Insect Endocrinology

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Ann. Rev. Physiol. 1980. 42:511-28 Copyright © 1980 by Annual Reviews Inc. All Rights reserved INSECT ENDOCRINOLOGY: Action of Hormones at the Cellular Level Lynn M Riddord +1283 Department of Zoology, University of Washington, Seattle, Washington 98195 The endocrine regulation of the life of the insect is based on the ecdysteroids (ecdysone, 20-hydroxyecdysone), juvenile hormone (JH), and a myriad of neurosecretory peptide hormones,most of which have yet to be purified and sequenced. Ecdysteroids and JH control both growth and development and later reproductive maturation. The neurohormones regulate the release of these two hormones and also a variety of homeostatic activities and behav- ior. The preceding review in this volume (47) discusses the regulation of the endocrine glands and the chemistry and metabolism of the hormones. This review is conceed with the hormonal regulation of physiological pro- cesses. Since there have been many recent reviews of various aspects of insect endocrinology and insect hormone action (13, 26, 36,48, 50-52, 60, 99, 111, 112, 120) I concentrate here on a few systems that promise to better our understanding of the action of ecdysteroids and juvenile hormone at the cellular level, in both morphogenesis and reproduction. I then discuss the actions of two identified peptide hormones. MORPHOGENETIC ACTIONS OF ECDYSONE AND JUVENILE HORMONE Since an insect lives within a rigid exoskeleton or cuticle, growth necessi- tates the periodic shedding of this cuticle and the production of a larger one. This process of molting is controlled by ecdysone from the prothoracic glands, which is converted to 20-hydroxyecdysone, the active hormone, by 511 0066-4278/80/0315-0511$01.00 Annu. Rev. Physiol. 1980.42:511-528. Downloaded from www.annualreviews.org by Universidade Estadual de Feira de Santana on 11/19/12. For personal use only.

Transcript of Insect Endocrinology

Ann. Rev. Physiol. 1980. 42:511-28 Copyright © 1980 by Annual Reviews Inc. All Rights reserved

INSECT ENDOCRINOLOGY:

Action of Hormones

at the Cellular Level

Lynn M Riddiford

+1283

Department of Zoology, University of Washington, Seattle, Washington 98195

The endocrine regulation of the life of the insect is based on the ecdysteroids (ecdysone, 20-hydroxyecdysone), juvenile hormone (JH), and a myriad of neurosecretory peptide hormones, most of which have yet to be purified and sequenced. Ecdysteroids and JH control both growth and development and later reproductive maturation. The neurohormones regulate the release of these two hormones and also a variety of homeostatic activities and behav­ior.

The preceding review in this volume (47) discusses the regulation of the endocrine glands and the chemistry and metabolism of the hormones. This review is concerned with the hormonal regulation of physiological pro­cesses. Since there have been many recent reviews of various aspects of insect endocrinology and insect hormone action (13, 26, 36,48, 50-52, 60, 99, 111, 112, 120) I concentrate here on a few systems that promise to better our understanding of the action of ecdysteroids and juvenile hormone at the cellular level, in both morphogenesis and reproduction. I then discuss the actions of two identified peptide hormones.

MORPHOGENETIC ACTIONS OF ECDYSONE

AND JUVENILE HORMONE

Since an insect lives within a rigid exoskeleton or cuticle, growth necessi­tates the periodic shedding of this cuticle and the production of a larger one. This process of molting is controlled by ecdysone from the prothoracic glands, which is converted to 20-hydroxyecdysone, the active hormone, by

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512 RIDDIFORD

the peripheral tissues (47). Since growth always occurs in the larval stages and is terminated at metamorphosis, a second hormone, juvenile hormone, from the corpora allata ensures larval molting. When the insect approaches its maximum size, the JH titer declines, allowing the ecdysteroids to initiate metamorphosis. The cellular changes associated with larval molting occur primarily in the epidermis; but at metamorphosis internal changes also occur.

Epidermis and Cuticle Formation The insect epidermis is a single cell layer of electrically coupled cells (21) that produces the overlying cuticle, each cell molding the surface pattern of the cuticle lying above it (127). Although epidermal cells are regarded as differentiated cells that make the cuticle, they can further differentiate into specialized structures such as bristles or hairs at the time of the molt (128). Most insect epidermal cells can produce various kinds of cuticle­i.e. larval, pupal, or adult, depending on the hormonal milieu.

