A factor causing stable stimulation of juvenile hormone synthesis by Diploptera punctata corpora allata in vitro

Co-incubation of corpora allata (CA) from the cockroach, Diploptera punctata, with ovaries, fat body or muscle but not brain or testis, leads to a substantial increase in juvenile hormone synthesis. Incubation of the glands in medium pre-conditioned with ovaries also stimulates JH synthesis. The ovary was used as a convenient source of stimulatory factor for a detailed analysis of its physiological effects on the CA. The increase in JH synthesis is stable, maintained over 24h after exposure to the stimulatory factor. Stimulation is dose-dependent, and the corpora allata show an exquisite relationship between sensitivity to this factor and developmental stage. Day 0 and day 1 glands, as well as glands from post-vitellogenic females, are sensitive to stimulation, whereas glands from vitellogenic females are not sensitive. Corpora allata attached to the brain do not respond to the stimulatory factor, and denervation in vivo leads to an increase in JH synthesis by the glands and a loss in sensitivity to the factor. These data suggest that glands from pre- and post-vitellogenic females are inhibited by their nervous connection to the brain. In contrast, glands from vitellogenic females are normally responding to the endogenous stimulatory factor and are thus no longer stimulated in vitro. Co-incubation of CA with allatostatin and conditioned medium still leads to a stimulation of JH synthesis, suggesting that the restraining effect of the nervous connections to the brain is not caused by allatostatin. The CA cell number increases between emergence and day 2, then remains stable until after oviposition. The stimulatory factor accelerates the increase in cell number in young adult females. The results are interpreted as providing evidence for a constitutive change in CA activity caused by a humoral factor produced by various tissues including the ovary, and modulated by nervous connections to the brain.     

Cell size control by ovarian factors regulates juvenile hormone synthesis in corpora allata of the cockroach, Diploptera punctata

The corpora allata synthesize and release juvenile hormone (JH) that in turn regulates insect growth, metamorphosis and reproduction. In the corpus allatum (CA) of the female adult cockroach Diploptera punctata, cyclic rise and decline in JH synthesis rates occur concurrently with cyclic growth and atrophy during an ovarian cycle. Here, we report that protein content decreases, whereas Golgi population, lysosomal content and autophagic activities increase with decrease in CA cell size. Also, the concentration of cyclic GMP (cGMP) is low in large cells and high in small cells. Results of treating CA with ovarian tissue suggest that a putative peptidergic growth regulator released from mature ovaries acts directly on active CA cells and induces the elevation of intracellular cGMP content. Consequently, elevated cGMP may inhibit protein synthesis or trigger massive and synchronous autophagic activities, resulting in cell atrophy and reduction of protein content. As a result of the depletion of cellular machinery, CA glands exhibit long-term depression in JH synthesis.     

The effect of l-methionine concentration on juvenile hormone biosynthesis by corpora allata of the cockroach Diploptera punctata

The effect of varying l-methionine (l-met) concentration on rates of juvenile hormone (JH) biosynthesis/release by corpora allata of females of the viviparous cockroach Diploptera punctata has been studied using a radiochemical assay. Both high activity glands (corpora allata from day 5 females) and low activity glands (corpora allata from day 11 females) were used to study the dose dependence of JH biosynthesis on l-met concentrations, under both de novo (spontaneous) conditions of JH biosynthesis and stimulated conditions (in the presence of the exogenous JH III precursor farnesoic acid). Maximal rates of JH biosynthesis/release were observed at l-met concentration of 20 μM (spontaneous) and 40 μM (stimulated). Below these concentrations, rates of JH biosynthesis declined linearly with decreasing l-met concentration. Optimal concentration of l-met appeared to be similar for both high and low activity corpora allata, under spontaneous and stimulated conditions of biosynthesis. Above 40 μM l-met, no increase in rates of JH biosynthesis was observed. It appears that the corpora allata of D. punctata are efficient scavengers of l-met and are able to utilize even low concentrations of the substrate for JH biosynthesis. The corpora allata of D. punctata may prove useful for the biosynthesis of authentic JH III, radiolabelled in the methyl position using as methyl donor, l[methyl-³H]met of high specific activity.     

