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.     

Effect of injection of synthetic Hyalophora cecropia juvenile hormone in Locusta migratoria L]

The synthetic racemic C18 Hyalophora cecropia juvenile hormone (JH-I) is injected at does of between 10 and 200 mug/animal at the end of the fourth instar of Locusta migratoria. The effects on mortality, length of the fourth and fifth instars, pigmentation and morphogenesis are reported. Higher doses of JH-I produce a higher mortality than lower doses. But mortality can also occur following the injection of oil which sometimes takes place only a few hours before the ecdysis. In no case is JH-I able to shorten the length of the instar. Many animals moult at the same time as the controls, but some of them, both in the fourth and fifth instars, show an important increase in the length of the instar because of an inhibition of the ecdysis. The effect of JH-I on pigmentation is very important and doses higher than 50 mug/animal present a greater effect than an implantation of one pair of corpora allata, both on the number of insects which turn green and on the intensity of this green pigmentation. At the metamorphosis the larvae injected with JH-I produce imperfect imagos and supernumerary larvae, the number of which depends upon the dose. Nevertheless the morphogenetic effect is considerably lower than that of one pair of corpora allata. We have reason to think that this is only due to the time of injection and not to the activity on morphogeneis of the injected hormone. JH-I is injected at the dose of 200 mug in young females which were allatectomized beforehand to prevent oocytes maturation. The hormone completely counter-balances the lack of the corpora allata and some days after the injection the oocytes are in the same state of development as those of the controls. All the results indicate that the synthetic racemic C18 juvenile hormone of Hyalophora cecropia shows a quite similar activity to the secretion of the corpora allata on Locusta migratoria although it has been said for some time that this hormone was not the principal one in locusts.     

Induction of supernumerary larval moulting in the tobacco hornworm Manduca sexta: interaction of bisacylhydrazine ecdysteroid agonists with endogenous Juvenile Hormone

Abstract When given in a critical dietary dose range, the insecticidal bisacylhydrazine ecdysteroid agonists RH‐5849 or tebufenozide (RH‐5992) cause fifth stage Manduca sexta (L.) larvae to moult to a supernumerary sixth‐stage giant larva. The effect is dependent on exposure to the chemicals immediately after the previous ecdysis. Previous removal of the corpora allata does not interfere with the induction of premature moulting by RH‐5849 but completely prevents the formation of supernumerary larvae. The juvenilizing effect is therefore due to the interaction of the moult‐promoting effect of the ecdysteroid agonists with the high titre of endogenous Juvenile Hormone that is present just after ecdysis to the fifth stage in this insect. The ecdysteroid agonists themselves appear to have no intrinsic Juvenile Hormone‐agonist properties. Sixth‐stage larvae resulting from exposure to critical dietary concentrations of RH‐5849 are morphologically completely larval in character. When transferred to diet without the ecdysteroid agonist, they feed normally and gain weight, growing much larger than control fifth stage insects. At the end of the supernumerary stage, they cease to feed, wander in the usual way, and form a normal pupal cuticle but then die as pharate pupae without shedding the sixth‐stage larval cuticle.     

Azadirachtin affects growth and endocrine events in larvae of the tobacco hornworm, Manduca sexta

Injection of azadirachtin in freshly emerged last-instar larvae of Manduca sexta elicited different reactions according to the dose administered. At low doses, pupation occurred in most of the cases, but the resulting pupae were defective for the most part. Individuals treated with higher doses usually did not fully complete development, moulting to supernumerary larvae or dying as larvae (sometimes at the wandering stage) after varying periods of survival. The haemolymph ecdysteroid titre of individuals treated with 2 μg azadirachtin/g bodyweight showed characteristic changes which are presumed to cause the disorders in the last stages that normally lead to pupation. Injection of moulting hormone in azadirachtin-treated individuals at certain times during the penultimate stage elicited no reduction of the azadirachtin-induced effects. It is shown that azadirachtin is able to inhibit development even when individuals performed a complete moult after the treatment.     

Quantitative determination of the juvenile hormones in the haemolymph of Locusta migratoria during normal development and after implantation of corpora allata

Simultaneous quantitative determination of the three naturally occurring juvenile hormones in insects (JH-I, JH-II and JH-III) was performed on haemolymph samples of both normally developing locusts and locusts implanted with active corpora allata, using capillary gas chromatography with electron capture detection.In fourth instar female larvae, 24–48 hr after the third ecdysis, as well as in adult females, 18 days after the imaginal ecdysis, only JH-III was detected. In fifth instar female larvae JH-III was present in very low concentrations, if at all.After implantation of four pairs of corpora allata taken from young fourth instar female larvae or one pair or corpora allata taken from adult females into fifth instar female larvae 0–24 hr after ecdysis, an elevation of the JH-III titre was observed. Neither JH-I nor JH-II could be detected. The amount of JH-III, already elevated 2 hr after implantation, remained high for several days in comparison to that of control insects. On the third day after the subsequent moult the JH-III level was comparable to that of normally developing fifth instar larvae. Factors involved in the achievement of the haemolymph JH-titre are discussed.     

