Advances in Insect Physiology, Volume 21

Advances in Insect Physiology

Volume 21

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Advances in Insect Physiology edited by

P. D. EVANS

and V. B. WIGGLESWORTH Department of Zoology, The University Cambridge,England

Volume 21

1988

ACADEMIC PRESS Harcourt Brace Jovanovich, Publishers London

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ACADEMIC PRESS LIMITED. 24j28 Oval Road London N W I 7DX United Siotes Edition published by ACADEMIC PRESS INC. San Diego, CA 92 I0 I

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British Library Cataloguing in Publication Data Advances in insect physiology Vol. 21 1. Insects. Physiology. Serials 595.7'0 1'05 ISBN 0-12-024221-4

Filmset by Eta Services (Typesetters) Ltd. Beccles, Suffolk and printed in Great Britain by St Edmundsbury Press, Bury St Edmunds, Suffolk

Contributors E. A. Howes

AFRC Unit of'Insect Neurophysiology and Pharmacology, Department of Zoology. University of Cumbridge, Downing Street, Cumbridge CB2 3ET, U K A. M. Lackie

Department oj Zoology, The University, Glasgow G12 8QQ, Scotland, UK P. J. S . Smith

A FRC Unit Insect Neurophysiology and Pharmacology, Department of Zoology, University of Cumbridge, Downing Street, Cambridge CB2 3ET, U K M. Sugumaran

Department of Biology, University of Massachusetts at Boston, Dorchester, Massachusetts 02125, USA J. E. Treherne

AFRC Unit qflnsect Neurophysiology and Pharmacology, Department of Zoology, University (if Cumbridge, Downing Street, Cambridge CB2 3ET, U K J. W. Truman

Depar tment qf Zoology, University of ' Washingt on, Seut tle, Washington 98195, USA

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Contents Contributors

V

Hormonal Approaches for Studying Nervous System Development in Insects J. W. TRUMAN

1

Neural Repair and Regeneration in Insects J. E.TREHERNE, P. J. S. SMITH and E. A. HOWES

35

Haemocyte Behaviour A. M . LACKIE

85

Molecular Mechanisms for Cuticular Sclerotization M. SUGUMARAN

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Subject Index

233

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Hormonal Approaches for Studying Nervous System Development in Insects James W . Truman Department of Zoology, University of Washington, Seattle, Washington 98195, USA

1 Introduction 2 Patterns oPnervous system development 2 . I Development of the embryonic nervous system 2.2 Postembryonic development of the nervous system 3 Endocrine regulation of insect development 3.1 Hormonal regulation of development 3.2 Techniques for endocrine manipulation of development 4 Hormones and embryonic development 5 Hormones and postembryonic development 5. I Ganglion migration 5.2 Development of the sensory system 5.3 Neurogenesis and neuronal differentiation 5.4 Restructuring of larval neurons 5.5 Control of neuronal death 6 Conclusions and future directions Acknowledgements References

1 Introduction

The hallmark of the nervous system is the specificity of interconnections made between cells. Many neurons are produced as unique individuals and these in turn make selective connections with other unique individuals. The mechanisms which produce this specificity are gradually being elucidated and include a variety of local signals. For example, the selection of a cell to become a neuroblast in the central nervous system (CNS) (Doe and Goodman, 1985) or a bristle mother cell in the periphery (Wigglesworth, 1940) occurs through local interactions within small groups of potential precursor cells. Similarly, axon guidance in the developing CNS appears to depend on the recognition of cell-surface molecules encountered along the path of outADVANCES I N INSECT PHYSIOLOGY VOL. 21 ISBN U 124242214

Copyright 01988 Academic Press Limited A// rights of reproductionin any form reserved

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growth (e.g. Bastiani et al., 1985). Besides these local signals and interactions, the nervous system also makes use of “global” cues such as those provided by the endocrine system. Hormones are potent intercellular signals that initiate or modulate numerous physiological and developmental processes. Their widespread presence in the blood allows coordination of developmental responses of diverse tissues. They can also have selective effects on a particular tissue or subset of cells depending on the distribution of their receptors. Extensive studies have been carried out on the effects of hormones such as the gonadal steroids on the development of the CNS of various vertebrates (Arnold and Gorski, 1984). Their presence during specific critical periods early in life results in dramatic effects on size, dendritic branching, and survival of neurons in selective regions of the brain and spinal cord. Such changes are stable and persist through the life of the animal. In insects, the role of hormones in the development of the embryonic nervous system is largely unexplored. Their effects during postembryonic life, by contrast, have received more attention and are better understood, especially for the holometabolous forms. These insects produce a simplified larval stage with a reduced CNS and reduced sensory systems. Much of their nervous system development has been transferred to postembryonic stages, thereby coming under control of the endocrine cues that regulate larval growth and metamorphosis. The purpose of this review is not to provide a catalogue of examples documenting that hormones can trigger various aspects of nervous system development. From our knowledge of moulting, metamorphosis and their endocrine control, one could justifiably assume that this would be the case. Rather, I hope to illustrate how hormones can be used to study the cellular responses and cellular interactions that underlie the development of the nervous system. Also, hormones serve as an important entry point to the molecular events that mediate these changes. The scope of this article has been narrowed to systems in which some knowledge of the cellular changes are known, and I have confined my considerations to juvenile hormone and the ecdysteroids. In interests of space, I will not deal with peptide hormones, such as eclosion hormone, which also have developmental effects on the CNS (e.g. Levine and Truman, 1983).

2 Patterns of nervous system development

2. I

DEVELOPMENT OF THE EMBRYONIC NERVOUS SYSTEM

Our knowledge of the development of the insect nervous system comes pri-

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00 00 00000 0 00000 0000000000000 0000 0000 00000 0 00000 00000 8 00000 0000 r\\ 0000

Fig. 1 An early embryo of a grasshopper showing the set of 61 neuroblasts (NB) and 7 midline precursor cells (MP) that generate the neurons of a segmental ganglion. Each NB undergoes a series of unequal divisions to produce a chain of ganglion mother cells, each of which divides equally to form two neurons; the MPs divide only once to produce two daughter cells.

marily from studies on the optic lobes (Meinertzhagen, 1973), the antenna1 lobes (Hildebrand, 1985), and the segmental ganglia. Most hormonal studies have focused on the latter ganglia so I will confine my comments to them. Work late in the last century (e.g. Wheeler, 1891) established that the segmental ganglia are produced from a set of stem cells (termed neuroblasts) that segregate from the ventral ectoderm after germ-band formation. It was not until 85 years later, however, that a key observation was made with Locusta embryos that these neuroblasts are arranged in a stereotyped array and that individual stem cells have a fixed position in this array (Fig. I; Bate, 19764. This segmental pattern repeats with minor variations throughout the ventral CNS so that ganglia as different as those in the thorax and abdomen are nevertheless produced from a similar set of neuroblasts. Each neuroblast produces a lineage of neurons by a repeated series of unequal divisions. The smaller product of each division, a ganglion mother cell (GMC), subsequently undergoes an equal division, thereby producing two daughter cells which then differentiate into neurons. For at least the early neurons produced in each lineage, the cellular phenotype of a given cell is determined by its parent neuroblast and the order of birth of its GMC (e.g. Taghert and Goodman, 1984). The mechanism by which the two daughter

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cells of a given GMC may assume different fates apparently involves interactions between the two cells after their birth (Kuwada and Goodman, 1985). Each segment also contains a small number of median precursor cells. Unlike the neuroblasts, these cells undergo a single, symmetrical division to produce two daughter neurons (Bate and Grunewald, 1981). These cells are among the first neurons to be born. They, along with early neurons produced by selected neuroblast lineages, extend out the first axons in the CNS and act as central pioneer neurons, establishing the characteristic pattern of longitudinal tracts and transverse commissures that make up the segmental architecture of the neuropil. These initial pathways are then used to guide the growing axons of the neurons that are to arise later in development (e.g. Bastiani et al., 1985). A typical segmental ganglion in Locusta is produced by 61 neuroblasts. Similar segmental arrays of neuroblasts are seen in embryos of the hawkmoth, Manduca sexta, and the fruit fly, Drosophila melanogaster (Thomas et al., 1984). The pattern of early neuronal development in these three species is strikingly similar, such that neurons which come from homologous stem cells show identical patterns of early axon outgrowth (Fig. 2 ) . Thus, there appears to be a basic plan for the early development of the CNS that is conserved in widely divergent groups of insects. How this basic plan is then modified to produce the wide diversity of insect nervous systems and behaviours is one of the intriguing problems for the future. Sensory neurons arise in the periphery in the developing ectoderm of the embryo. The first cells to be born are peripheral pioneer fibres that arise at the tips of embryonic appendages such as the legs, antennae (Bate, 1976b) and cerci (Edwards and Chen, 1979). The cues used by the axons of these pioneer neurons during their growth to the CNS have received extensive attention (see Palka, 1986, for a review). Sensory neurons arising later in development then follow the pre-existing axon tracts to the CNS. Unlike the central neurons that arise from specialized sets of stem cells, the sensory neurons typically arise from the general epidermal pavement. A short series of divisions produce the sensory neuron as well as cells such as the trichogen and tormogen cells which produce the hair and the socket of the associated sensillum (Bate, 1978). 2.2

POSTEMBRYONIC DEVELOPMENT OF THE NERVOUS SYSTEM

All nervous systems change during postembryonic life, but the extent and nature of such changes vary widely amongst the insects. Most immature forms add new sensory neurons with each successive instar, as seen, for example, for appendages such as the cerci of crickets (Murphey, 1981) and

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Fig. 2 Cameru lucidu drawings of homologous neurons in (from largest to smallest in each set) Sehistocerca, Manduca and Drosophila. The neurons that are compared are (a) the anterior (aCC) and posterior (pCC) corner cells; (b) the dorsal (dMP2) and ventral (vMP2) progeny of midline precursor 2 and the left daughter cell of MPI; and (c) the G-neuron. Homologous neurons in these three insects show remarkably similar early growth responses. (From Thomas et a f . , 1984.) for the general body surface of Manduca larvae (Levine et al., 1985). A notable exception to this pattern is seen in the higher Diptera where all of the larval sensory neurons appear to be present by the time of hatching (Hertweck, 1931; Campos-Ortega and Hartenstein, 1985). Complex sensory structures such as the compound eyes of hemimetabolous insects may also add new receptor units as the larva grows (e.g. Anderson, 1978). The holometabolous insects show the most dramatic changes in their sensory systems. The simple eyes and antennae of the larvae are replaced by the complex compound eye and antenna1 systems of the adult. Moreover, other adult structures such as the legs, wings and genitalia that arise from imaginal discs or imaginal placodes also contribute massive numbers of new sensory neurons. As was seen in the embryo, the earliest neurons to grow out axons in the imaginal discs form pathways down which later cells will grow (e.g. Sanes and Hildebrand, 1975). In terms of the central nervous system, there is a dramatic difference between the Hemimetabola and the Holometabola. In hemimetabolous forms, the adult nervous system is largely established by the end of embryogenesis. Many of the central neurons of the hatchling can be readily recognized because their pattern of central branching is very similar to that seen in

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Fig. 3 Addition of new ommatidia and lamina neurons to the visual system of Schistocerca during the fourth larval stage. Left, drawing of a horizontal section through the visual system; L, lamina; LO, lobula; M,medulla; OA, outer optic adage; PZ, profiferation zone. Right, graphs showing variation throughout the fourth instar in (A) mitotic activity within the proliferation zone; (B) mitotic activity in the outer optic anlage of the lamina; and (C) the amount of degeneration among the new lamina neurons. (Modified from data in Anderson, 1978.) the adult (e.g. Raper et al., 1983; Shankland and Goodman, 1982). Likewise, neuronal numbers remain quite stable after hatching (Gymer and Edwards, 1967; Sbrenna, 1971), giving no evidence that either neuronal birth or neuronal death plays a significant role in the postembryonic life of the ventral ganglia. Thus, the dramatic increases in the number of sensory cells added during larval life are not matched by the addition of new interneurons in the CNS. Importantly, the brain does not conform to this conservative scheme presented by the ventral ganglia. For example, in Schisrocerca (Fig. 3) the new ommatidia that are produced at each larval moult are accompanied by new neurons added to the lamina, medulla and lobula layers of the optic lobes (Anderson, 1978). Cell death also plays a role in shaping this area of the brain during each larval instar. In contrast to that of the Hemimetabola, the CNS of holometabolous insects undergoes profound changes during postembryonic life. Neurogenesis, programmed cell death and the remodelling of existing neurons interact to transform the larval CNS into that of the adult. Most larval neurons persist into the adult in a reorganized form associated with new adult functions. In Munducu sexta, for example, the skeletal muscle motoneurons of the adult are all recycled larval motoneurons (Taylor and Truman, 1974; Casaday and

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Camhi, 1976; Levine and Truman, 1982, 1985; Kent and Levine, 1988). A similar conservation of motoneurons through metamorphosis is seen in Drosophilu (C. M. Bate, unpublished), and in the beetle Tenebrio (Breidbach, 1987). The transition of neurons from their larval to their adult forms is a complex process involving first the loss of larval-specific branches (e.g. Weeks and Truman, 1985) followed by the outgrowth of the adult-specific neurites (Truman and Reiss, 1988). As described in Section 5.4, these two processes are under separate endocrine control. Metamorphosis also involves the programmed death of a subset of larval neurons. Some degenerate after the larval-pupal transition (Weeks and Truman, 1985) whereas others function through metamorphosis but then degenerate after the emergence of the adult (Truman, 1983). These latter cells are primarily those involved in maintaining pupal behaviour while the rest of the CNS is being rebuilt. Besides the death of larval neurons, there are numerous new neurons that degenerate at metamorphosis rather than maturing into functional nerve cells (Booker and Truman, 1987a). Neurogenesis plays a prominent role in forming parts of the adult CNS, especially the brain (Nordlander and Edwards, 1969a,b; White and Kankel, 1978), and the thoracic ganglia (Booker and Truman, 1987a; Truman and Bate, 1988). The segmental ganglia of larvae possess a stereotyped array of neuroblasts (Fig. 4). These become mitotic early in larval life, undergoing the division patterns typical of insect neuroblasts (Edwards, 1970). Each generates a lineage of up to 100 cells, but, unlike in the embryo, the progeny arrest their development soon after they are born. With the onset of metamorphosis, they then mature into functional adult neurons. In Manduca between 2000 and 3000 new neurons are added to each thoracic ganglion and about 50-100 cells to each unfused abdominal ganglion. Accordingly, about 6& 70% of the adult ventral CNS is produced postembryonically (Booker and Truman, 1987a); in Drosophila, the proportion is over 90% (Truman and Bate, 1988).

3 Endocrine regulation of insect development 3.1

HORMONAL REGULATION OF DEVELOPMENT

At the tissue level, growth and metamorphosis is regulated by two classes of hormones: the ecdysteroids and the juvenile hormones (JH). The ecdysteroids are a family of polyhydroxylated steroids that are the moulting hormones of arthropods (Fig. 5). Ecdysone is secreted from paired glands that are generally located in the first thoracic segment. It is hydroxylated by peripheral tissues to form 20-hydroxyecdysone (20-HE), which then acts on

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8 W

L Iv-0 2

v-0 2

w-0

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Fig. 4 The distribution of neuroblasts in the segmental ganglia of larval Manduca sexfa. Left: the open circles show the distribution of neuroblasts in the second thor-

acic (T2) and fourth abdominal (A4) ganglia. The solid circles are the M and X neuroblasts. Right: the number of progeny associated with selected thoracic (T) and abdominal (A) neuroblasts during the last half of larval life and metamorphosis. I V - 0 and V - 0 , freshly ecdysed fourth- and fifth-stage larvae respectively; W - 0 , wandering stage larvae; P-0, fresh pupa; A, adult. (From Truman and Booker, 1986.)

target tissues such as the epidermis or the nervous system (see Hoffmann, 1986, for a review). During the course of each moult the role of the ecdysteroids changes from stimulatory to inhibitory and this change is reflected in the shape of the hormonal blood titres (Fig. 6). The events early in the moltapolysis, cell division and the onset of cuticle synthesis-are stimulated by ecdysteroids and occur during the rising phase of the titre. As the moult progresses, however, the later phases of cuticle synthesis (Fristrom et d.,1982), the production of pigment (Curtis, el al., 1984) and events associated with the digestion of the old cuticle and ecdysis (Slama, 1980; Schwartz and Truman, 1983), are all inhibited by ecdysteroids. Consequently, the blood titre must decline before these events can occur. Although most moults are accomplished through a single surge of ecdysteroids, the moult to the pupal stage requires two ecdysteroid releases (Fig. 6). The first release, often called the “wandering” peak or the “commitment peak”, is generally quite small and causes the termination of feeding behaviour and the initiation of specialized behaviours such as burrowing or cocoon construction (Dominick and Truman, 1985). This ecdysteroid release is notable because it is the first time that the larva is exposed to reasonable

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Fig. 5 Chemical structures of (A) ecdysone; (B) 20-hydroxyecdysone; (C)juvenile hormone I; (D)juvenilehormone 11; (E)juvenile hormone 111. levels of ecdysteroids in the absence of JH. Studies by Riddiford and colleagues (see Riddiford, 1985, 1986) on the cellular and molecular responses of the epidermis of Manduca have shown that the commitment peak causes the termination of synthesis of larval-specific proteins and renders the tissue insensitive to JH, thereby committing it to pupal differentiation. There then follows a larger release of ecdysteroids, the prepupal peak, which promotes the differentiation of the pupal stage. Recently, it has become evident that ecdysteroids have roles in larvae beyond that of stimulating moulting. Low levels of steroid during the intermoult period have been implicated in changing patterns of cuticular protein synthesis (Wolfgang and Riddiford, 1986) and in stimulating epidermal mitoses (Hirn et al., 1979). The second class of hormones relevant to this article are the JHs. There are a number of naturally occurring JHs that are variants on the basic structure of an epoxyfarnesol methyl ester (Fig. 5). JH-111 has been found in all insect orders that have been examined; all of the other variants are found only in Lepidoptera (Schooley et al., 1984; Schooley and Baker, 1985). The JHs are synthesized in the corpora allata (CA), paired glands located just posterior to the brain. In the larva, their presence during each ecdysteroid release ensures the production of another larval stage. Their disappearance allows metamorphosis. In hemimetabolous insects, the first moult in the absence of JH produces the adult. In holometabolous insects, by contrast, the first such moult results in the pupal stage, and a second moult in the absence of J H is required for the formation of the adult. Interestingly, during the larval-pupal transition, the absence of JH is only required during the commitment peak of ecdysteroids which then renders the general epidermis insensitive to JH. Some tissues, such as the imaginal discs, require the presence of JH during

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Fig. 6 Haemolymph ecdysteroid titres as determined by RIA. (A) Relative ecdysteroid titres for the period spanning from the beginning of the fourth larval stage until the emergence of the adult in Manduca sexta. Arrows identify the beginning of each stage. (Modified from data in Bollenbacher et al., 1981; Curtis et al., 1984; and Wolfgang and Riddiford, 1986). (B) The ecdysteroid titre during the last larval stage of Locusta migra/oria. (Redrawn from data in Hirn et al., 1979.)

the subsequent prepupal ecdysteroid peak to prevent their precocious adult differentiation (Kiguchi and Riddiford, 1978). An important aspect of the action of JH in larvae is that the hormone is required for only a few hours at the beginning of each moult in order to ensure the nature of the stage to be produced. JH can then be used later in the moult to regulate caste polymorphisms in the social insects or polymorphisms in pigmentation (Nijhout and Wheeler, 1982). 3.2

TECHNIQUES FOR ENDOCRINEMANIPULATIONOF DEVELOPMENT

The initial indication of the involvement of a hormone with a particular developmental process is often the temporal correlation of that process with changing hormonal titres. The development of a number of sensitive and

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selective rddioimmunoassays (RIAs) for the ecdysteroids has made it possible to determine ecdysteroid levels in microlitre quantities of haemolymph (see Hoffmann and Hetru, 1983). Consequently, detailed ecdysteroid titres are now available for a number of insects. In some cases, separatory techniques such as high-performance liquid chromatography (HPLC) are employed in conjunction with the RIAs (e.g. Warren and Gilbert, 1986) to determine the exact nature of the ecdysteroids present (ecdysone, 20-HE, etc.). Blood titres of JH are more fragmentary because RIAs appropriate for measurement of JH in biological samples have become available only relatively recently (Strambi et al., 1984). Consequently, biological assays or physicochemical methods are generally employed, These latter techniques often require millilitre quantities of blood as compared to the microlitre quantities that are typically needed for ecdysteroid RIAs. Extirpation-replacement paradigms are often used to establish a causal relationship between a hormonal change and a developmental response. While it is preferable to remove only the glands of interest, in many cases the surgery is not suitable for mass experimentation and one resorts to ligation. A blood-tight ligature placed between the head and thorax deprives the latter of further exposure to brain neuropeptides and JH. (It should be cautioned that in some insects, such as the higher flies, the CA extends through the neck and remains after such a ligation.) A ligature between the thorax and abdomen in larvae deprives the abdomen of further ecdysteroids. Developmental arrests resulting from ligation should be overcome by administering the relevant hormone. Single injections of ecdysteroids are often ineffective because of rapid excretion of the steroid. Attempts to overcome this problem by injecting massive doses of steroid often results in abnormal development (“hyperecdysonism”, Williams, 1968). Consequently, the optimal method for ecdysteroid replacement is infusion of low doses of steroid over extended periods of time. Juvenile hormone replacement is easier because of the lipid nature of JH and JH mimics. When injected in an oil vehicle into the haemocoel, the JH slowly partitions out of the oil droplet into the blood giving a sustained release of hormone. Also, JH is active when topically applied to the cuticle. In the latter case, low doses give only local effects whereas with higher doses sufficient hormone leaches into the circulation to produce systemic effects. The production of animals that are mosaics of tissues having different developmental capacities has been a valuable approach for studying cellular interactions during development. Genetic techniques are often employed to generate such mosaics but endocrine approaches are also possible. The ability of JH to have local effects after topical application allows the production of juvenilized patches of tissue in an insect that has otherwise progressed t o the next developmental stage. Another approach is to take advantage of the

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Fig. 7 The fluctuation in free ecdysteroids extracted during various times through embryonic development of Locusta migratoria. Solid circles, ecdysone; open circles, 20-hydroxyecdysone. (Redrawn from Lagueux et al., 1979.)

fact that all cells do not necessarily respond to a hormonal signal at the same time. Consequently, by treating with exogenous hormone or by removing hormone at various times through the critical period, one can often find a time when one tissue or cell has received sufficient exposure to be committed to a particular developmental fate whereas other tissues have not. In both cases, the result of interaction of cells at different developmental stages can then be studied. Examples of these various approaches will be given in the sections that follow. 4

Hormones and embryonic development

Lagueux et al. (1979) first showed in Locusta that there are prominent fluctuations in free ecdysteroids correlated with various phases of embryonic development (Fig. 7). The free ecdysteroids that appear early in development are cleaved from ecdysteroid conjugates which were deposited in the yolk during oogenesis (Hoffmann and Lagueux, 1985). The late peaks may be due to the activity of the embryo’s own endocrine glands but this issue has not been conclusively resolved. Neurogenesis in embryos does not seem to have an absolute requirement for ecdysteroids. Cultured neuroblasts from embryonic Drosophila show relatively normal mitotic rates and produce normal-sized lineages even if cul-

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tured in the absence of exogenous ecdysteroids (Furst and Mahowald, 1985a, b). Assuming that some other cell types (or even the neuroblasts themselves) were not adding ecdysteroids to the medium, these results argue against an obligate role for ecdysteroids in embryonic neurogenesis but they do not exclude a modulatory role. In grasshoppers there is some evidence that ecdysteroids may be involved in regulating neuronal death (Harris and Goodman, 1983). The neuron QI extends an axon that pioneers an axon bundle in the posterior commissure of the ganglion but it later dies. Manipulations of embryos in short-term cultures suggest that ecdysteroids may be involved in this death. Obviously, much more work needs to be done to determine the role of these steroids during the embryonic development of the CNS. Besides the ecdysteroids, JH also appears late in embryonic development (Lanzrein el al.. 1984).This hormone may be involved in some aspects of the production of the first-stage larval cuticle but its impact on the nervous system is completely unknown.

5 5.1

Hormones and postembryonic development GANGLION MIGRATION

The CNS of most holometabolous insects undergoes a remarkable change in form during metamorphosis. Besides changing in size, certain ganglia may move en musse and fuse with other ganglia. In rarer instances, fused ganglia in the larva may separate to form distinct ganglia in the adult. The phenomenon of ganglion migration was examined by R. Pipa and colleagues in the wax moth, Galleria mellonellu. During metamorphosis of this insect, ganglia T2 to A2 fuse together to form a compound ganglionic mass. Likewise, ganglion A6 migrates posteriorly to fuse with the terminal ganglion. These migrations begin at pupation and are accomplished through the dramatic shortening of the interganglionic connectives. The neuroglia in the connectives appear to be responsible for this shortening process (Pipa, 1967; Tung and Pipa, 1972). Evidence for hormonal stimulation of connective shortening was provided by implanting sections of connectives into metamorphosing hosts and finding that the implant shortened in concert with the connectives of the host (Pipa, 1967). Subsequently, injections of ecdysone proved effective in causing the shortening response (Pipa, 1969). These in vivo studies were then followed up by in vifro experiments in which isolated connectives were cultured in the presence or absence of 20-HE (Robertson and Pipa, 1973; Robertson, 1974).

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Interestingly, the steroid could not initiate the process in vitro, but it could maintain shortening in culture if the process had already begun in vivo. 5.2

DEVELOPMENT OF THE SENSORY SYSTEM

5.2.1 Regulation of types and numbers of sensory neurons In most insects, new sensory neurons are added with each successive larval moult. The factors that regulate this addition were first explored by Wigglesworth ( 1 940) in the bug Rhodniusprolixus and current concepts are still based largely on his original insights. Based on his experimental findings, he proposed a working hypothesis for how the production of new bristles might come about. Upon stimulation by ecdysteroids, the bristle-forming cells drew from their neighbours a factor essential for bristle formation. Cells near existing bristles were depleted of this factor and inhibited from transforming into bristle mother cells. Cells distant from existing bristles would retain sufficient factor and could transform into a new bristle. By manipulating the size of the larvae produced after various moults, he concluded that the number of cells rather than absolute distance was the major factor in determining the spacing of new hairs. The first indication that receptor density might also be affected by the hormonal milieu also came from the Rhodnius studies (Wigglesworth, 1940). During the moult from the third to the fourth instar, the number of new bristles increases by 50% whereas it increases by only 27% for the moult from the fourth stage to the fifth. When fourth-stage larvae were exposed to the hormonal environment characteristic of an earlier instar by parabiosis to a moulting third-stage larva, their percentage increased to 36%. More dramatic examples of hormones, specifically JH, regulating receptor density are seen during the adult moult in many insects. For example, the antennae of the cockroach LPucophaea maderae (Schaffer and Sanchez, 1973, 1974) increase greatly in size through the seven larval instars but the density of olfactory sensillae that they bear remains relatively constant at about 400 sensilla per mm2. At the moult to the adult stage olfactory sensillar density abruptly increases by over 50%. When the adult moult was prevented and supernumerary eighth- and ninth-stage larvae were produced by treatment with JH mimics, the receptor density remained at the larval level. By contrast, allatectomy of sixth-stage larvae resulted in the production of miniature adults that precociously adopted the adult-specific density. The mechanism by which JH might regulate sensillar density was considered by Lawrence (1969) in his study of bristle and hair formation in the milkweed bug, Oncopeltus faciatus. The abdominal sternites of Oncopeltus

HORMONE ACTION ON THE NERVOUS SYSTEM

15

larvae have a low density of innervated bristles to which are added a high density of uninnervated hairs in the adult. The spacing of bristles and hairs in the overall pattern indicate that the two elements interact in an equivalent manner. Juvenile hormone application early in the moult to the adult stage inhibits hair formation as long as it is applied prior to the epidermal divisions that give rise to the hairs. Based on the Wigglesworth model, Lawrence considered two possible hypotheses to explain how the disappearance of J H results in an increase in hair and bristle density. The JH loss might enhance the production of the bristle-forming factor or it could reduce the thresholds of the epidermal cells so that lower levels of factor would allow them to transform into hair mother cells. Larvae receiving topical application of JH or low levels of injected J H mimics often had islands of adult cells in a surround of larval cells. In all such cases, the high density of bristles was confined to the adult cells and did not spread out to surrounding larval cells. This result is more consistent with the hypothesis that JH alters the threshold of the epidermal cells to the hypothetical morphogen. An interesting aspect of the effect of JH on the epidermal cells of Oncopeltus is that a number of features of the cells change during their transition to the adult stage. In the absence of JH these cells produce melanin granules and they also alter the surface sculpturing of the cuticle they secrete. Application of JH at different times showed that these characters are independently regulated by the hormone (Lawrence, 1969). Moreover, melanization is affected in an all-or-none fashion while the effects of JH on surface sculpturing are graded. 5.2.2

Shifting of sensory projections

Existing sensory neurons may shift their postsynaptic targets during the course of postembryonic life. For example, in the cricket, Acheta domesticus, the sensory neuron associated with the X hair on the cercus synapses onto the ascending giant interneuron MGI during the early instars. This contact is then lost as the animal becomes an adult and the sensory neuron now only excites smaller ascending interneurons (Shepard and Murphey, 1986). A similar shift in postsynaptic targets apparently occurs for the sensory neurons that mediate the pupal gin-trap reflex in Manduca sexta (Bate, 1973b; Levine et al., 1985). The regulation of this shift has been studied using JH-induced mosaics. The insect sensory system is especially amenable to manipulation by JH because the peripheral location of sensory neurons allows the local alteration of the environment around the cell body while leaving the CNS unaffected. Hence, one can examine the outcome of confronting the sensory neurons of one stage with the CNS of the next. In Manduca the gin-traps are pupal-specific, cuticular pits located on the

16

JAMES W. T R U M A N

Fig. 8 Hormonal regulation of the form of the terminal arbors of the gin-trap afferents in Manduca sexta. (A) The pupal stage showing the position of the abdominal gin-traps (GT). (B) Schematic representation of the circuitry mediating the gin-trap closure response; the gin-trap afferents project to the next anterior ganglion. ISM, intersegmental muscles. (C) Axonal arborizations of a sensory neuron treated with a JH mimic and unable to evoke the gin-trap reflex (left) and a control neuron from the normal contralateral gin-trap of the same animal (right). Ganglion cross-sections of a similar preparation were made at the levels of the arrows. The numbers on the two sides refer to the total length of terminal processes and the number of branch points.

anteriolateral margins of segments A5-A7 (Fig. 8). Each trap contains approximately 20 peg-like hairs, the deflection of which causes a robust contraction of the ipsilateral intersegmental muscles in the next anterior segment (Bate. 1973a; Levine and Truman, 1983). Although the gin-traps are not present in larvae, the sensory neurons that come to reside in the traps are found in the last-stage larva and innervate larval filiform hairs on the anteriolateral margins of the respective abdominal segments (Bate, 1973b; Levine et al., 1985). During the larval-pupal transition, these sensory neurons become associated with the peg-like pupal hairs, and their terminal axon arbors increase about 3-fold in length and 5-fold in the number of branches. The development of the gin-trap system was perturbed by treating laststage larvae with JH just prior to the commitment peak of ecdysteroids (Levine et al., 1986). Topical application to a presumptive gin-trap area resulted in the formation of pupae which carried a patch of larval cuticle at the site of treatment. The sensory neurons associated with the larval hairs in the patch responded to hair deflection but they did not initiate a gin-trap reflex. Importantly, their axon terminals retained the larval-like arbor rather than showing the branching expected for the pupal cell (Fig. 8). The effects of the JH on the axon morphology of the sensory neurons were not due to hormone leaking from the site of application and acting on the CNS. As seen in Fig. 8, axons that projected from the larval patch showed

H O R M O N E ACTION ON THE NERVOUS SYSTEM

17

larval-like terminals whereas those projecting to the same ganglion from the untreated side had pupal arbors. In addition, application of JH to only one half of a presumptive trap area resulted in a region that formed half a gintrap next to a larval patch (Levine et al., 1986). Neurons associated with hairs in the gin-trap half triggered a gin-trap reflex and showed a pupal arborization pattern whereas those associated with the larval hairs had a larval-like arbor and did not cause the reflex. These results argue that the pupal environment of the CNS does not force sprouting and growth of the sensory terminals. Rather, the environment of the cell body seems to be the crucial one. Further insight into the development of the gin-trap circuit was provided by the observation that some treated animals made morphologically normal gin-traps which did not trigger a closure reflex. Moreover, the axonal arbors of these sensory neurons were larval-like. Such a result would be expected if the cells that make the cuticular hairs were already pupally committed at the time of treatment and, hence, were insensitive to exogenous hormone. The sensory neurons were presumably still sensitive and their pupal sprouting was blocked. Irrespective of mechanism, this result shows that the pupal growth of the sensory neuron is not caused by the cuticular structure with which it is associated. Pupae bearing larval patches often acquired the ability to show a gin-trap reflex by 3 4 days after pupal ecdysis. This ability of the hairs to trigger the reflex was accompanied by the sprouting of the sensory neurons to form a pupal-like terminal arbor (Levine et al., 1986). After pupal ecdysis, the insect is again exposed to ecdysteroids in the absence of JH to initiate adult development. Elimination of this ecdysteroid by isolation of the pupal abdomen just after ecdysis prevented both the acquisition of gin-trap function and the terminal sprouting (R. B. Levine, personal communication). The above experiments show that neither the pupal environment of the CNS nor the nature of the cuticular hairs is sufficient to induce the terminal sprouting of the gin-trap afferents. Recent experiments by Levine (personal communication) indicate that neither condition is even necessary for this growth to occur. Abdomens isolated from fifth-stage larvae survive for a number of weeks as permanent larval abdomens. Using such abdomens, he applied ecdysteroids topically to the presumptive gin-trap area, thereby causing a local region of epidermis to be exposed to ecdysteroids in the absence of JH. The sensory neurons at that site of application showed axon sprouting even though in a permanently larval CNS. These results further support the conclusion that the hormonal environment around the cell body of the sensory neuron is the paramount factor in determining the growth response of its axon. A similar conclusion was reached for the metamorphosis of the visual system in frogs where local application of thyroxin to the eye of the

J A M E S W . TR U MA N

18

tadpole caused ganglion cells to show an adult pattern of growth over the larval tectum (Hoskins and Grobstein, 1985). 5.3 5.3. I

NEUROGENESIS AND NEURONAL DIFFERENTIATION

Neurogenesis

There is no evidence that hormones direct neurogenesis in postembryonic stages. In the optic lobes of Schistocerca, new neurons are being born at a constant rate through the moult and intermoult periods (Fig. 3; Anderson, 1978). Similarly, in the central brain and optic lobes of the monarch butterfly, Danaus plexippus, neurogenesis begins early in larval life and continues without interruption into metamorphosis (Nordlander and Edwards, 1969a, b). A similar picture is seen for neurogenesis in the ventral ganglia of Manduca (Booker and Truman, 1987a). Interestingly, despite the dissociation of neurogenesis from the ecdysteroid fluctuations associated with moulting, the production of glia in both the brain (Nordlander and Edwards, 1969a) and the ventral ganglia (Truman, unpublished) is synchronized with the moults. Although ongoing neurogenesis appears not to be under ecdysteroid regulation, the factors involved in initiating neurogenesis in the early larva and in terminating it at metamorphosis are not well understood. 5.3.2 Diferentiation of postembryonic neurons

In the embryo, the maturation of a neuron typically follows close on the heels of its birth. During the postembryonic period, by contrast, a neuron’s maturation may be delayed by a substantial period of time. In Manduca, for example, neurons born at any time during larval life arrest their development soon after their birth and collect into clusters of immature postmitotic neurons (Booker and Truman, 1987a). This arrest is then terminated at the onset of metamorphosis. During the larval-pupal transition substantial cell death occurs within selected lineages, with the survivors showing modest nuclear and cytoplasmic growth (Booker and Truman, 1987b). The pupal-adult transition then results in further cytoplasmic proliferation including the establishment of mature transmitter phenotypes (Witten and Truman, 1987). Both of these phases are under endocrine control. Ligation experiments (Fig. 9) showed that the cell death and early maturation that occurs during the larval-pupal transition is blocked by the absence of ecdysteroids but is restored by infusion of 20-HE (Booker and Truman, 1987b; see also Section 5.5.2). As might be expected, the ability of ecdysteroids to promote this early maturation is blocked by treatment with JH mimics. The final maturation

HORMONE ACTION O N THE NERVOUS S Y S T E M

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that occurs during the pupal-adult transition likewise requires the reappearance of ecdysteroids (J. L. Witten and J. W. Truman, unpublished). 5.4

RESTRUCTURING OF LARVAL NEURONS

Neurons can change dramatically during the transition from larva to adult. To this point mainly morphological changes have been documented but one would also expect changes in transmitter systems and in electrical properties

20

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Fig. 10 The effect of steroid manipulations on dendritic reduction in the abdominal motoneuron PPR. Left, camera lucidu drawing of a cobalt-filled PPR. Right-top, the ecdysteroid titres during the larval-pupal transition in Manduca. Bottom, drawings of PPR's contralateral arbor in each of five experimental cases; numbers are the mean ( *SEM) dendritic density values. (A) and (E) are fills from normal day 2 fifth-stage larvae (L2) and freshly pupated animals (PO) respectively. (B) and (C) are neurons from isolated abdomens, the number of days between ligation and filling are as indicated. In (D) the ligated abdomens were infused with 20-HE over a 12-h period. (From Weeks and Truman, 1985.) of metamorphosing neurons. Although the most dramatic examples have been reported from the Holometabola, it is likely that such changes also occur on a more restricted scale during postembryonic life in the Hemimetabola. An especially intriguing example is the recent report in dragonflies of descending visual interneurons that are present but electrically silent in immature stages (Olberg, 1986). They then become functional in the adult. The changes in neuronal form during the metamorphosis of holometabolous insects appears to occur in two steps, with first the loss of larval neurites followed by the outgrowth of adult-specific processes. Thus far, the endocrine regulation of these changes have been examined only for Manduca.

5.4.I

Hormonal control of neurite regression

Many larval motoneurons show some degree of neurite loss during the larval-pupal transition. The regulation of this loss has been examined in detail for motoneuron PPR (Fig. 10; Weeks and Truman, 1985) which innervates an abdominal proleg retractor muscle. This muscle degenerates early in the larval-pupal transition and PPR shows a dramatic reduction in its dendritic tree coincident with the muscle loss. A few days later, the neuron itself then dies. The loss of dendrites in PPR occurs during the prepupal ecdysteroid peak

HORMONE ACTION

O N THE NERVOUS SYSTEM

21

that triggers pupal differentiation. When larval abdomens were isolated prior to this peak, PPR retained its larval morphology (Fig. 10). Ecdysteroid infusion into such abdomens triggered dendritic loss followed by the death of the cell a few days later (Weeks and Truman, 1985). Using the wax moth, Galleria mellonella, Runion and Pipa (1970) first showed that ecdysteroids cause the death of proleg muscles during the larvalpupal transition. Since the same relationship holds for Manduca, the dendritic regression in PPR might result from the death of its target. This hypothesis was tested by surgically removing its target muscle prior to the commitment peak of ecdysteroids. This manipulation neither caused PPR to lose dendrites when maintained in the absence of ecdysteroids nor prevented dendritic loss in response to the prepupal ecdysteroid surge (Weeks and Truman, 1985). Thus, dendritic loss in PPR seems independent of the fate of its target. Whether it is likewise independent of changes in presynaptic cells is not known. The other hormone regulating the fate of PPR is JH, but JH can only act during the small commitment peak. The low levels of ecdysteroids normally present during this peak are not sufficient to cause neurite loss, but regression can be forced experimentally by infusing large doses of 20-HE (Weeks and Truman, 1986). Treatment with JH prior to steroid infusion prevents this neurite loss. After exposure to a normal commitment peak, however, the responsiveness of PPR is markedly altered. The effects of 20-HE infusion can no longer be blocked by JH, and PPR shows an enhanced sensitivity to ecdysteroids. In these respects, the response of PPR to ecdysteroids during the larval-pupal transition has many similarities to that seen for the epidermis of Munduca (Riddiford, 1985). In the latter, the commitment peak renders the epidermis insensitive to JH and the prepupal peak then induces the production of pupal-specific proteins (Kiely and Riddiford, 1985). In the epidermis, this loss of responsiveness to JH is correlated with the loss of JH receptors (Riddiford rt al., 1987). It is not yet known whether the same is true for neurons such as PPR. The dendritic loss in PPR is not simply a symptom of its imminent death. This conclusion is suggested by the fact that many of the motoneurons that persist into the adult also show a transient loss of dendrites in the pupa (e.g. Truman and Reiss, 1988). Thus, dendritic loss is not always followed by death. In the case of PPR, direct evidence for a dissociation of these two processes comes from the finding that the dendritic loss and the death responses have different endocrine requirements. By isolating abdomens at various times during the prepupal peak and, hence, curtailing the ecdysteroid exposure, Weeks ( 1987) showed that early ligations resulted in dendritic regression but not death. Only if the ligations were delayed for an additional day did neuronal death then follow the regression.

22

J A M E S W. T R U M A N

Fig. I I Changes in the dendritic arbor of the motoneuron MN-I during metamorphosis in Manduca. The neuron shows the growth of a new dendritic arbor ipsilateral to the soma during the transition from larva to adult. Insets show the progression of this growth which is quantified in terms of the dendritic extent. Each dot represents data from a cobalt-filled neuron. (Data from Truman and Reiss, 1988.)

5.4.2 Hormonal regulation of neurite outgrowth The outgrowth of new neurites occurs during the transformation of the pupa into the adult and this also requires the presence of ecdysteroids and the absence of JH. In the motoneuron MN-I, this outgrowth includes an extensive new arbor ipsilateral to the cell body (Fig. 1 I). Neurite extension starts on day 3 after pupal ecdysis and continues through the next 8-10 days. The start of outgrowth coincides with the rise in ecdysteroid which brings about adult development. Substantial outgrowth does not occur under conditions of pupal diapause during which time endogenous ecdysteroid secretion is suppressed. Infusion of 20-HE into diapausing pupae provokes the onset of neurite outgrowth by 24-36 h after the end of the infusion (Truman and Reiss, 1988). The adult-specific growth of MN-1 is also sensitive to JH. Treatment with JH mimics up to 2-3 days after pupal ecdysis blocks adult outgrowth. However, after adult outgrowth has commenced, the cell rapidly loses its sensitivity to JH. It is of interest that all neurons do not lose their sensitivity to JH mimics at the same time. In contrast to MN-I, the adult outgrowth of motoneuron MN-3 can be prevented by JH treatment up through 5 days after ecdysis. Consequently, pupae treated with JH at 4-5 days after pupal ecdysis develop into morphologically normal moths whose nervous system is a mosaic of pupal and adult cells (e.g. pupal MN-3s and adult MN-1s). Under normal conditions the adult-specific neurites of MN-1 and MN-3 invade the same

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23

region of the dorsal neuropil but after this treatment paradigm, only MN-I does so. Such a restricted response suggests that JH could be acting at the level of the respective neurons themselves, a hypothesis consistent with the situation for sensory neurons (Section 5.2.2). It is interesting that the same hormone, the ecdysteroids, causes opposite responses (regression versus growth) depending on when it is given. It is not yet known whether it is possible to make a larval cell skip the regression phase by exposing it to an endocrine environment characteristic of adult development. Obviously, the developmental history of the cell is an important variable in determining the cell’s response to endocrine signals.

5.5

CONTROL OF NEURONAL DEATH

Cell death is an important process in shaping developing systems. It figures prominently during the embryogenesis of the insect CNS (e.g. Bate et al., 1981) and also postembryonically in the developing optic lobes of hemimetabolous insects (Anderson, 1978), and in the brain (Nordlander and Edwards, 1969a, b) and ventral ganglia (Booker and Truman, 1987a) of the Holometabola. In these cases, the cells that die are immature and death may result from interactions with pre- or postsynaptic targets (e.g. Macagno, 1979). Cell death has a second role that is seen primarily in holometabolous insects, namely, that of removing larval neurons that are not needed in the adult. Hormones are involved in triggering both types of degeneration. 5.5.1

Death of immature neurons

The optic lobes of larval locusts show both neurogenesis and neuronal death during the course of an instar (Fig. 3; Anderson, 1978). Neurogenesis occurs at a constant rate throughout (Section 5.3.1). The frequencies of neuronal deaths, though, are erratic and it is not clear whether or not some of the deaths might be linked with the moult. In the ventral ganglia of Munduca, ecdysteroids are clearly involved in triggering the death of immature neurons at the outset of metamorphosis (Booker and Truman, 1987b). In this case, however, the factors that determine which cells will mature and which will die in response to the ecdysteroid signal are not yet known. The basic endocrine experiments showing the involvement of ecdysteroids are described in Section 5.3.2 (Fig. 9), but a few additional points are worth considering in the present context: (1) All of the immature neurons in a given lineage d o not die at the same time; a few die after the commitment pulse, the majority die during the prepupal peak and a few die after pupal ecdysis. Each wave of death seems to depend on a separ-

24

JAMES W. TRUMAN

ate ecdysteroid exposure (Booker and Truman, 1987b). (2) The death or differentiation of the immature neurons can still be blocked by the application of JH mimics after the commitment peak. Thus, these cells are unlike motoneurons (Section 5.4.1) and sensory neurons (Section 5.2.2), which are rendered insensitive to JH by this ecdysteroid exposure. Consequently, J H treatment at this time can block the development of the immature cells even though the remainder of the nervous system progresses to the pupal stage (Booker and Truman, 1987b). This result suggests that the developmental responses of these cells are not an indirect result of other changes induced by ecdysteroids in the CNS. (3) JH treatment can prevent the death of the immature neurons but the saved cells persist in their immature condition. Thus, the decisions to survive and to mature appear to be two distinct developmental decisions. 5.5.2 Death of larval neurons Metamorphosis also signals the death of many functional larval neurons. Major bouts of death occur after ecdysis to the pupal stage and after ecdysis of the adult. The death of PPR during the larval-pupal transition has been briefly considered in Section 5.4.1. Its death is triggered by the prepupal peak but, as indicated above, the duration of steroid exposure required to cause death is greater than that required for dendritic regression (Weeks, 1987). In many insects the emergence of the adult is followed by the death of specialized muscles that were used during ecdysis and the expansion of the new cuticle. The endocrine regulation of this death varies amongst the insects. In blowflies, the tanning hormone, bursicon, is likely involved in muscle death (Cottrell, 1962). In the giant silkmoth, Antheraeapolyphemus, death is caused by eclosion hormone acting on muscles that have been “primed” by the decline in haemolymph ecdysteroids (Schwartz and Truman, 1984). In Manduca, the ecdysteroid decline alone is sufficient to bring about intersegmental muscle degeneration (Schwartz and Truman, 1983). The muscle death in Manduca is accompanied by degeneration of motoneurons and interneurons (Truman, 1983). The motoneurons die in a stereotyped sequence with each cell dying at a characteristic time relative to ecdysis. This death is regulated by ecdysteroids, but by the disappearance of the steroid rather than its appearance (Truman and Schwartz, 1984). This relationship was first indicated by the observation that isolation of abdomens late in adult development caused a premature decline in ecdysteroids and also hastened neuronal death. Prevention of the ecdysteroid decline by infusion of 20-HE blocked neuron death for the duration of the infusion. Similarly, injections of 20-HE caused a dose-dependent delay in the time of onset of death.

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25

TIME OF TREATMENT

Fig. I2 The ability of injections of 20-HE to delay the degeneration of the D-IV motoneurons (triangles) and the ventral intersegmental muscles (squares) that they innervate. Animals were injected at the times indicated and examined 24 h after adult emergence. Motoneuron delay was the percentage of animals in each group that showed delayed neuron death. The muscle delay is expressed in hours delayed and based on a comparison of the dry weight of muscles from treated animals with those of controls. The arrow indicates the time of adult emergence; open and filled bars represent day and night respectively.

Infusions of 20-HE late in adult development spare muscles as well as motoneurons. Consequently, it was not clear which tissue was the primary target of the steroid. As seen in Fig. 12, by applying 20-HE at specific times prior to adult ecdysis, it was possible to define for both muscles and neurons a time before which steroid treatment saved the cells but after which it was ineffective. For the intersegmental muscles, the 50% commitment time was about 13 h before ecdysis, whereas that for the D-IV neurons, which innervate these muscles, was 2 h after ecdysis. Hence, 20-HE given a few hours before ecdysis spares the motoneurons even though their muscles die on schedule. Although the above results show that muscle death is not suficient to cause motoneuron degeneration, they do not exclude the hypothesis that muscle death is necessary for subsequent motoneuron death. This latter question was addressed by making mosaics by topical JH application to part of an abdominal segment early in adult development (Truman, unpublished). The resulting adults had a patch of pupal cuticle and the intersegmental muscles underlying this patch did not degenerate. Nevertheless, the D-IV moto-

26

JAMES W. TRUMAN

neurons innervating these spared muscles died along with those in the rest of the animal. Thus, the fates of the neurons and their muscles seem to be completely independent. The direct action of ecdysteroids on the CNS was demonstrated by culturing ganglia in the presence or the absence of 20-HE (Bennett and Truman, 1985). Culturing with physiological levels of 20-HE prevented neuronal death whereas the absence of steroid led to cell degeneration. Importantly, the cells that died under the latter conditions were the same individuals that normally die in vivo. These experiments show that 20-HE acts on the CNS but they do not prove that its action is directly on the cells that die. Indeed, recent experiments on Manduca suggests that trans-synaptic factors as well as the steroid decline may be necessary for the death of at least some of these cells (Fahrbach and Truman, 1987a). The last issue of concern is the mechanism of the death. In the nematode, Caenorhabdites elegans, two genes have been identified, ced-3 and ced-4, whose wild-type function is required for the normal programmed degeneration of neurons in the CNS (Ellis and Horvitz, 1986). Although such genes have yet to be identified in insects, it is likely that similar genes are involved in the degeneration responses seen in the CNS and muscle. Indeed, Lockshin (1969) showed that blockers of RNA and protein synthesis can prevent the programmed death of muscles in Antheraea polyphemus. In Manduca, blockers of RNA synthesis can inhibit motoneuron death if given up to about the time of commitment; protein synthesis inhibitors are effective until a few hours later (S. Fahrbach and J. W.Truman, unpublished). Such results are consistent with the hypothesis that the degeneration programme is initiated by new genetic transcripts.