The cytological events occurring in the epidermis in preparation for the molt have been described in detail (reviewed in 73, 86, 133). The following sequence of events generally occurs: The cells detach from the overlying cuticle (apolysis); an ecdysial membrane is secreted that separates the old cuticle from the one to be made; the molting gel is secreted, followed by the new epicuticle, consisting primarily of proteins and lipids, and the new procuticle of chitin and protein (4). Usually late in this sequence the en­zymes in the molting fluid, often including enzymes in the cuticle itself (7), are activated and digest the old procuticle leaving the epicuticle to be shed at ecdysis. After ecdysis the new cuticle is sclerotized by a cross-linking of the proteins with each other and with chitin (4), a process often accelerated by the hormone bursicon (86, 99). Thus the insect epidermal cell is primar­ily a secretory unit that makes and releases various products in a defined sequence in response to a hormonal stimulus. The cell must also undergo any cell division and/or differentiation dictated by the hormonal milieu at the beginning of this sequence. Ecdysone, which initiates cuticle formation and directs its progress, is present in the insect through the beginning of procuticle deposition (35, 99).

The hormonal control of this cuticular deposition is now being studied primarily in tissue culture. Irrespective of whether the epidermis is from embryos (16, 17), body wall (20,39, 78, 82, 83, 101), or from imaginal discs or their derivatives (76, 78, 81, 85, 88, 129), cuticle formation complete with differentiated bristles requires exposure to about 10--6 to 10-7 M 20-hydroxyecdysone (,B-ecdysone or ecdysterone) [similar to the titers being reported in the hemolymph (126)] for a defined length of time. In most systems a definite concentration-time relationship governs cuticle formation (20, 78,82, 85). Furthermore, ecdysone (a-ecdysone). the hormone secreted

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CELLULAR ACTIONS OF INSECT HORMONES 513

by the prothoracic glands (47, 48), appears to be about 1 % as effective as 20-hydroxyecdysone on epidermis (20, 39, 76, 78, 98), which indicates that it is a prohormone (47, 48). Whether ecdysone has any role itself in activat­ing the epidermis is still an open question; several workers have suggested that it may be important in stimulating very early cellular changes-e.g. DNA synthesis (18, 67), appearance of rough endoplasmic reticulum, and increased mitochondrial number (20). Low concentrations of 20-hydroxy­ecdysone can also cause these changes. (20). Thus before any definite conclusions can be drawn the role of the epidermis in the metabolism of ecdysone to 20-hydroxyecdysone must be clarified.

The biochemistry of 20-hydroxyecdysone-stimulated cellular events lead­ing up to cuticle production is being pursued profitably in the imaginal disc system (discussed below). The hormonal regulation of the type of cuticle deposited can be studied best in other kinds of epidermis. Thus far, two lepidopteran systems seem ideal for studying the morphogenetic role of JH in cuticle deposition: pupal wing epidermis (129) and larval abdominal epidermis (83, 101). In response to 20-hydroxyecdysone the former forms either adult cuticle with scales and hairs in the absence of JH or pupal cuticle in its presence. Similarly, the larval epidermis forms either new larval cuticle in the presence of JH or pupal cuticle in its absence. In both cases, the pattern of proteins synthesized in the presence of JH and 20-hydroxyecdysone differs from that synthesized in the presence of 20-hydroxyecdysone alone (100, 129). What these differences mean remains unclear, especially since some do not seem to be cuticular proteins (129).

Sclerotization and Tanning of the Cuticle After ecdysis the newly formed cuticle hardens and often darkens in a process called sclerotization (3, 4). This process is governed usually by neurosecretory hormones, though the ecdysteroids may also be involved in some instances (59, 99).

At the time of metamorphosis in flies, the last stage larval cuticle hardens and darkens to form the puparium within which the pupa develops. At first the ecdysteroids were thought to initiate this process by turning on the gene for dopa decarboxylase (59), a key enzyme in quinone tanning (3). Yet dopa decarboxylase activity increases before the release of ecdysone to initiate puparium formation (109). This apparent paradox is resolved by the finding that 20-hydroxyecdysone serves mainly to increase the rate of synthesis of this enzyme (42) in the epidermis, probably by increasing the rate of mRNA synthesis. &dysone then later causes the release into the hemolymph of a neurosecretory hormone, "puparium tanning factor" (PTF), which initiates the tanning process (109). PTF appears to act via cAMP (41, 109), which can substitute for the factor in the presence of an RNA synthesis inhibitor (actinomycin) but not in the presence of protein synthesis inhibitors. Since

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the addition of dopa or dopamine but not of tyrosine has the same effect, Fraenkel et al (41) have speculated that PTF acts somehow on the initial step in the pathway to convert tyrosine to dopa.