A stage-specific ovarian factor with stable stimulation of juvenile hormone synthesis in corpora allata of the cockroach Diploptera punctata

This is a study of a feedback loop from a stimulated organ to glands that produce the stimulatory hormone in the cockroach Diploptera punctata. In this insect as in many others, juvenile hormone (JH) produced by corpora allata (CA) stimulates vitellogenesis. In our previous studies, transplantations of ovaries at certain stages of development into ovariectomized mated females stimulated JH synthesis within 24h. An in vitro study by other investigators showed that all stages of ovaries release a stimulatory factor into culture medium that was not retained on a solid-phase extraction column but occurred in the aqueous flow-through. The present study is a comparison of the effect of medium conditioned with ovaries from days 1-4 and 8 of the first reproductive cycle, to the effect of the flow-through of that medium on members of a pair of CA from day 3 females. Results provide evidence for an ovarian factor that stimulates JH synthesis by CA in vitro after removal from the conditioning medium (i.e., stable stimulation). Only medium conditioned with ovaries from days 2 or 3 females significantly stimulated CA more than flow-through. Stimulation was dose dependent, sensitive to trypsin, and survived freezing. These results indicate that CA can be directly and stably stimulated by a stage-specific peptidergic ovarian factor.     

Farnesoic acid stimulation of C16 juvenile hormone biosynthesis by corpora allata of adult female Diploptera punctata

Biosynthesis of C₁₆ juvenile hormone (C₁₆JH) by isolated corpora allata (CA) of the viviparous cockroach Diploptera punctata has been studied by a radiochemical assay. This assay uses the incorporation from methyl labelled l-methionine in the methyl ester moiety of C₁₆JH. The optimal concentration of l-methionine in the medium has been determined. When varying proportions of (methyl-³H)- and (methyl-¹⁴C)-labelled l-methionine are used in the assay, no isotope effect was observed indicating that labelled l-methionine can be used as a mass marker to quantify C₁₆JH synthesis in D. punctata. C₁₆JH synthesis was stimulated by addition of farnesoic acid (FA) to the medium (maximum at 30–40 μM). When stimulated with 30 μM FA, the C₁₆JH release rate was directly dependent on C₁₆JH synthesis rate, and no intraglandular accumulation of either C₁₆JH or its immediate precursor methyl farnesoate were observed. Spontaneous and FA-stimulated rates of C₁₆JH release were studied during the first reproductive cycle of the adult female. The two final steps of C₁₆JH biosynthesis are not physiologically rate-limiting, as the ratio of spontaneous to FA-stimulated C₁₆JH release (fractional endocrine activity ratio) is always lower than unity. There is a precise relationship between the length of the basal oöcytes and the rate of C₁₆JH release.     

The effects of octopamine on juvenile hormone biosynthesis,electrophysiology, and cAMP content of the corpora allata of the cockroach Diploptera punctata

Summary Juvenile hormone production by the corpora allata of the adult female cockroach, Diploptera punctata, can be modulated by treatment with the biogenic amine, octopamine. Endogenous octopamine has been identified within the CA, using HPLC and electrochemical detection. Treatment with octopamine results in a sinusoidal, dose-dependent inhibition of JH biosynthesis by CA from day 2 virgin females, with maximal inhibition occurring at 10^-10 M and 10^-4 M. In day 4 and day 8 mated female corpora allata octopamine inhibited JH biosynthesis at 5·10^-5 M. Although the elevation of either cAMP or cGMP within the CA is known to be associated with an inhibition of JH biosynthesis, treatment with high concentrations of octopamine results in an increase in the level of cAMP but not cGMP. This effect is both dose- and time-dependent.Octopamine treatment also initiates changes in the passive membrane responses of the CA. Superfusion of CA with octopamine results in a pronounced hyperpolarization of CA cells and an increase in the electrotonic potential (indicative of the degree of electrical coupling between CA cells). This effect could be blocked by the octopamine receptor blocker phentolamine. Treatment with octopamine or phentolamine also blocked the hyperpolarization of CA cells normally associated with electrical stimulation of the axon tracts innervating the CA.We hypothesize that octopamine may be a natural neuromodulator of JH production by CA, regulating ion channels in CA cells themselves as well as release of the inhibitory neuropeptide, allatostatin, from the terminals within the CA.Abbreviations 4-AP 4-aminopyridine - CA corpora allata - CC corpora cardiaca - cAMP cyclic adenosine monophosphate - cGMP cyclic guanosine monophosphate - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N2-ethanesulfonic acid - HPLC high pressure liquid chromatography - IBMX 3-isobutyl-1-methylxanthine - JH juvenile hormone - ms millisecond - nA nanoampere - NCA I nervi corporis allati I - OCT octopamine - TEA tetraethyl ammonium     