6 Conclusions and future directions

Studies of the action of JH and ecdysteroids started more than 50 years ago with the pioneering studies of V. B . Wigglesworth on Rhodnius, and then with those by C. M. Williams on Hyalophora cecropia. Since that time we have acquired detailed information on the action of these hormones on a number of tissues, especially the epidermis (see Riddiford, 1985). The nervous system has been the last to receive detailed attention, largely because we only recently have had techniques to permit the analysis of development of individual identified neurons or identified lineages. Thus far, the action of these two hormones on the CNS is in line with their actions on other tissues. Of the two, JH is the more enigmatic. In the immature stages, JH appears to have no action on its own, but only when paired with ecdysteroids. Many of

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27

the processes discussed in this reviewdhanges in form of sensory neurons and motoneurons, maturation of immature neurons, and some aspects of neuronal death-are prevented if JH is given along with the ecdysteroids. Thus, in a very real sense, JH is indeed a “status-quo” hormone (Williams, 1961) for the nervous system. Juvenile hormone is most likely involved in the early determinative events that occur during the cell’s response to ecdysteroids. This early action is evident from the example of neurite outgrowth which occurs over the span of 8-10 days but which can only be blocked by JH at its onset. Thus, once a developmental programme has been initiated, JH appears to have no involvement in the execution of that programme. Importantly, the studies on Oncopeltus (Lawrence, 1969) show that various aspects of a cell’s response may be regulated by different programmes which are played out in parallel and which may have diverse endocrine requirements. It should also be noted that not all ecdysteroid related processes are modulated by JH. The degeneration of motoneurons after adult ecdysis cannot be prevented by JH treatment at the time of steroid withdrawal. Interestingly, however, JH treatment 3 weeks earlier at the outset of adult development can render the neurons aloof to the effects of the subsequent ecdysteroid decline (Truman, unpublished). In terms of both JH and the ecdysteroids, a major question relates to whether these hormones are indeed acting directly on the cells which show the respective changes. The best case for a direct response can be made for the gin-trap afferents (Section 5.2.2). Likewise neurite regression and death in motoneurons appear independent of responses in their target muscles but actions mediated through presynaptic elements are still a real possibility. An approach that has been used to great advantage in studies of steroid action on the vertebrate CNS is the localization of radiolabelled steroids (Arnold and Gorski, 1984). This technique has recently been adapted successfully to the CNS of Manduca using [3H]ponasterone, an active ecdysteroid analogue (Fahrbach and Truman, 1987b). As seen in Fig. 13, incubation of ganglia with [3H]ponasterone results in the concentration of label in neuronal nuclei. This labelling is abolished by preincubation of tissue with an excess of unlabelled steroid, and only a subpopulation of the interneurons and motoneurons bind steroid at any particular developmental stage. For example, at the time of adult emergence, motoneurons that will survive in the adult do not show ecdysteroid binding whereas binding is still seen in many of the celis that will die (S. E. Fahrbach and J. W. Truman, unpublished). These studies are still in their early stages, but it is hoped that the determination of the temporal and spatial patterns of ecdysteroid binding will aid us in understanding the complex pattern of ecdysteroid action in the insect CNS. In terms of its action on the CNS, the primary effects of ecdysteroids seems to be in permitting neurons to undergo any sort of change. This is best seen in

28

J A M E S W. TRUMAN

Fig. 13 Autoradiograms of sections through an abdominal ganglion of Manduca se.rta. Ganglia were removed from larvae on the afternoon of wandering, incubated for I h in 50nM [3H]-ponasterone, frozen, and processed for steroid autoradiogrdphy. (A) Low-power view showing accumulation of silver grains over a number of motoneuron somata indicating uptake of the radiolabelled steroid. (B) High-power view showing the concentration of silver grains over the nuclei of a motoneuron (arrow) and of interneurons (arrowheads). (S. E. Fahrbach, unpublished.)

the case of the topical JH treatment to the sensory neurons. It is highly likely that local interactions in the ganglion are responsible for shaping the axon arbor (see Murphey, 1986), but ecdysteroids (in the absence of JH) are required in order for the cell to undergo the growth which would make such an interaction possible. Likewise, the preganglion cells in the abdominal lineages of Manduca are presumably poised for either maturation or death but this decision is not made until the ecdysteroid signal is given. There are undoubtedly local cues and interactions that are important but the neurons must be exposed to ecdysteroids before they can take advantage of these cues or signals. An intriguing aspect of the effects of ecdysteroids on the insect CNS is that the same hormone can have so many different effects on the same cell. At one time a neuron loses dendrites when confronted with ecdysteroids, the next exposure might cause it to grow dendrites, and the next may cause it to die. Indeed, one aspect of the action of ecdysteroids appears to be to modify the cell so that it will respond differently when next confronted with 20-HE. How this comes about, whether it is intrinsic to the cells in question or related to

HORMONE ACTION O N THE NERVOUS SYSTEM

29

changing environments as growth and metamorphosis proceeds, is not known. A key to understanding how ecdysteroids promote change within the C N S undoubtedly lies in the pattern of gene expression evoked by these hormones. These include not only the structural genes through which changing phenotypes are established, but also the regulatory genes that presumably orchestrate the patterns of structural gene expression. For some tissues, putative regulatory genes that mediate ecdysteroid action are already known (Cherbas et ul.. 1986), and it is hoped that these will also provide the entry point for eventually understanding the molecular aspects of ecdysteroid action on the CNS.

Acknowledgements

I thank Professor L. M. Riddiford for helpful discussions and for a critical reading of this manuscript and Dr S. E. Fahrbach for allowing me to use Fig. 13. References Anderson, H. (1978). Postembryonic development of the visual system of the locust, Schistocerca gregaria. I . Patterns of growth and developmental interactions in the retina and optic lobe. J . Emhryol. exp. Morph. 45,55-83. Arnold, A. P. and Gorski. R. A. (1984). Gonadal steroid induction of structural sex differences in the central nervous system. A . Rev. Neurosci. 7,413442. Bastiani, M . J . . Doe, C. Q., Helfand, S. L. and Goodman, C. S. (1985). Neuronal specificity and growth cone guidance in grasshopper and Drosophila embryos. Trends Nrurosci. 8, 257-266. Bate. C. M . (1973a). The mechanism of the pupal gin trap. 11. The closure movement. J . exp. Biol. 59, 109-1 19. Bate, C. M. (1973b). The mechanism of the pupal gin trap. 111. Interneurones and the origin of the closure mechanism. J . exp. B i d . 59, 121-135. Bate, C. M . ( I 976a). Embryogenesis of an insect nervous system. I. A map of the thoracic and abdominal neuroblasts in Locustu migratoria. J . Emhryol. exp. Morph. 35, 107-123. Batc. C. M. (1976b). Pioneer neurons in an insect embryo. Nature 260,5456. Bate, C . M. (1978). Development of sensory systems in arthropods. In “Handbook of Sensory Physiology”, Vol. 9, pp. 2-53. Springer-Verlag, New York/Berlin. Bate, C. M . and Grunewald, E. B. (1981). Embryogenesis of an insect nervous system. 11. A second class of precursor cells and the origin of the intersegmental connectives. J . Emhryol. exp. Morph. 61,317-330. Bate, M., Goodman, C. S. and Spitzer, N. C. (1981). Embryonicdevelopment ofidentified neurons: segment-specific differences in the H cell homologies. J . Neurosci. 1, 103-106. Bennett, K . L. and Truman J. W. (1985). Steroid-dependent survival of identifiable neurons in cultured ganglia of the moth Manduca sexta. Science, N . Y . 229,58-60.

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Bollenbacher, W. E., Smith, S. L., Goodman, W. and Gilbert, L. I. (1981). Ecdysteroid titer during the larval-pupal-adult development of the tobacco hornworm, Manducu sexta. Gen. comp. Endocr. 44,302-306. Booker, R. and Truman J. W. (l987a). Postembryonic neurogenesis in the CNS of the tobacco hornworm, Manduca sexta. I. Neuroblast arrays and the fate of their progeny during metamorphosis. J. comp. Neurol. 255,548-559. Booker, R. and Truman J. W. (l987b). Postembryonic neurogenesis in the CNS of the tobacco hornworm, Manduca sexta. 11. Hormonal control of imaginal nest cell degeneration and differentiation during metamorphosis. J. Neurosci. 7,410741 14. Breidbach, 0. ( I 987). The fate of persisting thoracic neurons during metamorphosis of the meal beetle Tenebrio molitor (Insecta: Coleoptera). Wilhelm Roux’s Arch. Dev. Biol. 1%,93-100. Campos-Ortega, J. A. and Hartenstein, V. (1985). “The Embryonic Development of Drosophilu melanogaster”. Springer-Verlag, Berlin, 227 pp. Casaday, G. B. and Camhi, J. M. (1976b). Metamorphosis of flight motor neurons in the moth, Manducasexta. J. comp. Physiol. 112,143-158. Cherbas, L., Benes, H., Bourouis, M., Burtis, K., Chao, A., Cherbas, P., Crosby, M., Garfinkel, M., Guild, G., Hogness, D., Jami, J., Jones, C. W., Koehler, M., Lepesant, J.-A., Martin, C., Maschat, F., Mathers, P., Meyerowitz, E., Moss, R., Pictet, R., Rebers, J., Richards, G., Roux, J., Schulz, R., Segraves, W., Thummel, C. and Vijyraghavan, K. (1986). Structural and functional analysis of some moulting hormone-responsive genes from Drosophila. Insect Biochem. 16,241-248. Cottrell, C. B. (1962). The imaginal ecdysis of blowflies. Observations on the hydrostatic mechanisms involved in digging and expansion. J. exp. Biol. 3 9 , 4 3 1 4 8 . Curtis, A. T., Hori, M., Green, J. M., Wolfgang, W. J., Hiruma, K. and Riddiford, L. M. (1984). Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and black mutant Manduca sexta larvae. J . Insect Physiol. 30,597406. Doe, C. Q. and Goodman, C. S. (1985). Early events in insect neurogenesis. 11. The role of cell interactions and cell lineage in the determination of neuronal precursor cells. Devl Biol. 111,206-219. Dominick, 0. S. and Truman, J. W. (1985). The physiology of wandering behaviour in Manduca sexta. 11. The endocrine control of wandering behaviour. J. exp. Biol. 117,45-68. Edwards, J. S. (1970). Postembryonic development and regeneration in the insect nervous system. Adv. Insect Physiol. 6,97-137. Edwards, J. S. and Chen, S.-W. (1979). Embryonic development of an insect sensory system, the abdominal cerci of Acheta domesticus. Wilhelm Roux’s Arch. Dev. Biol. 186,151-178. Ellis, H. M. and Horvitz, H. R. (1986). Genetic control of programmed cell death in the nematode C . elegans. Cell 44,8 17-829. Fahrbach, S. E. and Truman, J. W. (1987a). Possible interactions of a steroid hormone and neural inputs in controlling the death of an identified neuron in the moth Manducu sexta. J. Neurobiol. 18,497-508. Fahrbach, S. E. and Truman, J. W. (1987b). Autoradiographic studies of ecdysteroid binding in the nervous system of Manduca sexta. Soc. Neurosci. Abstr. 13, 1518. Fristrom, J. W., Doctor, J., Fristrom, D. K., Logan, W. R. and Silvert, D. J. (1982). The formation of the pupal cuticle by Drosophila imaginal discs in vitro. Devl Biol. 91,337-350. Furst, A. and Mahowald, A. P. (1985a). Differentiation of primary embryonic neuroblasts in purified neural cell cultures from Drosophila. Devl Biol. 109, 184-192.

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Levine, R. B. and Truman J. W. (1983). Peptide activation of a simple neural circuit. Brain Res. 279,335-338. Levine, R. B. and Truman J. W. (1985). Dendritic reorganization of abdominal motoneurons during metamorphosis of the moth, Manduca sexta. J. Neurosci. 5,2424243 I . Levine, R. B., Pak, C. and Linn, D. (1985). The structure, function and metamorphic reorganization of somatopically projecting sensory neurons in Manduca sexta larvae. J . comp. Physiol. A. 157, 1-13. Levine, R. B., Truman, J. W., Linn, D. and Bate, C. M. (1986). Endocrine regulation of the form and function of axonal arbors during insect metamorphosis. J. Neurosci. 6,293-299. Lockshin, R. A. (1969). Programmed cell death. Activation of lysis by a mechanism involving synthesis of protein. J. Insect Physiol. 15, 1505-1 516. Macagno, E. R. (1979). Cellular interactions and pattern formation in the development of the visual system of Daphnia magna (Crustacea, Branchiopoda). Devl Biol. 73,206238.

Meinertzhagen, I. A. (1973). Development of the compound eye and optic lobe of insects. In “Developmental Neurobiology of Arthropods” (Ed. D. Young), pp. 51104. Cambridge University Press, Cambridge. Murphey, R. K. (1981). The structure and development of a somatotopic map in crickets: the cercal afferent projection. Devl Biol. 88,236-246. Murphey, R. K. (1986). The myth of the inflexible invertebrate: competition and synapse remodeling in the development of invertebrate nervous systems. J. Neurobiol. 17,585-591. Nijhout, H. F. and Wheeler, D. E. (1982). Juvenile hormone and the basis of insect polymorphisms. Q. Rev. Biol. 57, 109-133. Nordlander, R. H. and Edwards, J. S. (1969a). Postembryonic brain development in the monarch butterfly, Danaus plexippus plexippus, L. I. Cellular events during brain morphogenesis. Wilhelm Roux Arch. Dev. Biol. 162, 197-217. Nordlander, R. H. and Edwards, J. S. (196917). Postembryonic brain development in the monarch butterfly, Danus plexippus plexippus, L. 11. The optic lobes. Wilhelm Roux Arch. Dev. Biol. 163, 197-220. Olberg, R. M. (1986). Metamorphosis of identified visual interneurons which steer flight in the dragonfly. SOC.Neurosci. Abstr. 12,927. Palka, J. (1986). Neurogenesis and axonal pathfinding in invertebrates. Trends Neurosci. 9,482-485. Pipa, R. L. (1967). Insect neurometamorphosis-111. Nerve cord shortening in a moth, Galleria mellonella (L.), may be accomplished by hormonal potential of neuroglial motility. J. exp. 2001.164,47-60. Pipa, R. L. (1969). Insect neurometamorphosis-IV. Effects of the brain and synthetic a-ecdysone upon interganglionic connective shortening in Galleria mellonella (L.) (Lepidoptera). J . exp. Zool. 170, 181-192. Raper, J. A., Bastiani, M. and Goodman, C. S. (1983). Pathfinding by neuronal growth cones in grasshopper embryos. I. Divergent choices made by the growth cones of sibling neurons. J. Neurosci. 3,20-30. Riddiford, L. M. (1985). Hormone action at the cellular level. In “Comprehensive

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Neural Repair and Regeneration in Insects J. E. Treherne, P. J. S. Smith and E. A. Howes AFRC Unit of Insect Neurophysiology and Pharmacology, Department of Zoology, Cambridge CB2 3EJ. UK

1 Introduction 2 Degenerative responses 2.1 Central nervous system 2.2 Peripheral nerves 2.3 Secondary effects of axotomy 3 Regenerative responses of insect neurons 3.1 Interneurons 3.2 Motor neurons 3.3 Sensory neurons 4 Role of neuroglia and exogenous cells 4.1 Evidence for the involvement of exogenous reactive cells 4.2 Regeneration of the blood-brain barrier 4.3 Cell recruitment and interactions during glial repair 4.4 Long-term changes in glial repair 5 Concluding remarks References

1

Introduction

Nerve repair and regeneration is an intricate and frequently protracted business, involving a variety of specialized cell types and often producing widespread cellular responses beyond the lesion. The difficulty of studying these processes is compounded by the complexity of the nervous systems of higher vertebrates which are conventionally used as models for mechanisms of human brain repair. Surprisingly, in view of the contributions which they have made to our understanding of many other crucial aspects of neural function, invertebrate nervous systems have been relatively little exploited to elucidate the basic strategies of nervous repair and regeneration. Notable exceptions are pionADVANCES IN INSECT PHYSIOLOGY VOL. 21 ISBN 0-12-0242214

Copyrighr 0I988 Acudemic Press Limited AN righrs u/rrprudurlion in myform reserved

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eering studies on annelids and the nervous systems of some crustacean species which show highly-developed powers of neural regeneration far surpassing those of mammals. Despite their proven value in many aspects of physiological research and in the study of neural development, insects have not figured prominently in the literature on neural repair. The impressive regenerative capacity of insect nerves was first recognized by Bodenstein (1955, 1957) and since then the value of insect nervous systems for the study of nerve repair and regeneration has been gradually realized as is witnessed by an increasing trickle of publications. An undoubted experimental advantage, which insects share with the few invertebrates that have been studied so far, is the regenerative powers of their central nervous systems (CNS). They are capable of extensive axonal regrowth and accurate re-formation of synaptic connections which contrasts with the more limited potential of the brains of higher vertebrates. Furthermore, the experimental accessibility and the relative anatomical simplicity of the increasingly well-understood insect CNS provides valuable opportunities for the elucidation of cellular reactions in vivo. Such studies in mammalian species have been largely confined to in vitro studies on cultured cells in incompletely-defined media. The increasing success with in v i m culturing of insect nervous tissues ranging from whole abdominal nerve cords to conventional cell cultures provides opportunities for relating the results obtained on isolated cells to their performance in the reality of the repaired nervous system in the whole animal.

2

2.1

Degenerative responses CENTRAL NERVOUS SYSTEM

Surgical transection of insect axons causes degenerative changes analogous to those of mammalian neurons. Severance of central nervous connectives, for example, induces rapid ultrastructural changes at the cut ends of both proximal and distal axonal segments (Hess, 1960; Boulton, 1969; Meiri et al., 1983). These commence within 24 h and, characteristically, include disruption of neurotubules, the rapid appearance of amorphous material in the axoplasm and a slower accumulation of large numbers of mitochondria, various-sized vesicles and quantities of smooth endoplasmic reticulum. The observation that, in cockroach giant axons, these changes are similar at the severed ends of both the distal segment and the proximal one (which is still

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attached to the remainder of the neuron) suggests that they do not depend upon the presence of the soma and are, thus, locally-mediated (Meiri et al., 1983), as was originally proposed in the locust CNS (Boulton, 1969). At this stage, extensive changes also occur in the surrounding tissues of connectives or nerves when they are cut or crushed. These include damage to the superficial neuroglial layer, the perineurium, which constitutes the insect blood-brain barrier (cf. Treherne, 1985), as shown by the penetration of ionic lanthanum into the underlying extracellular system (Treherne et al., 1984) and the rapid effects of tetrodotoxin, which is excluded by the intact perineurial blood-brain barrier (Leech and Treherne, 1984). A notable feature of the early responses to mechanical damage associated with axonal severance is the swift accumulation of haemocytes at the damaged regions of nerves or connectives (Bodenstein, 1957; Boulton, 1969; Treherne P I a/., 1984). Another consistent response is the appearance of cells containing electron-dense granules, the so-called granule-containing cells, within damaged regions of the perineurium as well as between undamaged cells in the underlying glial layer (Treherne ef a/.,1984). These cells, together with haemocytes and, according to Boulton (1969), neuroglia contribute to the formation of scar tissue. This forms relatively slowly over the lesion, only after 4 months, for example, is the reorganization of such repaired regions of the blood-brain interface sufficient to exclude intercellular access of extraneously applied ionic lanthanum (Treherne et al., 1984). The neuroglia also show early responses to surgical lesioning. Within 24 h, glial processes become associated with the invaginations of the axolemma, which form at both proximal and distal axonal tips, (Meiri et al., 1983). By 4 days many of the glial cells in the lesion zone are clearly disorganized and there is also an enlargement of the extracellular spaces and marked changes in the appearance of the intercellular matrix, which is now highly fragmented and separated from the glial membranes (Boulton, 1969; Treherne et al., 1984). The immediate electrical responses of severed axons depends upon the position and the mode of lesioning. Cutting of cockroach central nervous connectives may be followed by inadequate axonal re-sealing, in which case there is a rapid decline in both resting and action potentials at recording sites close to the lesion. In such instances it is impossible to restore action potential production by hyperpolarizing the axonal membrane (Meiri er al., 1981; Leech and Treherne, 1984). In other cases, when the cut ends of the axons reseal adequately, normal resting and action potentials can be recorded, in vitro, for several hours. In vivo, segments of cockroach giant axons separated from their cell bodies retain their excitability for up to 8 days, and for 20 days if the connectives are also ligatured before cutting (Farley and Milburn, 1969; Leech and Treherne, 1984).

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P.J . S . SMITHand E . A . HOWES

Besides these essentially local responses, axonal transection in insects can, as in crustacean and mammalian neurons (e.g. Kuwada and Wine, 1981; Heyer and Llinas, 1977; Gustafsson, 1979), also produce more widespread effects within the neurons, including changes in the electrical properties of their associated cell bodies. It is well established that the cell bodies of some insect neurons do not normally generate action potentials (cf. Kerkut et al., 1969; Hoyle and Burrows, 1973; Burrows, 1977). However, after axotomy, inexcitable cell bodies in the ganglia of some cockroaches and grasshoppers, and a cricket (Fig. 1 ) develop regenerative, overshooting action potentials within 6 h to 4 days (Pitman et al., 1972; Goodman and Heitler, 1979; Roederer and Cohen, 1983b). The response depends upon the site of lesioning. In the cricket giant interneuron, for example, axotomy at 1 mm or more from the cell body is without effect, whereas closer lesioning produces excitability in the soma (Roederer and Cohen, 1983b). In such preparations, somatic excitability disappears after 2 days and the electrical properties of the membrane return to normal. This decline in transient excitability is associated with marked ultrastructural changes in the soma (Roederer and Cohen, 1983b). Membrane-bound vesicles (> 5.0 nm) appear after 2 days and begin to disappear after 6, and arrays of densely-packed microtubulelike structures accumulate in the soma, especially near the neurite. These structural changes in the insect soma parallel equivalent degenerative changes which occur in axotomized vertebrate neurons, where there is both vacuolation and the appearance of membrane-bound vesicles (cf. Barron et ~ l .1971, , 1973) and apparent effects upon microtubule metabolism (Heacock and Agranoff, 1976; Burrell et al., 1979). The distal segments of transected axons in the central nervous systems of insects show variable rates of degeneration. Unligatured cockroach giant axons, for example, degenerate relatively rapidly, within 3-8 days (Hess, 1958, 1960; Farley and Milburn, 1969) when separated from their cell bodies, whereas apparent distal segments in the grasshopper Laplatacris disper (Melamed and Trujillo-Cenbz, 1962) and the locust, Schistocerca gregariu (Rowell and Dorey, 1967; Boulton, 1969; Boulton and Rowell, 1969) have been described as showing only minimal degenerative changes after 10 days. Furthermore, in the cricket CNS, the dendritic arborization and associated axon of an identified motor neuron have been shown to survive for as long as 168 days when isolated from the cell body, although the distal axon segment by itselfdegenerates, physiologically and morphologically, within 4 1 5 days (Clark, 1976a, b). The survival in insects of some isolated distal segments can thus approach those recorded for annelid and crustacean species where axonal segments are maintained for several months in the absence of their cell bodies (Hoy et at., 1967; Wine, 1973; Frank ez al., 1975; Eirse and Bittner, 1976). It has been postulated that this is a consequence of direct transfer of

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h

NORMAL 20mV lOnA

L 40msec

-

24 HOUR A XOTOMY

- -

"5% L mrec

Fig. 1 The effects of axotomy o n the electrical responses of the membrane of the soma of the median giant interneuron of the cricket. Action potentials could not be induced from the soma in intact neurons (a<:) with increasing levels of injected current. Fast action potentials, in this case of 90mV amplitude, could, however, be evoked 24 h after axotomy (e and f). Note difference in calibration in the two cases. (From Roederer and Cohen, 1983b.)

essential metabolites to the axoplasm by the associated neuroglia (Meyer and Bittner, 1978a, b), an explanation which has been invoked to account for the prolonged survival of the isolated arborizations of the cricket motor neuron (Clark, 1976a, b; Edwards and Meyer, 1985). The degeneration of isolated insect axonal segments, for example those of cockroach giant axons, is characterized by swelling of mitochondria, extensive vacuolation, appearance of lysosomes and absence of neurotubules in the

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axoplasm. As the segments collapse, the extracellular space becomes filled with what are described as hypertrophied glia (Meiri et al., 1983). 2.2

PERIPHERAL NERVES

The degenerative responses of insect peripheral nerves, following surgical lesioning, share many of the features of those following,axotomy of central neurons: the sensory axons (with peripherally-located cell bodies) showing regressive changes in the proximal stump and motor neurons (with centrallylocated soma) in the distal stump. In the proximal segment of the severed metathoracic nerve 5 of the cockroach, Periplaneta americana, as in lesioned central connectives (Boulton, 1969; Treherne et al., 1984) and the cut nerves of transplanted ganglia (Marks et al., 1968), there is a swift accumulation, within 24 h, of haemocytes (Bodenstein, 1957; Blanco, 1987). This is followed by the transient appearance of granule-containing cells among the damaged perineurial and underlying glia. Changes also occur in the neural lamella, which, within 4 days, becomes darker and thinner. There is considerable glial disruption, although the perineurial cells close to the larger severed axons retain their characteristic appearance, but with greatly enlarged intercellular clefts. At this stage, in the severed cockroach metathoracic nerve, many of the smaller axons have already degenerated, the remaining cavities being filled with cellular debris, glial folds and quantities of matrix material (Blanco, 1987) The rates of degeneration of insect neuromuscular junctions are temperature dependent. At 20"C, transmission at excitatory synapses on locust skeletal muscles fails at between 9 and 24 days, after sectioning of the innervating metathoracic nerve, and after only 2 days at 30°C (Usherwood et al., 1968). Similar rates have been recorded for the failure of cockroch neuromuscular transmission at equivalent temperatures (Roeder and Weiant, 1950; Bodenstein, 1957; Jacklet and Cohen, 1967b; Guthrie, 1962, 1967; Washio, 1986). These rates of conduction failure are considerably faster than those recorded for severed crustacean motor axons (Bittner, 1973; Bittner and Johnson, 1974; Hoy et al., 1967; Norlander and Singer, 1972) some of which survived for as long as 368 days, at 20"C, in the leg nerve of the crayfish, Procambarus clarkii (Atwood et al., 1973). As with axotomized neurons of crustacean central nervous systems, it is suggested that such astonishing survival times result from appreciable trophic support provided by glial elements (Meyer and Bittner, 1978a, b) which must be lacking for the insect motor axons investigated. However, distal axonal segments of locust metathoracic nerves continue to conduct action potentials for some time after neuromuscular transmission

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ceases (Usherwood, 1963b), suggesting that in this species conduction failure primarily results from synaptic rather then axonal breakdown (Usherwood et ul., 1968). In denervated coxal muscles of the cockroach, the cessation of spontaneous miniature end-plate potentials, after 48-96 h at 25-26”C, is accompanied by the first signs of ultrastructural degeneration of the terminals: a clumping of synaptic vesicles around the mitochondria and an increase in the electron density of the terminal axoplasm (Wood and Usherwood, 1979; Washio and Nihonmatsu, 1987). Subsequently, glial cells move into the synaptic clefts to engulf the degenerating axon terminals (Fig. 2). At 5 days after nerve section, the closely packed vesicles form electron-dense, honeycomb-like structures and at the same time lamellar bodies, characteristic of lysosomal activity, appear within the surrounding glial cells. Neuromuscular degeneration in locust skeletal muscle differs from that of denervated cockroach coxal muscle in the appearance of bursts of so-called “giant” miniature end-plate potentials which coincide with transmission failure and the ultrastructural demonstration of appreciable agglutination of synaptic vesicles, to an extent which is not seen in the cockroach preparation (Usherwood et ul., 1968). Such “giant” spontaneous potentials could thus result from the synchronous release of large numbers of transmitter quanta from the degenerating terminals or, conceivably, from transmitter release from the proliferating glia, which are filled with large vesicles at this stage (Usherwood et ul., 1968), perhaps by a mechanism analogous to that proposed for the Schwann cells at denervated frog neuromuscular junctions (Dennis and Miledi, 1974).

2.3

SECONDARY EFFECTS OF AXOTOMY

Axotomy also induces other extensive changes which suggest the existence of powerful contact, trophic and/or neurohormone influences on the associated postsynaptic elements. In locust and cockroach skeletal muscles, for example, there is a swift onset of atrophy following cessation of spontaneous miniature discharge (Usherwood, 1963a, b; Usherwood et al., 1968; Wood and Usherwood, 1979). In locust muscle fibres, this is associated with dramatic changes in the extrajunctional sensitivity to the putative excitatory transmitter, glutamate. Following denervation, there is an approximately 100-fold increase in sensitivity, largely due to an increase in the depolarizing (D) receptors (Cull-Candy, 1975, 1978). Partial denervation of a target muscle in the cricket, by removal of the fast axon input, increases the twitch tension induced by the slow axon, by 5-10 times, and the tetanic tension by 10-30 times (Donaldson and Josephson, 198I): an example of synaptic plas-

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ticity by enhancement of synaptic transmission following removal of a parallel pathway. In the CNS, axotomy or peripheral deafferentation may profoundly affect the metabolism and function of target interneurons. In the cricket, for example, cercal deafferentation of the developing interneurons of the terminal abdominal ganglion reduces protein metabolism of the somata and within the dendrites of target interneurons (Meyer and Edwards, 1982). It can also affect elements of the cholinergic system, altering acetylcholinesterase levels and reducing the densities of both muscarinic and cholinergic receptors in the terminal abdominal gangiion of adult insects (Edwards and Meyer, 1985; Meyer et al., 1986). These examples serve to emphasize the complexity of the degenerative responses to neural lesioning which involve not only the injured neurons, but other excitable cells, neuroglia, extracellular elements and exogenous cells all of which are recruited and integrated in repair and-when they occur-in the subsequent regenerative processes. 3 Regenerative responses of insect neurons

The capacity for neural regeneration in invertebrate animals can be broadly related to their overall potential for bodily regeneration (cf. Edwards and Palka, 1976; Anderson et a[., 1980). Viewed in this light the regenerative powers of insect nervous systems is limited, for example, in comparison with those of coelenterates (which can regenerate whole organisms from single fragments) or of annelid species (which can replace extensive portions of their bodies). Nevertheless, in comparison with the modest capabilities of higher vertebrates the regenerative potential of insects is impressive; enabling the regeneration of entire appendages and extensive neural restructuring at successive developmental stages.

Fig. 2 Electronmicrographs of cross-sections of degenerating and regenerating nerve terminals from coxal depressor muscles of the cockroach, Periplunrtn americnna, following crushing of the 5th nerves from either meta or mesothoracic ganglia. The first signs of terminal degeneration are seen after 48 h. By 4 days (A and B) the accumulations of synaptic vesicles have disappeared and glial processes have moved into the synaptic cleft, where they are in close contact with the muscle fibre. After 18 days (C and D) the glial processes have drawn back from the synaptic cleft and by 24 (E and F), and to a greater extent after 36 days (G and H), synaptic vesicles accumulate at the presynaptic membrane. By 37 days the structure of the regenerating terminals is essentially similar to that of normal junctions. (From Washio and Nihonmatsu, 1987.)

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A notable feature of central nervous regeneration in insects is the vigorous axonal regrowth which can occur following neuronal lesioning. In higher vertebrates, transected axons show only very limited, and usually undirected, extension within the brain and spinal cord (cf. Aguayo et al., 1981), a limitation which was originally attributed to intrinsic properties of mammalian neurons (Cajal, 1928). In the insects which have been examined, on the other hand, lesioned central neurons are capable of considerable axonal extension. In the cockroach CNS, for example, regenerating giant interneurons can traverse extensively along the abdominal ganglionic chain (Fig. 3) (Meiri et al., 1981; Leech and Treherne, 1984; Spira et al., 1987). Furthermore, as in some other invertebrates, notably the leech (Chiquet and Nicholls, 1987; Muller et a!., 1987), insect neurons have the capacity to re-establish precise functional connectives within the CNS. This is particularly apparent with sensory neurons in the regeneration of abdominal cerci of the house cricket where appropriate terminations are made with identified giant interneurons after prolonged periods of postembryonic development in the absence of such connections (Edwards and Palka, 1976; Murphey et al., 1984; Kamper and Murphey, 1987). The regenerative capacities of insect peripheral nerves share, in broad terms, many of the features of those of higher vertebrate animals. However in this case, unlike the situation in the CNS, the speed of axonal regrowth in mammalian peripheral nerves can exceed those recorded for insect species. The motor axons from the metathoracic ganglion supplying the coxal depressor muscles in the cockroach, for example, grew from a crushed stump at an average rate of 0*9mm/day,at 22-23°C (Denburg et al., 1977). This approximates to the initial rate of axonal growth in crushed sciatic nerve of the rat, but falls far short of the subsequent maximal rate of 9.2 mm/day observed in some of the smaller axons (Olson, 1969). There is no evidence in insects that severed peripheral axons from the proximal stump can fuse with the surviving axonal segments from the distal stump, and then become functional again, such as has been shown to occur in crayfish motor neurons (Bittner, 1973). In insects, therefore, as in higher vertebrates (cf. Kline and Hudson, 1981), reconnection of severed motor axons with their target sites is achieved by regrowth from the proximal stump. Where lesioning is by nerve crush, guidance for axonal regrowth can be provided by residual basal laminae, extracellular matrices and/or surviving neuroglia. This could be the case, for example, in the regeneration of the axons innervating the coxal depressor muscle from the crushed metathoracic nerve root 5 of the cockroach, where the final pattern of axonal distribution is essentially similar to that of normal, unoperated, individuals (Denburg et al., 1977). However, specific neuromuscular reconnection can also be achieved in insects following complete nerve severance (Bodenstein, 1957; Guthrie, 1962,

Aiii

Ai

.i-AiiuG1‘7 n

Bi

Bii

Biii

Fig. 3 Cobalt-fillcd giant interneurons in ganglion A, of the cockroach abdominal nerve cord. In normal preparations (Ai-Aiii) a single, cychndricdl, giant axon sends out one (as in Ai and Aiii) or two (Aii) neurites which branch into the neuropil. Following crushing of the abdominal connectives (between ganglia A, and A,, 92103 days previously) the regenerating sprouts send out several neurites which branch more extensively and occupy a larger field than in the ganglia from normal preparations (Bi-Biii). Even when there are major errors in axonal regrowth (as in Biii) the elongating sprouts still send out neurites into the neuropil. (The bottom of the drawing corresponds to the posterior region of the nerve cord.) (From Spira et al., 1987.)

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1967; Pearson and Bradley, 1972; Blanco, 1987). This precludes the possibility of immediate contact guidance of regenerating axons by pre-existing structures associated with the distal stump and suggests an additional involvement of long-range factors in guidance to the appropriate target muscle. 3. I

INTERNEURONS

The regenerative responses of insect interneurons depend upon both the site and the mode of lesioning. Axotomy of giant interneurons, at a distance from their cell bodies, induces axonal elongation from the lesioned proximal stump in the severed or crushed connectives of the cockroach (Fig. 3) (Meiri et af., 1981, 1983; Leech and Treherne, 1984; Spira et af., 1987) and cricket abdominal nerve cords (Roederer and Cohen, 1983a). However, severance of the axon close to the cell body of the cricket giant median interneuron results in extensive dendritic sprouting with only limited axonal extension (Roederer and Cohen, 1983a). In this preparation, there seems to be a reciprocal relationship between the extents of dendritic and axonal sprouting. This, it is suggested, is associated with the virtually complete retrograde degeneration of the proximal axonal stump produced by lesioning close to the cell body (p. 38). The appearance of the numerous arrays of densely-packed microtubules in the vicinity of initial neurites after 2 days (when the transient excitability of the soma disappears) suggests that they may be critically involved in dendritic growth (Roederer and Cohen, 1983b). Research on vertebrate and molluscan neurons has focused on the role of calcium entry in triggering neurite extension (Llinas, 1979; Llinas and Sugimori, 1979; Anglister et af., 1982; Kater et al., 1988). Calcium action potentials have been recorded, for example, from growth cones of cultured neuroblastoma cells, while extracellular recordings along the neurites indicate that voltage-activated calcium channels predominate in the growth cone (Anglister et af., 1982). Digital imaging techniques using calcium-indicating dyes have also revealed that the generation of neuronal cytoarchitecture, associated with the growth cone, is apparently regulated in quite specific ways by Ca2+(Kater et al., 1988). An involvement of calcium ions in axonal sprouting has also been proposed for insect central neurons, following the report of a transient appearance of calcium-dependent action potentials in the vicinity of the tips of regenerating cockroach giant interneurons. These were reported to be present only between 7 and 60h after severance, from apparent resting potentials of only - 10 to -30mV (Meiri et al., 1981). Subsequent investigation failed to confirm the presence of primarily calcium-dependent action

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potentials, which were shown to be blocked by tetrodotoxin and unaffected by cobalt ions (Leech and Treherne, 1984). However, there is evidence for a contribution of voltage-dependent calcium channels in regenerating cockroach giant interneurons (Spira et al., 1987). This was shown by the intracellular injection of tetraethylammonium ions, which produced a large increase in spike duration due to reduced potassium conductance (cf. Pelhate and Pichon, 1974; Pichon, 1976; Pelhate and Sattelle, 1982). The prolonged action potential produced under these circumstances was abolished by tetrodotoxin, indicating that its initiation probably requires activation by voltagedependent sodium channels (cf. Narahashi, 1966). However, when barium ions were substituted for those of sodium, a long-lasting action potential was elicited which consisted of an early overshooting plateau (originating from the axonal membrane) and a later, smaller, one attributed to the axonal sprouts (Fig. 4). These responses-which were rarely seen in control preparations, were blocked by cobalt and cadmium ions and persisted in sodium-free saline-indicate the appearance of voltage-dependent calcium channels in the regenerating cockroach giant interneurons (Spira et al., 1987). An increased influx of calcium ions has also been invoked to account for the ultrastructural changes in median giant interneurons of the cricket following close axotomy (Roederer and Cohen, 1983b) on the basis of the similarity of the large vacuoles seen in axotomized cricket soma to those induced in molluscan neurons by intracellular injection of the cation (Nicaise and Meech, 1980). Changes in dendritic sprouting can also occur in the absence of direct neuronal damage. Postembryonic denervation of cricket auditory interneurons, for example, reduces the extent of the normal neurite, but induces growth of an aberrant one which branches into the contralateral, instead of the ipsilateral side of the prothoracic ganglion (Hoy et al., 1985). Such observations suggest the existence of local trophic factors, provided by afferent terminals, which maintain and determine the form of dendritic sprouting in the postembryonic CNS. Despite the vigorous regenerative capacity of some insect interneurons, the morphological redifferentiation of lesioned cockroach giant axons differs in a number of respects from that of normal ones (Spira et al., 1987). The dimensions of the regenerating axonal segments are much smaller, both in connectives and ganglia, than in the normal interneurons with consequent reductions in conduction velocity. In regenerating segments, conduction is less than 2m/s (Spira et al., 1987), as compared with 3-6m/s along intact giant axons (Spira et al., 1969; Dagan and Parnas, 1970). Changes also occur in a region of low safety factor for impulse propagation at the branching points of the sprouts from the proximal axonal segment (Spira et al.. 1987). This, it is proposed, results from impedance mismatching of the geo-

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D

A S

V

I

I

B

E

100 ms

h

so 111s

loll m s

Fig. 4 Overshooting Ba2 action potentials in the regenerating segment of a crushed cockroach giant axon. (A) Schematic diagram, showing the position of the voltagerecording (V) and the current-injecting elecrodes (I), close to the site of crushing, and the stimulating electrode (S) placed on the connective between ganglia A, and A,. (B) Action potential in normal physiological solution. (C) Prolonged action potential after intracellular injection of tetraethylammonium. (D) Application of tetrodotoxin ( mol/l) blocked the prolonged action potentia!. (E and F) Ba2+action potential after substitution of 100 mmol/l BaZ+for Na'. Intracellular stimulation produced an overshooting action potential followed by a smaller potential (E). Intracellular stimulation of the sprouts produced a small response, which, with increased stimulation intensity (F), increased to an overshooting spike. (From Spira et al., 1987.) +

metry of the regenerating axons and sprouts (cf. Spira et al., 1976; Parnas and Segev, 1979) which can prevent the propagation of action potentials in this region, even at moderate frequencies (Spira et al., 1987). Such atypical structure and function contrast with the relatively precise arborizations and apparent synaptic reconnections established by regenerating sensory neurons on regrowth into the CNS (see p. 55). This could result merely from disorganization of the scar tissue in which the axon sprouts arise or, conceivably, from the lack of appropriate guidance for regenerating interneurons in the adult CNS. The importance of the ganglionic microenvironment in providing directional cues for axonal regrowth is indicated by the effects of cellular destruction, produced by repeated freezing and thawing of cockroach abdominal ganglia (Spira et al., 1987). Under these circumstances, the regenerating

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sprouts never extend branching neurites, as in normal ganglia. Furthermore, the re-establishment of acetylcholine receptors, which in both intact and regenerating giant interneurons are located only on the neurites, was virtually abolished in freeze-thawed ganglia (Fig. 5). This could again indicate the existence of localized guidance cues, provided by the neuroglia and/or their associated neurons and extracellular matrices, both for neurite formation and the expression of acetylcholine receptors.

3.2

MOTOR NEURONS

As in vertebrate preparations, there is an appreciable lag period before detectable axonal growth occurs from peripheral axons of motor neurons in the distal stump, following crushing or nerve severance. In the cockroach metathoracic nerve, axonal sprouting occurs from the distal stump at 13-14 days after lesioning (Jacklet and Cohen, 1967b; Denburg el al., 1977) as compared with the 4-10 day lag described in equivalent mammalian preparations (cf. Sunderland, 1978). Evidence from the cockroach metathoracic neuromuscular preparation indicates that the lag period is associated with changes in neuronal protein synthesis. This is suggested by the appearance, within 12 h, of a dense concentration of RNA, which encircles the nucleus and reaches a maximum after 2-3 days (Cohen and Jacklet, 1965), and a subsequent increase in [3H]leucine incorporation in the cell bodies of axotomized motor neurons (Denburg and Hood, 1977). The increase in [3H]leucine incorporation occurs after 12 days (Denburg and Hood, 1977) and immediately precedes sprouting from the proximal stump (Jacklet and Cohen 1967b; Denburg et al., 1977). The perinuclear accumulation of RNA in the insect neuron is attributed to an aggregation of ribosomes (Cohen and Jacklet, 1965); a response which differs from that described for vertebrate neurons where there is a breakdown of the dense RNA aggregates in the Nissl bodies as ribosomes disperse following neuronal injury (Porter and Bowers, 1963). Growth of axonal sprouts to their neuromuscular targets depends upon the form of lesioning. When motor axons are severed by nerve crush, guidance is provided by the surviving nerve stump (Denburg er al., 1977). When the nerve is cut, and the ends remain well aligned, the regenerating axonal sprouts from the proximal stump can re-link to the distal one by connective (scar) tissue (Jacklet and Cohen, 1967b), as in equivalent vertebrate preparations (Liu, 1981; Lundborg and Hansson, 1981). However, when the cut ends retract, regenerating insect axons frequently follow tortuous routes through the haemocoel before joining the distal stump (Jacklet and Cohen, 1967b).

Fig. 5 Maps of the acetylcholine sensitivity, at various locations within ganglion A, from the cockroach nerve cord, constructed from the responses to iontophoretic application of acetylcholine (A). In a control preparation (B), responses were restricted to the central field of the ganglion, whereas in regenerating ones (Ci-Ciii) (6090 days after crushing of the connectives between ganglia A, and A3) there is a wider distribution of positive responses, corresponding to the enlarged fields occupied by the neurites (see Fig. 3). Giant axons regenerating through ganglia, in which the cells were destroyed by freezing and thawing, rarely exhibit acetylcholine sensitivity (DiDiii). (Small circles, no responses; large circles, positive responses; squares, iontophoretic electrode penetrated the giant axon.) (From Spira et al., 1987.)

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Innervation of cockroach coxal muscles can also be achieved from transplanted metathoracic ganglia (Jacklet and Cohen, 1967a) or when thoracic ganglia are rotated (Bate, 1976). Reconnection to appropriate muscles can, in addition, be made by serially homologous motor neurons when a mesothoracic leg is transplanted to the adjacent, metathoracic, segment (Young, 1972). Innervation in the absence of immediate contact guidance provided by the distal stump might imply the existence of diffusible factors to direct the regrowth of the regenerating axonal sprouts from the proximal stump, such as is suggested by recent research on frog sciatic nerve (cf. Kuffler, 1987). There is, however, no evidence from any insect preparation for the involvement of such factors. The persistence of neuroglia, both in the proximal and distal stumps (Blanco, 1987). as well as in the vicinity of the nerve terminal itself (p. 52) (Washio and Nihonmatsu, 1987), is likely to be a critical factor in the reinnervation of insect muscles, not only in providing contact guidance, but also for the possibility of releasing nerve growth factors such as has been recently demonstrated in regenerating mammalian sciatic nerve (cf. Heumann, 1987). Following severance of the cockroach metathoracic nerve 5, there is, in fact, proliferation of the glia both in the vicinity of the nerve terminals (Washio and Nihonmatsu, 1987) and the regenerating proximal stump (Blanco, 1987).In the proximal stump this was shown by an increase in [3H]thymidine labelling of the glial nuclei which reached a maximum a t between 2 and 3 weeks (Blanco, 1987), a timing which corresponds with the onset of axonal sprouting (Jacklet and Cohen, 1967b; Denburg et al., 1977). The axon sprouts in the regenerating cockroach proximal stump are entirely ensheathed by glial processes and are contained in an encapsulating extracellular matrix (Fig. 6). The extensive glial proliferation and ensheathment of the axon sprouts in the cockroach preparation would accord with the postulated role of the Schwann cells in regenerating mammalian sciatic nerve, where an up-regulation of nerve growth factor has been demonstrated in these non-neuronal cells (Heumann, 1987). By analogy with vertebrate systems, glial proliferation in the insect preparation could be initiated either by contact-mediated stimulation by axonal sprouts, such as has been demonstrated in cultured vertebrate Schwann cells (Salzer and Bunge, 1980; Salzer et al., 1980a, b), or, conceivably, from diffusible glial mitogenic factors released from exogenous reactive cells (see p. 72). The proliferation of neuroglia following the disappearance of miniature end-plate potentials at denervated nerve terminals of cockroach coxal muscle (Washio and Nihonmatsu, 1987), also implies that these non-neuronal cells

Fig. 6 Diagrammatic representation of the regenerating proximal stump of the cockroach metathoracic nerve 5 four weeks after cutting at the position indicated. (Re-drawn from Blanco, 1987.)

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may play a role in axonal extension and guidance. At 18 days after axotomy in this preparation the glial cells draw back from the synaptic cleft, prior to neuromuscular reconnection (see Fig. 2). At this stage the presynaptic axoplasm contains numerous, large, dense-core vesicles. These are also seen in regenerating terminals in the flight muscles of the moth, Antheraea polyphemus (Niiesch and Stocker, 1975), where they have been attributed a trophic function (Stocker and Nuesch, 1975). In addition to potential guidance cues provided by the neuroglia and associated extracellular matrices in the distal stump, specific muscular protein fractions have been invoked as recognition signals for re-innervation: a supposition based on the demonstration of unique protein fractions in each of the six coxal depressor muscles of the cockroach (Denburg, 1975, 1978). After denervation, the biochemical identity of these fractions is maintained, unlike the situation in mammalian skeletal muscle where the biochemical parameters of fast and slow fibres tend to regress to a common, intermediate, type (Gutmann, 1976). The maintenance of the characteristic complement of muscle proteins in the insect preparation is thus attributed to the presence of specific recognition molecules in the denervated coxal muscles (Denburg, 1975). However, it has not been demonstrated whether the proteins are cytoplasmic or associated with the surfaces of the muscle cells or their basal lamina as might be expected for putative recognition molecules. Unlike the cockroach coxal proteins, denervation altered glycoproteinlectin binding. However, it is still possible to distinguish the six different muscles from their binding of concanavalin-A and wheat germ agglutinin (Denburg et al., 1983a, b). On the basis of these results, it is proposed that the denervated muscles present different “cell surfaces” to regenerating motor neurons (Denburg et ul., 1983b), although it is not clear whether the lectin receptors are, in fact, located on the basal lamina or the cell membranes. Nor is there any additional evidence for the role of such putative receptors in the re-establishment of neuromuscular connections in insects such as exists for the agrin molecules in the basal lamina of frog skeletal muscle fibres-which induce the aggregation of acetylcholine receptors and acetylcholinesterase in the synaptic clefts of regenerating muscle fibres (M. A. Smith et al., 1987). Despite the precise pattern of motor re-innervation which can occur following normal crushing or severance of insect peripheral nerves-or even when nerves are re-routed, or ganglia and limbs are transposed (e.g. Young, 1972; Bate, 1976; Fourtner et af.. 1978)-regenerating motor axons can make inappropriate connections when normal target muscles are excised. This was shown with the fast excitatory coxal depressor motor neuron of the cockroach which, following severance and in the absence of the coxal depressor muscles, established functional connections with femerol levator and tibia1 flexor muscles (Whitington, 1977). The re-establishment of synaptic connec-

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tions by regenerating axon terminals is thus not necessarily uniquely specified at the cellular level. Reconnection could, thus, be achieved by relatively non-specific inducer molecules, perhaps analogous to those of agrin (M. A. Smith et al., 1987). In this case, it follows that the so-called speciJic muscular re-innervation observed in insect preparations following conventional nerve lesioning would result primarily from the direction of axonal regrowth, presumably by diffusible and/or contact factors associated with the residual glial and acellular elements of the proximal stump. Unfortunately, there appear to have been no attempts to elucidate the nature of such factors in any insect preparations. In addition to the bizarre neuromuscular connections which can be established in the absence of target muscles, some misdirection of regenerating motor axons can occur even when the muscles and their proximal stump remain. Following crushing of the motor nerve supplying the cockroach coxal depressor muscles it was found, after 20-44 days, that there was an average of 3.5 unidentified neurons, from the metathoracic ganglion, that were not normally associated with a particular nerve branch (branch 5 re) (Denburg et a[., 1977). The number of such apparent errors declined, 10-fold, between 45 and 90 days (to an average of 0.33 neurons per ganglion), to leave a pattern of re-innervation that was closely similar to that in undamaged preparations. In this case, the final high specificity of regenerated neuromuscular connections appears to be achieved by an appreciable degeneration of misdirected axons. Besides the direct effects of axotomy on target muscles, denervation can also produce dramatic changes in neuromuscular responses mediated by alternative synaptic inputs. Severance of the fast axon, for example, increases the excitatory junctional potentials evoked by the slow axon to the extensor tibiae muscle in both locust and cricket (Usherwood, 1963b; Donaldson and Josephson, 1981). In the cricket, such partial innervation induces a progressive increase in the mechanical response initiated by the slow axon: twitch tensions attaining 5-10 times and tetanic tensions 10-30 times those recorded in fully innervated muscles (Donaldson and Josephson, 1981). Such enhancement of synaptic transmission, by removal of a parallel pathway to the target cells, has been attributed to an unmasking of ineffective synapses so as to partially compensate for the loss of normal input as has been proposed for the sensory afferent cells of cat spinal cord (cf. Wall, 1977) and salamander neuromuscular systems (cf. Bennett and Raftos, 1977; Genat and Mark, 1977; Dennis and Yip, 1978). Unlike the situation with insect interneurons (p. 46), direct surgical damage of motor neurons appears to have little effect on dendritic sprouting. Thus axotomy, by peripheral nerve severance, of the fast coxal motor neuron in the metathoracic ganglion of the cockroach, produced no detectable

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changes in dendritic branching (Tweedle et al., 1973). However, removal of synaptic input to the motor neuron (by severance of other nerve trunks to the ganglion), resulted in extensive dendritic sprouting (Pitman and Rand, 1982). The sprouts appeared to grow preferentially into regions containing degenerating nerve fibres, often into those which the neuron would not normally occupy. It is not known whether this is a direct cellular response to loss of synaptic contacts or is caused by local release of factors from adjacent damaged neurons and/or from neuroglia. Nor is it known whether the dendritic sprouts serve any functional role in synaptic transmission.