In most insects, the tanning of the Dew cuticle after ecdysis is controlled �

by another neurosecretory hormone, bursicon (86, 99). In the tobacco homworm, Manduca sexta, bursicon is released from two identified neu­rons in each abdominal ganglion (P. Taghert, personal communication) into the hemolymph immediately after adult eclosion (95). It causes the in­creased plasticization of the wings that allows full wing expansion (93). This plasticization is then followed by tanning of the wings. Bursicon from M. sexta has now been purified to near homogeneity and appears to be a peptide of about 9000 daltons (P. Taghert, personal communication).

The precise mode of action of bursicon is not known, but it is thought to affect transport of tanning precursors into the hemocytes and/or the epidermal cells (86). It appears to increase cAMP levels in both of these cells (34, 86), though in the epidermal cells the evidence is only correlative. The critical experiments await a pure hormone preparation whose action on isolated cells can be defined.

Cellular Reprogramming of the Epidermis The larval epidermis of Lepidoptera and probably also Coleoptera seems ideal for the study of the hormonal control of cellular reprogramming. In these insects metamorphosis begins in two discrete steps: a decline in JH, followed by two releases of ecdysone (35, 55, 99). In the tobacco horn worm and probably in other species, the first release initiates metamorphosis by changing the commitment of the epidermis from that for larval differentia­tion to that for pupal differentiation (98, 122). The second and larger release promotes pupal cuticle synthesis (122). Both actions of 20-hydroxyecdy­sone are also seen when M. sexta larval epidermis is exposed to the hormone in vitro (82, 83, 98). Juvenile hormone can prevent the change to pupal commitment only if given during the initial exposure to 20-hydroxyecdy­sone (83, 98, 101, 122). After an exposure to 20-hydroxyecdysone in the absence of JH, the epidermis becomes insensitive to JH and produces pupal cuticle irrespective of the presence or absence of JH. Before such an expo­sure the epidermis remains capable of producing larval cuticle when in­duced to molt by 20-hydroxyecdysone in the presence of JH (98, 101). Thus, contrary to a recent suggestion (55), neither the decline of JH nor the appearance of "JH-specific" esterases (47, 48) causes the reprogram­ming of the epidermis. Rather this reprogramming must be initiated by 20-hydroxyecdysone in the absence of JH.

The cellular and molecular events that cause the change in commitment are only superficially understood at present. In Tenebrio molitor epidermis at the time of the ecdysone releases (35), changes in ionic coupling occur (21)

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CELLULAR ACTIONS OF INSECT HORMONES 515

that can also be induced by 20-hydroxyecdysone in vitro (22). These changes may be important for the regulation of the development of pupal cuticular pattern (21, 22). In M sexta DNA synthesis in the epidermis peaks during the first rise in ecdysteroids (100, 126), but this also occurs on the day preceding the ecdysteroid rise (126). Furthermore, similar in­creases in DNA synthesis leading to octaploidy are seen in epidermis cul­tured in vitro in both the presence and absence of hormone (100). Inhibition of this DNA synthesis by cytosine arabinoside does not inhibit the 20-hydroxyecdysone-induced change of commitment (100). This process of DNA synthesis and subsequent cell division (125) is undoubtedly important for the surface morphology of the new pupal cuticle (104), but it seems unnecessary for the switching of the cellular commitment.

By contrast, the altered patterns of RNA and protein synthesis initiated by 20-hydroxyecdysone in M sexta epidermis are necessary for the change in commitment (97, 1(0). At least one or two new mRNAs and proteins appear, whereas at least six or seven mRNAs and proteins present in the larvally committed cell are no longer synthesized. Quantitative changes in other mRNAs and proteins also occur. Presumably some of these latter proteins are larval cuticular proteins, for major endocuticular synthesis ceases when the animal stops feeding (97, 125). The new mRNAs appearing during the change in commitment do not appear to be for pupal cuticular proteins (A. C. Chen, L. M. Riddiford, manuscript in preparation). Whether some of the changes seen in response to 20-hydroxyecdysone in the absence of JH involve nuclear regulatory proteins, as might be expected for a change in programming, awaits further investigation.

Imaginal Discs Imaginal discs are groups of relatively undifferentiated cells found in holometabolous larvae. These discs grow during larval life but at metamor­phosis undergo differentiation into adult structures such as eyes, wings, legs, genitalia, etc (46). Their growth appears to be continuous throughout larval life; it stops soon after the beginning of pupal development (37, 67, 89, 110). Apparently JH has a permissive effect on this growth; it seems necessary for growth in vitro (32), though the critical concentrations and times of exposure have not been ascertained. Visible evagination and differentiation do not occur until 20-hydroxyecdysone in the absence of JH initiates pupal development.

