Regulation of the corpora allata in the black mutant of Manduca sexta

Regulation of corpus allatum activity in the black mutant strain of Manduca sexta was studied in vivo and in vitro. Allatectomy, denervation, and implantation studies demonstrated that black mutant corpus allatum activity remains low in both wild-type and black mutant host larvae. Attempts to distinguish humoral control mechanisms versus mechanisms dependent on intact allatal nerves indicated that intact allatal nerves were not required for the reduced black mutant corpus allatum activity in vivo. Incubation of corpora allata, using [1-¹⁴C]propionate as a juvenile hormone biosynthetic precursor and haemolymph as culture medium, confirmed that black mutant corpora allata are suppressed by a factor(s) in the haemolymph. Under identical conditions wild-type corpora allata were unaffected. Finally, the lowered black mutant corpus allatum activity in haemolymph in vitro correlates with the lowered juvenile hormone titre in black mutant larvae.     

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.     

Precocenes as pro-allatocidins in adult female Diploptera punctata: A functional and ultrastructural study

Adult mated females of the viviparous cockroach Diploptera punctata are moderately sensitive to precocenes. Oöcyte growth is inhibited and oviposition is delayed in insects topically treated with precocene II or precocene III. C₁₆ juvenile hormone release by corpora allata of precocene-treated insects is markedly inhibited when compared to corpora allata of acetone-treated controls. Electron microscopy of the corpora allata reveals that precocene treatment results in a disorganisation of the intracellular organelles. Topically applied precocene II reaches a high concentration in the haemolymph (0.5 mM 2 hr after topical application of 250 μg). C₁₆ juvenile hormone release by isolated corpora allata is inhibited by precocenes in vitro; half-maximal inhibition over a 3 hr period is obtained at 0.4 mM precocene II. In vitro inhibition of corpora allata by precocene II concentrations higher than 1 mM rapidly destroys the glands as evidenced by electron microscopy (total disintegration of cellular organelles) and by the virtual cessation of C₁₆ juvenile hormone synthesis by the corpora allata. Inhibition of C₁₆ juvenile hormone release by precocene is time-dependent and is not reversible over the short-term incubation in vitro. This inhibition does not appear to be related to the spontaneous activity of the glands in vitro, and it can be reduced by two epoxidase inhibitors. Precocenes are pro-allatocidins in this species: they are bioactivated within the corpora allata to cytotoxic epoxides.     

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.     

Biological,radiochemical and physicochemical evidence for the low activity of disconnected corpora allata in locust

The rate of juvenile hormone biosynthesis by locust corpora allata after transection of the nervi corporis allati 1, was measured in vitro using both radiochemical assay and gas chromatography—mass spectroscopy analyses. Incubations in different culture media or in pure haemolymph result in a low rate of juvenile hormone biosynthesis by disconnected glands. In vivo studies using juvenile, chromatotropic and gonadotropic effects of the corpora allata confirm the low activity of the disconnected glands. Furthermore, animals with disconnected corpora allata appear to be more sensitive to corpora allata implantation than control hosts.     

The activity of the corpora allata in the fourth and fifth larval instars of the migratory locust

The presence of juvenile hormone in the haemolymph of larvae of Locusta has been detected by a modified Galleria bioassay and these results are compared with indirect methods of estimating corpus allatum activity. Juvenile hormone is present in the haemolymph during the fourth larval instar except on the last day of the instar, and is absent from the haemolymph of the fifth and final larval instar except on the last day of the instar. Changes in the volumes of the corpora allata simply reflect changes in the growth of the whole insect and are of no value in predicting endocrine activity. Changes in the size of the cells of the corpora allata can be correlated with the presence of juvenile hormone in the haemolymph in the fourth larval instar, but similar changes in cell size occur in the fifth larval instar when no juvenile hormone is present in the haemolymph. The effects of the implantation of corpora allata are unreliable as estimates of corpus allatum activity as isolated corpora allata from fifth instar larvae release juvenile hormone. Indirect methods of measuring corpus allatum activity are thus shown to be unreliable. The R_f value of Locusta juvenile hormone as determined by thin-layer chromatography differs from that of Roeller's juvenile hormone, suggesting that the two hormones might be chemically distinct.     

