An active principle from the corpora cardiaca,corpora allata and suboesophageal ganglion of the locust,inducing egg diapause in Bombyx mori

The activity of the substance(s) which are contained in the cephalic endocrine organs of the locust which induce egg diapause in Bombyx mori was examined by implantation and injection of saline extracts of these organs. Extracts from the median and lateral neurosecretory parts of the locust brain were not effective in inducing egg diapause. Extracts of the corpora cardiaca, corpora allata, and suboesophageal ganglion of the locust induced diapause eggs in Bombyx pharate adults from which the suboesophageal ganglion had been removed. The first two extracts could induce egg diapause even in isolated abdomens of pharate adults of Bombyx. In the locust corpora cardiaca, the activity was present only in the glandular lobe and not in the nervous region. This activity decreased when the nervi corporis cardiaci I and II and of nervi corporis allati I were cut. Allatectomy also brought about a decrease in the activity in the glandular lobe which could not be restored by the injection of juvenile hormone. The activity in the corpora allata was enhanced slightly by the disconnection though not significantly.From these results, it is assumed that the corpora cardiaca, corpora allata and suboesophageal ganglion of the locust contain and active principle(s) capable of inducing egg diapause in Bombyx mori. The nervous connections between the brain, corpora cardiaca, and corpora allata are essential for the accumulation of the active substance(s) in the glandular lobes of the corpora cardiaca.     

20-hydroxyecdysone stimulation of juvenile hormone biosynthesis by the mosquito corpora allata

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. JH titer decreases in the last instar larvae allowing pupation and metamorphosis to progress. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again “competent” to synthesize JH, which plays an essential role orchestrating reproductive maturation. 20-hydroxyecdysone (20E) prepares the pupae for ecdysis, and would be an ideal candidate to direct a developmental program in the CA of the pharate adult mosquito. In this study, we provide evidence that 20E acts as an age-linked hormonal signal, directing CA activation in the mosquito pupae. Stimulation of the inactive brain-corpora allata-corpora cardiaca complex (Br-CA-CC) of the early pupa (24 h before adult eclosion or −24 h) in vitro with 20E resulted in a remarkable increase in JH biosynthesis, as well as increase in the activity of juvenile hormone acid methyltransferase (JHAMT). Addition of methyl farnesoate but not farnesoic acid also stimulated JH synthesis by the Br-CA-CC of the −24 h pupae, proving that epoxidase activity is present, but not JHAMT activity. Separation of the CA-CC complex from the brain (denervation) in the −24 h pupae also activated JH synthesis. Our results suggest that an increase in 20E titer might override an inhibitory effect of the brain on JH synthesis, phenocopying denervation. All together these findings provide compelling evidence that 20E acts as a developmental signal that ensures proper reactivation of JH synthesis in the mosquito pupae.     

Ecdysis triggering hormone ensures proper timing of juvenile hormone biosynthesis in pharate adult mosquitoes

Juvenile hormones (JHs) are synthesized by the corpora allata (CA) and play a key role in insect development. A decrease of JH titer in the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again “competent” to synthesize JH, which would play an essential role orchestrating reproductive maturation. In the present study, we provide evidence that ecdysis triggering hormone (ETH), a key endocrine factor involved in ecdysis control, acts as an allatotropic regulator of JH biosynthesis, controlling the exact timing of CA activation in the pharate adult mosquito. Analysis of the expression of Aedes aegypti ETH receptors (AeaETHRs) revealed that they are present in the CA and the corpora cardiaca (CC), and their expression peaks 4 h before eclosion. In vitro stimulation of the pupal CA glands with ETH resulted in an increase in JH synthesis. Consistent with this finding, silencing AeaETHRs by RNA interference (RNAi) in pupa resulted in reduced JH synthesis by the CA of one day-old adult females. Stimulation with ETH resulted in increases in the activity of juvenile hormone acid methyltransferase (JHAMT), a key JH biosynthetic enzyme. Furthermore, inhibition of IP₃R-operated mobilization of endoplasmic reticulum Ca²⁺ stores prevented the ETH-dependent increases of JH biosynthesis and JHAMT activity. All together these findings provide compelling evidence that ETH acts as a regulatory peptide that ensures proper developmental timing of JH synthesis in pharate adult mosquitoes.     

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.     

