
Fig. 3 Changes in the concentrations of Ayellow and A+ during AKH action (after Wheeler and Goldsworthy, 1983a). Serial haemolymph samples from 10 adult male 15-day-old Locusfa (mean k s.e. shown) injected with 2pmol natural AKH at t,, were treated with heparin (see Mwangi and Goldsworthy, 1977b. 1978). The immediate precipitate (closed circles) is Ayellow, and A+ precipitates from the supernatant by addition of EDTA (open circles)
lipoprotein after adipokinetic hormone injection can be measured routinely (Fig. 3); Ayellow decreases in amount as it participates in formation of A+ (as predicted by the Mwangi and Goldsworthy model) and this is dose-dependent (Fig. 4). Interestingly, decreases in Ayellow concentration are always less than the amounts of A+ formed and, according to the model, this represents the contribution of the CL-proteins. Indeed, radiolabelled CL-proteins become incorporated into lipoprotein A+ during adipokinetic hormone action (Fig. 5 ) , and Wheeler and Goldsworthy (1983a,b) have shown that lipoprotein A+ binds radiolabelled CL-proteins rapidly in vivo and in vitro. Additionally, at least some of the bound radiolabelled CL-protein is displaced in vitro by an excess of
THE ENDOCRINE C O N T R O L O F F L I G H T M E T A B O L I S M
+\+
C
.-0
I
C
I \
2
I
C
169
-8
0 0
c .Q, I
2 a
.-
-16
Fig. 4 Dose response relationships for the decrease in Ayellow and production of A+ during A K H action (after Wheeler and Goldsworthy, 1983a). At least 5 adult male 15-day-old Locusta ( m e a n & s.e. shown) were injected at each dose of synthetic AKH, and Ayellowl and A + were determined by heparin treatment of haemolymph at to and t,, (see Fig. 3). Identical results are obtained with natural AKH
non-labelled CL-protein (Wheeler, 1981; Goldsworthy and Wheeler, 1982a; Wheeler and Goldsworthy, 1982, 1983b; Goldsworthy et al., 1982). It is not known, however, if more than one pool of CL-protein is associated with A + , and whether all of this protein is readily exchangeable with free CL-proteins; some of the CL-proteins may prove more rigorously involved in the structural integrity of the A+ complex. A large number of problems remain concerning lipoprotein A+ formation. As demonstrated in Figs 3 and 6, after hormone injection Ayellow lipoprotein contributes its protein and diacylglycerol moieties to A+ (Mwangi and Goldsworthy, 1977b; Van der Horst et al., 1981b), but only after a delay of 10-15min (Mwangi and Goldsworthy , 1977b; Wheeler and Goldsworthy , 1983a). Increases
170
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0’
, 0
I
30 Time after AKH injection min
1
60
Fig. 5 Association of injected ‘H- C,;proteins with Ayellow and A + after injection of 2 pmol of natural A K H (Wheeler and Goldsworthy, 1983a). Purified C,-proteins from the C peak off Ultrogel AcA22 were radiolabelled using N-succinimidyl (2,3-”) propionate (Wheeler, 1981) and injected 30 min before AKH. The radioactivity associated with the AyeNaw and A+ was determined for 12 adult male 15-day-old Locusfa (mean F s.e. shown) using the hepariniEDTA technique (see Fig. 3). The amount of radiolabel associated with A + after 60 rnin corresponds to c. 4~gip.1of C,-protein
in total protein, CL-protein and lipid content of A+,however, occur without delay (see Figs 3, 5 and 6). We cannot explain these observations fully, but the earliest increases in protein, lipid loading, and associated CL-proteins of the heparin-EDTA precipitates appear to represent changes in a group of lipoproteins which migrate rather slowly (relative to Ayellow) on polyacrylamide electrophoresis gels and elute at the very leading edge of the A peak on Ultrogel AcA 22. These lipoproteins are present in low concentrations in resting haemolymph. They stain weakly with Lipid Crimson and Coomassie Blue on polyacrylamide gels, increase in their lipid loading within minutes of AKH injection (Wheeler and Goldsworthy, 1983a,b), and may represent an intermediate stage between Ayellow and A + , but no further information is available at present. The diacylglycerol/protein ratio of Ayellow in resting
171
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150dpm/pl 100-
500’
I
0
I
30
I
60 Time after AKH injection min
I
90
Fig. 6 Changes in IT-lipid associated with A+ after AKII injection (Wheeler and Goldsworthy, 1983a). Seven adult male 15-day-old Locusta (mean ? s.e. shown) were prelabelled with IT-palmitic acid 24 h before injection with 2 pmol natural AKH. The mobilization of diacylglycerol is indicated by the changes in IJC-label associated with Ayellow and A + , which were separated by the hepariniEDTA technique (see Fig. 3)
haemolymph is c. 0-2 (Peled and Tietz, 1975; Mwangi, 1977; Van der Horst et al., 1979) but after injection of adipokinetic hormone this increases to 0.3 according to Mwangi (1977), although Van der Horst and his colleagues (1979) found lower ratios during hormone action and flight. Nevertheless, A+ has a diacylglyceroliprotein ratio of c. 1.8 after A K H injection (Mwangi, 1977) or c. 1.4 after flight (Van der Horst et al., 1979). Clearly, we need to know more about the lipid loading of all the lipoproteins in the A peak from Ultrogel AcA 22. Van der Horst and his co-workers (1979, 1981a,b) have substantiated much of the chromatographic data obtained by Mwangi and Goldsworthy (1976, 1977, 1978) and provided important corroborative evidence to support the model depicted in Fig. 2 for the formation of A+ (see also Table 2). By the use of radioiodinated Ayellow and CL-protein, and immunochemical procedures, they have shown that both proteins are involved in the formation of the A + lipoprotein (Van der Horst et al., 1981a,b). However, in addition they find that a group of lipoproteins which are voided on Ultrogel AcA 22, and have a molecular weight in excess of 50 X lo6, change in amount during hormone action and flight. They describe
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this voided material from Ultrogel AcA 22 as being opalescent; the light-scattering being due presumably to suspended particulate 0 and O+ lipoproteins, which they believe are polymers of Ayellow and A+ respectively (Van der Horst et al., 1979, 1981a,b). In our laboratory, fresh haemolymph taken directly from locusts is introduced immediately into saturated ammonium sulphate and mixed regularly during collection until the volume of haemolymph equals that of the original ammonium sulphate solution, and the mixture is then centrifuged (Mwangi and Goldsworthy, 1977b). This results in a clear supernatant. Van der Horst and his co-workers (1979) collect haemolymph into tubes, centrifuge, and dilute the supernatant with elution buffer before fractionation. In our experience, this procedure leads to formation of a turbid solution, whereas fresh haemolymph is clear (unpublished observations). Fractionation of haemolymph treated with ammonium sulphate in the manner described by Mwangi and Goldsworthy (1977b), yields hardly any voided material on Ultrogel AcA 22 (see Fig. 2), although we can produce this in large quantities by deliberately omitting the mixing procedure; collected haemolymph floats at the surface of the ammonium sulphate solution and forms opalescent material which is voided on Ultrogel AcA 22 (unpublished observations). Chino and Kitazawa (1981) also describe the appearance of turbidity in dialysed haemolymph and the irreversible aggregation of Ayellow under some conditions in vitro (see also Brehelin, 1979). In addition, Candy (1978) describes a method of extracting locust lipoproteins using EDTA treatment of fresh haemolymph, which yields clear preparations resembling the appearance of the original haemolymph sample. We have recently confirmed (unpublished observations) that treatment of fresh haemolymph with an equal volume of 0.01 M EDTA yields preparations which, after centrifugation, separate on Ultrogel AcA 22 in an identical manner to those obtained by ammonium sulphate treatment; hardly any voided material is obtained from resting or elevated haemolymph. The voided 0 and O+ material described by Van der Horst et al. (1979) is thus absent normally from separations of haemolymph performed in two different laboratories and Goldsworthy and Mwangi (1981) question its existence in vivo (see also the evidence obtained from electron microscope studies of haemolymph particles discussed in Section 4.2.1). The 0 and O+ voided materials appear to be artifacts of the handling procedures for the haemolymphs employed by Van der Horst et al. (1979). A further problem concerning formation of lipoprotein A+
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concerns the exact role played by adipokinetic hormone. Mwangi and Goldsworthy (1977b) envisaged an indirect action of the hormone; increased lipid-loading of Ayellow leading to A+ formation without further direct hormonal intervention. Lipoprotein A+ is present in large amounts in haemolymph of starved Locusta (Mwangi and Goldsworthy, 1977b,c) in which the titre of AKH is no greater than that in rested fed locusts (Cheeseman and Goldsworthy, 1979), but the basis of hyperlipaemia during starvation is unknown and may prove yet to be endocrine. It is therefore not possible to exclude direct effects of the endocrine system, during starvation or flight, on the process of A + formation. Indeed, Van der Horst and his colleagues (1979) believe that AKH has a primary action in causing the association of haemolymph proteins to form A+. There is no direct evidence for this view, however, and no molecular mechanism has been proposed. The full details of the roles of AKH and the fat body during A + formation remain to be determined. Using gel filtration on Ultrogel AcA 22, the molecular weight of lipoprotein A + is estimated by Mwangi (1977) and Wheeler (1981) to be between 0.6-1.7 x lo6, but Van der Horst and his colleagues (1979) report a value of 3.5 x lo6. Dextran blue (molecular weight 2 X lo6) is voided on Ultrogel AcA 22 whereas A+ is partially included, which would suggest a molecular weight for the lipoprotein of less than two million. Nevertheless, when a possible molecular weight is calculated from the characteristic size of the A+ particles and their hydrated density (see Section 4.2.1), a value of 8.0 x lo6 is obtained. For an understanding of the molecular mechanism of A + formation it is clearly important that a more precise determination of molecular weight is obtained. A final interesting feature of the actions of adipokinetic hormone is the marked change in magnitude of the lipid mobilizing response during development. Fifth instar nymphs show a poor response to hormone, in which modest amounts of diacylglycerol are mobilized, but lipoprotein A + is not formed (Mwangi and Goldsworthy, 1977a,b). In fledglings, this poor response persists for 2-3 days but then improves rapidly until by day 8 of adult life a near maximal lipid response can be demonstrated and lipoprotein A+ is formed. The mechanisms controlling development of the full adult response are uncertain, but appear to occur independently of adult corpora allata, although these glands do exert a more long-term effect (Mwangi and Goldsworthy, 1980). It has been suggested that the poor lipid-mobilizing response and inability to form A+ in fledglings
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is correlated with an absence of C-proteins from their haemolymph (Mwangi and Goldsworthy, 1977b; Wheeler and Goldsworthy, 1983a), but Ayellow is present also in only low amounts in these locusts (see also Gellissen and Emmerich, 1978) and increases rapidly during adult development (Wheeler and Goldsworthy, 1983a). It is intriguing that age-related changes in response to adipokinetic hormone correlate with variations in flight performance; perhaps these changes are part of a common pattern of development in adult locusts which will form a useful basis for a study of jgeing in these insects (see Mwangi and Goldsworthy, 1980).
3.5.2
Glycogenolysis in the fat body
Extracts of the glandular lobes from locust corpora cardiaca are potent in causinghypertrehalosaemia in cockroaches and in activating locust fat body phosphorylase (Goldsworthy, 1969, 1970; Mordue and Goldsworthy, 1969). It seems that this activity is due mainly to the presence of A K H (Jones et al., 1977; Holwerda et al., 1977; Gade, 1981a) and to a lesser extent to compound I1 (see Van Norstrand et al., 1980; Gade, 1981b). The release of compound I1 has not been established, but a role for AKH in controlling glycogenolysis in the fat body seems possible. Indeed, Van Marrewijk and his colleagues (1980) have shown that glycogen phosphorylase in thoracic fat body is activated during flight; the amount of total enzyme which is in the active form increases from c. 10% in resting locusts to 25% within 5 min, and remains at c. 30% for up to 2 h of flight. Comparison of dose-response data for phosphorylase activation in response to synthetic A K H or extracts of corpora cardiaca (Gade, 1981) with the haemolymph titre of AKH (Cheeseman and Goldsworthy, 1979), suggests that AKH may maintain high levels of glycogen phosphorylase in the fat body during flight. But the activation within 5min of flight shown by Van Marrewijk et al. (1980) appears to require a greater and more rapid release of A K H than is thought to occur. Nevertheless, glycogenolysis is stimulated during flight and Van Marrewijk and his co-workers (1980) have shown that glycogen reserves in the fat body are depleted by c. 75% during a 2 h flight. Despite rapid activation of phosphorylase, however, glycogen contributes to the energy budget only after 30 min of flight (see Jutsum and Goldsworthy, 1976; Van der Horst et al., 197%) but the reasons for this apparent delay are unknown. We can calculate, however, that during the period from
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30 min onwards, when the haemolymph carbohydrate concentration is in steady state, the breakdown of fat body glycogen could contribute c. 25 pg/min of carbohydrate for oxidation by the flight muscles. This is considerably less than the rate of trehalose oxidation (40 pgimin) proposed by Van der Horst et al. (1978b). Presumably other tissues could contribute their stored glycogen but this has not been investigated. It has been suggested that during flight the contribution of fat body glycogen reserves may help to maintain haemolymph trehalose levels and therefore sustain a small but essential supply of carbohydrate to the flight muscles (Goldsworthy and Gade, 1982). This could be important because Robinson and Goldsworthy (197721) showed that in vitro flight muscle requires a supply of trehalose for continued contraction even when diacylglycerol is the predominant fuel. On the other hand, increased glycogenolysis could provide an important source of glycerophosphate for re-esterification of fatty acids produced during lipolysis, but it appears that glycerol returning from the flight muscles (see Section 2.3.4) more than adequately fulfills this requirement, because 14C-glycerol injected into flying locusts is recovered not only as 14C-diacylglycerol but also as 14C-trehalose (Candy et al., 1976). Indeed, Van der Horst and his colleagues (1983) believe that about 8 % of the trehalose oxidized during prolonged flight could be derived from glycerol liberated at the flight muscles (see Section 2.3.4). The mechanism of action of A K H in activating glycogen phosphorylase is uncertain, although it is generally assumed that it will prove to be similar to the mechanism determined for the action of glucagon in mammals. That is, it stimulates adenylate cyclase, elevates cyclic AMP levels and thus activates protein kinase which phosphorylates regulatory proteins. Cyclic nucleotide activated protein kinase has been found in locust fat body (Pines and Applebaum, 1978; Van Marrewijk et al., 1980) and is apparently activated by A K H (Pines et al., 1981) but it is uncertain whether this is concerned with stimulation of only lipolysis, or of only glycogenolysis, or of both processes.
3.5.3
Flight muscle metabolism
The metabolic changes in the flight muscles during the transition from rest to flight have been discussed fully in a number of recent reviews (Sacktor, 1975; Steele, 1981; Beenakkers et al., 1981) and will not be dealt with further in this account. Instead, we shall
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concentrate on examining the role which adipokinetic hormone plays in influencing substrate oxidation. There is much evidence to support the idea that, at least during the first few minutes of flight, trehalose and diacylglycerol compete as fuels for the flight muscles. If the concentration of either substrate in the haemolymph is increased experimentally, its rate of oxidation during the first few minutes of flight is increased above normal levels. Thus, if 6 m g of trehalose is injected into Schistocerca, so that its concentration in the haemolymph is doubled, trehalose decreases in the haemolymph at c. 570kgimin during a 20-min flight compared with c. 230 pgimin in control locusts receiving a saline injection (Robinson and Goldsworthy, 1976). Similarly, trehalose-injected Locusta show an average decrease in haemolymph trehalose during a 30-min flight of c. 310pgimin compared with c. 130 pg/min in saline-injected controls (Cheeseman et al., 1976). If, however, 6 m g of trehalose is injected into Locusta immediately after a 30-min flight and the locusts are flown again without delay, the trehalose remains largely in the haemolymph; the concentration decreasing only by c. 30 kg/min (Cheeseman et al., 1976). Thus, in this latter situation, the flight muscles use other substrate(s), presumably diacylglycerol, rather than the large quantity of injected trehalose. This suggests that AKH, which is at this time present in the haemolymph in large amounts, may play a positive role in determining which of the two major available substrates is oxidized. Data obtained from experiments in which extracts of corpora cardiaca are injected simultaneously with trehalose suggest that this is true (Robinson and Goldsworthy, 1976; Cheeseman et al., 1976), but studies involving injections of emulsions of dipalmitin suggest that the effect (=inhibition) of the hormone on carbohydrate oxidation is indirect; its prime effect is to stimulate disappearance of dipalmitin from the haemolymph during flight. Indeed, these experiments suggest also that injected dipalmitin is capable of inhibiting the utilization of trehalose sufficiently to impair flight performance but, in the absence of hormone extract, is unable to serve as an adequate substrate for the flight muscles. This led Robinson and Goldsworthy (1976) to suggest that adipokinetic hormone plays a positive role in favouring lipid oxidation in the flight muscles and thus inhibits to a large extent the further oxidation of trehalose. It is proposed that part of the function of the hormone is to conserve carbohydrate, and as a result only about half of the available haemolymph trehalose is utilized (see Section 2.1). The studies described above involve the injection of rather large
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amounts of trehalose or of dipalmitin, the latter being normally only a minor component of the natural diacylglycerol pool (Spencer and Candy, 1974; Jutsum and Goldsworthy, 1976; Robinson and Goldsworthy, 1976) and having to be presented as an emulsion. For these reasons, an attempt has been made recently to take a different approach to the study of fuel utilization in vivo. In these studies locusts are manipulated only by flight on roundabouts and/or by injection of hormone extracts (Goldsworthy et al., 1979). Locusts were flown for 30 min and then rested for 2 h before a second flight of 30min. They moved the roundabouts by their own activity, and the effects of injection of extracts of corpora cardiaca on the utilization of haemolymph carbohydrate during the two flights were studied. Hormone injection causes a small but significant reduction in the rate of disappearance of trehalose from the haemolymph during the first flight, when the locusts start with high (resting) levels of trehalose but low levels of diacylglycerol. The effect is more dramatic, however, when hormone is injected before the second flight, when the locusts start with moderate haemolymph levels of trehalose but high levels of diacylglycerol; there is hardly any change in the concentration of haemolymph carbohydrate during this second flight. In these experiments it is also noticeable that injection of hormone extracts before either flight brings about changes in the speed with which the locusts turn the roundabouts compared with controls; typically to reduce it in the first few minutes but to increase it in the last 15 min of the 30-min test flight (Goldsworthy et al., 1979). From these studies it is concluded that injection of hormone in vivo brings about an earlier utilization of diacylglycerol than in normal flown locusts. Thus, when the hormone is administered prior to the first flight, fatty acid oxidation in the flight muscle is stimulated but at a time when insufficient is available in the haemolymph to support the muscles fully, although trehalose oxidation is inhibited by increased oxidation of lipid; the initial period of slow flight represents the delay while lipid mobilization is accelerated. When hormone is injected prior to the second flight, haemolymph lipid levels are already high; lipid oxidation powers the flight muscles, very little trehalose is oxidized, and flight speed is relatively constant. In order to test these concepts in a more controlled situation, Robinson and Goldsworthy (1977a) studied the effects of corpus cardiacum extracts on flight muscle metabolism .in vitro, using a “half-thorax’’ perfusion system described originally by Candy (1970). They were able to confirm their interpretations of the
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studies in vivo. Trehalose utilization in working flight muscle preparation is reduced by c. 50% (compared with the situation when it is supplied alone) in the presence of haemolymph lipoproteins but is restored by increasing the concentration of the carbohydrate; an effect directly comparable with experiments in vivo when trehalose injected immediately before flight is utilized at a higher rate than normal (Cheeseman et al., 1976). Under conditions in which trehalose and extracts of haemolymph are included together in the perfusion medium, utilization of diacylglycerol from preparations of lipoprotein with a high lipid :protein ratio (from elevated haemolymph), but not ones with a low lipid : protein ratio (from resting haemolymph), is stimulated by crude extracts of glandular lobes containing physiological amounts of AKH. Indeed, pure natural AKH isolated by the method of Cheeseman et al. (1977) is equally effective (Robinson and Goldsworthy, 1977a). However, under these conditions, when lipid oxidation is stimulated by hormone extracts, trehalose utilization is minimal; a situation comparable perhaps with prolonged flight (see Section 2.1). More importantly, however, inhibition of trehalose utilization under these conditions is non-competitive because increasing the concentration of trehalose in the perfusion medium has no effect on its rate of oxidation (Robinson and Goldsworthy, 1977a); a phenomenon comparable perhaps to experiments in vivo where trehalose injected into locusts in the middle of a 60-min flight (Cheeseman et at., 1976) is hardly utilized. Oxidation of diacylglycerol from resting haemolymph, or of diacylglycerol from Ayellow isolated by gel filtration (see Section 3.5.1), is unaffected by addition of glandular lobe extracts to the perfusion fluid. Nor does addition of hormone affect the oxidation of trehalose when it is supplied alone or as a mixture with resting lipoprotein. But importantly, as we have said, the presence of resting haemolymph itself suppresses glycolytic flux by c. 50%. When elevated haemolymph, or A + lipoprotein isolated by gel filtration (see Section 3.5.1), is used as a source of diacylglycerol in flight muscle perfusions, addition of gland extracts containing AKH stimulates lipid oxidation and reduces glycolytic flux by c. 90%. If, however, 2-bromostearic acid is included in the perfusion medium, the rate of trehalose oxidation (in the presence of lipoproteins from elevated haemolymph and added AKH) is similar to that when trehalose is the sole substrate. Bromostearic acid, which has no effect on glycolytic flux when trehalose is the only substrate, inhibits mammalian mitochondria1 carnitine acyl transferase (Chase and
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Tubbs, 1972). It appears, therefore, that suppression of glycolytic flux in vitro is a direct consequence of increased oxidation of fatty acids derived from lipoprotein diacylglycerol. An exact mechanism and site of action for A K H on flight muscle metabolism has not been determined. In Locustu, injections of corpus cardiacum extracts, even in high doses, do not have any effect on the levels of cyclic AMP (Gade and Holwerda, 1976) or of cyclic GMP (Worm, 1980) in the flight muscles. Nor is there any significant change in the levels of either nucleotide in the flight muscles during flight (Worm, 1980). On the other hand, uptake of diacylglycerol by a single working flight muscle preparation can be stimulated by glandular lobe extracts, although 2-bromostearic acid prevents the response (Robinson and Goldsworthy, 1977b). This latter observation suggests that hormone extracts stimulate entry of diacylglycerol indirectly by increasing the flux of fatty acids into mitochondria, and Robinson and Goldsworthy (1977b) speculated that AKH could exert a direct action on carnitine acyl transferase activity. The possibility of direct control of the activity of flight muscle mitochondria by A K H requires further investigation. Robinson and Goldsworthy (19774 showed that the quality and quantity of lipoprotein preparations is important in demonstrating an effect of adipokinetic hormone on lipid utilization in perfused flight muscle. Progressive dilution of lipoprotein from elevated haemolymph produces graded attenuation of the inhibition of glycolysis, even in the presence of hormone, whereas a fourfold concentration of lipoprotein from resting haemolymph (with or without hormone) does not suppress glycolytic flux beyond that found with normal haemolymph concentrations of resting lipoprotein. Thus in vitro, flight muscles appear to require a particular type of lipoprotein, the A+ lipoprotein, but its concentration is also important. These observations may explain the failure of Candy (1978) to substantiate ail of our findings, especially the stimulatory effect of adipokinetic hormone on lipid oxidation; he was able to show a 50% inhibition of glycolysis by the addition of lipoprotein preparations to the perfusion medium, but there was no further suppression when ‘extracts of corpora cardiaca were added. There is then agreement concerning the inhibition of glycolysis when lipoprotein preparations are included with trehalose as substrate for flight muscle preparations in vitro. This is directly comparable to the situation in vivo during prolonged flight (Jutsum and Goldsworthy, 1976; Robinson and Goldsworthy, 1976, 1977a; Goldsworthy et ul., 1979) and provides a plausible explanation of
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why trehalose oxidation decreases dramatically. But how is such an inhibition brought about? Robinson and Goldsworthy (1977a) showed that while the oxidation of trehalose was reduced during diacylglycerol utilization, that of glycerol was not affected, suggesting that inhibition of glycolysis occurs before formation of triose phosphates. Ford and Candy (1972) suggest that inhibition of glycolytic flux brought about in vitro by the presence of butyrate in the flight muscle perfusion system, is most likely exerted at the aldolase reaction, and locust flight muscle aldolase has now been shown to be sensitive to inhibition by both citrate and palmitoyl-carnitine (Storey, 1980). There is some uncertainty about whether citrate increases in the flight muscles (see Ford and Candy, 1972; Rowan and Newsholme, 1979; Worm and Beenakkers, 1980), but the concentration of acyl-carnitine does increase steadily during flight and is already raised five-fold in amount within the first 30min (Worm et al., 1980). Accumulation of this intermediate during increased fatty acid oxidation could therefore regulate glycolytic flux and explain the observed decrease in trehalose utilization characteristic of prolonged flight; this is a third answer to the question posed in Section 2.2.1 concerning the phenomenon of trehalose conservation. 3.5.4 Protein synthesis A K H appears to exert a direct inhibition of protein synthesis in the fat body in vivo and in vitro. Quantities of synthetic AKH, or extracts of corpora cardiaca containing amounts of natural AKH comparable with those released during flight, inhibit by c. 40% the incorporation of "-leucine into total fat body protein (Carlisle and Loughton, 1979). Carlisle and Loughton (1979) found no evidence of inhibition of specific proteins, the effect being to decrease the incorporation of 'H-leucine into all proteins equally. The full physiological significance and mechanism of action of this effect remain to be determined.
Flight speed The possibility that adipokinetic hormone could influence flight speed arose from the results of early experiments, when it was observed that locusts in which hormone release is prevented by surgical interference, propelled their roundabouts at a slower rate than intact control locusts (Goldsworthy et al., 1972b, 1973a). Flight 3.5.5
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speed of these operated locusts was increased, however, by injection of gland extracts containing AKH in physiological amounts (Goldsworthy et al., 1973a; Goldsworthy and Coupland, 1974; Jutsum and Goldsworthy, 1977). In addition, it was proposed (Goldsworthy et al., 1973a) that the typical pattern of change of flight speed exhibited by normal adult laboratory locusts (but see Jutsum et al., 1982) is causally related to the change in substrate utilization; the initial period of high-speed flight is powered by oxidation of carbohydrate but subsequently locusts settle down to a lower lipid-fuelled “cruising” speed. However, the experiments (Goldsworthy et al., 1979) discussed in Section 3.5.3 in which locusts were flown, rested, and flown again, suggest that a strict relationship between flight speed and the nature of the energy substrate does not exist. Flight speed is not necessarily related directly to the qualitative nature of the substrate being utilized, but rather to its metabolic availability; the latter being determined by a number of factors including substrate concentration, concentration of competitive substrates, and the presence of hormones. Injection of adipokinetic hormone clearly does induce changes in flight speed, as does injection of trehalose (Goldsworthy and Coupland, 1974; Cheeseman et al., 1976), and this is not by changes in wingbeat frequency (Goldsworthy and Coupland, 1974), but must involve changes in power output per wing cycle. Octopamine (see next section) has been shown to have such an effect in vitro on locust flight (Candy, 1978) and skeletal (Evans and Siegler, 1982) muscle. 3.6
OCTOPAMINE
3.6.1 Changes in haemolymph titre Octopamine is present in haemolymph of resting locusts and is known to increase in concentration under certain conditions. In resting Schistocerca haemolymph, only the D-isomer is present at a concentration of c. 5 pg/kl. It increases dramatically during the first 10min of flight to reach a peak value of c. 26pg/pl, but then declines rapidly towards resting levels as flight continues (Goosey and Candy, 1980a). The site of release or origin of this octopamine is not certain, although Goosey (1981) and Goosey and Candy (1982a) have suggested that it is released from nerves, originating from thoracic ganglia, which innervate the flight muscles directly; during flight local concentrations of octopamine around the flight muscles may, therefore, increase more rapidly and reach higher levels than those measured in the general haemolymph.
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In Locusta, reported titres of octopamine in resting locusts are c. 11 pg/pl (David and Lafon-Cazal, 1979) and c. 33 pg/pl (Orchard et al., 1981). These values are considerably higher than those reported in Schisrocerca, but it is not clear whether this represents differences in methodology between the studies, or true species and/or strain differences. Excitation of Locusta by handling, results in a rapid increase in haemolymph octopamine to c. 54 pg/pl within 15 min, which persists for some time and only falls noticeably between 1 and 2 h after excitation (Orchard et al., 1981). In Schistocerca, however, turnover of octopamine appears to be far more rapid both during flight (Goosey and Candy, 1980a) and rest (Goosey, 1981) than it would seem to be in Locusta. It is likely that the Malpighian tubules are responsible for inactivation and excretion of octopamine in locusts (Goosey, 1981; Goosey and Candy, 1982a), and it could be variations in their activity with age, sex and rearing conditions which contribute to these apparent species or strain differences.