In the Lepidoptera these imaginal structures (or parts of them) respond to the early decline in JH titer in the last larval instar by becoming commit­ted to pupal differentiation before the first release of ecdysone [(64, 66, 87); L. M. Riddiford, unpublished studies], thus behaving somewhat differently from the abdominal epidermis discussed above (98). Yet no visible differen­tiation of the discs is seen until the wandering stage, when tracheolar

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migration occurs (89), presumably in response to the first release of ecdy­sone. An increase in DNA synthesis in lepidopteran (66, 67) and dipteran discs (124) occurs before the wandering stage and may be a consequence of the earlier decline in JH. Pupal differentiation of these structures, at least in some Lepidoptera, must occur in the presence of JH: Removal of the corpora allata during the last larval instar permits precocious adult devel­opment of some of these structures [(64) and references therein]. Pupal cuticle can be formed by lepidopteran wing discs in vitro in response to 20-hydroxyecdysone when no JH is present (85, 88, 89). The concentration of 20-hydroxyecdysone and the time of exposure appear to determine whether the cuticle formed is pupal or adult (85). Unfortunately, normal metamor­phosis (i.e. pupal then adult cuticle formation) by these discs has not yet been achieved in vitro. Further in vivo and in vitro investigation will settle whether or not the apparent differences between the responses to hormones of imaginal discs and abdominal epidermis are real.

By contrast to the Lepidoptera, the complete sequence of development of Drosophila melanogaster imaginal discs from larval to pupal to adult can be induced by two days' exposure of larval discs to 20-hydroxyecdysone in vitro (76, 81). Furthermore, since these discs can be readily mass-isolated, much is known about the early cellular and biochemical changes that occur in response to 20-hydroxyecdysone and culminate in evagination and the beginning of pupal cuticle synthesis (76, 107, 110). This material has been reviewed recently in detail (110, 111). 20-Hydroxyecdysone stimulates DNA, RNA, and protein synthesis in discs ready to metamorphose, and JH appears to prevent these hormone-induced increases. Furthermore, the pattern of protein synthesis that occurs in discs exposed to JH and 20-hydroxyecdysone differs from that in discs exposed to either hormone alone or to no hormone. This indicates that JH is not simply inhibitory; it directs cellular processes in discs as it does in other types of epidermis (see above).

Cellular receptors for the ecdysteroids have been obtained recently from imaginal discs (132). About 1000 receptors per cell seem to be associated primarily with the crude nuclear fraction. Thus they appear to differ from the predominant cytoplasmic steroid receptors of vertebrates. More work is necessary with purified nuclei to confirm this difference.

Salivary Glands and Chromosome Puffs In 1960 Clever & Karlson (27) demonstrated that ecdysone induced specific "puffs" in the polytene chromosomes of dipteran salivary glands. These puffs have been used ever since in the study of ecdysteroid action. At the end of larval life the salivary glands synthesize a glycoprotein glue that fixes the old larval skin (which will become the puparium) to the substrate; the glands then degenerate (9). A specific puff seen early in the third (final)

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CELLULAR ACTIONS OF INSECT HORMONES 517

larval instar of Drosophila melanogaster appears to be responsible for one of these proteins (65); this puff disappears when the ecdysone titer rises prior to pupariation.

More interest has recently been centered on the sequence of puffs that appears around the time of pupariation (5). In D. melanogaster this whole sequence can be induced by two exposures of isolated salivary glands to 20-hydroxyecdysone separated by a minimal 3-hr period in hormone-free medium (reviewed in 5, 99). The early puffs are thought to be induced directly by 20-hydroxyecdysone and to code for proteins that induce the late puffs and repress the early puffs (6). The synthesis of these proteins is postulated to occur only in the presence of 20-hydroxyecdysone so that the later obligatory 3-hr absence of hormone is necessary to derepress some of these genes. Thus the temporal appearance and disappearance of 20-hydroxyecdysone regulates the normal puffing sequence. JH has no appar­ent effect on the early sequence induced by 20-hydroxyecdysone (5, 96) but inhibits the late sequence when given during the hormone-free period (96).

The identity of the proteins encoded by the two early puffs is unknown. Newly synthesized RNA from both salivary glands and imaginal discs exposed to 20-hydroxyecdysone has been hybridized to the salivary gland chromosomes (15). The RNA from salivary glands hybridizes to the sites of the early puffs and also to some other regions (both hormone-insensitive puffs and unpuffed regions). The RNA from the imaginal discs does not hybridize to the early puff sites. Thus if the products of the early puffs are regulatory proteins they must differ from tissue to tissue. Recent studies on another dipteran, Acricotopus lucidus, which has two cell types in its sali­vary gland, have suggested that 20-hydroxyecdysone can have differential effects on RNA synthesis in the two cell types (90).