Endocrine regulation of terminal muscle differentiation: Atrophy and degeneration of the intersegmental muscles of Lepidoptera

The intersegmental muscles of the Lepidoptera pass through three separate, sequential differentiated states during pharate adult development: status quo; atrophy; degeneration. Each of these developmental programs is characterized by a distinct morphology, physiology and endocrine responsiveness. The factors responsible for regulating these differentiative changes are ecdysteroids. In Manduca sexta, the haemolymph ecdysteroid titre declines in a circadian-modified fashion during the last three days of adult development, which parallels the maturation of the intersegmental muscles. Abdomen-ligation, which causes a precipitous decline in the ecdysteroid titre, causes the precocious atrophy and degeneration of these muscles, whereas injection of, or infusion with, 20-hydroxyecdysone greatly delays such changes. While the terminal differentiation of the epidermis and nervous system is also regulated by ecdysteroids, endocrine manipulations have suggested that the development of the intersegmental muscles is independent of these tissues.In the silkmoth Antheraea polyphemus, ecdysteroids are also responsible for regulating intersegmental muscle differentiation, but eclosion hormone (a peptide) acts as the proximal trigger for the activation of the degeneration program. The declining ecdysteroid titre initiates the atrophy program and subsequently determines the timing of both release of eclosion hormone and intersegmental muscle sensitivity to the peptide. Eclosion hormone then acts directly on the muscles, via cGMP, to activate the degeneration program. Ecdysteroids appear to prevent premature muscle degeneration by regulating a biochemical step distal to both the eclosion hormone receptor and the rise in cGMP.     

Evidence for a crustacean hyperglycemic hormone-like molecule in the nervous system of the stick insect,Carausius morosus

Summary Histological sections of the brain, suboesophageal ganglion, and the corpora cardiaca/corpora allata complex were examined for the presence of crustacean hyperglycemic hormone-like immunoreactive material. With the use of an antiserum directed against the hyperglycemic hormone of Carcinus maenas, immunofluorescence was found in the median portion of the pars intercerebralis, and the corpora cardiaca. Extracts of corpora cardiaca were examined by radioimmunoassay for competitive binding to the antiserum; one pair of corpora cardiaca contains at least 7 pg crustacean hyperglycemic hormone-like material.     

Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and Black mutant Manduca sexta larvae

The absence of juvenile hormone at the time of head cap slippage during the last-larval moult of the tabacco hornworm, Manduca sexta, causes deposition of premelanin granules into the outer regions of the newly forming endocuticle beginning 13 h later. These granules were found to contain an inactive phenoloxidase which becomes activated about 9 h later, 4 h before body melanization begins. The onset of melanization was not accelerated by melanization and reddish colouration hormone from Bombyx heads, extracts of pharate-adult corpora cardiaca or pharate-larval ventral nerve cords (sources of eclosion hormone), or extracts of pharate-larval suboesophageal ganglia or corpora cardiaca-corpora allata complexes. Instead the fall of the ecdysteroid titre to below 250 ng/ml 20-hydroxyecdysone equivalents appeared to be the cue that allowed melanization about 4.5 h later. Up to, but not after, this time both melanization and ecdysis could be delayed by exogenous 20-hydroxyecdysone in a dose-dependent fashion above 0.1 μg per larva. In vitro studies published elsewhere indicate that 20-hydroxyecdysone prevents the activation of the premelanin granules. Thus the granules can be deposited at the proper time in the newly forming endocuticle but their melanization is regulated by the declining ecdysteroid titre and it thus synchronized with other events occurring just before ecdysis.     

Eclosion hormone activity in developing embryos of the silkworm, Bombyx mori

The ultimate timing of hatching in the silkworm, Bombyx mori, is controlled by a circadian oscillator. The presence of eclosion hormone in developing embryos of the silkworm is demonstrated. Eclosion hormone activity first becomes detectable in embryos which have developed almost to the stage of the differentiation of the neuroendocrine system. Hormonal activity increases sharply to a maximum level 1 day before hatching and falls by about a half in the newly hatched larvae. Eclosion hormone was partially purified from the pharate first-instar larvae and approx, a 2100-fold purification was achieved. The molecular weight of the embryo eclosion hormone is estimated to be 7000 ～ 9000 Daltons by gel-filtration on Sephadex G-50 (superfine). The role of eclosion hormone on hatching behaviour of the silkworm, Bombyx mori, is discussed.     

Polyoxin N,a New Member of the Polyoxin Family

Eclosion hormone was purified 5,000-fold from the extracts of male adult heads of the silkworm, Bombyx mori. The fourteen-step purification procedure consisting of solvent extractions, fractional precipitations and chromatographies afforded a partially purified preparation of eclosion hormone, 1.8 μg of which showed activity in a Bombyx pharate adult. The hormone was inactivated by proteolytic enzymes. The molecular weight of eclosion hormone was estimated to be 8,400 ± 1,000 daltons by gel-filtration on Sephadex G-50.     