3.6.2 Actions of haemolymph octopamine Circulating octopamine may act both on fat body and flight muscles. Orchard and his colleagues (1981) describe a small but rapid and relatively long-lived hyperlipaemic octopamine-mediated response to stress in Locusta. This is not dependent on octopamine-induced hormone release from the glandular lobes of the corpora cardiaca (see Orchard and Loughton, 1981b), but appears to be a direct effect on the fat body because injected octopamine potentiates hyperlipaemia in neck-ligated locusts. Indeed, Orchard and his colleagues (1982) have demonstrated a stimulation by octopamine of cyclic AMP production in Locusta fat body in vitro. These authors believe that flight-induced increase in haemolymph octopamine may initiate rapid mobilization of diacylglycerol during the earliest moments of flight, because Jutsum and Goldsworthy (1976) found that only 2 min of tethered flight initiated an immediate (and sustained) mobilization of diacylglycerol. Such a role for octopamine is an intriguing concept and may well be valid. It may, for example, explain the very small residual increases in haemolymph lipid (c. 1 pg/pI) observed in locusts in which adipokinetic hormone release is prevented surgically (Goldsworthy et al. , 1972b, 1973a; Jutsum and Goldsworthy, 1977). Nevertheless, there is good evidence for a very rapid release of adipokinetic hormone; within 5 min of the onset of flight evidence of increased exocytosis can be observed in the glandular lobes (Rademakers and Beenakkers,
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1977), and haemolymph titre of hormone increases markedly within 15 min (Houben, 1976; Cheeseman and Goldsworthy, 1979). Perhaps octopamine and adipokinetic hormone act together on the fat body during the first few minutes of flight. Information concerning haemolymph titres of octopamine during starvation, and possible effects of trehalose injection on its release during flight, would be useful in assessing the physiological significance of its hyperlipaemic action. At the moment, it appears that a clearer role for haemolymph octopamine could lie in its ability to influence flight muscle metabolism. Candy (1978) has shown that octopamine stimulates the oxidation of a variety of substrates in working perfused locust thoracic muscles; it increases also the size of muscle contraction. The response of the flight muscles occurs at concentrations of octopamine comparable to those found in haemolymph after a few minutes of flight (Candy, 1978; Goosey and Candy, 1980a,b) and appears to be a specific for the naturally occurring D-isomer (Goosey and Candy, 1980b). According to Candy (1978), octopamine at a concentration of 50 p M (7.7 pgipl) stimulates glucose oxidation in vitro by c. 100%, but stimulation of butyrate or diacylglycerol oxidation is less pronounced (c. 25 and 50% respectively). As with its actions on fat body, octopamine appears to exert its influence over flight muscle metabolism via an increase in cyclic AMP (Worm, 1980); theophylline and dibutyryl cyclic AMP have a similar although slightly weaker effect on flight muscle in vitro than octopamine (Candy, 1978). Nevertheless, in flight muscles, as in fat body (Van Marrewijk, personal communication) , octopamine does not activate glycogen phosphorylase (Goosey, 1981), and the exact mechanism by which cyclic AMP, or octopamine, stimulate flight muscle oxidation is unknown. The full physiological significance of the effects of octopamine on flight muscle metabolism has yet to be established. But it is tempting to imagine that it could play a vital role in maintaining the oxidation of glucose in the early stages of flight at a time when increasing concentrations of diacylglycerol in the haemolymph compete more effectively with trehalose-especially as this decreases in concentration rapidly during the first few minutes of flight. Thus, the increase in diacylglycerol utilization would be gradual (see Mayer and Candy, 1969a; Van der Horst et al., 1980) and allow a smooth transition from mainly carbohydrate to mainly lipid oxidation when sufficient diacylglycerol is present in the haemolymph to support flight metabolism adequately.
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3.7
NEUROSECRETION A N D OTHER FACTORS
The possible involvement of cerebral neurosecretory factors in the direct control of flight metabolism or behaviour has been discussed in earlier reviews (Mordue and Goldsworthy, 1974; Goldsworthy, 1976) but no recent advances appear to have been made in this area; flight is a strong stimulus for the release of stainable material from the endocrine system (see Goldsworthy and Mordue, 1974) but we still have no clear idea of its physiological significance. In Locusta, however, recent studies by Orchard and Loughton (1980), indicate the existence of a hypolipaemic factor in the storage lobes of the corpora cardiaca whose release in vitro can be brought about by electrical stimulation of the NCC I1 (but not the NCC I), although release in vivo has not been demonstrated. Interestingly, porcine insulin has hypolipaemic activity in Locusta, and insulin-like immunoreactive material is apparently present in the corpora cardiaca of locusts (Orchard and Loughton, 1980) but it is not known whether locust hypolipaemic factor and porcine insulin merely share a common pharmacology or whether they are related chemically. Orchard and Loughton (1980) suggest that the hypolipaemic factor may have important functions: during starvation, where its withdrawal could account for the observed hyperlipaemia ( Jutsum et al., 1975); in the first moments of flight, where it could overcome the inhibition of glycolysis brought about by haemolymph diacylglycerol; or after flight, where it could speed restoration of haemolymph lipid levels. Interestingly, Goosey and Candy (1982b) have found a factor in fresh extracts of corpora cardiaca from Schistocerca which overcomes in vifro the inhibitory effect of butyrate on glucose oxidation in flight muscle. Perhaps this is the hypolipaemic factor described by Orchard and Loughton (1980). These interesting possibilities remain to be investigated. 4
4.1
A comparative overview of the hormonal control of flight metabolism in locusts METABOLIC CHANGES
In a previous review, Gold&vorthy and Cheeseman (1978) drew attention to some parallels between hormonal and metabolic events occurring during flight in locusts and those in some exercising vertebrates. For example, physical exercise leads initially to an increased utilization of ‘blood” carbohydrate by the muscles (and
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mobilization of liver or fat body glycogen) but, as activity is prolonged, eventually lipid mobilization (from adipose tissue or fat body) is stimulated, and a greater proportion of muscle energy is derived from oxidation of fatty acids. Consequently during exercise, the changes in tissue and “blood” metabolites of these animals are remarkably similar (Fig. 7). Indeed, these comparisons can be FAT BODY
fft*t****i***
**********HAEMOLYMPH *** FLIGHT MUSCLE ****
1‘^
-GLUCOSE
/
GLUCOSE
A
\
1-J
,ENERGY
******* LlVER/ADIPOSE
TISSUE ***+**BLOOD
. t**t***li**** MUSCLE **+a***
Fig. 7 Changes in tissue metabolism and metabolites during exercise and starvation (after Goldsworthy and Cheeseman, 1978). The scheme refers primarily to locusts, but the changes in “blood” metabolites and tissue metabolism are qualitatively similar in both locusts and mammals. Changes in “blood” amino acids and rates of gluconeogenesis have been omitted (see text Sections 2.2.2 and 4.1)
extended to the fasting situation, when changes in tissue metabolites and metabolism are not only similar in these diverse animals but also resemble qualitatively those observed during sustained exercise. It may appear surprising initially that metabolic adaptations to exercise and starvation in animals as disparate as higher vertebrates and locusts should be similar, but their requirements in both situations are identical. They need to prevent a too rapid depletion of carbohydrate fuel because some tissues (at least in mammals) appear to have a continuous requirement for glucose; to achieve this, they provide an alternative and more ergonomic fuel in the form of lipid, and may increase the production of glucose from amino acids. O n this last point, however, although the rate of
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gluconeogenesis increases during starvation in locusts, we have found no evidence that it does so during flight (Davies, 1982; Goldsworthy et al., 1982). An essential part of the strategy concerning provision of lipid as an alternative fuel appears to be that glycolysis in the flight muscles is inhibited during their period of increased fatty acid oxidation (see Fig. 7). The glucose-fatty acid cycle theory proposed for mammals by Randle and his colleagues (1963) is an attempt to explain the mechanism by which glycolysis could be inhibited. It seems likely that the point of inhibition is not the phosphofructokinase reaction, as it is in mammals, because this enzyme from locusts (Walker and Bailey, 1969) and from a variety of other insects (Newsholme et al., 1977) is insensitive to citrate, but control of glycolysis is probably exerted at the aldolase reaction in insects (see Section 3.5.3). The conservation of carbohydrate during locust flight is thought by Robinson and Goldsworthy (1976) to be concerned with maintenance of a suitable source of energy for tissues other than flight muscles; the nervous system is considered, by analogy with mammals, to be a prime candidate (but see Strang and Clement, 1980; Strang, 1981). 4.2
LIPID TRANSPORT
In mammalian adipose tissue, lipid which is mobilized during exercise is in the form of non-esterified fatty acids and is carried bound to albumin in the blood. In contrast, as we have discussed in Sections 1.1 and 3.5.1, insects mobilize lipid as diacylglycerol bound to haemolymph lipoproteins. Although they appear at first sight to function in completely different ways and perform different roles, it is of interest to compare locust lipoproteins with those in mammals. 4.2.1 Chemical composition and physical properties of locust lipoproteins The detailed chemical composition of insect lipoproteins has been discussed in comparison with those of other invertebrates and of vertebrates by Chapman (1980). In general terms of gross chemical composition, locust lipoproteins are similar to those of mammals although the neutral lipid is mainly diacylglycerol, and only small amounts of triacylglycerol, cholesterol esters, and phosphatidylethanolamine are present (Peled and Tietz, 1975; Chino and Kitazawa, 1981).
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There is some conjecture as to whether insect lipoproteins are arranged in a similar manner to that proposed for man; that is, whether they consist of particles with polar proteins and phospholipids forming an outer hydrophilic shell surrounding a hydrophobic core of neutral lipids and cholesterol esters (Shen et al., 1977). Indeed, Pattnaik and his co-workers (1979) have concluded from calculations based upon the particle size and composition of haemolymph lipoproteins from larvae of the tobacco hornworm, Manduca sexta, that the core of the particle is too large for the small amount of non-polar lipids present. Furthermore, they suggest that a small subunit apoprotein occupies the central hydrophobic core and is therefore essentially immune to tryptic digestion while a larger subunit is susceptible because it is located mainly on the surface (Pattnaik et al., 1979). On the basis that diacylglycerol is relatively more polar than triacylglycerol, they argue that diacylglycerol may lie at the surface of the Manduca lipoprotein particle; this would allow more readily its utilization by tissues without need for particle degradation. In this context, however, it should be realized that mammalian VLDL (very low density lipoprotein) particles can be delipidated progressively and rapidly by lipolytic enzymes of the capillary endothelium in adipose tissue and muscle, without immediate degradation of lipoprotein particles (see Eisenberg and Levy, 1975; Nilsson-Ehle et al., 1980, and Fig. 9). Van der Horst and his colleagues (1981b, 1982) have suggested that locust lipoprotein Ayellow, A + , 0 and Of particles may be similarly constituted to those in Manduca. Their argument is based on an analogy with the Manduca lipoprotein and on their conclusion that there is a high rate of exchange of diacylglycerol between different locust lipoprotein species (see Van der Horst et al., 1981b), for which they believe diacylglycerol would need to be peripherally distributed in the particles (but see above). Nevertheless, if 0 and Of are artifacts (see Section 3.5.1) and do not exist in vivo (see below), then there is no evidence for an exchange of diacylglycerol between lipoproteins in vivo, except that Ayellow is converted together with its lipid into A+ (see Section 3.5.1). There is, nevertheless, evidence for rapid exchange between diacylglycerol of lipoproteins Ayellow and A+,and fat body or flight muscle, but this could simply be a function of specific membrane receptors for apoproteins in these tissues which facilitate access of membrane associated lipolytic enzymes to a neutral lipid core. Ayellow is particulate (see Fig. 8) with a mean diameter of c . 11nm (Wheeler, 1981) or 13 nm (Chino and Kitazawa, 1981).
Fig. 8 Lipoprotein particles from Locustu haemolymph (from Wheeler, 1981; Goldsworthy and Wheeler, 1982a). Samples of lipoproteins from whole fresh haemolymph, or from ammonium sulphate treated haemolymph separated on Ultrogel AcA22 were diluted in 0.05 M ammonium acetate to a final concentration of c. 100 pgipl and applied to colloidon coated carbon grids. Samples were allowed to settle for 1 min (preparations for shadowing were first fixed in unbuffered 1% OsO, for 5 min) and excess liquid removed carefully with paper tissue. Grids were stained in 1% phosphotungstic acid (1 min) or allowed to air dry and shadow cast at 60" using 80% platinumi20% gold wire. A and B are negatively stained preparations of A+ from Ultrogel AcA22 chromatography and whole fresh haemolymph respectively. The arrows indicate surface projections and apparent links between A+ particles in "chains". Note the large A + particles in B. Such particles are found also in chromatographically purified A+ and may be up to l l 0 n m in diameter. C is a shadowed preparation of chromatographically purified A + . For comparison, a shadowed preparation, D, and a negatively stained preparation, E, of Ayellow from Ultrogel AcA22 is included. Note the size difference between Ayellow and A+ particles, and their different appearance when negatively stained. Scale bars for A, B and E represent 30nm, whereas those for C and D represent 150nm
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The particles resemble diacylglycerol carrying lipoprotein particles described for other insects (see Chino et al., 1969), but their hydrated density of c. 1.05 g/ml (Wheeler and Goldsworthy, 1982; Goldsworthy et al., 1982) places them at the bottom of the H D L (high-density lipoprotein), or the top of the LDL (low-density lipoprotein) range as specified for mammalian lipoproteins. Particles of this size range and density have an estimated molecular weight of c. 4.2-6.9 x 1OS (using the formula M = 1.33 r3. N p, where N = Avogadro’s number, and p = density) which appears to be in reasonable agreement with estimates obtained by other means (see Section 3.5.1). Lipoprotein A + , however, consists of generally larger particles, usually 19-40 nm in diameter, although particles up to 110nm are seen occasionally (see Fig. 8). Lipoprotein A + has a hydrated density of less than 1-01giml (Wheeler, 1981; Goldsworthy and Wheeler, 1982a,b; Goldsworthy et al., 1982) and could be classified at the top of the VLDL or bottom of the LDL range in mammalian terms. When negatively stained and viewed in the electron microscope, lipoprotein A+ particles are remarkably similar in their appearance to LDL particles described in human blood by Forte and Nichols (1972); they are similar in size and general appearance, and tend to form chains of particles interconnected by short processes (Wheeler, 1981; Goldsworthy and Wheeler, 1982a). The lipoprotein A+ particles shown in Fig. 8A were obtained by chromatography of ammonium sulphate treated haemolymph on Ultrogel AcA 22, but identical particles are present when whole fresh haemolymph from adipokinetic hormone-injected, starved, or flown locusts is examined after negative staining (unpublished observations; see Fig. 8B). They are thus not artifacts of ammonium sulphate or chromatographic treatment of haemolymph. Larger, rather amorphous particulate material found at the void volume of AcA 22 columns (see Section 3.5.1) was not detected in samples prepared from whole fresh haemolymph (Wheeler, 1981; Goldsworthy and Wheeler, 1982b). This is further evidence to suggest that the 0 and O+ material of Van der Horst et al. (1979) does not occur in vivo. From the hydrated density and the most characteristic particle size, we estimate a possible molecular weight of the A + lipoprotein to be c. 8.1 x lo6, assuming that the A + particle is a simple sphere. In Fig. 8 it can be seen that the A+ particle is neither spherical nor simple. Many free particles possess a short projection while other particles aggregate into short chains connected by such processes. The particles also appear to have a dense core surrounded by a more diffuse halo. These observations suggest that this
.
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approximation of the molecular weight for A + could be an overestimate but, if chain formation occurs in free solution, so also could the values obtained from gel filtration studies (see Section 3.5.1).
4.2.2 Lipoprotein metabolism and the involvement of hormones in lipid transport In general, mammalian lipoproteins do not carry lipids directly for energy metabolism, but function in distributing lipid throughout the body, especially in transporting triacylglycerol from intestine and liver to peripheral tissues. The lipoproteins of locusts, however, carry diacylglycerols for direct use by flight muscles during sustained flight. In mammals, this role is fulfilled by non-esterified fatty acids carried in the blood as a complex with albumin. Correspondingly, points at which hormones affect lipid mobilization are primarily similar (see Section 3.5.1), but secondarily they affect different systems. Thus, in mammals, lipolytic hormones are concerned with mobilization of fatty acids but do not appear to be implicated directly in loading or unloading of neutral lipids associated with lipoproteins, whereas in locusts AKH plays a vital role in influencing lipid loading of Ayellow and A + lipoproteins (see Fig. 9). Despite these fundamental differences, comparisons between lipoprotein metabolism in mammals and locusts as shown in Fig. 9 may still give some useful indication of possible functions of the binding of CL-proteins to A + . In particular, we are intrigued by the part played by mammalian C-I1 apoprotein in activating membrane-bound lipoprotein lipase to effect unloading of neutral lipids from lipid-rich lipoprotein particles (see Nilsson-Ehle et al., 1980). Perhaps the presence of the CL-proteins bound to locust A+ particles (see Section 3.5.1) may activate lipases, or direct other specific interactions between lipoprotein A + and receptors in fat body and flight muscle to facilitate rapid loading and unloading of diacylglycerols. Although this suggests a possible function of CLproteins analogous to that of apoprotein C-I1 in mammals, the similar terminology is purely coincidental. The nomenclature for locust haemolymph lipoprotein and protein fractions separated on Sepharose 6B or Ultrogel AcA 22 arose from an alphabetical description of their sequence of elution (Mwangi and Goldsworthy, 1976, 1977b) and no functional or structural analogy with mammalian studies is implied. The locust CL-proteins which contribute to A + formation consist of two major protein bands when separated on polyacrylamide gels, and both appear (in SDS electrophoresis) to
MAMMALS TISSUE
'
ALBUMIN
-
FFA
LIVER
MUSCLE
LOCUSTS
Fig. 9 A simplified comparison of lipoprotein metabolism in locusts and mammals (after Goldsworthy and Wheeler, 1982a). In mammals, triacylglycerol (TGL)-rich lipoproteins (chylomicrons, CHYLO; and very low-density lipoproteins, VLDL) are synthesized in the gut and liver and released with their neutral lipid complement intact. In the blood they bind apoprotein C which is in equilibrium with a large pool carried on high-density lipoprotein (HDL) particles. Interaction of apoprotein C with membrane-bound lipases causes a loss of T G L to the tissues, and the lipid-depleted lipoprotein is released into the circulation as low-density lipoprotein (LDL) particles; T G L unloading is accompanied by loss of apoprotein C from the particles. LDL can further interact with tissue lipases to give up more T G L and the lipoprotein remnant is eventually removed from the blood by endocytosis. In locusts, lipoproteins synthesized in the fat body take up and release diacylglycerol (DGL) at the tissues and form a continuous shuttle. At times of high lipid turnover (due to AKH action or starvation), D G L is loaded initially onto Ayellow but eventually A + forms and this binds C,;proteins. D G L of the A+ particles is used by the flight muscles for energy metabolism and the C,-proteins and Ayellow are released again to continue a cycle of loading and unloading. Open arrows indicate lipoprotein synthesis; FFA is free fatty acid; A L is lipid-loaded Ayellow; A is Ayellow; the points at which hormones affect these systems are indicated
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have a molecular weight of c. 30 000 (unpublished observations). They are therefore larger than the C-apoproteins in mammals (see Eisenberg and Levy, 1975; Jackson et al., 1976). Nevertheless the phenomenon of their reversible binding to locust VLDL-like particles is intriguing.
4.3
THE ROLE OF HORMONES I N ENERGY METABOLISM
Until quite recently, A K H was the only hormone known to play a role in the control of locust flight metabolism, and a comparison in endocrine mechanisms operating to control energy metabolism in locusts with those in mammals suggested that although there are superficial similarities between actions of AKH and mammalian glucagon and growth hormone, the hormones themselves are quite dissimilar chemically. Additionally, because of our lack of detailed knowledge for locusts, the comparison suggested that endocrine mechanisms operating in locusts during flight were remarkably simple compared with those in vertebrates (see Goldsworthy and Cheeseman, 1978). Recent evidence would suggest, however, that a number of hormones may operate to control locust flight metabolism: there is the possibility of release of a second adipokinetic hormone, compound 11; there are the known changes in the titre of octopamine in the haemolymph, and its effects on muscle metabolism in vitro; and there is the possible involvement of a hypolipaemic hormone (see Fig. 10). One particularly intriguing aspect of these recent developments is the analogy which can be drawn between octopamine in insects and catecholamines in vertebrates. Octopamine is chemically related to the catecholamines, being very similar structurally to noradrenaline, and is found in large amounts in insect nervous tissue (Robertson and Juorio, 1976; Evans, 1978). Octopaminergic cells in the locust metathoracic ganglia innervate the extensor tibiae muscles (Hoyle, 1975; Evans and O’Shea, 1978), where octopamine is thought to play a neuromodulatory role; it potentiates the strength of contraction of these muscles in vitro (O’Shea and Evans, 1979; see also Evans and Siegler, 1982). This is comparable with the role of noradrenaline in the mammalian sympathetic nervous system. Pharmacologically, however, insect octopamine receptors resemble mammalian a-receptors, although they work through increases in intracellular cyclic AMP rather than Ca2+ (see Candy, 1981). Octopamine receptors in locust flight muscle appear to be different from those in the fat body and in the glandular lobes of the corpora
THE E N D O C R I N E C O N T R O L O F F L I G H T M E T A B O L I S M
*+****+****FAT
B O D Y *t*****+****
H A E M O L Y M P H ******FLIGHT
193
MUSCLE ******t*t**
Fig. 10 The endocrine control of flight metabolism in locusts (modified from Goldsworthy and Cheeseman, 1978). It should be appreciated that the temporal aspects of hormone actions, which are not included here, are important in the integrated control of energy metabolism. For a detailed explanation see text
cardiaca, because the a-blocker phenoxybenzamine prevents release both of lipid from fat body (Orchard et al., 1981b) and of adipokinetic activity from corpora cardiaca (Orchard and Loughto n , 1980), but has an agonistic effect on flight muscle in v i m (Goosey and Candy, 198Ob). Similarly, the P-blocker propranolol has no effect on flight muscle or its response to octopamine (Goosey and Candy, 1980b), but may bind to a-receptors in the corpora cardiaca and activate rather than block them (Orchard and Loughton, 1981b). Clearly, there is no reason to expect locust octopamine receptors to be identical, either chemically or pharmacologically, to a-receptors in mammals, and different locust tissues may contain heterogeneous populations of octopamine receptors which resemble to varying degrees the a-receptors described in mammals. The concept that, during locust flight, octopamine plays a variety of roles similar to those of catecholamines in mammals during exercise, begins to put flesh on the skeleton of the scheme proposed by Goldsworthy and Cheeseman (1978) and has been included in Fig. 10. Octopamine may occupy an important position in directing energy metabolism during the first few minutes of flight. Indeed, Orchard and his colleagues (1981b) suggest that it may mediate a “flight or fight” response. It now seems only a matter of time before insulin-like hormones (in actions, if not in chemical structure) will be considered as part of such a scheme.
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Other insects may not fall readily into the scheme illustrated in Fig. 10 because they use only a single energy substrate for flight and/or they fly for only short periods. Nevertheless, there appears to be a group of closely related insect peptides which influence the mobilization of energy reserves; in some insects they affect predominantly carbohydrate metabolism, while in others they affect lipid metabolism (see Goldsworthy and Gade, 1982). At least one beetle, Tenebrio rnoliror (Goldsworthy et a l . , 1972a), and three lepidopterans, Danaus plexippus (Dallman and Herman, 1978), Manduca sexta (Beenakkers et al., 1978), and Vanessa cardui (Herman and Dallman, 19Sl), possess adipokinetic hormone(s). It seems likely that Danaus (Dallman and Herman, 1978) and Manduca (Ziegler and Schulz, 1981) release their adipokinetic hormone(s) during flight. The hypertrehalosemic factor in blowflies may also be released in flight (Vejbjerg and Normann, 1974). Further, more detailed, studies on the endocrine control of flight metabolism in these and other insects are awaited anxiously. 0
Acknowledgements
I am grateful to colleagues who have given helpful criticism and advice or allowed me to refer to their unpublished work. My special thanks are due to Chrissie Ware for typing the manuscript and Mrs J. Mundy for taking the photomicrographs. Original research described in this review is supported by grants from The Royal Society and S.E.R.C. References Bailey, E . (1975). Biochemistry of insect flight. Part 2-Fuel supply. I n “Insect Biochemistry and Function” (Eds D. J. Candy and B. A . Kilby), pp. 89-176. Chapman and Hall, London. Beenakkers, A . M. Th. (1969). The influence of corpus cardiacum on lipid metabolism in Locusta migratoria. Gen. comy. Endocr. 13, Abstract 12. Beenakkers, A . M. Th. (1973). Influence of flight on lipid metabolism in Locustu migratoria. Insect Biochem. 3, 303-308. Beenakkers, A . M. Th.. Van der Horst, D. J. and Van Marrewijk, W. J. A . (1978). Regulation of release and metabolic function of the adipokinetic hormone in insects. In “Comparative Endocrinology” (Eds P. J. Gailard and H. H. Boer), pp. 445-448. Elsevier, Amsterdam. Beenakkers. A. M. Th.. Van der Horst, D. J. and Van Marrewijk. W. J. A. (1981). Role of lipids in energy metabolism. In “Energy Metabolism in Insects” (Ed. R. G. H . Downer). pp. 53-100. Plenum Press, New York.
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Rowan, A . M . and Newsholme, E . A . (1979). Changes in the contents of adenine nucleotides and intermediates of glycolysis and the citric acid cycle in flight muscle of the locust upon flight and their relationship to the control of the cycle. Biochenz. J . 178, 209-216. Sacktor, B. (1975). Biochemistry o f insect flight. Part 1-Utilization of fuels by muscle. In “Insect Biochemistry and Function” (Eds D . J. Candy and B. A . Kilby), pp. 1-88. Chapman and Hall, London. Shen, B. R . , S c a m , A . M. and Kezdy, F. J. (1977). Structure of human serum lipoproteins inferred from compositional analysis. Proc. Nut. Acad. Sci. USA 74, 837-841. Smith. D. S. and Sacktor, B. (1970). Disposition of membranes and the entry of haemolymph-borne ferritin in flight muscle tibers of the fly Phormia regina. Tissue and Cell 2, 355-374. Spencer. I . M. and Candy. D. J. (1974). The effect of flight on the concentrations and composition of haemolymph diacylglycerols in the desert locust. Biochem. Soc. Truns. 2, 1093-1096. Spencer. I. M . and Candy, D. J. (1976). Hormonal control of diacylglycerol mobilization from fat body of the desert locust. Sci+tocerca gregaria. Insect Biochem. 6, 289-296. Steele, 3 . E. (1981). The role of carbohydrate metabolism in physiological function. In “Energy Metabolism in Insects” (Ed. R . G . H . Downer), pp. 101-133. Plenum Press, New York. Stone, J . V. and Mordue, W. (1979). Isolation of granules containing adipokinetic hormone from locust corpora cardiaca by differential centrifugation. Gen. comp. Endocr. 39, 543-547. Stone, J . V. and Mordue, W. (198Oa). Adipokinetic hormone. In “Neurohormonal Techniques in Insects” (Ed. T. A . Miller), pp. 31-80, Springer-Verlag, Berlin. Stone. J . V. and Mordue, W. (198Ob). Isolation of insect neuropeptides. Insect Biochern. 10, 229-239. Stone, J . V.. Mordue. W.. Batley, K . E. and Morris. H. R. (1976). Structure of locust adipokinetic hormone. a neurohormone that regulates lipid utilization during flight. Nriture, Lond. 263, 207-21 1. Stone, J. V., Mordue, W., Broomfield, C . E . and Hardy, P . M . (1978). Structure-activity relationships for the lipid-mobilizing action of adipokinetic hormone. Synthesis and activity of a series of hormone analogues. Eur. J . Biochern. 89, 195-202. Storey, K. (1980). Kinetic properties of purified aldolase from flight muscle of Schistocrrca anzericuna gregaria. Role of the enzyme in the transition from carbohydrate to lipid-fuelled flight. Insrcr Biochern. 10, 647-655. Strang, R. H . C. (1981). Energy metabolism in the insect nervous system. I n “Energy Metabolism in Insects” (Ed. R. Ci. H . Downer). pp. l6Y-206. Plenum Press, New York. Strang, R. H. C. and Clement, E. M. (1980). The relative importance of glucose and trehalose in the nutrition of the nervous system of the locust Schistocerca aniericana gregciria. Insect Biochern. 10, 155- 16 1. Thomas. K. K. and Gilbert, L. I. (1968). Isolation and characterization of haemolymph lipoproteins of the American silkmoth, Hvalophora cecropia. Arch. Biocheni. Biophys. 127, 5 12-52], Tietz. A . (1967). Fat transport in the locust: the role of diglyceride. Eur. J . Biochern. 2, 236-242.