3.3

SENSORY NEURONS

The impressive regenerative capacities of insect sensory neurons are beautifully exemplified by the responses of the cercal giant interneuron pathway of the cricket to surgical manipulation. Thus, for example, when cercal tissue from one species (Cryflusbimaculatus) was grafted to a homologous position on the cercus of another (Acheta domesticus), the subsequent afferent projections of the transplanted neurons were found to be identical, in both arborization and position, to those of normal neurons in the host CNS (Murphey et al., 1984; Kamper and Murphey, 1987). Furthermore, when fully differentiated sensory neurons were transplanted to a non-homologous position on the cercus, the regenerating cell still arborized in typical fashion within the host ganglion (Fig. 7). However, removal of one cercus, which causes rapid axonal degeneration (Edwards and Meyer, 1985), results in expansion of the remaining cercal afferent terminals into regions normally occupied by the now absent contralateral neighbours (Murphey et al., 1984). Despite the developmental plasticity of the cricket cercal sensory system (in which appreciable, systematic variations in synaptic strength occur during development), normal synaptic efficiencies are eventually re-established after severance and regeneration of the cercal nerve (Chiba et af., 1988). Since, in contrast to the developing system, the regenerating axons arrive in the CNS at approximately the same time (ca 14 days), this suggests that the sensory neurons have specific preferences for particular interneurons and that these are retained during regeneration. The cellular mechanisms involved in the precise re-establishment of the synaptic projection by the regenerating cercal neurons in the CNS remain to be elucidated. However, the available evidence suggests that simple contact guidance is unlikely to be an important factor. This is deduced from the observation that the neuronal processes reached their normal target sites, often by circuitous routes, after various experimental manipulations (Murphey et al., 1983, 1984). Murphey et al. (1984) favour a “chemoaffinity model” which

Fig. 7 Regeneration of sensory neurons from the cercus of a black cricket (Gryllus bimaculutus) when transplanted to a non-homologous position on the cercus of a second species, the tan cricket (Achetu domesticus). (A) The result when a fully differ-

entiated neuron is transplanted to a non-homologous position on a second cricket. The transplanted neuron regenerates into the host tissue and arborizes in its usual ganglionic location in spite of the transplant (Az). The axonal arborization of the homologous control neuron has a similar arborization (A,). (B) A new neuron derived from repairing host tissues after the transplantation, arborizes in a novel site in the CNS. The neuron, which is recognized as graft-derived because of the colour of the hair it innervates arborizes in a location atypical for neurons usually produced by the transplanted tissue (Bz).A neuron homologous to those normally produced by the transplanted tissue is shown in B,. The lower panels in A and B are cross-sectional views of the neurons taken from the region of ganglion indicated by the bracket. (From Murphey ef al., 1984.)

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assigns neurons an identity based on their position in the CNS which “allows them to seek out partners in a corresponding array even if their location or time of arrival is disturbed”, together with “competitive cues” provided by neighbouring synaptic terminals. As with other neuronal systems which impinge upon insect central neurons (pp. 47 and 58), elimination of synaptic input from the cercal sensory cells in the cricket preparation induced changes in the target interneurons. Deafferentation throughout postembryonic development resulted in diminished growth of both the primary dendritic processes and the dendritic spines of the target giant interneurons (Murphey et al., 1975; Murphey and Levine, 1980), effects which could be reversed by allowing a return of sensory innervation (Murphey et al., 1977). These morphological responses were paralleled by reinforcement of the synaptic input from the remaining cercus when the other one was removed during postembryonic development, most probably due to removal of inhibitory input (Murphey and Levine, 1980). The reduction of synaptic input to the target interneurons, following prolonged cercal deafferentation, has also been shown to produce marked effects on metabolism of the terminal ganglion in the cricket preparation. The in virro incorporation of [3H]leucine into chronically deafferentated ganglia was markedly reduced compared with that observed in normal terminal ganglia (Meyer and Edwards, 1982). This effect was most evident in the primary dendrites of deafferented median giant interneurons (which contained 50% less labelled protein than their contralateral controls) and, to a lesser extent, in the somata (which incorporated 22% less radiolabelled leucine than did control cell bodies). No significant difference occurred, however, in the incorporation of radiolabelled leucine into the interneurons (Meyer and Edwards, 1982). It is concluded from these results that, during development, an increasing number of terminals might signal to the perikaryon to synthesize and transport greater amounts of protein needed for dendritic growth and function and that, conversely, reduced intraneuronal protein synthesis occurs in deafferented median giant interneurons (Edwards and Meyer, 1985). Studies with labelled ligands have, in addition, revealed that prolonged deafferentation of the cricket cercal giant interneuron system reduces the density of both muscarinic and nicotinic binding sites as well as the overall acetylcholinesterase activity in the terminal ganglion (Meyer et al., 1986). Unlike the cercal giant interneuron system, the sensory neurons of the cricket tympanal organ are unable to regenerate effectively (Ball, 1979; Biggin, 1981) due, it is suggested, to the late postembryonic development of the cricket tympanal organ (Ball and Young, 1974; Ball and Hill, 1978). This inadequacy is compensated by extensive dendritic outgrowing of the auditory interneurons that occurs following deafferentation, and which establishes novel functional connections with afferent neurons in the contralateral

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auditory neuropil (Hoy et af., 1978, 1985; Hoy and Moiseff, 1979; Pallas and Hoy, 1984; Schildberger et af., 1986). The contrast between this dramatic response and the more modest morphological changes induced in the cercal interneurons by prolonged deafferentation, suggests that the effects of synaptic deprivation might be mediated by different cellular mechanisms in different classes of interneurons. Chronic deafferentation of insect visual systems also produces dramatic effects on the subsequent neuronal organization of the developing CNS (cf. Nassel and Geiger, 1983). In the fleshfly, Sarcophaga buffata,for example, larval central nervous systems were transplanted-with and without imaginal eye discs-into freshly pupated individuals. After metamorphosis and eclosion, it was found that optic lobes, which had been transferred with eye discs, differentiated fully and possessed optic lobes with normal neuronal organization (Nassel and Sivasubramanian, 1983). In transplants without discs, however, the outer optic neuropil (the lamina), did not differentiate at all while the three more central ones (medulla, lobula and lobula plate) were substantially reduced in volume. The importance of retinal innervation for normal lamina development has also been demonstrated in other insect species (see Nassel and Sivasubramanian, 1983). However, in Musca (Fischbach, 1982) and Drosophifa (Nassel and Sivasubramanian. 1983), some neurons in the optic lobes survived deafferentation, presumably as a result of remaining synaptic contacts from the midbrain or, conceivably, from an intrinsic capacity of some cells to differentiate in the absence of afferent input. Certainly, some insect central nervous structures can differentiate adequately in the absence of sensory innervation. This has been demonstrated in deafferentated antenna1 lobes of the moth, Manduca sexta, where the distribution of putative acetylcholine receptors (as revealed by binding of l 2 SI-labelled a-bungarotoxin) appears to be unaffected by chronic differentation (Hildebrand et af., 1979).

4

Role of neuroglia and exogenous cells

The neuroglia of insects serve a variety of crucial physiological roles, ranging from transmitter inactivation (Salpeter and Faeder, 1971) and protein synthesis and transfer to the neurons (Smith and Howes, 1986) to the direction of extracellular current flow (Shaw, 1984) and the ionic homeostasis of the brain microenvironment (Coles and Tsacopoulos, 1981; Treherne and Schofield, 1981). Clearly, therefore, the restoration of normal neuronal function following neural damage must involve effective mechanisms of glial repair and regeneration. This is particularly evident with the nervous systems of some herbivorous species of bizarre blood composition (Shaw and Stobbart,

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1963) in which neuronal excitability depends upon the maintenance, by the neuroglial blood-brain barrier (see Fig. 9), of extracellular cation concentrations that can be quite different from those of the blood plasma (Treherne and Maddrell, 1967; Pichon et al., 1972). Furthermore, it is becoming increasingly recognized from research on vertebrate preparations that the glial environment plays a crucial role in neuronal regeneration (Aguayo et af.,1981; Bray et af., 1987), both in the provision of contact stimuli (Bunge and Bunge, 1983; Bunge, 1957) and the release of diffusible growth factors (Heumann, 1987). Recent research on an insect central nervous preparation has revealed that glial repair and regeneration involves an intimate participation of exogenous reactive cells derived from circulating haemocytes (P. J. S . Smith et al., 1987, 1988; Treherne et al., 1988b). For this reason, consideration will be given in this section of what is in effect an interplay between two organ systems-the blood and the brain-with each apparently stimulating and controlling the other, in the orchestration of glial regeneration.

4.1

EVIDENCE FOR THE INVOLVEMENT OF EXOGENOUS REACTIVE CELLS

Haemocytes accumulate at the cut ends of both peripheral nerves and central nervous connectives (see p. 37). They also appear, within 24 h, on the surface of cockroach central nervous connectives following selective glial disruption (Fig. 8); a preparation in which the DNA-intercalating agent, ethidium bromide, is used as a gliotoxin and in which the axons and neural lamella remain intact (Smith et al., 1984). Haemocytes have also been observed, apparently traversing the neural lamella of such preparations (Smith et al., 1984). In ethidium-treated connectives, this initial phase of repair is followed by the appearance of the so-called granule-containing cells (Fig. 10) in the disrupted perineurial and subperineurial tissues (Smith et al., 1984).These cells, never seen in normal nervous connectives, contain characteristic electrondense granules and are strikingly similar in ultrastructural appearance to the circulating blood granulocytes. It was suggested, on this circumstantial evidence, that they were in fact derived from blood cells (Smith et al., 1984). This supposition is supported by several subsequent lines of evidence (Smith et a/., 1986; Howes et al., 1987a), most convincingly by using monoclonal antibodies raised against haemocytes (Howes et al., 1987b). In this study, no antibody-binding cells were seen within normal abdominal connectives. However, within 24 h of selective glial destruction, monoclonally-labelled cells appeared in disrupted perineurial and subperineurial tissues (Fig. 1 1). The available evidence thus indicates that neural damage is associated with

Fig. 8 (A) Haemocyte (h) on the surface of a cockroach abdominal connective. In contrast to lesioned connectivcs, these are only very occasionally seen associated with the neural lamella (nl) of control preparations. pn, perineurium; g, glia; a, axon; m, matrix material within extracellular space. (From Treherne et al., 1984.) (B) Scanning electron micrograph of a haemocyte on the surface of a cockroach connective, 24 h after selective glial disruption with ethidium bromide, showing pitting of the neural lamella (arrowheads). (From Treherne et af., 1987.) Scale bars = 5 pm.

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a swift mobilization of blood-borne haemocytes which give rise to granulecontaining cells within the disrupted tissues. With severed cockroach abdominal connectives (in which there is disruption of the neural lamella, neuroglia, axons and extracellular matrices) the granule-containing cells appear throughout the damaged tissue, and among adjacent, undamaged, tissue. At the cut ends they form loosely-organized scar tissue (Treherne et al., 1984). With selective glial disruption, the granule-containing cells are effectively confined to the lesion zone (Smith et al., 1984). In these ethidium-treated preparations there is no direct access to the disrupted neuroglia, which are still overlaid by inract neural lamella (see Fig. 8B). Yet the haemocytes accumulate quickly on the surface of the connectives over the lesion zone and then are able to penetrate the neural lamella to reach the underlying disrupted perineurial and subperineurial tissues. The ability of the haemocytes to seek out the lesioned cells must result either from their responses to a release of diffusible cellular factors, able to pass through the neural lamella overlying the damaged neuroglia, and/or from the detection of changes in the neural lamella itself. The second possibility is not improbable, for there is an abrupt reduction in the rate of incorporation of labelled leucine (after 2 h of incubation) by the neural lamella proteins following selective glial disruption (Smith and Howes, 1986). 4.2

REGENERATIONOF THE BLOOD-BRAIN

BARRIER

An immediate consequence of perineurial damage, whether by surgical or chemical lesioning, is an increased accessibility of water-soluble ions and molecules to the underlying extracellular fluid which bathes the neuronal surfaces (Fig. 9). The regenerative responses of the damaged glia depend, however, on the form of lesioning. With mechanical disruption, such as cutting central nervous connectives, repair is complex and protracted. There is extensive proliferation of glial processes and dramatic extracellular changes, including massive secretion of intercellular matrix (Treherne et al., 1984). There was only slow restoration of blood-brain barrier properties in this cockroach preparation, as indicated by the penetration of ionic lanthanum which was not excluded from the extracellular system until after 4 months. In contrast, selective glial disruption, with ethidium bromide, resulted in a speedy and ordered repair (Smith et al., 1984). This suggests that the undamaged axons and/or extracellular matrices probably exert a profound influence on the processes of glial repair. In selectively lesioned cockroach connectives, the haemocytically derived granule-containing cells appear within 24 h of ethidium treatment and begin to form discrete layers within the disrupted perineurial glia (Fig. 10). They also appear scattered among the subperineurial tissues in the lesion zone. The

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numbers of granule-containing cells increases during the next 3 days, the majority being involved in the structural replacement of disrupted glia, although some are occasionally seen engaged in phagocytotic activity. By 4 days after lesioning, the cells beneath the neural lamella begin to form layers of compact cell processes, similar to those of normal perineurial cells and the proportion containing characteristic lysosomal granules has decreased (Smith et al., 1984). At this stage, extraneously applied ionic lanthanum can be excluded from the extracellular system, although the axon surfaces in the lesioned zone are still relatively accessible to small, water-soluble cations, as judged by the rapid axonal depolarization and conduction block produced by elevated external potassium concentration (Smith et af., 1984). By 14 days, the potassium impermeability of the blood-brain interface is reestablished and the perineurial and subperineurial glia are indistinguishable from those of unoperated connectives (see Fig. 9). Despite the rapid repair of the cockroach perineurium and the restoration of potassium impermeability, the original electrophysiological properties of the blood-brain interface were re-established more slowly (Smith et af., 1984). Even after one month the potassium-induced extraneuronal potentials, corresponding to the depolarization of the outwardly-directed perineurial surfaces, were smaller than in unoperated connectives (Fig. 12). This implies that, although repair appears structurally and functionally complete after 14 days, there must be longer-term changes in the cellular properties of the blood-brain interface (see p. 71). 4.3

CELL RECRUITMENT AND INTERACTIONS DURING GLIAL REPAIR

The regeneration of the perineurial and subperineurial glia is accompanied

Fig. 9 (A) Semi-diagrammatic drawing of a portion of the cockroach abdominal nerve cord, with a transverse section through the connective between the fourth and fifth abdominal ganglia. The surface of the central nervous system is delimited by the acellular neural lamella, which is leaky to small water-soluble cations and molecules. Beneath the neural lamella is a superficial layer of flattened glia, the perineurium, which (due to junctional complexes at the inner margins of the tortuous intercellular clefts) constitutes the blood-brain barrier. The underlying axons are closely wrapped by subperineurial glia which delineate a complex three-dimensional network of extracellular channels containing matrix material. (B) Effects of high-potassium saline on relative spike amplitude using sucrose-gap recordings. Due to the blood-brain barrier there is no appreciable effect on the action potential amplitude ( 0 ) .The change in d.c. potential (0)represents the depolarization of the outwardly directed perineurial membranes, access to the inwardly directed ones being restricted by the perineurial junctional complexes. The vertical lines through the data points indicate the extent of the standard error of the mean (n= 3). (From P. J. S. Smith et al., 1987.)

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by dramatic a n d relatively long-lasting changes in cell numbers in selectively lesioned cockroach connectives (Treherene ei al., 1987, 1988a). Initially, there is a n increase in the perineurial layer of the lesion zone, as would be expected from the haemocyte invasion of the damaged tissues (Fig. 13). By 4 days, a marked asymmetry develops with significantly higher numbers of cells anterior to, as well as within, the lesion. The asymmetry disappears within 7 days so that the increased cell numbers are again confined to the site of the original lesion (Treherne ei af., 1988a). The transitory increase in cell numbers outside the lesion zone, a t 4 days, is unlikely to have arisen from invading haemocytes for granule-containing cells are rarely seen in electron microscopic examination of control sections adjacent to the lesioned tissues (Smith et af.,1984). This implies that the increase in cell numbers outside the lesion zone is most likely t o involve endogenous reactive cells. There is also evidence that the recruitment of such endogenous reactive cells is initiated by invading haemocytes. This was shown in vitro with cultured nerve cords (Howes et af.,1987a) a n d following injection of physiologically inert particles (fluorescent carboxylated microspheres) into the blood which are taken up by the blood cells (Fig. 14) (Smith et al., 1986); treatments which preclude haemocyte involvement in repair. In both cases, there was n o increase in cell numbers equivalent to those in normal, selectively lesioned connectives in which there was a n entry of haemocytes and subsequent formation of granule-containing cells (Smith ei al., 1984). An unresolved question is the fate of the granule-containing cells that are derived from invading haemocytes and which play such a prominent role in the initial structural reorganization following selective disruption. This can be seen most clearly a t the blood-brain interface where these exogenously derived cells quickly assume the morphological characteristics of perineurial glia (Smith ei uf.,1984). They a r e present in maximal numbers a t 2-3 days

Fig. 10 ( A ) Semi-diagrammatic representation of the ultrastructure of a penultimate abdominal connective at 48 h after selective glial disruption with ethidium bromide. At this stage haemocytes accumulate in relatively large numbers and then penetrate the neural lamella. The perineurial layer now contains numerous granule-containing cells, derived from the invading haemocytes. (B) Effects of high-potassium saline on the electrical responses of the connectives 48 h after exposure to ethidium bromide. Unlike the situation in normal connectives (Fig. 9B) there is now a rapid collapse of the action potentials ( O ) ,the change in d.c. potential (0)now corresponding to axonal depolarization due to the relatively rapid penetration of potassium ions. The perineurial blood-brain barrier is now disrupted. The vertical lines through the data points indicate the standard errors of the means (n = 5 ) . (From P. J. S. Smith eral., 1987.)

Fig. 1 I (A) Heat-fixed cockroach haemocytes showing a positive peroxidase reaction following incubation with monoclonal antibody 3A7/F9. A few cells show no reaction (arrows). (B) Transverse section of the edge of a penultimate abdominal connective. 1 day after selective glial disruption by ethidium bromide, showing peroxidase-labelled haemocytes (h) accumulating on the outside of the neural lamella (nl). (C) Transverse section of a connective 2 days after selective glial disruption showing labelled haemocytes on both sides of the neural lamella. (D) 4 days after glial damage peroxidase-labelled haemocytes occupy the perineurial region beneath the neural lamella. A labelled haemocyte can also be seen, subperineurially, adjacent to a giant axon (*). Scale bars= 20 nm. (From Howes et al., 1987b.)

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Fig. 12 Electrophysiological responses to pulses of high-potassium saline from cockroach penultimate abdominal connectives at various stages of repair following selective glial disruption. (A) Control connectives showing persistent action potentials during application of high-potassium saline. The d.c. potential changes result largely from depolarization of the outwardly directed perineurial membranes (n= 3). (B) At 24 h post-lesion, the disruption of the perineurial glial cells results in the rapid access of potassium ions to the axon surfaces and a swift decline in the action potential amplitude. At this stage the recorded d.c. potential change results largely froin axonal depolarization (n= 4). (C) At 1 1 days post-lesion, there is now restricted access of potassium ions to the axon surfaces as shown by the maintenance of action potential amplitude. The d.c. potential changes, corresponding to perineurial depolarization, are, however, much smaller than in control preparations (n= 3). (D) At 28 days post-lesion, the postassium-induced d.c. potential changes have increased but are closer to the control preparations (n= 3). Sucrose-gap recordings. The vertical lines through the data points indicate the extent of the standard error of the mean. (From Smith et id., 1984.)

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Fig. 13 Cell fluxes in repairing cockroach connectives at various times after selective glial lesioning. The lesion zone is indicated by the shaded areas on the schematic drawings of the connectives. The cell numbers were estimated from nuclear counts made on stained 12pm light-microscope sections. The vertical lines through the data points indicate the extent of twice the standard error of the mean. (From Treherne et al., 1988a.)

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69

Haernocytesblocked

Fig. 14 Effects of haemocyte perturbation on the cell fluxes in repairing cockroach abdominal connectives following selective glial disruption with ethidium bromide. ( 0 ,perineurial numbers; 0, subperineurial numbers). Reduction of haemocyte participation was achieved by prior injection of carboxylated microspheres (0.27 pm diameter) into the haemolymph. These were taken u p by the haemocytes, which were then effectively prevented from entering the lesion. Under these circumstances there was a substantial reduction in cell numbers in the lesion zone, after 4 days and 7 days, as compared with control preparations. (From Treherne et al., 1988a.)

following selective glial lesioning, but after 4 days the proportion of cells containing characteristic intracellular granules declines abruptly; a stage at which the superficial repairing cells begin to divide, as indicated by their nuclear uptake of labelled thymidine (Fig. 15). There are two possible explanations for the apparent disappearance of the

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Days after treatment with ethidium bromide Fig. 15 Numbers of granule-containing cells plotted with the number of nuclei labelled by tritiated thymidine, counted from I-pm sections (A) through the lesion zone of the connective, at various stages of repair. The arrows in A indicate labelled cells outside the nervous system; B shows the distribution of tritiated thymidine in electronmicroscope section. Labelled thymidine was continuously present during the experimental periods. The dotted, broken line, is approximately the number of nuclei present in control sections from undamaged connectives. Standard errors are plotted on either side of mean values. (From Smith and Howes, 1986; Smith et al., 1988.)

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granule-containing cells: they could be transformed-changing both their cytoplasmic appearance and antibody-binding properties-or replaced by cells of glial origin (P. J. S. Smith et al., 1987; Treherne et al., 1988b). Both possibilities would account for the increased thymidine labelling that accompanies the decline in the proportion of granule-containing cells in the repairing connectives, for proliferation of exogenous cells could involve cellular transformation, while replacement by endogenous reactive ones is likely to involve cell division. The endogenous cells certainly have some capacity for repair, as is shown, in vitro, in repairing cultured cords (Howes et al., 1987a) and in vivo, in experiments where haemocyte involvement is substantially reduced by prior injection of microspheres into the blood (Smith et al., 1986). In both these cases, glial repair occurs but with delayed restoration of the perineurial blood-brain barrier by sparsely deployed glia of abnormal morphology. Aberrant repair also occurs after injection of the DNA-scission drug, bleomycin, which reduces the appearance of granule-containing cells within the lesion following selective glial lesioning (Treherne et al., 1986). Such aberrant repair could result from the necessity of the exogenous cells not only to initiate but also to organize their subsequent replacement by endogenous reactive cells.

4.4

LONG-TERM CHANGES IN GLlAL REPAIR

Despite the relatively swift cellular reorganization obtained following selective glial lesioning, repair is, in fact, a protracted process. This is revealed not only by the slow re-establishment of the full electrical characteristics of the repairing perineurium (p. 63), but, also, by a continuing increase in overall cell numbers in repairing cockroach connectives (Treherne et al., 1987, 1988a). In the subperineurial glia, for example, the cell numbers at 28 days are more than twice those after 7 days of selective lesioning (Fig. 13). What is more, the increase in cell numbers now spreads beyond the limits of the original lesion (Treherne et al., 1988a). Similar, long-term changes in glial cell numbers, extending beyond the lesion zone, have also been observed in cultured leech central nervous connectives following local crushing (Morgese et al., 1983). There are two possible explanations for the substantial increase in perineurial and subperineurial cell numbers seen in repairing cockroach connectives. It could be that the slower phase of increase could result from a population of progenitor cells which are smaller than those of the original neuroglia in the lesion. Alternatively, it could be a consequence of the fact that, following chemical lesioning, glial proliferation occurs within existing structural compartments delimited by the extracellular matrices and axons. If

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the mitogenic signals responsible for this proliferation are powerful and sustained, then this could result in an increased population of smaller cells which spill over from the limits of the original lesion zone. 5 Concluding remarks

The value of insect preparation in the study of neural repair and regeneration has been repeatedly emphasized-in research papers and grant applications-since the pioneering work of Bodenstein ( 1955) on insect peripheral nerve regeneration. A virtue most frequently extolled is their use as potential model systems, by which is usually implied their contribution to the understanding of the regenerative processes in the more complex mammalian central and peripheral nervous systems. In the case of glial repair, this optimism seems justified, for recent research has revealed a number of striking similarities with what is known of the equivalent events that occur in vertebrate preparations. A notable similarity is the initial invasion of the lesion by haemocytes (p. 59) which parallels the entry of the blood monocytes into mammalian brain tissues following surgical damage (Adrian and Schelper, I98 1). Even the timing of these events can be closely similar. As with cockroach haemocytes, the mammalian blood monocytes arrived within 2 days of lesioning and by 5 days transform to macrophage cells (DuBois et al., 1985): a timing which corresponds to the transformation or replacement of the insect granule-containing cells (p. 65). The cell division of the astrocytes, which commences at 2 days and continues for up to 7 days, also coincides with the onset of the proliferation of insect perineurial glia, as revealed by thymidine labelling (p. 69). Another point of similarity with the mammalian system is the role of haemocytically derived cells in triggering cell recruitment (p. 65), for it is now known that mammalian blood cells, principally T-lymphocytes, can release glial growth factors (Fontana et al., 1980; Benveniste el al., 1985). The possibility of endogenous activation of glial proliferation, which in the insect CNS could provide the stimulus for the prolonged increase in glial cell numbers in repairing tissues (p. 68), is indicated by the recent demonstration of the presence of glial morphogenic and mitogenic factors in the mammalian brain (Giulian and Baker, 1985; Giulian and Young, 1986; Giulian et al., 1985, 1986). Furthermore, the release of such endogenous factors in the insect CNS could be triggered by the arrival of exogenous reactive cells. This is suggested by the discovery that mammalian microglia, which are probably derived during development from monocytic stem cells (Rio-Hortega, 1932; Perry et al., 1985), can release growth factors that act selectively on populations of microglia (Giulian and Baker, 1985).

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An important feature of repair in the insect preparation, which appears not as yet to have been observed in mammalian brain, is the role of the granule-containing cells in the structural replacement of damaged glia (p. 69). It remains to be discovered whether these exogenously derived cells are transformed or are replaced by endogenous cells. An exciting implication of the recent insect and mammalian studies is that we can no longer consider the brain as a separate entity, structurally and functionally isolated from the blood, for in both animal groups there is now evidence for intimate interactions between the two organ systems in glial repair. Indeed, the mammalian brain, formerly thought to have no immune protection, has recently been discovered to be routinely patrolled by activated T-lymphocytes (Wekerle et al., 1987). There is no evidence, from any insect preparation, of the influence of the exogenous cells on neuronal growth or regeneration, although it is now known that in severed rat peripheral nerve there is an invasion of macrophages which produce signals that increase the synthesis of nerve growth factor (see Heumann, 1987). Mammalian Schwann cells have also been shown to release nerve growth factors following neural lesioning (Heumann, 1987). Again, there is no information on the role of equivalent glial-derived factors in nerve regeneration in insects. The regeneration of insect motor axons also takes place in a complex microenvironment: in severed peripheral nerve in a freshly-secreted matrix, with closely associated haemocytes, neuroglia and fat-body cells, and in crushed preparations initially through scar tissue and then between surviving neuroglia and extracellular matrices of the distal stump (p. 51). The importance of these elements in axonal regeneration was first recognized by Cajal (1928), yet it is only relatively recently that the nature of the physiological signals involved has begun to emerge. In vitro and in vivo experiments have shown, for example, that vertebrate Schwann cells can release diffusible factors that promote neurite extension (Varon and Adler, 1981; Bard et al., 1983; Berg, 1984) and that the extracellular matrix which they synthesize is also critically involved (cf. Bunge, 1987). Besides stimulating axonal extension, the extracellular environment also provides directional guidance. This is most obvious in crushed preparations where the regenerating proximal axonal segments can follow the nerve trunks of the surviving proximal stump (p. 51). There is also the possibility of longdistance signals for directing the growth of severed axons to the denervated muscle target, as has been indicated in an amphibian preparation (Kuffler, 1987), as well as the presence of synaptic-organizing molecules which have recently been discovered in the basal lamina of the regenerating amphibian neuromuscularjunctions (M. A. Smith et al., 1987). The vigorous regenerative capacity of insect central neurons, like those of

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other higher invertebrates, contrasts with the very limited axonal regrowth in the mammalian CNS; a limitation, which rodent grafting experiments have revealed, results not from innate properties of the neurons, but primarily from the influence of the glial microenvironment (see Aguayo et al., 1981). According to this interpretation the extensive axonal regeneration in the insect CNS, and in the mammalian PNS, would be facilitated by the neuroglia, although it is not clear whether this might be due merely to an absence of axonal “stop mechanisms”, such as has been suggested to be mediated by mammalian astrocytes (Liuzzi and Lasek, 1987) or from more adequate provision of nerve growth factors. Whatever mechanisms facilitate neuronal regrowth in the insect CNS, the available evidence suggests that, as in vertebrate neurons, axonal sprouting involves an increase in voltage-dependent calcium channels and, it is deduced, a rise in free intracellular calcium (p. 46). Dendritic sprouting in insect motor neurons (p. 55) and sensory neurons (p. 46) and in interneurons (p.47) can be induced by reduction in the synaptic input to them. Such deafferentation also produce dramatic changes in protein metabolism, in the distribution of muscarinic and nicotinic binding sites and in ganglionic acetylcholinesterase activity (p. 57). It remains to be discovered whether these effects are direct cellular responses to loss of synaptic input or result from local release of diffusible factors by adjacent neurons and/or neuroglia. Neurons within the central nervous systems of insects have a high capacity to regenerate accurate functional connections after injury. This is seen most clearly in the cricket cercal sensory system where the afferent reconnections with the central interneurons are virtually indistinguishable from the original synaptic inputs (p. 55). However, with this, and the other insect preparations which have been used, the crucial questions concerning the cellular and molecular mechanisms that achieve such impressive neurospecificity remain to be explored. Lack of knowledge of diffusible growth factors is an obvious omission. Neither is there any conclusive information as to the role of extracellular substrates in initiating and guiding neuronal growth. Yet recent work, notably that on cultured leech neurons, has clearly demonstrated the dependence, and specificity, of neurite growth on the substrate (cf. Chiquet and Nicholls, 1987). With leech Retzius cells, for example, one substrate (the matrix from the ganglia capsule) can induce rapid neurite extension, while another (concanavalin-A) induces extensive branching of the neurites. It seems inconceivable that equivalent contact stimuli will not turn out to be involved in insect nervous systems and, indeed, current research has already focused on the potential importance of the neuronal microenvironment in the regeneration of cockroach giant interneurons (Spira et al., 1987). In this system it is feasible that the rapid, essentially linear, sprouting of lesioned axons in the connectives, which contrasts with the dendritic arborization that

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occurs in the adjacent ganglia (p. 4 9 , could be a consequence of compartmentalized substrate cues. Such mechanisms would thus determine the form of neuronal regrowth and, in a general way, would ensure that it would be appropriate for regeneration in that domain of the nervous system. It is unlikely, however, that they could, by themselves, account for the accurate synaptic re-targeting observed in the regenerating cricket cercal system, where, for example, neurons in inappropriately positioned grafts can form virtually exact reconnections following abnormal and very convoluted axonal growth through host ganglia (p. 55). Such precise re-targeting has been attributed to “specific positional clues” as originally proposed by Sperry ( 1 963) in his chemoaffinity theory. Accordingly, the tortuous pathways followed by the axons of some grafted neurons would be attributed to a hunting of regenerating neurites through a notional three-dimensional array of elements-of yet unspecified properties-to locate regions of appropriate chemotactic attributes. Preliminary studies on developing insect nervous systems are, in fact, already revealing glycoprotein surface recognition molecules-in grasshopper and Drosophila embryos-and focusing on the importance of neuroglia in growth cone guidance (cf. Bastiani et al., 1986, 1987; Doe et al., 1986; du Lac et al., 1986; Patel et al., 1987). It is a chastening conclusion to realize, however, that despite the frequently vaunted merits of insect preparations for the study of peripheral and central nerve regeneration, no concerted efforts have yet been made to elucidate the cellular and molecular mechanisms involved in synaptic re-targeting. It is hoped that future research will realize the very real potential offered by insects for the understanding of this and all aspects of neural repair and regeneration.

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ful synapse regeneration in the leech: adding insult to injury. J . exp. Biol. 132,207-221. Murphey, R. K. and Levine, R. B. (1980). Mechanisms responsible for the changes observed in the response properties of partially deafferented insect interneurons. J . comp. Physiol. 119,267-283. Murphey, R. K., Mendenhall, B., Palka, J. and Edwards, J. S. (1975). Deafferentation slows the growth of specific dendrites of identified giant interneurons. J . comp. Neurol. 159,407-418. Murphey, R. K . , Matsumoto, S. G . and Mendenhall, B. (1977). Recovery from deafferentation by cricket interneurons after re-innervation by their peripheral field. J . comp. Neurol. 169,335-346. Murphey, R. K., Johnson, S. E. and Sakaguchi, D. S. (1983). Anatomy and physiology of supernumary cercal afferents in crickets: implications for pattern formation. J . Neurosci. 3,312-325. Murphey, R. K., Walthall, W. W. and Jacobs, G. A. (1984). Neurospecificity in the cricket cercal system. J . exp. B i d . 112,7-25. Narahashi, T. (1966). The physiology of insect axons. In “The Physiology of the Insect Central Nervous System” (Eds J. E. Treherne and s. W. L. Beament), pp. 1-20. Academic Press, London. Nassel, D. R. and Geiger, G. (1983). Neuronal organization in fly optic lobes altered by laser ablations early in development or by mutations of the eye. J . comp. Neurol. 217,86102. Nassel, D. R. and Sivasubramanian, P. (1983). Neural differentiation in fly CNS transplants cultured in vivo. J. exp. Zool. 225,301-310. Nicaise, G . and Meech, R. W. (1980). The effect of microinjection on the ultrastructure of an identified molluscan neuron. Bruin Res. 193,549-553. Norlander, R. H. and Singer, M. (1972). Electron microscopy of severed motor fibres in thecrayfish. Z . Zellforsch. 126, 157-181. Nuesch, H. and Stocker, R. F. (1975). Ultrastructural studies o n neuromuscular contacts and the formation of junctions in the flight muscle of Antheraea polyphemus (Lep.). 11. Changes after motor section. Cell Tiss. Res. 164,331-355. Olson, L. (1969). Intact and regenerating sympathetic noradrenalin axons in rat sciatic nerve. Histochemistry 17,349. Pallas, S . L. and Hoy, R. R. (1984). Effects of regeneration of auditory afferents on dendritic sprouting of an identified auditory interneuron. Neurosci. Abstr. 10, 1032. Parnas, I . and Segev, I. (1979). A mathematical model for conduction of action potentials along bifurcating axons. J . Physiol., Lond. 295,323-343. Patel, N. H., Snow, P. M. and Goodman, C. S. (1987). Characterization and cloning of Fasciclin 111: a glycoprotein expressed on a subset of neurons and axon pathways in Drosophila. CeN 48,875-988. Pearson, K. G . and Bradley, A. B. (1972). Specific regeneration of excitatory motoneurons to the leg muscles in the cockroach. Bruin Res. 47,492-496. Pelhate, M. and Pichon, Y. (1974). Selective inhibition of potassium current in the giant axon of the cockroach. J . Physiol., Lond. 242,90P. Pelhate, M . and Sattelle, D. B. (1982). Pharmacological properties of insect axons: a review. J . Insect Physiol. 28,889-903. Perry, V. H., Hume, D. A. and Gordon, S. (1985). Immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain. Neuroscience 15,3 13-326. Pichon. Y. ( 1 976). Pharmacological properties of the ionic channels in insect axons.

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In “Perspectives in Experimental Biology” (Ed. P. Spencer-Davies), pp. 297-3 12. Pergamon, Oxford. Pichon, Y., Sattelle, D. B. and Lane, N. J. (1972). Conduction processes in the nerve cord of the moth Manduca sextu in relation to its ultrastructure and haemolymph ionic composition. J. exp. Biol. 56,717-734. Pitman, R. M. and Rand, K. A. (1982). Neural lesions can cause dendritic sprouting of an undamaged adult insect motoneurone. J . exp. Biol. 96,125-130. Pitman, R. M., Tweedle, C. D. and Cohen, M. J. (1972). Electrical responses of insect central neurones augmentation by nerve section or colchicine. Science, N . Y . 178, 507-509. Porter, K. R. and Bowers, M. B. (1963). A study of chromatolysis in motor neurons of the frog Ranapipiens. J. Cell Biol. 19,56A. Rio-Hortega, P. del. (1932). Microglia. In “Cytology and Cellular Pathology of the Nervous System”, Vol.2 (Ed. W. Penfield), pp.481-584. Paul B. Hocker, New York. Roeder, K. D. and Weiant, E. A. (1950). The electrical and mechanical events of neuromuscular transmission in the cockroach, Periplaneta americana. J. exp. Biol 27, 1-13. Roederer, E. and Cohen. M. J. (1983a). Regeneration of an identified cell neuron in the cricket. I. Control of sprouting from soma, dendrites and axon. J. Neurosci. 3, 1835-1 847. Roederer, E. and Cohen, M. J. (1983b). Regeneration of an identified central neuron in the cricket. 11. Electrical and morphological responses of the soma. J. Neurosci. 3,1848-1859. Rowell, C. H. F. and Dorey, A. E. (1967). The number and size of axons in the thoracic connectives of the desert locust, Schistocerca greguriu. Forsk. Z . Zellforsch. 83, 282-294. Salpeter, M. and Faeder, I. R. (1971). The role of sheath cells in glutamate uptake by insect nerve muscle preparations. Prog. Bruin Res. 34,103-1 14. Salzer, J. L. and Bunge, R. P. (1980). Studies of Schwann cell proliferation. I. An analysis in tissue culture of proliferation during development, Wallerian degeneration and direct injury. J. Cell Biol. 84,739-752. Salzer, J. L., Bunge, R. P. and Glaser, L. (1980a). Studies of Schwann cell proliferation. 111. Evidence for the surface localization of the neurite mitogen. J. Cell Biol. 84,767-778. Salzer, J. L., Williams, A. K., Glaser, L. and Bunge, R. P. (1980b). Studies of Schwann cell proliferation. I. Characterization of the stimulation and specificity of the response to a neurite membrane fraction. J. Cell Biol. 84,753-766. Schildberger, K., Wohlers, D. W., Schmitz, B., Kleindienst, H.-U. and Huber, F. (1 986). Morphological and physiological changes in central auditory neurons following unilateral foreleg amputation in larval crickets. J. comp. Physiol A . 158, 29 1-300. Shaw, J. and Stobbart, R. H. (1963). Osmotic and ionic regulation in insects. Adv. Insect Physiol. 1,315400. Shaw, S. R. (1984). Early visual processing in insects. J. exp. B i d . 112,225-251. Smith, M. A., Yao, Y.-M. M., Reist, N. E., Magill, C., Bruce, G. and McMahan, U. J. (1987). Identification of agrin in electric organ extracts and localization of agrinlike molecules in muscle and central nervous system. J. exp. Biol. 132,223-230. Smith, P. J. S. and Howes, E. A. (1986). Glial toxin effect on protein synthesis in an insect connective. J. Cell Sci.70,83-92.

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Usherwood, P. N. R. (1963b). Response of insect muscle to denervation. 11. Changes in neuromuscular transmission. J. Insect Physiol. 9,811-825. Usherwood, P. N. R., Cochrane, D. G. and Rees, D. (1968). Changes in structural, physiological and pharmacological properties of insect excitatory nerve-muscle synapses after motor nerve section. Nature, Lond. 218,589-591. Varon, S. and Adler, R. (1981). Trophic and specifying factors directed to neuronal cells. Adv. Cell Neurobiol. 2, 115-163. Wall, P. D. (1977). The presence of ineffective synapses and the circumstances which unmask them. Phil. Trans. R. SOC.(Lond.)B278,361-372. Washio, H. (1 986). Spontaneous miniature potentials in denervated coxal muscle fibres of the American cockroach. J. exp. Biol. 120, 143-1 51. Washio, H. and Nihonmatsu, I. (1987). Structural changes in the denervated neuromuscular junction in coxal muscles of the cockroach Periplaneta americana. Comp. Biochem. Physiol. 86A, 643-647. Wekerle, H., Sun, D., Oropeza-Wekerle, R. L. and Meyermann, R. (1987). Immune reactivity in the nervous system: modulation of T-lymphocyte activation by glial cells. J. exp. Biol. 132,43-57. Whitington, P. M. (1977). Incorrect connections made by a regenerating cockroach motoneuron. J. exp. Zool. 201,339-344. Wine, J. J. (1973). Invertebrate central neurones: orthograde degeneration and retrograde changes after axotomy. Expl Neurol. 38,157-169. Wood, M. R. and Usherwood, P. N. R. (1979). Ultrastructural changes in cockroach leg muscle following unilateral neurotomy. I . Degeneration. J. ulrrasrruct. Res. 68, 265-280. Young, D. (1972). Specific re-innervation of limb transplanted between segments in the cockroach, Periplaneta americana. J. exp. Biol. 57,305-316.

Haemocyte Behaviour Ann M. Lackie Department of Zoology. The University, Glasgow G 12 800,Scotland, UK

1 Introduction 2 The haemocytes 2.1 Haemocyte classes 2.2 Other criteria for characterizing haemocytes 3 The origin and longevity of haemocytes 3.1 Haemopoietic organs 3.2 Mitosis in circulating haemocytes 3.3 In vitro culture of haemocytes 3.4 Haemocyte lineages 3.5 Haemocyte longevity 4 Haemocytic defence mechanisms 4.1 Phagocytosis 4.2 Nodule formation 4.3 Encapsulation 4.4 Haemocytic killing mechanisms 5 Humoral defence mechanisms 5.1 Antibacterial proteins 5.2 Serum lectins 6 Phenoloxidase and the prophenoloxidase-activation system 7 Changes in the haemocyte population 7.1 Developmental status of the insect 7.2 Stress 1.3 Wounding 7.4 Infection 7.5 Particulate material 7.6 Molecules in solution 7.7 Conclusions 8 Haemocytic involvement in wound-healing: the primary defence mechanism 8.1 Requirements for wound-healing 8.2 Recognition or response? A consideration of why haemocytes attach to a wound 8.3 Wound factors and their effects on haemocytes 8.4 Later events in wound-healing 8.5 Summary: the effects of wounding on haemocyte behaviour 9 Recognition of non-self ADVANCES I N INSECT PHYSIOLOGY VOL. 21 ISBN 0-12-0242214

Copyrithi 0 1988 Academic Press Limited A / / righis of repruduriion in m y form reserved

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The basement membrane Do haemocytes recognize inherent alterations in the basement membrane? Physical and biochemical properties that distinguish self from non-self Mediation of recognition by plasma components Can wound-healing and encapsulation be accounted for by similar recognition mechanisms? 10 Recruitment and cessation 1 1 Alterations in recognition and response Acknowledgements 9.1 9.2 9.3 9.4 9.5

References

1 Introduction

In the past ten years, insect immunology has moved dramatically from the description of phenomena towards the use of biochemical, immunological and molecular techniques to investigate the molecules produced in both “humoral” and “cellular” immunity. Despite this, we are still remarkably ignorant about how the cells involved in the immune response-the haemocytes-respond to wounds and “non-self ’, and interact with each other. Many of the earlier investigations were hampered by the inability to avoid haemolymph coagulation, and thus the difficulty in producing clean, singlecell suspensions or monolayers. With improvements in the handling, separation and maintenance of haemocytes in vitro, it is now becoming possible to investigate many different aspects of cell behaviour. A large number of books, conference proceedings and reviews concerned with haemocyte classification and insect immunity have been produced recently, many of which are strongly biased in favour of a particular hypothesis. In this review, however, my aim has been to look at haemocytes from a slightly different and, I hope, unbiased, point of view: one important role for haemocytes is aggregation around and sealing of wounds, and there are many aspects of this behaviour that are also fundamental to haemocytic immune responses. Thus, haemocyte numbers and subpopulations change in response to both wounding and the introduction of foreign material into the haemocoel. The mechanism whereby haemocytes recognize wounded self may be the basis upon which more refined recognition mechanisms have been added. Haemocytes show a gradation of response towards wounded self and towards non-self, indicating that the system can discriminate degrees of difference from self; the extent of the haemocytic response is clearly under careful control. After a description of the various types of cellular immune response and how they may relate to the “humoral” response, three particular aspects of haemocytic behaviour are examined: (1) changes in populations, subpopula-

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tions and in-individual haemocytes as a result of wounding and the introduction of foreign material into the haemocoel; (2) the initiation of these changes either directly, by haemocytic recognition of non-self or wounded self, or indirectly, by the interaction of haemocytes with soluble factors deriving from the area of non-self or wounded self; and (3) the controlled augmentation of the resultant haemocytic response. Since the degree of recognition and response is also affected by the insect’s prior experience, several factors which suppress or enhance the response are also briefly discussed.