Interendocrine control by 20-hydroxyecdysone of the corpora allata of Manduca sexta

A positive interendocrine control by 20-hydroxyecdysone regulates the corpora allata (CA) and thus the hemolymph titers of the juvenile hormones (JHs) during the fourth and fifth larval stadia of the tobacco hornworm, Manduca sexta. The effect of 20-hydroxyecdysone is exerted via the brain-corpora cardiaca (Br-CC), and the kinetics of stimulation by the hormone suggest that its control of the CA is indirect, possibly involving neural effectors. Co-incubation of precommitment CA with Br-CC resulted in a stimulation of the synthesis of JH I acid equivalent to that achieved by the incubation of brain-corpora cardiaca-corpora allata (Br-CC-CA) with 20-hydroxyedcysone, suggesting that a diffusible regulatory factor may be involved in the stimulation. During pharate pupal development, a close temporal relationship exists between the drop in the ecdysteroid titer and declines in CA activity and the JH hemolymph titer, suggesting that 20-hydroxyecdysone may now inhbit the CA. Incubations of day 6 Br-CC-CA with physiological concentrations of 20-hydroxyecdysone resulted in an apparent dose-dependent inhibition of CA activity. Thus in Manduca, 20-hydroxyecdysone may exert varying stage-specific interendocrine effects on the CA which direct the postembryonic development of this species.     

Re-evaluation of the role of corpora cardiaca in calling and oviposition behaviour of giant silk moths

Re-investigation of the role of the corpora cardiaca in the reproductive behaviour of the giant silkmoths, Hyalophora cecropia and Antheraea polyphemus, showed that this pair of glands plays no essential role, either in “calling” behaviour by virgin females or in increased oviposition due to mating. Removal of corpora cardiaca-corpora allata complexes, either from diapausing pupae or from freshly eclosed adult females, had no effect on the calling behaviour or on its timing in either species. Moreover after mating, these operated females laid eggs in the typical mated oviposition pattern. Furthermore, females in which there was only a nervous connection between the brain and the abdomen but no haemolymph circulation called normally and oviposited after mating.Although the corpora cardiaca were not essential for calling behaviour, hormogenates of corpora cardiaca-corpora allata complexes and blood from calling or ovipositing females induced a typical “calling” response in 30–60% of the isolated virgin H. cecropia abdomens tested. This activity was not species-specific as it was also found in Manduca sexta, but the restriction of major activity to corpora cardiaca extracts and haemolymph suggested that a neurosecretory factor may modulate the normal neural control of calling behaviour.     

Effect of endocrine glands on the unfixed total haemocyte counts of the bug Halys dentata

Changes in total haemocyte counts in relation to the endocrine glands have been studied in Halys dentata. Unfixed total haemocyte counts were taken after extirpation and implantation of three endocrine structures. It is concluded that haemocyte count decreases after the extirpation of the corpora allata in the early life span of adults, while an abrupt increase was noted after the 7th day. When both the corpora allata and corpora cardiaca are extirpated, the count was significantly decreased throughout the life span of adults. However, when only the corpora cardiaca were removed there was no significant variation up to the 7th day, but later on the count gradually decreased. The implantation of brain, corpora cardiaca and allata is followed by a significant rise in the haemocyte count throughout the life span. Total haemocyte count also fluctuated after the implantation of corpora allata and brain, separately. After implantation of corpora cardiaca of one-day-old adult insects there was no variation in the total haemocyte count while the corpora cardiaca of 6-day-old adults when implanted in one-day-old adults cause a significant rise in the total haemocyte count. Therefore, it is concluded that the corpora cardiaca influences the total haemocyte count after a critical period.     

Endocrine events during pre-diapause and non-diapause larval-pupal development of the tobacco hornworm, Manduca sexta