Development-activity relationships in nymphal corpora allata of the cockroach, Diploptera punctata

Abstract. In females of Diploptera punctata the corpora allata undergo a gradual increase in volume during most of the second nymphal stadium. In the first half of the stadium, steady growth of the glands results from a progressive increase in the size of constituent cells. Late in the stadium, cell size declines but the volume of the glands continues to rise due to an increase in cell number. Changes in cell size during the stadium displayed a distinct pattern in relation to Juvenile Hormone (JH) synthesis. Both cell size and activity increased during the first two-thirds of the stadium, peaked early in the last third of the stadium, and decreased before the moult. The rise in cell numbers late in the stadium corresponded to a wave of cellular mitosis and occurred after a steep decline in the rate of JH biosynthesis. Exposure of late second instars to fenoxycarb. a JH analogue, depressed mitosis significantly, suggesting autocrine regulation of cell proliferation in the corpora allata. Possible mechanisms modulating sequential cycles of growth and atrophy of cells and cell proliferation in these glands are discussed in relation to temporal patterns of JH and ecdysteroid titres in nymphs.     

Ultrastructural changes in corpora allata during a cycle of juvenile hormone biosynthesis in embryos of the viviparous cockroach, Diploptera punctata

We have observed changes with time in the fine structure of corpora allata (CA) during a known cycle of increasing and decreasing juvenile hormone (JH) synthesis in late embryos of Diploptera punctata. A previous report showed that rates of JH release were relatively low in 28-day-old embryos, but CA activity subsequently rose linearly to a peak on about day 42, and thereafter steadily declined to a low level on day 64 just before birth (Holbrook et al., 1997). We now show that, regardless of rate of JH synthesis, CA cells are large and replete with organelles which nevertheless exhibit variable morphology in embryos of different age. Highly active CA cells on day 40 contain abundant ring-form mitochondria, whereas CA cells of low activity on days 28 and 64 contain mitochondria that are rod-shaped or globular. Mitochondrial cristae were scarce and indistinct on day 28 but numerous and well developed on day 64. Endoplasmic reticula (ER) are rare on day 28 and appear in increasing numbers when CA activity rises. On day 40, ER are abundant and often exhibit a whorl-like appearance which is not observed on day 28. After day 44, when biosynthetic activity is declining, whorls of ER gradually decrease in number and are ultimately replaced by vesicular smooth ER on day 64. Neurosecretions are found only after day 38, by which time rates of JH synthesis have increased substantially from those of day 28. Except for membranous autophagic vacuoles, which are frequently found when ER whorls disintegrate as rates of JH synthesis decline toward birth, most autophagic vesicles such as multivesicular vesicles and dense bodies occur only sporadically among CA cells at all examined ages. We conclude that synchronous autophagy of exhausted organelles, which results in atrophy of CA cells and long-term arrest of JH synthesis in adult females of D. punctata, does not occur in embryos. The slow cyclic change in rate of JH synthesis in embryonic CA is most likely due to asynchronous autophagic activity and to alterations in certain unique features of intracellular organelles.     

Distribution of allatostatin in the adult cockroach,Diploptera punctata and effects on corpora allata in vitro

Direct radiochemical measurements of juvenile hormone synthesis showed that corpora allata from adult female Diploptera punctata can be inhibited in vitro by neuropeptides extracted from several ganglia of the central nervous system of females at many stages of the reproductive cycle. Extracts of protocerebra, corpora cardiaca, suboesophageal, thoracic and ventral ganglia all elicited dose-depedent reductions in juvenile hormone synthesis. On a ‘per organ’ basis, the protocerebrum contains the most extractable material. Inhibitory activity of extracts of suboesophageal, thoracic and 6th abdominal ganglia, like that of protocerebra (Rankin et al., 1986) was trypsin sensitive.Glands of high activity were less sensitive to protocerebral extract than those of low activity. The inhibitory effect on glands of low activity was maximal within 1 h, persisted in the presence of protocerebral extract for at least 46 h, and was abolished within 1 h after corpora allata were placed in normal medium. The inhibitory effect of protocerebral extract was not altered by the addition of magnesium to the medium. The extract had a specific effect on synthetic step(s) prior to methylation and epoxidation as demonstrated by enhanced juvenile hormone synthesis in the presence of inhibitory factor and the juvenile hormone precursor, farnesoic acid.     