Mode of action of the venom of the ectoparasitic wasp,Euplectrus comstockii, in causing developmental arrest in the European corn borer,Ostrinia nubilalis

Euplectrus comstockii Howard (Hymenoptera: Eulophidae), is an ectoparasitic, gregarious wasp which parasitizes the larval stage of several important lepidopteran pests. Parasitization of both natural and unnatural hosts prevents molting in the parasitized instar. Here we report the effect of wasp venom on the European corn borer (unnatural host), an important pest of corn and other vegetables. Venom collected from venom glands of adultE. comstockii, when injected intoO. nubilalis 5th instars, inhibited the growth rate, development and molting of the injected larvae. The observed effect on molting was dose and age dependent. When 3rd, 4th and 5th instarO. nubilalis were envenomated by adult wasps, the larvae also were developmentally arrested and failed to undergo a molt. However, 3rd and 4th instars underwent apolysis (separation of the epidermis from the old cuticle) and produced new cuticle. Fifth instars did not. A premolt hemolymph ecdysteroid peak was not observed in these experimental 5th instars, but injections of 20-hydroxy-ecdysone induced apolysis and new cuticle formation. Envenomated 4th instars (on becoming pharate 5th instars) exhibited a premolt hemolymph ecdysteroid peak. HPLC/RIA revealed that 20-hydroxyecdysone was present in the hemolymph of these pharate 5th instars. Thus, in the European corn borer, the mode of action of the venom depended upon the instar parasitized. Our results support the presence of a venom component(s) that, in 4th instar hosts, inhibited ecdysis, but did not prevent hemolymph ecdysteroid levels from increasing sufficiently to stimulate apolysis. In 5th instars, the same, or perhaps, a different component(s) ofE. comstockii venom prevented the synthesis/release of ecdysteroid by inhibiting a previously unknown molt-regulating physiological event that occurs between days 3 and 4 of the instar. Deceased     

Supernumerary instars produced by chilled wax moth larvae: Endocrine mechanisms

Young last instar larvae of Galleria mellonella underwent supernumerary ecdyses within 3 to 6 days after being chilled at 0 to 1°C for 30 min. The frequency diminished from 89 ± 9.4% for the survivors of those that were chilled <16 hr after their last ecdysis, to 25 ± 11.2% for those 46 to 88 hr old, and was no longer evident beyond 123 hr.Irrespective of their ages, the larvae never became “superlarvae” unless they had fed after they had been chilled. This was unlike the requirement for metamorphosis, when a feeding period of 40 to 48 hr immediately following ecdysis allowed half the larvae that were subsequently chilled and starved to pupate. The propensity to become superlarvae could be extended by starvation. Chilling signaled the occurrence of the larval moulting program, but its expression was held in abeyance until the larvae had fed.Brains from chilled or unchilled donors were equally effective initiators of supernumerary larval apolyses. The capacity to respond to chilling was abolished following bilateral extirpation of the corpora cardiaca and corpora allata, but not after the corpus cardiacum and corpus allatum of one side were removed. This effect of bilateral cardiacectomy and allatectomy could be remedied by applying Altosid, a juvenile hormone analog. Potentiation of the larval-larval apolysis by chilling and by JH may involve separate mechanisms, for the analog was less effective on unchilled larvae than on those that had been chilled. The results are discussed with reference to the hypothesis that the brains of young larvae produce an “allatotropic hormone”.     

Anteroposterior gradient during nymphal-adult moulting cycle of the tropical bont tick,Amblyomma variegatum (Acarina: Ixodidae)