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Tietz, A . and Weintraub. H . (1978). Hydrolysk of glycerides by lipases of the fat body of the locust. Locusta migratorin. Insect Biochern. 8, 11-16. Tietz, A . and Weintraub, H . (1080). The stereospecific structure of haemolymph and fat body 1.2-diacylglycerol from Locusta niigratoria. Insect Biocheni. 10, 61-63. Tietz. A , , Weintraub. H. and Peled, Y. (1975). Utilization of 2-acyl sn-glycerol by locust fat body microsomes: specificity of the acyltransferase system. Biochirn. Biophys. A c ~ u388, . 165170. Van den Hondel-Franken, M. A . N., Van den Broeck, A . Th. M. and Beenakkers, A . M. Th. (1980). Flight muscle development in Locusta migratoria: effects of implantation of corpora allata on the attainment of metabolic enzyme activities. Gen. comp. Endocr. 41, 477-486. Van der Horst, D . J . (1982). Resources and substrate transport. In “Exogenous and Endogenous Influences on Metabolic and Neural Control” (Eds A . D. F. Addink and N . Spronk), pp. 243-255. Pergamon Press, Oxford. Van der Horst, D . J . , Baljet, A. M. C.. Beenakkers. A . M. Th. and Van Handel. E. (1978a). Turnover of locust haemolymph diglycerides during flight and rest. Insect Biochem. 8, 369-373. Van der Horst. D . J . . Van Doorn, J. M. and Beenakkers, A . M. Th. (1978b). Dynamics in the haemolymph trehalose pool during flight of the locust. Locicstn migratoria. Insect Biochem. 8, 413-416. Van der Horst, D . J.. Van Doorn, J. M . and Beenakkers, A . M. Th. (1979). Effects of the adipokinetic hormone on the release and turnover of haemolymph diglycerides and on the formation of the diglyceride transporting system during. locust flight. Insect Riochern. 9, 627-635. Van der Horst, D . J . . Houben, N. M. D . and Beenakkers. A . M. Th. (19x0). Dynamics of energy substrates in the haemolymph of Lomstrr nzigrrrtoriri during flight. J . Insect Physiol. 26, 441448. Van der Horst, D . J . , Stoppie, P.. Huybrechts, R . . De Loof. A. and Beenakkers. A . M. Th. (1981a). Immunological relationships between the diacylglycerol-transporting lipoproteins in the haemolymph of Loritsto. Cornp. Biochem. Physiol. IOB, 387-392. Van der Horst. D . J . . Van Doorn, J. M.. De Keijzer, A . N. and Beenakkers. A . M . Th. (1981 b). Interconversions of diacylglycerol-transporting lipoproteins in the haemolymph o f LOCLLW.Insect Riochrw. 11, 717-723. Van der Horst, D . J . , Abbink, J. H . M.. Van Doorn. J . M., Van Marrewijk. W. J. A . and Beenakkers, A . M. Th. (1983). Glycerol dynamics and metabolism during flight of the locust, L o c k t u rnigrutorim Insect Hiocheni. 13, 45-55, Van Marrewijk, W. J. A.. Van Den Broek. A . Th. M. and Beenakkers, A . M. Th. (1980). Regulation of glycogenolysis in the locust fat body during flight. Irisecr Biochern 10, 675-679. Van Norstr;.nd, M . D.. Carlsen, J . B.. Josefsson. L. and Herman. W. S. (1980). Studies on a peptide from the cephalic endocrine system of the honey bee. Apis mellijeru. Gerz. conip. Endocr. 42, 526S33. Vejbjerg, K. and Normann. T. C. (1974). Secretion of hyperglycaemic hormone from the corpus cardiacum of flying blowflies. Culliphorrr er~throc~ci~~lirrl~i. J . Insect Physiol. 20, 1189-1 192. Wajc, E. (1973). The effect of the corpora allata o n flight activity of Lociista migratoria migratorioirks (R&F). Ph.D. Thesis. University of London.
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Wajc, E. and Pener, M. P. (1971). The effect of the corpora allata on the flight activity of the male African migratory locust, Locusta migratoria migratorioides (R&F). Gen. comp. Endocr. 17, 327-333. Walker, P. R. and Bailey, E. (1969). A comparison of the properties of the phosphofructokinases in the fat body and flight muscle of the adult male desert locust. Biochem. J . 111, 365-369. Wheeler, C. H. (1981). Haemolymph proteins and lipid transport in Locusta migratoria. Ph.D. Thesis, University of Hull. Wheeler, C. H. and Goldsworthy, G. J. (1982). Interaction of haemolymph proteins in Locusta during the action of AKH. Gen. comp. Endocr. 46, 382. Wheeler, C. H. and Goldsworthy, G. J. (1983a). Qualitative and quantitative changes in Locusta haemolymph proteins and lipoproteins during ageing and adipokinetic hormone action. J . Insect Physiol. 29, 339-347. Wheeler, C. H. and Goldsworthy, G . J . (1983b). Protein-lipoprotein interactions in the haemolymph of Locusta during the action of adipokinetic hormone: the role of CL-protems. J. Insect Physiol. 29, 349-354. Worm, R. A: A. (1980). Involvement of cyclic nucleotides in locust flight muscle metabolism. Comp. Biochem. Physiol. 67C, 23-27. Worm, R. A. A. and Beenakkers, A. M. Th. (1980). Regulation of substrate utilization in the flight muscle of the locust, Locusta migratoria, during flight. Insect Biochem. 10, 53-59. Worm, R. A. A . , Luytjes, W. and Beenakkers, A. M. Th. (1980). Regulatory properties of changes in the contents of coenzyme A, carnitine and their acyl derivatives in flight muscle metabolism of Locusta migratoria. Insect Biochem. 10, 403-408. Ziegler, R. and Schulz, M. (1981). Hinweise auf ein Adipokinetisches Hormon bei Manduca sexta (L.). Verh. Dtsch. Zool. Ges. p. 281.
T he Ne u ros ec reto ry- Ne u roha e ma I Sy st e m of Insects ; An atom ica 1, St ructuraI and Physi o logica I Data M. Raabe Laboratoire de Neuroendocrinologie des Insectes, Universite P. et M. Curie, CNRS, Paris, France
1 Introduction 2 Neurohormone production 2.1 Neurosecretory cell morphology and function 2.2 Neurosecretory cell types 2.3 Neurosecretory cell location 2.4 Immunoreactive ns cells 2.5 Catecholamines and indolamines 3 Neurohormone release 3.1 Transport and release of neurosecretory material 3.2 Corpora cardiaca 3.3 Perisympathetic organs 3.4 Neurohaemal areas 3.5 Neuroeffector junctions 3.6 Terminals in nervous system 4 Regulation of neurohormone production and release 4.1 Histophysiological studies 4.2 Significance of loaded neurosecretory cells 4.3 Storage places 4.4 Nuclei and organelles 4.5 Investigations with labelled cysteine 4.6 Autoregulation 4.7 Rhythmic functioning 4.8 Environmental and internal inputs 4.9 Feedback 5 Neurohormones, a brief survey 5.1 Neurohormonal activities 5.2 Origin and release sites 5.3 Mode of action 5.4 Purification and identification
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1 introduction
As is often the case, the observation that nerve cells contain chromophilic particles was made several years before the concept of neurosecretion was formulated (Scharrer and Scharrer, 1937). This concept was originally based on the presence of secretory activity in nerve cells; but the idea that neurosecretory (ns) products are transported along the axon quickly acquired predominant importance, and these products were shown to accumulate in specialized regions called neurohaemal organs, located close to blood vessels from which the ns products are released. Another important feature which has been stressed about ns cells was that they form no close efferent synaptic junctions or contacts with other cells. Considerable progress was achieved by applying to ns cells the staining techniques devised for the pancreas, i.e. chrome haematoxylin-phloxine and then paraldehyde fuchsin. The staining obtained through such techniques, which proved to be very effective, especially for detecting ns pathways, were considered characteristic of ns products. However, the advent of electron microscopy, provided a new criterion for neurosecretion-the presence in ns cell bodies and axons of elementary membrane-bound electron-dense granules, 100-300 nm in diameter (Scharrer, 1963). Soon after the corpora cardiaca were identified as the neurohaemal organs of insects, other neurohaemal organs were discovered, the perisympathetic organs, corresponding to the ventral nerve cord (Raabe, 1965). At about the same time, ns endings were described in several organs and were termed neurosecretomotor junctions (Knowles and Bern, 1966). This contradicted previous concepts, arid demonstrated that all ns products were not transported via the bloodstream. Ns cells located outside the central nervous system were found among the peripheral sensory cells (Finlayson and Osborne, 1968), as well as neurohaemal areas located on various nerves and involved, at least partly, in the release of peripheral ns products. Other hypotheses that have been advanced recently suggest that ns products sometimes possess a modulatory function, and it was
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also proposed that more than one substance may be synthesized and transported within a single neuron. Thus, neurons containing conventional neurotransmitters such as catecholamines, serotonin or acetylcholine may also contain gastrointestinal peptides like substances P or vasoactive intestinal peptide (VIP). In addition, hypothalamic neurons containing ACTH or T R H may also include other peptides like @-endorphinor substance P. During the past few years, many investigations have been made in insects where new methods have been introduced to detect both amines and vertebrate peptides. To sum up the present state of knowledge, ns cells may be considered as neurons synthesizing products released by the neurohaemal organs and transmitted by the bloodstream or occasionally transported along the axon directly to target organs. Ns cells can be visualized by histology, electron microscopy, immunocytology or specific histochemical techniques for amines. Since amines are sometimes transported by the bloodstream, they act like neurohormones, and may be examined together with ns cells.
2
2.1
Neurohormone production N E U R O S E C R E T O R Y C E L L M O R P H O L O G Y A N D FUNCI’ION
2.1.1 Neurosecretory cell characteristics Histologically, ns cells are visualized by the occurrence of cytoplasmic chromophilic inclusions of very varied density and shape. Some ns cells contain round granules whereas others are filled with small irregularly-shaped grains or with large droplets. However, histology is not really sufficient to identify ns material, because lipofuchsin pigments and certain cytoplasmic organelles may react like ns material. Definite identification requires electron microscopy which, as already mentioned, reveals the presence of the dense elementary granules which characterize ns cells. Except for their secretory functions, the ns cells behave like other neurons. They are synaptically controlled, produce action potentials and transport nerve impulses. Electrical recordings of pars intercerebralis ns cells and their axons demonstrate that they generate electrical activity and transport nerve impulses. They exhibit a resting potential of 20-50 mV and an action potential similar to that of non-neurosecretory neurons (about 60 mV) but much slower (3-6 ms compared to O.6-2.5nis) (Gosbee et at., 1968; Normann,
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1973; Finlayson and Osborne, 1975). When the release of ns material is accelerated, bursts of impulses replace the normal spiking pattern. Upon arrival at nerve terminals, the nerve impulses trigger the release of neurohormones. 2.1.2 Morphology of ns cells The morphology of ns cells does not differ very much from that of other neurons: the cell body is not necessarily large, and the nucleocytoplasmic ratio is variable. The dendrites, like those of all insect neurons, originate in the proximal part of the axon and are difficult to distinguish from the collaterals. They penetrate the neuropil, where they make synaptic contact with interneurons running from the sensory or associated centres. Synaptic contact may also occur elsewhere along the axon pathway (axo-axonic contact) or within the neurohaemal organs. The ns cell axons often run long distances before entering neurohaemal organs, where they branch repeatedly. At each distal end they form a pre-terminal swelling which precedes the ending proper. The latter is in contact with a thin connective tissue sheath which separates the axon terminals from the extracellular fluid into which the ns products are released. In other places, a thick glial investment forming a number of folds surrounds the perikaryon, dendrites, collaterals and axon. To conclude, it should be stressed that the ns products released into the haemolymph are considerably diluted, especially compared to the degree of dilution of conventional neurotransmitters in the narrow synaptic cleft. In addition, none of the material secreted can be recovered as it can be in the synapse. Consequently, a large amount of material has to be manufactured in the cell body and discharged at the axon endings which implies a large increase in the number of these endings. 2.1.3 Synthesis ojneurosecretory products Synthesis of ns material is initiated in the rough endoplasmic reticulum and continues in the Golgi apparatus. Ns material is gradually condensed, and electron-dense membrane-bound granules bud off from the extremities of the Golgi saccules. These processes have been clearly demonstrated in the A cells of the pars intercerebralis of Locustu rnigrutoriu by experiments involving incorporation of 3%-cysteine. The label appears, first in the rough
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endoplasmic reticulum, then in the Golgi apparatus (15-30 min) and lastly after 1 to 2 h in the granules (Girardie and Girardie, 1972). The diameter of the granules ranges from 100 to 300nm. Their density also varies and several reports describe large transparent vesicles. One may wonder what the advantage is for the ns material to be enclosed in membrane-bound granules, and what exactly these granules contain. The sequestration of ns material probably protects it within the perikaryon and axon. Moreover, the granule may be a convenient structure for transporting the material over long distances, and for avoiding loss of molecules. The content of the elementary granule apparently consists of small neurohormones and large carrier proteins. The exact relationships between these two substances is not clearly understood. The neurohormones may be a fragment of the carrier protein, or both substances may have a common precursor, as has often been suggested for vertebrates. In insects, the presence of carrier-proteins has not been demonstrated conclusively, but is suggested by various observations. First, it has been shown in several cases that ns cells which stain in different ways when techniques other than paraldehyde fuchsin are used, stain in the same way with paraldehyde fuchsin, suggesting that paraldehyde fuchsin is retained by some substance common to the various ns cells (Mahon and Nair, 1975). It was further shown that hormonal activity does not always correspond to the staining intensity of the ns cells supposed to be involved in a given process (Meola and Lea, 1971). In such cases, the material stained may be assumed to be a carrier protein rather than the hormone itself. Finally, a putative neurophysin was isolated from the brain and corpora cardiaca of locusts (Friedel et al., 1980). Its molecular weight was 11092 and its components were (Met)], (Try,Phe)*, ( A h ,Ile,Arg)4, (Val ,Leu)S, (Thr ,Ser ,Prol),, (Gly ,half-Cys)g, and (Lys)l4. The preparation of an antiserum against this protein enabled it to be localized by immunochemistry. It was found only in the paraldehyde fuchsin-positive ns cells of the pars intercerebralis. Subsequent experiments proved that this putative neurophysin is released together with the diuretic hormone (Orchard et af.,1981a). Related to these problems is the question of the maturation of granule contents during axonal transport. A change in the staining properties of ns material during transit from the brain to the corpora cardiaca has been reported by Gabe (1972). Using the alcian blue-alcian yellow technique, which involves the reaction of weakly
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acid groups to form a blue-green product and of stronger acids to produce a yellowish colour, he detected a decrease in the acidity of the ns product during distal transport in the cerebral ns cells of 10 species of pterygote insects. This observation is consistent with the finding that the number of 1-2 glycol groups increases progressively during migration. Some experimental data also indicate that the physiological activity of nerve ganglia is not always the same as that of the neurohaemal organs. In a study devised to purify the diuretic hormone of Rhodnius prolixus, Aston and White (1974) isolated different substances from the ganglia and neurohaemal organs, and demonstrated that the latter contained factors with a lower molecular weight. 2.1.4 Resorption and storage of neurosecretory material All the ns material synthesized in the perikaryon is not transported to the axon terminals; thus, any excess material synthesized is destroyed in the cell body by lysosomes or multivesicular bodies. These organelles are generally considered to be involved in the resorption processes but they have also been reported to play a part in the process of maturation of granules after their release from the Golgi apparatus into the cytoplasm (Morris and Steel, 1977). Ns material is also destroyed at the axon endings in the pre-terminal swellings. The pre-terminal swellings may, however, intervene in a different process-that of granule storage; the granules stored are thus available if the fast release of a large amount of material is required.
2.2
NEUROSECRETORY CELL TYPES
Since neurosecretion was first investigated, various staining properties have been observed in ns cells, which were seen to resemble glandular cells.
2.2.1 Chrome haematoxytin and paraldehyde fuchsin The first technique extensively used to study ns cells was chrome haematoxylin-phloxine staining. Another technique, which also came into use rapidly, was paraldehyde fuchsin staining. Both techniques involved a strong oxidation procedure which acts upon basic ns products; some of them became acidic, while others remained basic. Thus, the subsequent use of two stains, the first
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basic (chrome haematoxylin and paraldehyde fuchsin) and the second, acidic (phloxine and light green or picroindigo carmine), made it possible to distinguish two kinds of ns products, the acid ones, termed A products, and the basic one, known as B products. These techniques are not histochemical, but colouration by basic product staining is often due to the presence of SS or SH groups in the ns products. Further, aldehyde and dicarboxylic groups stain intensely with paraldehyde fuchsin. The results of this method are therefore not identical to those of chrome haematoxylin, and paraldehyde fuchsin has been suggested to stain the ns material at an earlier stage of its production than chrome haematoxylinphloxine (Raabe, 1965; Mahon and Nair, 1975). In any case, it reveals a number of ns cells that are not stained by chrome haematoxylin-phloxine, such as lateral protocerebral and tritocerebral ns cells and glandular cells of the corpora cardiaca. The physiological significance of A and B cells has often been debated. Some authors have stated that B cells are inactive A cells, while others consider that two different kinds of cell are involved. In fact the pars intercerebralis contains not only A and B cells, but also other ns cell types.
2.2.2
A zocarmine
Certain ns cells can only be visualized by azocarmine, using material fixed in Helly’s fluid (Raabe, 1964). This method revealed bright red cells in the pars intercerebralis, the tritocerebrum and all the ganglia of the ventral nerve cord (Raabe, 1964; Delepine, 1965; Chalaye, 1967; de B e d , 1967; Baehr, 1968; Ramade, 1969). These cells, which were at first designated by the letter C, are now termed Cr (Raabe et al., 1979; Raabe, 1982), to avoid confusion with other C cells belonging to the A type.
2.2.3
Other cell types and detection techniques
For many years, investigators have noticed the presence of distinct categories among A and even B ns cells. The cells in these categories are recognizable by their location, size and morphology, and by the appearance of their ns products. Thus, according to Johansonn’s study on the bug Oncopeltus faxiatus (1958), the pars intercerebralis contains four distinct ns cell
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types: B cells, and A, C and D cells, all three possessing the staining properties of the A type. In the locust, C cells belonging to the A type are described together with A and B cells (Girardie and Girardie, 1964). The occurrence of several distinct A types has been confirmed in a number of insects, and the application of new staining techniques allows better distinction between some of them. The new techniques which are worth mentioning here are Victoria blue and fuchsin resorcine staining employed in toto. They have the great advantage of revealing the entire tract of each ns cell, but they only stain A type ns cells. Acridine orange is also an in toto method which stains different types of ns products. Its results require observation by fluorescence microscopy, as is also the case for the Sterba pseudoisocyanine method. For accurate identification of type A ns cells, the methods using alcian blue-alcian yellow and those using paraldehyde fuchsin are very useful, because they make it possible to distinguish between two kinds of A cells (A and A ' , also called A1 and A2) which look very much alike and are located close to one another in the pars intercerebralis. When stained with chrome haematoxylin-phloxine, they can usually be distinguished, because A cells are heavily loaded, whereas A ' cells contain little neurosecretory material (Baehr, 1968). Further observation of both types during an intermoult period in bugs showed that their activity cycles are asynchronous (Robert, 1979; Furtardo, 1979). The presence in the pars intercerebralis of two types of A ns cells has been reported in several orders (Raabe et al., 1979). Thus, if alcian blue-alcian yellow or thionine phloxine or paraldehyde thionine-paraldehyde fuchsin are used, instead of chrome haematoxylin or paraldehyde fuchsin , clear-cut differences in colour may be observed between A and A' cells (A1 and A2) in many insects (Raabe et al., 1979). The A 1 cells are stained by thionine because they contain strong acids, whereas A2 cells are stained by fuchsin because they contain weak acids. The same observations were made in Locusta, but using other methods. Two distinct types of A ns cells were shown by alternate staining with paraldehyde fuchsin and Victoria blue. While all the type A cells were stained by paraldehyde fuchsin, only some of them appeared with Victoria blue (Ganagarajah and Saleuddin, 1970). Both types demonstrated by this method appeared significantly different in electron microscopy (round granules 140-200nm in diameter in A1 ns cells and ovoid granules with a diameter of 250-300nm in A2 ns cells (Girardie and Rossi, 1978).
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2.2.4 Histochemical investigations The observations made with diverse staining methods suggest that better separation of ns products should be attempted by histochemical techniques. Such techniques have been applied to many species by Arvy and Gabe (1962) and also by several other authors (see Raabe, 1982). The general features which emerge from the literature indicate that ns cells do not contain lipids or carbohydrates, but do contain large amounts of protein, as confirmed by enzymatic destruction experiments. Despite contradictory data, it appears that in the pars intercerebralis, the A cells and probably also the B cells contain cysteine and/or cystine amino acids, which is not the case for the Cr cells. Indol and pyrrol groups occur in A cells, but are hardly present in B cells; they are more abundant in Cr cells, which, like certain B cells, contain basic amino acids. In the ventral nerve cord, the techniques which reveal SS and SH groups react strongly with the medio-ventral ns cells of the suboesophageal ganglion, and with the lateral abdominal ns cells; however, the thoracoabdominal ns A cells respond to these techniques in an inconsistent fashion, which is surprising, since they display the same staining reactions as the ns A cells of the pars intercerebralis. The Cr ns cells of the ventral nerve cord behave like the Cr cells in the brain; besides indol groups, they contain basic amino acids but no SS or SH groups. The glandular cells of the corpora cardiaca have not been extensively studied. They seem to contain pyrrol and indol groups but no SS or SH groups. In conclusion, it may be said that SS and SH groups are not present in all ns cell types, but only in certain A and B ns cells. Among these, lateral ns cells and the ns cells of the optic lobes do not react to SS and SH groups. These SS and SH groups are generally considered to belong to carrier proteins, and one would thus expect to find them regularly. The inconsistent results obtained may be partly due to the methods used, which are difficult to apply. Negative results, therefore, need not necessarily be considered significant, the more so as they may be due to the temporary absence of ns products in the cells under study.
Electron microscopy Do the electron microscope studies which demonstrate the presence 2.2.5
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of elementary granules in ns cells and neurohaemal organs allow better understanding of ns types? Elementary granules are of various sizes, ranging from 100 to 300 nm in diameter. Their form also varies: often they are round and sometimes roughly oval. Most elementary granules are electrondense but some are electron-transparent (Scharrer, 1963, 1968; Normann, 1965; Smith and Smith, 1968; Cassier and Fain-Maurel, 1970a; Cazal et al., 1971). In addition to electron-dense and electron-transparent granules or vesicles, pale granules have also been described. This aspect is considered as a phase in the synthetic activity cycle, or in the release of ns material (Schooneveld, 1974a). The ultrastructural features of ns cells depend on the different cell types. However, different insect species, even related ones, whose pars intercerebralis display histologically similar A1 and A2 ns cells, do not always exhibit the same characteristics in electron microscopy. Despite certain parallel features, it remains difficult to detect identical ns cell types on both electron microscopical and histological pictures. Sometimes histology reveals fewer cell types than electron microscopy. For example, amid the histological Cr type located in the abdominal ganglia of Locusta, electron microscopy revealed three to five subtypes (Chalaye, 1974a). Electron microscopy also detects those cells or axons containing vesicles less than 100 nm in diameter. which are considered as amines. 2.2.6 Multiplicity of neurosecretory cell types-nomenclature For obvious reason, the question of the nomenclature of ns cells is a complicated matter. For cells other than the A and B types, many authors used a nomenclature of their own. In addition, the staining techniques are difficult to apply and any slight variations in the origin of the staining agents, the p H of the water or the duration of various phases of the staining process may radically alter the picture (Raabe, 1980). For instance, the chrome haematoxylin-phloxine technique comprises three main steps-oxidation, CH staining and differentiation of the stains-and their duration determines the final result. Therefore, if the time allowed for each stage varies from 1 to 5 min, distinctly different results are obtained for the same tissue. If oxidation only lasts for 1min, type A cells will exhibit only a slight affinity for chrome haematoxylin and if the actual staining step is brief A2 ns cells will not be stained. If oxidation lasts longer (5 min), the affinity for phloxine of type B ns products will dwindle and disappear. Unduly deep phloxine colouration after medium or weak
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oxidation will, on the other hand, yield uniform staining for a large number of cells, some of which are not ns. Depending upon the way the staining technique is applied, the number of B ns cells which stain will vary, and either one or two different B cell categories will be revealed. In several instances, ns cells distributed in the same fashion appear to behave differently as regards their staining properties. Such is the case for the lateral abdominal ns cells of Locusta which are phloxinophilic, whereas the lateral abdominal ns cells of cockroaches, stick insects and other insects stain with chrome haematoxylin (Raabe, 1965; Chalaye, 1967; D e Besse, 1967). Such is also the case for the medioventral ns cells of the suboesophageal ganglion, which are strongly stained by chrome haematoxylin in stick insects and many other species but are phloxinophilic in cockroaches. It is, therefore, impossible to give an overall picture of the staining reactions of ns cell types. What is certain, however, is that a large number of cell types are revealed, even if the presence of immunoreactive cells and aminergic neurons is not taken into account. A comparative study of the pars intercerebralis of several cockroaches fixed in Bouin’s or Helly’s fluid and stained with paraldehyde thionine-phloxine, paraldehyde thionine-paraldehyde fuchsin and azan revealed the presence of six different cell types: A l , A2, A2‘, B, C , and D (Davydova, 1981). Type A l was stained by paraldehyde thionine while A2 and A2’ were stained by paraldehyde fuchsin; B cells were phloxinophilic, and the C and D cells, which stained with azan, were respectively bright red and lilac. The use of both histological methods and electron microscopy revealed the presence of four cell types in the pars intercerebralis of the dragonfly Aeschna cyanea (Tembhare, 1980); whereas five types were distinguished in the bug Roscius elongatus (Robert, 1979), and in the lepidopterans Manduca sexfa (Borg and Bell, 1977), and Pectinophora gossypiella (Raina and Bell, 1978). In the cricket Acheta domesticus, six types were recognized (Geldiay and Edwards, 1973). The cell types distinguished in the suboesophageal ganglion of stick insects included two medioventral A cells, four lateral A’ cells, and numerous Cr cells, some of which displayed histological and ultrastructural differences. This multiplicity of ns types is not surprising in view of the number of neurohormones identified. It also occurs in invertebrates
M. RAABE
216
other than insects, for example, the pond snail Lymnea stagnalis, in which alcian blue-alcian yellow stains seven different cell types. In the light of the above observations, it is proposed to designate the ns cells by their location and basic type as A , B, or Cr, additional categories within each basic type being designated by numbers, e.g., A l , A2, A3, B1, B2, C r l , Cr2, etc. 2.3
NEUROSECRETORY CELL LOCATION
In insects, the best known ns cells are located in the pars intercerebralis but such cells are also present in other parts of the brain, in the ventral nerve cord ganglia (Fig. 1) and even in the ganglia of the anterior sympathetic nervous system. Moreover, some other ns cells were discovered among the peripheral sensory cells. When the question of the number of ns cell types was discussed (Section 2.2), it was concluded that the pars intercerebralis contains five to six different cell types. In the ventral nerve cord, types A and C are the most common, but several A and C subtypes are also found. Large variations in the number of ns cells have been observed among different species (see Rowell, 1976). In insects like dictyopterans, phasmopterans and orthopterans, the type A ns cells in the pars intercerebralis are small and very numerous (1000-2000). In other orders like lepidopterans, dipterans, heteropterans, there are, on the contrary, only a few large ns cells (24) (Panov and Kind, 1963). According to Panov and Biryukova (1973), the number of these cells depends on the size of the insect. In seven cockroaches of different sizes, it varied from 500 to 2000. The protocerebrum is the part of the central nervous system containing the most ns cells. It includes the pars intercerebralis, located at the junction of the protocerebral lobes, and the protocerebral lateral ns cells located in the lateral parts in front of the pedunculate bodies.