Allatostatin-immunoreactive neurons projecting to the corpora allata of adult Diploptera punctata

Summary A monoclonal antibody against allatostatin I was used to demonstrate the allatostatin-immunoreactive pathways between the brain and the corpus cardiacum-corpus allatum complex in the adult cockroach Diploptera punctata. The antibody was two to three orders of magnitude more sensitive to allatostatin I than to the other four known members of the allatostatin family. Whole and sectioned brains in which immunoreactivity was localized with horseradish peroxidase-H₂O₂-diaminobenzidine reaction showed strongly immunoreactive cells in the pars lateralis of the brain with axons leading to and arborizing in the corpus cardiacum and the corpus allatum. Although many neurosecretory cells of the pars intercerebralis project to the corpora allata only, four strongly immunoreactive cells were evident here (two pairs on either side), and these did not project to the corpus cardiacum and corpus allatum but rather terminated within the protocerebrum in areas in which lateral cells also formed arborizations. Immunoreactivity was found in many other cells in the brain, especially in the tritocerebrum.     

In vitro inhibition of the corpora allata by a larval brain factor in the cockroach Diploptera punctata

The inhibitory activity of extracts of brains from final instar male and female larvae on release of newly synthesized juvenile hormone (JH) was examined, as was the responsiveness of corpora allata (CA) from males and females to brain extract. Aqueous extracts of protocerebra from day 0 and day 14 final instar larvae (in which the CA are active and inactive, respectively) were tested on CA from adult females in vitro. For comparison, protocerebra from day 0 adult males and females were also tested. Dose responses were similar for protocerebra from all animals tested. In each case a dose of 1–2 protocerebral equivalents was required for maximal response.CA from penultimate instar females were inhibited to a greater extent by brain extract than those of males. The response of CA from final instar larvae to brain extract declined in the first few days of the stadium as the ability of the glands to biosynthesize JH declined.     

Terminal stages in juvenile hormone biosynthesis in corpora allata of Diploptera punctata: Developmental changes in enzyme activity and regulation by allatostatins

The O-methyltransferase, which is responsible for the methylation of farnesoic acid in the corpora allata of Diploptera punctata, is a cytosolic enzyme. The activity of O-methyltransferase closely parallels JH biosynthesis in last instars and adult females. Because allatostatin 4 (AST 4) from D. punctata and callatostatin 5 (CAST 5) from Calliphora vomitoria can inhibit juvenile hormone biosynthesis, their effects on the activity of O-methyltransferase and epoxidase, the enzymes involved in the final two steps of juvenile hormone biosynthesis, were investigated in vitro. AST 4 can inhibit methyltransferase activity whereas CAST 5 stimulates it. AST 4 inhibits epoxidase activity slightly whereas CAST 5 inhibits it significantly (36%). Treatment of corpora allata with farnesoic acid (40 μM) can reverse the inhibitory effect of AST 4 and CAST 5 on JH release by corpora allata. Thus, allatostatins appear to exert their inhibitory effect on JH biosynthesis at least partially through inhibition of the activity of terminal enzymes. Two biosynthetic pathways for the conversion of farnesoic acid to JH may exist in corpora allata of D. punctata: the predominant pathway is farnesoic acid to methyl farnesoate, then to JH whereas the other, representing about 5–10% of total JH production, is farnesoic acid to JH III acid, then to JH.     

Humoral inhibition of the corpora allata in larvae of Diploptera punctata: Role of the brain and ecdysteroids

Juvenile hormone synthesis by adult female corpora allata was inhibited following implantation into final-larval-instar males; inhibition was prevented by decapitation of the larval hosts on day 11 (prior to the head critical period for moulting), but not by decapitation on day 13. Implantation of one larval protocerebrum restored inhibition of implanted corpora allata, demonstrating that the brain releases an inhibitory factor. Corpora allata implanted into larvae decapitated on day 11 were inhibited by injections of 20-hydroxyecdysone. Since treatment of corpora allata with 20-hydroxyecdysone in vitro did not inhibit juvenile hormone synthesis, ecdysteroids probably act indirectly on the corpora allata. Juvenile hormone synthesis and haemolymph ecdysteroid concentration were measured following implantation of corpora allata along with two larval brains into larval hosts. Brain implantation did not affect ecdysteroid concentration, but did inhibit juvenile hormone synthesis, even in animals with low haemolymph ecdysteroid concentration. Incubation with farnesoic acid stimulated juvenile hormone synthesis by corpora allata from males early in the final larval stadium, but not after day 8, showing that one of the final two reactions of juvenile hormone synthesis is rate-limiting in larval corpora allata at this stage. Adult female corpora allata which had been humorally inhibited by implantation into larvae were stimulated by farnesoic acid.     