2 The haemocytes 2.1

HAEMOCYTE CLASSES

Classification of insect haemocytes is an issue that stimulates an instantaneous loss of good manners and an induction of bad temper at any conference on insect physiology or immunity. The subject has been reviewed several times and will not be discussed here; instead, the reader is referred to the basic classification scheme outlined by Rowley and Ratcliffe (1981). Other schemes have also been proposed by Gupta (1979) and BrehClin and Zachary (1986). 2.1.1 Circulating haemocytes Not all classes of haemocyte are present in all insect families, or even in all species within a family (see Arnold, 1982, for a description of Noctuid haemocytes). However, the main classes of circulating haemocyte involved in internal defence are: (1) Prohaemocytes (PRs):Small, round cells, more common in the haemolymph of larvae than in adult insects and considered to be stem cells from which certain other classes develop (see Section 3.4). (2) Plasmatocytes (PLs):Agranular (e.g. Lepidoptera) or displaying few to many granules (e.g. Orthoptera, Dictyoptera). Lysosomal enzymes present. Usually the most numerous class of circulating haemocyte. Round or spindle-shaped in suspension, but adhesive, flattened and often motile over substrata both in vivo and in vitro. The cells are phagocytic (= “macrophageous PL” of BrehClin and Zachary, 1986) and involved in encapsulation (= “typical PLs” of BrehClin and Zachary, I986), nodule formation and woundhealing. Within this class can also be included lamellocyres, large, flattened cells

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into which PLs “transform”, and which are involved in encapsulation reactions in Drosophila larvae (Fig. 3 , Section 4.3). (3) Granular cells (GRs): Confusion may arise here since the cystocytes of Lepidoptera (that are highly granular in contrast to lepidopteran PLs) have more recently become known as GRs (Rowley and Ratcliffe, 1981). In contrast, where all haemocytes display varying degrees of granularity as in some Orthoptera and Dictyoptera, the term GR has been used to refer to cells that are adherent by fine filopodia but are rounded and phase-bright in vitro. The morphology may be related to the state of activation of the cell (Arnold, 1959a; Takle and Lackie, 1986) and GRs in these species may constitute a behavioural subclass of PLs. (4)Coagulocytes (COs) (also referred to as cystocytes): Granular cells, equivalent to the GRs of Lepidoptera, that lyse to produce a gel or granular coagulum (Grtgoire and Goffinet, 1979) and become phase-bright and nonadherent, with the nucleus surrounded by a thin rim of vacuolated cytoplasm. Other types of circulating haemocyte include: ( 5 ) Crystal cells: Found in larval Drosophila, and so-called because they contain a prominent crystalline array that is rich in prophenoloxidase; known to play a role in melanization (Rizki et al., 1980). (6) Spherule cells (SPs): Containing large spherical phase-bright inclusions, that stain for acid mucopolysaccharides (Ashhurst, 1982b; Ratcliffe, 1975) and incorporate [3 SS]sulphateinto a sulphated glycosaminoglycan-like molecule (Cook et al., 1985). Their role in defence, if any, is unknown. (7) Oenocytoids (OE): Very large cells that are fairly uncommon in the circulation, and whose role is uncertain although they apparently contain prophenoloxidase (see Section 6). (8) Thrombocytoids of Calliphora are apparently important in wound-healing and encapsulation (Zachary et al., 1975), whereas unusual cells such as the spindle cells of pharate tsetses (East et al., 1980; Kaaya and Ratcliffe, 1982) are unlikely to have any role in defence. 2.1.2 Sessile haemocyres It is unlikely that all haemocytes are freely circulating in an unstimulated insect at any one time. Many will be found amongst the lobes of the fat-body (Faye, 1978), or sometimes as accumulations within the haemocoel (Shrestha and Gateff, 1982a) or transiently adherent to haemocoelic surfaces (Arnold, 1959a; Gunnarsson, 1987). The rapid increase in the number of circulating haemocytes that may occur in response to some stimuli must result from the rapid mobilization of these “resting” cells (see Section 7). More permanently sessile cells are the pericardial cells alongside the dorsal

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vessel (Crossley, 1975) and the sessile haemocytes of the “haemopoietic organs” (see Section 3). In locusts, for example, large amounts of injected particulate material are trapped in the dorsal diaphragm by these cells (Brehelin and Hoffmann, 1980). 2.2

OTHER CRITERIA FOR CHARACTERIZING HAEMOCYTES

Although classes of haemocytes can be distinguished on morphological and ultrastructural grounds, this may give only a very crude indication of their functions. As with mammalian lymphocytes, which can be divided and subdivided into subpopulations with very different functions, so, too, some functional subdivision of haemocyte classes is apparent. Different subpopulations may be identified according to physical criteria such as their density; biochemical criteria such as enzyme content; behavioural criteria that include adhesion and locomotion, or according to their affinity for lectins or antibodies. By utilizing the differential adhesion of PLs and GRs from Leucophaea, Bohn (1977a) physically separated these two classes of haemocyte onto separate coverslips on each side of a glass chamber. Visual discrimination, but not physical separation, of subpopulations within one class was made possible by analysis with time-lapse cinephotography of the locomotory behaviour of cockroach haemocytes in filming chambers; the PL class comprised subpopulations of adhesive, highly-spread, non-motile cells and rounded, fast-moving cells (Section 8.3.3; Baerwald and Boush, 1971; Takle and Lackie, 1986). The successful physical separation of crustacean haemocytes into hyaline, semi-granular and granular classes by centrifugation on continuous Percoll density gradients, was pioneered by Soderhall and Smith (1983). Modifications of this method for haemocytes of different insect species were developed and assessed by Mead et al. ( I 986); continuous gradients allowed separation of PLs and GRs of larval Manduca and Galleria to give purities ranging from approximately 70 to 95%, dependent on cell class and species, and produced 90% pure COs for Blaberus. Weisner (1986) used continuous gradients to produce 90% pure GRs and PLs from Galleria, and lepidopteran SPs were isolated by Cook et al. (1985) on discontinuous gradients. These methods have been successful in separating haemocyte classes and will be invaluable for investigating cellular and intercellular behaviour within and between haemocyte classes. Quantitation of cytochemical differences in enzyme content can provide information about haemocyte classes, but is particularly useful when it distinguishes different subpopulations within a particular class. This method

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has already been used successfully in studies on molluscs (Granath and Yoshino, 1983; Dikkeboom et al., 1984). Analysis of the staining patterns of cockroach and locust PLs for acid phosphatase and “peroxidase” (M. Carr, unpublished results) and phenoloxidase (Huxham and Lackie, 1986, 1988) revealed marked differences in enzyme content between different cells within the PL class. In an attempt to avoid using morphological criteria, Peake (1979) used a combination of techniques to distinguish between the PRs and differentiating PLs of Culliphoru larvae. Haemolymph from [3H]thymidine-injected larvae was centrifuged on discontinuous Ficoll gradients, and the most dense cells were found to be thymidine-labelled but negative for acid phosphatase. Labelled cells became less dense, stained increasingly strongly for acid phosphatase, and showed greater protease activity as they developed from PRs to PLS. When held in anticoagulant and therefore stable, haemocytes of Schistocercu show a range of granularity and are difficult to separate on a continuous gradient; however, by using a discontinuous Percoll gradient, Huxham and Lackie (1988) separated a 95% pure band (band 5 ) of dense, heavilygranulated, PL-like haemocytes, that adhered and spread in vitro. Band 4 contained PLs, but cells that were less dense and less granular, and band 3 contained intact COs and agranular PLs. Thus the PLs were separated into three subpopulations, which were found to show marked differences in phenoloxidase content and behaviour. Band 4 cells were highly phagocytic whereas band 5 cells were not; band 5 cells showed chemokinetic behaviour in the presence of laminarin-activated haemocyte lysate supernatant (see Section 10) and aggregated in the presence of laminarin, whereas band 3 PLs did neither; high phenoloxidase activity was associated with cell lysates and intact, living cells from band 5, whereas that of band 3 was negligible. Another method of distinguishing between haemocyte types is by the use of antibodies or lectins conjugated to fluorescent dyes for light microscopy or to electron-dense markers such as colloidal gold or ferritin for electron microscopy. Fluorescent lectins have been used to distinguish between haemocyte classes in larvae of Antheraea pernyi larvae (Beaulaton, 1985) and PIodia interpunctella (Beeman et al., 1983). The morphologically distinct lamellocytes and PLs of Drosophila melanogaster larvae have each been shown, by their affinity for the N-acetylglucosamine-specificlectin, wheat germ agglutinin (WGA), to comprise distinct subpopulations. In the tu-Sz‘* mutant, T. Rizki and Rizki (1983) found that WGA bound specifically, in a speckled pattern (spk’ cells) to one subpopulation of each cell type; the proportion of spk’ cells increased during melanotic tumour formation, with which lamellocytes are involved, and during encapsulation of xenogeneic implants. This increase in spk’ cells during successful encapsulation re-

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actions was confirmed by Nappi and Silvers (1984) for the turn mutant of Drosophila and, more recently, Nappi and Christensen (1986) found that the proportion of WGA-reactive cells in adult Aedes aegypti increased during successful melanization reactions against microfilariae. Where it is possible to collect sufficient cells, physical separation of such labelled subpopulations should be possible by using a fluorescence-activated cell sorter. Monoclonal antibodies against surface epitopes of different subpopulations of haemocytes will also provide most useful tools and have already proved helpful in characterizing subpopulations of mollusc (Yoshino and Granath, 1983; Dikkeboom et al., 1985) and tunicate (Schlumpberger et al., 1984) leucocytes. Now that it is possible to collect and handle haemocytes, to maintain them in vitro for longer periods and, for some species at least, to physically separate classes or subpopulations, we can hope that attention will be turned from morphological and ultrastructural criteria to characterization of haemocyte subpopulations with respect to their behaviour, biochemistry and surface determinants. 3 The origin and longevity of haemocytes

The structure of haemopoietic tissue has been reviewed fairly recently in a useful article, liberally illustrated with micrographs, by Hoffmann and his colleagues ( 1979), and the literature on the multiplication of haemocytes has been reviewed by Feir (1979). It is clear that a considerable amount of confusion exists, and most of the available information is based on histological and ultrastructural work. Understanding the rate and mechanism of production of haemocytes is a very basic step in understanding the process of “immunization”. That we require much more information on the control of mitotic rates and on the longevity of haemocytes should become clear from the brief review presented here.

3.1

HAEMOPOIETIC ORGANS

Although relatively few studies have been carried out, the presence and the site of the haemopoietic organs (HPO) varies with an insect’s phylogenetic position and developmental stage. HPOs also vary in complexity from small clusters of cells to highly organized tissues. Earlier work, reviewed by Jones (l970), relied mainly upon histological studies, but more recently an experimental approach has been adopted; ligation or ablation of the HPO and determination of the effect upon the concentration of circulating cells, or the

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induction of haemorrhage followed by examination of the suspected HPO, have all proved useful. 3.1.1 Exopterygota The haemopoietic organs of Gryllus and Locusta have been investigated and described in great detail by Hoffmann and his colleagues (1 979, review). The most highly organized tissues are found in the housecricket, Gryffus,connected to each side of the dorsal vessel in the second and third abdominal segments. The organs are delimited by connective tissue and fibroblast-like cells, and the cortical region comprises a meshwork of reticular cells; these cells, in common with vertebrate reticuloendothelial cells, originate from embryonic mesoderm and have both an endocytic and a haemopoietic function. Some of the progeny of dividing reticular cells become haemocytoblasts, and these divide to produce “isogenic cell islets”, that include either PLs, GRs or COs. Differentiated cells are found free in the lumen of the organ and presumably then pass into the haemolymph via the dorsal vessel. In Periplaneta and Schistocerca (A. Lackie and E. Currie, unpublished observations) and Locusta, the HPO is less well organized and is found as irregular accumulations of cells along the dorsal vessel. The tissue contains reticular cells and islets of dividing cells, which presumably pass into the pericardial sinus after differentiation (Hoffmann, 1970a). Corroboration of haemopoietic function comes from experiments in which the HPOs of nymphal and adult Locusta were selectively irradiated to interrupt mitosis; 24 h later the number of circulating haemocytes had dropped by 50%. However, by 72 h, the cell count returned to normal, coincident with hypertrophy of the HPOs and massive proliferation of reticular cells and isogenic islets (Hoffmann, 1972). If the problem is approached from the other direction, by drastically depleting the number of circulating cells through experimentally induced haemorrhage, the result is an increase in mitoses within the HPO of Grylfus and Locusta (Hoffmann et al., 1979). Additional evidence for the haemopoietic function of these tissues has been obtained from experiments in which the immune system has been challenged by injections of large doses of iron saccharate (BrehClin and Hoffmann, 1980) or low doses of Bacillus thuringiensis (D. Hoffmann et af., 1974); these particles are phagocytized by the reticular cells, as well as the circulating haemocytes, and as a result the reticular cells do not divide and the total haemocyte count is reduced for several days. During the early stages of infection of Schisrocerca by the fungal pathogen, Metarhizium anisopliae, the total haemocyte count decreases (Gunnarsson et af., 1988), and the HPO becomes hypertrophied (S. Gunnarsson and A. Lackie, unpublished information).

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In these species, haemopoietic tissue is found in adults as well as nymphs. In endopterygotes, sites of haemopoiesis appear to be lost at the last moult. 3.1.2 Endopterygota

In Cafliphora larvae, haemopoietic tissue containing reticular cells and islets of dividing haemocytes is found dorsally in abdominal segments 5-8; clumps of dividing cells are released into the circulation and continue to divide and differentiate in the adult insect (Hoffmann et al., 1979). The HPOs, or “lymph glands” of larval Drosophila are found as 4-6 pairs of lobes in the thorax, posterior to the brain (Nappi and Carton, 1986). Careful comparison of circulating haemocytes and lymph gland cells, for the number and type of cytoplasmic inclusions, and the presence and enzyme cytochemistry of primary and secondary lysosomes, strongly suggests that the lymph glands are haemopoietic (Shrestha and Gateff, 1982a). Indirect evidence comes from studies of the tumour mutant [1( l)mbn] in which the lymph glands are hypertrophied and full of PRs, and the number of circulating haemocytes rises without control throughout the short life of the larva (Shrestha and Gateff, 1982b). In Lepidoptera, HPOs are associated with the imaginal wing discs (Hinks and Arnold, 1977; Monpeyssin and Beaulaton, 1978) and, in Euxoa decfarata (Hinks and Arnold, 1977), comprise a cortical zone of dividing cells and a medullary zone of differentiated cells. These authors, in common with Akai and Sato (1971) for Bombyx larvae, suggested that mature haemocytes were released through breaches in the cortical surface. In Euxoa, the HPO atrophied from the end of the sixth instar and, in common with the situation in other endopterygotes, apparently disappeared at the imaginal moult. Beaulaton (1979, 1980) confirmed the haemopoietic function*of the thoracic organs described by Monpeyssin and Beaulaton (1978) for larvae of Antheraea pernyi; surgical removal of the organs resulted in a large drop in haemocyte count compared with sham-operated controls. 3.2

MITOSIS IN CIRCULATING HAEMOCYTES

The ability of circulating haemocytes to divide and replenish or increase their numbers is an important question, yet this topic has been largely ignored and no consensus has been reached. In general, the methodology has been unsophisticated, relying on examination of fixed, stained blood smears from untreated (Arnold and Hinks, 1976; Feir and McLain, 1968) or colchicineblocked (Baerwald and Boush, 1970) insects. Pulse-labelling with [3H]thymidine which is incorporated into dividing chromosomes has been used only

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M . LACKIE

rarely (Feir and Pantle, 1971; Peake, 1979; Peake and Crossley, 1979; Lea, 1986). Postmoult changes in the haemocyte mitotic rate of larval Oncopeltus were followed by Feir and McLain (1968), who found the rate increased to 4% by 30 h postmoult, remained high until 74 h, then decreased to 1 ?LOor less. In larvae pulse-labelled with [jHIthymidine, peak incorporation was found to occur mid-instar, coincident with the peak in ecdysone titre (Feir and Pantle, 1971). According to these authors, if monolayers of haemocytes were incubated with radioisotope in vitro, as many as 7% of the cells incorporated the label-a surprisingly high figure in view of the difficulty generally found in persuading haemocytes to divide in vitro (Section 3.3). In adult Orthoptera and Dictyoptera, in which the HPOs persist, mitosis of circulating haemocytes is rarely seen, even after injection of bacteria (Ryan and Nicholas, 1972) and seems to be mainly confined to the PLs. Replenishment or increase of the number of circulating cells is presumably determined by the activity of the HPO (Section 3.1.1 and Section 7). Haemocyte incorporation of [3H]thymidine was used by Howcroft and Karp (1987) as the basis of an assay designed to show rejection of allogeneic grafts in adult Periplaneta. Pieces of filter paper were implanted into cockroaches, removed 24 h later and incubated with [3H]thyrnidine in vitro; large amounts of radioactivity became associated with the haemocytic capsules. Since, by 24 h, haemocyte recruitment to capsules has ceased and production of the external “coating material” (Section 10) has begun (Hillen, 1977; Ennesser and Nappi, 1984; Lackie et al., 1985), the implication that the encapsulating haemocytes have undergone a high rate of mitosis is surprising. In adult Lepidoptera and Diptera, in which the HPOs disappear at pupation, haemocyte numbers must presumably be maintained by mitosis of the circulating haemocytes. Even in larval endopterygotes, mitosis of circulating cells has been frequently reported; however, opinion is divided as to which classes of cell undergo mitosis. There seems to be some agreement (Shrivastava and Richards, 1965; Jones, 1970; Arnold and Hinks, 1976) that PLs do not divide in circulation and are probably “end cells”, like mammalian neutrophil leucocytes. This has recently been disputed by Pelc (1986) for Mamestra. The most complete histological study, carried out by Arnold and Hinks (1976) on six larval instars of the noctuid Euxoa declarata, showed that mitosis occurred amongst PRs, GRs and SPs. Calculations based on the total and differential cell counts, the mitotic index and the blood volume (which was, unfortunately, derived from weighing larvae before and after “totally expressing” the haemolymph) showed that a maximum of 2.5 x lo5 new haemocytes could be produced daily. More recent estimates, based on different staining techniques, indicated that the mitotic index of GRs may be as high

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as 6%, compared with other cell classes, and it was calculated that SPs could replace themselves in 24 h (Arnold and Hinks, 1983). In contrast, Lea (1986) found that very few Galleria SPs incorporated [3H]thymidine or were observed in mitosis on blood smears; the label was, however, incorporated by PRs by 6 h post-injection. In diapausing pupae, the number of free haemocytes is very low but increases after wounding (Section 7.3); part of this increase can be attributed to an increase in the number of mitotic cells that are found free in the circulation (Lea and Gilbert, 1961). Whether this response also occurs in adult moths is unknown. It is thus extremely difficult to draw any firm conclusions about the mechanisms for maintenance or expansion of the haemocyte population. In general, most of the exopterygotes are probably able to rely, throughout their lives, on the continued production of haemocytes by the dorsal HPO; when the immune system is challenged, or when the haemocyte population is depleted, haemopoiesis is stimulated. In those endopterygotes in which the HPO disappears at pupation-and too few species have been studied to know if this is a general phenomenon-it is possible that the circulating cells can divide and maintain the level of the population. There is thus an implicit assumption that higher mitotic indices will be observed in animals in which the total cell count has been induced to rise. 3.3

IN VITRO CULTURE OF HAEMOCYTES

Various media have been developed that are suitable for short-term maintenance of haemocytes, but culture of replicating cells has met with little success because mitosis occurs rarely, if at all. Despite this major problem, a line of haemocyte-like cells was developed from embryonic Periplaneta americana (EPa) and showed many characteristics of PLs in vitro, such as the ability to phagocytose bacteria. More recently, strains HPa33 from male nymphs and HPa34 from adult females were established and Landureau (1976) notes that “they are completely comparable to the embryonic haemocytes”. Apparently these cells, derived directly from haemolymph, resemble PLs but “their ultrastructure modulates considerably according to culture conditions, ranging from that of GRs, COs and even PRs”! Haemocytes from adult Periplaneta have been maintained in culture for up to 4 months in a medium [Lackie and Huxham’s medium, in Crompton and Lassiere (1987), modified from that of Quiot et af. (1985)], containing 10% foetal calf serum. The cell density scarcely altered and there was little evidence of either mitosis or cell death, although the majority of the PLs degranulated (M. Huxham, unpublished information).

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Early attempts to culture haemocytes of Malacosoma disstria resulted in a mixture of haemocyte types that only began to grow after 3-6 months in culture, and which was, unfortunately, infected with a microsporidian parasite (Sohi, 1971). However, other attempts gave rise to persisting mixtures of PRs, PLs and GRs, of which one line had been subcultured more than 100 times at the time of report (Arnold and Sohi, 1974). Large poiyploid haemocytes began to appear with time, and many cells took on the highly vacuolated appearance that seems to be a characteristic of long-term haemocyte culture. Shozawa et ul. (1985) developed a line of haemocytes from caterpillars of Mamestra brassicae, that were phagocytic in vitro, and which appeared to comprise a mixture of cell types. Production of haemocyte lines for continuous in vitro culture has thus been largely unsuccessful, and the value of the existing cultures for studies on haemocyte behaviour, rather than providing host cells for viral or protozoan parasites, is questionable. The determination of optimal conditions for cell culture is laborious, but in future attempts, the use of haemopoietic tissue that has been previously stimulated in vivo (Section 3.1) might be considered. The addition of ecdysone to the cultures might prove beneficial (Marks, 1980), and the incorporation of less adhesive substrata such as collagen (Elsdale and Bard, 1972; Kaplan and Gaudernack, 1982) or polyHEMA (Folkman and Moscona, 1978) might influence the mitotic rate. 3.4

HAEMOCYTE LINEAGES

That PRs are stem cells and divide to produce PLs is almost universally agreed on the basis of morphological (Gupta and Sutherland, 1966; Marschall, 1966; Beeman et al., 1983), ultrastructural (Shrestha and Gateff, 1982a) and radioisotopic (Shrivastava and Richards, 1965; Peake, 1979; Peake and Crossley, 1979; Lea, 1986) studies. Gupta (1979), on the basis of his own work and a review of the literature, believes that PLs then give rise to GRs, from which all other types develop. The derivation of lepidopteran GRs ( = COs) from PLs is also supported by Lai-Fook (1973), Monpeyssin and Beaulaton (1977) and Beeman et al. (1983), but the origin of orthopteran or dictyopteran COs is unknown. Nor is it clear whether PLs dividing in the circulation give rise to other PLs in addition to other cell types. Ultrastructural investigations of haemopoietic tissue in Antheruea led Monpeyssin and Beaulaton (1978) to suggest that SPs derived from PLs, but Beeman and colleagues (1983) and Arnold and Hinks (1983), on examination of circulating cells, would argue for a separate line of development.

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The progenitors of Drosophila crystal cells seem to be distinct from PRs (Shrestha and Gateff, 1982a; T. Rizki et al., 1980) and lepidopteran OE may also have a separate origin from the PR-PL lineage (Beeman et al., 1983). The origin of COs/GRs and of PLs-whether from other circulating PLs or from PRs-is an important problem, since it determines which cell type must be stimulated to divide when the haemocyte population needs to be increased. As an example, division of PLs might be stimulated by contact with “non-self ’, whereas PR division might require hormonal signalling.

3.5 HAEMOCYTE LONGEVITY The interrelationship between the mitotic rate, length of life and state of activation of haemocytes will affect the outcome of contacts between the immune system and “antigen”. The overall state of enhanced responsiveness (Section 11) might be due to an increase in size of a reactive subpopulation either by mitosis or by transformation of previously quiescent cells to an activated state, or both; maintenance of enhanced responsiveness will depend partly on whether the activated cells revert to a quiescent state and partly on the lifespan of the haemocytes. One of the basic assumptions of this argument is best illustrated by considering the result of an infection in which the infective organism (a bacterium or protozoon) is able to replicate within the insect host. One possibility is that there is a rapid rise in parasitaemia that stimulates an increase in effector cell (reactive haemocyte) density; these cells are able to overcome the parasite infection so that it is virtually eliminated. If the life expectancy of the effector cells is long relative to the lifespan of the host, the insect is effectively immune to reinfection and the defence system is provided with the equivalent of a “memory component” (Fig. 1; Anderson, 1986). Although information on the rate of turnover of effector cell subpopulations-presumably PLs and COs-is relevant to both the persistence of enhanced responsiveness and experiments in which the haemolymph is transferred (Shrivastava and Richards, 1965; Karp and Rheins, 1980; BurnsWeatherby and McCroddan, 1982; Ham, 1986), there appears to be little published work on this topic. The most widely quoted data are those of Shrivastava and Richards (1965), who found that rather high doses of [3H]thymidine injected into Galleria larvae labelled PRs then PLs (24h), then adipohaemocytes ( = GRs). By the time the adipohaemocytes had been labelled, after 3 days at 35”C, the larvae had pupated, and all labelled cells disappeared by 6 days post-infection. On the basis of persistence of [jH]thymidine-labelled haemocytes in Galleria kept at 28”C, Lea (1986) calculated

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Time in arbitrary units Fig. 1 Predicted pattern of changes in parasite (dotted line) and haemocyte (solid line) densities, with the assumption that effector cells are long-lived. A “challenge” infection introduced at time t = 2 is rapidly eliminated.(From Anderson, 1986.)

that PLs and adipohaemocytes were able to persist in the circulation for at least 9 and 17 days respectively. Careful work on Calliphora by Peake and Crossley (1979) showed that the low-dose pulse label of [3H]thymidine was only available for about 2 h to PRs in viva, The cell cycle time was determined to be 9-1 h at 25”C, and Peake’s (1979) density gradient measurements showed that labelled PRs took approximately 24 h to differentiate into PLs. The longevity of the PLs was not investigated. So far, then, there is little solid information on either effector cell longevity or the fate of effete haemocytes. In mammals, leucocytes are lost daily through migration into the gut and secretory fluids, and effete or damaged cells are removed by neutrophil leucocytes, or by macrophages in the mononuclear-phagocyte system of the liver and lungs. During insect metamorphosis haemocytes are found containing cellular debris such as muscle fragments (Crossley, 1968; Whitten, 1969), so that it is possible that haemocytic housecleaning duties could extend to daily removal of their moribund colleagues. Kislev et al. (1969) occasionally found haemocytes, in Spodoptera littoralis infected with nuclear polyhedrosis virus, that were apparently ingesting virus-infected haemocytes and Wigglesworth (1933) reported that haemocytes, apparently containing the remains of other haemocytes, settled upon

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the epidermal basement membrane of Rhodnius during the process of cuticle formation. Do the reticular cells of the dorsal diaphragm in locusts and cockroaches trap and destroy senescent haemocytes? How is a senescent haemocyte recognized as such? Does the fat-body participate in removal of the bacteria-laden haemocytes found associated with it (Faye, 1978)? These questions urgently require answers.

4

Haemocytic defence mechanisms

Many functions have been ascribed to haemocytes, including the transport of lipids (Chino, 1985), proteins (Geiger et al., 1977) and tyrosine (Post, 1972; Vandenberg and Mills, 1974), coagulation of haemolymph and wound-healing (see Section 8), formation of connective tissue (Section 9.1.2), secretion of “humoral” antibacterial factors (Section 5) and cellular immune responses. The involvement of haemocytes in coagulation, wound-healing and immunity ensures an efficient means of internal defence. The mechanisms of the immune response for which the circulating haemocytes are responsible are briefly described in this section.

4.1

PHAGOCYTOSIS

Small biotic particles such as bacteria and yeast, and abiotic particles such as latex beads and colloidal carbon are internalized by both circulating haemocytes and sessile reticular cells; the role of the reticular and pericardial cells (Crossley, 1975) will not be considered further. Careful quantitative work by Ratcliffe and Walters (1983), using various pathogenic and non-pathogenic bacteria in Galleria larvae, showed that low doses of bacteria-less than 1O3/p1-were removed by phagocytosis. Above this threshold level, nodule formation (Section 4.2) was a more efficient clearance mechanism. With the E. coli K , * strain, however, phagocytosis was the most important clearance mechanism, irrespective of dose. Attempts to draw conclusions about the classes and numbers of haemocytes involved in phagocytosis are hampered by occasional confusion over haemocyte classification and, in many cases, results expressed in terms of the whole haemocyte population. Although there are exceptions, most authors concur that PLs are the predominant class of phagocyte both in vivo and in v i m . A small proportion of GRs/COs also appear to phagocytose or associate with particles both in vivo (Wago, 1980; BrehClin and Hoffmann, 1980; Abu-Hakima and Faye, 1981; Guzo and Stoltz, 1987; M. Carr and A.

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Lackie, unpublished) and in vitro (Rowley and Ratcliffe, 1976a; Huxham and Lackie, 1988). Since the GRs/COs degranulate in vitro, producing a sticky coagulum (GrCgoire and Goffinet, 1979) and “acid mucopolysaccharide” (Marschall, 1966; Anderson and Chain, 1986), it may be difficult with in vitro analyses to discern whether particles have been internalized or have adhered to extracellular material; the use of fluorescently labelled particles of which the fluorescence, if external to and thus unprotected by the phagocyte, can be quenched (Hed, 1977), or the use of particles coupled to indicator dyes that change colour within the phagosome (Jensen and Bainton, 1973) can be helpful. Studies of the GRs of Galleria using scanning electron microscopy showed that bacteria became trapped within an amorphous material surrounding the cell (Leonard et af., 1985a). The proportion of the haemocyte population that is phagocytic depends on the dose and type of particle (Rabinovitch and de Stefano, 1970; Hanschke et af., 1980; Ratcliffe and Walters, 1983), the time post-injection at which the haemolymph is examined and the prior experience of the insect (Table 1). Mohrig and colleagues (1979) found that prior injection of Gafferia larvae with latex beads increased the ability of the haemocyte population to ingest a subsequent dose of Bacillus thuringiensis subtoxicus; this enhanced phagocytic ability was also stimulated after injection of diluted, centrifuged haemolymph collected from animals that had themselves been injected with beads. Phagocytosis has also been “non-specifically” stimulated by injection of saline into Schislocerca (Table 1; Gunnarsson, 1988a) or of Sepharose beads into Peripfaneta (Dularay and Lackie, 1987) (Section 11). In Crustacea, Smith and Soderhall and their colleagues (reviewed in Smith and Soderhall, 1986) have shown that @1,3-glucansor bacterial lipopolysaccharides, both of which activate the prophenoloxidase system (Section 6), increase the phagocytic competence of the hyaline haemocytes in vitro. They suggest (Soderhall et af., 1986) that these specific stimuli induce the semigranular and granular cells (depending on the species of crustacean) to release the prophenoloxidase system which is then activated and opsonizes particles for uptake. In insects, glucans increase the proportion and phagocytic competence of haemocytes in vivo (Gunnarsson, 1988a; Table 1) and in vitro (Ratcliffe et af., 1984; Leonard et al., 1985a; Huxham and Lackie, 1988); in all cases, the PLs were the predominant phagocyte, although latex beads (Gunnarsson, 1988a) and bacteria (Leonard et al., 1985a) were also associated with COs/GRs. Evidence for intercellular cooperation and opsonization is at present circumstantial (Section 10). There have been several histological and ultrastructural examinations of phagocytosis by haemocytes; the mechanism is essentially similar to that seen

TABLE 1 The proportion of haemocytes, and their classes, involved in phagocytosis in vivo Species

'?LOHaemocytes

Choristoneura 6th instar Orgyia 3rd instar Galleria larvae Periplaneta nymph, adults adults adults Schistocerca adults

Conditions

Cell class

phagocytic

Authors

1&11

PL, GR

lo6 E. coli or B. cereus: 24 h

Dunphy and Nolan (1982)

12

PL, GR

Controls: 3 h

Guzo and Stoltz (1987)

28 30 5 50

PL, GR

1

Not COs

10 10 20 10


1825 40

PL, co PL

Parasitized: 3 h Fixed horse erythrocytes: 1 h Sheep erythrocytes: 1 h Latex particles: 2 h (large dose, many free particles at 24 h) Formalinized yeast: 3 h : 12h Viral polyhedra: 3 h : 12h Latex beads (high dose): 24 h Heat-killed Saccharomyces: 3 h Latex beads (low dose): 3 h Latex beads: 4 h Latex beads: but insects injected 24 h earlier with: Saline Zymosan supernatant 24 h fungal infection, percutaneous

Rabinovitch and de Stefan0 (1 970) Hanschke et al. (1980) Ryan and Nicholas ( 1972)

Smith et al. (1986) M. Carr (pers.comm.) Gunnarsson (1 988a)

Gunnarsson et al. (1988)

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in other types of phagocytic cell and the process is examined in greater detail by Ratcliffe and Rowley (1979). Pinocytotic uptake into coated pits has been described by Crossley (1968) and Rowley and Ratcliffe (1979), but seems not to have been confirmed for phagocytosis by haemocytes. Lysosomes fuse with the phagosome (Rowley and Ratcliffe, 1976a,b; Brehilin and Hoffmann, 1980) and acid phosphatase, an enzyme characteristically found within lysosomes, is released into the resultant secondary lysosome (Rowley and Ratcliffe, 1979), Death of the enclosed microorganism is usually, but not always, the outcome (see Section 4.4). In the case of tubercle bacteria in Galleria (Cameron, 1934), various viruses (Devauchelle et al., 1961; Kislev et al., 1969; Younghusband and Lee, 1970; Leutenegger, 1967) and trypanosomatid protozoa such as Trypanosoma rangeli in Rhodnius prolixus (Tobie, 1970; Takle, 1988) and Crithidia fasciculata in Drosophila (Schmittner and McGhee, 1970; reviewed in Molyneux et ~ l . 1986), , the organism may survive and even replicate. The means whereby these parasites evade phagocytic killing are particularly interesting but, perhaps because large numbers of infected haemocytes are not readily obtainable, have been little studied in comparison with the intracellular parasites of mammalian macrophages. The biochemistry of phagocytosis has also been neglected. Working with haemocytes of Blaberus craniifer, Anderson and colleagues (1973a) found that the energy for phagocytosis was provided by glycolysis, but that the “respiratory burst” and stimulation of the hexose monophosphate shunt, characteristic of oxygen-dependent killing by mammalian macrophages and neutrophils, was lacking (Section 4.4). 4.2

NODULE FORMATION

The formation of nodules-aggregates of haemocytes entrapping particulate material-has long been known as a defence mechanism (Salt, 1970). The trapping of bacteria within haemocytic aggregates is a particularly effective and rapid means of clearing the haemolymph and it is considered to be of greater importance than phagocytosis for removing bacterial doses of greater than 103/pl(Ratcliffe and Walters, 1983). Depending on the bacterial dose, this mechanism of clearance is usually very rapid (Gagen and Ratcliffe, 1976). In larvae of Lepidoptera, such as Galleria and Pieris, degranulating GRs are found associated with live or dead Bacillus cereus, E. coli or Sarcina lutea within minutes of bacterial injection; the bacteria are apparently trapped within the coagulum, around which PLs start to aggregate. The mixture of bacteria, coagulum and necrotic GRs becomes melanized and the surrounding PLs, some of which have phagocytosed bacteria, flatten and form a

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capsule-like structure which usually adheres to surfaces of tissues within the haemocoel (Ratcliffe and Gagen, 1976, 1977). Preliminary observations suggested a similar sequence-trapping by GRs/COs followed by PL adhesion-in Tenebrio and Clitumnus (Ratcliffe and Rowley, 1979). Whether or not this sequence of events is the same in other species is not yet known and, in those species where it is difficult to distinguish cell classes on the basis of ultrastructure, may have to await the use of suitable markers for haemocyte subpopulations. It is worth noting that melanization is not always a consequence of nodule formation (Gunnarsson and Lackie, 1985; Guzo and Stoltz, 1987). Nodule formation is stimulated by injection of bacteria (Hanschke et al., 1980; Dunphy and Nolan, 1982; Horohov and Dunn, 1983), viral polyhedra (Ryan and Nicholas, 1972), vertebrate erythrocytes (Ryan and Nicholas, 1972), yeasts (Guzo and Stoltz, 1987), fungal spores (Vey et al., 1985; Gunnarsson and Lackie, 1985), protozoan parasites (Takle 1986, 1988; Molyneux et al., 1986) and xenogeneic leucocytes (Lackie, 1986a), as well as a variety of abiotic materials (reviewed in Salt, 1970; Ratcliffe and Rowley, 1979). Interestingly, nodules are also formed in response to solutions of bacterial lipopolysaccharide (endotoxin) (Schwalbe and Boush, 1971; Smith et al., 1984; Gunnarsson and Lackie, 1985), laminarin and zymosan supernatant (Smith et al., 1984; Gunnarsson and Lackie, 1985; Gunnarsson, 1988a) and in Periplaneta, galactose-rich glycoproteins such as porcine stomach mucin (Lackie and Vasta, 1988; see Section 9.3.2). Prior injection of zymosan supernatant stimulates the immune system of Schistocerca so that, 24 h later, even wounding and injection of endotoxin-free saline stimulates large numbers of nodules (Gunnarsson, 1988a). Solutions of dextran (Smith et al., 1984; Gunnarsson and Lackie, 1985) or of bovine serum albumen (Lackie and Vasta, 1988) are without effect. The mean size of the nodules is affected by the type of molecule injected (Gunnarsson and Lackie, 1985). Thus, aggregation-inappropriate as it is in these circumstances-occurs in response to soluble and not only surface-bound molecules (see also Section 8.3.3.3).

4.3

ENCAPSULATION

Encapsulation is the sequestration, within multilayered aggregates of haemocytes, of objects too big to be phagocytosed or trapped within nodules (Fig. 2). Investigation of capsule formation is aesthetically and intellectually pleasing in that three distinct questions can be asked of this closely defined system: (1) What stimulates a circulating haemocyte to adhere to the foreign surface?

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ANN M. LACKIE

(2) How are other circulating cells recruited to the capsule? and (3) Why does capsule formation cease? Histological and ultrastructural studies provide some clues, although details of capsule structure vary according to the insect species and the authors’ terminology. In general, a completed capsule comprises an inner layer of rounded, often necrotic and melanized, haemocytes that are surrounded by layers of flattened interdigitated cells. There may also be an outermost layer of more rounded cells. (Matz, 1965; Grimstone et al., 1967; Brehelin et al., 1975; Sat0 et al., 1976; Schmit and Ratcliffe, 1977; Ennesser and Nappi, 1984; Lackie et al., 1985). Ultrastructural studies of the time-course of capsule formation in Galleria showed GRs adhered to and degranulated on the foreign surface within minutes of implantation (Schmit and Ratcliffe, 1977). Similar primary involvement of COs or GRs was shown in Clitumnus (Schmit and Ratcliffe, 1978), Locusta and Melolontha (Brehklin et al., 1975) and Bombyx (Sato et al., 1976). Early degranulation of COs was not found in Periplaneta by Lackie et al. (1983, although there was some evidence that cell lysis occurred in Schistocerca (Lackie, 1976; Lackie et al., 1985). Massive and continued lysis of Periplaneta “GRs” around pieces of glutaraldehyde-fixed nerve cord was found by Ennesser and Nappi (1984); the haemocyte classification adopted by these authors is difficult to interpret, since they report the haemocyte population to contain at least 90% GRs, the remainder comprising PLs. Despite this confusion the reason for the discrepancy between the results of these authors and those of Lackie’s group (1985) are not clear. The multivesicular bodies observed in encapsulating haemocytes by Ennesser and Nappi (1984) have only been seen in adherent (Takle, 1986) or phagocytosing (Rowley and Ratcliffe, 1979) haemocytes in vitro. Ratcliffe (1 986) has proposed a working model for a biphasic process of encapsulation, in which GRs/COs discharge components of the prophenoloxidase system onto foreign surfaces; PLs are then able to adhere to form a capsule (see Section 10). With the development of surface and enzymic markers for haemocyte subpopulations this hypothesis can now be tested more vigorously. One question which requires investigation is why some surfaces, but not others, stimulate production of melanin at the core of the capsule (Lackie, 1983a; Ennesser and Nappi, 1984). Capsules are not all of the same thickness, but vary between species depending on the total number and the proportions of haemocyte types available (Gotz, 1986; Lackie et al., 1985). For example, the median number of cell layers around positively-charged Sepharose beads is 17 in Periplaneta but only 3 in Schistocerca; locusts have relatively and absolutely fewer PLs than cockroaches (Lackie et al., 1985), and this may also be reflected in the proportions of reactive subpopulations within the two cell classes. Capsule

H A EM O C Y T E

B EH A V I 0 U R

105

thickness also varies within a species depending on the surface propertiessuch as surface-charge or hydrophobicity (Lackie, 1983a; 3986b; Takle, 1986; Vinson, 1974) or carbohydrate composition (Lackie and Vasta, 1988-f the implanted object (Section 9.3). The thickness is due to the number of haemocytes recruited, thin capsules having fewer layers of cells (Lackie et af., 1985). Recruitment has usually ceased by 18-24h (Hillen, 1977; Takle, 1986) and the layers of haemocytes are flattened and interdigitated (Fig. 2). Several different types of intercellular junction form between the flattened cells, mainly of the gap (“E-type”, because the intramembranous particles remain adherent to the extracellular fracture face after freeze fracture) and desmosomal type (reviewed by Baerwald, 1979). Flattening of haemocytes probably requires microfilament assembly, since cytochalasin B interferes with spreading and capsule compaction in vitro (Davies and Preston, 1987). Apparently intercellular communication within the capsule is possible; Caveney and Berdan (1982), in preliminary experiments of which the full details were not recorded, found that there was ionic coupling between cells as far as 600 pm apart in 72 h capsules from cockroaches, and the dye carboxyfluorescein could cross the gaps from cell to cell. Completed capsules become coated by a layer of extracellular material (Hillen, 1977), initially laid down as plaques (Takle, 1986), that stains with Alcian blue (Lackie et al., 1985) (Fig. 7 ) ; once this stage has been reached, capsules gradually become compacted, and presumably remain in the haemocoel until the insect dies. In the Diptera, capsule formation is much more variable, either involving disintegration of “thrombocytoids” as in Caffiphora(Zachary et al., 1975) or the transformation of PLs to lamellocytes (Nappi, 1975; R. Rizki and Rizki, 1980; Nappi and Carton, 1986) (Fig. 3). In some insects either only small numbers of haemocytes may be involved (as in Anopheles quadrimaculatus; Chen and Laurence, 1985), or an acellular capsule of melanin may be deposited without apparent haemocyte involvement (Chen and Laurence, 1985; Kaaya et al., 1986; review, Gotz, 1986). Comparisons between the type of capsule formed-humoral or cellular-and the number of haemocytes available within an insect species suggested that those Diptera that relied on humoral melanization had fewer blood cells (Gotz, 1986). Recently there has been some success in devising methods to test encapsulation in vitro. As anyone working with haemocytes knows, haemocytes become very adhesive once removed from the insect. Ratner and Vinson (1983) devised a method for studying encapsulation of cotton loops by Hefiothis virescens haemocytes in a mixture of haemolymph and medium. This technique was subsequently modified by Davies and Vinson (1986) to investigate evasion of haemocytic encapsulation by the parasitoid Cardiochiles in Hefiothis virescens haemolymph; allogeneic tissues and mature

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coated eggs from the wasp’s calyx were not encapsulated in vitro whereas immature eggs were, showing that the system still possessed discriminatory ability in vitro. R. Holt and A. Lackie (unpublished information), modifying Davies’ technique for use with anticoagulant-collected Periplaneta haemocytes, have also developed the system such that allogeneic ovarioles, which are not encapsulated in vivo (Lackie, 1979) remain uncapsulated in vitro, whereas pieces of nylon ligature are encapsulated both in vivo and in vitro. Recently, Davies (Davies and Vinson, 1988) has used the in vitro system to investigate the ability of a plasma-derived peptide, “encapsulation-promoting factor” (EPF), to restore the encapsulation capability to lightly-trypsinized Heliothis haemocytes (Section 10). Finally, with the development of methods of separating haemocytes into different classes or subpopulations (Section 2.2) the ability of separated fractions to adhere to foreign surfaces in vitro can be tested, and any cooperative roles investigated. Thus, crayfish semigranular cells but not hyaline cells adhered to a variety of particles in vitro (Persson et al., 1987) (see Section lo). 4.4

HAEMOCYTIC KILLING MECHANISMS

Mammalian leucocytes possess a wide range of intra- and extracellular killing mechanisms. These include the oxygen-dependent killing (superoxide anion, myeloperoxidase-hydrogen peroxide-halide system, hydroxyl radical, and singlet oxygen) of neutrophils and activated macrophages; oxygenindependent mechanisms such as the battery of proteinases, esterases, acid phosphatase, and carbohydrate-splitting enzymes including lysozyme, found in lysosomes and secreted into phagosomes and to the exterior; and molecules such as Major Basic Protein and Cationic Protein secreted by eosinophils and responsible for killing parasites such as schistosomulae in vitro (McLaren, 1980). It is therefore surprising that so little is known about haemocytic killing and digestion of ingested microorganisms. Cytochemical tests for myeloperoxidase in Blaberus craniifer haemocytes were negative (Anderson et al., 1973a) and the dye nitroblue tetrazolium (NBT) was not reduced either in Blaberus cells phagocytosing zymosan

Fig. 2 Haemocytic capsules around DEAE-Sepharose beads, 24 h after implantation into Peripfaneta. (a) Scanning electron microscope picture shows several rounded cells adherent to the underlying flattened layer. Bar = 10pm. (b) Transmission electron microscope picture through the inner layers of a capsule (B = Sepharose bead) shows inner melanized layer of disintegrated cells, covered by many layers of extremely flattened interdigitated haemocytes. Bar = 5 pm. (From Lackie et al., 1985.)

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Fig. 3 Haemocytic capsule formed in Drosophila around egg of the parasitoid Leptopilina heterotorna. Note large and extremely flattened lamellocytes. Bar = 10 pm. (Photograph courtesy of Dr A. J. Nappi.)

(Anderson et a[., 1973a) or Schistocerca cells phagocytosing promastigotes of Leishmania hertigi (Molyneux et al., 1986), indicating the absence of hydrogen peroxide. However, by careful choice of optimum fixation methods, M. Carr (unpublished information) has found that PLs of Periplaneta and Schistocerca stain with NBT for hydrogen peroxide and are positive with the diaminobenzidine test for “peroxidase”. Further investigation and use of more sensitive techniques such as chemiluminometry should be useful. The role of the melanization process in killing is controversial. The observation that the core of capsules and nodules is usually melanized led Taylor ( I 969) to propose that some of the variety of phenolic compounds that are produced during melanin formation might be microbicidal. Evidence in favour of this hypothesis derives from experiments by Soderhall and Ajaxon (I 982), who found that dihydroxyindole, but not melanin itself, suppressed growth of mycelia of the crayfish fungal pathogen Aphanomyces astaci. However, melanized cell-free haemolymph of the crab Carcinus maenas (White et al., 1985) and of Galleria (Walters and Ratcliffe, 1983) had no bactericidal activity and immune Galleria haemolymph does not melanize (Pye, 1978). Thus, although various humoral antibacterial molecules have been described (Section 5), we still do not know how intracellular killing operates. Some of the antibacterial proteins of Lepidoptera, at least, are now known to be pro-

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duced by the haemocytes (Trenczek, 1986; and personal communication); whether this is the case for other insects remains to be seen. That bacteria can be killed is quite clear (Anderson et al., 1973b). Whether extracellular killing, by exocytosis of enzymes or other damaging molecules, occurs as with mammalian leucocytes is unknown. The idea that a multilayered capsule kills the enclosed organism by cutting it off from oxygen and nutrients (Salt, 1970) is probably no longer tenable in the light of Caveney and Berdan’s (1982) observations that the gap junctions confer “global coordination” of encapsulating cells. Moreover, helminth larvae may remain alive and unmelanized within capsules for many days (A. Lackie, unpublished information), and bacteria such as Bacillus cereus (Walters and Ratcliffe, 1983) and fungi such as Metarhizium anisopliae (Fargues et al., 1976; Gunnarsson, 1987) can escape from thick haemocytic nodules. The swelling and disintegration of ingested E. coli within phagolysosomes in Calliphora PLs is presumably due to their digestion by lysosomal enzymes (Ratcliffe and Rowley, 1979). The presence of lysosomal “marker” enzymes has been shown by cytochemical, ultrastructural and biochemical means; these include acid phosphatase (Crossley, 1968; Rowley and Ratcliffe, 1979; Peake, 1979; Shrestha and Gateff, 1982a) and b-glucuronidase and b-glucosaminidase (Walters and Ratcliffe, 1981). Lysozyme is also found within haemocytes (Anderson and Cook, 1979), localized within granules (Zachary and Hoffmann, 1984; Trenczek, 1986; and personal communication), and is released in the haemolymph after “immunization” of the insect (Croizier and Croizier, 1978; Anderson and Cook, 1979; Ourth and Smalley, 1980; D. Hoffmann, 1980; Scheider, 1985; Glinski and Jarosz, 1986; Chadwick and Dunphy, 1986; Spies et al., 1986a). Considering the importance of haemocytic killing in cellular defence, our ignorance of the mechanisms involved is surprising. 5 Humoral defence mechanisms As with mammalian immunity, the division of the response into “cellular” and “humoral” components is artificial, since many of the humoral factors are actually produced by the cellular components and react with foreign surfaces to damage or “mark” them for subsequent cellular attack. Humoral components comprise lysozyme and the inducible antibacterial proteins; the serum lectins will also be included here for convenience. 5.1

ANTIBACTERIAL PROTEINS

Synthesis of antibacterial proteins is induced by wounding and, to a greater

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extent, by injection of non-pathogenic bacteria; the concentration of the proteins within the haemolymph rises over a period of several days, the timing depending on the insect species. One of the induced proteins in H . cecropia, P4 (for which a biological role has not yet been found) itself appears able to induce synthesis of immune proteins (Andersson and Steiner, 1987); the factors responsible for switching off the system have not yet been identified. The pioneering work of Boman and his colleagues has been responsible for identifying, in the haemolymph of Hyalophora cecropia pupae, at least 15 different inducible proteins, of which the two main groups comprise the cecropins (basic proteins, molecular weights approximately 4000; active against Gram-positive and -negative bacteria) and the attacins (a group of larger proteins, active against the outer membrane of E. coli). The considerable amount of work in isolating, characterizing and sequencing these molecules is reviewed by Boman (1986). Molecules with similar characteristics to cecropins and attacins have now been found in Galleria (D. Hoffmann et al., 1981), Manduca sexta (Spies et al., 1986a), several species of Diptera (Okada and Natori, 1985; Keppi et al., 1986; Robertson and Postelthwaite, 1986; Flyg et al., 1987; Ando et al., 1987; Kaaya et al., 1987) and in a coleopteran (Spies et al., 1986b) but appear to be absent from the cockroach Periplaneta (G. Takle, personal communication), the cricket Gryllus (Schneider, 1985) and Locusta (D. Hoffmann, 1980). In Locusta (D. Hoffmann, 1980; Lambert and Hoffmann, 1985) and in Rhodnius (de Azambuja et al., 1986), antibacterial proteins of a different type are synthesized in response to infection. A comprehensive review of antibacterial molecules is provided by Boman and Hultmark (1987). Lysozyme, which is also inducible (Chadwick, 1970; Ingram et al., 1984; Spies et al., 1986a; Boman and Hultmark, 1987), was first purified from Galleria (Powning and Davidson, 1976) and more recently two lysozymes with different pH optima have been purified from Gryllus (Schneider, 1985). Whether lysozyme released into the haemolymph is likely to be effective against bacteria has not really been considered although Chadwick (1970) found that antibacterial immunity in Galleria was not correlated with increased lysozyme levels. Since attacins have been shown to attack the outer membrane of bacteria such as E. coli and since cecropins, being amphipathic, have a detergent-like activity on membranes, Boman and Hultmark (1 987) consider that these two classes of molecule attack bacterial cell walls synergistically, leaving the peptidoglycan layer to be digested by lysozyme. The lepidopteran fat-body has been shown to be the main site of synthesis of antibacterial proteins (Faye and Wyatt, 1980) and can be induced to synthesize the proteins in vitro (Dunn et al., 1985; Trenczek and Faye, 1988). Bacterial peptidoglycan (Dunn et al., 1985) or LPS (Trenczek and Faye, 1988) both act as inducers. In H. cecropia pupae, Faye (1978) and Abu-Hak-

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ima and Faye (1981) found that haemocytes that had phagocytosed bacteria became associated with the fat-body, and these authors suggested that these haemocytes might stimulate the fat-body to produce proteins. Subsequently, it has been found that haemocytes from injured or immunized pupae, but not naive pupae, enhance synthesis of immune proteins by fat-body in vitro (Trenczek and Faye, 1988). If fat-body from Galleria larvae was incubated with LPS and whole haemolymph in vitro, greater antibacterial activity was produced than if cell-free haemolymph was used (De Verno et al., 1984). Of great importance, with respect to haemocytic involvement in the humoral response, is the work of Trenczek ( I 986; and personal communication), who has shown both by radioisotopic labelling and by immunoblotting, that haemocytes of H . cecropia pupae can also synthesize cecropins and attacins. Thus, during phagocytosis and nodule formation, antibacterial proteins will also be released where they are most needed. It will be interesting to see if this is also true for haemocytes of Diptera and other Lepidoptera.

5.2

SERUM LECTINS

Whether the serum lectins of arthropods are also present in the plasma and are not merely released from haemocytes is not entirely clear, since most methods for obtaining haemolymph lectin involve collecting haemolymph and allowing it to coagulate before centrifugation. Pistole (1978) was able to identify an agglutinin in both the serum and the plasma of horseshoe crabs, and similar work is required for insects. The occurrence of arthropod lectins and the characteristics of the few lectins that have been purified are discussed in two useful reviews by Stebbins and Hapner (1986) and Hapner and Stebbins (1986). Despite continued optimism and a considerable amount of research, there is still no clear understanding of the role of arthropod lectins. That they have a role in immunorecognition has long been hoped, especially since fungal glucans and bacterial lipopolysaccharides are known to stimulate activation of the prophenoloxidase system (Section 6). However, the majority of the insect lectins that have been purified have galactose specificity, as in Periplaneta (Lackie and Vasta, 1986, 1988; Kubo and Natori, 1987), certain grasshoppers (Stebbins and Hapner, 1985, 1986), Sarcophuga (Komano et a[., 1980), Spodoptera (Pendland and Boucias, 1986) and H . cecropia (Castro et al., 1987). One problem is that, since even the major haemolymph lectin is often present in only small amounts, it is more difficult to purify minor lectins of other specificities, even though cross-adsorption and inhibition studies suggest they are present ( G . Vasta and A. Lackie, unpublished information; Renwran tz, 1986).