Fat Body Among the viscera, the larval-pupal transformation of the fat body offers a good experimental system in which to study the role of the two morpho­genetic hormones. The insect fat body is essentially equivalent to the liver and the adipose tissue of vertebrates. During larval life it processes incom­ing nutrients and, in preparation for metamorphosis, stores material not needed for growth. In several Diptera and Lepidoptera the larval fat body synthesizes and secretes several large proteins ("'500,000 daltons) that accumulate in the hemolymph; these are then taken back into the fat body and are concentrated into granules at the cessation of feeding (130). The disappearance of these proteins during metamorphosis indicates their con­sumption during this process.

Recent studies on the blowfly, Calliphora vicina, show that calliphorin is synthesized by the fat body up to two days before pupariation; then

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synthesis ceases but its mRNA remains until shortly before pupariation (108). Since the highest rate of synthesis occurs early in the last larval stage, JH may direct this synthesis (108). By contrast, in both Bombyx mod (116) and Manduca sexfa (A. C. Chen, unpublished studies in this laboratory) the storage proteins appear to accumulate in the hemolymph in response to a declining JH titer. Although fat body is notorious for its rapid metabo­lism of JH (47, 48), the role of JH in the synthesis of these proteins should be readily clarified using an in vitro system.

That the reuptake of these proteins and the formation of storage granules are induced by ecdysteroids at metamorphosis is most convincingly demon­strated in Drosophila melanogasfer (72, 102). In the temperature-sensitive ecd1 mutant, which cannot pupariate at the restrictive temperature due to lack of sufficient ecdysone (45), the addition of exogenous 20-hydroxyecdy­sone causes the larval serum proteins to be taken up into the fat body (72). The hormone also initiates the synthesis of several new proteins by the fat body at this time.

During the onset of metamorphosis, autophagic vacuoles appear in the fat body (33, 106, 123). This appearance can be induced precociously by 20-hydroxyecdysone in vivo only after the decline of JH (106) or in vitro at the very end of the instar (33, 106, 123).

Cells In Vitro Hormonally responsive insect cell lines have been developed recently and utilized to study the biochemical mechanism of action of the ecdysteroids (29, 69, 77). A cell of Drosophila melanogaster Kc line responds to ecdys­teroids by cessation of DNA synthesis and mitosis (105); a series of morpho­logical changes occurs, consisting first of elongation of cellular processes and then of clumping; finally, within three to four days, the cell dies (10, 25, 29). During this hormone-induced differentiation, the spectrum of sur­face glycoproteins synthesized changes (80). Other new proteins are pro­duced (10), including acetyl cholinesterase (11, 25) and ,a-galactosidase (12). The physiological basis for these enzyme inductions is unclear. Yet they provide a ready means for studying the molecular action of ecdys­teroids in a morphological system.

Ecdysteroid-binding protein(s) has been found in the cytosol of these D. melanogastercells (79). Apparently the receptors translocate rapidly to the nucleus. Mostrof the labeled hormone is found in the crude nuclear pellet after a 30 min incubation. The binding data agree remarkably well with those obtained for the ecdysteroid receptors in imaginal discs (132), but the question of the normal cellular location of these unbound receptors has not been resolved.

Juvenile hormone and certain JH mimics inhibit the morphological differentiation induced by 20-hydroxyecdysone in several of the Drosophila

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Kc cell lines (30, 131). Although the mechanism of action of JH has not been elucidated, the D. melanogaster system has been effectively used re­cently in the search for a cellular receptor for JH (23). The preliminary results suggest the presence of cytoplasmic receptors (about 2000 sites per cell).

An interesting sidelight on JH action is its ability to inhibit phytohemag­glutinin phorbol ester (12-0-tetradecanoylphorbol-13-acetate) induced mitogenesis in bovine lymphocytes (62), apparently by preventing the phor­bol ester induced ornithine decarboxylase synthesis (63). It is unknown whether JH prevents the action of the phorbol ester on the cell membrane or acts later in the sequence of events leading to increased enzyme activity. The effective JH concentrations (5 X 10--5 M or higher) were at least 100-to WOO-fold above the apparent physiological level of the hormone in insects.