Juvenile hormone III biosynthesis by the larval corpora allata of Manduca sexta

A radioimmunoassay (RIA) for juvenile hormone III has been established which quantifies the biosynthesis of this hormone in vitro by the corpora allata of larvae and pupae of the tobacco hornworm, Manduca sexta. The specificity of the RIA for homologues and metabolites of juvenile hormone III was determined and it was found that the antibody was specific for juvenile hormone III and its acid. The juvenile hormone III RIA activity synthesized in vitro by corpora allata from day-5 last-instar larvae was identified as juvenile hormone III by high pressure liquid chromatography. The kinetics of hormone synthesis by corpora allata from selected stages during larval-pupal development revealed differential rates of synthesis, suggesting that juvenile hormone III may have a hormonal function in the larva and that regulation of its synthesis may occur. The significance of these developmental fluctuations in rates of juvenile hormone III synthesis by the corpora allata is discussed in relation to the haemolymph titres of the hormone.     

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.     

The effects of juvenile hormone, 20-hydroxyecdysone and precocene II on activity of corpora allata and the mode of negative-feedback regulation of these glands in the adult Colorado potato beetle

When the titre of juvenile hormone III in female Leptinotarsa decemlineata was elevated by the implantation of supernumerary corpora allata or by the injection of the hormone, the rate of endogenous hormone production by the host glands was significantly restrained, as determined by the short-term in vitro radiochemical assay. From denervation studies, it is suggested that during phases of elevated juvenile hormone titre, the corpus allatum activity is regulated via humoral as well as neural factors requiring intact nerve connections. Restrainment of gland activity appears to be mainly via the neural pathway. Isolated corpora allata were not influenced by 10^?5 M juvenile hormone III added to the incubation medium in vitro.Studies with farnesenic acid revealed that the final two enzymatic steps in the biosynthetic pathway of juvenile hormone are also diminished during prolonged neural inhibition of the corpora allata.20-Hydroxyecdysone and precocene II had no apparent effect on the corpus allatum activity of Leptinotarsa decemlineata.     

The mode of regulation of the corpus allatum activity during starvation in adult females of the Colorado potato beetle, Leptinotarsa decemlineata (Say)

When two-day-old female Leptinotarsa decemlineata were starved, their corpus allatum activity, as measured by the radiochemical in vitro assay, was significantly reduced after 24 hr. Such a reduction was not observed when the nerve connections between the central nervous system and the retrocerebral complex were severed and the beetles starved up to 5 days. In some experiments, the rate of juvenile hormone biosynthesis in vitro, was substantiated by measurement of the juvenile hormone titre in the haemolymph by physico-chemical methods. It is concluded that intact nervous connections between the central nervous system and the corpora allata are essential for restraining the juvenile hormone biosynthesis during the initial stages of starvation.Corpora allata from 1-day starved insects were considerably stimulated in vitro by farnesenic acid indicating that juvenile hormone synthesis is controlled enzymatically at a stage prior to the final two steps in the pathway. However, on day 5 of starvation, rate-limitation may occur after formation of this intermediate, since farnesenic acid stimulation was much less at this time.Corpora allata of adult females newly emerged from the soil were activated within 4 hr regardless of feeding.     

Inhibition of the corpora allata of last-instar male larvae of the cockroach, Diploptera punctata

Normal rates of juvenile hormone synthesis, cell number and volume of corpora allata were measured in penultimate and final-instar male larvae of Diploptera punctata. The rate of juvenile hormone synthesis per corpus allatum cell was highest on the 4th day of the penultimate stadium, declined slowly for the remainder of that stadium, and rapidly after the first day of the final stadium.Regulation of the corpora allata in final-instar males was studied by experimental manipulation of the corpora allata followed by in vitro radiochemical assay of juvenile hormone synthesis. Nervous inhibition of the corpora allata during the final stadium is suggested by the observation that rates of juvenile hormone synthesis increased following denervation of the corpora allata at the start of the stadium; this operation induced a supernumerary larval instar. Juvenile hormone synthesis by corpora allata denervated at progressively later ages in the final stadium and assayed after 4 days decreased with age at operation. This suggests an increasingly unfavourable humoral environment in the final stadium, which was confirmed by the low rate of juvenile hormone synthesis of adult female corpora allata implanted into final-instar larvae. Thus, inhibitory factors or lack of stimulatory factors in the haemolymph may act with neural inhibition to suppress juvenile hormone synthesis in final-instar males.     