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Much early work concerned the role of “serum agglutinins” in opsonizing erythrocytes or bacteria for phagocytosis by haemocytes; all such experiments with insect serum proved negative (Scott, 1971b; Rowley and Ratcliffe, 1980). In some insect species the ability to encapsulate certain parasites in vivo was found to coincide with the ability of serum to agglutinate the parasites in vitro as, for example, with oncosphere larvae of the tapeworm Hymenolepis diminuta in Periplaneta; Schistocerca, on the other hand, neither encapsulates nor agglutinates the larvae, which develop normally within its haemocoel (Lackie I976,1981a, b). Since the major lectin in Periplaneta haemolymph is galactose-specific, it was of interest to investigate the haemocytic response to galactose-rich glycoproteins and it was found that these molecules stimulated thick encapsulation reactions and the production of large numbers of nodules in vivo (Lackie and Vasta, 1988; see Section 9.3.2). Recently, Natori and his colleagues have found that the Sarcophaga galactose-specific lectin is synthesized in response to wounding and to injection of sheep erythrocytes, which are lysed by the lectin (Komano and Natori, 1985), and they consider it likely that the lectin plays a role in the haemocytic recognition and removal of damaged or foreign material (Takahashi et al., 1986). If lectins should prove to be important in immune recognition, one important problem concerns the discriminatory ability of the system-since different species differ in their acuity of recognition (Lackie, 1986b), then lectins with different specificities and/or different binding avidities must also be present (see Section 9.4). As yet, we are a long way from solving these problems. 6

Phenoloxidase and the prophenoloxidase-activation system

Phenoloxidases are found within the cuticle (reviewed by Brunet, 1980, Andersen, 1985) and haemolymph of insects and are involved in protein sclerotization by quinone-bonding in the cuticle and in the production of melanin in haemocytic nodules and capsules and haemolymph clots. Brunet (1980) has written a very informative and critical review of the similarities and differences between insect cuticular and haemolymph phenoloxidases (PO), and of the mechanisms of PO production. The haemolymph enzyme (EC 1.14.18.1) oxidizes mono- and diphenols (dihydroxyphenylalanine (DOPA) is used as substrate for spectrophotometric tests of enzyme activity) and melanin production is inhibited by the use of the competitor, phenylthiourea (PTU). The important work of Seybold and colleagues (1975) using homogenates, and thus mixtures of haemolymph and cuticular enzymes, of enormous

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numbers of Drosophila larvae and pupae, identified at least six components in the production of PO. This led them to propose that a cascade of reactions was involved, in which the latent activator S was converted to active S, which in turn converted P to P’ and this, in association with prophenoloxidase (PPO), resulted in production of PO. This is similar to the activation cascades of the mammalian coagulation, fibrinolytic and complement systems where there are several steps, in each of which a precursor,zymogen is activated to acquire proteolytic activity, and in turn activates its substrate by limited proteolysis, releasing a biologically-active peptide in the process. Evidence for the involvement of a similar type of sequence for activation of Bombyx haemolymph PPO derives from the work of Ashida and Dohke (1980). It is unfortunate that the authors use an unusual method of plasma preparation, involving injecting “cane sugar factor” (Ashida, 198 l), which drastically reduces the haemocyte count and thus leads to some difficulties in interpretation. Despite this drawback, it was shown that purified hemolymph PPO underwent limited proteolysis by a purified activating enzyme (PPAE) isolated from cuticle (Dohke, 1973) and released a 5000 Dalton molecular weight peptide. PPAE was shown to be a serine proteinase. Suggestions that a PPAE might be involved derived originally from the work of Pye (1974), investigating PPO activation in LPS-immunized and naive Galleria larvae. Recently, Yoshida and Ashida (1986) found that addition of zymosan to Eombyx plasma sequentially activated a serine esterase, then PPAE (tested for activity on purified PPO), then PO itself; results of inhibition studies suggested that both activating enzymes were present as zymogens before activation. Anderson et al. (1986; and personal communication) have now purified PPAE and PPO from H. cecropia haemolymph, and have confirmed that PPAE is a serine proteinase. so clearly seen, Since the effects of PPO activation-melanization-are and since at least one peptide with as yet unknown biological activity is produced during activation, it is clearly important to know how the components of the activation sequence are partitioned within the haemolymph. Phenoloxidase aggregates and adheres to foreign surfaces (Unestam and Beskow, 1976; Ashida and Dohke, 1980; Soderhall, 1981; Dularay and Lackie, 1985), thus the observation that the core of capsules becomes melanized need not necessarily imply that PO is released by adherent haemocytes. Moreover, the phenomenon of “humoral melanization” (Section 4.3) needs to be explained. Saul et al. (l987), using Ashida’s (1981) “cane sugar factor” and the low-pH anticoagulant of Leonard et al. (1985b), are convinced that PPO is found mainly in the plasma rather than the haemocytes of Manduca; if this is the case, lack of haemocytic involvement in melanization is possibly easier to understand. However, in Crustacea, if crude lysate supernatant is prepared from haemocytes that have been collected and washed in low-pH anticoagu-

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lant, and is treated with fl1,3-glucans or bacterial lipopolysaccharide (LPS) then serine proteinase activity appears prior to PO activity (Soderhall, 1983; Soderhall and Hall, 1984). In insects, crude lysate supernatant from anticoagulant-washed haemocytes can be activated by b1,3-glucans to produce phenoloxidase; the activation is inhibited by serine proteinase inhibitors (Dularay and Lackie, 1985; Leonard et al., 1985b), suggesting, but by no means proving, that serine proteinase and PPO are associated with the cells. No activity was found in the anticoagulant-diluted plasma (M. Huxham and A. Lackie, unpublished observations), even after dialysis (Leonard et al., 1985b). Persistent attempts have been made to draw analogies between the PPO activation system of arthropods and the alternate complement pathway of mammals (Soderhall, 1982; Ratcliffe, 1986); one major difference seen at present is that the former system seems to be contained predominantly within the cells, whereas the latter system is found within the plasma. Such compartmentalization is likely to impose very different constraints upon the activation sequences (see also Section 9.4). Any activation process that relies on proteolysis must also incorporate inhibitors that will down-regulate the system so that it does not run out of control. Thus, plasma factors I and H control the production of C3-convertase in the alternate complement pathway (Whaley, 1986). Proteinase inhibitors have been purified from crayfish haemocytes (Hall and Soderhall, 1982) and cuticle (Hall and Soderhall, 1983), but they inhibit the subtilisin rather than the chymotrypsin or trypsin type of serine proteinase. However, inhibitors with anti-trypsin and anti-chymotrypsin activity were isolated from Bombyx haemolymph (Sasaki and Kobayashi, 1984). Molecules with a,-macroglobulin-like activity that inhibit proteolysis by trypsin have been found in horseshoe crabs and Crustacea (Armstrong et d., 1985; Hergenhahn and Soderhall, 1985); the activity is released from Limulus haemocytes by exocytosis (Armstrong and Quigley, 1985); nevertheless, a,-macroglobulin does not inhibit PPAE (Soderhall et al., 1986). In insects, some advance has been made as an inhibitor of cuticular PPAE has been isolated from Manduca haemolymph (Saul and Sugumaran, 1986). The relationship between the cuticular and haemolymph PPO systems is not at all clear, and it should not be forgotten that PO is a multifunctional enzyme which is involved in pathways other than melanization in the haemolymph. It is highly probable that activation may be regulated in a number of ways, depending on how the product is to be used (K. Anderson, personal communication). The PPO system of insect haemolymph can be activated, in the presence of calcium ions, by fll,3-glucans such as laminarin and by supernatant from zymosan (Pye, 1974; Soderhall, 1981; Ashida et al., 1983; Dularay and Lackie, 1985; Leonard et al., 1985b) that contains a mixture of fl-glucans and mannans (Bacon et al., 1969; Soderhall and Unestam, 1979). There are some

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intriguing differences between species, in that laminarin is a good activator of Schistocerca haemocyte lysate, but has no observable activating effect on that from Periplaneta (Huxham and Lackie, 1986; Takle, 1986). Unlike the situation in Crustacea (Soderhall and Hall, 1984; Soderhall et al., 1986), the insect PPO system is not activated by bacterial LPS (Dularay and Lackie, 1985; Leonard et al., 1985b; Yoshida and Ashida, 1986; Takle, 1986) (see also Section lo), although bacterial peptidoglycan apparently activates the Bombyx system (Yoshida and Ashida, 1986). Inulin and other polysaccharides such as dextran, cellulose and chitin failed as activators but, interestingly, denatured plasma proteins and denatured plasma lipophorin were stimulatory (Ashida et al., 1983). Some similarities with the mammalian alternate complement pathway are thus apparent, since this can be activated by zymosan and LPS although laminarin and pure inulin are ineffective (Czop and Austen, 1985). Additionally, PPO from haemocyte lysate can be activated at a low level by denaturing effects such as heat and by the withdrawal of haemolymph (Ashida and Siiderhall, 1984). Results of cytochemical studies on the occurrence of PO within haemocytes are conflicting-GRs in Galleria (Schmit et al., 1977), crystal cells in Drosophila (T. Rizki et al., 1980), oenocytoids in mosquitoes (Drif, 1983) and Lepidoptera (Monpeyssin and Beaulaton, 1977; Horohov and Dunn, 1982; Stoltz and Guzo, 1986), and COs of Locusta (Hoffmann et al., 1970). That the oenocytoids of Bombyx contained PPO was shown imrnunochemically by Iwama and Ashida (1986). The association of PO with oenocytoids is perhaps rather surprising since they are a relatively uncommon cell type whose function is unknown (Section 2.1). Haemocytes collected in anticoagulant are more stable, and little or no phenoloxidase is associated with them until they are washed and stimulated; Soderhall and Smith (1983) separated crustacean cells on Percoll gradients and found that PO was associated with the granular cells. Similarly, PO is associated with separated GRs from Galleria (Ratcliffe, 1986), and with a separated subpopulation of densely granular haemocytes from Schistocerca (Huxham and Lackie, 1988). Haemocytes held in balanced salt solutions tend to be unstable and to release a considerable amount of protein, so that the majority of haemocytes turn black if DOPA is added as substrate for PO; if the stability is improved by the use of specially formulated media and the addition of foetal calf serum, PO remains localized and only d small proportion of cells turn black (Huxham and Lackie, 1986). Since PO and presumably other granule contents are released into the medium, the possibility has been explored that some of these molecules are opsonic. Although it aggregates and adheres to foreign surfaces, PO itself is not opsonic for locust (Dularay and Lackie, 1985) or crustacean (Soderhall rt al., 1984) haemocytes. However, bacteria incubated in LPS-activated hae-

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mocyte lysate supernatant are phagocytosed 3 4 times more effectively by separated crustacean hyaline haemocytes in vitro (Soderhall et al., 1986) and thicker capsules are formed in crayfish around fungal spores pretreated with activated lysate supernatant (Soderhall et al., 1984). More important, glucan-activated crustacean haemocytes release factors that enhance the phagocytic competence of hyaline cells in vitro (Soderhall et al., 1986). Although circumstantial evidence suggests components of the PPO activation sequence are involved, there is always the possibility that completely unrelated molecules may play a role. In insects, laminarin-stimulation of haemocyte monolayers from Leucophaea, Blaberus and Galleria enhanced the phagocytosis of Bacillus cereus by PLs and, to a lesser extent, by COs/GRs. Stimulated GRs became surrounded by a coating of material to which bacteria adhered (Leonard er al., 1985a) and it should be remembered that GRs/COs are also the cells that produce the “haemocyte gel” during haemolymph coagulation (Section 8.3). In separated subpopulations of Schistocerca cells, the PPO-containing, densely granular subpopulation shows little association with fluorescent latex beads unless stimulated by laminarin (Huxham and Lackie, 1988). Finally, haemocyte lysate supernatant in which PPO has been activated by glucan treatment dramatically stimulates chemokinesis in vitro of Periplaneta PLs and of the separated subpopulation of very granular Schistocerca haemocytes (Takle and Lackie, 1986; Huxham and Lackie, 1988; see Section 10). In contrast, separated crustacean granular cells adhere more readily to glass coated with activated lysate (Johansson and Soderhall, 1986). The role of the PPO activation sequence in these phenomena remains to be ascertained (Section 10). 7 Changes in the haemocyte population

As one might expect, neither the concentration nor the absolute number of haemocytes remains constant throughout an insect’s life. The factors that affect cell number are very diverse, dependent on endogenous cues such as the developmental and reproductive status of the insect and exogenous insults such as cuticular wounds or invasion by parasites and pathogens. A very helpful and comprehensive review of the literature concerned with changes in haemocyte populations within several insect orders has been provided by Shapiro (1979), and the reader should consult this for a detailed list of references. Rather than reiterate that information here, my aim is instead to note the factors known to alter the numbers of haemocytes and the proportions of haemocyte subpopulations, and to discuss the relevance of these alterations to the cellular immune response.

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DEVELOPMENTAL STATUS OF THE INSECT

In general terms, it appears that the cell numberlyl (=cell count) increases with the larval or nymphal instar of the insect. For example, in their studies on haematopoiesis in larvae of the noctuid Euxoa declarata, Arnold and Hinks (1 976; see also review by Arnold, 1979) found that the cell count more than tripled between the second and sixth instar, although the PL population showed a relative decrease. In larvae of Bombyx mori, reared under specificpathogen-free conditions and sampled rather soon (3 h) after each ecdysis, there was a marked increase in cell count from first to fifth instar (Wago and Ishikawa, 1979) and Dunphy and Nolan (1980) found that both the cell count and the absolute Dumber of cells (calculated from measured changes in blood volume) increased from third to sixth larval instar of the spruce budworm Choristoneura fumiferana. Adult Locusta (Webley, 195 1) and fifth-instar Rhodnius (Jones, 1967) had a higher haemocyte count than the previous instar. However, there are large discrepancies between the results of different authors for any particular species of insect. These discrepancies are due partly to different sampling techniques and partly to the time, within any instar, at which the insect was sampled. As an example of variation within one instar, examination of Shapiro’s (1979) data for Galleria larvae shows major changes in cell count, ranging from 32,5 13 f 3765 through 38,130 f 338 1 to 47,220 f 3553, throughout the development of the seventh instar. It is almost impossible to generalize about the changes in haemocyte count during any ecdysial period, although several authors note a decrease in cell count just prior to ecdysis: in Rhodnius (Wigglesworth, 1955), in Periplaneta (Patton and Flint, 1959); in Schistocerca (Lee, 1961); in Halys dentata (Pathak, 1986) and in Pieris brassicae (Breugnon and Le Berre, 1976). In Holometabola, whether or not a postecdysial increase also occurs, as in the hemimetabolous Periplaneta (Patton and Flint, 1959), may also depend on whether the moult was pupal or imaginal-in the former case, numbers tend to remain low throughout the pupal stage as, for example, in Sarcophaga bullata (Jones, 1956), Ephestia (= Anagasta) kuehniella (Arnold, 1952), Hyalophora cecropia (Walters, 1970) and Mamestra brassicae (Pelc, 1986). The count subsequently increases after the imaginal moult (Walters, 1970). Finally, the whole blood picture is further complicated because the volume of blood per unit mass is not always constant (Shapiro, 1979; Wag0 and Ishikawa, 1979; Pathak, 1986); thus, an apparent increase in cell count might be due to decrease in blood volume, as shown by Wheeler (1963) in Periplaneta, resulting in the absolute number of haemocytes per insect remaining approximately constant. That large variations in blood volume occur, dependent on the reproductive status of the insect (Hill er al., 1968) or the avail-

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ability of moist food (Wharton et al., 1965) is well known. Rhodnius provides a good illustration of the complex relationship between ingestion of a bloodmeal, haemolymph volume, ecdysis and haemocyte count-after a bloodmeal, haemocytes are more easily obtainable (Takle, 1988; and personal communication), the haemolymph volume is increased (Maddrell and Gardiner, 1980) and haemocytes are found undergoing mitosis (Wigglesworth, 1933). Why there should be an overall increase in the haemocyte count throughout juvenile development is not entirely clear since, on first consideration, a first-instar larva is just as much in need of a competent cellular defence system as is a sixth instar; moreover, the surface area of cuticle and gut, and hence the area for invasion by pathogens, is larger relative to volume in the smaller individual. It is interesting to speculate about the reasons for the high mortality of young instars and the importance of partitioning energy resources into an effective defence system in the small proportion of later instars that remain. Nor is it clear how ontogenetic changes in the concentration and absolute number of haemocytes influence the efficacy of the immune response. It is worth noticing that most investigations into cellular immunity have been carried out on larval holometabolous but adult hemimetabolous insects. It would be valuable, therefore, to have comparative information for total and differential haemocyte counts for all stages throughout a life cycle and, as far as technically possible, to compare the relative efficacy of a defined parameter of the immune response for the different developmental stages. Thus, Peake (1979) found that there was an increase in the proportion of phagocytic haemocytes in Culliphoru with age. In Bombyx, Wag0 and Ishikawa (1979) found that GRs increased from approximately 20% to 60% of the total population between first and sixth instar and, since GRs appeared to be the main cell type that adhered to the test particles, erythrocytes, these authors suggested that the increased ability of developing larvae to respond to erythrocytes was due to the increase in the G R subpopulation. More information is required to help explain the differential response with age of hosts to parasites and pathogens (Salt, 1968; Lynn and Vinson, 1977; Payne et ul., 1981). Decrease in haemocyte count prior to a metamorphic ecdysis may be due to changes in haemocyte adhesion and haemocytic involvement in removal of redundant tissues. These changes in haemocyte behaviour may be hormonally induced and might increase the responsiveness of the cells to non-self or altered self. Crossley ( 1968), studying muscle autolysis and regeneration during metamorphosis of larvae of Culliphoru, found that injected crustecdysone stimulated an increase in the relative proportion of phagocytes (= PLs?). Moreover, the proportion of these cells that were “engorged” increased from 3 % to 29% within 24 h of the hormone injection. This coincided with the

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prematurely-induced breakdown of the thoracic muscles, and Crossley suggested, on the basis of histological investigations, that the phagocytic haemocytes were required to remove degraded tissues. Whether the increase in phagocytic activity was due to increased activity of a pre-existent haemocyte subpopulation, or to an increase in the number of phagocytic cells, was not determined. However, the locomotory activity of Manduca haemocytes is increased in the presence of ecdysone (Judy and Marks, 1971), and G . Takle (unpublished information) found that this hormone stimulated an increase in both the speed and directional persistence of movement of Periplaneta haemocytes in vitro. The increase in proportion of PLs switching to lamellocyte morphology and behaviour that occurs at the end of larval development in Drosophila (Rizki, 1957) is thought, on the basis of experiments in which the corpus cardiacum was ligatured (Nappi, 1975), to be stimulated by changes in hormonal levels. A review of the hormonal control of haemocyte populations is provided by Crossley (1975). Careful and coordinated investigations of the developmental changes in cell count, blood volume, differential haemocyte count and cell behaviour, appear to be needed.

7.2

STRESS

Stressed insects, whether starved, overcrowded or subjected to non-physiological temperatures, are less immunocompetent (Steinhaus, 1958; Steinhaus and Dineen, 1960) than unstressed controls. This is hardly surprising, since the body’s resources will have to be shared out in different ways according to the prevailing conditions, a greater share of the available energy being directed to growth or reproduction. Decreased immunocompetence could be due to changes in production of “humoral” factors or changes in the number of reactive cells. Prolonged starvation reduced the cell count in Tenebrio (Jones and Tauber, 1952) and starvation for a period of 10 days produced a significant decrease in cell count in Galleria larvae, although the blood volume remained comparable to that of the control larvae (Shapiro, 1967). In starved Locusta, there was not only a decrease in cell count, but also a dramatic decrease in size of the coagulocyte population, which fell by 74% after 8 days of starvation (Hoffmann, 1970b).

7.3

WOUNDING

Haemocytes are involved in wound-healing (Section 8) and thus it might be

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expected that wounding might induce some change in the total number of cells and proportions of cell types. Information on population changes derives not only from direct investigations into the mechanism of cuticular wound-healing but also from the controls-pricked or saline-injected insects-for experiments in which the effect of injected particulate material on total or differential haemocyte count has been investigated. Thus, Harvey and Williams (1961) pricked brainless diapausing pupae of Hyalophora cecropia and found that the cell count increased by approximately 10 times within 30 min, and continued to increase with each successive sample over the next 6 days. Walters (1970), during an examination of haernocyte behaviour in diapausing saturniid moth pupae, produced large-scale wounds by removing the brains and the cuticle covering face and legs; the superficial wounds were treated, according to the methods of Harvey and Williams (1961) with PTU, antibiotics and Ringer’s solution, before being sealed with wax or plastic coverslips. Despite this horrendous treatment, and even though only a small number of pupae were compared, it was quite clear that the cell count had increased by 12-18 times, 72 h post-operation. In diapausing pupae, the circulating haemocyte count is very low (e.g. for H. cecropia, 10 per mm3, Harvey and Williams, 1961; total available approximately 3 x lo5, Trenczek, personal communication); presumably, as with the induction of synthesis of antibacterial proteins in response to wounding (Section S), the cellular response can also be activated through production of new haemocytes by mitosis (Lea and Gilbert, 1961) and/or the return of sessile haemocytes to the circulation. In holometabolous larvae, or hemimetabolous nymphs and adults, the situation is possibly rather different, since these stages in the life cycle are not quiescent. Nevertheless, an elevated cell count was found 4 h after cuticular puncture in third-instar larvae of the tussock moth, Orgyia leucostigma, and persisted above that of control larvae for 24 h. A more rapid increase in cell count also occurred after injecting saline, the number approximately doubling within 3 h (Guzo and Stoltz, 1987). These third-instar larvae moulted 4 days after injection and throughout this period the cell count in control, uninjected larvae quadrupled-this illustrates the importance of taking samples from control insects throughout the experimental period and not relying on a comparison with the value at zero time. In Drosophila melanogaster and D. yakuba, cuticular puncture alone and saline injection both provoke an increase in cell count and an increase in the proportion of lamellocytes (BrehClin, 1982) In contrast to these reports of an elevated cell count after wounding, there appear to be cases in which no change occurs at all, as in Manduca larvae (Horohov and Dunn, 1982) or Periplaneta nymphs (Ryan and Nicholas, 1972).

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These conflicting results become more explicable when the time-course of the response is examined in much finer detail during the first 1-2 h postinjection-ertain interesting events then become apparent. The careful quantitation, by Gagen and Ratcliffe (1976), of the process of noduleformation in response to injected bacteria, showed that the cell count in 200 mg “control” Galleria larvae dropped by approximately 30% within 5 min of saline injection or cuticular puncture; after 60 min the cell count had returned to the starting level. Similar decreases in cell count in Galleria in response to wounding or saline injection have also been recorded by Dunphy et al. (1986), Dunphy and Webster (1985) and Ratcliffe and Walters (1983). In adult male Schistocerca injection of 25 pl of saline (approximately 10% of the total blood volume) caused the cell count to drop by 35% within IOmin, but to return to a level similar to that in uninjected controls by 60 min (Gunnarsson, 1987). This transient decrease would be overlooked if sampling was not carried out until several hours had elapsed. Leaving aside the question of changes in blood volume, few investigations have been carried out to determine whether or not this decrease in cell count in response to wounding is due to a decrease in the entire cell population or to a decrease in a particular class. Again in Galleria larvae, Dunphy and Webster (1985) found that the proportion of PLs dropped from 40% to 2% within a few minutes of making a puncture wound or injecting saline; in punctured animals, the PL count returned to control levels within 2 h, but that of saline-injected larvae remained low for about 5 h post-injection, during which time the total cell count was also low. In contrast, Chain and Anderson (1982) found that the proportion of PLs in Galleria injected with tissue-culture medium did not differ from that in controls at 1 h postinjection. One possible explanation for this discrepancy is that Dunphy and Webster (1985) used rather small (120mg) larvae with a much lower cell count (8 x 106/ml compared with 22 x 106/ml in the 200-mg larvae of Ratcliffe and Walters, 1983) than that of other workers; the effec? of injection might thus appear disproportionately great. In summary, puncturing the cuticle causes a short-term change in cell count: in pupae and, surprisingly, in Manduca larvae, the cell count increases; in lepidopteran larvae and adult locusts, the count decreases. In Galleria at least, most of the decrease seems to be due to a decrease in size of the PL subpopulation; whether this is true for other species remains to be investigated. Information on the changes that occur within the haemocyte population in response to wounding requires expansion and clarification because it will ultimately help to provide insights into the primary events in the cellular immune response. In mammals, the events that follow upon wounding the epidermis, basal membrane zone and dermis serve to bring the various classes of leucocyte into the very area in which they may be required to deal with invad-

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ing microorganisms; it is not unlikely that in insects, damage to the cuticle, epidermis and basement membrane could also trigger a series of cellular interactions that resulted in changes in the PL subpopulation, bringing them into a state of readiness to deal with invaders (see Section 8.3.3.2).

7.4

INFECTION

There have been many investigations of changes in differential or total haemocyte count induced by viral (Lea, 1986; Davies et al., 1987), protozoal (ZClCdon and de Monge, 1966; Laigo and Paschke, 1966; Ibrahim et al., 1986; Takle, 1986), fungal (Gunnarsson, 1987), helminth (Andreadis and Hall, 1976; Dunphy and Webster, 1985; Nappi and Christensen, 1986) and parasitoid (Nappi, 1975, 1981; Nappi and Stoffolano, 1972; Carton and Kitano, 1979; Guzo and Stoltz, 1987; Davies et al., 1987) infection. As with bacterial infections, the observed haemocytic response is a result of a complex interaction between the evasive mechanisms of the infective agent and the response mechanisms of the host and, as such, is not easily interpreted. In contrast to work using bacteria, the response to killed or fixed viruses or eukaryotic organisms has rarely been investigated and would probably not, in any case, be very informative. Because of the complexity of the interrelationship the effect of infections on haemocyte number and class will not be discussed here, but examples will be used to illustrate other arguments where relevant.

7.5

PARTICULATE MATERIAL

There are numerous reports on the phagocytic clearance of injected live or heat-killed bacteria, yeasts and protozoan parasites, or of injected abiotic material such as latex beads, colloidal gold or carbon particles. Since the phagocytic capabilities of the haemocyte population are dependent on both the inherent phagocytic capability of each cell and the numbers of phagocyticcompetent cells, many of these investigations have also been concerned with measuring changes in the total or differential haemocyte count. Some typical results are summarized in Table 2; for references up until 1979 the reader should consult Shapiro (1979). In those cases where the insect is able to sequester the injected material, the cell count drops and-unlike the transient decrease shown in response to wounding (Section 7.3)-remains low for several hours before returning to and often surpassing the normal

TABLE 2 Examples of changes in cell count after injection of particulate material Insect Galleria larvae

Pieris brassicae larvae Orgyia larvae Manduca larvae Periplaneta nymphs adults Locusta adults Schistocerca adults

Change in cell count

Stimulant

Authors

Bacillus cereus Fixed E. coli E. coli, low doses B. cereus Heat-killed virus (S.I.V.) Heat-killed Pseudomonas Some Pseudomonas strains

75% 1; still low at 60 min transient 1; normal by 3 h

Gagen and Ratcliffe (1976) Gagen and Ratcliffe ( 1976)

1, recovery by 6 h 1,at 24h 1 1 1

Ratcliffe and Walters (1983) Lea (1 986) Stephens (1963) Dunphy et al. (1986) Gagen and Ratcliffe (1976)

Saccharomyces cerevisiae Pseudomonas E. coli D3 1 Various particles and bacteria Saccharomyces cerevisiae Latex beads B. thuringiensis (non-lethal dose) Iron saccharate Trypanosomatid protozoan spp.

1, then t from 60 min

Guzo and Stoltz (1 987)

1at 60min t

Horohov and Dunn (1983) Ryan and Nicholas (1972)

1

M. Carr (pers. comm.)

1, then t at 24h

D. Hoffmann et al. (1974)

}

1

1, for 48 h, then recovery

Molyneux et al. (1986)

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M . LACKIE

levels. This reduction in cell count is partially due to trapping of injected material in haemocytic nodules, whereby haemocytes are removed from circulation. The most interesting observations are on the effects of injected particulate material on the different haemocyte classes. Hanschke et al. (1980), using Galleria larvae, noted that injection of latex beads or formalinized E. coli caused a rapid decrease in the number of PLs and GRs within 5 min of the injection. More recently, Chain and Anderson (1982) confirmed this effect for the same species, using a variety of pathogenic, non-pathogenic and heatkilled bacteria; the PLs were particularly affected-in haemolymph taken 1 h after injection of heat-killed Bacillus cereus, the mean proportion of PLs had dropped from 46% to 7%, and the cell count had correspondingly decreased. Since nodule formation alone could not account for this marked change, the authors suggested that a factor was released by stimulated cells (plasmatocyte depletion factor, PDF) that caused temporary removal of PLs from the circulation, possibly by changing their adhesive properties (Chain and Anderson, 1983a) (Section 8.3). Heat-killed Bacillus thuringiensis stimulated a large decrease in the number of COs (functionally equivalent to GRs in Lepidoptera) in Locusta, and also had some depressive effect on the number of PLs (D. Hoffmann et al., 1974). In both insect species, the cell count eventually returned to normal, and dividing haemocytes were found in the circulation of Galleria (Hanschke et al., 1980) and the haematopoietic tissue of Locusta (D. Hoffmann et al., 1974). In contrast to this frequently reported decrease in cell count, some authors have found that injected particles stimulate an increase. In one recent example Horohov and Dunn (1982) found that E. coli and heat-killed Pseudornonus aeruginosu stimulated an increase in cell count, particularly in the proportions of GRs and SPs, by 60 min post-injection. Brehelin (1982) has found that the cell count, and particularly the proportion of PLs, is increased by 3 h after injection of iron saccharate into Drosophila; by 24 h, the lamellocyte population has also increased. The increase in PLs was thought to be due to release of sessile cells. On the basis of their results for Galleria, Ratcliffe and Walters (1983) suggested that the degree of alteration in cell count was influenced by the pathogenicity of the bacterial species injected, the more pathogenic strains (B. cereus) stimulating the greater depletion, but the results of Dunphy et al. (1986), using a range of lipopolysaccharide mutants of Pseudomonas, indicate that the relationship may not be so simple. Clearly these discrepancies cannot be accounted for until we know much more about the nature of intercellular cooperation, but the effect of injection on the proportion and behaviour of PLs, the cells most heavily involved in effecting phagocytosis and nodule formation, requires more detailed investigation.

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7.6

125

MOLECULES IN SOLUTION

Many years ago, Schwalbe and Boush (1971) showed that an injected solution of bacterial lipopolysaccharide (LPS; endotoxin) stimulated nodule formation in larvae of Galleria and this was subsequently confirmed for Schistocerca and Periplaneta by Gunnarsson and Lackie (1985). Other molecules of microbial origin, the B1,3-glucans found in fungal cell walls, also produced a similar effect in these two insect species (Gunnarsson and Lackie, 1985) and in Crustacea (Smith et al., 1984) (see also Section 4.2). Concomitant with nodule formation, the cell count decreases by approximately 75% in locusts injected with zymosan supernatant (Gunnarsson, 1987). This decrease is too large to be accounted for by haemocyte aggregation and may be due to a transient increase in haemocyte adhesion to haemocoelic surfaces (see Section 8.2).

7.7

CONCLUSIONS

Rather than despairing over our present inability to provide a simple and generally applicable interpretation of the observed changes in haemocyte numbers in response to various stimuli, we should instead try to decide what we need to know, and why. Investigation of the response of haemocytes to sterile wounds should provide valuable information about the ability of these cells to detect and cooperate in the repair of damage. Any expansion of the available information on the transient (up to 60min) changes in cell numbers, whether of the total population or within particular classes, in response to sterile wounding would thus help our understanding of this very basic defence mechanism. Assessing changes in total cell number is probably too crude a method, in most cases, to detect changes in cell behaviour. With the development of internal and external markers (Section 2.2) for haemocyte subclasses, it is becoming possible to detect much more subtle numerical changes within the total population. It is only necessary to look at the information that was provided by the use of markers for T-lymphocyte subclasses in AIDS infections, thus detecting changes in the proportions of T4/T8 subclasses, to have an example of the usefulness of this technique. The significance of the numerical changes within the haemocyte population in response to infection, or injection of particulate material also needs careful questioning. Thus, if injected colloidal carbon or iron saccharate produces a decrease in cell count and “blockade” of phagocytosis, for how long is the immune system compromised and when does the blood picture return to normal? In chronic infections, are the reactive haemocytes gradually depleted or are they re-

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placed? After an effective response to an acute infection, does the number of effector cells return to a normal level or is it elevated, thus permitting a more rapid “secondary” response (Anderson, 1986)?

8 Haemocytic involvement in wound-healing; the primary defence mechanism

8.1

REQUIREMENTSFOR WOUND-HEALING

Haemocytes operate both in immunological defence and in wound-healing; whether these two activities require different triggers and whether the mechanisms of the response are essentially the same in both cases is at present unclear (Lackie, 1986e) although there are strong similarities between certain aspects of wound-healing and encapsulation. Since most experimental studies of wounding have been carried out under non-sterile conditions, the results obtained will contain an inextricable mixture of information on both wounding and immunological defence; despite this, a considerable amount of useful information about cellular events in wounding derives from studies with an immunological rather than a physiological bias. With the exception of the midgut wall, all penetrative wounds damage the cuticle and its underlying epidermis and basement membrane layer, and may lead to haemolymph leakage. Internal wounds also occur occasionally, arising either from morphogenetic errors at ecdysis or damage during oocyte release; the lesions found in the fat-body of Drosophilu larvae in certain types of tumour mutant (R. Rizki et ul., 1983) also come into this category. The main requirements for wound-healing are (1) rapid clotting of blood at the site of the lesion to prevent leakage; (2) strengthening of the clot by the addition of cells; (3) repair of the underlying layer of tissue cells; and (4) repair of the basement membrane or connective tissue coating. In mammals, skin wounds involve damage to epithelium, basement membrane zone (BMZ) and the dermis, within which the blood is confined to endothelium-lined capillaries. Mechanical damage, even if it does not sever capillaries, causes release of vasoactive amines, which increase the permeability of the endothelium, permitting leakage of plasma proteins into the tissue fluid. Plasma kininogens are activated, mainly by the activation of Hageman factor in contact with tissue collagen, and a fibrin clot results (see Taussig, 1984, for details). Blood leucocytes, especially neutrophils, are stimulated to adhere to the endothelium (a process termed “margination”) and to migrate into the damaged area through endothelial junctions. A complex series of intercellular reactions occurs, whereby granular leucocytes respond to molecular stimuli produced in the wound area, migrate through the dermal

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matrix and, in turn, produce a variety of factors that recruit other leucocytes to the area. In lesions formed within nervous tissue there is evidence that blood monocytes become involved in repair and the granulomatous tissue formed in response to internal wounds is well described, but whether leucocytes are also involved in the repair of skin wounds is at present unclear; it is likely that the principal reason for leucocytic infiltration is immunological defence and the removal of damaged tissue and the clot. Fibroblasts are actively involved in repair of connective tissue and epithelial cells migrate across the extracellular matrix and restore the integrity of the surface. In comparison, “skin” wounds in insects require a simpler solution. The subepidermal basement membrane is in direct contact with the circulating haemolymph and only in the wings is the haemolymph confined to “veins”; rapid clotting of haemolymph at the wound-site is a necessity and, since there is no dermis, the only cells immediately available to strengthen the clot are the haemocytes (Fig. 4). Epithelial cells then spread over the haemocytic layer. As with the mammalian BMZ, the origin of the subepithelial basement membrane is still a matter for dispute. Since it does not show the stratification and ultrastructural characteristics of an epithelium-derived basement membrane, it has been suggested, on the basis of histological (Wigglesworth, 1979). radioisotopic and histological (Shrivastava and Richards, 1969, immunological and ultrastructural (Ball et al., 1987) and genetic (Knibiehler et al., 1987) evidence, that haemocytes also secrete components of this layer. This is hotly disputed by, for example, Ashhurst (review, 1985) on the basis of biochemical and histochemical studies. Modern radiochemical and separative techniques should permit resolution of the controversy (Section 9.1.2). In addition to their involvement in cuticular wound-healing, haemocytes aggregate at the site of internal damage, such as the cut ends of implanted tissues (reviewed by Lackie, 1986b), the perineurium of damaged nerves (Shivers, 1977; Treherne et al., 1987) and at lesions caused by invasion of parasites through the gut wall (Cawthorn and Anderson, 1977) or Malpighian tubules (Harry and Finlayson, 1976).

8.2

RECOGNITIONOR RESPONSE? A CONSIDERATIONOF WHY HAEMOCYTES

ATTACH TO A WOUND

There is an important distinction between the inherent ability of a haemocyte to recognize damage and the necessity for a haemocyte to be primed by “wound factors” (this vague term is used throughout this section merely for convenience). In the first case, the haemocyte is itself observant; in the second, it is oblivious until the damage is pointed out. In terms of the initiation of the cellular response to wounds there are thus

Fig. 4 Haemocytic aggregation on the basement membrane beneath a puncture wound. The cuticle and epidermis of adult Schisrocerca were punctured, and these scanning electron microscope photographs were taken from material fixed 6 h after wounding. (a) A very localized aggregate has formed; the surface of the inner region is very flattened and apparently covered with “coating material”, whereas several haemocytes at the periphery are still fairly rounded. Bar = 25 pm. (b) Higher magnification of edge of aggregate, to show haemocytes with filopodia. (c) Note “lumpy” surface of plasmatocytes, indicative of presence of underlying granules. (Photographs courtesy of Dr S.G . S. Gunnarsson.)

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two main possibilities: ( I ) recognition of damage by randomly contacting haemocytes, which then adhere to that region; or (2) release of soluble wound factors which stimulate changes in haemocyte behaviour, so that the cells respond by, for example, adhering at the site of wound-factor production. The first situation is the simplest to organize as it relies merely on random contact, recognition of non-self and then adhesion by the haemocytes. The cells that adhered would then have to release “recruiting factors” so that more haemocytes were caused to adhere. The second situation is more complex since it need not require that haemocytes recognize damaged self but rather that they recognize the soluble wound factors, reception of which must cause the cell to change from being non-adhesive and freely circulating to being adherent, although not necessarily sessile. However, soluble molecules will not remain localized unless they are continuously produced and locally inactivated, and low levels of wound factors within the haemolymph may promote a general change in adhesion so that stimulated haemocytes attach randomly to any haemocoelic surface (as in margination). According to this hypothesis, adhesion should persist only where the wound factors are most concentrated; further haemocyte aggregation at the wound-site could result from the secondary production of recruiting factors by these persistently adherent cells. Thus, in the case of cuticular wounds adjacent to the circulating haemolymph, it is possible to account for haemocyte accumulation at a wound in terms of changes in adhesive behaviour alone. However, this behavioural change cannot account for haemocytic infiltration of deeper tissue wounds, such as those of the gut (Day and Bennetts, 1953; Kawanishi et al., 1978) and nervous system (Smith et al., 1986). Chemotaxis is probably one of the most widely misused terms in this context and, so far, there is no clear evidence either for or against chemotactic behaviour by insect haemocytes, mainly because suitably objective assays developed for mammalian cells (Wilkinson, 1986) have not been considered and modified. Both chemotaxis and chemokinesis require that the responding cell is adherent to and moving over a substratum; neither term can be applied to non-adhesive and circulating cells. Chemokinesis is a response to a soluble molecule “which involves a change in the rate or,frequency of movement, or a change in the frequency or magnitude of turning behaviour” (J. Lackie, 1986). Thus, a cell showing positive chemokinesis may be stimulated to move faster and may also turn more frequently-such behaviour would ensure that it remained within a small area, whereas a cell that moved faster and turned less would be able to scan a large area. Chemokinetic behaviour would be suitable for a marginated haemocyte in the vicinity of a wound, especially if high concentrations of wound factor increased adhesion to such an extent

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that locomotion then ceased. Since speed of locomotion is related to the extent of cell-substratum adhesion (Wilkinson et a f . , 1984; Section 8.3.3) then a chemokinetic stimulus issuing from a wound would be sufficient to cause haemocyte accumulation. Chemotaxis, in contrast, is the response of a motile cell to a “concentration gradient of a soluble chemical in the environment which affects the direction in which a turn is made, and thus the direction of movement” (J. Lackie, 1986). Thus, chemotactic attraction of haemocytes by a wound factor requires that a gradient of the factor be maintained, so that a motile cell in the vicinity would receive and respond to the molecules by turning up-gradient. It would also be necessary for the cell to adhere firmly and stop moving at the source of the gradient. A gradient of chemotactic factor cannot be maintained where fluid is circulating and therefore this mechanism can only operate within tissue wounds. Finally, although it has been argued here that changes in adhesion and locomotion alone could account for haemocyte accumulation at a wound, haemocytic recognition of damaged self tissues at the wound-site need not, of course, be excluded. In the following subsections, the nature of the clotting stimulus, and the evidence for wound factors and for changes in haemocytic adhesion and locomotion after wounding, will be examined. The nature of the differences between intact and wounded self that might be recognized by haemocytes are dealt with in Section 9.

8.3

WOUND FACTORS AND THEIR EFFECTS ON HAEMOCYTES

8.3.1 The clotting stimulus According to current theories, haemolymph clotting requires the interaction of a plasma coagulogen and a haemocyte-derived coagulogen (Brehklin, 1979; Barwig, 1985). Most biochemical investigations have used locusts (Brehtlin, 1979; Gellissen, 1983) or cockroaches (Bohn, 1986) since in these species, provided all manipulations are carried out at 4”C, it is possible to produce separate plasma and haemocyte fractions, the important criterion being that the separated haemocytes have not degranulated and can be resuspended. This distinction between plasma (where the cells are intact) and serum (where the cells lyse or degranulate and form a sticky coagulum) is important. I t has only fairly recently been recognized that the plasma coagulogen is probably plasma lipophorin; lipophorins (also known as diacylglycerol lipoproteins or high-density lipoproteins) are high molecular weight lipoproteins,

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usually comprising two subunits, that contain from 30 to 50% lipid and function as carriers of diacylglycerols and other lipids (reviewed by Chino, 1985). Haemocyte coagulogen is released during lysis of Leucophaea haemocytes in vitro and forms a gel that is insoluble in a mixture of urea and detergents, but can be solubilized by a mixture of urea and dithiothreitol and digested by proteinases, suggesting that it is a disulphide-linked protein (Bohn and Barwig, 1984). Immunofluorescent-antibody tests provide some evidence that the coagulogen is contained in the granules of GRs, PLs and even SPs (Bohn el al., 1981). When the haemocyte gel is mixed with plasma coagulogen, a stable clot is formed. By studying the effect of a range of proteinase inhibitors on this clotting process, Bohn and Barwig (1984) concluded that a serine proteinase was not involved (unlike the clotting of vertebrate plasma) but that a haemocyte-derived cysteine proteinase was important in stabilizing the clot. Whether this was the same as the haemocyte coagulogen could not be determined for technical reasons. Stability of the clot depends on formation of crosslinks between the two molecules; such crosslinking is inhibited by hydroxylamine and hydrazine, both of which are known to be inhibitors of crosslinking in collagen (Bohn and Saks, 1986). It would thus seem that, in some species at least, the plasma requires haemocyte coagulogen for a stable clot to be formed. This biochemical evidence fits with earlier histological and ultrastructural studies in which lysis or degranulation of COs or GRs has been shown to produce a localized coagulum (reviewed by Gregoire and Goffinet, 1979; Rowley and Ratcliffe, 1981). With the use of modern haemocyte separation techniques it should now be possible to state unequivocally which haemocyte class is involved in the reaction. However, there still remains the problem of explaining observations on species in which haemocytes are not found in association with plasma clots (Day and Bennetts, 1953, for Aedes aegypti; Locke, 1966, for RhodniusproLxus); are these always the same species in which “humoral melanization” (Section 4.3) occurs? A rather different problem, in terms of our attempts to identify the stimulus for haemocyte involvement in wound-healing, is to determine how clotting is initiated; in other words, what stimulates the CO or G R to release coagulogen? The clot is local, so release must be local-and it is unrealistic to assume that a sufficient number of coagulogen-containing cells are damaged in the wounding process and drop out of circulation at the wound-site. Instead, the possibility of either local alterations of tissue surfaces-either by mechanical damage, enzyme release from the wound or interaction of wounded surfaces with plasma components-or the release of wound factors affecting the haemocytes, needs to be considered again. In several species of Endopterygota, the production of antibacterial proteins is stimulated by wounding (Trenczek and Faye, 1988) and, more dramati-

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cally, by injection of non-pathogenic bacteria. In Manduca larvae, a large number of new proteins appear in the haemolymph (Hurlbert et al., 1985) and one of these, M 13, has been found recently to provoke coagulation of PLs and GRs in vitro, such that long filaments of coagulum were produced (Minnick et al., 1986). Whether this is also produced locally during wounding and sufficiently rapidly to initiate clot formation is obviously an interesting problem. It is also particularly interesting that plasma lipophorin of Locusta and Periplaneta has been found to aggregate spontaneously in haemolymph that is collected through a wound without the use of anticoagulants (Chino et al., 1987), and that native lipophorin can be induced to aggregate in vitro by the action of phenoloxidase (Chino, personal communication). In addition, denatured lipophorin has been shown to activate the haemolymph prophenoloxidase (PPO) system of Bombyx (Ashida et al., 1983). In conclusion, identification of the stimulus that initiates clotting is particularly important, since this is also likely to be the stimulus that initiates the haemocytic response. 8.3.2 The nature of wound,factors In the older literature on mammalian cells, the term wound or injury factor was used to indicate release of unspecified molecules, from damaged epithelia, that were thought to induce changes in adjacent epithelial cells in order that they might migrate and heal the wound (Banda et al., 1982). The term has also been used in work with insects and, although vague, is useful in that it identifies a concept if not a molecule. Cherbas (1973) wounded pupae of H . cecropia, Philosamia Cynthia and A . polyphemus moths and observed the effect of wounding on the spreading behaviour of PLs in vitro. On the basis of her observations (described in greater detail in Section 8.3.3), she suggested that both the wounded epidermis and the plasma from wounded animals contained an injury factor, that she termed “haemokinin”. Examination of her experimental methods shows that the origin of haemokinin is not clear; “epidermal fragments” and “homogenized epidermis” included cuticle, and “plasma” was obtained by centrifuging undiluted haemolymph at high g forces or by filtration through a Millipore filter, thus causing considerable haemocyte lysis. Nevertheless, her observations on changes in haemocyte behaviour are very pertinent (Section 8.3.3.2). During an investigation of injury metabolism in H . cecropia pupae, Harvey and Williams (1961) found, using Warburg manometry, that the oxygen consumption of a wounded pupa increased in relation to the circumference of the cuticular wound and, on the basis of this and other evidence, they suggested that a wound factor was released from the margin; again, the cuticular or epidermal origin of the factor cannot be differentiated.

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The cuticle, in addition to structural proteins and chitin, also contains the constitutive molecules of the epidermal pore canals and their associated hydrocarbons, and a variety of enzymes such as peroxidase (Locke, 1969), laccases and phenoloxidase (Brunet, 1980; Andersen, 1985). The distinction between haemolymph and cuticular prophenoloxidase (PPO), and their activation sequences, has been discussed in Section 6; suffice it to mention here that the proenzyme is activated by a serine proteinase in both cases (Dohke, 1973; Anderson et al., 1986; Aspan et al., 1986). Activation of the haemolymph PPO system produces factors which affect haemocyte adhesion (Johansson and Soderhall, 1988), locomotion (Takle and Lackie, 1986; Huxham and Lackie, 1988) and exocytosis (Ratcliffe et al., 1984), and it is obviously very important to determine whether such factors might also be produced during activation of the cuticular PPO sequence. Recent work by Natori and his colleagues has shown that production of the galactose-binding lectin found in the haemolymph of Sarcophaga peregrina larvae is induced when the larval cuticle is punctured with a syringe needle (Komano et al., 1980, 1983); the lectin gene is activated transiently after puncture and to a greater extent if sheep erythrocytes-which are cleared by the immune system (Komano and Natori, 1985)-are also injected. Lectin is produced by the fat-body, but whether or not it originates from other tissues as well has not so far been tested. One interesting feature is that the purified lectin activates murine and human macrophages and neutrophils in vitro to produce cytotoxic factors (Itoh et al., 1984; Tamura and Natori, 1984). Whether this Sarcophaga lectin can, like Manduca protein M 13 (Section 8.3. I), stimulate behavioural changes in haemocytes has not been investigated, and it is debatable at present whether it falls into the category of wound factor. 8.3.3 Changes in haemocyte adhesion und locomotion after wounding 8.3.3.1 The unstimulated haemocyte Arnold (1959a, b; 1961) was the first to observe and analyse the behaviour of haemocytes in vivo by photographic techniques. He immobilized adult Blaberus giganteus and observed the passive circulation and the active locomotory behaviour of haemocytes within the wing veins. In these unstimulated (although undoubtedly stressed) animals, some differences in PL behaviour were observed with age of the insect. In young adults, the majority of the PLs were flat or spindle-shaped, circulated freely and rarely adhered to obstructions within the veins; in older animals, in which wing veins were often occluded and circulation was slow, haemocytes adhered more frequently and moved over the substratum in an “amoeboid” manner. Speeds of up to 5 pm/

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min at 25°C were observed (Arnold, 1959b). Permanent adhesions sometimes occurred, but in other cases the cell might move over the surface of the vein for varying periods of time before returning to the circulation (Arnold, 1959a). Arnold could only distinguish between PRs and spindle-shaped or round granular cells, all of which he termed PLs, adding (1959b) that the changes in cell form associated with independent movement probably account for some of the discrepancies in haemocyte classification. Similarly, the GR and PL morphologies of Periplaneta haemocytes have been found to be interchangeable, depending on the locomotory state of the cell in vitro (Takle and Lackie, 1986). Obviously, collection of haemolymph itself involves production of a wound and the layering of cells onto any substratum in vitro presents them with a non-self surface. Despite this, observations of haemocyte locomotion and cellular interactions are valuable and informative. In vitro, cells of the GR/CO class usually degranulate and/or lyse; stability can be maintained for longer periods if haemocytes are collected in anticoagulant, washed and resuspended in an appropriate medium with foetal calf serum as stabilizer. Under these conditions, COs of Schistocerca and Periplaneta tend to remain rounded, only slightly adhesive and non-motile, whereas the majority of the PLs adhere and spread (Huxham and Lackie, 1988). In diapausing saturniid pupae, if the undiluted haemolymph is collected in such a way as to minimize contact with the cut edge of the wound (Cherbas’ Method 11, 1973) and mixed with phenylthiourea to inhibit melanization, the PLs adhere but remain round or spindle-shaped, and do not clump (Fig. 5). This is somewhat surprising in view of observations on PLs from other species, but is an important observation since the humoral immune response, at least, is switched off in these diapausing pupae (Boman, 1986, for review); the haemocytes, too, are thus probably in a relatively unactivated state, a condition almost impossible to achieve in larval or adult insects. In all other systems examined in vitro, whether using diluted haemolymph or anticoagulant-washed haemocytes, PLs adhere and spread, and exhibit ruffling lamellopodia. According to Davies and Preston (1985, 1987), working with lepidopteran larvae, all PLs became spread in vitro, and locomotory activity was only stimulated by cellkell contact during spreading-the cells became polarized, with anterior lamellae, and moved away from each other (“evasive translocation”). In these experiments, haemolymph was allowed to drip or was squeezed from a cuticular puncture, into medium and thus might have come into contact with putative wound factors. However, the relationship between this type of in vitro behaviour and that which might be expected in vivo is difficult to interpret since plasmatocytes which aggregate at a wound clearly d o not avoid each other; some other stimulus must be required. Spread Periplaneta (Takle, 1986; Takle and Lackie, 1986) or Schistocerca

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Fig. 5 Haemocytes from “wounded” (a and c) and unwounded (b and d) pupae of Hyakiphora cecropia The “wounded” pupa was punctured 3 days before the haemolymph was sampled. (a, b) Cells immediately after placing on the microscope slide. Note how the haemocytes from the wounded pupa (a) are aggregated. (c, d) The same cells 90 rnin later. Those from the unwounded pupa (d) remain rounded, whereas the

aggregated cells from the wounded pupa (c) have spread extensively. (Photographs courtesy of Dr T. Trenczek.)