Summary The structure of the extensible (alloscutum) and inextensible (scutum) integument of the nymph, Amblyomma variegatum was examined during the whole bloodmeal and the nymphal-adult moulting cycle. Integumental events were tentatively correlated with the ecdysteroid levels measured by radioimmunoassay. We observed that all the integumental events were realised along an anteroposterior gradient. During the 5 days corresponding to the bloodmeal, although the hormone concentration was low, a new endocuticle was deposited on both the alloscutum and scutum. Furthermore, mitoses were initiated in the capitulum. On days 1–2 after the meal, ecdysteroid titres began to increase and reached a first peak corresponding to 4.1 ng 20-hydroxyecdysone equivalents/tick on the 4th day after the ticks dropped off their host. At this time the epidermis of the capitulum was detached and the outline of the adult capitulum was already visible. Mitotic activity in the alloscutum was initiated. On day 6 post-drop, the frontal apolysis was achieved and the ecdysteroid titres declined to basal values. A second peak much higher than the first one (maximum value of 33.7 ng/tick) and identified principally as 20-hydroxyecdysone by HPLC/RIA was noted on the 13th day post-drop. During the period of increase in the ecdysteroid levels (days 9–10 post-drop), the mitotic phase ended in the alloscutum and the apolysis began. Epicuticle was deposited after day 12 postdrop. Then, while the titre fell to low values (about 1.6 ng/tick, days 16–20 post-drop), the exocuticle was deposited and the nymphal cuticle was digested. All adult structures were functional 3 days before ecdysis. In young male as in female adults the mean value of the ecdysteroid levels corresponded to about 2.5 ng/tick. Finally, hydrolysis of tick whole extracts with esterase demonstrated a low increase of RIA-positive material, demonstrating the probable presence of natural ecdysteroid fatty-acid conjugates in this species.     

Hormonal basis of adult eclosion in the gypsy moth (Lepidoptera: Lymantriidae)

Eclosion hormone was found to control the stereotypic adult eclosion behaviour of Lymantria dispar, the gypsy moth. A bioassay for hormonal activity was developed utilizing pharate adult females, and comparisons were made with the Manduca wing assay. The distribution of eclosion hormone activity was confined to the central nervous system tissues including the protocerebrum, corpora allata/corpora cardiaca complex, thoracic and the last abdominal ganglion. Haemolymph ecdysteroid titres were determined daily throughout pupal-adult development, and the peak activity period was found in 3–4 day pupae. Eclosion hormone activity in the brain and corpora allata/corpora cardiaca complex started to increase when the ecdysteroid titre dropped to background levels. Eclosion hormone in the brain peaked in the pharate adult stage, was released in the haemolymph 1 h prior to eclosion, which coincides with the depletion of activity in the retrocerebral complex, and fell to undetectable levels after the adult emerged.     

ACTIVATION OF THE CORPORA ALLATA IN RELATION TO OVARIAN MATURATION IN THE SEASONAL FORMS OF THE BUTTERFLY, POLYGONIA C-AUREUM L.

In Polygonia c-aureum , there are two seasonal forms, viz. , the summer form and the autumn form. Previous experiments (E ndo , 1970) showed that the corpora allata of the summer form accelerate the maturation of ovarian follicles soon after imaginal ecdysis. On the other hand, the corpora allata of the autumn form do not stimulate ovarian maturation during a period of 2 to 3 weeks after the emergence.
In the summer form (S) as well as in the autumn form (A), the corpora allata remained in an inactive state for about 15 days after emergence when they had been isolated microsurgically from the brain and the corpora cardiaca during the larval period. Further, when separation of the brain into the right and left hemispheres or ablation of the medial neurosecretory group cells of the pars intercerebralis had been carried out on S-pupae of 28 hr after pupation or of earlier ages, they developed into autumn form in respect to wing pattern and their corpora allata did not stimulate ovarian maturation until 2 to 3 weeks after emergence. On the other hand, when the above operations had been performed 34 hr after the pupation or of later ages, they developed into summer form in respect to wing pattern and ovarian maturation was evident soon after the emergence. In these cases, stimulation of the corpora allata is closely connected with the development of seasonal-forms of wing pattern.
From these experiments, it is clear that the medial neurosecretory group cells of the pars intercerebralis in S-insects stimulate the corpora allata about 30 hr after pupation by way of the nervi corporis cardiaci and the activated corpora allata promote ovarian maturation throughout adult life. In A-insects, on the other hand, the medial neurosecretory group cells of the brain are inactive and fail to activate the corpora allata which in turn have no influence on ovarian maturation.     

Prothoracic gland involution related to moulting hormone levels during the metamorphosis of Tenebrio molitor L.

Prothoracic gland (PG) of Tenebrio shows ultrastructural changes which can be correlated with ecdysteroid levels (measured by radioimmunoassay) during larval-pupal development. However, the gland cells begin to degenerate before pupal-adult ecdysis: the PG involution is completed before the moulting hormone peak which triggers pupal-adult development. These facts strongly suggest that another endocrine organ produces moulting hormone needed for adult development.     

A possible role of ecdysteroids in pre-ecdysial tanning in larvae of Sarcophaga bullata (Diptera: Sarcophagidae)

The levels of ecdysteroids in Sarcophaga bullata were determined by radioimmunoassay (RIA) from the time of larviposition (0 hr) to after the 2nd ecdysis and from late larval to pupal development. Two distinct peaks of ecdysteroid activity were recorded mid-way through the first and second stadia (14 and 34 hr) and two smaller peaks occurred a few hours prior to each ecdysis. A large release of ecdysteroids occurred from 8 hr before and up to 18 hr after formation of the white prepupa. This peak initiated the formation of the prepupa, the tanning of the puparium, larval/pupal apolysis and secretion of the pupal cuticle.Assays for the cuticle tanning hormone, bursicon, in pre-ecdysial larvae were not positive and a possible role for ecdysone in pre-ecdysial tanning of larval cuticular structures is proposed.     