The haemolymph ecdysteroid titre and in vitro capacities of prothoracic glands and corpora allata to synthesize ecdysone and juvenile hormone, respectively, during the last-larval instar of diapause-destined (short-day) and non-diapause-destined (long-day) Manduca sexta were investigated. In general, the ecdysteroid titres for both populations of larvae were the same and exhibited the two peaks characteristic of the haemolymph titre during this developmental stage in Manduca. The only difference in the titre occurred between day 7 plus 12 h and day 7 plus 20 h, when the short-day larval titre did not decrease as quickly as the long-day titre. The in vitro synthesis of ecdysone by prothoracic glands of short- and long-day larvae during the pharate pupal phase of the instar were also essentially the same. Activity fluctuated at times which would support the idea that ecdysone synthesis by the glands is a major contributing factor to the changes in the haemolymph ecdysteroid titre. There was one subtle difference in prothoracic gland activity between the two populations, occurring on day 7 plus 2 h. By day 7 plus 10 h, however, rates of ecdysone synthesis by the short- and long-day glands were comparable. This elevated activity of the short-day glands occurred just prior to the period the haemolymph ecdysteroid titre remained elevated in these larvae. The capacities of corpora allata to synthesize juvenile hormone I and III in vitro were not markedly different in long- and short-day last-instar larvae. At the time of prothoracicotropic hormone release in the early pupa, activity of corpora allata from short- and long-day reared animals was low and also essentially the same. There were a few differences in the levels of synthesis at isolated times, but they were not consistent for both homologues. Overall, there are no compelling differences in the fluctuations of ecdysteroids and juvenile hormones between diapause-destined and non-diapause-destined Manduca larvae. Since these hormones do not appear to play any obviously significant role in the induction of pupal diapause in this insect, the photoperiodic induction of diapause in Manduca appears to be a predominantly brain-centred phenomenon not involving endocrine effectors.     

Induction of egg diapause in Bombyx mori by some cephalo-thoracic organs of the cockroach, Periplaneta americana

Induction of egg diapause in the silkworm, Bombyx mori by some cephalo-thoracic organs of the cockroach, Periplaneta americana was examined. All tissues tested such as brain, corpora cardiaca, corpora allata, suboesophageal and thoracic ganglia and nerve cords between thoracic ganglia were able to produce diapause eggs in non-diapause egg producers both by transplantation and injection of their crude homogenates. The homogenate of thoracic ganglia was effective even in pharate adults with the suboesophageal ganglion removed or in isolated abdomens of pharate adults.From these results, it was surmised that some endocrine organs, as well as the central nervous system in the cephalo-thorax of Periplaneta americana, contained the active principle responsible for egg diapause in Bombyx mori.     

NADPH diaphorase and nitric oxide synthase in the corpus cardiacum-corpus allatum of the cockroach Diploptera punctata

Juvenile hormone synthesis by corpora allata is regulated partly by allatostatin containing nerves from the brain that innervate the corpora cardiaca and the corpora allata. To investigate whether NO also participates in the regulation of juvenile hormone synthesis, antibody against NO synthase and the histochemical test for NADPH diaphorase activity, a marker for NO synthase, were applied to the corpora cardiaca-corpora allata of Diploptera punctata. Strong NADPH diaphorase activity occurred in corpus allatum cells but not in nerve fibers in the corpora allata or corpora cardiaca. In contrast, NO immunoreactivity occurred in nerves in the corpora cardiaca but not within the corpora allata. NO and allatostatin were not colocalized. NO synthase and NADPH diaphorase activity were localized in similar areas of the subesophageal ganglion and cells in the pars intercerebralis of the brain. Positive correlation of the quantity of NADPH diaphorase activity with juvenile hormone synthesis during the gonadotrophic cycle and lack of such correlation in subesophageal ganglia suggest that NADPH diaphorase activity reflects the necessity of NADPH in the pathway of juvenile hormone synthesis. These data suggest that NO is unlikely to play a significant role in the regulation of the corpora allata.     

Lack of humoral control in calling and pheromone release by brain,corpora cardiaca,corpora allata and ovaries of the female gypsy moth, Lymantria dispar (L.)

Investigations into the physiological regulation of calling behaviour and pheromone release by the adult female gypsy moth, Lymantria dispar, showed that the brain, corpora cardiaca, corpora allata and ovaries play no significant endocrine role for either calling behaviour or pheromone release. Removal of corpora allata, corpora cardiaca-corpora allata complex and ovaries from last-instar larvae had no effect on calling behaviour nor on the pheromone release in the resulting moths. Severing the suboesophageal connectives had no effect on calling, but effectively eliminated pheromone release. Since severing nerves should not remove humoral output from the brain, the regulation on pheromone release appears neural.     