(Huxham and Lackie, 1988) PLs are either non-motile or only move slowly and for short distances. In contrast, the small subpopulation of cockroach PLs that move actively over protein-coated glass (medium containing foetal calf serum, bovine serum albumen, o r dilute haemolymph) tend to be rounded and phase-bright, with a small leading lamellopodium. Under these conditions, cells moved at approximately 1 ,um/min a t 22°C (Takle and Lackie, 1986). By manipulating the substratum, in such a way as to alter haemocyte adhesion, the speed of the locomotory cells can also be altered. Schistocercu haemocytes, in medium containing foetal calf serum, move only slowly on glass but can attain speeds of 5 prn/min at 28OC on siliconized glass in the same medium (Huxham and Lackie, 1988). Similarly, Periplunetu cells

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adhere and spread less on polyHEMA-coated glass (Takle, 1986; Lackie, 1986b) and move 3 times faster than on protein-coated glass (M. Huxham and A. Lackie, unpublished information). It can thus be seen that locomotion of insect haemocytes can be affected by altering cell-substratum adhesion. This is a useful experimental tool since, by choosing suitable substrata, it becomes possible to maximize the differences in behaviour between stimulated and unstimulated cells, permitting greater accuracy in quantitative assays. 8.3.3.2 Haemocytes from wounded insects If an insect is wounded by removing part of its integument, and the hole is covered by a plastic or glass coverslip, the wound-healing response of the haemocytes can be observed in vivo. Thus, Clark and Harvey (1965) sealed the wounded facial area of diapausing H . cecropia pupae onto a plastic observation chamber, and surrounded the pupae with a physiological salt solution containing antibiotic and phenylthiourea. Immediately after the chamber was set up, several sizes and shapes of PLs could be seen circulating in the blood, some of which were small and rounded, occasionally occurring in clumps. Other cells were larger and spindle-shaped and, with time, increasing numbers of very large, hyaline PLs were observed. As haemocytes contacted the coverslip, they adhered and flattened, and showed some apparently random migratory behaviour. It is not easy to judge the rate at which haemocytes accumulated, but coverslips became completely coated within 2-10 days. Walters (1970), using a similar system, found that plastic coverslips were completely coated by 48 h. The impression is gained, from Clark and Harvey’s description ( 1 9 6 9 , that haemocytes often arrived at the wound in an activated state, as judged by their clumping within the circulation and intracellular vacuolation. Also using saturniid pupae, and having observed the behaviour of unstimulated PLs, Cherbas (1973) was able to show that PLs in haemolymph that was squeezed out through a slit in the cuticle (=Method 1) rapidly adhered and spread in vitro (Fig. 5 ) . Since PLs collected by the two different methods from the same pupa, at the same time, displayed the two different types of behaviour, it is clear that the behavioural change was due to the method of collection rather than differential sampling of different subpopulations. In one experiment, cells collected by method I1 were induced to spread when co-cultured with pieces of wing cuticle and epidermis. Cherbas thus postulated the release of an injury factor (haemokinin) from the cut tegument and suggested-although unfortunately without much evidence-that it might have some effect on activation of prophenoloxidase. When Walters (1970) collected pupal haemolymph through an integumental slit, he also

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found that PLs adhered and spread, and were able to move at speeds of up to 2 pm/min in haemolymph on glass; intercellular connections were continuously “established, ruptured and renewed”. GRs remained spherical, although adherent by means of fine filopodia, and did not contribute to formation of the haemocyte network. The locomotion of Periplaneta cells in vitro was examined, using time-lapse cinephotography, by Baerwald and Boush (1970). These authors injected cockroaches with Ringer solution 6 5 h before collecting haemolymph from a severed antenna and thus they would have been using haemolymph from a wounded animal. The PL class comprised spread, sedentary cells and cells that were more rounded, phase-bright and with small leading lamellae; such cells moved at speeds of 0.5-3 pm/min in diluted haemolymph at room temperature. Unfortunately, no numerical data were presented to indicate the proportion of these motile cells. In our own laboratory, the heat-inactivated PPO system from a haemocyte lysate (in which a small amount of PPO is non-specifically activated; Soderhall et af., 1986) stimulates a slightly higher proportion of PLs to assume the rounded, “fast-locomotor’’ morphology and chemokinetic behaviour (Takle and Lackie, 1986). Although most work on the effect of the activated PPO system on cell behaviour in vitro has utilized PPO derived from glucan- or lipopolysaccharide-activated haemocyte lysate supernatants (see Sections 6 and 10) and is thus not relevant in the context of “non-specific” responses, Johansson and Soderhall(l988) have found that calcium-activated lysate supernatant from crayfish haemocytes promotes adhesion and spreading of crayfish granular cells in vitro. The relationship between adhesive and chemokinetic factors for arthropod haemocytes is at present unknown. 8.3.3.3 Nodule formation in response to wounding Changes in the haemocyte count in response to wounding were discussed in Section 7.3; the low resting count in lepidopteran pupae dramatically increased, but in most larval and adult insects the count showed a transient decrease. That this might be due to a process similar to leucocyte margination in mammals, whereby leucocytes exhibit a transient increase in adhesion to the lining of the capillaries, is an intriguing possibility, which it should be possible to test by filming haemocyte behaviour in wing veins in vivo (Section 8.3.3.1). The.loss of a small proportion of the haemocytes from the circulation might also be due to changes in haemocyte-haemocyte adhesion and the formation of small aggregates or nodules. Wounding of adult Schistocerca or Peripfaneta by the injection of a balanced salt solution stimulates a transient decrease in cell count (M. Carr, personal communication; Gunnarsson,

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1988a), and a behavioural change resulting in the formation of a small number of haemocytic nodules (Gunnarsson and Lackie, 1985; Lackie, 1986a). This effect persists in that a greater proportion of haemocytes from saline-injected locusts are capable of phagocytosing fluorescent latex beads as late as 24 h after the saline injection (Gunnarsson, 1987); whether this represents an activation of pre-existent circulating haemocytes, or the entry into the haemolymph of a previously sessile subpopulation of phagocytes, is not yet known. Interestingly, if locusts are wounded by saline injection 24 h after an injection of a p1,3-glucan solution, a large number of new nodules are formed, despite the continuing depression of the haemocyte count (Gunnarsson, 1988a). This nodule-stimulating effect of a saline injection has also been observed in Periplanera, 24 h after the cockroaches had been injected with grafts of Blaberus tissue (A. Lackie and B. Dularay, unpublished information). Since it is unlikely that a small volume of balanced salt solution of correct pH and osmolarity could itself stimulate nodule formation, it is possible that the cells, activated by the prior stimulus of tissue implantation or glucan injection, had become more sensitive to factors derived during puncture of the cuticle and epidermis. Although haemocyte lysate supernatant, containing the glucan-activated PPO system, has been shown to cause degranulation of crustacean (Soderhall et al., 1986) and lepidopteran (Leonard et al., 1985a) haemocytes, and G R degranulation has been shown to be a central event in nodule formation against bacteria by lepidopteran larvae in vivo (Ratcliffe and Gagen, 1977), the putative role of the haemolymph or cuticular PPO system in stimulating changes in haemocyte behaviour in response to wounds has not been determined. For such investigations, non-specific activation of the system-by denatured lipophorin, for example (Ashida et al., 1983)-would be required; the role of any plasma components of the PPO system requires careful attention. 8.4

LATER EVENTS IN WOUND-HEALING

The multilayered aggregate of haemocytes at the site of cuticular wounds appears to provide a substratum over and within which epidermal cells can subsequently migrate. In wounded Galleria larvae, epidermal cells started to migrate across the mixed plug of coagulated haemolymph and necrotic fatbody cells within 12h and formed a confluent monolayer by 24h. At all times, the epidermal cells appeared to be closely associated with the aggregated haemocytes (Rowley and Ratcliffe, 1978). Similar migration of epidermal cells over a haemocyte “deposit” beneath cuticular wounds was

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observed by Williams (1946) in pupae of Platysamiu cecropia. The epidermis of nymphal Periplaneta grows across the layer of haemocytes adherent to rejected cuticular xenografts (equivalent to a large cuticular wound) and forms a continuous sheet of epithelium (Lackie, 1983b). Bohn (1975, 1977b) has suggested, on the basis of the results of in vitro culture of Leucophaea tissue, that haemocytes produce “conditioning factors” that facilitate migration of epidermal cells across glass coverslips; in the absence of this factor, which was apparently not proteinaceous, outgrowth from epidermal explants occurred only slowly. Late stages in the healing of wounded tissues do not appear to have been examined in detail. During neural repair in Periplaneta, haemocytes adhere to and apparently invade the perineurium (Treherne et al., 1987), and it has been suggested, on the basis of several pieces of experimental evidence, that the granule-containing cells subsequently involved in glial repair may derive from haemocytes (Smith et al., 1986; Howes et al., 1987). It seems unlikely that haemocytes could actively contribute to epithelial regeneration, so the significance of these interesting results in other aspects of wound-healing or encapsulation is unclear. Haemocytes trapped between the epidermis and the clot are usually melanized and necrotic, whereas the intact PLs adherent beneath the newly formed epidermis had disappeared by 72 h after wounding in Galleria (Rowley and Ratcliffe, 1978), presumably losing their adhesions and returning to the circulation. In these circumstances, the haemocytes must presumably receive a signal that they are no longer required, or fail to receive a signal for continued adhesion.

8.5

SUMMARY: THE EFFECTS OF WOUNDING ON HAEMOCYTE BEHAVIOUR

The clotting of haemolymph at the site of a wound involves crosslinking plasma coagulogen ( = lipophorin) with haemocyte-derived coagulogen; since the latter must be released locally, the stimulus for release must also be localized. Wounding induces a transient haemocytopaenia; whether this is due to a phenomenon similar to margination, whereby haemocytes show transient adhesion to the basement membrane/connective tissue that lines all haemocoelic surfaces, is as yet unknown. The accumulation of haemocytes at the wound-site could be accounted for by induced changes in their adhesive and locomotory behaviour. Chemokinetic behaviour alone could explain haemocyte accumulation, although chemotaxis would improve the efficiency of the response. Whether factors that stimulate release of haemocyte coagulogen and that cause alteration in haemocyte adhesion originate from the cuticle and/or

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tissue itself, or whether the factors derive from local alteration or activation of soluble plasma molecules in contact with altered self, remains to be ascertained.

9

Recognition of non-self

In the previous section the accumulation of haemocytes at a wounded surface was discussed in terms of the effect of “wound factors” on haemocyte behaviour. Although this accumulation can be explained in terms of changed adhesivity, such behaviour is insufficient to explain the accumulation of haemocytes around abiotic objects introduced into the haemocoel. Since wound factors cannot be released from such a surface, it becomes necessary to invoke haemocytic recognition of non-self, due either to differences inherent in the non-self surface, or to adsorption and alteration of plasma components upon that surface. All insect tissues that lie within the haemocoel are coated with a basement membrane, which is usually thicker and more complex than epithelialderived basement membrane and should perhaps be referred to more correctly as a connective-tissue layer. However, since connective tissue is also found as a support within tissues, “basement membrane” will be used here. Cuticular wounds and internal lesions usually also damage the basement membrane, so the molecular characteristics of this layer that could describe itself as “self’ will be discussed here.

9.1 THE BASEMENT MEMBRANE 9.1.1 Structure The last decade has seen an explosion of interest in the biochemistry and molecular architecture of mammalian basement membranes, particular attention being focused on the glycoproteins and glycosaminoglycans (GAGS), but this interest has only recently been extended to insects. Ashhurst (1982a, 1985) has reviewed information on the structure of insect basement membranes and connective tissues, and Fessler (Fessler et al., 1984) provides an overview of his group’s work on the collagen and glycoproteins of Drosophila basement membrane. The current view of the vertebrate basement membrane is that it comprises a network of non-fibrillar (type IV) collagen, to which proteoglycans and glycoproteins are attached (Timpl et al., 1981). This layer, by reason of vari-

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ations in pore size and charge, exerts a controlling influence on the exchange of metabolites between tissue and fluid. In insects, the presence of type IV collagen has been suggested by electronmicroscopy (for example, de Biasi and Piloto, 1976) and, more recently, by biochemical (Ashhurst and Bailey, 1980) and molecular and immunochemical (Natzle et al., 1982; Fessler et al., 1984; Knibiehler et al., 1987) techniques. At present, there is not much information about the types of glycoproteins in insect basement membranes. Cells of the Drosophila KC line produce laminin, a high molecular weight glycoprotein that may assist in binding the collagenous matrix to the cell surface; antibody to this laminin stains the basement membranes of Drosophila larvae (Fessler et ul., 1984). An entactinlike sulphated glycoprotein is also released by the KC cell line (Fessler et al., 1984). Fibronectin is found in the plasma and on cell surfaces in mammals, and has several important biological activities which include cell adhesion and binding to collagen and certain GAGs (reviewed by Yamada, 1983). Radioimmunoassay of detergent-solubilized homogenates of a variety of invertebrates has shown fibronectin to be present in crustaceans-insects were not tested (Akiyama and Johnson, 1983). However, no fibronectin-like proteins have yet been identified in Drosophila (Fessler et al., 1984; Naidet et af., 1987) or associated with four insect cell lines (Goldstein and McIntosh, 1980). A large sulphated proteoglycan is released by KC cells in culture and is also found in Drosophilu basement membranes (Fessler et al., 1984). The occurrence of glycosaminoglycans in insect basement membranes has long been suggested by histochemical studies using either Alcian blue, which binds to “acid mucopolysaccharides”, or the cationic dye, Ruthenium red; the histochemical techniques and evidence are reviewed by Ashhurst (1982a). Analysis of the neural lamellae of Periplaneta and Locusta using the sensitive Critical Electrolyte Concentration method for Alcian blue suggests the occurrence of poorly-sulphated GAGs like chondroitin and keratan sulphates (Ashhurst, 1984). The decrease in binding of Ruthenium red by Galleria neural lamellae after hyaluronidase treatment suggested to Dybowska and Dutkowski (1977) that hyaluronic acid was present. Cassaro and Dietrich (1977) purified sulphated GAGs from homogenates of whole Periplanetu and found that the preparation ran as one band on agarose gels, with a migration rate intermediate between heparan sulphate and chondroitin sulphate B. A single band displaying these unusual characteristics has also been found in basement membrane prepared from Periplaneta midgut (V. 0’Brien, A. Lackie and J. Kusel, unpublished information). A number of different functions have been ascribed to the various GAGs found in mammalian tissues; for example, heparan sulphate in glomerular basement membranes

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regulates the uptake of charged molecules (Kanwar et al., 1980); heparin regulates blood clotting and possibly the alternate complement pathway, and the sulphated proteoglycan released by natural killer cells may be involved in the mechanism of cytotoxicity (MacDermott et al., 1985); it is fortunate that interest in insect GAGs is increasing. The carbohydrates, usually associated with glycoproteins and proteoglycans, of insect basement membranes have been investigated mainly by the use of lectins; thus, peanut agglutinin (galactose-specific) binds specifically to the basement membrane of Periplaneta midgut but not Malpighian tubules or ovaries (R. Martin and A. Lackie, unpublished observations); wheat germ agglutinin (N-acetylglucosamine-specific)binds to Drosophila fat-body (R. Rizki et al., 1983; T. Rizki and Rizki, 1984). Perrone and colleagues (1986), using a panel of different lectins, found that the binding patterns for Aedes aegypti salivary gland varied with the lobe of the gland examined and suggested that malarial sporozoites, which penetrate only certain parts of the gland, might have specific receptors for the carbohydrates of the appropriate regions. The occurrence of sialic acid is problematical. Analysis of homogenates of whole insects (Warren, 1963) and of cell membranes from insect cell lines (Butters and Hughes, 1981) failed to demonstrate the presence of sialic acid. In contrast, the binding of the Ruthenium red to neural lamella is decreased after neuraminidase treatment (Dybowska and Dutkowski, 1977); it should be noted, however, that most commercial neuraminidases often contain proteinase. Finally, basement membranes have a net negative charge, generally illustrated by the binding of cationized ferritin (Perrone et al., 1986) or Ruthenium red (Dybowska and Dutkowski, 1977), and usually considered to be due to the presence of acidic sugars and sulphated GAGs. 9.1.2 Haemocyte involvement in the formation or repair of basement membranes Whether the haemocytes contribute to the repair or formation of the basement membrane that lines the haemocoel is a controversial subject (see Wigglesworth, 1979, and Ashhurst, 1979, for opposing arguments). The controversy has largely arisen from observations that (1) haemocytes are associated with the basement membrane during wound-healing and metamorphosis, and (2) haemocytes have been shown by histological techniques to contain acid mucopolysaccharide (Beaulaton, 1968; Costin, 1975). Spherule cells of Lepidoptera are particularly rich in this type of molecule, which is apparently a sulphated GAG-like polymer (Ashhurst, 1982b; Cook et al., 1985); however, the function of the SPs remains uncertain. Recently, Knibiehler and colleagues (1987) have shown that a DNA probe

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specific for Drosophilu basement membrane type IV collagen hybridizes with mRNA in haemocytes in the lymph glands and the caudal haemocoel, providing strong circumstantial evidence for a link between haemocytes and basement membranes. Immunogold labelling of monoclonal antibody raised against a glycoprotein-rich fraction from locust muscle sheaths stained the basement membranes of locust embryos and granules within haemocytes; haemocytes were found in close apposition to basement membranes and the investigators (Ball et ul., 1987) suggested this reflected a haemocytic contribution to basement membranes. The resolution of the controversy is not merely an intellectual exercise-it will provide important clues about haemocyte recognition since, if haemocytes are involved in the formation and repair of basement membranes, they must also be capable of recognizing alterations in that layer.

9.2

DO HAEMOCYTES RECOGNIZE INHERENT ALTERATIONS IN THE BASEMENT

MEMBRANE?

Most of our knowledge about the haemocyte response to basement membranes comes from work with tissue transplants, either in the course of studies on insect physiology or development, or from studies on insect transplantation immunology. Allogeneic implants or cuticular grafts are not recognized as non-self nor, in certain restricted species combinations (such as between closely related species, or where the recipient species has a low acuity of immunorecognition) are xenografts (reviewed by Lackie, 1986b). Thus, haemocytes do not adhere to and encapsulate the intact basement membrane covering these tissues; only when the surface is physically or chemically modified does encapsulation ensue (Salt, 1961; Scott, 1971; R. Rizki and Rizki, 1980). Also, haemocytes do adhere to the cut surfaces of the transplants, where both tissue and basement membrane have been damaged (Salt, 1970; Lackie, 1986b). Further interesting evidence in favour of haemocytic recognition of abnormalities in the basement membrane comes from the work of the Rizkis on various Drosophilu tumour mutants. In the Tu-SZ" temperature-sensitive mutant, for example, larvae develop melanotic tumours at 25 C but not at 18°C. At the higher temperature, the basement membrane overlying the atypical cells of the caudal fat-body becomes abnormal, and the lamellocytes adhere to and encapsulate this tissue (R. Rizki el ul., 1983; T. Rizki and Rizki, 1984). Haemocytes also invade the apparently intact basement membrane surrounding muscles of Culliphora, autolysing at metamorphosis (Crossley, 1968). and the intact neural lamella surrounding chemically damaged neuroglia in Periplunetu (Treherne et al., 1987). However, subtle differences in

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molecular architecture or charge of the basement membranes will not be seen by electron microscopy and are likely to arise as the result of alterations to the enclosed cells or, as in the case of the work on Peripfaneta, the handling technique. Similarly, since the cuticle is intimately associated with the epidermal cells, damage resulting from intracuticular growth of the fungal pathogen, Metarhizium anisopliae, is very likely to affect the epithelial cells of Schistocerca and thus the basement membrane, resulting in the observed accumulation of haemocytes on the haemocoelic surface of the basement membrane beneath the site of fungal invasion (Gunnarsson, 1988b). Taking the hypothesis that alterations in basement membrane are recognized and responded to by the haemocytes, it then becomes necessary to consider which of its properties allows the basement membrane to be distinguished as self or non-self. It has been argued elsewhere, with reference to transplantation immunology (Lackie, 1986b), that the characteristics that describe self lie within a certain .range, the extent of which varies for a species and an individual insect; thus Schistocerca fails to recognize a wide variety of interspecific grafts as non-self, whereas the discriminatory ability of Peripfaneta is much higher, presumably because the thresholds beyond which recognition of foreign-ness occurs are much closer together than in Schistocerca. The properties of a basement membrane that describe it as self are most likely to be related to its charge and carbohydrate composition. The ability of haemocytes to recognize and respond to differences in these parameters is discussed below.

9.3

PHYSICAL AND BIOCHEMICAL PROPERTIES THAT DISTINGUISH SELF

FROM NON-SELF

9.3.1 Physicochemicalproperties That the net charge of foreign surfaces affects the extent to which they are encapsulated by haemocytes has been known for some time: Vinson ( 1974) in Heliothis larvae, Dunphy and Nolan (1 982) in Choristoneura larvae, and Lackie (1983a, 1986e) in adult Periplaneta and Schistocerca, have found that differently charged ion-exchange beads are encapsulated to different extents. Negatively charged beads are not encapsulated at all by the lepidopteran larvae in vivo, or in vitro (Walters and Williams, 1967; Davies and Vinson, 1986). In recent work investigating the encapsulation of differently charged beads by separated classes of crayfish haemocyte, it has been shown that cells of the semigranular class adhered more readily to neutral than to positive or negative Sepharose (Persson et a f . , 1987).

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In Periplaneta and Schistocerca, the magnitude of the in vivo response appears to correspond rather well to the adhesive behaviour of the haemocytes in vitro; using polystyrene substrata with a range of negative charge and hydrophobicity, it was found that a greater proportion of anticoagulantwashed (i.e. haemolymph-free) haemocytes from Periplaneta adhered as the substratum negativity increased and hydrophobicity decreased. In vivo, polystyrene beads were encapsulated more thickly the greater their negativity, and CM-Sepharose beads provoked thicker capsules than neutral Sepharose. In contrast, only a low proportion of Schistocerca haemocytes adhered to the polystyrene surfaces in vitro, irrespective of their charge, and negatively charged CM-Sepharose or polystyrene beads were not encapsulated at all (Lackie, 1983a). By injecting agarose beads with different negativities into Schistocerca, it was found that the encapsulation response was not switched on until the surface charge was close to neutrality (Lackie, 1986~). The influence of substratum surface charge on haemocyte adhesion and encapsulation irz vivo might be due either to its effect on adsorption of soluble intermediaries whose presence is necessary for adhesion to occur (Section 9.4) or to a direct effect on cell adhesion itself, the cells being attracted or repelled according to their own surface charge. According to the latter hypothesis, and with regard to the differences in their adhesive behaviour towards charged substrata, the haemocytes of Periplaneta and Schistocerca might themselves be expected to have rather different surface charges. By using cell electrophoresis, and by measuring the thickness of bound cationized ferritin, it was found that locust cells were significantly more negatively charged than those of the cockroach (Takle and Lackie, 1985). On the basis of theories of electrostatic repulsion and attraction in cell-substratum adhesion (Curtis, 1972; Pethica, 1980), it could be surmised that contact between locust haemocytes and the predominantly negatively charged surfaces of experimental biotic implants or invading parasites would present some problems. Certainly, as discussed in greater detail elsewhere, the locust acts as host to a wide range of parasitic species (Lackie, 1986d) and tissue transplants (Lackie, 1986b). However, the net surface charge of haemocytes and parasites cannot readily account for the effective interaction between Schistocerca haemocytes and the protozoon, Trypanosoma rangeli (Takle and Lackie, 1988), nor is it likely to provide the complete explanation for the greater subtlety of the recognition mechanism in Periplaneta. Other mechanisms must be invoked (Section 9.4). The relative hydrophobicity of the surfaces of a leucocyte and a foreign particle affect whether cell-particle interaction occurs (Edebo et al., 1980; Magnusson et a f . , 1980). The hydrophobicity of a foreign surface is also an important factor in haemocyte adhesion in vitro (Lackie, 1983a; Takle, 1986) and in vivo. In the latter case, thick capsules are formed around paraffin wax

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(Salt, 1970), and glass beads coated with hydroxyl-treated polyvinyl acetate, a substratum presenting a very hydrophobic surface, are more thickly encapsulated than are uncoated glass beads or beads coated with untreated polyvinyl acetate (Takle, 1986; Lackie, 1986b). It appears, therefore, that haemocytic recognition of and response to a surface is affected by the physical properties, such as charge and hydrophobicity, of that surface. 9.3.2 Carbohydrate composition as a recognition signal Arthropod haemocytes respond to fungal /?1,3-glucansand bacterial lipopolysaccharides or peptidoglycans by releasing a mixture of molecules, including phenoloxidase (Section 6), and there is some preliminary evidence to suggest that receptors specific for PI ,3-glucans are associated with Bombyx haemocyte membranes (Ashida et al., 1986). The haemolymph of insects contains lectins (Section 5 ) that, in some cases, appear also to be associated with the haemocytes (Amirante and Mazzalai, 1978; Lackie and Vasta, 1988). It is, therefore, a tempting hypothesis that differences between self and damaged self/non-self are recognized by differences in carbohydrate composition or concentration. That the extent of the haemocytic encapsulation response in vivo is influenced by the carbohydrate composition of a foreign surface has been shown for Periplaneta by Lackie and Vasta (1988). Sepharose beads conjugated to galactose-rich glycoproteins such as porcine stomach mucin (PSM), desialized PSM, desialized bovine submaxillary mucin (BSM) and desialized fetuin provoke significantly thicker capsules than d o beads conjugated to sialic acid-rich glycoproteins such as BSM or fetuin, or to the protein bovine serum albumen. The differences in encapsulation between sialo- and asialoglycoproteins were not due merely to differences in charge, since the hierarchy of the response was in the opposite direction from that expected on the basis of charge alone. The major serum lectin of Periplaneta, which is a galactose-specific molecule with subunits of molecular weight of approximately 30,000 daltons ( G . Vasta and A. Lackie, unpublished information)-and which, incidentally, binds only poorly to crosslinked Sepharose-has also been shown by immunofluorescence to be associated with the haemocytes (Lackie and Vasta, 1988) and it is at least possible that it may influence immunorecognition of the glycoprotein-conjugated beads. Within an individual insect, however, the carbohydrate composition of the haemocoelic basement membrane is not constant, different tissue surfaces showing different lectin-binding affinities (Section 9.1.1) and, if insects really do lack sialic acid, how are we to explain the observations that most insect lectins so far purified are galactose-specific (Section 5.2)?

HAEMOCYTE BEHAVIOUR

9.4

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MEDIATION OF RECOGNITION BY PLASMA COMPONENTS

In the previous section, discussion centred on haemocytic recognition of intrinsic differences between self and non-self/damaged self; another possibility is that soluble molecules might become bound to surfaces that are non-self or damaged self and these molecules, which might be denatured, stabilized or activated on transition from the soluble to the solid phase, might mediate recognition. A miscellany of observations will be presented in this section. Some phenomena may be related, some may prove to be irrelevant, but it is hoped that they will at least provide basis for argument! In an earlier discussion it was shown that coagulation required crosslinking of lipophorin and haemocyte coagulogen; denatured lipophorin activated the haemolymph prophenoloxidase system (Section 8.3.1). Since lipophorin is present at such high concentration in the haemolymph (50% of total plasma protein in Peripfuneta, Chino, 1985), it is plausible that it might adsorb to damaged self or to foreign surfaces entering the haemocoel and, in doing so, present an altered configuration that would then be recognized by the haemocytes and would stimulate the release of coagulogen. It can be assumed that differences in charge or hydrophobicity would affect this nonspecific adsorption. Lectins, if indeed they are found in the plasma and are not entirely cellassociated, may bind specifically to carbohydrates newly exposed during damage or newly introduced during invasion, and may opsonize the surface for haemocyte attachments; haemocyte-lectin interaction could itself be mediated through receptor-ligand interactions (Coombe et uf., 1984), as with Fc, receptors on mammalian neutrophils. The galactose-specific lectin induced in Surcophaga in response to injury or injection might possibly work in this manner, although it would need to be continuously present at low levels in order to ensure a rapid response. Lackie and Vasta’s results (1988) on haemocytic encapsulation of beads coated with galactose-rich glycoproteins could also be explained by postulating opsonic involvement of a plasma lectin, but this would not explain the observed nodule-promoting effect of injected glycoprotein solutions. In view of the differences in the carbohydrate composition of basement membranes overlying different tissues, it is unlikely that all haemocoelic surfaces would lack the carbohydrates for which the haemolymph lectins were specific. Nor can this particular problem be readily solved by postulating a low affinity of plasma lectins for “self’ carbohydrates (Coombe et ul., 1984; Renwrantz, 1986) or by postulating that the alterations in cell responsiveness observed after wounding cause alterations in the distribution of haemocyte surface ligands or receptors; neither of these postulates can overcome the possibility that intact self might also be encapsulated. In-

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stead, a battery of soluble lectins of different affinity and avidity would be necessary. Another possibility is that haemolymph lectins are not normally found in plasma at all but, as with the coagulation-promoting protein M 13 in Manduca (Minnick et al., 1986), are induced; coagulation must be locally confined, so the lectin must only attach to damaged self or non-self. Alternatively, Mosser and Edelson (1980), in a review of mechanisms for macrophage adherence and endocytosis, point out that “an individual particle can bind to a phagocyte by more than one mechanism, and that cooperation between binding mechanisms can cause quantitative and qualitative alterations in the physiology of phagocytosis”. This statement was based partly upon the work of Ellenberger and Nussenzweig (1977), who observed that occupation of receptors for Fc, (i.e. specific IgG, normally with a low affinity for macrophages) and C3b (“non-specific”, surface-bound opsonin) had a synergic effect on macrophage uptake of erythrocytes. The argument could possibly be extended, although without any experimental evidence as yet, to include interactions between specific and non-specific, soluble and surface-bound, factors in haemocytic reactions. An interesting line of enquiry is opening up with investigations into the role of the Arg-Gly-Asp (RGD) sequence of amino acids in cell adhesion. This triplet lies at the cell-binding site of vertebrate fibronectin-a molecule which attaches to collagen and to cell surfaces (Yamada, 1983)-and of adhesion-promoting molecules in E. coli and Dictyostelium (Ruoslahti and Pierschbacher, 1986). It has recently been shown that peptides containing the RGD sequence prevent gastrulation in Drosophila embryos; the specificity of the inhibitory activity suggests that this sequence may also be used by insect cells to mediate attachment phenomena (Naidet et al., 1987). The existence of fibronectin, or fibronectin-like molecules, in insects is at present uncertain (Section 9.1.1) but if a soluble molecule containing this RGD sequence were to be found in haemolymph, this would have exciting implications for the mediation of haemocyte attachment at the site of wounds. The prophenoloxidase-activationsystem has been referred to as a recognition system (Soderhall, 1982; Ratcliffe et al., 1984). However, the evidence available at present indicates that this description is incorrect-the observed phenomena, such as the release of putative opsonins by stimulated haemocytes (Leonard et al., 1985a) are secondary events, responses. For the system to provide recognition, plasma components or soluble molecules released during cuticular damage would need to be involved initially. Consideration of the mammalian alternative complement pathway provides an example: C3b-like activity is generated continuously in the plasma, at a low level, and is normally rapidly inactivated by factors I and H. However, the labile molecule is stabilized by adsorption to certain types of surface, such as those low in sialic acid content, and can then combine with factor B to form the C3-

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convertase complex of the alternate pathway, splitting more C3. This locally activates the complete sequence of the alternate pathway and, in doing so, produces factors such as C3b that opsonize the stabilizing surface for adhesion by neutrophils or macrophages (see Whaley, 1986, for review). The recent observations by Phipps et al. (1987) that Galleria haemolymph has C3-convertase activity similar to that of cobra venom factor may well be relevant here. Because of the difficulty in working with insect plasma and because we know so little about early steps in the PPO-activation sequence, we can only speculate upon the existence of soluble, continuously turning over, components. The role of soluble plasma factors in recognition deserves considerable attention in the future.

9.5 CAN WOUND-HEALING AND ENCAPSULATION BE ACCOUNTED FOR BY SIMILAR RECOGNITION MECHANISMS?

It has been argued that “recognition” of cuticular wounds need not occur; haemocyte aggregation and production of extracellular material can be explained by leakage of soluble wound factors that alter haemocyte adhesion and locomotory behaviour (Section 8.3.3). Haemocytic aggregations around wounds in nerve cords, midgut or Malpighian tubules require, on this basis, that similar wound factors are produced by these non-cuticular tissues. Although it can be argued that parasites entering the haemocoel become coated, during penetration, with wound factors that stimulate cell adhesion, this explanation does not account for haemocoelic encapsulation of objects that are injected directly into the haemocoel. In such cases, both recognition of difference and stimulation of adhesive behaviour are required. Since puncturing the cuticle is sufficient to stimulate a transient increase in haemocyte adhesivity (Section 8.3.3) and bring haemocytes into a state of readiness (Section 8.3.3.3), this may increase the efficiency of recognition and response (Section 1 I).

10 R e c r u i t m e n t and c e s s a t i o n

In an earlier section (4.3), two of the questions asked about encapsulation were: how are haemocytes recruited, and why does capsule formation cease? The same questions apply to the formation of nodules and of aggregates around wounds. Adherence of one layer of cells (referred to below as primary cells) will conceal the surface previously recognized as foreign or will reduce the outward diffusion of putative wound factors (Section 8.3.2), so it becomes

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necessary to think of other mechanisms to explain the adhesion of the second layer of cells. Three possible explanations suggest themselves: (1) that haemocytes recognize and respond to differences in each other; the “dorsal” surface of a primary adherent cell appears different because there has been redistribution of particular surface epitopes, charged molecules, etc. Random contact by another haemocyte would lead to recognition, adhesion, and slightly less modification of the secondary cell’s surface, and so on; (2) that adherent primary cells secrete adhesive materials so that other randomly contacting haemocytes either become physically trapped or adhere via specific or non-specific adhesions; or (3) that primary cells release “recruiting factors” that stimulate changes in adhesion (and locomotory behaviour) of the secondary cells.

Evidence in favour of the first suggestion is lacking, because the appropriate experiments do not seem to have been carried out and would, in any case, be difficult to interpret. Moreover, any transiently adhesive cell might be in danger of recruiting an aggregate. The second suggestion is favoured by Ratcliffe and co-workers, who have produced histological and ultrastructural evidence to show that GRs/COs release material that traps bacteria within nodules (Ratcliffe and Gagen, 1977), adheres to the surface of biotic and abiotic implants (Schmit and Ratcliffe, 1977, 1978) and coats GRs when they are stimulated in vitro (Leonard et al., 1985a). GRs/COs are the cells involved in plasma clotting, presumably because they contain the necessary coagulogen (Bohn, 1986; Section 8.3.1); GRs are also associated with phenoloxidase (Schmit et al., 1977) and with acid mucopolysaccharide (Chain and Anderson, 1983b; Anderson and Chain, 1986). Ratcliffe (1986) has suggested a model for haemocyte cooperation in insects in which activated GRs release the activated PPO system (Section 6), components of which coat the foreign surface so that PLs then adhere to and phagocytose or encapsulate the invader. Using separated classes of crustacean haemocytes, Soderhall and colleagues (1986) have shown that the exocytosis of granule contents from the PPO-containing semigranular and granular cells results, as might be expected, in release of several proteins into the medium; since the phagocytic uptake of bacteria by hyaline cells is enhanced when these so-called “sticky proteins” are released, it is thought that the proteins have an opsonic function. Also in Crustacea, activation of the full PPO-activation sequence (that is, including the serine proteinase(s)) in lysate supernatant from granular or semigranular cells, results in production of a glycoprotein that stimulates adhesion and spreading

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of granular cells in vitro (Johansson and Soderhall, 1988). Whether these proteins also play a role in cellkell adhesion is as yet unknown. With the development of in vitro encapsulation techniques (Ratner and Vinson, 1983), it is becoming possible to investigate cellkell interactions under more controlled conditions. Haemocytes aggregate in diluted haemolymph, but if a cotton loop is introduced into the culture vessel within 10 min of haemolymph dilution, cells adhere to it and form a capsule with a similar structure to that found in Heliothis in vivo. Encapsulation was inhibited by the addition of various compounds to the medium, including propranolol and trypsin; however, the trypsin effect was reversed if fresh haemocyte lysate but not serum was added, leading Ratner and Vinson (1983) to suggest that encapsulation-promoting factors (EPFs) might originate from the haemocytes. Propranolol treatment caused degranulation and “deterioration” of the GRs, and it may be relevant that both propranolol (Fisher and Brady, 1983) and trypsin (Soderhall and Smith, 1983) activate the PPO pathwayprior rather than simultaneous activation of the PPO pathway fails to enhance phagocytosis of bacteria by PLs in vitro (Leonard et al., 1985a). Subsequently, Davies et al. (1988) partially purified an EPF from plasma of Heliothis, and showed that it restored cohesive ability to the haemocytes. The data suggest EPF is a small (less than 3500mol. wt), heat-stable peptide. Since the method of plasma collection involved brief centrifugation of diluted haemolymph at 8000g,the authors conceded that EPF might also be derived from haemocytes. It is, of course, still too early to determine the exact origin of EPF, but the use of separated cells in these in vitro assays is likely to be informative. As an example, crayfish semigranular cells, which contain components of the PPO system, encapsulate a variety of objects in vitro, but hyaline cells d o not adhere to these preformed aggregates or to the uncoated object (Person et al., 1987), suggesting that the cell adhesion molecule derived from semigranular or granular cell lysate (Soderhall et al., 1986) is not involved in intercellular co-operation in this system. The third possible explanation for cell-cell adhesion in multicellular responses is that the primary cells release recruiting factors that alter adhesion and locomotory behaviour (chemotaxis, chemokinesis) of other haemocytes. Chain and Anderson’s (1983a) observations that the supernatant from in vitro haemocyte-bacteria interactions, when injected into Galleria, caused a marked depletion in the number of plasmatocytes, may have some relevance here. The authors postulated that the plasmatocyte-depletion factor (PDF) might stimulate increased adhesiveness in PLs. A change in haemocyte locomotory behaviour would be inappropriate for objects free in the haemocoel such as injected beads or larvae of helminths because the haemocytes are in suspension, but it would be an efficient means of ensuring that haemocytes aggregate at the site of invasion (Section 8.2). Investigations in our own

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(a)

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f-

Fig. 6 Tracks from a time-lapse cine film of cockroach haemocytes moving on glass in vitro (a) In cockroach haemocyte medium containing zymosan-activated haemocyte lysate supernatant (phenoloxidase activity equivalent to 0.007 units); (b) control, in cockroach haemocyte medium. Note smaller step lengths. The tracks for the 10 fastest cells in each test have been drawn. Each dot represents the position of the nucleus at 400-s intervals of real time, and the cell outline represents the position of each haemocyte at the start of filming. Bar = 50 pm. (From Takle and Lackie, 1986.)

laboratory have so far failed to find evidence for chemotactic behaviour of haemocytes in response to glucan-activated lysate supernatant or to known chemoattractants for mammalian neutrophils. This is disappointing, since the phenomenon of chemotaxis has intellectual appeal; the negative result may indicate either that the cells lack this behavioural attribute or that our techniques lack suitability. However, haemocytes of Periplaneta and of Schistocerca can be stimulated to show chemokinetic behaviour. If Periplaneta cells are incubated in medium with or without zymosan supernatant (Zs), approximately 20% of the PLs are motile. Addition of Zs-activated haemocyte-lysate supernatant results in mobilization of nearly all the PLs and an overall highly significant increase in speed and directional persistence (Fig. 6). The increases in speed

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and persistence are correlated for the individual cells in activated lysate supernatant; each cell thus scans a larger area in a shorter period of time and if this behaviour were to occur in vivo, it would increase the chance of a cell contacting a site of invasion or a wound (Takle and Lackie, 1986). In separated subpopulations of locust cells, chemokinetic activity in the presence of laminarin-activated haemocyte-lysate supernatants was confined to a small subset of heavily granulated, PO-rich plasmatocyte-like cells (band 5 cells) (Huxham and Lackie, 1988). Activated lysate supernatant prepared from this PO-rich subpopulation was also a strong inducer of chemokinesis in band 5 cells but not in agranular plasmatocytes from band 3 (M. Huxham and A. Lackie, unpublished observations). Although the role of band 5 cells in the cellular immune response is not yet known, it is clear that subpopulations within a particular class respond in different ways. There is thus strong circumstantial evidence that the chemokinins originate from the activated PPO sequence, but identification and purification of the molecule is now required. However, there is also evidence for the involvement of factors other than those from the PPO system in cell aggregation and recruitment. It was noted in Section 6 that bacterial lipopolysaccharide (LPS) does not activate the insect PPO system, but LPS does affect the behaviour of both the insect (Bronstein and Comer, 1984) and its haemocytes. Injected LPS induces nodule formation (Schwalbe and Boush, 1971; Gunnarsson and Lackie, 1985) and lysozyme release (Anderson and Cook, 1979), even if the viscous solution is centrifuged before injection. Incubation of haemocyte monolayers (PLs and GRs/COs) in the presence of LPS increases their phagocytic capability (Ratcliffe er al., 1984) and stimulates PLs of Periplaneta to round up and start moving more rapidly and with greater directional persistence (Takle, 1986). Thus LPS stimulates marked changes in haemocyte behaviour, such that both activation and recruitment appear to occur, presumably due to molecules released from stimulated cells. We have, then, evidence in favour of two possible, and not necessarily mutually exclusive, explanations to account for the early stages of recruitment. However, not all haemocytic aggregates are the same size or thickness: capsules around Sepharose beads in Periplaneta vary from 12 to 30 pm thick, depending on the bead’s charge (Lackie, 1983a); the thickness of the haemocyte aggregates beneath xenografts (Jones and Bell, 1982) or around xenogeneic implants (Lackie, 1979) depends on the phylogenetic relatedness of the donor species; and the size of nodules depends on the molecular stimulus (Gunnarsson and Lackie, 1985). The thickness of the completed response is a function of the number of cell layers (Jones and Bell, 1982; Lackie et al., 1985) and we may fairly safely assume that thick capsules represent recognition of a greater departure from “self’ characteristics, since grafts from phylogenetically distant species are more thickly encapsulated than those from

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closer relations (Bell, 1972; Jones and Bell, 1982; reviewed by Lackie, 1986d). Thus, the extent of recruitment varies according to the strength of the stimulus and could, for example, depend on the number of receptors occupied upon interaction with the foreign surface and/or the extent of rearrangement of the haemocyte surface by non-specific adhesive interactions (Section 9.3), both events controlling the release of recruiting molecules. Cell-substratum adhesion has been shown to be an important trigger for the release of tumoricidal molecules by macrophages (Friedman and Beller, 1987). To account for continued recruitment after the primary cell layer has been covered, and taking into account the fact that capsules can be very thick, it must be postulated that (a) recruited cells also release recruiting or adhesive factors, or (b) capsules are initially rather leaky structures so that recruiting factors continue to diffuse outwards, or (c) cells attach to the altered dorsal surfaces of other attached cells, as suggested earlier. Caveney and Berdan’s (1982) observation that there was ionic coupling between encapsulating cells, and that injected carboxyfluorescein passed from cell to cell, argue for tight intercellular communication rather than leaky gaps, but these authors were not working with early capsules. There is evidence neither for nor against the first postulate since we are still ignorant about the nature of factors produced by PLs, the main cell type involved in encapsulation and wound-healing. However, the following observations might provide some clues. Capsules formed in Periplaneta around DEAE-Sepharose beads or polyHEMA-treated glass beads are considerably thicker than those around neutral Sepharose or untreated glass beads (Lackie, 1983a; Takle, 1986). Even at 24 h, the outermost cells of the thicker capsules are not completely flattened (Fig. 2) whereas the surfaces of capsules around glass are fairly smooth with plaques of coating material (Takle, 1986). One plausible explanation is that thickness depends on a balance between the strength of the recruiting signal and the extent of cell spreading-strong recruiting signals cause cells to arrive rapidly so individual cells initially have less room to spread [flattening and consolidation appears to be a later stage in capsule formation (Davies and Preston, 1987)l. Recruiting factors could thus continue to leak out between the cells, but their strength would decrease with time and with the addition of more cell layers; encapsulation would eventually cease. Where the signal is strong, a proportion of the cells recruited could also include some which themselves would be stimulated to release recruiting factors, thus maintaining the signal for a longer period. In thin capsules, the recruiting signal is weaker, the rate of recruitment lower, and the individual cells can more readily spread and cut off the source of recruitment. Cessation of capsule building could thus depend on physical blocking of recruiting factors. However, it is more likely that an active mechanism for down-regulation is also included in the process. There is some evidence to

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suggest that PLs release material that might be designed to slow down capsule formation; rate of recruitment would thus be influenced by the relative balance between secreted recruiting factors and “cessation factors”. In early studies of capsule ultrastructure a homogeneous, electron-dense material was noticed between the flattened cells (Grimstone et af., 1967) and subsequent workers have reiterated this view (Hillen, 1977; Ennesser and Nappi, 1984). Completed capsules become coated in a sheet of homogeneous material (Hillen, 1977) that stains for glycosaminoglycans with Alcian blue (Lackie et al., 1985) (Fig. 7). The completed capsules provoke the same degree of response as if they were intact tissue transplants when they are transferred to allo- or xenogeneic recipients (Salt, 1960; Lackie, 1986d); the coating material would thus appear to share properties with its donor’s basement membranes. Peripiuneta haemocytes, cultured for 24 h in a specially-formulated medium containing [3sS]sulphate, secrete a high molecular weight, sulphated molecule containing protein and carbohydrate. This molecule runs at the same position on agarose gels as does the unusual GAG from the basement membrane of Peripfaneta midgut (V. O’Brien, A. Lackie and J. Kusel, unpublished observations). Whether or not the haemocytic product is a GAG has not yet been determined. It is interesting that Kramerov and colleagues (1986) have recently found a new type of glycoprotein, secreted by a Drosophifu cell line; these molecules, sulphated “chitinproteins”, have also been detected in larval fat-body and intestine. If, as suggested, PLs release “cessation factors” (perhaps they are merely trying to repair what appears as damaged basement membrane?), they will have more chance to do so when recruitment is slow, and the outer surface of the capsule will more rapidly look like “self’. 11 Alterations in recognition and response

The survival of a parasite population within that of its host relies on the existence of a critical threshold density of susceptible hosts, below which the infection is unable to persist within the host community. Anderson (1986) has explored this interaction by means of various mathematical models and one conclusion which emerges is that “high rates of host recovery (an effective immune defence system) increase the critical density of susceptible hosts necessary for persistence of the infection within the host population”. If the hosts that recover also acquire a degree of immunity to reinfection, parasite persistence is made even less likely. The ability of a host to overcome or prevent infection by a particular parasite may depend on its genetic background (Carton and Bouletreau, 1985), but is also influenced by its prior immunological experience.

Fig. 7 The surface of completed haemocytic capsules around Sepharose beads, taken from Periplunetu. (a) Haemocytes are covered in a sheet of coating material. Bar = 50 pn. Inset shows small area in which coating material has been torn away to reveal underlying cells. (b) Section through 6-week capsule to show coating material overlying surfxe of flattened haemocytes. Bar = 0.5 pm.(c) Section through Alcian blue-stained capsule: dye has bound in electron-dense aggregates to layer of coating material. (From Lackie et a[., 1985.)

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Thus, some parasites may suppress the host’s immune response so that other parasites or particles, introduced later, fail to provoke a response. The number of papers on this topic is enormous, so a few examples will have to suffice: parasitoid infection decreases the cellular response of various lepidopteran larvae to injected particles or molecules, other parasites or tumorous cells (Nappi, 1975, 1977; Stoltz and Guzo, 1986; Davies and Vinson, 1986) and reduces the number of plasmatocytes (Davies et al., 1987) or lamellocytes (Nappi, 1981; R. Rizki and Rizki, 1984); larvae of the helminth Moniliformis moniliformis reduce the phagocytic capability of Periplaneta haemocytes in vivo (A. Lackie, unpublished information) and permit a greater proportion of larvae of the tapeworm, Hymenolepis diminuta-to which Periplaneta is not usually susceptible-to develop within the haemocoel (Holt and Lackie, 1986); Schistocerca infected with the nematode Mermis nigrescens are susceptible to infection by two species of trypanosomatid protozoan (Ibrahim et al., 1986-note that cell count should read “per ml”; D. Molyneux, personal communication), and secondary metabolites (destruxins) produced by the fungal pathogen Metarhizium reduce the ability of insects to produce nodules in response to spores of other fungi (Vey et al., 1985) or /31,3-glucans(Huxham et al., 1988), and suppress the PPO-activating effect of /31,3-glucans on locust or cockroach haemocytes in vitro (Huxham and Lackie, 1986; Huxham et al., 1988). The dangers of immunosuppression for the survival of parasites with a long developmental period are discussed by Lackie (1986d) and Yoshino and Boswell(1986). In contrast, immunocompetence may be increased if the immune system has recently been stimulated. Thus, the haemocytes of Galleria larvae can be persuaded to ingest a normally non-phagocytosable strain of Bacillus thuringiensis, if the larvae have been previously injected with latex beads (Mohrig et al., 1979). Larvae of Moniliformis invade the hafmocoel of their usual host, Periplaneta, by penetrating the midgut wall-larvae of Hymenolepis enter the gut wall but cannot penetrate it. If Periplaneta are fed on a mixed dose of eggs from the two parasites, the number of Moniliformis that develop within the haemocoel is considerably reduced, apparently because they are encapsulated and melanized as they enter the haemocoel (R. Holt and A. Lackie, unpublished information)-the response stimulated against Hymenolepis attacks Moniliformis as well. The immune system of Periplaneta does not usually recognize or respond to tissue implants (Bell, 1972; J. Lackie, 1975; A. Lackie, 1979) or cuticular grafts (Lackie, 1983b) from the closely related species, Blatta orientalis-if cuticle from Blatta nymphs is grafted onto nymphal Periplaneta, the donor epidermis heals in and produces new cuticle of its own type in synchrony with the recipient’s moult. However, if Periplaneta is first implanted with Blaberus tissue or with Sepharose beads, both of which provoke a strong encapsula-

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tion response, and then BIuttu cuticle is grafted onto these primed animals between 1-3 days later, the majority of the cuticular grafts are rejected, and replaced with Periplunetu cuticle at the moult. Prior implantation of Peripluneta or BIuttu tissue, neither of which are encapsulated, does not have this stimulatory effect (Dularay and Lackie, 1987). Within these 3 days of heightened responsiveness, the in vivo phagocytic and nodule-forming ability of the recipient Periplunetu’s haemocytes is also enhanced (B. Dularay and A. Lackie, unpublished information). Thus, not only is the responsiveness of the system increased but also, more interestingly, its ability to recognize a previously unrecognized foreign surface as non-self. The mechanisms whereby this change is effected remain to be explored. Does the cell count alter? Is a particular subpopulation expanded, and how? Is the adhesive and locomotory behaviour of haemocytes enhanced so that they search out “non-self” more efficiently? Is there altered expression of putative membrane receptors on the haemocytes responsible for recognition? Are soluble recognition molecules induced? Some of the answers may be hinted at in the previous sections.