2.3.1 Pars intercerebralis The pars intercerebralis has been investigated by many authors in many insect species and all these studies cannot be reported here (see Raabe, 1982). The major works dealing with the most common laboratory insects concerned the flies (Thomsen, 1965; Bloch et al., 1966; Panov, 1976, 1979), the bugs (Johansson, 1958; Baehr, 1968; Morris and Steel, 1975; Panov, 197%) and the beetle Leptinotarsa
THE N E U R O S E C R E T O R Y - N E U R O H A E M A L S Y S T E M O F
INSECTS
217
(Schooneveld, 1974a) and the locust Locusta (Girardie and Girardie, 1967). As already mentioned, the pars intercerebralis contains several ns cell types, and the number and size of individual ns cells vary considerably, depending on the size and order of the insect. The pars intercerebralis is not always homologous from one species to another or from one instar to another. In a number of insects, the ns cells of the pars intercerebralis, which form a single group in the adult, are divided into three distinct groups in the larva, and migration occurs during development. This is the case for lepidopterans, trichopterans and dipterans (Panov and Kind, 1963; Dogra and Tandan, 1965; Gieryng, 1976). The axonal pathway from the pars intercerebralis is not as simple as was initially believed. It has long been assumed that the two fibre bundles originating from the right and left lobes of the pars intercerebralis cross in front of the brain, just before emerging from the latter and then form the two voluminous nerves, the nccI, each of which branches into the contralateral corpus cardiacum. While most fibres originating in the pars intercerebralis follow this pathway, some of them together with their collaterals, follow a different one (see Section 3.6). 2.3.2
Other protocerebral neurosecretory cells
Some median ns cells located outside the pars intercerebralis were described a long time ago in dipterans and lepidopterans. Although these cells have not been identified in all insects, they were recently shown to be present in several species in which they were designated under various names (Dogra and Ewen, 1970, Melanoplus sanguinipes; Girardie, 1970, Locusta; Geldiay and Edwards, 1973, Acheta; de Besse, 1978, Leucophaea maderae; Anwar and Jsmail, 1979, Gryllus bimaculatus). Thus, three groups of such cells were identified in dragonfly larvae-one mediodorsal group consisting of four type A cells, and two paramedian groups, each including both A and B cells (Charlet et al., 1974). Little is known about the axonal pathways of the protocerebral median and paramedian ns cells. The only available data were supplied by Geldiay and Edwards (1973), who reported that in Acheta, these pathways terminated in a neurohaemal area of the brain. Neurosecretory cells are present in the lateral parts of the protocerebrum, near the c a k e s of the pedunculate bodies. Few in number, they often form two distinct groups on each side. Their
M. RAABE
218
staining properties have been described as corresponding to those of the Cr cell type (Raabe, 1965), but according to Girardie (1973), they are paraldehyde fuchsin-positive in Locusta and belong to several types. The axons of the lateral protocerebral ns cells run along a sinuous path and emerge in the ventral part of the brain forming the nccII, which in more highly evolved species fuse with the nccI. The collaterals of these cells follow a complicated pathway (see Section 3.6). 2.3.3
Deutocerebrurn, tritocerebrurn and optic lobes
Deutocerebral ns cells have been described in a few cases and correspond to a distinct pair of corpora cardiaca nerves, the nccIV (Browse-Gaury, 1967). The tritocerebrum, investigated in many species (Dupont-Raabe, 1957; Raabe, 1964), always contains a small number of type Cr ns cells. According to ultrastructural studies, five distinct types are present in the tritocerebrum of Locusta (Girardie, 1975). Some of the axons of the tritocerebral ns cells enter the nccIII (Raabe, 1964), whereas others join the nccI (Mason, 1973, Girardie, 1975). The presence of ns cells has been reported in the optic lobes of termites (Noirot, 1957), moths (Mitsuhashi, 1963), flies (Thomsen, 1965), cockroaches (Beattie, 1971; Khan, 1976b) and coleopterans (Barde, 1981). As regards their type, it is suggested that they often escape attention because they do not belong to the A type. In cockroaches, Beattie (1971) demonstrated that these cells were paraldehyde fuchsin-negative and azocarmine-positive. In coleopterans, however, they were shown to belong to the A and B types (Barde, 1981). The destination of the axonal tract of optic lobe ns cells is not well known. It seems to run towards the brain, where ns axons form synapses with microgranular axons (Beattie, 1971). 2.3.4 Ventral nerve cord Neurosecretory cells are present in the various ganglia of the ventral nerve cord (see Fig. 1). However, since they were discovered 40 years ago, systematic exploration of all the ganglia have not often been performed. The most thorough investigations were conducted on dragonflies (Charlet et al., 1974, Tembhare and Thakare, 19771, stick insects (Raabe, 1965), cockroaches (de Besse, 1967), locusts (Freon, 1964; Delphin, 1965; Chalaye, 1967), various orthopterans (Panov, 19781, the beetle Blaps rnucronata (Fletcher, 1979), the
THE N E U R O S E C R E T O R Y - N E U R O H A E M A L S Y S T E M O F I N S E C T S
br
SO
219
(i) '":/""? v .. ea
"8"
**.ee
R hodnius prolixus .O
CH or PAF + phloxine + CH + phloxine + azocarmine +
000 I
Locusta migratoria
n
Fig. 1 Neurosecretory cells in the central nervous system of the cockroach Periplaneta americana, the locust Locusta migratoria and the bug Rhodnius prolixus. a , abdominal ganglion; br, brain; CH, chrome haematoxylin; so, suboesophageal ganglion; PAF, paraldehyde fuchsin; th, thoracic ganglion (from data of de BessC, 1967; Chalaye, 1967; Baehr, 1968, Baudry, 1968)
moth Galleria mellonella (Delepine, 1965), the fly Phormia regina (Hsiao and Fraenkel, 1966) and several bugs Oncopeltus fusciatus (Johansson, 1958), Zphita lirnbata (Seshan, 1968), Rhodnius prolixus (Baudry, 1968), Eurygaster integriceps (Tisiachnuk, 1980). Although the problems of nomenclature and staining methods complicate a synthetic approach to the study of ventral nerve cord ns cells, some features emerge fairly clearly. The ns cells are fewer in number and more widely dispersed than those of the brain, but they belong to several types and form rich diversified systems. Four major categories of ns cells are to be found in the ventral nerve cord of almost all the insects studied.
220
M. RAABE
The first category consists of a single pair of A type ns cells, located medioventrally in the suboesophageal ganglion, and is present in almost all species. The axons of these cells follow a particular path. which will be described below (Section 3.6). Another type A ns cell category is exemplified by the thoracoabdominal ns cells first described in the very early studies of Geldiay (1 959, BIaberus craniifer) and Fuller ( 1960, Blatta orientalis, Periplaneta americana, Chaoborus crystallinus corethra) . In certain insects, these cells are confined to the thoracic region, but in others they may extend to the suboesophageal ganglion where they are more numerous, since they comprise a posterior pair in addition to the anterior one. In the abdominal ganglia, these thoracoabdominal ns cells are either present at all levels or only in the first four ganglia, as in cockroaches and stick insects. Their distribution is often regular, but may be less so in evolved fused central nervous systems, like those of dipterans and heteropterans. These cells are large pyriform neurons containing round A type granules. Several insects, e.g. dragonflies, cockroaches and stick insects possess two anterior pairs, the first being of the A1 type and the second, of the A2 type. The third kind of ventral ns cell is confined to the abdominal part of the central nervous system. It consists of rather small laterally located A or B cells which form small paired groups of two to five ns cells each. In heteropterans these cells are conspicuous and lie close to one another, due to the fusion of the abdominal ganglia. The paired groups include two types of ns A cells instead of one (Baudry, 1968). The fourth kind of ns cell is the azocarminophilic Cr cell described in Section 2.2. The ventral nerve cord contains a large number of these cells, which are present in all the insect species investigated so far. They form large ventromedian areas in the suboesophageal ganglion and abdominal ganglia, and small lateral groups in the thoracic ganglia (Raabe, 1965, stick insects; Chalaye, 1967, locusts; de Bess6, 1967, cockroaches; Baudry, 1968, heteropterans). The pathways and release sites of the ventral nerve cord ns cells have not yet been completely elucidated. As will be seen, a large number of ns products, mostly of the Cr type, are released by the metamerical perisympathetic organs. Some of the products of ganglionic ns cells are released in the neurohaemal areas of various nerves. These products may also be transported directly to the target organs and released in neuroeffector junctions.
THE NEUROSECRETORY-NEUROHAEMAL S Y S T E M O F I N S E C T S
2.3.5
221
Anterior sympathetic nervous system
Neurosecretory cells are not often found in the sympathetic nervous system. However, certain histological studies and most electron microscope investigations reported the presence of either peptidergic or aminergic ns cells in the sympathetic ganglia. Peptidergic ns cells have been shown to occur in the frontal and hypocerebral ganglia in cockroaches, (Chanussot, 1972). Conspicious ns cells have also been described in the frontal ganglion of moth larvae, namely Manduca sexta (Borg et al., 1973; Bell et al., 1974); Diatraea grandiosella (Yin and Chippendale, 1975); Achoea janata and Philosamia ricini (Awasthi, 1980). The final destination of the ns products of the sympathetic ganglia is not known precisely. Those of the frontal ganglion run either towards the brain or towards the recurrent nerve, from where they probably reach oesophageal targets. Neurosecretory granules have been reported in the oesophageal nerves of Melanoplus (Dogra and Ewen, 1970) and Calliphora (Thomsen, 1969).
2.3.6
Peripheral neurosecretory neurons
Numerous peripheral sensory neurons have been described in insects, and it is only recently that the neurosecretory nature of some of them was demonstrated. Surprisingly, they appear in some cases to be anatomically isolated from the central nervous system. The peripheral ns cells are multipolar neurons. Their secretory products invade the different branches whose terminal portion is often devoid of glial sheaths. These products stain with paraldehyde fuchsin .(Finlayson and Osborne, 1968), azocarmine and acridine orange (Hinke, 1975). Ultrastructurally , they appear as dense granules. These multipolar neurons are found in the vicinity of the muscles of the heart (Miller and Thomson, 1968), midgut (Wright et al., 1970), the rectum (Nagy, 1978), and alary muscles (Hinks, 1975). In addition, they are found along various nerves (Finlayson and Osborne, 1968), especially neurohaemal nerves like the lateral cardiac nerves of aphids (Bowers and Johnson, 1966) and cockroaches (Johnson, 1966; Miller and Thomson, 1968). According to Finlayson and Osborne (1968); Hinks (1975); Grillot (1977); Fifield and Finlayson (1978), these neurons enter the perisympathetic organs in stick insects, certain lepidopterans, and dipterans. This observation, together with the fairly frequent
~
M .RAABE
222
occurrence of ns cells in the perisympathetic organs, suggests that the peripheral ns cells were gradually incorporated into the neurohaemal organs during evolution. This hypothesis led to the idea that the glandular cells of the corpora cardiaca may be in fact transformed peripheral cells. Peripheral ns cells differ from non-ns peripheral cells in their electrical activity, which resembles that of ganglionic ns cells (Orchard and Finlayson, 1977). Nevertheless, it is possible that the peripheral cells fulfil a double sensory and secretory role. In vertebrates, such dual processes are known, since primary sensory neurons producing substance P are involved both in the sensory processing of pain and in the secretory control of skin vasodilatation. 2.4
IMMUNOREACTIVE NEUROSECRETORY CELLS
It is less than 10 years since investigators first attempted to identify insect ns cells, using vertebrate antibodies raised against vertebrate peptides. The positive results obtained in several cases will be listed here (Table 1). After a review of the data supplied by immunocytochemistry and radioimmunoassay with vertebrate antibodies we shall examine the results obtained with antibodies raised to insect peptides.
2.4.1 Neurophysins-vasopressin No neurons reacting to neurophysin I or I1 were found in the brain or suboesophageal ganglion of Thaumetopoea pityocampa (Remy et al., 1978) or in the brain of Locusta mzgratoria, but two medioventral ns cells of the suboesophageal ganglion proved to be neurophysin 11-positive, both in Locusta and in a stick insect, Clitumnus extradentatus (Remy et al., 1977, 1979). Clitumnus (Remy et al., 1977), Locusta (Remy et al., 1979) and Acheta domesticus (Strambi et al., 1979) were all tested with vasopressin antibodies. No reactive neurons were present in the brain of any of these insects, but the medioventral cells of the suboesophageal ganglion, which reacted with neurophysin, also reacted strongly with vasopressin. These cells have very distinct pathways that were revealed by immunocytochemistry (see Section 3.6). In Locusta, radioimmunoassay confirmed the presence of a vasopressin-like peptide in the central nervous system, rectum
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223
Malpighian tubules and haemolymph. The same peptide was also observed in the brain and suboesophageal ganglion of Acheta (Strambi et al., 1979). In the cockroach Ler4cophaea maderae, material immunoreactive to both vasopressin and oxytocin was shown in the neurohaemal part of the corpora cardiaca (Hansen et al., 1982). In the lepidopteran Thaumetc~poea,neither vasopressin nor oxytocin-like material was discovered (Remy et a l . , 1978). 2.4.2 Somatostatin The presence of several neurons reacting to somatostatin antibodies has been demonstrated in the pars intercerebralis of Locustu (Doerr Schott et al., 1978) and Periplaneta (Fujita et ul., 1981). In another cockroach Leucophaea the glandular part of the corpora cardiaca was also seen to contain immunoreactive material (Hansen et ul., 1982) as did the brain of Eristalis aenus (El-Salhy et a l . , 1980). However, the results for the brain and suboesophageal ganglion of the lepidopteran Thaumetopoea were negative (Remy et al., 1978). In Periplaneta midgut cells and nerve fibres located in the outer muscles of the digestive tract proved to be immunoreactive (Iwanaga et al., 1981) and somatostatin-like cells were found in the midgut and Malpighian tubules of the bee (Bounias and Dubois, 1982).
2.4.3 Opioids The presence of opioids (met-enkephalin, leu-enkephalin and endorphin) was investigated in the lepidopterans Thaumetopoea (Remy et al., 1978) and Bombyx (Remy et al., 1979). No reactive cells were found in the brain of either species, but their suboesophageal ganglion possessed two groups of 4 and 9 azocarmine-positive and paraldehyde fuchsin-negative neurons containing a-endorphin-immunoreactive material. In the brain of Locusta, four met-enkephalin-immunoreactive neurons were identified, two of them located among the lateral ns cells, and the other two at the bases of the optic lobes. Their fibres branch profusely in an area located along the tracts of nccI and nccII (Remy and Dubois, 1981). In Leucophaea, the neurohaemal part of the corpora cardiaca contains met-enkephalin and (3-endorphin-like material (Hansen et al., 1982). The opioids were also investigated by radioimmunoassay in locust tissue extracts, in which met-enkephalin was found to be more
224
M. RAABE
abundant than leu-enkephalin. The former is present in the brain, namely in the optic lobes, in the suboesophageal ganglion and in the corpora cardica. Small amounts are also found in the Malpighian tubules of this insect (Gros et al., 1978). 2.4.4 Bombesin, neurotensin, VIP) substance P Bombesin and neurotensin-like peptides were reported to occur in glandular parts of the corpora cardiaca (Hansen et a l . , 1982). The occurrence of VIP immunoreactive material was demonstrated in the cockroach Periplanetu both in the brain (Fujita et al., 1981) and in the nerve fibres located in the outer layer of midgut muscles (Iwanaga et al., 1981). A substance P-like immunoreactive material has been described in the brain of Eristalis (El-Salhy et al., 1980) and Locusta, where it is located in small neurons surrounding the pars intercerebralis, and in the fibres of the central and pedunculate bodies (Benedeczky et al., 1982). In the cockroach Leucophaea, a material exhibiting the same characteristics is present in the glandular part of the corpora cardiaca (Hansen et ul., 1982). 2.4.5 Insulin, Glucagon, Gastrin Neurons reacting to insulin have been found in the brain of Calliphora vornitoria (Duve and Thorpe, 1979) (six ns cells corresponding to the A 2 cells described by Panov, 1976), in the brain of Eristalis (El-Salhy et a [ . , 1980) and of Bornbyx (Yui et a l . , 1980) (eight large ns neurons whose fibres extend to the corpora cardiaca and corpora allata). Insulin-immunoreactive material is also present in Manduca sexta corpora cardiaca-corpora allata complexes (Tager et al., 1976). The gut of hymenopterans was shown by radioimmunoassay to contain an insulin-like material (Ishay et ul., 1976; Moreau et af., 1981). Glucagon-immunoreactive cells were not observed in Bombyx (Yui et al., 1980) but were discovered in the brain of Eristalis (El-Salhy et al., 1980), in the corpora cardiaca-corpora allata complexes of Manduca (Tager et al., 1976) and in the midgut of the cockroach Periplaneta (Iwanaga et al., 1981). Gastrin/cholecystokinin-like immunoreactive neurons were present in the brain of Calliphora erythrocephula (Duve and Thorpe, 1981), Eristalis (El-Salhy et al., 1980) and Periplaneta (Fujita et al., 1981). Gastrin-like immunoreactive neurons were found in Bornbyx
T H E N E U R 0 s E C R E T O RY- N E U R 0 H AE M A L S Y STE M 0 F I N S E C T S
225
both in the pars intercerebralis (10 paraldehyde-negative fuchsin neurons) and in the prothoracic ganglion (two neurons). In Manduca brain-corpora cardiaca-corpora allata complexes, a gastrin-like peptide was found (Kramer et al., 1977b). The presence of gastrinlike substances was investigated in the gut of several insects (Manduca, Dermestes maculatus and Musca domestica) but the results were negative (Kramer et al., 1977b). Products immunoreactive to insulin and glucagon, but not to gastrin, were found in the haemolymph of Manduca (Kramer et al., 1980) and insulin-like material was observed in A p i s (Kramer et al., 1982). Several attempts have been made to identify the structural similarities between vertebrate and insect hormones. Partially purified extract of bee royal jelly exhibited several insulin-like peptides whose major component had a molecular weight similar to that of bovine insulin (Kramer et al., 1977a). Further investigations in A p i s showed that this insulin-like substance is very abundant in the midgut (250 p,U/100 mg). Its molecular weight was estimated at 18000, i.e. three times heavier than mammalian insulin (Moreau et al., 1981). Further, Kramer et al. (1982), who purified insulin-like peptides from the haemolymph of Manduca, showed that these peptides resemble vertebrate insulin as regards solubility, chromatographic, immunological and biological properties. The amino acid composition of these vertebrate and insect hormones was comparable except for three amino acids. When glucagon was purified from Manduca midguts, the substance appears to be a peptide with a molecular weight of 15000 (Tager and Kramer, 1980). Gastrin was purified from 270 neuroendocrine complexes of Manduca. After filtration, immunoreactive gastrin was detected as a single peak. Its molecular weight appears to resemble that of vertebrate gastrin (Kramer et al., 1977b). 2.4.6 Pancreaticpolypeptide The presence of neurons immunoreactive to pancreatic polypeptide was demonstrated in the central nervous system of several insects, including the larval brain of the silkworm Bombyx (Yui et al., 1980) and the brain and ventral nerve mass of Eristalis larvae (El-Salhy et al., 1980). Twelve such neurons were found in the brain of Calliphora erythrocephala and 10-12 in its suboesophageal ganglion (Duve and Thorpe, 1980). The ventral nerve mass of Calliphora
TABLE 1 The distribution of iminunoreactive molecules in insects that resemble vertebrate Deptides Order
Insect
Phasmoptera Orthoptera Lepidoptera
Clirumtm exrradetztarus Locusfa migruroria Thuumetopoeu pityocanipu
Nervous bystem
+ +
Corpora nh.
Cardiaca gl .
Gut
Reference
Neurophysin R i m y e r a l . . 1977. 1979
0
R i m y er al.. 1978 Varopressin
Dictyoptera Phasmoptera Orthoptera Lepidoptera
Liwcophaea maderue Cliriirnniis extradenratus Locirsta migratoriu Thuurnetopoeu piryocarnpa
0
Dictyoptera
Perijhneiu arnericutzu
+
Lepidoptera Diptera Hymenoptera
Leucophaea maderae Thuumeropoeu piryocutnpu Erisialis aenus Apir mellificu
+ +
0
+
Somarosrutin
Hansen ei al.. 1982 Remyrtul.. 1977 Remy et ul.. 1979 Remy et al.. I978
+
Fujitacral., 1981: Iwanagaet u / . ,
+
Hansen urnl.. 1982 Remy er 01.. 1978 El-Salhy er ul. 1980 Bounias and Dubois. 1982
1981
+
+
Op ioids Dictyoptera Orthoptera
Leucopharu muderae Locus ru migruroria
Lepidoptera Diptera
Thuumetopoeu piryocanipu Bonibyr rnori Erisralis aetius
+ + + +
Dictyoptera
Prriplaneru americutia
+
+
Vip
Hansen ei ul.. IY82 Gros er a l . . 1978; R t m y and Dubois. I98 1 Rtmyerul.. 1978 RCmyeial., 1979 El-Salhy eral.. 1980
+
Fujitaerul.. 1981:Iwana~a~i ul.. 19x1
Subsrunce P Dictyoptera Orthoptera Diptera
Leucophaea maderae Lociista migratoria Eristalis aenus
Lepidoptera
Bomhyx mori Manduca sexta Calliphoru erythrocrphola Eristalis aenus hymenopterans
Diptera Hymenoptera
Hansen et al.. 1982 Benedeczkyetal.. I982 El-Salhy e t a / ., 1980
+ + Insulin
+ + +
+
+
+ Glucugon
Dictyoptera Lepidoptera Diptera
Periplanetn umrricanu Bomhyx mori Manduca sexta Erista/i.r aenus
+
+
+ t
Gastrinich olecyst ok in i n e
Dictyoptera Coleoptera Lcpidoptera Diptera
Periplane tu arnericuna Dermestes macularus Manduca sexta Bornhyx rnori Musca domesticu Calliphora eryrhrocephula Eristalis aenus
+
++ + +
+
Pancrearic polypeptide
Dictyoptera
Periplaneta americum
Lepidoptera Diptera
Bombyx mari Calliph o ra i'omiro ria Calliphoru erylhrocephnln Eristalis uenus
C . +.
+ no.i a: fcw.
++ ++ ++ ++ +
several immunorcactive cells: gl: glandular: n h : neurohaemal
t
0 0 0
+ ++
Yuietal.. 1980 Tager et ul., 1976 Duve and Thorpe. 1979 El-Salhy et al.. 1980 Ishay et al. , 1976: Moreau rt ul.. 1981 Iwanagaetal.. 1981 Yui e t u l . . 1980 Tager et a [ . , 1976 El-Salhy e t a / . . 1980 Fujita ei a/. . 1981 Kramer et a / . . 1977b Kramer e t a l . . 1977h Yui et a / . . 1980 Kramer et al.. 1977b Duve and Thorpe, 1981 El-Salhi et (11.. 1980 Endo rt a / . , 1982: Iwanaga et 01.. 1981 Y u l e r a l . . 1980 Duve and Thorpe. 1982 Duve and Thorpc. 1980 El-Salhi eral.. 1980
M . RAABE
228
vornitoria also contained these neurons (Duve and Thorpe, 1982); three thoracic and six abdominal pairs of immunoreactive neurons were seen to send their axons out into the dorsal ganglionic sheath which constitutes the neurohaemal organ of flies, as described in the tsetse fly (Grillot and Raabe, 1973; Baudry-Partiaoglou and Grillot, 1975). In Periplaneta (Endo et al., 1982), a study of the entire central nervous system demonstrated the presence of numerous immunoreactive neurons, located as follows: six paired groups in the brain, three pairs in the suboesophageal ganglion and each thoracic ganglion, one pair in the first abdominal ganglion and four to six pairs in the terminal ganglion; abdominal ganglia two to five are devoid of immunoreactive neurons. The pathways in Periplaneta run from the brain towards the corpora cardiaca, the ventral nerve cord and the sympathetic nervous system. Pancreatic polypeptide-reactive material is not restricted to the central nervous system, since Duve and Thorpe (1982) observed 20 to 30 immunoreactive neurons in the hypocerebral ganglion of Calliphora vorniforia. Furthermore, several small pancreatic polypeptide-immunoreactive cells are present in the midgut of the same insect (Duve and Thorpe, 1982), as well as in that of Periplaneta (Iwanaga et al., 1981). In Calliphora erythrocephala, purification of the pancreatic polypeptide-like substance and the study of its physicochemical properties, as revealed by Sephadex-gel filtration and polyacrylamide-gel electrophoresis, showed that they are almost identical to those of bovine pancreatic polypeptide. Determination of the amino acid composition of this substance showed a close resemblance between insect and vertebrate pancreatic polypeptides-(Duve et al., 1982). /'
2.4.7
lnsect neurohormones
It is difficult to obtain antibodies to specific insect neurohormones since only two neurohormones have been synthesized. Nevertheless, several attempts have been made to raise antibodies against ns products located in the pars intercerebralis and corpora cardiaca. The tissue antigens thus obtained from the brain and corpora cardiaca corresponded to certain paraldehyde fuchsin-positive ns products (Eckert et al., 1971; Eckert, 1976). Further purification resulted in the separation of distinct factors, one of which appears to
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be the activation factor I1 which stimulates prothoracic gland (Gersch et al., 1977). O n the other hand, antibodies raised to brain ns products appear to inhibit allatotropic activity in Locusta (Rembold et al., 1980). In the frontal ganglion of the cockroach Periplaneta, immunoreactive products, probably originating from the brain, were observed in one type of fibre but there were no products in the cell bodies (Ude et al., 1978). After the identification and synthesis of proctolin, a visceral musculature neurotransmitter or neuromodulator (Brown and Starratt, 1975; Starratt and Brown, 1977), a specific antiserum was raised and its immunocytochemical localization investigated. In the cockroach Periplaneta, proctolin-like immunoreactivity was detected in the terminal abdominal ganglion-proctodeal nerve-hindgut system by Eckert et al. (1981). A strong proctolin-like reaction was also found in the proctodeal nerve and nerve terminals of the hindgut musculature. Four types of neurons and a number of neurons whose pathways were determined using cobalt chloride filling appear to contain proctolin-like material. It appears that type 1 proctolin-like neurons form small ventral and dorsal groups, whereas type 3 neurons are a pair of large lateral neurons. Both types which ramify in the ganglion neurophil might play a modulatory role. Other proctolin-like neurons project into the proctodeal nerve, hindgut and rectal muscles: the type 2 neurons are grouped together in the posterior part of the ganglion, while type 4 comprise three pairs of lateral neurons. O’Shea and Adams (1981) studied one pair of lateral neurons termed LW, located in the abdominal ganglia of the cockroach Periplaneta and the cricket Gryllus bimaculatus. These neurons stain with neutral red, suggesting an aminergic content. Bioassays for proctolin were performed using a locust leg extensor muscle preparation that generates a heat-like myogenic rhythm of contractions. These assays demonstrated that proctolin was associated specifically with the LW neurons, which contained 0.05-0.1 pmole proctolin each. Using HPLC, LW neuron extracts were also shown to contain a bioactive component whose molecular weight was in the same range as that of proctolin (648.6 daltons). Comparison of these two sets of results for LW neurons suggests that they may be homologous to type 4 neurons, which have been shown to innervate the hindgut, and give rise to immunofluorescent fibres. Their cell bodies are probably the same as those of the A type ns cells described by de Besse (1967).
,
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2.4.8. Conclusion A number of vertebrate peptides are found in insects, as in other
invertebrates. The major problem now seems to be the understanding of the physiological significance of the relevant data. The occurrence in haernolymph of molecules such as insulin, glucagon and vasopressin certainly suggests a possible physiological role. Moreover, there are several fairly reliable indications as to the possible role of such vertebrate peptide-like molecules in insects. Vasopressin, which is found in the haemolymph, appears to control water metabolism. Its level was seen to vary both in the haemolymph of Locusta (Proux and Rougon-Rapuzzi, 1980) and suboesophageal ganglion of Achefa (Strambi et al, 1978), depending on the state of hydration. Somatostatin, whose occurrence has been reported in cockroaches, lepidopterans and hymenopterans, inhibits the hypoglycaemic function of insulin, thus acting as in vertebrates (Bounias and Dubois, 1982). With regard to insulin and glucagon, it seems that these molecules may be true insect neurohormones. Injection experiments have since shown that insulin reduced fat body lipid release in Hyalophora cecropia (Bhakthan and Gilbert, 1968) and decreased haemolymph trehalose in Vespa (Ishay et al., 1976), Manduca (Tager et al., 1976) and Apis (Bounias and Pacheco, 1979). Glucagon decreased fat body glycogen in Manduca (Tager et al., 1976). In Locusta, it raised the trehalose content of the haemolymph as corpora cardiaca extracts do, whereas insulin or the intestinal insulin-like molecule lowered the blood trehalose level (Lequellec et a/., 1982). Therefore, some vertebrate peptides appear identical or similar to insect neurohormones. They are released in the blood but, in some cases, there are indications that peptide-like molecules occur in the neuropil where they may play a role in neurotransmission or neuromodulation . Finally, in insects as in other organisms the same peptide may be detected both in the gut and the nervous system.
2.5
CATECHOLAMINES A N D INDOLAMINES
Since biogenic amines and neurohormones have several points in common, the data concerning amines will be briefly reviewed here. For more detailed information, the reader is referred to the reviews by Klemm (1976) and Evans (1980).
THE
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The methods used to investigate biogenic amines have progressed rapidly over the last two decades. The first modern technique to be extensively used was the Flack and Hillarp fluorescence method, which allows distinction of catecholamines and indolamines. To some extent electron microscopy permits the detection of terminals containing amines from the appearance of the vesicles (often dense-cored), but biogenic amines appear to be stored in vesicles of different size ranges. According to Richardson (1966) the vesicles containing amines can be identified following permanganate fixation that reveals their dense core aspect. Reserpine may be used to verify thehisappearance of suspected amines, and a good way of identifying amines is by the neutral red technique. Autoradiography and radioenzymology, which are far more sensitive, are also used for this purpose. 2.5.1 Central nervous system-corpora cardiaca The presence of biogenic amines in the nervous system of insects was shown by several authors, mostly working with cockroaches and locusts (Table 2). Serotonin was first considered to be the most common of these amines (Gersch et al., 1961; Colhoun, 1963). However, using radioenzymatic assay, Robertson (1976), Evans (1978), and Dymond and Evans (1979) demonstrated the presence of octopamine in the various parts of the central nervous system of Periplaneta and Locusta. This amine is very abundant in the brain, fairly abundant in the optic lobes, suboesophageal ganglion and thoracic ganglia but only a little is present in the abdominal ganglia. The amounts of the other biogenic amines are not very large. Noradrenaline is present only in small amounts whereas serotonin and dopamine are present in larger amounts, especially in the brain. In Manduca sexta, octopamine and dopamine are found extensively throughout the entire central nervous system, but serotonin exhibits a distinct regional distribution: there is almost none in the brain, suboesophageal ganglion or thoracic ganglia, but fairly large amounts are present in the optic lobes and abdominal ganglia (Maxwell et al., 1978). Biogenic amines have mostly been identified in the neuropil of the brain especially in the central body and pedunculate bodies (Mancini and Frontali, 1970; Klemm, 1968~1, 1974; Plotnikova, 1969), and in the ventral nerve cord. In addition to aminergic fibres, aminergic neurons have also been detected. In the pars intercerebralis, microgranular neurons have been discovered in flies (Bloch et al., 1966; Ramade and l’Hermite, 1971)
TABLE 2 Identification of biogenic arnines in organs and tissues Insects
Organ
Trichopterans brain
Biogenic amines Noradrenaline Dopamine Serotonin
+
Locusts brain optic lobes brain pi neurons D U M neuron terminals cc neurohaemal lobe glandular lobe central nervous system sympathetic nervous system haemol ymph heart Cockroaches central nervous system brain ventral nerve cord (+).