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.     

Haemolymph juvenile hormone III esterase activity during adult life of female and male crickets,Gryllus bimaculatus de Geer (Ensifera,Gryllidae)

Using an in vitro method, juvenile hormone III degradation was studied in the plasma of adult female and male crickets, Gryllus bimaculatus. The primary route of juvenile hormone III metabolism in cricket haemolymph is ester hydrolysis to juvenile hormone III acid by juvenile hormone III esterase. Juvenile hormone III esterase activity in females' haemolymph is low just after imaginal moulting. A sharp peak of enzyme activity is observed on day 3 after emergence, and two subsequent peak values occur in older animals. Plasma juvenile hormone III esterase activity in freshly ecdysed males is also low, but increases rapidly thereafter. Another increase in enzyme activity is observed in older males. The fluctuations in juvenile hormone III esterase activity are discussed in correlation with changes in haemolymph volume, haemolymph protein content, haemolymph juvenile hormone III titer, and the rates of juvenile hormone III biosynthesis in vitro of the corpora allata.     

Improved assay conditions for measurement of corpus allatum activity in vitro in the adult Colorado potato beetle, Leptinotarsa decemlineata

Assay conditions for the short-term, radiochemical, in vitro determination of the spontaneous rate of juvenile biosynthesis by isolated corpora allata from Leptinotarsa decemlineata have been further improved, permitting the measurement of juvenile hormone biosynthesis by individual pairs of corpora allata. The final incubation product has been identified as juvenile hormone III with the aid of High-performance liquid chromatography (HPLC) and juvenile hormone esterase degradation. Using the new assay conditions, the activities of adult corpora allata during maturation were found to be significantly higher in reproductive, long-day animals than in pre-diapause, short-day beetles. During diapause no activity was detectable, whereas corpora allata from post-diapause beetles were reactivated totally after 5 days. Simultaneous determination of the in vitro rates of juvenile hormone biosynthesis and corpus allatum volumes revealed no clear correlation although the results suggest that the volume may be indicative of the maximal capacity for juvenile hormone production. Corpora allata from a population of beetles did not display any synchronous diurnal rhythmicity.     

Regulation of juvenile hormone synthesis during pregnancy in the cockroach, Diploptera punctata

Regulation of juvenile hormone synthesis during pregnancy was investigated after determining the normal rates of synthesis in pregnancy and the second gonadotrophic cycle in Diploptera punctata by direct in vitro radiochemical assay.The low rate of juvenile hormone synthesis during early pregnancy is maintained by three factors: (1) the small ovary which is incapable of eliciting increased rates of juvenile hormone synthesis (2) an inhibitory centre in the brain acting via intact nerves to the corpora allata (similar to that in virgin females) and (3) an inhibitory centre in the brain acting via the haemolymph (elicited by embryos in the brood sac).The existence of two inhibitory centres in the brain is supported by the additive effect of denervating the corpora allata and removing embryos. Whereas these operations alone activated the corpora allata in 54 and 31% of the females, respectively, together they activated 87%, similar to the 91% activated by denervation alone in late pregnancy.The inhibition which remains after denervation of the corpora allata can be removed by decapitation and restored by implantation of the protocerebrum from a pregnant female but not from one developing oöcytes.The inhibition elicited by embryos in the brood sac can be overcome by introduction of a stimulatory ovary and/or substitution of active corpora allata.     