HORMONAL CONTROL OF REPRODUCTION

Unlike the vertebrates, the insects have utilized the hormones that govern growth and development to control reproduction in adult life. In the adult the corpora allata again secrete JH, which usually directs some aspect of oogenesis in the female (36, 99) and in a few cases is important in the maturation of accessory glands (36, 71, 99) or reproductive behavior (120) in the male. The prothoracic glands usually degenerate during adult devel­opment. The surprise of the past few years, however, is the finding that the adult ovary becomes an endocrine organ and secretes ecdysone, either into the hemolymph or into the egg (47, 111) where it may be used to control oogenesis or embryonic development, respectively.

Juvenile Hormone and Oogenesis The role of JH in oogenesis varies but may affect one or more of the following processes: (a) maturation of pre vitello genic oocytes; (b) initiation of female-specific yolk protein (vitellogenin) synthesis in the fat body; (c) induction of vitellogenin uptake into the oocyte.

In some insects (2, 52, 84, 115) JH stimulates previtellogenic growth of the follicles so that they become competent to take up the vitellogenin at a later stage. The cellular events that occur in this development of compe­tence have not been described exactly.

The hormonal regulation of vitellogenesis may involve the synthesis and! or uptake of vitellogenin into the oocytes. Since this subject has been re­viewed recently (52, 130), a brief summary of two systems that promise more information about the mode of the gonadotropic action of JH suffices to illustrate the complexities involved. In the locust Locusta migratoria JH appears to be necessary for the normal maturation of the female fat body

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520 RIDDIFORD

into a protein-secreting organ (24, 28). In allatectomized females, a JH mimic causes the initiation of vitellogenin synthesis within 48 hr after th(: first exposure and nearly immediately after subsequent exposures (24), a phenomenon similar to primary and secondary hormonal stimulation in vertebrates. Also, a cytosolic receptor for JH has been found in vitello genic fat body (P. Roberts, G. R. Wyatt, personal communication). Thus it appears that JH may directly induce mRNA synthesis in this system, but this conclusion cannot be confirmed until the whole process can be per­formed in vitro. One major problem here (as in using the fat body to study the action of JH in larval life) is rapid metabolism of the added hormone (47).

In Rhodnius prolixus JH is important in oogenesis, primarily in promot­ing vitellogenin uptake into the oocyte (31). Ovarian follicle cells incubated in vitro with JH I exhibit an increase in the size of intercellular spaces due to a 50% decrease in cellular volume (2). Thus JH is postulated to stimulate a membrane-related pump to expel fluid from the cell, but no definitive experiments as to the nature of this pump or its interaction with JH have been reported. Inhibitor studies suggest that RNA and protein synthesis are not involved in this response to JH (1), but no direct measurements have been made. Thus this effect of JH could be entirely different from its induction of vitellogenesis in the fat body.

Role of Ecdysone in Female Reproduction It was first shown in the mosquito Aedes aegypti that ecdysone played a direct role in insect reproduction (see 52 for review). After a blood meal ecdysone is released from the ovary and converted to 20-hydroxyecdysone, which acts on the fat body to stimulate vitellogenin synthesis and release. Whether this stimulation involves initiation of vitellogenin mRNA synthe­sis or some aspect of translation or processing [as recently shown for JH in Oncopeltus (61)] has not .been resolved (52). Prior exposure of the fat body to JH is necessary to render it competent to respond to 20-hydroxyecdysone (40). JH may initiate changes similar to those seen in locust fat body (24, 28), possibly including synthesis of vitellogenin mRNA; it may thus act as the primary stimulus for vitellogenesis in this as in most other insect systems. This interesting two-hormone system requires further ,exploration.

Since these initial findings in mosquitoes an increasi�g number of adult female insects ,have been found to contain ecdysteroi(�ts ,(47). So far ecdys­teroids have been implicated in the process of egg maturation in only one other insect, Drosophila melanogaster. The temperature-sensitive ecd1 mutant matures eggs only at the permissive temperature (45). Also, 20-bydroxyecdysone injected into isolated female Drosophila abdomens causes about a three-fold increase in newly synthesized vitellogenin in the hemo-

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lymph but does not permit its uptake into the oocyte (53). JH is necessary for the latter process (91) and by itself can stimulate both the synthesis and uptake in the isolated abdomen (53). Consequently JH is thought to stimu­late a tissue in the abdomen to secrete ecdysone, which then increases vitellogenin synthesis in the fat body (53). This tissue could well be the ovary, for mature adult ovaries stimulate vitellogenesis when implanted into males [(57) but see (52) for a critique]. This role of the ovary has yet to be tested by experiments to determine whether isolated ovariectomized female abdomens synthesize vitellogenin in response to JH.