+. ++. +++, very slight. slight.
(+)
+
Octopamine
+
Klemm. 1968a
+ + +
++
300 neurons
1 neuron
+
+
0 ++and(+)
+ +
+ +++ ++ ++ +++
(+I
(+I
References
+ ++
+++ ++
+++ +
+++ + ++ +++ +++
Robertson, 1976 Hiripi and S.-R6sza. 1973 Klcmm and Fnlck. 1978 Evans and O’Shea, 1978: Hoyle and Barker. 1975 Lafon-Cazal and Arluison, 1976: David and LnfonCazal. 1979 Evans. 1978 Klemm. 197!: Chanussot and Pentreath. 1973 Goosey and Candy, 1980: Orchard el nl.. 1981 Hiripi and S.-R6sza. 1973 Dymond and Evans. 1979 Sloley and Owen. 1982
medium. abundant: cc. corpora cardiaca; D U M . dorsal unpaired medial; pi. pars intcrcerehralis
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as well as in locusts (Girardie and Girardie, 1966), crickets (Geldiay and Edwards, 1973) and in the coleopteran Leptinotarsa decemlineatu (Schooneveld, 1974a). Falck and Hillarp’s technique detected one serotonin cell body and several hundred dopaminergic neurons intermingled amongst the ns cells in the pars intercerebralis of locusts (Klemm and Falck, 1978). The occurrence of microgranular fibres in the corpora cardiaca has been reported by several authors (Normann, 1965; Johnson, 1966; Scharrer, 1968; Cassier and Fain-Maurel, 1970a,b; Cazal et al., 1971; Klemm, 1971; Gersch etal., 1974). An exhaustive study of both the glandular and neurohaemal lobes of the corpora cardiaca of locusts enabled a comparison to be made between the fibre types distinguished by electron microscopy and the amine locations revealed by autoradiography. The neurohaemal lobe axons, which contain dense granules 100 nm in diameter, appear to incorporate tritiated serotonin (Lafon-Cazal and Arluisson, 1976). The neurohaemal lobe also contains some octopamine, but the glandular lobe contains much more (David and Lafon-Cazal, 1979). In addition, cobalt chloride retrograde filling showed that the corpora cardiaca glandular lobe was innervated by lateral protocerebral neurons (Rademaker, 1977). They contain elementary granules about 100nm in diameter and form synaptic endings in contact with the glandular cells. It has been suggested that octopamine controls adipokinetic hormone release from the corpora cardiaca glandular cells. Examination of the effects of various aminergic agonists and antagonists on the release induced by electrical stimulation of nccII and by high potassium confirmed this hypothesis. Release was blocked by reserpine and the a-blocker phenoxybenzamine, but not by the P-blocker propranolol (Orchard et al., 1981b).
2.5.2
D U M neurons
The involvement of octopamine in the functioning of DUM (dorsal unpaired medial) neurons has been the subject of several investigations. D U M were demonstrated to occur in the metathoracic ganglion of Schistocerca, in Locusta, Periplaneta (Crossman et al., 1971) and other insects too. They were essentially studied by intracellular recording of electrical activity, and filling of the axon and consequently of its collaterals by procion yellow and cobalt chloride. The D U M form a distinct group of neurons on the mediodorsal
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side of the ganglia and have unusual features. Morphologically, their median neurites bifurcate, sending out paired axons on each side of the body. They exhibit an action potential, which invades the cell bodies, and is of longer duration than is usual in insect neurons (Crossman et al., 1971). There are several DUM neurons in the metathoracic ganglion, but only the DUMeti innervating the tibial extensor muscles of the locust jumping leg has been studied in detail as an identified neuron. Application of octopamine, neutral red staining and radioenzymatic assay showed the DUMeti to be octopaminergic (Hoyle and Barker, 1975; Evans and O’Shea, 1978). The amount of octopamine produced in each neuron was evaluated at 0.1 pmol. The DUMeti displays a typical modulatory action. It causes longterm inhibition of the intrinsic rhythm of tibial extensor contraction. Such inhibition was shown to facilitate the amount of transmitter released by another neuron, the SETi, through action on its presynaptic receptor (Evans and O’Shea, 1978; O’Shea and Evans, 1979). The terminals of DUMeti octopaminergic fibres contain dense-core granules, 60-190nm in diameter, which appear to be released in the vicinity of the muscle fibres but not in close contact with them (Hoyle et al., 1974). In this particular case octopamine acts as a local neurohormone. 2.5.3
Sympathetic tzewous system
The presence of amines has also been shown in the sympathetic nervous system in Schistocerca gregaria and Btabera craniifer (Chanussot et d . , 1969; Gersch et al., 1974). These amines are assumed to be noradrenaline or dopamine (Chanussot and Pentreath. 1973) or, again, serotonin, as proposed by Klemm (1971) and Pandley and Habibulla (1982). As far as the posterior sympathetic nervous system is concerned, intense fluorescence, probably dopaminergic, was reported in the locust metathoracic ganglion at the origin of the median nerves of the unpaired sympathetic nervous system (Plotnikova, 1968). 2.5.4 Haemolymph The haemolymph has rarely been examined for amines but recent studies showed octopamine to be present in the haemolymph of locusts (David and Lafon-Cazal, 1979; Goosey and Candy, 1980; Orchard et al., 1981). Octopamine titre was observed to increase
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during flight (Goosey and Candy, 1980) and in experimentally excited locusts with a rapidly rising lipid level (Orchard et al., 1981). Isolated pieces of fat body were seen to release lipids in response to low concentrations of octopamine, in a dose-dependent response with a threshold of about 2 x 1 0 V 7 ~and a maximum around 5 X 1 0 - 6 ~(Orchard et al., 1982). In such cases, octopamine behaves like a true neurohormone and exerts the same effect as adipokinetic hormone.
2.5.5
Salivary glands-firefly
lantern
Regulation of salivary gland activity was first investigated in the fly Calliphora. In this insect, the salivary gland is not innervated, and serotonin was demonstrated to be the regulator involved, thus acting as a true hormone (Berridge and Patel, 1968). In other species, whose salivary glands are innervated, serotonin has sometimes been suggested to play a part in the control of secretion (Whitehead, 1971). However, in later investigations, dopamine was shown to be the candidate in the cockroach (Bowser-Riley and House, 1976), locust (Klemm, 1972) and Manduca (Robertson, 1974). Like the salivary gland, the firefly or Photinus pyralis lantern lends itself to experimentation. Smalley (1965) demonstrated that normal neural excitation of the Photinus pyralis lantern was mediated by an aminergic transmitter identified as octopamine (Robertson and Carlson, 1976). I t is interesting that the firefly lantern was later shown to be innervated by DUM neurons, now known to be octopaminergic. Electron-dense granules 90-155 nm in diameter were observed in the nerve terminals. 2.5.6 Fat body
It is well known that in the locust, the initial fuel for flight is trehalose, whereas continuous flight depends on diglyceride, released from the fat body by the action of adipokinetic hormone (AKH) (see the review by Goldsworthy in this volume). As shown above, octopamine may control the release of material from corpora cardiaca glandular cells. In addition to this function, octopamine appears to act directly on the fat body. Thus, in Schisrocerca, the lipid blood level rose sharply during the first 10min of flight (Goosey and Candy, 1980), a period during which no AKH had
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been released, but the amount of lipids in the haemolymph continued to increase. Octopamine, therefore, apparently acts directly on the fat body to induce lipid production. It also appears to be involved in trehalose production as well as in fat body and nerve cord glycogenolysis in cockroaches (Robertson and Steele, 1973; Downer, 1979). In locusts, octopamine raises the rate of glucose oxidation in the flight muscles (Candy, 1978).
Visceral organs Numerous investigations have demonstrated that biogenic amines have a stimulatory effect on visceral organs, including the heart, the gut, Malpighian tubules and oviducts. Serotonin often appears to be more active in this respect than noradrenaline, dopamine or octopamine. However, it is still not known whether these effects are physiological or pharmacological. Several neurotransmitters were assayed on the isolated or semiisolated heart in cockroaches (Collins and Miller, 1977) and locusts (S.-Rozsa and Miller, 1981). In both insects, all the neurotransmitters assayed caused acceleration of the heartbeat. The most potent were serotonin and octopamine. In Lucilia larvae, the body-wall muscles, innervated by both fast and slow axons, also respond to biogenic amines. Octopamine, like the neurohormone proctolin, causes rhythmic depolarization which appears to be post-synaptic and mediated by receptors specific for each agonist (Irving and Miller, 1980). In Rhodnius, serotonin has the same effect on the Malpighian tubules as diuretic hormone. However, the amount present in the corpora cardiaca is too small to indicate that it actually regulates Malpighian tubule activity. 2.5.7
2.5.8 Physiological significance The effects of biogenic amines have been demonstrated in so many cases that it is now highly probable that catecholamines and indolamines have a role to play in controlling physiological processes in insects. Serotonin has been suggested to be involved in controlling circadian rhythms. In experiments on the lepidopterans Noctua pronuba and Agrotis ipsilon, serotonin injections enhanced the duration and amplitude of night flight, whereas other amines had no effect (Hinks, 1967). In Drosophila melanogaster (Fowler et al.,
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1972) the amount of serotonin varied over a 24-h period. In the cricket, Acheta domesticus, reserpine inhibited locomotor activity (Cymborowski, 1973) and serotonin levels in the central body displayed diurnal fluctuations (Muszynska-Pytel and Cymborowski, 1978). This amine is also involved in the variations in the circadian rhythm of spontaneous electrical activity of the frontal ganglion of Periplaneta (Pandley and Habibulla, 1982). Estimates of octopamine levels in hyper- and hypoactive ants showed that the former have a higher octopamine content (David and Verron, 1982). Variations in biogenic amine levels have been demonstrated in locusts both during growth and in relation to phase (FuzeauBraesch, 1977; Fuzeau-Braesch and David, 1978). The results show that gregarious insects contain more dopamine than solitary insects, but that the latter have twice as much octopamine as the gregarious insects. In Locusta, the octopamine content rises during middle larval life and adult life, but drops sharply at moulting (FuzeauBraesch et al., 1979). Amine involvement in growth and colour change has been shown in Oedipoda coerulescens (Moreteau and Janots, 1978) and Galleria mellonella (Glowacka and Dutkowski, 1979) by reserpine treatment that retards larval development and inhibits dark colour change in Oedipoda. In Mamestra configurata, a large rise in the octopamine level was shown to occur in the brain and optic lobes during metamorphosis. The functioning of the octopaminergic system may be correlated to changes in the sensory system which play a decisive role in triggering metamorphosis (Bodnaryk, 1980). Reserpine has been reported to inhibit vitellogenesis and oviposition in several insects, which suggests that amines are involved in these processes (Huot et al., 1960, ~ r i ~ o l i uconfusum; m Masner et al., 1970, Tenebrio molitor; Hentschel, 1972, Periplaneta americana). The effects of reserpine and amines on various physiological processes appear to arise indirectly, due to an inhibition of release of ns products by amines because the ns cells, after reserpine treatment, are overloaded with these products, in insects such as Tenebrio (Masner et al., 1970), Periplaneta (Hentschel, 1972) and Oedipoda (Moreteau and Janots, 1978). Apart from the histological data concerning the ns cells, the effects of reserpine have been shown by other methods, which demonstrated a decrease in both the fluorescence and octopamine level in cockroach and locust brain after reserpine treatment (Robertson, 1976; Sloley and Owen, 1982). In Galleria, reserpine
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treatment depleted the dense-core granules of adrenergic neurons of the pars intercerebralis, which confirms the role of amines in regulating the release of ns products (Warton and Dutkowski, 1978).
2.5.9 Conclusion In some cases, biogenic amines behave like true neurohormones and are transported by the bloodstream to the target organs. They also act like neurotransmitters or neuromodulators in the salivary gland, firefly lantern, corpora cardiaca glandular lobe, tibia1 extensor of the jumping leg and central nervous system. By these various modes of action, biogenic amines resemble neurohormones which not only act via the circulatory pathways but are also transported to their target organs, through a neural pathway.
3 Neurohormone release
The sites of neurohormonal release are now known to be many and varied. Instead of' a single place of release in the corpora cardiaca, there appear to be several distinct release systems. They may be centrally located in the corpora cardiaca and the perisympathetic organs or distributed more diffusely in neurohaemal areas and inside target organs (Fig. 2).
3.1
TRANSPORT A N D R E L E A S E OF N E U R O S E C R E T O R Y MATERIAL
The ns products contained in elementary granules are transported from the cell bodies along the ns axons to the terminals. This was demonstrated in experiments in which nerve tracts containing ns axons were ligated or sectioned. These procedures caused the accumulation of a large amount of ns material on the proximal side of the damaged part of the nerve. Neurotubules are probably involved in the transport of ns elementary granules. The latter are sometimes arranged in a row along the neurotubules and it has been demonstrated in vertebrates that colchicine, which alters microtubule function, inhibits the transport of ns material. The speed of elementary granule transport was calculated in
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1
1
neurohaemal organ
t--neurohaemal area
I
neuroeffector junction
central nervous system
Fig. 2 Schematic view of the different modes of release of ns products
Schistocerca adult females using ""Scysteine. During vitellogenesis, characterized by rapid release of ns material the latter was transported at a speed of 3.2 mm/h. In mature females, however, the rate was slower (1mm/h) (Highnam, 1976). Neurohormone release, extensively reviewed by Normann (1976) and Maddrell and Nordmann (1979) is elicited by the arrival of a nerve impulse which depolarizes the axon terminal. Depolarization of these terminals by high concentrations of external potassium or by electrical stimulation also leads to the release of ns material. Such release is detectable by histology, electron microscopy or the appearance in the incubation medium of physiologically active factors like the diuretic factors of Rhodnius and Glossina (Maddrell and Gee, 1974). Concomitantly with the depletion of ns material from the terminals (as indicated by the drop in elementary granules and the increased number of omega profiles and other figures relating to discharge of ns material-Scharrer and Kater, 1969), several neurohormones are released. They include the cerebral gonadotropic factor of the locust Schistocerca gregaria (Highnam, 1961b) and
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Locusta rnigratoria (Girardie and Girardie, 1977), the cardioaccelerators of the cockroach Periplaneta arnericana (Kater, 1968), the trehalosemic hormone of the fly Calliphora erythrocephala (Normann and Duve, 1969) or both (Gersch et al., 1970). The release of ns material depends partly on the action of acetylcholine and biogenic amines. Among the latter, octopamine was identified in neurohaemal organs of cockroaches and locusts where its concentration was 200 to 700 times higher than in a thoracic ganglion (Evans, 1978). As regards acetylcholine, it has been shown to enhance the action potentials of the perisympathetic organs of Carausius and Rhodnius (Finlayson et al., 1976). The ns products transported by axons to the neurohaemal organs are considered to be released at definite points. These are characterized, in addition to populations of elementary granules, by clusters of electron-lucent vesicles (30-40 nm in diameter and often resting against densely stained portions of the axon membrane). Such figures , which resemble synaptic sites, have been called synaptoid sites, and are considered to be regions of ns granule release. They are usually found isolated in connective tissue, in the bare peripheral axon endings-thus implying direct passage of the neurohormones into the haemolymph. However, they are sometimes in contact with glial cells or axons, particularly in the corpora cardiaca of cockroaches (Scharrer, 1968) and locusts (Cassier and Fain-Maurel, 1970a; Cazal et al., 1971), and in the median-type perisympathetic organs of coleopterans (Provansal-Baudez and Baudry-Partiaoglou, 1983) and heteropterans (Baudry-Partiaoglou, 1983a). Synaptoid sites are not permanent. Their number increases in axon endings during enforced release of ns material by stress, poisoning, or acetylcholine or potassium treatment. In such cases the axon membrane becomes irregular and exocytotic omega profiles appear, sometimes containing electron-dense material but more often devoid of dense content. Coated vesicles indicative of endocytosis are also found. The significance of synaptoid vesicles has been debated and is still controversial. They are widely believed to be products of membrane recovery, by endocytotic retrieval; they are destroyed in the terminals and seem to give rise to material conveyed back to the ns cell bodies following an antidromic flux. Other interpretations suggest that the synaptoid vesicles result from vesiculation of the ns granules or contain a neurotransmitter which could influence the release of ns material from electron-dense granules. The latter hypothesis is
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strengthened by the recent discovery that two distinct neurotransmitters or peptides may be present in the same axon. The mechanisms of release of ns products at the axon endings are still under debate. Some investigators believe that the entire granule passes through the axon membrane of the terminals before releasing its content, but there is little evidence to confirm this hypothesis. It has also been suggested that the granule content is released within the cytoplasm of the axon ending and then traverses its membrane. Some patterns of granule dedensification, fragmentation or budding (Scharrer and Wurzelmann, 1977) support this conception. In the bug Roscius elongatus such processes have been observed in axons containing irregularly shaped ovoid granules with a heterogeneous or slightly dense content (Baudry-Partiaoglou, 19834. Most authors support the view that the release process is one of exocytosis, by fusion of the elementary granule membrane with that of the axon ending, and dissolution of the entire granule content in the extracellular medium. The successive phases of this process (i.e. binding of the elementary granules to the axon terminal membrane, modification of the axon profile by the sporadic appearance of omega profiles, and an increase in the number of small vesicles) have been actually observed during active neurohormone release (Normann and Duve, 1969; Scharrer and Kater, 1969). Exocytosis involves a significant increase in axon terminal membrane surface, and requires regulatory mechanisms. I t is generally considered that a constant axon terminal surface is maintained, after the release of ns material, a micropinocytotic process giving rise to coated vesicles and numerous synaptoid vesicles characteristic of the release points. A variant of exocytosis is the compound exocytosis observed in the perisympathetic organs of the bug Roscius in which the ns axons lie very close to one another, leaving very little room for the release of ns material (Baudry-Partiaoglou, 1983a). As already mentioned, electrical stimulation or artificial depolarization by K+ rich solutions result in the release of ns material. This, however, cannot be achieved under any conditions and requires the presence of Ca2+ ions in the medium, as shown in the cockroach (Gersch et al., 1970), in Rhodnius (Maddrell and Gee, 1974) and in Calliphora (Normann, 1974). What is the mechanism of Ca2+ action after it has penetrated the axon membrane? This question cannot yet be precisely answered, but various hypotheses can be formulated. It is well known that the granule membrane, like the axon membrane, carries negative
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charges which prevent their fusion. Calcium ions, which have strong positive charges, may diminish electrostatic repulsion by aggregating against the axon membrane. In addition, the penetration of calcium ions within the axon terminals may alter cytoplasmic viscosity, thus increasing the mobility of the granules and their chances of encountering the axon membrane.
3.2
CORPORA CARDIACA
The most important release sites for brain ns products are the corpora cardiaca which have been extensively studied in insects since the pioneering work of Cazal (1948). Although these organs are mostly located in the head, behind the brain, they are sometimes found in the neck or anterior part of the prothoracic segment. The corpora cardiaca are paired but may fuse into a single unpaired organ. They often merge with the lateroventral walls of the aorta, but their links with the aorta are very varied. Sometimes they are only partly fused with it and sometimes they are not connected to it at all, as in certain lepidopterans, in which case their position becomes lateral instead of medial. The corpora cardiaca are connected to the sympathetic hypocerebral ganglion which innervates the gut. In certain insects, this ganglion is fused with the corpora cardiaca, whereas in other insects, it is located close to these organs, with which it exchanges nerve fibres; ns axons from the corpora cardiaca pass into the hypocerebral ganglion, whereas sympathetic ns and sensory fibres transit from the hypocerebral ganglion to the corpora cardiaca. In some cases, there are no links between the corpora cardiaca and the hypocerebral ganglion (which is not present in some insects) and the fibres pass from the corpora cardiaca to the recurrent nerve, which contains axons from the frontal ganglion and also from the brain. Like the corpora cardiaca, the corpora allata fuse in a number of species. In the larvae of higher dipterans, the corpora cardiaca, corpora allata and moulting glands are incorporated into a single organ, the ring gland or Weismann’s ring. Except for ephemeropterans, whose corpora allata are innervated by the suboesophageal ganglion only, constant connections exist between the corpora allata and corpora cardiaca, which provide the corpora allata with their cerebral innervation. The corpora cardiaca are innervated by three pairs of nerves (ncc) originating respectively in the pars intercerebralis, the lateral protocerebral ns cells and the tritocerebral ns cells. These nerves are sometimes partly or com-
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pletely fused, especially in highly evolved insects. The ncc contains ns axons as well as other axons, both aminergic and cholinergic, as demonstrated by Gersch (1974). There are several types of ns axon, as seen in cockroaches (Scharrer, 1963, 1968), the blowfly (Normann, 1965), stick insects (Smith and Smith, 1966) and locusts (Girardie and Girardie, 1967; Cassier and Fain-Maurel, 1970a,b). The nerve fibres entering the corpora cardiaca constitute a large bundle in the centre of the organ, which sends out a large number of branches. These branches divide several times and finally reach the outer connective membrane of the corpora cardiaca or its invaginations, where the ns products are released at synaptoid sites, as seen above. The glial cells are well developed. They were termed “chromophobic cells”, by the first investigators, to distinguish them from other chromophilic cells, namely the glandular cells of the corpora cardiaca. These glandular cells (also termed intrinsic or parenchymatous cells) are transformed unipolar neurons with short processes which come into contact with the connective sheath or its invaginations, where they release their secretory products. Histological staining of the glandular cells of the corpora cardiaca is not as pronounced as that of the ns cells or the pars intercerebralis. Glandular cell content is often paraldehyde fuchsin-positive but is not always stained by chrome haematoxylin-phloxine. Electron microscopy, on the other hand, clearly reveals the glandular cells of the corpora cardiaca which contain well-developed synthetic organelles, and electron-dense granules which may be markedly larger than the ns granules originating in the brain. The presence of two distinct types of glandular cells had been reported in Leptinotarsa decernlineata (Schooneveld, 1970), Oncopeltus fasciatus (Unnithan et al., 1971), Schistocerca gregaria (Krogh, 1973) and Aeschna cyaena (Tembhare, 1980). The glandular cells of the corpora cardiaca are often intermingled with the ns axons coming from the brain. In some insects, however, they are either more abundant in the posterior part of the corpora cardiaca or completely separated from it, forming a glandular lobe apposed to the neurohaemal storage lobe (Highnam, 1961a). In other insects, the corpora cardiaca contain glandular cells only, the ns endings being located in the aorta walls. The corpora cardiaca also contain a few neurons whose function is not quite clear. Perhaps they trigger or modulate the release of ns products, thus acting like certain nerve fibres coming from the brain.
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Although ns products release is elicited by the arrival at nerve terminals of bursts of nerve impulses transported by the ns axon itself, it is probably also influenced by other axons making axoaxonal contact. Other axo-axonal or axo-somatic synapses are present between ns fibres and the processes or somata of glandular cells, as observed among others, by Scharrer (1963, 1968) and Normann (1965). Not only peptidergic, but also aminergic axons have been reported to occur in the corpora cardiaca (Cazal et al., 1971) in which dopamine, serotonin and octopamine have all been observed-the latter mostly in contact with glandular cells (Klemm, 1971; LafonCazal and Arluison, 1976; Evans, 1978; David and Lafon-Cazal, 1979). The origin of the octopaminergic fibres which innervate the glandular lobe of the corpora cardiaca of Locusta was traced through the nccII to 15 small protocerebral lateral neurons (Rademakers, 1977). Finally, acetylcholine was also shown to be present in the corpora cardiaca and to play a part in the release of certain neurohormones from this organ. In Periplaneta the release of neurohormone D (Gersch, 1972) was elicited by electrical stimulation of the nccI, which involves cholinergic components, whereas (as seen above) electrical stimulation of the nccII induces release of the hypertrehalosemic factor by adrenergic components (Gersch, 1972). It is also worth noting that in the ventral nerve cord, cholinergic compounds stimulate the release of heart accelerators (Gersch, 1974).
3.3
PERISYMPATHETIC ORGANS
3.3.1 General features The perisympathetic organs perform the same function for the ns products of the ventral nerve cord as the corpora cardiaca for brain ns products (Raabe, 1965). Basically, they display two characteristic features: firstly, a metameric distribution in all the segments of the body (since they are connected with each segmental ganglion) and, secondly, the perisympathetic organs are linked to the sympathetic nervous system. These two features are also encountered in the corpora cardiaca, that is: one neurohaemal organ for each nervous ganglion responsible for releasing its ns material, and a relationship with the sympathetic nervous system which probably helps to
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regulate neurohormonal release. The perisympathetic organs owe their name to their location on the nerves of the unpaired sympathetic system. In highly evolved species, the above features are often modified. The ganglia of the central nervous system show a tendancy to fuse, which leads to joining of the perisympathetic organs. The sympathetic nervous system is gradually incorporated into the somatic nerves, and it is no longer possible to assume that the relationship between these organs and the sympathetic fibres is maintained. The perisympathetic organs-like other neurohaemal organscontain ns axons which repeatedly branch and form numerous terminals. Numerous glial cells ensheath these axons up to their endings; glandular cells occur irregularly. The neural lamella is very thin and may form deep invaginations, creating sinuses where the ns material is released through the neural lamella. Histological investigations show that the products contained in the perisympathetic organs are mostly of the Cr type, but B and A type products are also found. These products originate in the nerve ganglia as shown in experiments in which the median nerve was cut thus inducing an accumulation of ns material at the sectioned end and depletion of the perisympathetic organs (Raabe, 1965). The source cells of certain perisympathetic organs have also been traced by histology and the cobalt chloride technique in several species. They belong to the Cr type and are located in the ventral part of the ganglia (see Baudry-Partiaoglou, 1983b). The occurrence of neurons or glandular cells in the perisympathetic organs is not a general feature but both were reported in several species. It seems that the cell body of the peripheral ns cells terminating in the perisympathetic organs, may be located outside the perisympathetic organs, at various distances from them, as observed in a few cases (see Baudry-Partiaoglou, 1983b). 3.3.2 Morphological aspects The perisympathetic organs display great morphological variations, and a number of variants has been observed both among different insects and in the same insect, depending on the developmental stage and anatomical level (see Grillot, 1983). In primitive insects, these organs consist of swellings located on the median nerve, the transverse nerves or both. In more highly evolved insects, the perisympathetic organs migrate inside the segmental nerves or ganglia, where they constitute metameric-neurohaemal areas or one single neurohaemal area under the
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neural lamella. The neurohaemal organs located on the segmental nerves are often far from the ganglion and exhibit a particular structure since they constitute thin and fairly extensive plexuses (Baudry-Partiaoglou, 1983a). The swellings formed by the perisympathetic organs are apparent in varying degrees. In some cases they are long and very thin, and envelop either the sympathetic nerves, or the somatic nerves. In such cases they are difficult to detect histologically and have mostly been visualized by vital staining with methylene blue and electron microscopy. In other cases the perisympathetic organs are quite distinct and conspicuous. 3.3.3 Classijication The great diversity of the perisympathetic organs suggests the need to classify them and to examine their possible evolution (Grillot, 1976a,b; Grillot, 1983) (Fig. 3). On the basis of all the available data, two features were chosen to define the morphological types of perisympathetic organs: the position of the neurohaemal tissue on the median or transverse nerves and the degree of anatomical distinctness of these nerves. Three main categories of perisympathetic organs were established (according to whether the neurohaemal tissue was located on both the median and transverse nerves, or only on one or the other): mediotransverse, median and transverse. Within each category, primitive, intermediate and evolved subtypes were distinguished, depending on whether the transverse nerves were individualized and either partly or totally incorporated into the somatic nerves (see Fig. 3). The primitive median type of perisympathetic organs is located around the median nerve or at the origin of the transverse nerves. The intermediate median type has the same position but the sympathetic nerves are partly anastomosed with t h e connectives of the ventral nerve cord. The evolved median type of perisympathetic organs is apposed to or incorporated into dorsal part of the ganglion. The primitive mediotransverse type of organ is characterized by the presence of thin continuous neurohaemal tissue all along the sympathetic nerves, or by discontinuous tissue ensheathing the median and transverse nerves, and forming three distinct swellings. In the evolved mediotransverse type of organ, the neurohaemal tissue forms three distinct swellings but the lateral neurohaemal
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0 external organ
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3-rx T\ I n
r/
Jnr
intermediate perisympathetic organ
Periploneto omericano
Diprion pini
Gryllus domesticus
evolved perisympathetic organ
Diprion pini
1
Tenebrio molitor
Ltransverse -
Roscius elongatus
mediotransverse Fig 3 Morphological types and evolution of the perisympathetic o r g m s (from Grillot. 1083)
tissue may be located under the nerve sheath, and the median tissue, very close to the ganglion, externally or internally. The lateral tissue may also be far from the ganglia and form a plexus at the end of the somatic nerves, the median tissue forming a cluster of ns endings located within the ganglion itself. The primitive transverse type of organ exhibits two subtypes:
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those which extend from the median nerve to both transverse nerves, and the opposite form consisting of two separate organs located on the transverse nerves. The highly evolved, transverse type appear as distinct swellings which are flattened against the ganglia or located inside the segmental nerves. 3.3.4 Evolution The primitive perisympathetic organs appear to constitute extended neurohaemal tissues located on the median and transverse nerves; the other types are believed to originate from this tissue by fragmentation and, at the same time, come closer to the central nervous system. This hypothesis is supported by the following findings. Crickets (which are primitive insects) reveal a changing arrangement of their perisympathetic organs along the ventral nerve cord. They are thus found first on the transverse nerves, then partly fused with the segmental nerves, and subsequently incorporated into the segmental nerves (Thomas and Raabe, 1974). In the larva of the hymenopteran Diprion the perisympathetic organs form a continuous neurohaemal structure of the intermediate mediotransverse type, whereas in the adult, these organs belong to the evolved type and are divided into three parts-one median and two lateral (Provansal, 1971). In Dytiscus the perisympathetic organs, which are incorporated into the single thoracoabdominal ganglion, are differently arranged in the successive metameres presenting a range of evolutive steps (Grillot, 1976a). In heteropterans, transformations o f the perisympathetic organs were discovered when specimens from several families were studied. The perisympathetic organs migrate first from an external to an internal position within the ganglionic mass, and then into the nerves (Baudry, 1972; Baudry-Partiaoglou, 1978). 3.3.5 Ultrastructural duta The varied morphology of the perisympathetic organs suggests that the differences between them can be understood by ultrastructural study (see Baudry-Partiaoglou, 1983b). Both median and transverse organs have been thus investigated in species belonging to the mediotransverse type, to the median type or to the transverse type. Evolved mediotransverse perisympathetic organs were studied in Oryctes, Glossina and Roscius whereas intermediate mediotransverse perisympathetic organs were investigated in Vespa.