Acknowledgements

My grateful thanks go out to all the people who have, in various ways, helped with the production of this chapter: to Garry Takle, Bubbly Dularay, Rohan Holt, Margaret Carr, Vincent O’Brien and Max Huxham for their determination and cheerfulness and for allowing me to use unpublished results; to Kerstin Andersson for her valuable comments on Section 6; to Toni Nappi for Fig. 3; to Stefan Gunnarsson for Fig. 4; to Tina Trenczek for Fig. 5; to Gerardo Vasta and John Kusel for friendly and inspiring collaborations; to Liz Currie for looking after us all, including our insects; to Murthy for his expert typing of the manuscript; to John Lackie for patient discussions and tolerance, and to our daughters for reducing their background noise while I was writing. Thanks are also due to Oxford University Press and Professor Roy Anderson for permission to use Fig. 1; to Springer-Verlag for permission to use Figs 2 and 7; and to the Nuffield Foundation (grants SCI/I68/356; SCI/I68/ 441; SCI/168/705), the AFRC (AG 17/160) and the SERC (grants GRCOI 351 and GRC87959) for their financial support. Professor K. Vickerman and Professor R. S. Phillips, as Heads of Department, have been most generous with their support. (Received September, 1987).

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Rowley, A. F. and Ratcliffe, N. A. (1980). Insect erythrocyte agglutinins. In vitro opsonisation experiments with Clitumnus extradentatus and Periplaneta americana haemocytes. Immunology 40,483492. Rowley, A. F. and Ratcliffe, N. A. (1981). Insects. In “Invertebrate Blood Cells” (Eds N. A. Ratcliffe and A. F. Rowley), Vol. 2, pp. 471490. Academic Press, New York. Ruoslahti, E. and Pierschbacher, M. D. (1986). Arg-Gly-Asp: a versatile cell recognition system. Cell 44,s 17-5 18. Ryan, M. and Nicholas, W. L. (1972). The reaction of the cockroach Periplaneta americana to the injection of foreign particulate material. J . Invert. Path. 19,299-307. Salt, G . (1960). Experimental studies in insect parasitism XI. The haemocytic reaction of a caterpillar under varied conditions. Proc. R. SOC.Lond. B. 151,446-457. Salt, G . (1961). The haemocytic reaction of insects to foreign bodies. In “The Cell and the Organism” (Eds J. A. Ramsay and V. B. Wigglesworth), pp. 175-192. Cambridge University Press, Cambridge. Salt, G. (1968). The resistance of insect parasitoids to the defence reactions of their hosts. Biol. Rev. 43,200-232. Salt, G . (1970). “The Cellular Defence Reactions of Insects”. Cambridge University Press, Cambridge. Sasaki, T. and Kobayashi, K. (1984). Isolation of two novel proteinase inhibitors from hemolymph of silkworm larva, Bombyx mori. Comparison with human serum proteinase inhibitors. J. Biochem. 95, 1009-1017. Sato, S., Akai, H. and Sawada, H.(1976). An ultrastructural study of capsule formation by Bombyx haemocytes. Annot. Zool. Japon. 49, 177-188. Saul, S. J. and Sugumaran, M. (1986). Protease inhibitor controls prophenoloxidase Lett. 208, 113-1 16. activation in Manduca sexta. Fedn Eur. biochem. SOCS Saul, S. J., Bin, L. and Sugumaran, M. (1987). The majority of prophendoxidase in the hemolymph of Manduca sexta is present in the plasma and not in the hemocytes. Dev. comp. Immunol. 11,479486. Schlumpberger,J. M., Weissman, I. L. and Scofield, V. L. (1984). Separation and labeling of specific subpopulations of Botryllus blood cells. J. exp. Zool. 229,40141 1. Schmit, A. R. and Ratcliffe, N. A. (1977). The encapsulation of foreign tissue implants in Galleria mellonella larvae. J . Insect Physiol. 23, 175-1 84. Schmit, A. R. and Ratcliffe, N. A. (1978). The encapsulation of Araldite implants and recognition of foreignness in Clitumnus extradentatus. J. Insect Physiol. 24,511-521. Schmit, A. R., Rowley, A. F. and Ratcliffe, N. A. (1977). The role of Galleria mellonella haemocytes in melanin formation. J. Invert. Path. 29,232-234. Schmittner, S . M. and McGhee, R. B. (1970). Host specificity of various species of Crithidia Leger. J . Parasitol. 56,684-693. Schneider, P. M. (1985). Purification and properties of 3 lysozymes from hemolymph of the cricket Gryllus bimaculatus (De Geer). Insect Biochem. 15,463470. Schwalbe, C. P. and Boush, G. M. (1971). Clearance of [51Cr]-labelledendotoxin from hemolymph of actively immunized Galleria mellonella. J. Invert. Path. 18,8588. Scott, M. T. (1971a). Recognition of foreign-ness in invertebrates: Transplantation studies using the American cockroach, Periplaneta americana. Transplantation 11, 78-8 5.

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hi

Molecular Mechanisms for Cuticular Sclerotization" Manickam Sugumaran Department of Biology, University of Massachusetts at Boston. Dorchester, Massachusetts

02 125.USA

Introduction The components of sclerotized cuticle 2.1 Structural proteins 2.2 Chitin 2.3 Catechols 2.4 Enzymes 3 Dityrosine crosslinks 4 Quinone tanning hypothesis 4. I Do aryl-lysine crosslinks really exist? 4.2 Can carboxyl groups participate in quinone tanning? 4.3 Arc hydroxyl groups involved in quinone tanning? 4.4 Aryl-histidine adducts 4.5 Reactions of other amino acids 4.6 Melanization and its prevention 4.7 Peroxidasc participation in quinone tanning 5 /&Sclerotiza tion 5.1 a,/J-Sclerotization 5.2 Quinone methide sclerotization Differential mechanism of tanning Combined pathway and crosslinking mechanisms Frcc radical formation Concluding remarks Acknowledgements References Note added in proof I

7

1 Introduction

Insects are quite incredible animals and have attracted the attention of scientists for several decades. More than three-quarters of known animals are in* This ilrllcle I S dedicated to the l a k Professor Herbert Lipke, who introduced me to the fasrinaling field of cuticul;ii- hiochemistry. ADVANCES IN INSECT PHYSIOLOGY VOL. 21 ISBN Cb12424221-4

Copyrighf01988 Academic Press Limired All risks ofreproduction in uny form reserved

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MANICKAM SUGUMARAN

sects and there are at least five times as many different species of insects in the world today as there are of all the other animal forms combined. At present, nearly a million species of insects have been described, but an estimated two or three times this number may actually exist. Their populations often number many millions to the acre. There may be as little as 50,000 bees to a hive and a locust swarm covering hundreds of square miles may contain as many as 40,000 million individuals weighing approximately 80,000 tons which may eat their own weight in food in a day. Insects have also become adapted to innumerable different ways of life, and occur in arid and scorching deserts as well as inhospitable arctic and antarctic regions. Some live in salt marshes, some in fresh water but most insects are of terrestrial origin. The great success of insects as terrestrial animals is in part due to their integument. The integument is the outer layer of the insect comprising the epidermis and the cuticle. The cuticle, which is secreted by epidermis, affords protection for the soft-bodied insects from their harsh environment; it is also a means for maintaining the body shape; it provides points of attachment and leverage for muscles; it restricts water loss from the body surface; and it serves several other important functions necessary for growth, development and survival of insects. Since cuticle is hard, it does not allow continuous growth, hence insects often shed their old cuticle and replace it with new cuticle. Freshly synthesized cuticle is soft and flexible, but subsequently it becomes hard. Before it hardens, it is expanded to allow for growth. The process of hardening is known as sclerotization or tanning. Sclerotization also aids the hardening of the pupal cuticle and protects the pupae after larval-pupal transformation. Similarly, tanning of egg capsules affords protection to the eggs of several insect species. Thus the sclerotization process is an essential biological mechanism for the successful survival of insects. Naturally, several groups of workers have devoted their attention to the study of this single most important event. In order to understand this process, it is essential to study the components of cuticle first. The biochemical components that participate in the sclerotization process are proteins, chitin, enzymes and phenols. It will be beyond the scope of this review to cover the biosynthetic and biochemical aspects of these components. However, certain physicochemical properties of these components need to be considered in detail as they are essential to a better understanding of the sclerotization process and hence cuticular structure. It should be remembered that there may be other processes such as mineralization, wax deposition, etc., either operating in parallel or independent of sclerotization during cuticle synthesis which might involve other chemical components as well. Again, such processes will not be considered for discussion. The present paper addresses mainly the biochemical mechanisms of tanning. The reader may refer to several excellent re-

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181

view articles which have appeared in the literature from time to time for other aspects of sclerotization as well as for background information (Rudall, 1963. 1967, 1976; Andersen, 1966, 1976. 1977. 1979a, 1985; Brunet, 1967, 1980; Neville, 1975; Hepburn, 1976; Miller, 1980; Sherald, 1980; Lipke et al., 1983; Kramer et al., 1985; Kramer and Koga, 1986).

2 The components of sclerotized cuticle 2. I

S T R U C T U R A L PROTEINS

Egg capsules (ootheca) and egg shell (chorion) of insects are devoid of chitin and the only biopolymers found in them are proteins. However, in the case of other stages, insect cuticle contains both chitin and protein in varying proportions. Only a representative example of each stage will be dealt with in this section. The structural proteins of Periplaneta americanu ootheca have been well characterized (Pau et al., 1971, 1986). Based on their molecular weight, they are classified as oothecins A ( 1 3,000), B ( 1 3,500), C ( I 5,500), D (21 ,OOO), E (28,000) and F (39,000). While oothecin A and B are water soluble, oothecin C and D are water insoluble. The amino acid composition of the major protein oothecin F, differs from all others in that it has high valine and proline content. Others have high glycine content. Oothecins A, B, C , and D contain high tyrosine (12-20%). None of them have cysteine. Electronmicroscopic studies indicate that the insoluble oothecins form spherical particles and are found embedded in a matrix of soluble proteins. After tanning of ootheca, the above structure is still preserved. An interesting aspect of ootheca proteins is their hydrophobicity. Due to the very low hydrophilic amino acid content, these proteins do not have any hydrophilic regions in their primary structure (Fig. I ) . The chorion (egg shell) of silkmoth, Antheraea polyphernus contains five proteins. They are strikingly similar to each other and are characterized by a region of high cysteine content at the amino terminal side followed by a longer stretch of a low-cysteine region, rich in alanine. glycine, leucine and tyrosine (Regier et al., 1978). They are remarkably similar to oothecins in their hydrophobicity, but seem to be stabilized by disulphide bridges. In the case of larval proteins, considerable information is available only for Drosophilu melunogaster. The amino acid sequence of four of the five major third-instar larval cuticular proteins has been determined (Snyder et ul., I98 I , 1982). They are highly homologous to each other and exhibit considerable homology to the last-instar larval cuticular proteins from Sarcoplwgu hullutu (Lipke et al., 1983; Henzel e / a/., 1985). Interestingly these proteins do not resemble any of the chorion or oothecal proteins and exhibit \ti:.,

M A NI CKAM SUG UM A R A N

182

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Fig. 1 Hydrophilicity plots of some cuticular proteins. (A) Oothecin from P . americana; (B-E) larval cuticular proteins 1,2,3,4 from D . melanogaster; (F) pharate cuticular protein from L. migratoria. All determined on an IBM-XT computer using the commercially available "Microgenie" program from Beckman Instruments, CA. ( + ) Hydrophilic regions; ( - ) hydrophobic regions. Note proteins A and F d o not have any hydrophilic region at all, while 9-E have both hydrophilic and hydrophobic. regions on their primary structure.

alternate stretches of hydrophilic and hydrophobic regions in their primary structure (Fig. I ) . Recently, Hojrup et al. (1986a, b) determined the N-terminal sequence of a few of the pharate cuticular proteins from the migratory locust, Locusta migratoria. These proteins are high in alanine, proline, glycine and tyrosine and exhibit no homology to any of the other known cuticular protein sequences. However, they are highly hydrophobic like oothecins and chorion proteins (Fig. I ) . 2.2

CHITIN

Chitin is a 8-1 ,Chomopolymer of N-acetylglucosamine. Figure 2 summarizes the sequence of reactions leading to its biosynthesis starting from glucose.

CUTICULAR SCLEROTIZATION

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Fig. 2 Biosynthesis of chitin.

Kramer's group (Kramer et al., 1985; Kramer and Koga, 1986) has recently reviewed various aspects of chitin biochemistry. It is widely distributed in insect cuticle and contributes to the mechanical strength of the cuticle. Together with structural proteins, it forms the majority of cuticular components. Chitin is found in cuticle as microfibrils and is known to form covalent adducts with proteins to yield chitin--protein complexes (Rudall, 1963, 1967, 1976; Lipke et al., 1980, 1983; Sugumaran et al., 1981, 1982; Kramer and Koga, 1986; Schaefer et al., 1987). However, the chemical nature of such a linkage remains to be determined. Also the reactions of chitin with sclerotizing agents need to be studied in detail to understand their contribution to sclerotization. An interesting aspect to chitin chemistry is the fact that some of the acetamido groups of chitin may be found in the hydrolysed form. As much as 20% of amino groups may be present in free form making them likely candidates for nucleophilic addition reactions with sclerotizing agents. Apart from the amino group, the only other functional group on chitin capable of participating in any reaction is the hydroxyl group present at the C-3 and C-6 positions of the monomer units. Yet surprisingly, no one has ever determined the chemical nature of chitin-protein adducts or crosslinks.

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2.3

CATECHOLS

There is no doubt that catechols are widely used for the tanning reaction of insect cuticle. However, there exists a confusion as to whether they are sclerotizing agents or sclerotizing precursors. Catechols per se do not react with any biological molecule, but their oxidized products such as quinones can react spontaneously with various nucleophiles to form adducts. Therefore, catechols must be regarded as sclerotizing precursors rather than sclerotizing agents. The term sclerotizing agent should then be reserved for any reactive species capable of forming adducts and crosslinks with cuticular components. 3,4-Dihydroxybenzoic acid (1) was the first catechol to be identified as the tanning precursor for the egg capsule of cockroach (Pryor 1940a; Pryor et af., 1946). Subsequently, the P-glucoside of 3,4-dihydroxybenzyl alcohol was isolated as an additional precursor for tanning of Bfaberus ootheca (Pau and Acheson, 1968). Since 3,4-dihydroxybenzyl alcohol (2) can yield (1) as an oxidation product, it is not clear at present whether (1) is a by-product or an intermediate participating in sclerotization reactions. Recently, glucosides of N-acetyldopamine (3), N-P-alanyldopamine (4), N-(N-acetyl-P-alanyl) dopamine (S), and N-(N-malonyl-P-alanyl) dopamine (6) have been shown to be the tanning precursors for the egg cases of praying mantids (Kawasaki and Yago, 1983; Yago and Kawasaki, 1984; Yago e f af., 1984). In the case of insect cuticle, Karlson and his group identified N-acetyldopamine (3) as the tanning precursor for not only Caffiphorabut also many other insects (Karlson and Sekeris, 1962; Karlson and Herrlich, 1965; Sekeris and Herrlich, 1966). P-Alanine is an unusual amino acid widely found in sclerotized cuticle. Although the fact that the incorporation of P-alanine into cuticle is dependent on the incorporation of dopamine was known for quite some time, only recently, P-alanyldopamine (4) was characterized to be a tanning precursor for Manduca sexta and several other insects (Hopkins et af., 1982). Subsequently, N-8-alanylnorepinephrine(7) was also shown to be associated with the sclerotized cuticle (Sugumaran et af.,1986; Morgan et af., 1987). Apart from these three compounds, other catechols such as N-acetylnorepinephrine (8) and 3,4-dihydroxyphenethyl alcohol (9), 3,4-dihydroxyphenylacetic acid (10) may also contribute to the aromatic structure of sclerotized cuticle (Andersen, 1975; Andersen and Roepstorff, 1978; Peter, 1980; Lipke r f af.,1983). In the special case of the tanning of silk, Brunet and Coles (Brunet, 1967; Brunet and Coles, 1974) have identified 3-hydroxyanthranilic acid and gentisic acid to be the tanning precursors. Although these two compounds are not catechols, they yield quinonoid compounds readily upon enzymic oxidation and hence form adducts with cuticular components. However, the reaction

185

CUTICULAR SCLEROTIZATION

1

3

2

OH

OH

4

7 0

0

I

bH

9

10

OH

8

on HO

11

Fig. 3 Structure of some catecholic sclerotizing precursors.

products will differ in structure from those derived from catecholic compounds. On the other hand, the recently reported tanning precursor N-(3,4dihydroxyphenyllacty1)-dopa (1l), from the Japanese giant silkmoth, possesses the o-dihydroxy group similar to other catecholamine derivatives and can function like them (Kawasaki and Sato, 1985). The structure of some of these catecholamine derivatives are shown in Fig. 3. It is obvious from the above summary that our knowledge on the nature of catecholamine derivatives destined to become part of crosslink structure in insect cuticle is limited. Hence, a systematic search and characterization of catecholamine derivatives in a variety of insect species requires urgent consideration.

186

2.4

MANICKAM SUGUMARAN ENZYMES

Insect cuticle may contain several enzymes (Neville, 1975) but only those committed to tanning reactions will be considered in this section. Detailed summaries on these enzymes can be found elsewhere (Yamazaki, 1969, 1977; Andersen, 1976, 1978, 1979b; Brunet, 1980; Barrett and Andersen, 198I; Barrett, 1986). o-Diphenoloxidase, also known as tyrosinase, phenolase, catecholoxidase, polyphenoloxidase, phenoloxidase and dopa oxidase is a copper-containing monooxygenase widely found in cuticle. It exhibits wide substrate specificity and can oxidize a variety of o-dihydroxy compounds to o-benzoquinones. It has been characterized from several insect species (Yamazaki, 1969, 1977; Andersen, 1976, 1978, 1979b; Brunet, 1980; Barrett and Andersen, 1981 ; Barrett, 1986). Cuticle also possesses another phenoloxidase which shows specificity towardsp-diphenols (Brunet, 1980; Barrett, 1986). This enzyme, known as laccase, exhibits preference for the oxidation of p-diphenols over o-diphenols and oxidizes both to their corresponding quinone derivatives. The significance of this enzyme for cuticular tanning is not clear at present as p-diphenols do not appear to be sclerotizing precursors for cuticular tanning. Nevertheless, laccases may contribute to quinone tanning of cuticle, as they can oxidize o-diphenols. To account for p-sclerotization, Andersen and his school (Andersen and Roepstorff, 1982; Andersen, 1985) invoked the presence of a desaturase which converts N-acetyldopamine (3) to 1,2-dehydro-N-acetyldopamine (12). Remarkably, the nature of this enzyme resembles that of phenolases rather than desaturases like succinate dehydrogenase. Further, we feel that the dehydro compound may arise by a non-enzymatic route from the quinone methide derivative of (3) (Sugumaran, 1987b). Hence, further work on “desaturase” is essential to evaluate its role in tanning. Peroxidase is yet another enzyme which is present in cuticle. It is a hematin-containing oxidase which catalyses the oxidation of reduced compounds (AH,) using hydrogen peroxide as the oxidant: AH,

+ H,O,-+A + 2H,O

This enzyme is versatile in that it can use a wide range of reduced substrates. Of particular interest to the present discussion is its ability to oxidize phenols and catechols. Sawahata and Neal ( 1982) presented evidence for the peroxidase-catalysed oxidation of phenol to o,o‘- and pg’-diphenols. Interestingly, during their studies, these authors also observed covalent binding of radioactive phenols to proteins. Since peroxidase converts hydroquinone and catechol to their

C U T 1C U L A R S C L E R O T l Z A T l O N

187

corresponding quinones, it is likely that through quinone formation, covalent addition of radioactive phenol to proteins has occurred. Therefore, i t is probable that peroxidase can also participate in quinone tanning. Locke (1969) observed peroxidase in granules of epidermal cells of Culpocks cthlius during the formation of hard brown cuticle. According to him, peroxidase secreted by spine cells is taken up by neighbouring support cells during hardening and tanning of Culpocirs cuticle. During studies on the fate of dityrosine-containing peptides in Surcophugu hullutu cuticle, our laboratory reported thc further oxidation of dityrosine (13)-containing peptides (Sugumaran P I d., 1982). Since 13 is not oxidized by phenoloxidase, it is likely that cuticular peroxidase oxidized the dityrosine-containing peptides to form additional crosslinks of unknown structure. While the above studies indicate the probable involvement of peroxidase in quinone tanning of cuticle, evidence for this proposal must be obtained in the near future.

3

Dityrosine crosslinks

The insect cuticle is not uniform in appearance or in its physicochemical properties. I t may be rigid in certain areas and softer and flexible in some other places. Weis-Fogh ( 1960) identified resilin-a rubber-like proteinfrom the elastic cuticle of desert locusts. Subsequently Andersen (1963, 1964) isolated and characterized two unusual amino acids viz., dityrosine (13) and trityrosine (14) from the acid hydrolysates of resilin (Fig. 4a). Dityrosine and trityrosine can be synthesized from the monomeric tyrosine by the action of peroxidase (Gross and Sizer, 1959). Therefore, Andersen ( 1 966) suggested that these two novel amino acids are produced by the oxidative coupling of protein-bound tyrosine. Model studies with silk fibroin, hydrogen peroxide and peroxidase resulted in artificial crosslinking of silk fibroin. Such crosslinked proteins upon acid hydrolysis liberated both 13 and 14. At the same time, Coles (1966) identified a peroxidase in the epidermal cells which secreted resilin. From these studies, it was apparent that 13 and 14 are formed in vivo by crosslinking of a precursor protein rich in tyrosine by peroxidase. There are two biochemical mechanisms by which 13 can be synthesized de novo (Fig. 4b). While the first mechanism involving the linkage of free tyrosine to protein-bound tyrosine accounts for the formation of 13, it does not account for the elasticity of cuticle containing this amino acid. Whereas the second mechanism calls for true crosslinking of peptide-bound tyrosine residues and hence will account for the rubber-like nature of the linked proteins. In order to distinguish these two possibilities, we performed sequencing studies on peptides generated from elastic cuticle of S . hullata larvae (Sugu-

MANICKAM SUGUMARAN

188

on

OH

on

on

14

on 13

0 0 OH TVROSINE

OH

P R O T E I N BOUND TYROSINE

OH

P R O T E I N SOUND T Y R O S I N E

OH

OH

D l T Y R O S l N E ADDUCT

OH

OH

O I T Y R O S I N E CROSS L I N K

Fig. 4 Structures of dityrosine (13) and trityrosine (14) and alternate mechanisms for dityrosine crosslink formation.

maran er ul., 1982). Proteolytic digests of last-instar larval cuticle with pronase released several peptides containing 13; most of which exhibited multiple N-termini in accordance with the operation of the second mechanism. The structure of two peptides were elucidated by a combination of protein sequencing techniques and are illustrated in Fig. 5 . From the results, it is obvious that dityrosine residues are made by the actual crosslinking of cuticular proteins. However, our results do not distinguish between intermolecular and intramolecular crosslinking. Further studies are necessary to clarify this aspect.

189

CUT1 C U L A R S C L E R O T I Z A T I O N

PEPTIDE 1 Asp-Tyr- Pro-Ser

I

Gly-Tyr- Pro-Ser

OR

Gly-Tyr

I

Asp-Tyr

PEPTIDE 2 Asp- Pro-Tyr- Pro-Val

I

Gly-Tyr

Gly- Pro-Tyr- Pro-Val

OR

I

Asp-Tyr

Fig. 5 Structure of dityrosine-containing peptides from S. hullufa. (From Sugumaran el d..1982.)

In the case of Drosophilu, the chorion proteins are made insoluble by the action of a peroxidase which crosslinks their tyrosine residues to produce diand trityrosine residues (Petri et d., 1976; Mindrinos et al., 1980). We have identified 13 in cuticular proteins from dipteran larvae, S. hullutu, Tuhunus nigrovittutus and D . melunoguster. but were unable to detect it in the mosquito, Aedes uegypti. The discontinuous occurrence of 13 within the life cycle of a given species was evidenced by good recovery from aedine eggs but not aedine larval cuticle, from S . hullutu larval cuticle but not from puparial cases and from the larvae of M . sexfa but not from the eggs or pupal cuticle (Sugumaran rr ul., 1982). Since synthesis of 13 and 14 calls for free radical reactions which are nonspecific in nature, obviously one expects isomeric products being formed during this process. Theoretical considerations favour the formation of phenoxide as well as quinonoid radicals which on coupling yield isodityrosine (15) and 13 (Fig. 6). Although 15 has been characterized from plant cell wall glycoproteins (Fry, 1982), it remains to be identified from any insect cuticle. Isotrityrosine, however, has been characterized from the cuticle of parasitic worm, Ascuris lumhricoides (Fujimoto rt ul., 1981). Unlike phenoloxidase which uses molecular oxygen to oxidize its substrates, peroxidase requires hydrogen peroxide as an oxidant. Hence, cells synthesizing 13-containing proteins must possess the machinery to make hydrogen peroxide also. Candy ( 1 979) has described the presence of a glucose oxidase system in the locust cuticle which produces hydrogen peroxide as a by-product of glucose oxidation. However, it is not clear whether the sole purpose of this system being localized at the cuticle is to aid hydrogen peroxide production for 13 synthesis or for other purposes. Clearly a lot of work needs to be done in this area to resolve several unanswered questions. These

MANICKAM SUGUMARAN

190

6""' - 6""' - ,dNH2 coon

COOH

OH

COOH

0

FREE

TYR

P

coon

0

RADICALS

Hn2

OH

15 Fig. 6

on

OH

13

Mechanism of dityrosine (13) and isodityrosine (15) formation.

include, but are not limited to: (a) Are isodityrosine and isotrityrosine present in resilin and other insect proteins? (b) Does the crosslinking process occur at specific sites or randomly on the precursor protein? (c) Is crosslinking intramolecular or intermolecular? (d) Is cuticular glucose oxidase localized in the cuticle to provide hydrogen peroxide for crosslinking or for other purposes? (e) Are there any other sources of hydrogen peroxide? ( f ) What are the fates of di- and trityrosines? 4

Quinone tanning hypothesis

From the structure of sclerotizing precursors, and the presence of oxidizing enzymes in cuticle, one obviously expects the formation of quinones as the sclerotizing agents. Quinones are known for their notorious reactivities and hence they were easily recognized as sclerotizing agents as early as 1940. Working with cockroach ootheca which is devoid of chitin, Pryor (1940a) observed the production of protocatechuate quinone (16), by the interaction of the sclerotizing precursor (1) with phenoloxidase. Based on this finding, he proposed the quinone tanning hypothesis (Pryor, 1940a, b, 1962). According to him. quinones generated by the action of phenoloxidases on catechol derivatives, react spontaneously with cuticular proteins resulting in the formation of crosslinked proteins.

191

C U T I C U LA R S C L E R O T l Z A T l 0 N

Subsequently, Hackman and Todd (1953). as well as others (Mason, 1955; Pierpoint, 1969a.b; Horspool et al., 1971; Davies and Frahn, 1977) who studied the reactions of quinones with amines and amino acids, observed the facile nucleophilic addition of amines to quinones. These observations, coupled with the fact that during hardening the lysine content was reduced from 9.7 to 3.6% (Hackman and Goldberg, 1963) strengthened the quinone tanning hypothesis further. On these grounds and other evidences, the quinone tanning hypothesis gained wide acceptability and even found its way into textbooks of entomology. Using 3 as the sclerotizing precursor, a modified quinone tanning hypothesis is depicted in Fig. 7. According to this, cuticular phenoloxidase catalyses the conversion of 3 to its quinone, 17, which can undergo facile non-enzymatic Michael 1 ,Caddition reaction with avail-

17

3

I CMaCM2 NMCOCMa

RW2

CM2CM2 NMCOCM3

OM

OM

[a uIN o NE- P R OT EIN RWM2

ADDuc

TI(CAT ECHOL -P R o T E IN

A D D u c T)

1 CMtCM2 NMCOCMa

AND/ OR OM

(PROTEIN-PROTEIN

""ONR q2CM2MMCOCM3

0

CROSS LINKS)

Fig. 7 Quinone tanning hypothesis. N-Acetyldopamine (3) (or other sclerotizing catechols) secreted by the epidermal cells is oxidized to the corresponding quinone (17) by the cuticular phenoloxidase. Compound 17 undergoes non-enzymatic Michacl- 1 ,Caddition reaction with cuticular proteins forming catechol-protein adducts. These adducts, upon further oxidation and coupling to another protein, yield protein-protein crosslinks. The exact chemical structures of the crosslink and adducts are yet to be determined. Only the amino group of the proteins was considered. (From Sugumaran et al., 1987c.)

192

MAN I C KAM S U G U MAR AN

able amino groups on proteins yielding catechol-protein adducts. These adducts on further oxidation to quinone-protein adducts and coupling to other structural units produce protein-protein crosslinks. However, concrete structural proof for existence of adducts and crosslinks derived from quinones and cuticular components is still lacking. The instability, complexity and multiplicity of products formed from the reactions of sclerotizing agents with cuticular components have severely hampered the characterization of such adducts and crosslinks. Hence, in recent years, several scientists have resorted to the use of a chemically defined, in vitro model system to examine quinone tanning mechanisms. Earlier, from this laboratory, Strout and Lipke (1974) attempted to study the in vitro sclerotization reactions using cuticular proteins from S. hulfuta and phenoloxidase. They used dopamine as the precursor for in situ generation of quinone. Unfortunately, dopamine quinone prefers to undergo rapid intramolecular cyclization with the appropriately positioned internal amino group rather than reacting with external amino groups of the test proteins (see Section 4.6). As a result, numerous attempts made by these authors to demonstrate in vitro protein polymerization ended in vain. Grun and Peter ( 1 983), on the other hand, used N-acyldopamine derivatives and observed the oligomerization of arylphorins but failed to witness the polymerization of commercial proteins such as ferritin or bovine serum albumin. This result was puzzling, because it is generally believed that quinones are non-specific in their reactivity and should react with different proteins alike as long as potentially reactive nucleophiles are available on the proteins. Contrary to this, the model system seemed to exhibit specificity in polymerizing insect proteins. Furthermore, these authors carried out polymerization reactions for long times (24 h). By contrast, tanning reactions are complete in 3-4 h in nature. Hence, we re-examined this problem more carefully with a model system which consisted of mushroom tyrosinase/test pro1987~). teins/l and oxygen (Sugumaran et d., From a reaction mixture containing mushroom tyrosinase, lysozyme and 1, lysozyme slowly precipitated with time, indicating its polymerization. This was also confirmed by SDS-polyacrylamide gel electrophoresis of the reaction mixtures. Omission of any of the above three compounds or the replacement of native tyrosinase with heat-inactivated tyrosinase resulted in total failure to observe the protein polymerization. The model system not only polymerized lysozyme, ribonuclease and cytochrome-c individually, but also cross-polymerized bovine serum albumin and lysozyme. Crosslink formation as outlined in Fig. 7 calls for the generation of catechol-protein monomer adduct as a prerequisite for oligomerization. In order to visualize this adduct, radioactive 3 was incubated with tyrosinase and lysozyme and the resultant products were subjected to SDS-polyacrylamide gel

CUTICULAR SCLEROTIZATION

193

Fig. 8 Demonstration of lysozyme-N-acetyldopamine(3)adduct formation. A reaction mixture containing [ring3H]-3, lysozyme and catalytic amounts of mushroom tyrosinase was incubated for different time intervals and subjected to SDS-polyacrylamide gel electrophoresis. Gels A and B are duplicate runs. (A) represents autoradiography; (B) kenacid blue stain. Lane 1 : zero time; lane 2: 30 min reaction; lane 3: 60 min reaction. (From Sugumaran et ul., 1987c.) electrophoresis. Non-specific binding of radioactive 3 to lysozyme was monitored with appropriate controls. From Fig. 8 it is clear that the protein band corresponding to the monomer is heavily labelled with radioactive 3, while no such labelling is observed in the control, proving the formation of N-acetyldopamine-lysozyme adducts. In addition, the above experiment also reveals that the binding o f 3 to lysozyme is covalent in nature and not d u e to hydrophobic interactions. The time-course of radioactive incorporation studies showed that the monomer band becomes radioactive followed by dimer and trimer in the given order. The N-acetyldopamine-lysozyme adduct formed in the reaction mixture could be either catechol type or quinone type or both (see Fig. 7). In order to determine the ratio of these two adducts formed, the radioactive proteins were extensively dialysed against borate buffer, hydrolysed with 6N HCI a n d the hydrolysate was chromatographed on a dihydroxyboryl cellulose column to separate quinone-type adducts from catechol-type adducts (Sugumaran and Lipke. 1982b, 1 9 8 3 ~ ) The . results (Fig. 9) indicate that 80% of the adducts are catechol type while the rest are quinone type. From these experiments i t is clear that quinone tanning does occur in vitro.

194

MANICKAM SUGUMARAN

n I

L K

motbn Nucnkr

Fig. 9 Dihydroxyboryl cellulose chromatography of [3H]-%lysozyme adduct hydrolysate. The radioactive 3-lysozyme adduct formed in a 30-min reaction mixture (see Fig. 8) was dialysed and hydrolysed with 6N HCl. Following evaporation, the hydrolyste was chromatographed on a dihydroxyboryl cellulose column (which binds to catechols specifically) to separate catechol-type adducts from quinone-type adducts (Sugumaran and Lipke, 1983~).Elution of bound catechols was achieved with 1M acetic acid. (From Sugumaran el af., 1987c.)

4.1

DO ARYL-LYSINE CROSSLINKS REALLY EXIST?

Most proposals on quinone tanning invoke the participation of lysine in the crosslinking process. This is primarily based on the known reactions of quinone(s) with amines. Accordingly one expects a drastic reduction in the lysine content of pre-sclerotized tissues following sclerotization. Since the dipteran puparium is constructed from the last larval instar cuticle without significant addition of new macromolecules, we compared the amino acid content of the unsclerotized last instar larval cuticle and sclerotized puparial sheaths of D . melunogaster and S. bullatu and found no significant reduction in lysine content due t o tanning (Sugumaran et al., unpublished results). One possible explanation for this result is the unusual chemistry of aryl-lysine adducts. Amino quinones such as aryl-lysine derivatives are vinylous amides (Fig. 10). Hence o n e can expect them Eo behave like regular amides (Cranwell and Haworth, 1971). Therefore, on acid hydrolysis, they must liberate free lysine. As a result mass balance studies will not give correct information about the

C U T 1C U L A R S C L E R O T l Z A T l O N

195

RNH2

OH

Fig. I0

OH

Mcchanism of hydrolysis of quinone-lysine adducts.

existence of lysine crosslinking. To overcome this problem, we have performed non-invasive, solid-state NMR studies on the whole cuticle (Sugumaran et al., unpublished results). Figure 11 shows the NMR spectrum of [' 5N]lysine-labelled sarcophagid larval cuticle (A) and puparial cases (B). From the figure it is clear that less than 2% of total lysine nitrogen incorporated into the puparial cases ends up in aryl-amine adducts. Hence, at least in Sarcophuga, lysine contribution to quinone tanning seems to be minimal. This could be due to the following reasoning.

-I l y s - NH2

300

200

100

0

-100

PPM

Fig. I I Solid-state I5N-NMR spectrum of Sarcophagid cuticle labelled with [~-'~N]lysine. (A) Larval cuticle; (B) puparial cases. (Sugumaran et al., unpublished results.)

196

MANICKAM SUGUMARAN

At physiological pH, the &-aminogroup of lysine residues on cuticular proteins are protonated. While only the free base with its lone pair of electrons is suited for nucleophilic addition reactions with quinones, the positively charged - NH,' group is less likely to react with quinone. Ionization to free base might help such a nucleophilic condensation, but it may be suppressed in the microenvironment of the cuticle. Therefore, lysine-quinone crosslink formation may not occur in certain cuticles. Only further studies can resolve the existence of such crosslinks. In this context, the colossal but unsuccessful attempt made by British scientists to solve the structure of quinone-lysine adducts must be mentioned. During the studies on the effect of polyphenols on nutritionally important amino acids in plant proteins, Synge and his collaborators (Davies er d.,1975; Laird et ul., 1979; Eagles et al.. 1980) used hydrogenation as an approach to study the reactions of oxidized phenols with amino groups of lysine. After confirming that aryl-amino acid adducts are unstable to acid hydrolysis, these workers hydrogenated them prior to acid hydrolysis. The aliphatic products thus formed were subjected to gas chromatography-mass spectroscopic analyses. Although such an analysis indicated the presence of quinonoid-lysine coupled products, it did not lead to any conclusive identification of the putative adduct.

4.2

CAN CARBOXYL GROUPS PARTICIPATE I N QUINONETANNING?

The participation of carboxyl groups in quinone tanning has never been contemplated. Of the total amino acid composition of cuticular proteins 15-30% is due to aspartic and glutamic acids (Willis et ul., 1981; Lipke el ul., 1983; Henzel et ul., 1985; Hojrup et ul., 1986a). Although it is not known with certainty how much of these are present in amide form, the possibility of carboxylate groups participating in quinone tanning cannot be ignored. Evidence for this proposal comes from model studies. During the enzymatic oxidation of 3,4-dihydroxyphenyl propionic acid (18), the corresponding quinone (19) formed, proved to be extremely unstable and underwent ring (20) as the ini tial product closure to give 6,7-dihydroxy-3,4-dihydrocoumarin (Sugumaran et ul., unpublished results). 20 was further oxidized by tyrosinase to its quinone (21) (Fig. 12). Thus, an appropriately placed internal carboxylate group seems to participate in intramolecular nucleophilic addition with quinones. At the same time, it must be pointed out that we have not been able to observe such a nucleophilic coupling of quinones with exogenous carboxyl groups. This need not necessarily rule out the presence of carboxylate-derived ester crosslinks in insect cuticle. Cuticle might provide an appropriate environment for the nucleophilic addition of carboxyl groups

CUTICULAR SCLEROTIZATION

" HO O

z

W

18

o

197

-

1

'0

21

20

Fig. I 2 Mechanism of oxidation of 3,4-dihydroxyphenyl propionic acid (18) by tyrosinase. Tyrosinase converts 18 to its quinone 19 which undergoes facile non-enzyma(20) which tic intramolecular cyclization to give 6,7-dihydroxy-3,4-dihydrocoumarin is further oxidized to its quinone (21) either enzymatically or non-enzymatically. Quiunpublished results.) none 21 accumulates in the reaction mixture. (Sugumaran et d.,

to quinones (Fig. 13). Since the ester crosslinks formed are acid labile, acid hydrolysis will regenerate the parent amino acid and yield hydroxy quinone derivatives from the aromatic part of the crosslink. Hence, their detection in cuticle by destructive techniques is not possible.

OH

Fig. 13 Participation of carboxyl groups in quinone tanning. The carboxyl groups of aspartate, glutamate and C-terminals can add on to sclerotizing agents such as 17 ( R = COCH,; .Y= H ) to produce ester crosslinks.

198

4.3

MANICKAM SUGUMARAN ARE HYDROXYL GROUPS INVOLVED IN QUINONE TANNING?

Hydroxyl groups of threonine, serine, tyrosine and chitin constitute the bulk of available nucleophiles in cuticle. It is the most abundant functional group in cuticle, but totally ignored in mechanistic proposals of quinone tanning. Examples for the reactions of hydroxyl groups with quinones are readily available in the literature. An exhaustive coverage of this topic is beyond the scope of this article. Hence, only two examples are given below. While studying the oxidation of catechol by plant phenoloxidases, Kandaswami and Vaidyanathan (1973) reported a novel dimerization of catechol (22) to generate diphenylene dioxide quinone (23). The formation of 23 could not be possible without the participation of hydroxyl groups of 22 in Michael reaction with o-benzoquinone 24 (Fig. 14). One may argue that the phenolic group, but not a simple hydroxyl group possesses the potential for such a reaction. The following example, however, illustrates that even the hydroxyl group of water can react with quinones. During the oxidation of Q-methylcatechol (25), Kalyanaraman et al. (1987) observed the formation of 5-hydroxytoluquinol (26) through the quinone (27) (Fig. 15). This reaction is more favourable at alkaline pH confirming the nucleophilic nature of addition. It should be possible to observe such additions with cuticular components and sclerotizing quinones.

t yOH

22

[a:] 24

J 23 Fig. 14 Oxidation of catechol 22 to diphenylene dioxide-2.3-quinone (23), viz. o-benzoquinone(24). (From Kandaswami and Vaidyanathan, 1973.) 4.4

ARYL-HISTIDINE ADDUCTS

The imidazole group of histidine is ideally suited for coupling reaction with quinones but only in 1982 was this possibility realized (Sugumaran and

C U T I C U L A R S C L E ROT1Z A T l 0 N

25

199

27

26

Fig. I5 Formation of hydroxytoluquinol (26) from 4-methylcatechol (25), viz. nuclcophilic addition of water to the quinone (27). Lipke, 1982a). During our search for lysine-quinone crosslinks in sarcophagid cuticle, we evaluated the role of all amino acids in quinone tanning and found evidence for the existence of aryl-histidine adducts in sarcophagid cuticle. After incorporation into the puparial case, tyrosine and histidine administered to maggots at the threshold of puparium formation were converted to basic substances distinguishable from the parent amino acids. The metabolites could be resolved from tyrosine and histidine by chromatography on Dowex 50, Sephadex LH-20, cellulose thin-layer plates, and affinity supports retaining o-dihydric phenol derivatives. Products of similar or identical chromatographic properties were recovered from peptides generated from the sclerotized proteins by cleavage of unmodified tyrosyl residues with N-bromosuccinimide. This pattern of isotope distribution was in keeping with a mechanism wherein modification of histidyl residues is accomplished through arylation by a tyrosine metabolite after translation. That a substantial number of the histidine residues were modified by conjugation with carbon derived from the tyrosine ring was shown by the good recovery of radioactivity from tyrosine (62%) and histidine (40%) in the pH 12 fraction (Fig. 16A). This fraction seems to contain at least three components, as evidenced by further resolution on LH-20 columns (Fig. 16B). Currently, we are purifying these components in large scale for structural elucidation. Although our results indicate the formation of metabolites carrying radioactivity from both histidine- and tyrosine-derived metabolites, they do not shed any light on the structure of adducts. Following our work, Schaefer rt ul. (1987) used a non-destructive, solid-state NMR study of l s N - and I3Clabelled M. .sc'.utci cuticle and confirmed the presence of quinonoid-histidine adducts by double cross-polarization magic angle spinning (DCP-MAS) NMR technique. This technique specifically monitors the formation of bonds between 3C- and SN-containingprecursors. Hence, after providing I SN-labelled histidine (as a precursor for cuticular protein) and 13C-labelled dopamine (precursor for sclerotizing agent) to M . sexta, these authors specifically looked for the formation of aryl-histidine adducts in the cuticle and found evidence for the same. Furthermore, they believed that such adducts also contained chitin and proposed the structure 28 (Fig. 16). The

200

MANICKAM SUGUMARAN

Fraction Number PROTEIN

-

0

0 - CHITIN OH

28

Fig. 16 Demonstration of the presence of aryl-histidine adducts in S. bullata. S. bullata puparial cases doubly labelled with [3H]tyrosine and [14C]histidinewere hydrolysed and chromatographed on a Dowex 50 column employing step gradients (Sugumaran and Lipke, 1982a). At pH 7, free histidine and tyrosine elute off the column while aryl adducts are desorbed only at pH 12 (A). The pH 12 fraction can be resolved into at least three components on LH 20 columns (B). (Sugumaran et al., unpublished results.) Scheme shows the structure of histidine+atechol-chitin adduct (ZS),proposed by Schaefer et al., (1987) is also shown. formation of 28 was presumed to occur by the reaction of quinone of N-8alanyldopamine (4) with histidine ring nitrogens. T h e catecholic oxygen was assumed to form linkages with the chitin carbon atoms in the glucosamine ring (C-3, C-4 or other). However, as the authors admit, even with this

CUT1 C U L A R S C L E R O T I Z A T I O N

201

sophisticated technique, they could not derive the exact structure of the histidyl-catecholamine ring adduct, which illustrates the complexity of the chemical linkages in cuticle.

4.5

REACTIONS OF OTHER AMINO ACIDS

Cuticular proteins also contain other amino acids such as methionine, cysteine. proline. arginine and tryptophan which have side-chains capable of reacting with sclerotizing agents. Tryptophan content in cuticular proteins is insignificant and hence its contribution to the tanning reaction should be minimal or nothing. Moreover, the side-chain nitrogen of tryptophan forms part of the indole ring in this amino acid, which make it unavailable to react with quinones. Arginine, which has the guanido group on the side-chain, f'diled to react with quinones in model studies (Sugumaran et ul., unpublished results). Proline is an imino acid, and in proteins, the imino group is further deactivated by peptide bond formation. Since amide nitrogens do not react with quinones (Davies and Frahn, 1977), we can safely disregard proline as a potential candidate for crosslinking. Cuticular proteins in general contain a low titre of sulphur-containing amino acids. Even if cysteine is found in significant amounts, it readily forms cystine bridges and hence is diverted away from nucleophilic reactions. If cysteine did exist freely in cuticular proteins, it would be the most likely candidate to react with quinones because quinones add on to thiols at amazingly high rates. This reaction is known to play a major role in pheomelanin biosynthesis (Thomson, 1974; Prota and Thomson. 1976). Numerous studies on the reactions of cysteine residues with quinones have been conducted in the past and a voluminous literature is available on such additions. Cysteine itself reacts most readily with dopaquinone-forming cysteinyl dopas as well as dicysteinyl dopas (lto and Nicol, 1975; Ito and Prota, 1977; Ito et d., 1979). The addition occurs even o n protein-bound cysteine residues (Kato rt d., 1986). However. i t is disappointing to learn that this amino acid is found only in low levels in cuticular proteins. Methionine also reacts with quinones in a similar fashion (Vithayathil and Murthy, 1972; Vithayathil and Gupta, 1981), but again its level in cuticular proteins is too low to make its contribution significant for quinone tanning.

4.6

MELANIZATIONAND ITS PREVENTION

Melanins are high molecular weight pigments formed by the oxidative polymerization of simple catechol derivatives (Blois, 1978; Prota and Thomson.

202

MANICKAM SUGUMARAN

1976). The most widely studied melanins are eumelanin, derived from the polymerization of dopa (29)and pheomelanin formed by the polymerization of cysteinyldopas. To a certain extent the catecholamine derivatives are committed to melanin biosynthesis in some cuticle. Dopa (29)(and dopamine (30)) may constitute either the main or the sole starting material for insect melanins. The well-established pathway for melanin biosynthesis from 29 calls for the cyclization of initially formed dopaquinone (31). Following this, numerous nonenzymatic transformations take place to finally yield 5,6-dihydroxyindole (32)and melanin pigments (Fig. 17). While experimental evidence indicates that some of these transformations may be driven Fzister by enzymes, it is generally agreed that abiological and biological transformations follow exactly the same pathway. Dopamine (30)follows a similar route and gets converted to 32 before forming melanin. Since these catecholamine derivatives are found in cuticle, their enzymic oxidation can result not only in melanin production but also in adduct formation with cuticular components. In order to test this possibility, we

32

Fig. 17 Mechanism of melanin formation from dopa (29). Dopaquinone (31) formed by the action of tyrosinase on dopa (30) undergoes rapid intramolecular cyclization to produce leucoaminochrome which undergoes further transformation to produce 5,6-dihydroxyindole(32).Compound 32 gets converted to melanin through its quinonoid derivatives.

203

CUTICULAR SCLEROTIZATION

conducted model sclerotization studies with 29, 30 and norepinephrine (33) (Hasson and Sugumaran, 1987; Sugumaran ct d..1 9 8 7 ~ ) .We found in general, that quinones of 29, 30 a n d 33, due to the presence of an appropriately positioned internal nucleophile, v k . the side-chain amino group, preferentially underwent intramolecular cyclization, rather than reacting with external nucleophiles on protein. Hence, these three compounds d o not support protein polymerization, which explains the failure of Strout and Lipke (1974) to observe protein polymerization with 30. O n the other hand, their N-acetylated derivatives readily participate in protein crosslinking. From the above discussion, the biological significance of acylation of catecholamine derivatives used for sclerotization of insect cuticle also becomes clear. Although 29, 30 and 33 are converted to sclerotizing precursors in insects. they are not the immediate precursors of sclerotizing agents, as their quinones have the tendency to undergo internal reactions rather than forming adducts with cuticular components. Nature seems to have devised two ways to deactivate the reactive amino groups on these molecules. Removal of the amino group and conversion to 1 and 10 is one way to produce sclerotizing precursors that avoid intramolecular cyclization; acylation of these compounds to N-acetyl and N-8-alanyl derivatives is the alternate way. (Section 2.3: Fig. 3). As shown in Fig. 18, N-acylation of 30 not only reduces the nucleophilicity of the amino group, but makes the resultant amide nitrogen positively charged. The quinone of such a compound cannot undergo intramolecular cyclization but can only react with external nucleophiles. Accordingly. both Peter ct id. (1985) a n d Korytowski et (I/. (1987) did not find any cyclization of 17. From these considerations it is clear that sclerotizing precursors d o not indolize and hence crosslink formation from their indolized quinones is not possible.

3

3

17

Fig. I8 Prcvcntion of indolc ring formation by acylation of dopamine. The amidc group of N-acyldopamincs can be compared to peptidc bonds of proteins. Like the peptidc-bound nitrogen, the amide nitrogen in 3 ( R = C H , ) is deactivated by resonance; hence it cannot participate in intramolecular cyclization. Therefore, adduct formation between cuticular components and indolized quinones derived from sclcrotizing prccursors such as 3 and 4 is not possible.