Juvenile hormone paces behavioral development in the adult worker honey bee

Behavioral development in the adult worker honey bee (Apis mellifera), from performing tasks inside the hive to foraging, is associated with an increase in the blood titer of juvenile hormone III (JH), and hormone treatment results in precocious foraging. To study behavioral development in the absence of JH we removed its glandular source, the corpora allata, in 1-day-old adult bees. The age at onset of foraging for allatectomized bees in typical colonies was significantly older compared with that of sham-operated bees in 3 out of 4 colonies; this delay was eliminated by hormone replacement in 3 out of 3 colonies. To determine the effects of corpora allata removal on sensitivity to changes in conditions that influence the rate of behavioral development, we used "single-cohort" colonies (composed of only young bees) in which some colony members initiate foraging precociously. The age at onset of foraging for allatectomized bees was significantly older compared with that of sham-operated bees in 2 out of 3 colonies, and this delay was eliminated by hormone replacement. Allatectomized bees initiated foraging at significantly younger ages in single-cohort colonies than in typical colonies. These results demonstrate that JH influences the pace of behavioral development in honey bees, but is not essential for either foraging or altering behavioral development in response to changes in conditions.     

Adaptation of a radiochemical assay for juvenile hormone biosynthesis to study caste differentiation in a primitive termite

A radiochemical assay measuring juvenile hormone synthesis by corpora allata incubated in vitro was adapted for use with the termite Zootermopsis angusticollis. Corpora allata from 3–4-day old virgin female neotenic reproductives were used in these studies because this caste showed the highest rates of juvenile hormone synthesis (0.6 pmol h^?1 per pair corpora allata). Juvenile hormone-III synthesis was linear for up to 6 h over the range of concentrations of labelled l-methionine from 27–280 μM. Rates of juvenile hormone synthesis were stimulated up to 10-fold in a dose-dependent manner by the addition of farnesoic acid to the incubation medium. However, the relatively high concentration of 120 μM farnesoic acid reduced the rates of juvenile hormone synthesis. The radiochemical assay was used to determine rates of juvenile hormone synthesis in vitro by corpora allata from larvae with a queen and king vs orphaned larvae. The presence of reproductives resulted in a suppression of larval corpus allatum activity relative to orphaned controls.     

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.     

Juvenile hormone esterase activity during the last larval and pupal stages of Tenebrio molitor

In vitro analysis of juvenile hormone esterase activity of haemolymph of T. molitor was performed during the end of post-embryonic development. Weak activity was found in penultimate stage larvae as in the major part (except the last day) of last-larval instar, while very high activity was monitored in the early pupae (female or male).This pupal peak was the only one detected during development in the insect, coinciding with the pupal juvenile hormone sensitive period. The first juvenile hormone sensitive period, during the lastlarval instar, does not seem to be protected by any juvenile hormone esterase activity in contrast to other species. These results suggest a central control for the drop in juvenile hormone level ceasing synthesis by the corpora allata after integration of external stimuli. This hypothesis could explain the natural occurrence of prothetelic larvae, the absence of pupal adult intermediates and the variable number of instars in Tenebrio.     

On the relative importance of juvenile hormone and vitellogenin for oöcyte growth in the cockroach Nauphoeta cinerea

The corpora allata of castrated females of Nauphoeta grow only very slightly and do not reach a volume greater than that of the glands of normal females during gestation. These small corpora allata are, however, active and are responsible for the synthesis of vitellogenin (female specific protein) in large amounts. Besides vitellogenin the other haemolymph proteins are also synthesized and accumulated in the haemolymph in much higher concentrations than in normal females. Implanted oöcytes grow in castrated as well as in normal females at about the same rate until the tenth day of the oöcyte maturation period. Thereafter they only grow in castrated females. If castrated and normal females are decapitated, their protein content decreases. At the same time the growth stimulating capacity of their haemolymph decreases at a much faster rate. If oöcytes are implanted in castrated and decapitated females after 4 days they cannot grow any more although the vitellogenin titre of the haemolymph is still much higher than it is at any time in normal females. It can be concluded that vitellogenin alone cannot induce oöcyte growth and that juvenile hormone is necessary as well for vitellogenin synthesis as for its incorporation into the oöcytes. However, in insects rich in vitellogenin juvenile hormone leads to a more rapid oöcyte growth than in insects containing only small amounts of this protein.