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In Oryctes, the transverse organs are located inside the somatic nerves and the median organs, in the fused thoracoabdominal nerve mass (Provansal-Baudez and Baudry-Partiaoglou, 1983). The tsetse fly exhibits both median intraganglionic organ and widely dispersed distal neurohaemal tissues (Baudry-Partiaoglou and Grillot, 1975; Robert et al., 1981). In Koscius the perisympathetic organs are composed of three parts. First, the distal “in network” organs, whose thinnest branches contain only one or two ns axons and which are found at somatic nerve endings. Second, neurohaemal areas dispersed along the abdominal nerves. A third component, the “subjacent” organs is found laterally within the ganglionic mass. The latter are not superficial tissues, and a thick layer of perineurial cells isolates them from the circulating blood. In Vespu the perisympathetic organs include a globular median organ and two lateral voluminous swellings surrounding the somatic nerves (Provansal et al., 1970; Raabe et al., 1970). Transverse perisympathetic organs have also been investigated in species exhibiting a primitive transverse type such as Caruusius (Raabe and Ramade, 1967; Brady and Maddrell, 1967) and in evolved transverse type, as in Tenebrio (Provansal-Baudez and Baudry-Partiaoglou, 1983). Median perisympathetic organs have been studied in insects belonging to the median primitive or intermediate type, e.g. cockroaches (Scharrer, 1968), locusts (Chalaye, 1974b) and the coleopteran Chrysocarabus auronitens (Provansal-Baudez and Baudry-Partiaoglou, 1983). Despite the fact that these insects belong to different types, all the median and transverse perisympathetic organs have common structural features. Thus, transverse organs are characterized by bare axon terminals, a loose structure and numerous sinuses which are no longer present when the neurohaemal tissue occurs along great lengths of nerve. Median perisympathetic organs are globular and often composed of two parts: a central one containing the bulk of the ns axons, and a peripheral part, penetrated by short invaginations of the neural lamelia and consisting of bulbous ns terminals invested by glial infoldings. When in highly evolved perisympathetic organs the neurohaemal tissue is included in the nerve ganglion their structure is fairly homogeneous. Further exploration of ns products in the various perisympathetic organs, shows that, like the corpora cardiaca, these organs contain a number of axons with different types of granules (i.e. dense spherical granules with a diameter between 110 and 190nm, sometimes forming two different subtypes, irregular or ovoid dense
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granules and in a few cases electron-lucent vesicles whose diameter can reach 250 nm). Dense cored vesicles 80 nm in diameter can also be seen. Transverse perisympathetic organs were always observed to contain several types of axons, whereas median organs only contained a single type. The modes of release differ in median and transverse perisympathetic organs. In transverse organs, release occurs in places where the ns terminals are in direct contact with the basement membrane or its invaginations. In median perisympathetic organs, on the other hand, the neurosecretory products are discharged in front of glial cells or, in other terminals, into the intercellular spaces, sometimes at a certain distance of the neural lamella. The same process has been described for the corpora cardiaca. To conclude, there is no doubt that median and transverse perisympathetic organs differ in their structure, in the number of ns products they contain, and in their mode of releasing these products. These differences are independent of their degree of evolution and of the fact that they belong to insect species possessing either median and transverse structures, or only one type of structure. The physiological significance of these facts remain to be elucidated.
3.4
NEUROHAEMAL AREAS
Neurohaemal areas have been progressively discovered in several regions (see Raabe, 1982, 1983). They are thin structures that include the terminals of both ganglionic and peripheral ns cells. They sometimes recall certain perisympathetic organs. The corpora cardiaca are the main centre for the release of brain ns products, but may only be partly involved in this release-as in mosquitoes (Meola and Lea, 1972). In these insects, the neurohaemal terminals occur both in the posterior region of the corpora cardiaca' and in the aorta, where ns axons terminate between the muscle fibres. The same situation occurs in some heteropterans, although in others, release is completed inside the aorta walls (Johansson, 1958; Dogra, 1967; Unnithan etul., 1971). It seems that for the nccI, the release site is often the aorta, while the nccII terminate in the corpora cardiaca. The aorta wall constitutes a major or fairly important release site for brain ns products in insects belonging to other orders too, for instance in the dipteran Culli-
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phora (Normann, 1965), in dermapterans (Awasthi, 1975) and in a coleopteran (Ray et al., 1980). In other insects, a few ns axons terminate in various regions outside the corpora cardiaca, although these organs play a normal neurohaemal part. Thus, in Gryllus birnaculatus (Naik, 1978) certain axons terminate in the wall of the aorta, and in several species, ns endings were found against the sheath of the nerves leading from the brain to the corpora cardiaca (e.g. in Leptinotarsa, Schooneveld, 1974b; Locusta, Cassier and Fain-Maurel, 1970b and Calliphora, Kaiser, 1979). The oesophageal or cardiac nerves issuing from the corpora cardiaca are neurohaemal too in Periplaneta (Johnson, 1966), Blaberus craniifer (Gersch et al., 1974), Myzus persicae (Bowers and Johnson, 1966), Calliphora (Thomsen, 1969), and the primitive dipteran Chiroriorniis plurnosus in which the source cells of these neurohaemal areas were identified in the pars intercerebralis (Panov, 1979a). In cockroaches a neurohaemal commissural cardiaca-allatal plexus occurs (Adiyodi, 1974) and the cardiac nerves developed into true neurohaemal organs containing several types of ns endings, some of which appear to be peptidergic and others, aminergic (Johnson, 1966; Gersch et a l . , 1974). The nca2 joining the corpora allata to the suboesophageal ganglion form small neurohaemal organs in Periplaneta (Pipa and Novak, 1979) and rather well developed ones in crickets (Thomsen, 1943 and others). A surprising feature is that in some species, the ns endings are located in a peripheral area surrounding the corpora allata. This occurs in lepidopterans and coleopterans where corpora cardiaca only contain glandular cells (Kind, 1965; Tombes and Smith, 1970; Srivastava et al., 1975). This morphological arrangement explains the unexpected findings that in lepidopterans, the prothoracic gland was stimulated by corpora allata implants. The origin of the brain hormone located in the neurohaemal area surrounding the corpora allata was effectively demonstrated by Agui et al. (1980) in Manduca sexta. Finally, in the cricket A c h e p domesricus, where neurohaemal areas have already been localized in the aorta and the nca2, the ventral superficial part of the brain was found to have a neurohaemal structure (Geldiay and Edwards, 1973; Geldiay and Karagali, 1980). In this insect, the ns axons penetrate between the perilemma cells up to the basement membrane, and synaptoid sites characteristic of release areas can be observed. The source cells of this neurohaemal cerebral region are not precisely known, but
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several distinct types of nerve endings have been described in Melunogrylfus desertus (Geldiay and Karagali, 1980) and Teleogryllus commodus (Breitfuss et ul., 1980). They are supposed to originate in brain ns cells, but one substance at least originates in the suboesophageal ganglion, since the axonal pathway of the medioventral ns cells was traced to the brain through the circumoesophageal connectives, both by in toto staining in Teleogryllus (Weinbormair et al., 1975) and by immunochemistry in Acheta (Strambi et ul., 1979) and Locusta (Remy and Girardie, 1980). Moreover, sectioning of circunioesophageal connectives induced noticeable changes in the neurohaemal area (Breitfuss et uf., 1980; Remy and Girardie, 1980). Various release areas have been described along somatic or sympathetic nerves, sometimes in the vicinity of perisympathetic organs, often close to visceral organs like the heart or gut, and also to peripheral ns cells. Some of these neurohaemal areas such as those surrounding cockroach cardiac nerves, are large and well defined; others, on the contrary, are very narrow, such as the link nerve neurohaemal areas of stick insects. The ns products stored in the neurohaemal areas appear to originate both in the ventral nerve cord and peripheral ns cells. Few studies have been devoted to these questions, but important data have been supplied by the investigations of stick insects (Finlayson and Osborne, 1968; Fifield and Finlayson, 1978) and locusts (Remy and Girardie, 1980). In both species, neurohaemal areas have been shown to occur in the proximal part of the somatic nerves originating in the medial ns cells of the suboesophageal ganglion, which also sends out processes into the brain, as shown above. In the stick insect Curuusius, a large complex of neurohaemal areas has been described along the somatic nerves, the transverse nerves and the link nerves joining them. Eight types of ns products have been shown to be present; their origin was attributed to ganglionic ns cells and to the peripheral multipolar ns cells, which send out numerous processes in various directions (Fifield and Finlayson, 1978). Connections between neurohaemal areas and peripheral ns cells have also been observed in the proctodeal nerve of Oryctes (Nagy, 1978), in the nerve of the ventral intersegmental muscles (Anwyl and Finlayson, 1973), in the abdominal ns organs of Rhodnius (Kuster and Davey, 1981) (and in other cases as well). Electrical recording from neurohaemal areas have only been made in Curuusius. The bursts of activity recorded extracellularly seem to indicate that they are initiated both centrally and peripherally (Finlayson and Osborne, 1970, 1975).
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Do amines and acetylcholine play a part in neurohaemal areas? In the cardiac nerves of Blaberus these areas contained fluorescent material corresponding to dopamine and serotonin which disappears after reserpine application and exhibited a characteristic appearance in electron microscopy (Gersch et al., 1974). In Rhodnius, the abdominal ns organs were innervated by aminergic axons of central origin (Kuster and Davey. 1981). In addition, it has been shown that the frequency of peripherally originating spikes can be modulated by the application of acetylcholine (Finlayson and Osborne, 1975). To sum up, it appears that a certain dispersion of ns terminals may be the general rule and that the scant knowledge of them is simply due to lack of research and to the great difficulties inherent in demonstrating their presence.
3.5 N E U R O E F F E C T O R J U N C T I O N S Evidence has been accumulating that in several organs, ns axon endings have terminals which are in contact with either glandular or muscular cells, or with the connective tissue. Release sites have been observed in such organs and when contact is with the glandular or muscle cells, ns products obviously seem to play the part of neurotransmitters; when ns terminals are located against the connective tissue, their products behave like local neurohormones since they probably move about the organs in the connective tissue. The insect organs which receive neuroeffector innervation are mostly the corpora allata, the prothoracic gland, the visceral muscles of the heart, gut and spermatheca, the exocrine glands like the salivary glands, the pericardial cells, and the lantern of the firefly (see Raabe, 1982, 1983). Most of the nerves innervating the organs mentioned above originate in the brain and ventral nerve cord, but they sometimes issue from the corpora cardiaca or from the ganglia of the sympathetic nervous system. Certain nerves, like the cardiac ones, have a composite origin since they include axons from both the corpora cardiaca and ventral ganglia. It should be noted that some ns axons follow very long pathways before joining their target tissues or organs. The axons which make neuroeffector junctions do not necessarily release all the products they contain in the same way. A single axon may terminate both in a target and in nearby neurohaemal organs or areas. Such phenomena have been observed in molluscs and are well known in vertebrates.
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The substances released in this way seem to consist of biological amines and peptides. Electron microscope investigations have frequently demonstrated the presence of two types of granule in the same organ: one resembling the large peptidergic granules and the other (smaller and dense-cored) resembling amines. With respect to amines, application of the Falk and Hillarp technique to the midgut of Schistocerca (Klemm, 1972) and the heart of Blaberus (Gersch et al., 1974) confirmed that these substances occur. Amines are also known to occur in the firefly lantern and salivary gland (see Section 2.6). Octopamine is involved in the first case and, dopamine in the second. Thus, in some cases at least, we know that amines are present and which are involved. In other cases, we are aware of the presence of active factors both in the target organs and their nerves. Whereas extracts of stomodeal and proctodeal nerves (Brown, 1967; Holman and Cook, 1972) stimulate heart beat in cockroaches, gut extracts from Vanessa lo (Koller, 1948), Periplaneta (Brown, 1967), exert a myotropic effect. It is interesting to compare the ultrastructural features of certain organs where amines or peptides have been identified in order to see if there is a link between the ultrastructure and the substances involved. In the firefly lantern, whose control involves octopamine (Robertson and Carlson, 1976), the axons contain large dense-cored vesicles (90-135 nm) and smaller electron-lucent vesicles. They are wrapped in glial processes up to their distal part, where they enter into close contact with photocytes, forming synapses (Oertel et al., 1975). In the salivary glands, which are mostly controlled by dopamine, small electron-lucent vesicles are always observed, but the size and density of the large vesicles varies. In Nauphoeta cinera there are large electron-dense vesicles, 92 nm in diameter (Maxwell, 1978), whereas in Manduca sexta the large vesicles have a diameter of 50-100 nm and are all dense-cored (Robertson, 1974). After permanganate fixation, which stains the aminergic vesicles, the small vesicles only became electron-dense in Nauphoeta; moreover their great depletion by electrical stimulation suggested that amines are located in the small vesicles and not the large ones, whose significance, in that case, remains unclear (Maxwell, 1980, 1981). The ultrastructural appearance of the large vesicles (which are supposed to contain peptides like proctolin or other peptidergic products) varies according to the species studied, even when the
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same organ is considered, as for example, in the case of the ventral intersegmental muscles of Schistocerca, Carausius and Phormia terranova (Osborne et ul., 1971; Anwyl and Finlayson, 1973). This variation has also been observed in ns cells or neurohaemal organs; probably because of the methods of investigation used and also of species-specific properties. It therefore seems that ultrastructural studies cannot at present allow amines to be clearly distinguished from peptides in neuroeffector junctions. The mode of contact between neurosecretory terminals and target tissues or other tissues like connective tissue does not permit better recognition of the substances involved in such contacts. Aminergic axons like DUMeti, do not form synaptic structures with muscle fibres (Hoyle et al., 1980), which is not really surprising since their modulatory role has been demonstrated. Gut endings containing the peptide proctolin do, on the other hand, form synapses with the muscle cells (Brown, 1967), and the formation of other “peptidergic” synapses has been described with heart muscles (Johnson, 1966; Bowers and Johnson, 1966), rectal pads (Gupta and Berridge, 1966), and uterine muscles (Robert et al., 1981). In other cases, however, sites of release by peptidergic axons are described as being in contact with the connective tissue of various organs, namely the spermatheca (Gupta and Smith, 19693, intersegmental muscles (Osborne et al., 1971), and muscles of the dorsal diaphragm (Miller et al., 1979). Lastly, axon endings facing both target cells and connective tissue have been reported in termite sternal glands. (Quennedey, 1969) and in rectal pads (Oschmann and Wall, 1969). The significance of the presence of neuroeffectory junctions in addition to neurohaemal organs and neurohaemal areas may seem difficult to understand. When ns products are released in contact with connective tissue, it may be supposed that the active substances move around in the lacunae of this tissue and come within reach of a number of cells. The situation is reminiscent of the distal neurohaemal areas. The hormone is released in the vicinity of its target cells, allowing a reduction in the amount of substance necessary to transmit a message, since no dilution takes place in the haemolymph. This makes the transmission of information faster. These two advantages exist also when the nerve terminals form a synaptic or synaptoid contact with the target cells. However, this would require repeated branching of ns axons to establish contact with all the cell bodies.
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3.6
TERMINALS I N NERVOUS SYSTEMS
The occurrence in various organs of ns endings which form true synapses and release ns products into the connective tissue or make contact with other neurons or glial cells resembles that found in other parts of the nervous system. A number of the axons involved, which follow long pathways to reach their target tissue, send out collaterals along their entire route. Some axons send out two processes in different directions. Furthermore, ns cells have numerous branched collaterals and, whereas one process runs inside its nerve bundle, the collaterals branch out profusely towards ns cell bodies belonging to other groups of ns cells. These processes were discovered using histological techniques. Heavily stained droplets were observed in the connectives of the ventral nerve cord in Blaberus craniifer (Geldiay , 1959), Chaoborus crystallinus (Fuller, 1960) and the ganglionic neuropil of stick insects (Raabe, 1965). In some cases, the entire pathway of these particular ns cells has been traced. In the aphid M y z u s two cerebral ns cells were found to send out one of their two processes to the corpora cardiaca and the other through the circumoesophageal connectives all along the ventral nerve cord to the hindgut (Johnson, 1963). The same long-distance axonal transport was observed in another aphid Myzus vicia (Steel, 1977), as well as in the primitive dipterans Chaoborus (Fuller, 1960), Culex tarsalis, Culesita inormata (Burgess, 1971, 1973) and Chironomus plumosus (Panov, 1979a) (Fig. 4). In stick insects and locusts, two cells possess well-developed processes, as in aphids, but they are located in the suboesophageal ganglion instead of in the brain. These cells give rise to one process branching off profusely in the brain neurohaemal area (see Section 3.4) and to a second, running all along the ventral nerve cord and branching off in each ganglionic neuropil and segmental nerves, where the ns axons terminate in neurohaemal areas (Girardie and Remy, 1980). In the bug Zphita limbata, axonal pathways are shorter, and only extend from one ganglion to the next. Each ns cell bifurcates close to the cell body; one process branches out in its own ganglion while the other runs back to the next ganglionic mass and divides here, in the vicinity of the collaterals of the ganglionic ns cells proper (Seshan, 1968). The accumulation of swollen branches loaded with ns products in the brain and nerve ganglia has often been reported. Two types of
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Myzus persicae
Locusta migratoria
257
lphita limbata
C hironomus plumosus
Fig. 4 Schematic representation of n5 pathways and terminals within the central nervous system (from Johnson, 1963 for Myzus; Panov. 1Y7Ya for Chironornus; RCmy and Girardie, 1980 for Locusfa; Seshan, 1968 for lphita)
process seem to occur: storage, which takes place in axon loops or collaterals, observed in locusts along the axon bundles originating in the pars intercerebralis (Highnam and West, 1971), and a process occurring throughout the neuropil, which consists of establishing contacts between the collaterals of the ns cells of different groups. Thus, the lateral cells of Periplunetu send branches towards the pars intercerebralis in the vicinity of which they again branch off repeatedly in close contact with the collaterals of the pars intercerebralis ns cells (Fraser and Pipa, 1977; Rademakers, 1977; Pipa, 1978). Among the collaterals described, it was shown that metenkephalin collaterals occur in Locusta at the periphery of the tractus of the nccl and nccII (Remy and Dubois, 19Sl), and that proctolin-like terminals are abundant in the neuropil of the terminal
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ganglion of Periplaneta (Eckert et al., 1981). Of particular significance is the study by Schooneveld (1974b) of the brain of Leptinotarsa. The relationships of the ns processes with other neurons appear to be of two kinds: firstly, true synapses (observed between the ns processes and presynaptic neurons, the ns branches receiving presynaptic inputs) and, secondly, synaptoid sites formed by the ns processes; the formation of the latter sites is suggestive of release processes and suggests that ns products exert some kind of action, probably modulatory, in the central nervous system. Therefore it appears that in nervous ganglia (as well as in various other organs including neurohaemal organs) peptidergic and aminergic terminals have the role of neurotransmitters or neuromodulators, although at the same time they can exert a neurohormonal function involving the release of ns material into the blood circulation. Such findings have also been reported in vertebrates, in which niolecules like L H R H , T R H or somatostatin are released concomitantly into the blood stream and at the synapses inside the brain.
4
Regulation of neurohormone production and release
In an attempt to understand the physiological meaning of ns cells, numerous studies have been conducted, using various techniques. 4.1
HISTOPHYSIOLOGICAL STUDIES
The histophysiological method has been applied to many species during growth, diapause, o r reproduction, and to processes like diuresis, and circadian activity. Good correlations were often recorded, mostly for A type ns cells. An exhaustive review cannot be given here and only a few examples will be described. In Schistocerca gregaria, injection of salt solution caused the accumulation of ns material (Highnam et al., 1965) and treatment with distilled water led to its rapid release from the ns system. Similar observations were made in other species (Dogra and Ewen, 1969; Jarial and Scudder, 1971). In the stick insect Carausius morosus, two medioventral ns cells in the suboesophageal ganglion exhibited important modifications as regards colour change and hygrometric conditions. In green insects reared in a humid environment, the cell bodies are small and contain numerous granules. In black insects living in a dry environ-
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ment, the cytoplasm is, on the contrary, voluminous, and loaded with large granules and vacuoles (Raabe, 1966). In locusts and crickets, the same ns cells also appear to have a function both in diuresis (Proux and Rougon-Rapuzzi, 1980) and reproduction in Curausius and Teleogryllus, ovariectomy of the female results in a depletion of the content of these ns cells, followed by an increase in the size of the nucleus and in labelled cysteine incorporation (Mouton, 1968; Diirnberger et ul., 1978). In the insect PanstrongyEus megistus, the A and A' ns cells of the pars intercerebralis become depleted, with a 2-3-day interval during the last instar. The type A ns cells, which trigger gonial mitosis, release their products immediately after the blood meal, while the type A ' ns cells, which stimulate gonial meiosis, only discharge three days after the blood meal (Furtado. 1979). 4.2
SIGNIFICANCE OF LOADED NEUROSECRETORYCELLS
Despite the correlations established between several processes and the variations in the amount of ns products stored in some ns cells, the exact significance o f empty and heavily loaded ns cells is still not known. At the inception of this kind of study, it was tacitly admitted that heavily loaded ns cells are very active cells which both synthesize and release their products. Subsequently, however, the loaded cells proved in many cases to be in an inactive state, for example during diapause, whereas the empty cells corresponded to an active state during which ns products are released rapidly. In such cases these products are synthesized as fast as they are released. 4.3
STORAGE PLACES
Very few histophysiological studies have been made of the ns nerve tracts and neurohaemal organs, probably because it is very difficult to measure the amount of material present in such structures. However, despite the difficulties involved, it would be very useful to know the storage level of the terminal part of a complex in which the release process actually takes place. Closely connected with this problem is the question of identifying the storage sites of ns material, as they seem to differ from one insect to another. Highnam and West (1971) showed that in the pars intercerebralis, the axons of type A ns cells form loops along the
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nccI tracts where material is stored. These “reservoirs” are well developed in Locustu but smaller in Schisfocerca. General understanding of the whole process requires that the transport and release of ns material should be considered as distinct separately-controlled processes.
4.4
NUCLEI A N D ORGANELLES
The state of ns cell activity may be deduced not only from the amount of ns products in the cell body or axon but also from cell size, especially the size and appearance of the nuclei. Thomsen (1965) showed that meat-fed, maturing eggs Calfiphora have large nuclei and little ns material in the cell body. Sugar-fed flies (which do not produce eggs) have, on the contrary, small nuclei and heavily loaded cell bodies. Similar observations have been made in Locusta. Fasting insects have small nuclei and loaded ns cells, while eating ones have large nuclei and almost empty ns cells (McCaffery and Highnam, 197Sa). It is possible to examine the synthetic organelles, rough endoplasmic reticulum and Golgi bodies and the ns material, using electron microscopy. The granule content, number of Golgi apparatuses and area covered by the endoplasmic reticulum in the ns cell body all vary greatly. Further, during advanced storage stages, lysosomes and dense bodies are abundant. A good example in this respect is the blowfly Calliphora, which exhibits striking changes during its reproductive cycle, as explained above. In the latter, ns material accumulates in the cell body while the rough endoplasmic reticulum becomes disorganized, the Golgi bodies decrease in number and lysosomes become abundant. These changes indicate reduced hormone production and storage in the perikaryon. In meat-fed flies, on the other hand, the rough endoplasmic reticulum and Golgi apparatus are well developed, demonstrating increased synthetic activity (Bloch et al., 1966). In addition to the cell body, electron microscopy has led to an interpretation of neurohaemal ultrastructure. An increase in the number of synaptoid sites and omega profiles means that the release process takes place. It occurs after electrical stimulations which also enhance the release of cardioaccelerating and hypertrehalosemic neurohormones, as observed in Peripfanetu (Scharrer and Kater, 1969) and Culliphoru (Normann and Duve, 1969).
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INVESTIGATIONS WITH LABELLED CYSTEINE
As already specified, type A ns products, or their carrier proteins, contain cystine or cysteine. This suggests the use of 35S cysteine to investigate the functioning of the ns system. The rate of cysteine incorporation into the cell body reflects the level of synthetic activity, and the speed of transit towards the corpora cardiaca indicates the velocity of release. Several authors have used labelled cysteine to study ns processes. Siew (1965) showed in Galeruca tanaceti that ns labelling reaches a peak at 9 h in mature females, at 3 h in ovipositing females, and at 24 h in diapausing females. In Anacridiurn aegyptiurn, labelling of ns cells was examined in diapausing and reproductive females which were respectively raised with 9-h and 15-h photophases. In long-day animals, labelling was intense for 3-9 h after injection but, by 18 h, most of the newly-synthesized material had disappeared from the brain. In brains of short-day animals, the amount of cysteine rose much more slowly, and the peak occurred later. These autoradiographic results are therefore consistent with histological observations indicating rapid synthesis and transport by ns cells during ovarian development, and slow accumulation of material during reproductive arrest (Geldiay, 1970). In Schistocerca delivery of the label from the cell body to the corpora cardiaca takes 0.5 h in young females maturing their oocytes, 1.5 h in females with fully mature oocytes, and 4 h in fasting females in whom vitellogenesis has been arrested (Highnam, 1976). Similar results were obtained in Locusta (Girardie and Girardie, 1972).
4.6
AUTOREGULATION
Insect ns cells which appear to be continuously active, may also regulate their own activity in accordance with the load carried by their terminals. Thus, they are able to modify their synthetic rhythm, the speed of transport of ns material and the catabolic processes, in response to terminal information. These phenomena have been demonstrated in Locusta rnigratorza (Girardie et al., 1976). The experiments for this purpose were conducted using labelled cysteine as an indicator of ns activity, and electrostimulation to induce the release of ns material which was evaluated by the gonadotropic activity of the medium. It was also
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proposed that the most recently produced granules are released first, as occurs in vertebrates. The way in which synthetic processes in the cell body may be modified by events occurring in nerve terminals remains obscure. It has been suggested that antidromic nerve impulses are involved (Anwyl and Finlayson, 1974). 4.7
RHYTHMIC F U N C T I O N I N G
In some cases, ns cell functioning appears to be governed by an internal diurnal rhythm. In Acheta dornesticus, RNA and protein synthesis, as well as labelled amino acid incorporation, were measured throughout the 24-h cycle in the ns cells of the pars intercerebralis. A rhythmic pattern was observed, in which the RNA synthesis peak occurred a few hours before the protein synthesis peak (Dutkowsky et al., 1971). These results confirm other observations for several insects, including the beetle Carabus nernoralis, the fruit fly Drosophila rnelanogaster and the bee Apis rnellzfica. Circadian functioning of the ns cells in Acheta results from the action of a circadian pacemaker located in the pars intercerebralis (Cymborowski, 1981). 4.8
ENVIRONMENTAL AND INTERNAL INPUTS
Because ns cells trigger the most important events in insect life, they are regulated by coordinating brain centres which respond to various information. Temperature, photoperiod, feeding, mating, and incubation are known to have a decisive role in growth and reproduction, and the pathways of the sensory inputs involved have been traced to the brain. In the blood-sucking bug Rhodnius, the meal distends the digestive tract, resulting in the stimulation of the tension receptors from which the inputs run along the ventral nerve cord to the brain, which initiates the release of the ns material for moulting or reproduction. In the viviparous cockroach Leucophaea, oocyte maturation is arrested during incubation periods. Here also, tension receptors are involved in the internal inputs reaching the brain. This organ in turn inhibits and activates the functioning of the corpora allata, whose secretion is important for egg maturation. The control exerted by the brain on the ns cells involves various pathways and chemicals. The ns cells are certainly controlled by
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inhibitory or excitatory synapses from coordinating interneurons, which are related to the major sensory centres corresponding to the ocelli, the compound eyes, the antennae, etc. The existence of anatomical connections have been shown to exist between these centres and the pars intercerebralis. Another type of coordination exists between the ns centres, pars intercerebralis, lateral protocerebral and tritocerebral cells, and cells of the ventral nerve cord. The collaterals of the ns cells run from one to another of these ns centres, exchanging information. Along the tractus of the nccI there is, at least in some insects, a large area of contact between the collaterals of pars intercerebralis neurons and those of the ns cells of the pars lateralis (see Section 3.6). The relationships between the ns cells and the sympathetic nervous system must also be considered. The frontal ganglion transmits to the brain the sensory information provided by gut tension and osmoreceptors. Extirpation of this ganglion greatly perturbs the release of the ns material affecting growth, egg maturation, and protein and water metabolisms. Furthermore, the ns cells have numerous contacts not only in the brain but also along the ventral nerve cord. In the neurohaemal organs the release of ns products is modulated in axo-axonal synapses by input from the periphery, as well as from the central ganglia. The ns cell activity is regulated by neurotransmitters and modulators (i.e. acetylcholine, biogenic amines and probably peptides). Amine involvement has been shown by reserpine treatments, which cause striking changes in the amount of ns material contained in the cell body (see Section 2.5). In the pars intercerebralis of Galleria, injections of noradrenaline causes accumulation of ns material only in certain ns cells (Warton and Dutkowski, 1977), where didisulfiram, a specific inhibitor of noradrenaline synthesis, depletes the electron-dense material of the dense-core vesicles and reduces the amount of ns material (Warton, 1981).