Ionotropic glutamate receptors mediate juvenile hormone synthesis in the cockroach,Diploptera punctata

By monitoring changes in the cytosolic [Ca2+](i) and rates of juvenile hormone (JH) synthesis in response to L-glutamate agonists and antagonists, we identified and characterized glutamate receptor subtypes in corpus allatum (CA) cells of the cockroach, Diploptera punctata. During the first ovarian cycle, corpora allata exhibited a cycle of changes in sensitivity to L-glutamate correlated to cyclic changes in rates of JH synthesis. When exposed to 60 microM L-glutamate in vitro, the active corpora allata of day-4 mated females produced 60% more JH, while inactive corpora allata at other ages showed 10-20% stimulatory response. Pharmacological characterization using various L-glutamate receptor agonists and antagonists indicated that several ionotropic subtypes of L-glutamate receptors were present in the CA. The CA showed an increase in rates of JH synthesis in response to NMDA, kainate, and quisqualate, but not to AMPA in both L-15 medium and minimum incubation medium. In contrast, applications of the metabotropic receptor-specific agonist trans-ACPD failed to elicit a change in the cytosolic [Ca2+](i) and JH production.An elevation of cytosolic calcium concentration, followed by 20-30% rise in JH production, was observed when active CA cells were exposed to 10-40 microM kainate. Kainate had no stimulatory effect on JH synthesis in calcium-free medium. The kainate-induced JH synthesis was blocked by 20 microM CNQX but was not affected by 20 microM NBQX. Kainate-stimulated JH production was not suppressed by MK-801 (a specific blocker of NMDA-receptor channel), nor was NMDA-stimulated JH production affected by CNQX (a specific antagonist of kainate receptor). These data suggest that active CA cells are stimulated to synthesize more JH by a glutamate-induced calcium rise via NMDA-, kainate- and/or quisqualate-sensitive subtypes of ionotropic L-glutamate receptors. The metabotropic-subtype and ionotropic AMPA-subtype L-glutamate receptors are unlikely to be present on active CA cells.     

Methyl farnesoate and juvenile hormone production in embryos of Diploptera punctata in relation to innervation of corpora allata and their sensitivity to allatostatin

Corpora allata (CA) of embryos of Diploptera punctata have been previously shown to produce JH III. We have re-examined sesquiterpenoid biosynthesis throughout embryonic development and have found that early embryos produce both methyl farnesoate (MF) and JH III; as development proceeds, less MF and more JH is produced. The cockroach allatostatin peptide Dippu-allatostatin (AST) 7 inhibits sesquiterpenoid production by CA of mid to late embryos whereas it exerts a dose-dependent stimulatory effect in early embryos. This stimulatory effect is particularly apparent on MF biosynthesis. CA become innervated by allatostatin-containing nerves in early embryos (35% development). Shortly thereafter, the allatostatin-containing innervation of the CA appears complete.     

Localization and physiological effects of RFamides in the corpora allata of the cockroach Diploptera punctata in relation to allatostatins

The distribution of FMRFamide immunoreactivity in the brain-retrocerebral complex of adult female Diploptera punctata was examined. Immunoreactivity was observed in the brain and corpus allatum as well as in the corpus cardiacum. Immunoreactivity co-localized with allatostatin immunoreactivity within several lateral neurosecretory cells of the brain and in their endings within the corpus allatum. By in vitro radiochemical assay of juvenile hormone release, the effect of two native D. punctata RFamides, an FLRFamide (Leucomyosuppressin) and an FIRFamide were examined. The latter, for which the sequence (SKPANFIRFamide) is reported here, stimulated juvenile hormone release but acted only on corpora allata from females at the end of vitellogenesis (day 6). The interaction of these two RFamides and three D. punctata allatostatins, Dippu-AST 2, 5, and 7 were similarly examined. Only Dippu-AST 2 stimulated release of RFamides from the corpora allata and only on day 6 whereas both RFamides were able to attenuate the inhibitory activity of Dippu-AST 2.     