Effects of decapitation and ovariectomy on the regulation of juvenile hormone synthesis in the cockroach, Diploptera punctata

Corpora allata from Diploptera punctata females at adult ecdysis or at the end of the last-larval stadium, when implanted into decapitated females, underwent a cycle of juvenile hormone synthesis similar in timing and magnitude to that of glands implanted into control animals which had been starved and allatectomized. Starvation did not alter the cycle in rates of juvenile hormone synthesis of sham-operated animals.Decapitation of ovariectomized animals resulted in no cycle in rates of juvenile hormone synthesis by implanted adult corpora allata; however, implantation of an ovary along with the corpora allata into decapitated, ovariectomized hosts resulted in a cycle of juvenile hormone synthesis. In control animals, which retained their heads but were starved and allatectomized as well as ovariectomized, the implanted corpora allata showed a cycle of juvenile hormone synthesis only when implanted with an ovary. The maximal rates of juvenile hormone synthesis by the corpora allata in both experimental and control conditions were lower than normal, likely due to the repeated trauma of surgery. However, at no time from eclosion to the end of the first gonotrophic period was the brain necessary for the cyclic response of the corpora allata to the presence of the ovary.     

Dynamics and metamorphosis of an identifiable peptidergic neuron in an insect

Eclosion hormone (EH) is a 7000 Da peptide that triggers ecdysis behavior in insects. In the moth, Manduca sexta, EH is found in two pairs of ventromedial (VM) cells in the brain which send their axons down the ventral nerve cord to a neurohemal site in the proctodeal nerve in the larva and pupa. During adult development, these cells send axon collaterals to the corpora cardiaca where they form a new release site used for adult eclosion. Studies of bioassayable peptide during the 5th larval instar and the larval-pupal transformation revealed that after depletion at ecdysis, the VM cells showed a transient increase in EH found in their cell bodies and axons. By contrast, their terminals in the proctodeal nerve showed a gradual accumulation of peptide followed by a release of over 90% of the stored material at pupal ecdysis. In situ hybridization analysis on whole mounts of the brains showed that the VM cells always contained EH mRNA with increased accumulation during the larval and pupal molting periods with a slight decline just before ecdysis. High levels of EH mRNA were found in brains of diapausing pupae. During the first two-thirds of adult development, mRNA accumulated to high levels, then slowly declined until ecdysis. EH mRNA levels up to 3 days after adult eclosion. At no time was EH mRNA found in the lateral neurosecretory cell cluster previously reported to produce EH for adult eclosion. 1994 John Wiley & Sons, Inc.     

Disturbance of adult eclosion by fenoxycarb in the silkworm,Bombyx mori

Fenoxycarb treatment before and after pupal ecdysis of Bombyx mori disturbed adult eclosion and the animals were unable to escape from the pupal exuviae. This effect of fenoxycarb was dose and time dependent with the highest sensitivity around the pupal ecdysis. The sensitivity rapidly diminished within 20 hours of pupal ecdysis. Twenty-hydroxyecdysone (20E) produced similar effects. Fenoxycarb injection at the pupal ecdysis induced higher ecdysteroid production by the prothoracic glands and higher PTTH-secretory activity in the brain-corpora cardiaca-corpora allata complexes. As a result, the fenoxycarb treated pupae contained higher ecdysteroid titres in the haemolymph. Both the fenoxycarb and the 20E treatments resulted in the lack of development of the rectum in pharate adults. This was the main cause of high ecdysteroid titres in the pharate adult stage. This effect was mimicked by either removal of the rectum early in the pharate adult stage or a surgical extirpation of the hindgut at the time of pupal ecdysis. These results suggest that the disturbance of adult eclosion by fenoxycarb is due in part to the inability of the formation of the rectum in the pharate adult stage.     

Spatial expression of the mevalonate enzymes involved in juvenile hormone biosynthesis in the corpora allata in Bombyx mori

The developmental expressions of the mRNA of JH synthetic enzymes have been studied using homogenates of the corpora cardiaca-corpora allata (CC-CA) complexes in Bombyx mori [Kinjoh, T., Kaneko, Y., Itoyama, K., Mita, K., Hiruma, K., Shinoda, T., 2007. Control of juvenile hormone biosynthesis in Bombyx mori: cloning of the enzymes in the mevalonate pathway and assessment of their developmental expression in the corpora allata. Insect Biochemistry and Molecular Biology 37, 808-818]. The in situ hybridization analyses in the CC-CA complex showed that the distribution of the mRNAs of all the mevalonate enzymes and juvenile hormone (JH) acid O-methyltransferase occurred only in the CA cells, indicating that the fluctuations of the enzyme mRNA amounts in the CC-CA complexes were derived solely from the CA. In addition, the size of the CA and their nuclei was not associated with the JH synthetic activity by the CA until the pharate adult. Only female adult CA synthesized JH in B. mori, and the CA and the nuclei were significantly larger than those of male CA which do not synthesize JH.