4.9
FEEDBACK
It is now considered that the release of products from the ns cells and their synthetic activity may be affected by humoral factors in the haemolymph. Their effects are mediated at ns axon terminals or in cell bodies.
264
M. RAABE lateral neurosecretory
neuropil
external stimuli internal clock
nervous ganglion
\
L .
I
2 ’
ecdysone feedback
neurohaemal organ
+?I 4
/ peripheral
axon neuron
Fig. 5
Schematic representation of the functioning regulation of a ns cell
In vertebrates, there are many examples illustrating feedback processes. In insects, growth hormones, juvenile hormone and ecdysone have been shown to influence 11s activity both in the larva and adult insect.
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Ecdysone often modifies brain hormone release from the cell bodies, for instance, in the cockroach brain, in vitro (Marks et a f . , 1972) or in the brain of Mamestra in vivo or in vitro (Agui and Hiruma, 1977a,b). Extirpation of the moulting gland also causes changes in certain brain ns cells and in type A ns cells of the ventral nerve cord in Aeschna (Charlet et a f . , 1974). In addition, ecdysone acts on brain ns cells triggering the release of a myotropic hormone that causes ovarian contractions in Rhodnius prolixus (Ruegg et a f . , 1981). As regards juvenile hormone, it was shown that the activity of the ns cells of the pars intercerebralis is affected by the presence of this hormone in haemolymph, but its effects differ in the adult, pupa and larva. In the reproductive female, removal of the corpora allata was observed to depress the synthetic activity of pars intercerebralis ns cells in several insect species including Tenebrio (Mordue, 1965) and Leptinotarsa (de Wilde and d e Boer, 1969), and implantation of supernumerary corpora allata, or injection or application of juvenile hormone or its analogues stimulated the activity of pars intercerebralis ns cells, an effect substantiated by the nuclear enlargement observed in Calfiphora (Thomsen and Lea, 1969) and by load changes of type A ns cells in Locustu (McCaffery and Highnam, 1975a,b) . In Manduca larvae juvenile hormone was shown to exert a negative feed-back, since corpora allata removal cause anticipated release of brain hormone (Nijhout and Williams, 1974). In Calfiphora vicina, the observation of Scheller and Bodenstein (1981) illustrate the effects of ecdysone and of the juvenile hormone analogue methoprene on R N A and protein synthesis in ns cells of young and older third instar larvae. Their results vary according to the age of the recipient and might depend on the presence of receptors. Protein and R N A synthesis, which are correlated to ns material production, decline in young insects and rise in older ones under the influence of both ecdysone and juvenile hormone. It has been suggested that the hormones involved in feedback modulate the electrical activity of the ns cells at their terminals in the corpora cardiaca, because it is well known that neurohormonal release is accelerated by intensification of the electrical activity of the cells which generate bursts of impulses. In Rhodnius the electrical activity of the ns cells was monitored by applying suction electrodes to the corpora cardiaca. While in ovariectomized females the spike frequency recorded fell sharply, it rose when they were injected with ecdysone (Ruegg et al., 1982).
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5 Neurohormones, a brief survey
The ns cells are the source of various neurohormones which are released in different places. The occurrence of neurohormones has been demonstrated experimentally by several methods and bioassays. These hormones play a very important role in insect physiology. Their origin and release sites are not always precisely known. A certain amount of information has been obtained about their mode of action. Several neurohormones have been purified but only two have been completely identified and synthesized (see Locke and Smith, 1980; Miller, 1980; Raabe, 1982).
5.1
NEUROHORMONAL ACTIVITIES
A large number of hormonal activities has been detected in the central nervous system and neurohaemal organs. In point of fact, neurohormones have been shown to intervene in all the major physiological regulations, acting on a number of tissues including the endocrine glands, fat body, reproductive organs, and Malpighian tubules as well as visceral muscles of heart, gut and rectum. Neurohormones therefore play a role in growth, reproduction, and in different aspects of metabolism including pigment synthesis, tanning and diuresis, and also in rapid processes like ecdysis, contraction of the visceral muscles and pigment movements.
5.1.1 Development and reproduction Moulting gland functioning (i.e. the production of ecdysone) is of fundamental importance in growth regulation, which also depends on the juvenile hormone produced in the corpora allata. Moulting gland activity is triggered by the brain hormone, the first neurohormone demonstrated in insects (Wigglesworth, 1940). A particularly clear manifestation of the absence of brain hormone and consequently of ecdysone secretion is the diapause process occurring in pupae. Implantation of the brains of activated pupae into diapausing pupae induces termination of diapause, which is also ended by the implantation of a moulting gland. The mechanisms involved in embryonic diapause are different. In the silkworm Bombyx mori, the eggs laid may or may not undergo diapause depending on the environmental factors to which the mother is subjected. The onset of diapause is triggered by a diapause hormone from the suboesophageal ganglion whose pro-
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duction is stimulated by the brain (Hasegawa, 1951; Fukuda, 1952). In diapausing eggs, both the glycogen and 3-hydroxykinurenin content of the ovary are very high (Yamashita and Hasegawa, 1966). The data concerning the role of neurohormonal factors from the brain in controlling corpora allata functioning are less well known. Section of the nccI or 11, removal of the pars intercerebralis and other experiments have often demonstrated that the brain can both stimulate and inhibit the functioning of the corpora allata. The nervous or neurohormonal nature of the inhibiting factor involved is still uncertain, but the neurohormonal allatotropic function of the pars intercerebralis was shown in several species. Another way in which the brain is involved in regulating the juvenile hormone level is by stimulating juvenile hormone esterase production as shown in Lorustu and Galleria (Retnakaran and Joly, 1976; McCalef and Kumaran, 1980). The role of the brain in regulating the activity of the corpora allata is probably the same during growth and in the adult, during the reproduction period. Thus, a number of processes related to reproduction, such as vitellogensis, the functioning of male and female accessory glands, the production of pheromones, and sexual behaviour depend both on the corpora allata and on a neurohormone from the brain. Moreover, several stages of reproduction are directly or indirectly controlled by the brain. The first reproductive processes in which neurohormones are involved are gonial mitoses and the entry of the oogonies into meiosis, as observed in the insect Panstrongylus megistus. Whereas gonial mitosis depends directly on the A cells of the pars intercerebralis, meiosis is triggered by the moulting hormone, whose secretion is induced by the brain (Furtado, 1979). Brain factors or gonadotropins intervene in the initial growth of oocytes in Tenebrio (Laverdure, 1972) and in previtellogenesis (Mordue, 1965). In vitellogenesis they act indirectly, by triggering corpora allata activity, as shown above, but a direct action on the ovary was demonstrated in Cufliphoru (Thomsen, 1952), in Locusta (Girardie, 1966) and a few other species (see Raabe, 1982). Brain gonadotropins induce vitellogenesis in two different ways. In some species they appear to stimulate vitellogenin synthesis and deposition directly, whereas in others, like the mosquito Aedes aegypti, the brain factor EDNH, which stimulates vitellogenesis (Lea, 1972) acts on the ovary, and induces production of ecdysone.
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This ecdysone, in turn, stimulates fat body production of vitellogenins (Hagedorn. 1974). The imaginal diapause provides good examples of arrests in reproduction due to the cessation of activity of the corpora allata, itself caused by the arrest of brain stimulation. Thus, cauterization of the pars intercerebralis in Leptinotarsa induced the onset of diapause (de Wilde and de Boer, 1969), and electrical stimulation of the pars intercerebralis of Anacridium aegyptium induced diapause termination (Girardie et al., 1974). The control of ovulation has not been much investigated, but seems to involve both amines (Hentschel, 1972) and neurohormones (Chaudhury and Dhadialla, 1976; Mesnier, 1982). The latter also help to regulate oviposition, which depends on neurohormones from the entire central nervous system, as demonstrated by experiments performed on isolated oviducts (Girardie and Lafon-Cazal, 1972) and in vivo (Thomas and Mesnier, 1973, stick insects; Mesnier, 1972, Galleria Galleria). Truman and Riddiford (1972) showed its presence in the corpora cardiaca in Hyalophora. Among other neurohormonal effects, it is worth mentioning the role of the pars intercerebralis in the maternal behaviour of the dermapteran Labidura riparia (Caussanel et a/., 1978), as well as the role of certain brain ns cells in determining the morph of the offspring of the aphid Megoura viciae (Steel, 1978). 5.1.2 Metabolism Neurohormones have been shown to control various aspects of metabolism, mostly by acting on the fat body. 5.1.2.1 Carbohydrates Trehalose is the most important sugar in insect haemolymph. It is used as an energy source and originates from glycogen stored in the fat body. The existence of a hypertrehalosemic neurohormone was discovered in the corpora cardiaca of Periplaneta (Steele, 1963) and Locusra (Goldsworthy, 1969). This neurohormone activates a fat body phosphorylase which reduces its glycogen content and raises the haemolymph trehalose level. Since the discovery of hypertrehalosemic hormone, its presence has been demonstrated in many insects but species specificity occurs, in particular, in the stick insect (Gade and Lohr, 1982). Other neurohormones have also been shown to be active in carbohydrate metabolism. For instance, hypotrehalosemic hormone was discovered in flies (Normann, 1975; Chen and Friedman, 1977)
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and in Manduca (Tager et al., 1976), and the existence of a glycogenolytic neurohormone that reduces fat body glycogen has been proposed in Aedes (Van Handel and Lea, 1965). In the silkworm, the diapause hormone also affects carbohydrate metabolism by inducing a rise in ovary glycogen, whereas fat body glycogen and haemolymph trehalose decrease (Hasegawa and Yamashita, 1965). The role of biogenic amines in sugar regulation has not been extensively studied but in Periplaneta, octopamine appears to have a strong hypertrehalosemic effect (Downer. 1979). 5.1.2.2 Lipids Lipids are a major fuel in insects. They are stored in the fat body as fatty acids, mostly triglycerides, and discharged into the blood as diglycerides. Lipid metabolism depends on octopamine, on juvenile hormone and on a neurohormone produced in the glandular cells of the corpora cardiaca; the existence of this hormone was first shown in the locust during flight (Mayer and Candy, 1969; Goldsworthy et al., 1972). This neurohormone termed the adipokinetic hormone (AKH) causes the release of fat body diglycerides in vivo and in vitro (see the review by Goldsworthy in this volume). The presence of an adipokinetic factor has been demonstrated in Danaus (Dallmann and Herman, 1978), Carausius (Gade and Lohr, 1982) and Periplaneta (Downer, 1972), but curiously, in both these species it is only active in the locust and not in the donor insect. 5.1.2.3 Proteins As explained above, neurohormones from the brain induce vitellogenin synthesis either indirectly or directly. The direct action of these neurohormones on the fat body has been demonstrated by histophysiological and experimental methods, particularly in locusts (Hill, 1962), grasshoppers (Loher, 1966) and cockroaches (Scheurer, 1969). Two opposite factors might in fact be involved in such action since, in locusts, the storage lobe of the corpora cardiaca stimulates protein synthesis, whereas the glandular lobe exerts an inhibitory effect (Carlisle and Longhton, 1979). Further, the amino acid proline, the main substrate for flight in certain insects, is mobilized by a neurohormone from the central nervous system and the corpora cardiaca in Glossina morsitans (Pimley and Langley, 1982), and by adipokinetic hormone in Leptinotarsa (Weeda, 1981). 5.1.2.4 Proteases and amylases Midgut proteases and amylases appear to be controlled by neurohormones. This was demonstrated
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by the results of decapitation and ligature experiments, and, above all, by removal of the pars intercerebralis or corpora cardiaca-corpora allata, which caused a decrease in the protease content of the digestive tract in several insect species. A normal protease level was re-established by implanting the pars intercerebralis (Muraleedharan and Prabhu, 1979, Dysdercus fasciatus), corpora cardiaca (Hrubesova and Slama, 1967, Pyrrhocoris), or corpora cardiaca and corpora allata (Thomsen and Moller, 1963, Calliphora),or again by injections of brain extracts (Langley , 1967, Glossina morsitans). Increased amylolytic activity was obtained by the same injections in Tenebrio (Jankovic-Hladni et al., 1978). 5.1.2.5 Pigments Many insects are able to change their cuticular and epidermal pigments according to the environmental factors. Whereas the corpora allata stimulate production of green pigment, they inhibit synthesis of melanins and ommochromes (Hori and Riddiford, 1982, Manduca) a process stimulated by the neurohormones whose presence was demonstrated in the lepidopterans Pieris (Ohtaki, 1960), Papilio xuthus and pvotenor (Hidaka, 1961), Hestina japonica (Osanai and Arai, 1962), Bombyx (Hashiguchi, 1964), and in Carausius (Raabe, 1966), in Locusta, (Girardie, 1967) and Oedipoda (Moreteau-Levita, 1972a,b), and Leucania separatn (Ogura, 1975). 5.1.2.6 Tanning The tanning process takes place after moulting and is completed very rapidly. It was shown to be controlled by neurohormones, first in flies (Fraenkel and Hsiao, 1965) and then in cockroaches (Mills, 1965), locusts (Vincent, 1972), Manduca (Truman, l973), Tenebrio (Grillot et a l . , 1976). The neurohormone involved in tanning was termed bursicon. It seems to be at least partly involved in regulating the other processes occurring at moulting, i.e. plasticization (Reynolds, 1977), melanization, endocuticle secretion (Fogal and Fraenkel, 1969) and cytolysis of wing epidermis (Seligman and Doy, 1973). On the other hand, puparium formation and anterior retraction of the fly larvae are not due to bursicon but to other neurohormones, i.e. anterior retraction factor (ARF) and puparium tanning factor (PTF) (Zdarek and Fraenkel, 1969). 5.1.2.7 Water and ions Insect water balance is mostly ensured by the Malpighian tubules which remove excess salt and water, and by the rectum, which allows reabsorption to take place. As in other
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types of physiological regulation, two neurohormones acting in opposite ways are assumed to be involved. Histological investigation of various ns cells of insects subjected to hydrating or dehydrating conditions suggested that they may be involved in regulating water balance. Immunocytochemistry showed, moreover, that vasopressin-like immunoreactive cells in the suboesophageal ganglion are probably involved too (Section 2.4). Experimentally, a diuretic neurohormone was shown to be present in blood feeders, which have to eliminate excess water rapidly in order to lose weight (Maddrell, 1963, Rhodnius; Gee, 1975, Glossina austeni; Nijhout and Carrow, 1978, Anopheles freeborni). Other insects including cockroaches, stick insects, locusts, crickets, grasshoppers, bees, beetles, butterflies and bugs, also possess diuretic neurohormone. The existence of antidiuretic hormone has been demonstrated in several species namely in cockroaches, stick insects and locusts (Wall, 1965; Cazal and Girardie, 1968; de Besse and Cazal, 1968; Mordue, 1972). Furthermore, a factor from the corpora cardiaca was shown to stimulate electrogenic transport of C1- in the rectum of locusts (Phillips et al., 1980). 5.1.3 Ecdysis, muscle functioning, and pigment migration 5.1.3.1 Eclosion The time at which eclosion occurs is often species specific. In the saturnid moths Hyalophora cecropia and Antherea pernyi, the eclosion rhythm is suppressed by brain removal and re-established by brain reimplantation, when it becomes the rhythm of the donor insect (Truman, 1971). The active factor termed eclosion hormone stimulates the central nervous system, even in vitro, initiating, within a few minutes, the behaviour pattern characteristic of eclosion, followed by plasticization and wing spreading. Eclosion hormone also affects intersegmental rnuscle breakdown (Schwartz and Truman, 1982). Eclosion hormone was shown to be present in several insects (Taghert et al., 1980) and to regulate larval moulting of Manduca (Copenhaver and Truman, 1982). 5.1.3.2 Activity The part played by neurohormones in regulating flight and activity has been investigated in a number of species, the most extensively studied being locusts (Michel, 1972; Goldsworthy et al., 1973, 1977), cockroaches (Harker, 1956; Brady, 1969), crickets (Cymborowski, 1973; Sokolove and Loher, 1975) and
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Drosophila (Handler and Konopka, 1979). The results are often contradictory and it is not possible to define exactly how flight and activity rhythms are governed. It is certain, however, that the brain controls both processes and that it contains a “biological clock”. In this connection certain extracts have also been shown to affect endogenous nerve activity (Ozbas and Hodgson, 1958). 5.1.3.3 Visceral muscles The presence of myotropic factors acting on the visceral muscles of the heart, gut, Malpighian tubules and oviducts has been reported in cockroaches, stick insects, locusts, Chaoborus larvae, and the bug Iphita. The results, often obtained in vitro, are sometimes contradictory as regards the number of factors involved and their origin and release sites. The corpora cardiaca were at first shown to be myotropic in the cockroach (Davey, 1962), but further investigations demonstrated that the different ganglia of the central nervous system were also active (Gersch, 1955; Ralph, 1962; Rounds, 1963; Girardie and Lafon-Cazal, 1972), and that they often contained both cardioaccelerators and cardiodecelerators (Rounds, 1963; Banks, 1976). 5.1.3.4 Colour A few insects display rapid colour changes which involve either chromatophores, as in Corethru (Chuoborus) or epidermal ommochromes, as in the stick insect, Carausius. In both insects, darkening depends on a chromactivating factor originating in the brain and ventral nerve cord (Dupont-Raabe, 1949, 1951, 1957; Hadorn and Frizzi, 1949; Gersch, 1956). 5.2
SITES OF ORIGIN AND RELEASE
5.2.1 Sites of origin The origin of neurohormones has often been sought in the brain, particularly in the pars intercerebralis. The ventral nerve cord has been much less studied, so that the data relating to it are still fragmentary. The location of neurohaemal factors seems to vary sometimes according to the species studied. Certain neurohormones have been found in the pars intercerebralis or in the brain, but it is not known whether they occur in other ganglia. This is the case for the allatotropic factor, for the factors involved in reproduction and in the sugar and protein metabolism, and for those that stimulate enzyme production by the midgut. Other neurohormones are known to be present in the brain only,
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for instance, brain hormone (Gibbs and Riddiford, 1977; Mala et al., 1977) and EDNH which regulates vitellogenesis in mosquitoes (Hagedorn et al., 1979). Several neurohormones, like the myotropic and chromactivating factors, the factors involved in oviposition, diuresis, antidiuresis and tanning, or ARF and PTF, are present throughout the entire central nervous system or most of it. Bursicon, for example, is distributed throughout this system in Locusta, Periplaneta and Tenebrio, but in Sarcophaga it is only found in the brain and abdominal ganglia, and in Manduca it is predominantly located in the abdominal ganglia (Taghert and Truman, 19824. Other neurohormones are present only in a particular part of the central nervous system, for instance, the diapause hormone of Bombyx, which only originates in the suboesophageal ganglion. This ganglion appears to play a major part in regulating several other processes such as circadian activity, reproduction, water balance or darkening, but in the latter, the origin of the darkening factor varies according to the species. It may originate in the pars intercerebralis of the brain (as in Locusta and Oedipoda), in the suboesophageal ganglion (as in Carausius) or in various ganglionic complexes. These are formed by the brain and suboesophageal ganglion (in Leucania) or by the brain, suboesophageal ganglion and one or several thoracic ganglia (in Papilio, Pieris and Bombyx) or again, by several abdominal ganglia. Finally, the glandular cells of the corpora cardiaca are known to be the source of adipokinetic hormone in locusts (Goldsworthy et al., 1972), but this is probably not their only function. Besides identifying the nerve ganglia involved in neurohormonal activities, attempts have been made to locate the source cells of the neurohormones. The origin of the brain hormone-producing cells has been explored in several species, with irregular results. Recently, the use of an in vitro bioassay allowed identification of a single pair of lateral neurons involved in brain hormone production in Manduca (Agui et a/., 1979). Neurohormone-containing cells have been successfully identified in the brain in a few cases. For example, a factor stimulating ecdysis in Aeshna has been found in two anteromedial ns cells (Charlet, 1974), factors stimulating moulting and tanning in aphids are contained in two pairs of ns cells (Steel, 1978), and a factor triggering gonial mitosis in Panstrongylus, identified in type A ns cells of the pars intercerebralis (Furtado, 1979). The chromactivating factor of stick insects was shown to be absent from the
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pars intercerebralis and to originate in the tritocerebron (DupontRaabe, 1957). In locusts and cockroaches dinresis appears to depend on two pairs of ns cells in the anterior part of the brain (Girardie, 1970; Proux and Buscarlet, 1976; de Besse, 1978) but in other species, it also depends on ns cells located in other ganglia. Several neurohormones appear to originate in the two medioventral ns cells in the suboesophageal ganglion, e.g. the diapause hormone of Bornbyx and Orgyia (Fukuda and Takeuchi, 1967; Kind, 1969), diuretic factor (Proux and Rougon-Rapuzzi, 1980) and darkening factor (Raabe, 1965). In the thoracic and abdominal ganglia, the precise identity of the ns cells producing neurohormones is not always clear. In Rhodnius, the source cells of diuretic hormone have been found to be localized in the abdominal part of the thoracoabdominal mass (Baudry, 1968; Berlind and Maddrell, 1979). Attempts have been made to locate the origin of proctolin by immunochemistry, with conflicting results. However, neurons containing bursicon were identified as six lateral ns cells in Manduca (Taghert and Truman, 1982) and the ns cells releasing oviposition factor in stick insects appear to be the lateral A ns cells of the ventral nerve cord (Thomas and Mesnier, 1973).
5.2.2
Sites of release
The corpora cardiaca and perisympathetic organs have been frequently shown to be involved in neurohormone release. However, when neurohormone production is limited either to the brain or to the ventral nerve cord, it is normal to find neurohormones present only in the corpora cardiaca or in the perisympathetic organs. Thus, release sites may be arranged in different ways: hormones may be present in both the corpora cardiaca and perisympathetic organs, as occurs for bursicon (Grillot et al., 1976), antidiuretic factor (de Besse and Cazal, 1968) and myotropic factors (Raabe et a / . , 1966). Alternatively, they may only be present in either the corpora cardiaca or the perisympathetic organs; in Schistocerca, for example, diuretic hormone is found in the corpora cardiaca (Goldsworthy et al., 1972) whereas in Rhodnius and Glossina it is stored in the perisympathetic organs located in the somatic nerves (Maddrell, 1966; Maddrell and Gee, 1974). Again, the corpora cardiaca only release certain neurohormones such as eclosion hormone (Truman, 1973), PTF and hyper- and hypotrehalosemic neurohormones. Finally, certain neurohormones are released neither in the corpora
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cardiaca nor in the perisympathetic organs and their sites of release are still unknown. These include the chromactivating and oviposition factors of stick insects (Raabe et al., 1966; Thomas and Mesnier, 1973). As already emphasized, many target organs or tissues receive ns innervation. It is believed that in such cases, neurohormones are released within the organs themselves. Brain and allatotropic hormones might be released in this way, as well as the factor which triggers midgut protease production and the myotropic factors which have been observed in nerves and effector organs. Certain ns cells have been shown to follow very unusual pathways, which suggests that the neurohormones synthesiz’ed in these cells may be released at several distinct points, perhaps simultaneously.
5.3
MODE O F ACTION
The action of neurohormones has been proved to involve cAMP as second messenger. cAMP or other nucleotides, stimulate moulting gland activity, ovulation, darkening of the integument, diuretic activity of Malpighian tubules, etc. (Gersch and Birkenbeil, 1973; Denlinger et al., 1978; Matsumoto et af., 1979; Maddrell et al., 1971 among others). They also act on the fat body (causing hypertrehalosemia and adipokinesis) and trigger pupariation and eclosion. The cAMP level has been shown to rise in the target tissue under the action of neurohormones (brain hormone, eclosion hormone, hypertrehalosemic and adipokinetic neurohormones and bursicon). The way in which biogenic amines act on some of their effectors has been investigated too. Serotonin action upon Cuffiphorasalivary glands was shown to involve cAMP (Berridge and Prince, 1971). In metamorphosing Antheraea pernyi and Hyafophoru cecropia, serotonin appears to be the activator of brain adenyl cyclase (Rasenick and Berry, 1981). Adenylate cyclases have been characterized in the brains of Periplaneta, Locusta and Mamestra, and octopaminesensitive adenylate cyclases have been separated from dopaminesensitive ones (see Bodnaryk, 1979a,b). Octopamine receptors are sensitive to the a-adrenergic blocking agent phentolamine, but not to the P-adrenergic blocking agent propanolol. Thus, octopamine receptors may be differentiated from AKH receptors, since the latter are not inhibited by a-adrenergic receptor antagonists (Gole and Downer, 1979).
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5.4
PURIFICATION A N D IDENIIFICATION
Several teams have made considerable efforts to isolate and identify various neurohormones. The identification and synthesis of adipokinetic hormone and proctolin were the results of these efforts. At all events, neurohormones appear to be peptides, destroyed by proteolytic enzymes; their molecular weights range from SO0 to 40 000. Although interspecific activity was demonstrated for several neurohormones, species-specific differences nevertheless exist. Adipokinetic hormone (AKH) was isolated from 3000 glandular lobes of locust corpora cardiaca. It was identified using the lipid mobilization capacity of an adult male locust as a bioassay. AKH was shown to be a decapeptide with a molecular weight of 1158 and the formula Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NHl (Bloomfield and Hardy, 1977). A dose-response curve was established for this hormone, one unit being defined as the quantity of hormone causing the release of 1 p,g of lipid per microlitre of haemolymph in 1 h (Jones et al., 1977). Isolated locust A K H is hypertrehalosemic in the cockroach (Holwerda et al., 1977a,b) but not identical to cockroach factors. A second active peptide, AKHII, was isolated from locust corpora cardiaca (Carlsen et id.,1979). Like AKHI, it acts on crustacean chromatophores (Mordue and Stone, 1976) whose red pigment-concentrating hormone (RPCH) has a similar formula: RPCH: Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NHz AKHI: Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NHZ AKHII: Asp-Thr-Ser-Glu-Gly-Leu-Phe-Trp-NHl The identification of proctolin was the outcome of attempts by several teams to isolate the diuretic, hypertrehalosemic, chromactivating and myotropic factors from the corpora cardiaca and central nervous system (Gersch et al., 1960; Brown, 196.5; Gersch and Stiirzebecher, 1967; Mordue and Goldsworthy, 1969; Natalizi et al., 1970; Traina et al., 1976; Baumann and Gersch, 1982). Proctolin was identified as a basic pentapeptide with a low molecular weight of .500--700 (Brown and Starratt, 197.5). It was synthesized by Starratt and Brown (1977). Its formula is Arg-TyrLeu-Pro-Thr. The threshold of proctolin action on the proctodeal muscles in vitro is about 1 0 - " ~ .It also acts on heart and oviduct contractions, but a second myotropic factor (Holman and Cook, 1979) appears to exist. Brain hormone is certainly the neurohormone whose identifica-
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tion has been most often attempted despite numerous technical difficulties. The most recent results were obtained with Bombyx heads, using brainless Samia pupae as a bioassay. Brain hormone is a protein with the relatively high molecular weight of 4330-4740. A high degree of purification was achieved since 7ng of active substance was obtained starting from three million heads (Nagasawa et a l . , 1980; Suzuki et a / . , 1982). The diapause hormone of Bornbyx has been purified by several investigators. Two factors were obtained. One of them, DHA, contains 14 amino acids and two amino sugars; it has a molecular weight of 3300. The second factor, DHB, has a molecular weight of 2000, and also contains 14 amino acids but no amino sugars (Kubota et al., 1976). Darkening factors have been thoroughly investigated in lepidopterans, using Bornbyx as the donor for the isolation of melanization and reddish colouration hormone (MRCH), and the isolated abdomen of Leucania serving as the bioassay. The active darkening factor was purified, but has not yet been isolated (Suzuki et al., 1976; Matsumoto et a / . , 1981). The factors involved in sugar regulation are certainly distinct from myotropic factors. I t is conceivable that they may be identical to the vertebrate metabolic hormones, insulin and glucagon. Octopamine may also play a part in such regulation. The diuretic and antidiuretic factors have been investigated in Periplaneta, Rhodnius, Glossina and Danaus. The only known characteristics of antidiuretic hormones is the molecular weight, which in Periplaneta is between 8000-10 000. The molecular weight of diuretic hormone has been estimated at 30 000 for Periplaneta, 1200 for Glossina, 60000 and 2000 for Rhodnius and 3000 for Danaus (Goldbard et al., 1970; Gee, 1975; Aston and White, 1974; Dores et at., 1979). Bursicon, A R F and PTF have also been explored. Their molecular weights are respectively 38 000-40 000, 180 000 and 300 000 (Fraenkel et al., 1966; Mills and Nielsen, 1967; Sivasubramanian et at., 1974; Zdarek et al., 1981).