Programming,induction, or prevention of the breakdown of the prothoracic gland in the cockroach, Nauphoeta cinerea

Changes of the prothoracic gland (PGL) diameter and of the corpora allata (CA) volume during the second last and last larval instar, and transplantation experiments as well as juvenile hormone (JH) analogue applications, demonstrate that only an activated PGL seems to be competent to degenerate and that the breakdown of the activated PGL is programmed by the absence of JH for a few days. Then, some days later, at the time of apolysis induction which occurs 7 to 6 days before an ecdysis the breakdown of a programmed PGL is induced by factors present in the haemolymph and it is suggested that β-ecdysone in apolysis-inducing amounts could be responsible for the induction of degeneration. However, PGL-protecting factors released probably from the corpora cardiaca (CC) are capable of partially preventing the breakdown of PGLs that have already been induced to degenerate, and it seems that the actual degeneration process is initiated by the absence of PGL-protecting factors only.     

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.     

Azadirachtin,ecdysteroids and ecdysis in Locusta migratoria

1. The injection of azadirachtin into eight-day-old 5th instar nymphs of Locusta migratoria elicits different morphological effects dependent upon the timing of the injection relative to the major haemolymph ecdysteroid peak.2. Ecdysteroid levels can be drastically reduced, delayed and extended or not significantly affected by azadirachtin treatment dependent upon the timing of injection.3. Air swallowing to inflate the gut during the moult is significantly impaired in azadirachtin-treated insects resulting in both a reduced rate of inflation and a reduction in total air volume.4. The significance of azadirachtin treatment to the attainment of a “critical body mass”, the release of eclosion hormone and successful ecdysis is discussed.     

Developmental profiles of ecdysteroids, ecdysteroid receptor mRNAs and DNA binding properties of ecdysteroid receptors in the Ixodid tick Amblyomma americanum (L.)

Total body ecdysteroid titers were determined at specific stages during the larval and nymphal life of Amblyomma americanum (L.). One ecdysteroid peak was observed following the completion of larval apolysis. However, two distinct ecdysteroid peaks occurred at a comparable stage in the nymphal molting cycle. The first occurred following apolysis and the second peak occurred at about the time of ecdysis. When whole body profiles of EcR and RXR mRNAs were examined during the molting cycle using RT-PCR, the expression of both AamEcR and AamRXR mRNAs was shown to be correlated with the ecdysteroid titer. Using an electrophoretic gel mobility shift assay, it was demonstrated that AamEcR*AamRXR1, but not AamEcR*AamRXR2, exhibits broad DNA binding specificity, forming complexes with a variety of synthetic direct repeat and palindromic nuclear response elements with the half-site consensus AGGTCA. These data suggest that functional differences may exist between the AamRXR1 and AamRXR2 proteins.     

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.     

Quantitative studies on the activity of the corpora allata in adult male Locusta and Schistocerca

Juvenile hormone has been detected in the haemolymph and corpora allata of adult male Locusta and the haemolymph of adult male Schistocera by a modified Galleria bioassay. The hormone was readily detected in the haemolymph of insects immediately after the final ecdysis, but then became difficult to detect until 2 days prior to the onset of sexual maturation. In sexually mature insects the titre of juvenile hormone was maintained at a constant level. The corpora allata of adult male Locusta increased in size throughout adult life. The juvenile hormone content of the corpora allata was low during the period of somatic growth, but increased at the onset of sexual maturation. Sectioning of the nervi corporis allati I in insects immediately after the final ecdysis prevented the normal increase in size of the corpora allata, but did not render them inactive since juvenile hormone was detected in the haemolymph after the operation. The half life of juvenile hormone in the haemolymph of allatectomized adult male Locusta was 1 to 2 hr.     

Activity of the corpora allata in the adult female migratory locust

Juvenile hormone was detected in the haemolymph of adult female Locusta by a modified Galleria bioassay. The hormone was present in the haemolymph immediately after the final ecdysis, but could not be detected after this time until the end of the period of somatic growth just before the start of ovarian development. During the first gonotrophic cycle the levels of juvenile hormone in the haemolymph could be related to the growth of the proximal oöcytes. The volumes of the corpora allata could be related to haemolymph juvenile hormone levels during the first gonotrophic cycle. Ovariectomy had no effect on haemolymph juvenile hormone levels or on the volumes of the corpora allata.