Photoaffinity labeling of allatostatin receptor proteins in the corpora allata of the cockroach, Diploptera punctata.

Membranes of corpora allata, obtained from Diploptera punctata virgin females, were incubated for 45 min with a bioactive, radioiodinated azidosalicylamide photoaffinity analogue of allatostatin-1. Irradiation of the incubation mixture at 254 nm, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography, revealed the presence of two bands (59 and 39 kDa) whose specific labeling was demonstrated by addition of excess allatostatin. In brain preparations, clear competition by excess allatostatin was shown for a 41 kDa protein. The abdominal fat body also contained proteins (38, 41, 60 kDa) which bound specifically to the photoaffinity analogue. The proteins identified in corpora allata are likely involved in allatostatin recognition, but the functions of those identified in the brain and fat body are as yet undetermined.     

An allatostatic factor and juvenile hormone synthesis by corpora allata in Locusta migratoria

The hemolymph juvenile hormone (JH) titer in sexually immature female adults of Locusta migratoria (Ibaraki strain, Japan) was lower than in sexually mature females; nevertheless, JH synthetic activity by the corpora allata (CA) in vitro was considerably higher in immature females than in sexually mature females ([Okuda et al., 1996]). We carried out experiments to explain this contradiction. The CA activity of sexually immature female adults was very low when the CA were incubated as a complex together with the corpora cardiaca (CC) and brain. When the same complex was assayed after cutting the nerve cord connecting the CC and CA (NCA1), JH synthesis by the CA was enhanced tenfold. When this pair of CA was incubated in fresh medium without the CC and brain, JH synthesis was further increased. Therefore, the higher in-vitro JH production by CA from immature female adults was the result of isolation of the CA from the brain and CC. A methanolic extract of brain-CC complexes contained a factor that inhibited JH synthetic activity by CA in vitro in both immature and mature insects, and this inhibition was reversible. The factor was heat-resistant but lost allatostatic activity after pronase digestion. These results indicate that the allatostatic factor is probably a heat-stable peptide.     

Autophagy and acid phosphatase activity in the corpora allata of adult mated females of Diploptera punctata

The corpora allata exbibit cycles of synchronous cell growth and atrophy during ovarian cycles in adult females of the cockroach Diploptera punctata. In the present report, the process of synchronous autophagy of organelles which results in cellular atrophy was investigated. In general, unwanted organelles were sequentially sequestered by several different mechanisms and then targeted for destruction. Autophagy was initiated on day 4 when corpus allatum cells were largest and most actively synthesizing juvenile hormone. The first sign of the initiation of autophagy was aggregation of ribosomes in an isolation membrane. By day 5, many organelles were isolated in the autophagic vacuoles. The ribosomecontaining vacuoles were wrapped by flattened stacks of Golgi cisternae to form conspicuous whorl-like autophagosomes. This is a previously undescribed type of autophagic vacuole with the entire complex of Golgi cisternae forming part of the autophagic membranes. Smooth endoplasmic reticulum was wrapped into membranous autophagic vacuoles with concentric arrays of doubel membranes. Plasma membrane was invaginated and then isolated in a multivesicular body. These three different types of isolated vacuoles did not show acid phosphatase activity as indicated by histochemical staining with -glycerophosphate as substrate. Subsequently, these autophagosomes fused with each other and with 1° or 2° lysosomes to form giant autophagolysosomes. Some mitochondria appeared to have coalesced directly into autophagolysosomes. Golgi complexes were evident during this period; they actively participated in making lysosomal enzymes. Cytoskeletons were frequently observed in the vicinity of autophagic vacuoles and were presumably involved in the transport of the vacuoles. As a result of lysosomal degradation lipofuscins and dense bodies were frequently observed by days 9–12 indicating atrophy of corpus allatum cells. Structural parameters, especially those present early in autophagy, such as the isolation membrane, ribosome-containing vacuoles and whorl-like autophagosomes, can be used to search for potential growth regulators responsible for the induction of autophagy, of the corpora allata, and the subsequent termination in juvenile hormone synthesis.