In the Orthoptera and Lepidoptera (14, 54, 56, 68, 70) the ovary also produces ecdysone but not until the terminal phases of oogenesis just before chorion formation. In Locusta migratoria this ecdysone is produced by the follicle cells (49, 68), secreted into the egg, and apparently utilized to initiate the formation of the serosal cuticle (M. Lagueux, J. Hoffmann, personal communication). In the Lepidoptera and the cockroach most of the ecdys­teroids are also put into the egg (14, 54, 56, 70), but their function in embryonic development is unclear. In these species some ecdysteroids are also found in the hemolymph but have no apparent function.

NEUROSECRETORY HORMONES

Neurosecretory hormones abound in the insect (13, 51). They are released in response to environmental signals, either external or internal, to maintain homeostasis [regulation of carbohydrate (43, 60, 112) and lipid (50, 60) metabolism, water balance (75), etc]; to alter the structural properties of the cuticle (92, 93, 109); to initiate various behaviors (120); and in one well­documented case, that of the prothoracicotropic hormone (PITH), to di­rect the activity of an endocrine gland, the prothoracic gland (47). Presumably the activity of the corpora aHata is also controlled by neurohor­monal factors as well as by direct nervous signals (47). Yet in spite of their importance only one neurosecretory hormone, the adipokinetic hormone of locusts, a peptide, has thus far been isolated and sequenced. The action of this hormone and of the partially purified eclosion hormone are discussed here.

Adipokinetic Hormone (AKH) Flight in locusts relies on both carbohydrate and lipid as fuel (58). At the outset of flight, hemolymph trehalose (the insect blood sugar) is utilized. Within 5-10 min this carbohydrate reserve is depleted and AKH is released from the grandular lobe of the corpora cardiaca to mobilize the stored triglycerides from the fat body (8, 50). Diglycerides are then released into the hemolymph and taken up by the muscle.

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AKH was recently isolated and sequenced (PCA-Leu-Asn-Phe-Thr-Pro­Asn-Trp-Gly-Thr-NH2) (113). Synthetic AKH has been found to stimulate a two-three-fold increase in cAMP in locust fat body about 6 min after injection of 10 pmol (44). Presumably this increase in cellular cAMP causes the activation of a cAMP-dependent protein kinase (8), which in turn activates a triglyceride lipase (50), a sequence analogous to the stimulation of lipolysis in mammalian adipose tissue by epinephrine and glucagon (38). But this cascade of enzyme reactions has not yet been demonstrated with AKH, though cAMP is known to mimic the lipolytic action of the hormone (8).

In working flight muscle itself, AKH-containing extracts of the glandular lobes of the corpora cardiaca have been reported to cause an increase in fatty acid oxidation (103). AKH was postulated to act directly on the mitochondrial enzyme carnitine acyl transferase to stimulate lipid metabo­lism and thereby acyl group uptake into the mitochondria. Recently, how­ever, Candy (19) has been unable to repeat these results, though he did find that octopamine stimulated both lipid and carbohydrate metabolism in these muscles. These experiments must be repeated with pure hormone before any direct action on the muscle can be claimed.

Eclosion Hormone Eclosion, the emergence of adult holometabolous insects from their pupal cases, is initiated by a neurosecretory hormone from the brain. In moths this hormone is produced by certain medial neurosecretory cells in the brain during the latter part of adult development and sent to the corpora cardiaca for storage (117). On the final day of adult development in response to a circadian clock set by the photoperiod regime, the hormone is released into the hemolymph (95) and triggers a species-specific series of behaviors that result in the emergence of the moth and the assumption of adult behavior (118). The hormone has been partially purified and is thought to be a peptide of about 9000 daltons (94).

The purified ec1osion hormone has been shown to induce the stereotyped series of abdominal movements associated with eclosion by acting directly on the abdominal nervous system to elicit the readout of a behavioral program (119). The animal (presumably its nervous system) only becomes responsive to the hormone on the day before or the day of eclosion, depend­ing on the species (95, 118). In Manduca sexta the onset of responsiveness occurs suddenly about 4 hr before the hormone is released (95). Whether this is simply part of the developmental program of the adult nervous system or is due to some other neurohormonal release that synchronizes the synthesis of hormonal receptors or other cellular events necessary for the response is unknown. Once the animal or the isolated nervous system has

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been exposed to the hormone it permanently loses the ability to respond again to the hormone (119). This loss occurs immediately, which suggests a rapid loss of cellular receptors, followed in some species by degeneration of certain neurons that may or may not be the target cells for the hormone [(114); J. W. Truman, personal communication].