6 Conclusion
The field dealt with by this review of insect ns cells covers a large body of research. It includes the morphology of these cells as well as their functioning, location and release sites, their relationship with
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vertebrate peptides and biogenic amines, and the numeroiis physiological and biochemical processes regulated by neurohormones. Neurohormones obviously play a major part in regulating insect homeostasis. Their number is considerable, since some factors may fulfil several roles simultaneously. The action of all neurohormones involves CAMP as a second messenger. Their purification is being actively investigated, and two neurohormones have so far been isolated.
Acknowledgements
I wish to thank those co-workers from whom I received support during the writing of this review, Christine Vermeillet for typing the manuscript, and Daisy Chervin for drawing and reference work. Lastly, I am grateful to the Centre National de la Recherche Scientifique for sponsoring my work.
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Subject Index passim indicates here and there throughout. Page numbers in italics indicate references to figures or tables. A Absorption of tracheole fluid, 119123 muscular activity and, 124-127, 131-132 Adaptive responses of tracheoles, 1 I(k115 Adipokinetic hormone actions, 162-181, 190, 192, 194 chemical nature, 160-162 control of release. 158-160, 233 dynamics of release. 157-158 site o f synthesis and release, 156157 Air, tracheoles filling with. 119123,123-133,137 Amines (see also Octopamine) neurosecretory-neurohaemal system and. 207,230-238. 240,253,254-255.263 in sclerotization. 31-32, 35, 45, 52-72 passim Amino acid(s) in chitin-protein bonds. 46-47 composition of adipokinetic hormone, 16& 162 of apoproteins, 164-165 of cuticle proteins, 10-38 passim, 49 of neurohormones, 209, 225. 228
in crosslinking, 39-45,48.55,68 in substrate provision, 154 Anaerobic metabolism and tracheolar fluid, 130 Anal papillae respiratory function, 102-103 tracheole filling in, 127-128. 129 Anastomosis of tracheoles. 87-88. 109
Artefacts in haemolymph lipoprotein preparation, 172 Autoregulation in neurosecretory cells, 261-262 Autotanning, 5 1. 57-58
B Basal lamina. tracheoles and. 87. 135 C Capillarity and fluid level in tracheoles, 123-124. 125, 129.133-134. 137-138 Capture, tracheole, 115-1 19 Carbohydrate in haemolymph, 150, 151 mobilization of, 150, 174-175 neurohormones and. 268-26'1, utilization of. 152-1 53. 1 7 6 180. 181.183.184-186 305
306
Catechols (set. also Amines) in crosslinking, 52-72 passim in neurosecretory system, 23& 238 Cement rings in tracheoles. 9(W1 Central nervous system neurohormones in, 272-274 neurosecretory cells in, 2 1G229, 256258 amines i n , 231-234,237-238 and corpora cardiaca, 242-244 and neurohaemal areas, 250252 and perisympathetic organs, 244-249 regulation of, 262-265 oxygen supply, 101-102 Chitin, 32-36 alignment o f , 54 kinetics of deposition, 33,3('38 lifestage and. SO -protein bonds, 4 6 4 7 . 4 8 . 6 8 Chromatography in protein composition. 12. IS, 28 to identify crosslinking amino acids, 4(b42,43 ofapoprotein, 167. 171, 188-389 Comparative endocrine control. 184-194 Composition of adipokinetic hormone, 16(L 162 o f apoproteins. 164-165 o f cuticle proteins, 10b12. 14-38 o f sclerotized tissue, 38-5 1 Corpora cardiaca and adipokinetic hormone. 15& 157, 158-159. 160, 174 and control of flight metabolism. 184, 192-193 neurohaemal organs. 206. 209. 222,242-244
SUBJECT INDEX
amines in, 231-233,238 immunochemistry in 223-38 pussim and neurohaemal areas, 25025 1 neurohormone release in, 242, 243-244 oxygen supply, 101 Corpus allatum and adipokinetic hormone response, 173 and corpora cardiaca, 242,251 and flight muscle tracheoles. 112 neurohormones and, 267 oxygen supply. 10 I Crosslinking chemical mechanisms of, 51-72 in sclerotization, 3-4,s-6, 19, 38-46,48-49.50-51 Cuticle (see also Sclerotization) deposit ion kinetics of. 3G38 in tracheal system, 91 structure, 38-5 1 Cuticular proteins composition and preparation, 12-38,
[email protected],60, 6 1-63 quinone reactions, 5&57 structure. 18-19,32-35, 5 0 , 54 elastic forces and tracheole fluid. 129-130 synthesis, 8-12,33,36-38 Cyclic nucleotides and adipokinetic hormone, 162163, 175, 179 and neurohormones, 275 octopamine and, 182, 183, 192 Cytoplasm, tracheoblast, 94, 112 Cytoplasmic filaments in tracheole movement, 11&118
SUBJECT INDEX
inclusions in neurosecretory cells, 207. 260 sheaths of tracheoles, 118,130 vacuoles in tracheole formation, 88 D Deposition, cuticle, 33,36-38,91 Diacylglycerols in haemolymph, 151, 155 mobilization, 151, 153, 186-190 adipokinetic hormone and, 156, 158,159-160, 162-174 octopamine and, 182, 193 trehalose and, 159-160 utilization, 153, 17&180, 181, 183 Diffusion in respiration, 98. 99-100, 102- 104 Dimerization in sclerotization, 5-8, 19,23.57 mechanisms, 57.61-64.70 Dopamine (see a h Amines) in neurosecretory system, 234, 235,237 in sclerotization, 55.56.61-66. 68-7 1 DUM neurones, 233-234
E Ecdysis fluid absorbed at, 119-120 neurohormones and, 266.271 tracheoles in, 89-90, 94, 110 Ecdysone neurohormones and, 266 and neurosecretory cells, 264 tracheoblast and, 94, 112 Ecdysone-juvenile hormone control o f cuticle protein turnovcr, 38
307
Edman degradation, 24,30,36 Elastic forces of protein structure and tracheole fluid, 129-130 Electron microscopy of lipoproteins, 189 of neurohaemal organs, 243,254. 260 of neurosecretory cells, 206.208209, 212,213-214,215,231. 260 of tracheal gills, 102 of tracheoles, 87. 93, 97, 105. 109 Electrophoregis of apoprotein, 164. 167, 170-172. 173 in protein composition. 12-13, 14-15, 19-31 puJ,im Endocrine control (see a1.w Neurosecretory-neurohaemal system) of cuticle protein turnocer, 38 of flight metabolism in locu\ts, 149-151 basic features of flight metabolism. 152-155 comparative overview, 18 194 hormones and flight, 155- 84 Endoplasmic reticulum and neurosecretory material synthesis, 208-209, 260 tracheoblast and ecdysone. 94 Enzymic activity of cuticular proteins, 16-18,32, 33-36.5 1 in fat body, 174-175 in flight muscle mitochondria. 114 Epicuticle in tracheal system, 91-94 Epidermal cells and tracheole migration, 1 1 6 119
308
SUBJECT I N D E X
F Fat body fuel reserves in. 150 glycogenolysis, 174-175 mobilization of, 153. 156, 162163, 164-174 neurohormones and, 268-269 octopamine in, 182-183. 193, 235-236 oxygen supply, 101 protein synthesis in, 180 tracheoles in, 87, 94 Feedback in neurosecretory system. 263-265 Filaments taenidial, 9 1 , 9 3 4 4 in tracheole capture, 116-1 18 Flight metabolism, endocrine control of, in locusts, 149151 basic features of, 151-155 comparative overview, 184-194 hormones and flight, 155-184 Flight muscles juvenile hormone and, 155 metabolism, 175-180, 183, 186 octopamine in, 181, 183,192-193 tracheoles in, 92, 104, 105-110, 111-112,113-115,131-132 anastomosis in, 88, 109 oxygen and, 99-100,101 permeability, 138-139 transport system in, 15@151 Fluid absorption in tracheoles, 119-123 movement in tracheoles, 123133,137 Folds in tracheoles, 91,93 Formation matrix, in sclerotization, 5-9, 39-5 1 Dassim
plasma membrane imagination. 112-113 of tracheoles, 88-89 Fuels, respiratory in flight muscles, 150-151. 152 mobilization of, lS(b151, 162175,186-192 utilization of, 152-155, 17G180, 181 184-186
G Gluconeogenesis, 185-186 Glucose from amino acids, 185-186 in haemolymph, 154 octopamine and, 183 Glycerol release in flight, 154155, 175 Glycogen as energy reserve, 150, 151 glycogenolysis, 174-175 neurohormones and, 268-269 Glycolysis, adipokinetic hormone and, 178,179-180,186 Glycosylated components of cuticle, 32-36 Granules, secretory adipokinetic hormone, 15G157, 158 in neurosecretory cells, 206,207, 208-210,214,254,260 release of, 240-242,249-250 transport of, 238-239
H Haemol y mph adipokinetic hormone in, 157158,159,176 diacylglycerols in, 151,153,159160,164-174,176-179,186190 neurohormones in, 230
309
SUBJECT INDEX
octopamine in. 181-183, 192, 234-235 respiratory fuels in, 15Ob151. 154 184-185 trehalose, 151, 152-153, 159, 176178,269 Histochemistry of neurosecretory cells, 213 Histology of neurohaemal organs, 243.245 of neurosecretory cells, 207-217. 256 of tracheoles, 91-95 visualization, 95-98 Histophysiological studies of neurosecretory cells, 258259 Homology in cuticular proteins, 16, 21,24-26 Hypolipaemic factor, and flight metabolism, 184, 192, 193 I Immunological techniques on apoproteins, 171 on cuticular proteins, 16. 50 on neurosecretory cells, 209, 222-230 and protein synthesis, 9-10 Injection of dyes in tracheoles, 9598 Insecticides, action of, 37-38,49 Insulin-like activity, 184, 192, 193, 224 Intima of tracheal system, 91-93, 128-129,135 staining, 95, 98 Invasion of cells by tracheoles. 104113 Ion absorbing epithelia and tracheal gills, 102-104 tracheole filling in, 127-128, 129
J Juvenile hormone control of cuticle protein turnover. 38 and neurosecretory cells, 264265,267 K Ketone bodies. mobilization o f , and flight, 154 Kinetics of chitin-protein deposition, 33, 3638 of crosslinking, 46 of dimer assembly, 5-8 of polymer assembly, 12
L Lipids (see also Diacylglycerols) as respiratory fuel, 151-193
passim and tracheoles, 87,91-93,98 Lipoprotein carrier complexes, 15 164174,179, 186190 M Matrix formation in sclerotization 5-9,39-5 1 passim Mechanisms, crosslinking, 51-72 Metabolism flight, endocrine control of, 149151 basic features of, 151-155 comparative overview, 184194 hormones and flight, 155-184 neurohormones and, 268-271 Metabolites accumulation of in flight, 131-132 and tracheolar fluid absorption, 123-124,125126,129, 13C132 supply of, 15 1
310
Metamorphosis octopamine and, 237 tracheal system in. 89. 1 11-1 12, 113-1 14 Microtubules tracheoblast, 94 tracheolar. 95 Mi tochondria adipokinetic hormone and, 179 tracheoblast. and ecdysone, 94 and tracheoles, 87, 101-103 in flight muscle, 92, 105, 108109, 113-115,131 Morphology DUM neurones, 234 neurosecretory cells. 207-208 perisympathetic organs, 245-246. 247 tracheoblast, 94, 112 Movement o f fluid in tracheoles, 123-133, 137 by tracheoles, 95, 110-113, 115119 Muscular activity and fluid absorption in tracheoles, 124-127, 131-132 neurohormones and, 271-272 N Nervous control (see also Neurosecre tory-neurohaemal system) of adipokinetic hormone release, 158-159, 160 of flight metabolism, 184, 192-193 of fluid absorption in tracheoles, 122,133 of neurohormone release, 239240,244,253,262-263,265 by neurosecretory cells, 258 of photogenic cells, 134-136
S U B J E C T INDEX
Neuroeffector junctions, 253-255 Neurohaemal areas (see also Corpora cardiaca and Perisympathetic organs), 250-253 Neurohormones, 26&277 Neurosecretory-neurohaemal system, 205-207 and flight metabolism. 184 neurohormone production, 207238 neurohormone release, 238-258, 274-275 neurohormones, 266-277 regulation of neurohormones, 258-266 Nucleic acid synthesis, 9-10, 2&21, 36-37
0 Octopamine (see also Amines), 181-183.192 and adipokinetic hormone release, 159 and nervous system, 231-237,254 Osmotic pressure and fluid level in tracheoles, 123-129, 136, 137- 138 Ovary oxygen supply, 101 tracheoles in, 87 Oxygen in control of photogenic cells, 134136 lack and fluid absorption in tracheoles, 122, 124-127, 13@132 and tracheal supply, 116, 117 supply and permeability of tracheoles, 138-139
31 1
SUBJECT INDEX
uptake and mitochondria, 101-102 partial pressure and, 100 P Pars intercerebralis neurohormones in, 272,273 neurosecretory cells in. 208-209, 211-213,215-217 amines in, 23 1-233,238 immunochemistry in, 222-228 passim regulation of, 262,265 Peripheral neurosecretory cells, 22 1-222 amines in, 233-234 immunochemistry in, 222,224, 225,229 and neurohaemal areas, 2.52 regulation of, 263 Perisympathetic organs, 206,221222,244250 Permeability of tracheoles, 136, 137- 139 Photogenic organs, tracheolar supply to, 134-136 Phylogenetic considerations in study of sclerotization, 7-9 Pigmentation neurohormones and, 270,272 partial proteolysis of chitinprotein complex and, 34-36 sclerotization and, 43,44, SOM1,
54-55 Plasma membrane of tracheoblasts, 74 tracheoles and, 92, 10.5-106, 107, 112-1 13 Polymerization in sclerotization, 19,39-43.47 in tracheolar epicuticle, 93
Prawn red pigment concentrating hormone. 160- 162, 163 Preparation of cuticular proteins. 12-38,4G 42.47-S1,60,61-63 of tracheoles for visualization, 95-98 Protein synthesis adipokinetic hormone and. 180 neurohormones and, 269 tracheoles and mitochondria and. 101-102 Proteins, cuticular composition and preparation, 1238,40-42,47-51.60.61-63 quinone reactions, 5 6 5 7 structure, 18-19,32-35, SO, 54 elastic forces of. and tracheole fluid, 129-130 synthesis, 8-12,33,36-38 Proteol ysis partial in sclerotization. 8 in study of cuticular proteins, IS16,32,33-36,38,47-48.61 artefactual, 12-13 Protomer-matrix formation in sclerotization, 3-9 Puparium, sclerotization in, 7-8. 38-47
Q Quinones in crosslinking, 43,49. 52-60,71-72 methides, 6&70,71-72 R Rectal papillae, oxygen supply i n , 101, 102-103 Regulation of neurosecretory cells, 2.58-266 water, 27G271 Relatedness in cuticular proteins, 16,21,2626
312
Release adipokinetic hormone, 156-160, 233 glycerol, in flight, 154-155,175 neurosecre tory granule. 240-242. 249-250 octopamine, 181 Reproduction, neurohormones in, 267,268 Respiratory fuels in flight muscles, 150-151, 152 mobilization o f , 150-151, 162175,186-192 utilization of, 152-155. 17fj-180, 181,184-186 Respiratory function of tracheoles, 98-104,125-126 Restoration of tracheation. 115-1 19 S Sarcoplasrnic reticulum of flight muscles, tracheoles and, 105-106 Sclerotization in Dipterans, 1-3, 73-75 chemical mechanisms of crosslinking, 51-72 composition and preparation of proteins, 10-38 composition of sclerotized tissue, 38-5 1 protomer-matrix transformation, 3-9 Size limits in tracheoles, 104 Spectroscopy in protein structure, 18,63 Spiracles, 100. 102, 121, 125, 126 Staining of neurosecretory cells, 206.209212,214-216,231 in protein resolution, 21-22 of tracheoles, 90,Ol-93, 95-98
S U B J E C T INDEX
Storage adipokinetic hormone, 1 5 1 5 7 , 158 fuel, 152,174-175 of neurosecretory material, 210, 259-260 Structure of adipokinetic hormone, 1 6 s 162 of cuticle, 38-51 of cuticular protein, 18-19,3235, 50, 54 elastic forces of, and tracheole fluid, 129-130 of tracheoles, 87,91-96 Surface tension in tracheoles, 132133 Synaptic contacts of neurosecretory cells, 208,254,255,256-258 transmission, oxygen and, 102 Synthesis adipokinetic hormone, 156157 of neurosecretory products, 20821 0 nucleic acid, 9-10,2(&21,36-37 protein, 8-12,33,36-38, 101102, 180,269
T Taenidial filament in tracheoles, 91, 93-94 Tanning (see ulso Sclerotization) auto, 51,57-55 neurohormones and, 269 quinone, 59-60,7 1 Temperature and fluid absorption in tracheoles, 172, 125-126, 135 Time course of sclerotization, 6-7, 8-9,33,45 protein synthesis, 10,36-7
313
SUBJECT INDEX
Tracheae in ecdysis, 89-91 respiratory function and, 98, 99 restoration of, 115-116 staining, 95-97 tracheoles originating in, 86,88, 94 Tracheal gills, 102-104 Tracheoblasts, 8 6 8 7 , 88-89, 94, 105, 110-111,112-113 in photogenic organs. 135-136 Tracheoles, 85 adaptive responses, 110-1 15 air in, 119-123 definition and format, 8&91 to firefly photogenic organs, 134136 fluid in, 123-134 histology and histochemistry ,9195 intracellular, 104-1 10 permeability and, 137-139 respiratory functions, 98-104 restoration of, 115-119 visualization, 95-98 Transport of neurosecretory granules, 238239 of respiratory fuels, 150-151, 162-175,186-190,193-194 Trehalose and adipokinetic hormone release, 159
in haemolymph, 151,152,175 neurohormones and, 268-269 utilization of, 152-153, 176180, 181 Triacylglycerol as energy reserve, 150,162 Tubule system of flight muscles, tracheoles in, 105-110,111-112 V Ventral nerve cord ganglia neurohormones in, 273-274 neurosecretory cells in, 210,211. 213,215,216,218-220,256 257 immunochemistry in, 222-228 and neurohaemal areas, 252 and perisympathetic organs, 244-249 regulation of, 263,265
w Water regulation, neurohormones in, 270-27 1 sclerotization and, 5,39-40,42, 43,68-69
X X-ray analysis in cuticle structure, 46-47 in protein structure. 16, 19
Cumulative List of Authors Numbers in bold fuce indicute the vafume numbers of the series Aidley, D. J . , 4, 1 Andersen, Sven Olav, 2, 1 Ashburner, Michael, 791 Ashini, E., 6, 1 Baccetti, Baccio, 9, 315 Barton Browne, L., 11,l Beament, J . W. L., 2, 67 Beetsma, J . , 16, 167 Bernays, E. A., 16, 59 Berridge, Michael J . , 9, 1 Bodnaryk, Robert P., 13,69 Boistel, J . , 5 , 1 Brady, John, 10, 1 Bridges, R . G., 9,51 Burkhardt, Dietrich, 2,131 Bursell, E., 4, 33 Burtt, E. T . , 3, 1 Calhoun, E. H . , 1, 1 Carlson, A . D., 6,51 Catton, W. T., 3, I Chapman, R . F., 16, 247 Chen, P. S., 3,53
Cottrell, C. B., 2, 175 Crossley, A. Clive, 11, 117 Dadd, R . H., 1,47 Dagan, D., 8,96 Davey, K. G . , 2,219 Edwards, John S., 6, 97 Eisenstein, E. M., 9, 111 Elmer, Norbert, 13, 229 Engelmann, Franz, 14,49 Evans, Peter D., 15, 317 Gilbert, Lawrence I . , 4,69 Gilby, A . R . , 15, I Goldsworthy, G. J . , 17,149 Goodman, Lesley, 7, 97 Harmsen, Rudolf, 6, 139 Harvey, W. R . , 3, 133 Haskell, J . A . , 3, 133 Heinrich, Bernd, 13, 133 Henzel, W., 17, 1 Hinton, H. E., 5,65 314
Howells, A . J . , 16, I19 Hoyle, Graham, 7, 349 Jungreis, Arthur M., 14,109 Kafatos, Fotis C., 12, 1 Kammer, Ann E., 13, 133 Kilby, B. A , , 1, 111 Lane, Nancy J . , 15, 35 Lawrence, Peter A., 7, 197 Lees, A . D . , 3,207 Linzen, Bernt, 10, 117 Lipke. H . , 17, 1 Machin, John, 14, 1 Maddrell, S. H. P., 8, 200 Michelsen, Axel, 10, 247 Miles, P. W., 9, 183 Miller, P. L., 3,279 Morgan, E. David, 12, 17 Narahashi, Toshio, 1, 175; 8, 1 Nelson, Dennis R., 13, 1
CUMULATIVE L I S T O F AUTHORS
Neville, A. C., 4,213 Njio, K. Djie, 14, 185 Nocke, Harold, 10, 247 Palka, John, 14,251 Parnas, I . , 8,96 Pichon, Y., 9,257 Piek, Tom, 14, 185 Pyliotis, N. A., 16, 119 Poole, Colin F., 12, 17 Popov, Andrej V., 13,229 Prince, William T., 9, 1 Pringle, J. W. S . , 5, 163 Raabe, M . , 17,205 Reynolds, Stuart E., 15,475 Riddiford, Lynn M . , 10,297
Rowell, C. H. F., 8, 146; 12,63 Rudall, K. M., 1,257 Sacktor. Bertram, 7, 268 Sander, Klaus, 12, 125 Sattelle, David B., 15,215 Shaw, J . , 1,315 Simpson, S. J., 16,59 Skaer, Helen IeB., 15,35 Smith, D . S., 1,401 Staddon, Brian W., 14,351 Steele, J. E., 12,239 Stobbart, R. H., 1, 315 Sugumaran, M . , 17, 1 Summers, K. N., 16, 119 Telfer, William H., 11,223
315
Thomson, John A . , 11,321 Treherne, J. E., 1, 401 ; 9,257 Truman, James W., 10,297 Usherwood, P. N. R . , 6,205 Waldbauer, G. P., 5, 229 Weis-Fogh, Torkel, 2, 1 White, Richard H., 13,35 Wigglesworth, V. B., 2,247; 17,85 de Wilde, J., 16, 167 Willmer, P. G., 16, 1 Wilson, Donald M.. 5,289 Wyatt, G . R . , 4,287 Ziegler, Irmgard, 6, 139
Cumulative List of Chapter Titles Numbers in bold face indicate the volume number of the series Acetylcholine Receptors of Insects, 15, 215 Active Transport and Passive Movement of Water in Insects, 2, 67 Amino Acid and Protein Metabolism in Insect Development, 3, 53 Atmospheric Water Absorption in Arthropods, 14, 1 Biochemistry of Sugars and Polysaccharides in Insects, 4, 287 Biochemistry of the Insect Fat Body, 1, 111 Biogenic Amines in the Insect Nervous System, 15, 317 Biology of Eye Pigmentation in Insects, 16, 119 Biology of Pteridines in Insects, 6, 139 Biophysical Aspects of Sound Communication in Insects, 10, 247 Cells of the Insect Neurosecretory System: Constancy, Variability, and the Concept of the Unique Identifiable Neuron, 12, 63 Cellular Mechanisms Underlying Behaviour-Neuroethology, 7, 349 Chemoreception: The Significance of Receptor Numbers, 16, 247 Chitin Orientation in Cuticle and its Control, 4, 213 ChitiniProtein Complexes of Insect Cuticles, 1, 257 Choline Metabolism in Insects. 9, 51 Calour Discrimination in Insects, 2, 131 Comparative Physiology of the Flight Motor, 5, 163 Consumption and Utilization of Food by Insects, 5, 229 Control of Food Intake, 16, 59 Control of Polymorphism in Aphids, 3, 207 Control of Visceral Muscles in Insects, 2, 219 Cytophysiology of Insect Blood, 11, 117 Development and Physiology of Oocyte-Nurse Cell Syncytium, 11, 223 Effects of Insecticides in Excitable Tissues, 8, 1 Electrochemistry of Insect Muscle, 6, 205 Endocrine control of Flight Metabolism in Locusts, 17, 149 Excitation of Insect Skeletal Muscles, 4, 1 Excretion of Nitrogen in Insects, 4, 33 Extraction and Determination of Ecdysones in Arthropods, 12, 17 Feeding Behaviour and Nutrition in Grasshoppers and Locusts, 1, 47 Frost Resistance in Insects, 6, 1 316
C U M U L A T I V E L I S T O F CHAPTER TITLES
317
Function and Structure of Polytene Chromosomes During Insect Development, 7, 1 Functional Aspects of the Organization of the Insect Nervous System, 1, 40 1 Functional Organization of Giant Axons in the Central Nervous System of Insects: New Aspects, 8, 96 Hormonal Control of Metabolism in Insects. 12, 239 Hormonal Mechanisms Underlying Insect Behaviour, 10, 297 Hormonal Regulation of Growth and Reproduction in Insects, 2, 247 Image Formation and Sensory Transmission in the Compound Eye, 3, 1 Insect Blood-Brain Barrier, 9, 257 Insect Ecdysis with Particular Emphasis on Cuticular Hardening and Darkening, 2, 175 Insect Flight Metabolism, 13, 133 Insect Sperm Cells, 9, 315 Insect Visual Pigments, 13, 35 Insect Vitellogenin: Identification, Biosynthesis, and Role in Vitellogenesis, 14, 49 Intercellular Junctions in Insect Tissues, 15, 35 Integration of Behaviour and Physiology in Ecdysis, 15, 475 Learning and Memory in Isolated Insect Ganglia, 9, 111 Lipid Metabolism and Function in Insects, 4, 69 Long-Chain Methyl-Branched Hydrocarbons: Occurrence, Biosynthesis, and Function, 13, 1 Major Patterns of Gene Activity During Development in Holometabolous Insects, 11, 321 Mechanisms of Insect Excretory Systems, 8, 200 Mechanisms of Sclerotization in Dipterans, 17, 1 Metabolic Control Mechanisms in Insects, 3, 133 Microclimate and the Environmental Physiology of Insects, 16, 1 Morphology and Electrochemistry of Insect Muscle Fibre Metabolism, 14, 185 Nervous Control of Insect Flight and Related Behaviour, 5, 289 Neural Control of Firefly Luminescence, 6, 51 Neuroethology of Acoustic Communication, 13, 229 Neurosecretory-neurohaemal system of Insects; Anatomical, Structural and Physiological Data, 17, 205 Osmotic and Ionic Regulation in Insects, 1, 315 Physiological Significance of Acetylcholine in Insects and Observations upon other Pharmacologically Active Substances. 1, 1 Physiology of Caste Development in Social Insects, 16, 167 Physiology of Insect Circadian Rhythms, 10, 1
318
C U M U L A T I V E L I S T O F CHAPTER TITLES
Physiology of Insect Tracheoles, 17, 85 Physiology of Moulting in Insects. 14, 109 Polarity and Patterns in the Postembryonic Development of Insects, 7, 197 Postembryonic Development and Regeneration of the Insect Nervous System, 6, 97 Properties of Insect Axons, 1, 175 Regulation of Breathing in Insects, 3, 279 Regulation of Intermediary Metabolism. with Special Reference to the Control Mechanisms in Insect Muscle, 7, 268 Regulatory Mechanisms in Insect Feeding. 11, 1 Resilin. A Rubberlike Protein in Arthropod Cuticle, 2, 1 Role of Cyclic AMP and Calcium in Hormone Action, 9, 1 Saliva of Hemiptera, 9, 183 Scent Glands of Heteroptera, 14, 351 Sequential Cell Polymorphism: A Fundamental Concept in Developmental Biology, 12, 1 Specification of the Basic Body Pattern in Insect Embryogenesis, 12, 125 Spiracular Gills, 5, 65 Structure and Function of the Insect Dorsal Ocellus, 7, 97 Structure and Function of Insect Peptides. 13, 69 Theories of Pattern Formation in Insect Neural Development, 14, 251 Transpiration, Temperature and Lipids in Insect Cuticle, 15, 1 Tryptophan+Ommochrome Pathway in Insects, 10, 1 17 Variable Coloration of the Acridoid Grasshoppers, 8, 146
F
t