204

4.7

M A NI C KAM S U G U M A RA N PEROXIDASE PARTICIPATION IN QUINONE TANNING

As stated in Section 2.4, peroxidase participation in quinone tanning is a possibility which has never been considered before. In order to understand the role of peroxidase in quinone tanning, we carried out some in vitro studies with horseradish peroxidase, test proteins, catechols and hydrogen peroxide (Hasson and Sugumaran, 1987). Peroxidase-mediated protein polymerization strictly required the presence of catechols such as 1 or 3. Figure 19 gives the time-course of polymerization of lysozyme in a reaction mixture containing 1. H,O,, peroxidase and lysozyme. During the enzyme-catalysed poly-

TIME

100

-

1 MIN.

n

0

I00

< u

*

n

o

-

100

15 M I N .

n

0

n

n

in._, 30 MIN.

lo:

e (10 M I N .

lo0 o

TRIMER

MONOMER

TETRAMER

DIMER

Fig. 19 Peroxidase participation in in vitro quinone tanning. A reaction mixture containing lysozyme, hydrogen peroxide, 3,4-dihydroxybenzoic acid (1) and catalytic amounts of peroxidase was incubated for indicated time intervals and subjected to SDS-polyacrylamide gel electrophoresis. The gels were stained and various polymer bands appeared, were quantified using a densitometer. (Note the appearance of dimer, trimer and tetramer bands with progress of time.) (Modified from Hasson and Sugumaran, 1987.)

CUTICULAR SCLEROTIZATION

205

merization, quinone formation could be witnessed by visible spectroscopy. Amino acid analysis of polymerized proteins revealed the absence of dityrosine-type crosslinks, confirming our contention that the observed polymerization was due to quinone tanning and not due to dityrosine crosslink formation. Using radioactive 3, we could obtain evidence for the formation of both intermolecular and intramolecular crosslinking of lysozyme. While these studies certainly attest to the ability of peroxidase to participate in quinone tanning. in vivo occurrence of such a reaction awaits confirmation.

5 [j - S c Ie rot iza t ion The first indication for the operation of an entirely different type ofcrosslinking process came from the studies of Andersen and his co-workers. Andersen ( 1970)isolated a ketocatechol from the cuticular hydrolysates of Schisrocrrca grquria, Nqdirotomu suturulis. Culliphora eryrhrocephalu, Hyulophora cecropiri, Pcriplanc~raurnericana and Pieris hrassicae and identified it to be arterenone (2-amino-3',4'-dihydroxy acetophenone) (34). Subsequently, his group isolated and characterized additional catechols viz., N-acetylarterenone ( 3 9 , N-acetylnorepinephrine (8), norepinephrine (33), 3,4-dihydroxyphenylglycol (36), 3,4-dihydroxyphenylglyoxal (37) and 2-hydroxy-3',4'-dihydroxyacetophenone (38) from sclerotized tissues (Andersen, 197 I ; Andersen and Barrett, 1971; Andersen and Roepstorff, 1978). From these studies, it was clear that somehow the side-chain of the sclerotizing precursor is utilized for crosslinking cuticular components; and the crosslink structure 39 was proposed to account for this mode of tanning. Further studies confirmed the occurrence of this process in a number of other organisms as well (Andersen, 1970, 1972, 1974, 1975, 1976, 1977, 1978, 1979a; Andersen et al.. 1981; Barrett, 1977, 1980). In support of this process, Andersen ( 1 974) also obtained evidence for the release of tritium from the side-chain of radioactive 3 during enzymic oxidation and termed this process as 8-sclerotization. During these studies, Andersen's school (Andersen, 1972) isolated a dimeric product of 3 which produced equimolar amounts of 30 and 38 upon acid hydrolysis with 1 N HCI for 3 h at 100 C. However, if the hydrolysis was performed with 6N HCI for only 30 min, it gave a mixture of 3, 34 and 35. Based on these initial studies, structure 40 was proposed for the dimer. However, with the use of modern spectroscopic techniques, the structure of the dimer was finally established to be 41 (Andersen er ul., 1980). The structure of some of these compounds are illustrated in Fig. 20. Since several such dimers could be isolated from cuticle (Andersen and Roepstorff, 198I ) the same biochemical process which causes the dimer production was assumed to be responsible for observed sclerotization reactions and a mechanism for this mode of sclerotization was proposed

MANICKAM SUGUMARAN

206

-..

&i

bn

34

30

36

36

38

37

40

CH&ONH

n

$

y

d

n

~

;

~

~

;

OH

41

Fig. 20 Structures of some catecholic products associated with /hclerotization.

(Andersen and Roepstorff, 1982; Andersen, 1985) which will be called a$sclerotization hereafter. 5.1 GQ-SCLEROTIZATION

According to this mechanism, sclerotizing precursor 3 is enzymatically activated by a desaturase found in the cuticle to produce the dehydro compound 12. Cuticular phenoloxidase then oxidizes 12 to its corresponding quinone, 42. Reaction of 42 with 12 and 3 produces the dehydro dimer 43 and saturated dimer 41 respectively (Fig. 21). Further, 42 was assumed to react with cuticular components through its side-chain with the regeneration of catecholic structure to account for the use of the aliphatic side-chain in the crosslinking process (Andersen and Roepstorff, 1982; Andersen, 1985). The above proposal has several drawbacks which are summarized in a recent review (Sugumaran, 1987b). The most serious drawback is its mechanis-

C UTIC U LAR SC L E R O T lZATlO N

207

SCLEROTIZATION

Fig. 2 I Mechanism of sc,/j-sclerotization. N-Acetyldopamine (3) is converted by a cuticular desaturdse to 12. Cuticular phenoloxidase oxidizes 12 to its quinone (42) which is believed to be the causative agent for b-sclerotization. Its undefined reaction with cuticle generates /I-crosslinks. Its coupling with parent compound 12 seems to produce the observed dimer (43). (Adopted from Andersen and Roepstorff, 1982; Andersen. 1985.)

tic inability to account for crosslink formation with cuticular components. For instance, this mechanism does not elaborate how quinone (42) reacts with chitin and proteins through its side-chain. Again, it is not clear why 42 cannot exhibit simple 1,4-addition reactions on the ring. Finally it does not account for the enzymic oxidation of compounds which cannot form a double bond in the side-chain such as 10 and 25. In order to critically evaluate the cx,B-sclerotization mechanism we needed large amounts of 12. This compound was isolated originally from locust cuticle by alkali treatment, which is deleterious to catechols (Andersen and Roepstorff, 1982). Hence, instead of trying to isolate this novel intermediate from a biological source, we decided to synthesize it chemically. Using the scheme of reactions outlined in Fig. 22, 12 was synthesized (Ramamurthy and Sugumaran, 1987). Synthetic 12 exhibited the same physical and spectral properties as those reported for the biological isolate (Andersen and Roepstorff, 1982). In addition, synthetic 12 proved to be stable and did not decompose spontaneously as suspected earlier (Lipke ei al., 1983). If, as i t has been claimed, 12 is generated freely in cuticle, incubation of cuticular phenoloxidase with radioactive 3 should produce the radioactive 12 as a transient intermediate. The trace amounts of radioactive dehydro compound thus formed can be diluted by exogenous addition of the cold compound and its further oxidation can be prevented by arresting the reaction. Re-isolation of the dehydro compound from the reaction mixture and rddio-

MANICKAM SUGUMARAN

208 0

44

0

45

46

Y

12

0

48

t

47

Fig. 22 Synthesis of 1,2-dehydro-N-acetyldopamine(12) starting from dimethoxy cinnamic acid (44)(From Ramamurthy and Sugumaran, 1987.)

Fraction Number Fig. 23 Radioactive trapping experiment to characterize the intermediary formation of 1,2-dehydro-N-acetyldopamine (12). Cuticular phenoloxidase from S. bulluta was incubated with 10 pCi of [1ing-~H]-3(specific activity 100mCi/mmol) in 300 pl of 25mM sodium phosphate buffer, pH7.0. After exactly 15s, 1 mg each of 3 and 12 were added to the reaction mixture and the reaction was arrested by the addition of 3 0 0 ~ of 1 10 M acetic acid. The contents were filtered and chromatographed on a Biogel P-2column (30 cm x 1.5 cm) using 0.2 M acetic acid as the eluant. Fractions of 3.5 ml were collected and an aliquot from each tube (1 ml) was used for radioactive measurements. Peaks at fractions 14, 17 and 31 are due to 8, 3 and 12 respectively. (From Sugumaran 1986b, 1987a, 1988a.b.)

CUT1 C U L A R S C LE R O T l Z A T l O N

209

active analysis will then confirm the intermediary formation of this compound if it is radioactive. Figure 23 gives the results of such a trapping experiment (Sugumaran, 1986b, 1987a). Clearly no radioactivity was trapped in 12, thus ruling out this compound as a freely generated intermediate. However, the generation of radioactive 8 (Fig. 23) confirms the participation of the CI and not the B carbon (with respect to ring) of the side-chain of 3 in the oxidation process (Sugumaran, I986b, 1987a, 1988a,b).

5.2

OUlNONE METHIDE SCLEROTIZATION

The reactive species responsible for P-sclerotization remained elusive for nearly a decade because of the general assumption that the course and the mechanism of the reaction catalysed by cuticular phenolase is identical to that of other phenoloxidases such as mushroom tyrosinase. While mushroom tyrosinase is known to catalyse the oxidation of catechols to o-benzoquinones, it also performs certain unique reactions involving quinone methides as intermediates (Fig. 24). Thus, during the oxidation of 3,4-dihydroxyman-

0-

0-

A. OH

10

6;

H H

0

52

n+

53

CHO

OH

$on on

0

d4 50 61

49

Fig. 24 Proposed mechanism for the oxidation of 3,4-dihydroxyphenylacetic acid (10) to 3,4-dihydroxymandelicacid (49) to 3,4-dihydroxybenzaldehyde(50). Tyrosinase converts 10 to 52 which is highly unstable and probably gets converted to 53 before yielding 49. Earlier studies have reported the conversion of 49 to 50, viz. 51 (Sugumaran, i986a). Chemically synthesized 52 also produces 49 (Sugumaran et af.,

1988d.)

21 0

M A NI CKAM SUG UMARAN

delic acid (49), instead of converting it to the corresponding quinone, tyrosinase oxidized it to 3,4-dihydroxybenzaldehyde(50) through the quinone methide intermediate (51) (Sugumaran, 1986a; Sugumaran and Lipke, 1984). Recent studies from our laboratory indicate that suitably substituted quinones can isomerize to quinone methides. Thus, carboxymethyl-o-benzoquinone (52) formed from 3,4-dihydroxyphenylaceticacid (10) by tyrosinase action, is notoriously unstable and undergoes tautomerization to yield the quinone methide 53. The carboxylate and the quinone ring of 52 make the methylene side-chain acidic (active methylene group). This leads to the ionization of the methylene group and eventual transformation of 52 to 53. Quinone methide 53, being unstable, undergoes rapid hydration to produce 49. We have also confirmed this novel transformation of 52+53+49 in a totally non-enzymatic system (Sugumaran et al., 1988d). Thus it appears that enzymes capable of synthesizing quinones from 4-substituted catechols can produce tautomeric quinone methide as well, provided the suitable substrate is given. In addition it is also likely that substituted quinones can undergo isomerization to quinone methides.

1.2

W

V

,z

0.8

m

a

0 v)

m U

0.4

350

400

450

500

550

WAVELENGTH (nm) Fig. 25 Spectral changes occurring during the oxidation of catechols with mushroom tyrosinase (m-series) and cuticular polyphenol oxidase (c-series). Substrates used: ( I ) catechol; (2)4-methylcatechol;(3) N-acetyldopamine; (4) 3,4-dihydroxyphenethyl alcohol; and (5) 3,4-dihydroxyphenylaceticacid. Assay conditions: I mM substrate in 1 ml 50 mM sodium phosphate buffer (pH 6.0) and crystalline mushroom tyrosinase (I 8 pg) or cuticle powder (25 mg). (From Sugumaran and Lipke, 1983b.)

C U T 1C U L A R S C L E R O T l Z A T l O N

21 1

Indication for quinone methides production by cuticular phenoloxidase came from spectral studies associated with the oxidation of 4-alkylcatechols, such as 3,9, 10 and 25 (Sugumaran and Lipke, 1983a, b). During the mushroom tyrosinase-catalysed oxidation of these compounds, generation of quinones as the primary products of oxidation can be witnessed by monitoring the increase in absorbance at the visible region of spectrum (Fig. 25). However, a similar study with sarcophagid larval cuticular phenoloxidase failed to provide such visible spectral changes indicating that products other than quinones are formed in the reaction mixture. Chemical considerations indicted the generation of quinone methides rather than conventional quinones as the products of alkylcatechols. Although 4-methylcatechol (25) oxidation by the insect enzyme resulted in quinone (27) production as well, HPLC analysis of the reaction mixture revealed the presence of 3,4-dihydroxybenzaldehyde(50) and traces of 2 as additional products in the reaction mixture (Fig. 26). While quinone forma1

l

I

7.15

OK)

I

A

0

TIME ( m i d Fig. 26 HPLC analysis of cuticular phenoloxidase-catalysed 4-methylcatechol (25) oxidation. A reaction mixture containing S. bulfutu larval cuticle (500 mg) and 100pmol of 25 in sodium phosphate buffer, p H 7.0 was incubated for an hour and an aliquot was subjected to HPLC as outlined by Sugumaran and Lipke (1983b) (Trace A). The cuticle from the above reaction was isolated, washed and hydrolysed with 6 N HCI. The catechols liberated were analysed on HPLC (Trace B). The peak at 12.72 min has been characterized to be quinone (27). (From Sugumaran and Lipke, 1983b.)

21 2

MANICKAM SUGUMARAN

tion does not account for the generation of 50, the tautomeric quinone methide 54 readily explains the aldehyde synthesis (Fig. 27). Furthermore, cuticle treated with 25 showed the presence of covalently bound catechols when tested with Arnow’s ( I 937) reagent-a property consistent with production of quinone methides. Quinone methide 54 can react with cuticle by 1,6Michael addition to give catechokuticle adducts with the regeneration of the o-dihydroxyphenolic group. Such cuticle upon acid hydrolysis released 50 as the major product confirming our contention that 54 is involved in the oxidation process. Mechanistic studies on the enzymatic oxidation of 10 provided further proof for quinone methide production. Like 25, this compound cannot form

CHZCOOH

H

H

OH OH

0

OH

10

54

26

CH2OH

0

OH

OH

63

2

60

CHOH COOH

@OH OH

49

q

0 :

H

51

Fig. 27 Proposed mechanism for the oxidation of 4-methylcatechol (25) and 3,4dihydroxyphenylaceticacid (10) by cuticular phenoloxidase. Cuticular phenoloxidase from S. bullata oxidizes 25 to quinone methide 54,in addition to 27. Hydration of 54 yields 2 which is further oxidized by the enzyme to 50, viz. 51. Compound 54 also reacts with cuticular components to yield 3,4-dihydroxybenzylderivatives (not shown in figure) which on acid hydrolysis yield 50. The same enzyme oxidizes 10 to either 53 or 54. Both 53 and 54 are converted to 50 as shown.

CUT1 C U L A R S C L E R O T l Z A T l O N

21 3

a double bond on the side-chain, yet exhibits reactions characteristic of Bsclerotization. On treatment with cuticular phenoloxidase, this catechol did not produce any detectable quinone (52) (Fig. 25). However, it readily generated 50 as the major product (Fig. 28). The bulk of the substrate oxidized was bound to cuticle with the retention of o-dihydroxyphenolic function. These cuticular preparations also liberated 50 upon acid hydrolysis. Taking quinone methide as the central intermediate, the scheme of reactions shown in Fig. 27 accounts for the products observed on HPLC. Final proof for quinone methide production comes from tritium release studies using specifically tritiated 3. The principle behind this experiment is illustrated in Fig. 29. As indicated, generation of quinone methide 55, 12 and 17 from specifically labelled 3 by cuticular phenoloxidase requires selective release of tritium from the substrates. Therefore, by measuring the radioactivity released into water during enzymatic oxidation, one can identify the actual intermediate formed. With [w3H]-3 as the substrate, tritium release will be observed when both 55 and 12 are formed. However, if [P-3H]-3 is

r

I 0

I

,

I

2

4

6

8

10

Time (mid

Fig. 28 HPLC of products of 3,4-dihydroxyphenylaceticacid (10) oxidation by cuticular phenoloxidase from S. bullatu. A reaction mixture containing 1 pmol of 10 and 50 mg of cuticular phenoloxidase in 2 ml 50 mM sodium phosphate buffer, pH 6.0, was incubated at room temperature. At zero time (IOpl, trace I) and after 2 h of incubation ( 2 0 ~ 1trace , 11) supernatant from the reaction mixture was subjected to HPLC as outlined by Sugumaran and Semensi (1987). (From Sugumaran, 1987b.)

214

MANICKAM SUGUMARAN

a CIIT C H 2 NHCOCHj

BINDING TO -on

CUTICLE

-OH 0

~nco cn3 FURTHER

-rHTO on

REACT 10N S

@OH

3 CH2CH2 NH CO CH3

12 CH 2 CH2 NHCOCHj

T 0

3

HTO

BINDING TO CUTICLE THROUGH THE RING

17

Fig. 29 Proposal for characterization of reactive intermediates formed from N-acetyldopamine (3). Specifically tritiated 3 can be used to establish the nature of reactive species formed by cuticular phenoloxidase action. The formation of 55 and 12 from 3 is accompanied by specific release of tritium from [ u - ~ H ]into - ~ water. However, if one uses [fi-3H]-3 as the substrate, only the formation of 12 will accompany tritium release into water. Finally, if [ring3H]-3 is used, only when the corresponding quinone (17) binds to the cuticle through the ring, will release of tritium be observed.

used as the substrate, only 12 formation will release tritium. Figure 30 gives results of such an enzyme-catalysed tritium release from selectively tritiated 3. Since enzyme-catalysed tritium release was observed only with [w3H]-3 and not with [b-3H]-3, quinone methide 55 and not 12 generation is inferred. Moreover, tritium was not released from [ring-3H]-3. As the enzymecatalysed oxidation was accompanied by the covalent binding of 3 to the cuticle and neither 17 nor 12 accumulated in the reaction, the above results prove that quinone methide 55 and not 12 or 17 was formed as the initial product of oxidation of 3 by chitin-bound cuticular phenoloxidase of S . huflura larvae. We have demonstrated that quinone methide production occurs not only in S. hullata, but also in other insects such as M . sexta, D . melanogaster, P . americana and A . aegypti (Sugumaran and Semensi, 1987; Sugumaran, 1987c, 1988a; Sugumaran et al., 1988e). Thus, quinone methide formation

21 5

CUTICULAR SCLEROTIZATION

a

0

15

30

Time ( m i d 4 Fig. 30 Time course of tritium release from (A) [cr-jH]-, (B) [/3-3H]- and (C) [ring3H]-N-acetyldopamine catalysed by cuticular phenoloxidase. Sarcophagid cuticular phenoloxidase (25 mg) was incubated with 1 pCi of indicated tritiated 3 in 25 mM sodium phosphate buffer, pH 7.0 and the enzyme-catalysed tritium release was followed as outlined by Andersen (1974). (From Sugumaran, 1988b.)

does not seem to be a mechanism confined to a single species of interest, but is of widespread occurrence. Quinone methides are relatively short-lived, highly reactive intermediates (Wagner and Grompper, 1974). They react rapidly with any nucleophiles in the vicinity and form 1,6-addition products with the regeneration of the benzenoid ring (Fig. 31). The available nucleophiles in cuticle are the &-amino group of lysyl residues, the imidazoyl group of histidine residues, the hydroxyl groups of chitin backbone and serine and threonine residues, the carboxyl groups of aspartic and glutamic acids, the thiol group of cysteine, the thioether group of methionine, N-terminal and C-terminal residues of proteins and free amino groups of chitin. These reactions convert their unstable quinonoid ring structure to a stable benzenoid structure. Therefore, they are highly favoured over other reactions. It is this property that makes the quinone methides t he attractive sclerotizing agents for p-sclerotization. The quinone methide+xticle adducts formed will not have any absorption in the visible region in accordance with the property of related catechols. There-

21 6

MANICKAM SUGUMARAN

69 Fig. 31 Reactions of quinone methide with cuticular components. Quinone methides formed in cuticle by the action of phenoloxidase on catecholic derivatives such as 3,4,9or 10 can react with hydroxyl groups to produce ethers (S),cysteine residues to produce thioethers (57), methionine to produce the derivative (B),carboxyl groups to yield esters (59), amino groups to give secondary amines (60), and histidine and Nterminal prolines to give tertiary amine (61) type compounds. If no nucleophile is available, quinone methides can react with water (ROH = H,O) to give side-chain hydroxylated compounds (R= H in 56). The reactions are depicted for N-acetyldopamine quinone (55) (X= CH,NHCOCH,). Other quinone methides derived from 4 (X=CH,NHCOCH,CH,NH,), 9 (X=CH,OH) and 10 ( X = H or COOH) will also react the same way. fore, they will acknowledge the formation of sclerotized but colourless cuticle. Such cuticle-catechol adducts upon acid hydrolysis can release the catechols like 33-38. In addition, quinone methides can add on to water to yield side-chain hydroxylated compounds (Fig. 32). Thus hydration of 55 yields 8. Since this reaction is non-enzymatic, it is non-stereo selective and therefore yields both d and 1 isomers of 8 (Peter, 1980; Peter and Vaupel, 1985). Similar conversion accounts for the formation 7 from 4 (Sugumaran et a/., 1986; Morgan et af., 1987). Quinone methide 55 can also tautomerize to

21 7

CUTICULAR SCLEROTIZATION

41

43

Fig. 32 Proposed metabolism of N-acetyldopamine by quinone methide route. Cuticular phenoloxidase converts 3 to 55 rather than 12. Isomerization of 55 leads to the generation of 12 and hydration of 55 produces 8. The bulk of 55 participates in quinone methide sclerotization. Traces of 12 formed undergoes further oxidation to produceeither43or41.

12 in cuticles such as Locusta migratoria cuticle, accounting for Andersen's findings (Fig. 33). But this is a minor pathway (Fig. 32). Some of the adducts ( 5 M 1 ) on alkali treatment can undergo /%elimination and produce 12. Traces of 12 formed can be oxidized by phenoloxidase to produce dehydro dimer 43. Mushroom tyrosinase also catalyses this conversion (Sugumaran et al., 1987a, b). We could not visualize the formation of any 42 during the reaction, but had spectral evidence for 63, 64 or 62. Based on our observations, we proposed a quinone methide route for dimerization (Fig. 33). The same route can account for the saturated dimer 41 formation. We have also observed these transformations by a nonenzymatic route as well. (Sugumaran et al., 1988a). Thus, the quinone methide mechanism accounts for p-sclerotization much more precisely than the cr,f?-sclerotizationmechanism.

6

Differential mechanism of tanning

In all of the above mechanistic proposals, a single sclerotizing precursor was considered for different tanning pathways. However, recent studies from our

MANICKAM SUGUMARAN

21 8

NMCOCM,

I

b

@OM

QM

OH M+

12

\ s s

Fig. 33 Mechanism of formation of 12 in cuticle and its further reactions with phenoloxidase. The formation of 12 in certain cuticle could be due to the isomerization of quinone methide 55 as shown, and is unlikely to be due to the action of a specific desaturase. Compound 12 can also arise from cuticle55 adducts by j-elimination during alkali treatment. Oxidation of 12 does not produce 42 but produces either the free radicals (63 and 64) or the quinone methide 62 (Sugumaran et al., 1987a, b). Coupling of 63 and 64 yields the quinone methide 65, which rapidly cyclizes to give 43. Alternatively 62 can react with 12 to give 65. Even if 42 is formed, it is unlikely to condense with 12 by Diels Alder-type reaction in the aqueous environment. (From Sugumaran el NI.1987b.)

laboratory indicate that two different compounds such as 3 and 8 can be differentially used for different tanning modes as well. Compound 8 is synthesized from 3 by cuticular phenoloxidase (Sugumaran and Lipke 1983a, b; Peter, 1980). Thus 8 is both structurally and biologically related to 3. Compound 3 is oxidized by cuticular phenoloxidase from S. hullutu to the quinone methide 55. On similar grounds we expected 8 to be converted to the corresponding quinone methide 66 and participate in quinone methide scler-

21 9

CUTICULAR SCLEROTIZATION

otization. Contrary to this expectation and quite surprisingly, cuticular phenoloxidase readily oxidized 8 to its quinone 67 (Fig. 34). HPLC studies indicated that no catecholic product(s) was formed in the reaction mixtures containing 8 and cuticular enzyme. Moreover, cuticular samples treated with 8 neither retained covalently bound 8 nor released any catechols on acid hydrolysis, confirming our contention that not even traces of quinone methide 66 are formed during enzymatic oxidation of 8. Although it could be argued that cuticular preparations contained an additional phenoloxidase specific for quinone production, it does not account for the unique conversions of 3 to its quinone methide 55 and 8 to its quinone 67. Phenoloxidases are well known for their non-specific action and presence of a quinone-producing enzyme should have produced 17 from 3 as well. Moreover, typical phenoloxidase inhibitors such as phenylthiourea, potassium cyanide, sodium fluoride and sodium azide inhibited the oxidation of 3 and 8 by cuticular enzyme to the same extent. Thus one enzyme seems to catalyse both the observed reactions.

L

350

55 0

410

WAVELENQTH

(nm)

Fig. 34 Differential mechanism of oxidation of N-acetyldopamine (3) and N-acetylnorepinephrine (8) by cuticular phenoloxidase from S . bullutu. Compounds 3 and 8 (0.25mg) were incubated with 125mg of sarcophagid cuticle in 5 ml of 0.25 M sodium phosphate buffer, pH 7.0 and the visible spectral changes associated with the enzymic oxidation were recorded at various time intervals. (A) Oxidation of 3 (traces 1 4 were recorded at 10-min intervals after starting the reaction). (B)Oxidation of8ttraces 1-6 recorded at 0.75, 1.25,2.5, 5, 10 and 15min after starting the reaction). (From Sugumaran et al., 1988b,c.)

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MANICKAM SUGUMARAN

The reason for this differential mechanism of oxidation becomes obvious if one considers the structure of the products (Fig. 35). Compound 66 is a n enol and should exhibit typical keto-enol tautomerization. Since the double bond of the enol portion is conjugated to the dienone structure of quinone methide, tautomerization accompanies rapid aromatization of 66 t o yield N-acetylarterenone 35 as the sole product. This reaction is undoubtedly in favour of 35 production. We have already reported a similar tautomerization (51-40) during the enzymic oxidation of 49 (Sugumaran, 1986a). Hence, even if 66 is formed, it will be converted t o 35 rather than forming adducts

-

P PO

cutlclo

3

-

Cutlolo Adduct

(p

PPO

n

0

Ho&iL

3

66

OH

0

OH

8

17

67

4

f PPO

,;go-,-

$

o

H-0

\

QUINONE O & f on

I0

Z!L

TANNlNa

H.

06

36

Fig. 35 Proposed mechanism for the differential mechanism of oxidation of Nacetyldopamine (3) and N-acetylnorepinephrine (8). Cuticular phenoloxidase (ppo) oxidizes 3 to its quinone methide 55 which reacts rapidly with cuticular components. Since no quinone or quinone methide accumulates in the reaction, there is no visible spectral increase in this case (see Fig. 34A). However, the enzyme oxidizes 8 to its corresponding quinone (67)which accumulates in the medium (Fig. 34B). Formation of quinone methide 66 is not favoured because 66 will readily isomerize to 35 which is a poor substrate for phenoloxidases (Sugumaran, 1986a). Therefore, its formation is a wasteful reaction for tanning. However, if 67 is formed it can participate in quinone tanning. Therefore, the observed differential mechanism of oxidation seems to be advantageous for the insect. (From Sugumaran et d., 1988b.c.)

C U T I C U L A R S C LE R O T l Z A T l O N

221

with cuticle. Compound 35 is a poor substrate for phenoloxidases (Sugumaran, 1986a) and hence it will be wasteful to synthesize it. On the other hand, if 8 is converted to 67, the resultant quinone at least has the potential to form quinone-type crosslinks. Thus the observed differential mechanism seems to be an advantageous strategy adopted by cuticular phenoloxidase to maximally utilize the available catecholamine derivative for crosslinking process (Sugumaran et a/., 1988b,c).

7 Combined pathway and crosslinking mechanisms From the mechanistic considerations detailed in previous sections, it is obvious that cuticular proteins and chitin fibres form adducts with sclerotizing agents. In this section crosslinking of two different components is considered. This could occur either by a concerted mechanism or by a sequential mechanism. While the concerted mechanism readily produces the crosslink without the intermediary formation of a protomer-sclerotizing agent adduct, the sequential mechanism demands its production prior to crosslink synthesis. The crosslink formed could either be protein-protein or chitinprotein or chitin-chitin in nature. During our studies on the tanning of dipteran cuticle, we observed that the sclerotized matrix was assembled probably by a concerted bridging of protomers without accumulation of dimer, trimer or higher oligomer in the ureasoluble fraction of cuticular proteins (Lipke et al., 1981). Willis et al. (1981) also reported a similar observation. From these results, it appears that crosslinking of cuticular components may occur by a concerted mechanism. Both quinone and quinone methide can function as bifunctional reagents and crosslink two different structural protomers. But chemical and mechanistic considerations demand that the crosslink process must proceed through a sequential mechanism with the intermediary formation of catechol-protomer adducts. The reason for this is the obvious protonation of the carbonyl group of quinones and quinone methides, which results in the regeneration of benzenoid structure and hence the catechol-monomer adduct formation (see Figs 13, 17 and 3 1). Although this process can be depicted as a simultaneous addition of both base and an acid to the bifunctional sclerotizing agent, the acid portion is always the omnipresent protons rather than any other groups. Thus mechanistic considerations totally discount trimolecular reactions of sclerotizing agents with cuticular components. If this is the case, then one should be able to observe the sclerotizing agentmonomer adducts. Evidence for this proposal comes primarily from model sclerotization studies (Hasson and Sugumaran, 1987; Sugumaran et al., 1987~).During in vitro model studies, with both phenoloxidase and peroxi-

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MANICKAM SUGUMARAN

dase, we readily observed the formation 3-lysozyme adduct prior to the formation of crosslinks (see Fig. 8 for example). Currently, we are searching for such adducts in insect cuticle. Preliminary studies reveal that such adducts are indeed present not only in Sarcophaga cuticle but also in other insect cuticle. While the initial reactions of sclerotizing agent with cuticular components yield the monomer adducts, further reactions ensure crosslink formation. Such crosslinks could be solely quinone- or quinone methide-type or a mixture of both. Radioactive experiments indicate that quinone tanning and quinone methide sclerotization operate together in several insect cuticles (Andersen, 1974; Sugumaran and Lipke, 1983c; Sugumaran and Semensi, 1987). Thus these two mechanisms seem to occur simultaneously and independently of each other or operate in an interdependent manner. The latter possibility, as suggested in our earlier review (Lipke et al., 1983),can produce additional crosslinks. Only future studies can shed light on this aspect. 8 Free radical formation A major aspect of catecholamine chemistry for cuticular sclerotization may involve one electron oxidation of the parent compound to produce semiquinones as the sclerotizing agents. Yet this important realization has been totally ignored in the study of cuticular tanning. Peroxidase presence in cuticle has been demonstrated (Coles, 1966; Locke, 1969; Sugumaran et at., 1982). Peroxidase is known to produce free radicals (Yamazaki et af., 1%0). If it participates in quinone tanning, one can then obviously expect the generation and subsequent reactions of semiquinones in cuticle. Studies with phenoloxidase and laccase also indicate the likelihood of free radical formation. Using ESR studies, Mason et al. (1961) first reported that the primary product of catechol(22) oxidation by tyrosinase is the quinone 24. However, in the same paper, they registered that 24 formed may undergo reverse dismutation with 22 non-enzymatically to yield semiquinones as the products (Fig. 36). Free radicals are transient in nature and rapidly decay via disproportionation to give back the catechol and quinone (Fig. 36). But, in insect cuticle, this reaction may be delayed to allow radical reactions with cuticular components. If this occurs in the insect cuticle then the reactions of semiquinones formed, with cuticular components may be more complex than those of either quinones or quinone methides. Free radical formation and their subsequent decay can be monitored by ESR spectroscopy. In the past, due to the limitation in their concentration, it has been difficult to study them. However, with the development of spin sta-

CUTICULAR SCLEROTIZATION

223

4 Further React ions 0

Fig. 36 Free radical pathway for the oxidation of catechols. Catechols are oxidized by phenoloxidases to quinones. Non-enzymatic reverse dismutation of quinones and catechols generates semiquinones which can react with cuticular components by a complex free radical mechanism. On the other hand, cuticular peroxidase and laccase can directly produce semiquinones and provide them for further reactions.

bilization techniques, it has now become easier to monitor their reactions. Using this technique, Peter et al. (1985) identified the corresponding semiquinones during the oxidation of 3 and 4 in aqueous solutions. Interestingly, they did not observe any Michael-type addition reactions of N-acyldopamine quinones. However, Korytowski et al. (1 987) conducted a careful ESR study of radicals formed during tyrosinase-catalysed oxidation of catechols and reported the formation of semiquinones from 25 and 3. Further, they identified the addition of water to quinones which resulted in the formation of hydroxylated catechols. These studies are thus in conformity with the reaction pathways of quinones. We have detected by ESR spectroscopy, the presence of free radicals other than melanins in the cuticles of S . bullata, D . melanogaster, P . americana and M . sextu (Sugumaran et al.. unpublished observation). Currently, we are investigating their chemical nature. This field is only in its infancy and one may expect numerous applications of ESR spectroscopy to the study of semiquinones with biological molecules in the coming years.

9 Concluding remarks

In this article, I have attempted to present some of the likely biochemical mechanisms responsible for sclerotization reactions observed in insects. This

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work does not represent an exhaustive survey of literature, yet it clearly demonstrates the complexity of sclerotization reactions. Even after four decades since the first proposal of quinone tanning hypothesis appeared in the biochemical literature, we still do not have a definite structural proof for any type of adduct or crosslink other than the dityrosine type. As outlined in this article, the reaction pathways are complicated not only because of numerous reactive groups in cuticle, but also because of the heterogeneity of sclerotizing agents and the formation of hybrid adducts and crosslinks. Solving the structure of adducts derived from sclerotizing agents and cuticular components requires determined action on the part of scientists from different disciplines to unify the resources and techniques for the study of sclerotization. If this could be achieved, elucidation of crosslink structures found in insect cuticle should be possible in the near future. Ac knowIedgement s

It is a pleasure to acknowledge the valuable assistance provided by Mr Brian Hennigan, Mr Victor Semensi, Dr Hemalata Dali, Dr Steven Saul, Dr B. Ramamurthy, Ms Teresa Rivera, Ms Linda Burgio, Mr Chad Hasson, Mr Wayne Mitchell, Mr Jim Durkin, Ms Shareda Hosein and Mr Charlie King. I thank Drs Gary Jacob and Jack Schaefer for recording the solid-state NMR spectra. The kindness of Professor J. P. Anselme in allowing us to use his infra-red and NMR spectral facilities is greatly appreciated. Financial support for this project was provided by grants from NIH (R01AI-14753) and University of Massachusetts at Boston (Healey, BRSG, Educations Needs and Faculty Development Grants).

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NOTE ADDED IN PROOF We have recently identified a 4-alkyl quinone: 2-hydroxy-p-quinone methide isomerase in S. bullata, M. sexra and other insects. This enzyme converts quinone such as 17, and 52 into quinone methides 55, and 53 respectively. Therefore, quinone methide formation from 4-alkyl catechols can occur either directly by the action of quinone methide generating phenoloxidase or indirectly by the combined action of quinone producing phenoloxidase and quinone methide isomerase. The latter possibility also accounts for the differential mechanism of oxidation of N-acetyldopamine and N-acetyl norepinephrine observed in Figures 34 and 35, as the isomerase causes rapid conversion of 17 to 55 but acts sluggishly on 67. Therefore 67 accumulates in the reaction mixture while 17 does not.

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Acetylcholine sensitivity, 50 Acetyldopamine-lysozyme adduct, 193 oxidation, 219,220 Acheta domesticus (cricket), 15 giant interneuron, 38,57 Aedes aegypti, 9 1, 140 AIDS infections, 125 Alpha-beta-sclero tization, 206209 Amino acids reactions in quinone tanning, 201 Anopheles quadrimaculatus, 105 Antheraea polyphemus (silkmoth), 24,26, 53,90,93,96, 181 Antibacterial proteins, 109-1 11 Aphanomyces astaci, 108 Aryl-histidine adducts, 198-201 Aryl-lysine crosslinks, 194-196 Ascaris lumbricoides (worm), I89 Axonal regrowth, 44,46,48 Axons giant, 38 regeneration of motor, 73 Axotomy, 39,54 secondary effects, 41 Bacillus cereus, 109 thuringiensis,92, 100, 124 Basement membrane, 139-143 haemocyte involvement, 141, 142-143 structure, 139-141 Beta-scholerotization, 205-21 7 and alpha, 20&209 and mechanisms, 207 quinone methide, 209-21 7 Biosynthesis, chitin, 183 Blaberus, 89, 184 craniifer, 102, 107 giganteus, 133 Blatta orientalis, 156

Blood-brain barrier, regeneration, 61-63 Bombyx, 93, 113, 114,118, 132 Butterfly (monarch) (Danaus plexippus), 18

Caenorhabdites elegans, 26 Calcium ions, in axonal sprouting, 46,47 Calliphora, 88, 90, 93, 98, 109, 1 18, 142, 184 Calpodes ethlius, 187 Capsules, 104 haemocytic, 154 Carbohydrate composition, 144 Carboxyl groups, and quinone tanning, 196197 Carcinus maenas, 108 Catecholamine derivatives, acylation of, I84 Catechols, 1 8 4 185,223 Cells (see also Haemocyte) crystal, 88 exogenous, 58-7 recruitment during glial repair, 6371 ganglion mother (GMC), 3 granular, 88 plasmatocytes (PLS), 87-88 prohaemocytes (PRS),87 Schwann, 5 1,73 sessile, 88-89 spherule, 88 Central nervous sytem degenerative responses, 3 H O development, hormonal approaches, 1-34 endocrine (hormonal) regulation, 712 postembryonic, 4-7 patterns of, 2-7 embryonic, 2 4

234

‘Chemoaffinity model’, 57 Chitin, 182-183 biosynthesis, I83 Coagulocytes (COs), 88 Coagulogen, 131 Cockroach abdominal nerve cord, 63 connectives, 61, 65,67,68,69, 71 coaxal muscles, innervation of, 5 1,54 giant axons, 38 haemocytes, 66,67, I5 I (Leucophaea maderae). 14,37,53 metathoracic nerve, 49, 52 Periplaneta americana, 40,41 Connective shortening, 13 in cockroach 61,65,67,68,69,71 Coaxal muscles, cockroach, innervation of, 51, 54 Crayfish (Procambarus clarkii),40 Cricket (Acheta domesticus), 40 giant interneuron 38,57 Cryllus bimaculatus 56 Crosslinks aryl-lysine, 194-1 96 dityrosine, 197-1 90 pathway mechanisms, 221-222 Crustacea, 100 Crystal cells, 88 Cuticle, 132, 180 components of sclerotized, 181-187 catechols, 184-185 chitin, 182-183 enzymes, 186187 structural proteins, 181-182 peroxidase in, 222 Cuticular sclerotization, molecular mechanisms in, 179-230 (see Sclerotization, cuticular) Danausplexippus (monarch butterfly), 18 Defence mechanisms haemocytic, 99-1 12, I25 encapsulation, 103-107 killing mechanisms, 107-109 nodule formation, 102-103 phagocytosis, 99- 102 humoral, 109-1 12 antibacterial proteins, 109-1 11 serum lectins, 1 1 1-1 12

INDEX

Degenerative responses, in neural repair, 3M3 Dendritic sprouting, 46 Dictyoptera, 94 Diptera, 94, 105 Diphenoloxidase, I86 Dityrosine crosslinks, 187-190 Drosophila melanogaster (fruit tly), 4, 7, 12, 58, 75, 90, 91, 93, 102, 139, 140,141, 142,181, 189, 194 Ecdysis, 17 Ecdysteroids, 7,8,9, 12, 13, 16,23,26 effects on infants CNS, 28 haemolymph titres, 10 Embtyonic development, and hormones, 12-13 Embryonic nervous sytem, development, 2-4 Encapsulation, 103-107 recruitment and cessation, 148-1 55 and wound-healing, 148 Endocrine (hormonal) regulation, in insect development, 7-12 Endocrine manipulation techniques, 1012 Endopterygota, 93, I31 Enzymes, in cuticle, 1 8 6 187 Euxoa declarata 93,94, 1 16 Exogenous cells and neuroglia, 58-72 reactive, 59-61 recruitment during glial repair, 63-71 Exopterygota, 92-93 Extirpation-replacement paradigms, 1 1 Fat-body, lepidopteran, 110 Fly flesh (Sarcophaga bullata), 58, 112, 133, 146, 181, 187, 192, 194, 195, 21 1 fruit (Drosophilia melanogaster), 4, 7, 12, 58, 75, 90, 91, 93, 120, 139, 140, 141, 142, 181, 189, 194 Musca, 58 Free radical formation, 222-223 Fruit fly (Drosophila meIanogaster), 4, 7, 12, 58, 75, 90, 91, 93, 120, 139, 140, 141, 142, 181,189,194

INDEX

Galleria mellonellu (wax moth), 13, 21, 89,95,97, 104, 110, 113, 119, 120, 138, 147, 150 Ganglion abdominal, in hawkmoth, 4 migration, 13-14 mother cell (GMC), 3 Giant axons, 38 Giant interneuron, 38,57 Gin-trap reflex, 17 in Manduca sexta, 15, 16 Glial repair, 72 long-term changes in, 71-72 recruitment during, 63-71 Granule-containing cells, 37,88 Grasshopper (Laplatacris disper), 13, 38, 75 embryo, 3 Gryllus bimaculutus (black cricket), 56, I10

Haemocyte, 60 behaviour, 8 6 1 78 adhesion and locomotion, 133 alterations in recognition and response, 155-1 57 changes in population, 116125 count, 117, 118, 123 criteria for characterizing, 89-91 densities, 98 in vitro culture, 95-96 involvement of basement membrane, 141, 142-143 involvement in wound-healing, 125138 lineages, 9 6 9 7 origin and longevity, 91-99 phenoloxidase and prophenoloxidase-activation system, 112-1 16 recognition of non-self, 138-148 recruitment and cessation, 148-1 55 unstimulated, 133-1 36 classes circulating, 87-88 coagulocytes (COs),88 crystal cells, 88 oenocytoids, 88 granular cells (GRs), 88 mitosis in, 93-95

235

plasmatocytes (PLs), 87-88 prohaemocytes (PRs), 87 spherule cells, 88 thrombocytoids, 88 sessile, 88-89 cockroach, 66,67,51 Haemocytic capsules, 104 Haemokinin, 132 Haemolymph, 114, 133, 134 Haemopoietic organs, 91-93 endopterygota, 93, 131 exopterygota, 92-93 Hawkmoth (Manduca sexta), 4 abdominal ganglion, 28 gin-trap reflex, 15, 16 neuroblasts in, 8 neurogenesis in, 7 20-HE, 25 Heliothis viriscens, 105, 107, 149, 150 Hemimetabola, $ 6 Holometabola, 5 Hormonal regulation of insect development, 7-1 2 of neurite outgrowth, 22-23 Hormones ecdysteroids, 7,8,9, 12, 13, 16,23,26 and embryonic development, 12-1 3 and postembryonic development, 1326 juvemile (JH), 7,9, 13, 15, 16,21 Humoral defence mechanisms, 109-1 12 antibacterial proteins, 109-1 11 serum lectins, 111-1 12 Hyalophora cecropia pioneering studies, 26, 110,119, 135 Hymenolepsi diminuta, 1 12 Hydroxyl groups, and quinone tanning, 190 Hyperecdonism, 1 I Infection, 121-122 AIDS, 125 Insect developmental status of haemocyte in, 1 1 6 1 19 endocrine (hormonal) regulation in development, 7-12 regenerative responses of neurons, 43-58 stress in, 119

INDEX

236

Interneurons, 4 6 4 9 Isodityrosine, 190

reactions with cuticular components, 216 Milkweed bug (Oncopeltus faciatus), 14,

Juvenile hormones (JH), 7, 9, 13, 15, 16, 21

Molecular mechanisms in cuticular sclerotization, 179-230 Molecules in solution, 124-125 Moniliformis, 156 Moth, wax (Galleria mellonella), 13, 21, 89,95,97, 104, 110, 113, 119, 120, 138,147, 150 Motor neurons, 49-55 abdominal, 20 Moults, 8 Musca, 58

Lamellocytes, 87 Laplatacris disper (grasshopper), 13, 38, 75 Larval neurons death of, 2 6 2 6 restructuring, 19-23 Lectins, serum, 11 1-1 12 Leishmania hertigi, 108 Lepidoptera, 9,93,94 Lepidopteran fat-body, 110 Leucophaea maderae (cockroach), 14,89, 130 Limulus, 1 14 Locust (Schistocerca gregaria), 6, 38, 90, 92, 103, 114, 115, 121, 128, 134, 143, 144, 150 Locusta, 4,41, 131 embryos, 3 migratoria, 10, 12, 182,217 Lysine -aryl crosslinks, 194-196 quinone adducts, 195, 199 Malcosoma disstria, 96 Marmestra brassicae, 96 Manduca sexta (hawkmoth), 4, 5, 9, 10, 18, 20, 21, 22, 23, 24, 26, 58, 89, 114, 121,131, 133,146, 184, 199 abdominal ganglion, 28 gin-trap reflex, 15, 16 neuroblasts in, 8 neurogenesis in, 7 Manipulation techniques, endocrine, 1012

Melanins, 201 formation of, 202 Melanization, 1 13 prevention, 201-203 Metamorphosis, 7 Metarhizium anisoplae, 92, 142 Metathoracic nerve, 49, 52 Methide, quinine sclerotization, 209-21 7

15

N-acetylydopamine-lysozyme adduct, 193 oxidation, 219,220 Nerves metathoracic, 49,52 peripheral, 40-41 Neural repair and regeneration, 35-84 degenerative responses, 36-43 regenerative responses of insect neurons, 43-58 role of neuralgia and exogenous cells, 58-75 Neuri te outgrowth, hormonal regulation, 2223 regression, hormonal control, 20-22 Neuroblasts, 3, 8, 19 Neurogenesis and neuronal differentiation, 18-19 Neuroglia, 37, 51 and exogenous cells, 58-72 Neuronal death, control, 23-26 Neurons, 74 death of immature, 23-24 differentiation of postembryonic, 1819 homologous, 5 larval, restructuring, 19-23 regenerative responses of insect, 43-58 interneurons, 4 6 4 9 motor, 49-55 sensory, 55-58 regeneration, 56 sensory, regulation, 14-15

INDEX

Nodule formation, 102-103 and wounding, 136137 Non-self recognition, 138-148 basement membrane, 139-143 and self, 143-145 physicochemical properties, 143144

Oenocytoids, 88 Oncopeltus faciatus (milkweed bug), 14, 15,27 Ootheca, 18I Orgyia leucostigma, I20 Orthoptera, 94

Particulate material, 122-124 Pathway mechanisms, 221-222 Peripheral nerves, degenerative responses in, 4 W 1 Periplaneta americana (cockroach), 40, 92, 95, 103, 114, 117, 131, 134, 137, 140, 141, 143, 144, 145, 146, 150 Peroxidase, 186, 189 in cuticle, 222 and quinone tanning, 204-205 Phagocytosis, 99-102 Phenoloxidase, 186, 189 and prophenoloxidaseactivation system, 112-1 16 Plasma components, recognition mediation, 145-147 Plasrnatocytes (PLs), 87-88 Platysamia cecropia, 137 Plodia interpunctella, 90 Postembryonic development, 13-26 ganglion migration, 13-14 sensory system, 14-18 Postembryonic nervous system, development, 4-7 Procambarus clarkii (crayfish), 40 Prohaemocytes (PRs), 87 Prophenoloxidase-activation system, 112-1 16, 147 Proteins antibacterial, 109-1 I 1 in scleroticdl cuticle, 181-182 Pseudomonas aeruginosa, 124

237

Quinone methide sclerotization, 209-2 17 reactions with cuticular components, 216 tanning hypothesis, 190-205 carboxyl groups, 196-1 97 differential mechanism, 2 17-22 1 hydroxyl groups, 198 -1ysine adducts, 195-199 peroxidase participation, 204-205 Recognition and response, alterations in, 155-157 Recruitment and cessation, in encapsulation, 148-155 Regenerative responses, of insect neurons, 43-58 Rhodnius, pioneering studies, 26,99, 1 17 Surcophaga bullata (flesh fly), 58, 112, 133, 146, 181, 187, 192, 194, 195, 21 1 Schistocerca gregaria (locust), 6, 38, 90, 92, 103, 114, 115, 121, 128, 134, 143, 144, 150 Schwann cells, 51,73 Sclerotization, cuticular, molecular mechanisms for, 179-230 alpha, 206209 beta, 205-2 I7 components, 181-187 dityrosine crosslinks, 187-190 free radical formation, 222-223 pathway and crosslinking mechanisms, 221-222 tanning quinone, 19Ck205 differential mechanisms, 217-22 1 Sensitivity, acetylcholine, 50 Sensory neurons, 55-58 regeneration, 56 Sensory system development, 14-18 projections, 15-18 regulation of neurons, 14-15 Serum agglutinins, 1 I 1 lectins, 1 1 1-1 12 Sessile circulating cells, 88-89 Silkmoth (Antheraea polyphemus), 24 Spherule cells, 88

238

Spodoptera littoralis, 98 Stress, in insects, 119

Tanning, quinone, 19Ck-205 carboxyl groups, 196-197 differential mechanisms, 217-221 hydroxyl groups, 198 -1ysine adducts, 195, 199 peroxidase participation, 204-205 Tenebrio, 7, 103, 119 Thrombocytoids, 88 Thymidine, 70 Tritium release, time-course, 21 5 Trityrosine, I88 Tyrosinase, 197

INDEX

‘Wandering’or ‘commitment peak’ 8 Wax moth (Galleria mellonella), 13, 21, 89,95,97, 104, 110, 113, 119, 120, 138, 147, 150 Worm (Ascaris lumbricoides), 189 Wound factors, 127, 130-137 nature of, 132-133 haemocyte recognition or response, 127-1 30 Wound-healing, haemocyte involvement, 125-130, 137-138 and encapsulation, I48 requirements, 125-1 27 Wounding, I 19- I2 1 and nodule formation, 1 3 6 137