When crude corpora cardiaca extracts were used in this system, eclosion hormone appeared to act on the nervous system by triggering an increase in cAMP (121). With the purified hormone no increase in cAMP could be elicited (121a). Instead, a two-fold rise in cGMP was observed. Further­more, cGMP was found to be more than 100 times more potent in provok­ing eclosion than was cAMP. Therefore, it was concluded that the hormone acts via an increase in cGMP. Both the role of cGMP in triggering the behavioral program and the target cells for the hormone are unknown.

The eclosion hormone also acts on other target organs in the pharate moth, namely the wings and the intersegmental muscles. It acts on the wings to increase their extensibility by plasticizing the cuticle (93), a neces­sary preparation for their expansion immediately after eclosion. It also initiates the breakdown of the abdominal intersegmental muscles (L. Schwartz, personal communication), which are used in emergence and wing inflation, then degenerate within 24-28 hr (74). In both of these instances the hormone is thought to act via cGMP (J. W. Truman, L. Schwartz, personal communication), but the subsequent cellular events are unknown. Inhibitor studies indicate that both RNA and protein synthesis are neces­sary for the muscle breakdown (74).

ACKNOWLEDGMENTS

I wish to thank Professor James Truman for helpful suggestions and a critical reading of this manuscript, Ms. Jackie Cho for the typing, and Ms. Anna Curtis and Dr. Karen Dyer for the final proofreading. The unpub­lished work from my laboratory was supported by NSF (PCM 76-18800), NIH (AI 12459), and the Rockefeller Foundation (RF 73019).

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86. Neville, A. C. 1975. Biology of the Ar­thropod Cuticle. NY: Springer. 448 pp.

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89. Oberlander, H., Silhacek, D. L. 1976. Action of juvenile hormone on imaginal discs of the Indian meal moth. In The Juvenile Hormones, ed. L. I. Gilbert, pp. 220-33. NY: Plenum. 572 pp.

90. Panitz, R. 1978. Cell specific effect of ecdysone on RNA synthesis in the differentiated salivary gland of Acricotopus lucidus. Cell Dijf. 7:387-98

91. Postlethwait, J. H., Handler, A. M. 1978. Non-vitellogenic female sterile mutants and the regulation of vitello­genesis in Drosophila melanogaster. Dev. Biol 67:202-13

92. Reynolds, S. E. 1 976. Hormonal regula­tion of cuticle extensibility in newly emerged adult blowflies. J. Insect Physiol 22:529-34

93. Reynolds, S. E. 1977. Control of cuticle extensibility in the wings of adult Man­duca at the time of eclosion: effects of eclosion hormone and bursicon. J. Exp. Biol 70:27-39

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95. Reynolds, S. E., Taghert, P. H., Tru­man, J. W. 1979. Eclosion hormone and bursicon titer and the onset of hormonal responsiveness during the last day of adult development in Manduca sexta (L.). J. Exp. Biol 78:77-86

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97. Riddiford, L. M. 1976. Juvenile hor­mone control of epidermal commitment in vivo and in vitro. See Ref. 89, pp. 198-2 1 9

98. Riddiford, L . M . 1978. Ecdysone­induced change in cellular commitment

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1 18. Truman. J. W. 1976. Development and hormonal release of adult behavior pat­terns in silkmoths. J. Compo Physiol. 107:39-48

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122. Truman. J. W., Riddiford, L. M., Sa­franek. L. 1 974. Temporal patterns of response to ecdysone and juvenile hor­mone in the epidermis of the tobacco hornworm, Manduca sexta. Dev. Bioi. 39:247-62

123. Tysell, B., Butterworth, F. M. 1978. Different rate of protein granule forma­tion in the larval fat body of Drosophila melanogaster. J. Insect Physiol. 24: 201-6

124. Vijverberg, A. J. 1973. Incorporation of 3H-thymidine in the wing and leg discs of Calliphora erythrocephala. Short term effects of ecdysterone on DNA synthesis during larval and prepupal de­velopment. Neth. J. Zool 23: 1 89-2 14

1 25. Wielgus, J. J . , Gilbert, L. I. 1978. Epi­dermal cell development and control of cuticle deposition during the last larval instar of Manduca sexta. J. Insect Physiol 24:629-38

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129. Willis, J. H., Hollowell, M. P. 1 976. The interaction of juvenile hormone and ec­dysone: antagonistic, synergistic, or permissive. See Ref. 88, pp. 270-87

130. Wyatt, G. R., Pan, M. L. 1978. Insect plasma proteins. Ann. Rev. Biochem. 47:779-8 1 7

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