The effect of juvenile hormone on prothoracic gland function during the larval-pupal development of Manduca sexta: An in situ and in vitro analysis

The application of juvenile hormone I or ZR 512 to neck-ligated, day-5 fifth instar (V₅) larvae reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA) reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the ecdysteroid titres were similar to those of normally developing larvae although the ecdysteroid peak elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V₅ larvae that were untreated ultimately pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 μg/ml haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine whether this stimulation was direct or indirect, an in vitro approach was taken. Prothoracic glands from V₅, V₆ and V₇ larvae were incubated in vitro under conditions in which they could be stimulated by prothoracicotropic hormone, and were exposed to concentration of free juvenile hormones I, II, III or ZR 512 ranging from 10^?5M to 10^?10M. In no case were the prothoracic glands stimulated in a dose-dependent manner that would be indicative of hormone activation. Similar results were obtained when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with the acids of the three juvenile hormone homologues revealed them to be ineffective in activating prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from these data that juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so indirectly.     

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.     

Larval-pupal transformation of the prothoracic glands of Mamestra brassicae

The sensitivity of the prothoracic glands to juvenile hormone and prothoracicotropic hormone (PTTH) of penultimate (5th)-instar larvae of Mamestra brassicae was compared with that of the same-instar larvae destined for pupal ecdysis by allatectomy. The activity of the prothoracic glands was assessed using either moulting of isolated abdomens or ecdysone radioimmunoassay. Juvenile hormone application immediately after neck-ligation (which removes brain-corpora cardiaca-corpora allata complex) prevented prothoracic gland function in larvae at all stages. When larvae were allatectomized 12 hr after ecdysis, followed by neck-ligation at different times and given juvenile hormone immediately, the hormone inhibited the prothoracic glands of young larvae, but activated the prothoracic glands from day-5 or older larvae. Juvenile hormone I, juvenile hormone II and methoprene activated the prothoracic glands, but juvenile hormone III was relatively ineffective. Brain implantation instead of juvenile hormone application led to activation of the prothoracic glands at all stages.Allatectomy thus caused changes leading to metamorphosis including a transformation of the prothoracic glands from ‘larval’ to ‘pupal’ type. After this change these prothoracic glands were able to respond not only to PTTH but also to juvenile hormone just as in last-instar larvae.     

Regulation of insect prothoracic glands: Existence of a haemolymph stimulatory factor in Manduca sexta

The primary regulator of ecdysone biosynthesis by insect prothoracic glands is the prothoracicotropic hormone. However, it now appears that other factors, secondary regulators, may modulate prothoracic gland activity. One such factor has been isolated from the haemolymph of Manduca larvae. This haemolymph factor stimulates in vitro ecdysone synthesis by larval and pupal prothoracic glands by approx. 5-fold. It has an apparent mol. wt of ～330 kD, is protease-sensitive and is heat labile, the latter clearly distinguishing it from the prothoracicotropic hormone. Further, its steroidogenic effects and those of prothoracicotropic hormone are additive. Treatment of larval or pupal prothoracic glands with both moieties simultaneously effects an approx. 10-fold increase in ecdysone synthesis. The haemolymph titre of the stimulatory factor is low at commitment of the last-larval instar, then increases by approx. 3-fold later in the instar during pharate-pupal development. This increase in the titre is sufficient to effect a significant increase in prothoracic gland activity that could be physiologically important. Thus, it appears that the fluctuating level of this haemolymph stimulatory factor may act in conjunction with prothoracicotropic hormone to regulate the haemolymph ecdysteroid titre by modulating the ecdysone biosynthetic activity of the prothoracic glands.     

Switchover in the sensitivity of the prothoracic glands to juvenile hormone in the cotton leafworm, Spodoptera littoralis

Switchover in the sensitivity of the prothoracic glands to juvenile hormone analogue during the last-larval instar of Spodoptera littoralis occurs in the middle of the third scotophase i.e. at the end of phagoperiod when the body weight is maximal and the ecdysteroid is increasing in the haemolymph. Application of the analogue to larvae neck-ligated before the switchover completely inhibits or delays metamorphosis due to an inhibitory effect on the prothoracic gland cells and is not mediated by the nervous system. This inhibition by the analogue is dose-dependent, and when complete inhibition of metamorphosis occurs, the prothoracic glands cells degenerate. Treatment of neck-ligated larvae with the analogue after the switchover stimulates metamorphosis by accelerating the appearance of an ecdysteroids peak in the haemolymph. The stimulatory effect of the analogue to the prothoracic glands in neck-ligated larvae is not direct one, and some unknown factors seem to play a role therein.

The probale role of prothoracicotropic hormone as a synchronizing factor in the switchover in the sensitivity of the prothoracic glands to juvenile hormone is discussed.     

Factors affecting change in sensitivity of prophoracic glands to juvenile hormone in Mamestra brassicae

The prothoracic glands of the early last-instar larva of Mamestra brassicae (day 0–3) were found previously to be insensitive to stimulation by juvenile hormone, whereas those later in the instar (from day 4 on) were activated by this hormone. When neck-ligatured young larvae (day-1, day-2 and day-3) were given juvenile hormone 5–10 days after ligation, pupation was induced. Similarly, juvenile hormone induced pupation of isolated abdomens which contained prothoracic glands taken from neck-ligatured day-3 larvae 5 days after ligation. If the glands were exposed to prothoracicotropic hormone (PTTH) from implanted brains before they were transplanted to isolated abdomens, their sensitivity to juvenile hormone activation was enhanced. Ecdysone but not 20-hydroxyecdysone given every 3 hr for 12 hr also slightly enhanced sensitivity. These results suggest that prothoracic glands from either day-1, day-2 or day-3 larvae can slowly acquire a sensitivity to juvenile hormone activation by prolonged incubation in the absence of factors from the head. The acquisition of sensitivity occurs more rapidly in the presence of both a factor from the brain, presumably PTTH, and ecdysone released from the prothoracic glands themselves.     

An in vitro study on regulation of prothoracic gland activity in the early last-larval instar of the silkworm Bombyx mori

The endocrine mechanisms that regulate prothoracic gland (PG) activity in early stages of final larval instar of the silkworm Bombyx mori were investigated using a newly developed long-term cultivation system of the gland. The PGs dissected from day-0 fifth instar larvae did not secrete detectable amounts of ecdysone for the first 24 h in culture but started secretion within the next 2 days. The amount of secreted ecdysone increased day by day. When day-0 PGs were co-cultivated with corpora allata, however, they remained inactive for at least 8 days. PGs dissected from 1-day younger larvae (day-3 fourth instar larvae) secreted ecdysone for the first 24 h but stopped secretion for the next 24 h, followed by recovery of ecdysone secretory activity. By contrast, PGs from day-1 fourth instar larvae remained active throughout a cultivation period without any sign of inactivation. However, when the same glands were exposed to a high titer of 20-hydroxyecdysone for the second 24h in culture, they gradually lost their activity. These results indicate that PGs of fourth instar larvae are inactivated by ecdysteroid through a negative feedback mechanism and that thus inactivated PGs spontaneously recover ecdysone secretory activity in the early fifth instar unless inhibited by juvenile hormone.     

Stage dependent influences of polydnaviruses and the parasitoid larva on host ecdysteroids

In the solitary egg-larval parasitoid Chelonus inanitus (Braconidae) both polydnavirus and the parasitoid larva manipulate host development. Parasitization leads to a premature drop in juvenile hormone titre and a precocious onset of metamorphosis in the 5th larval instar. The C. inanitus bracovirus (CiBV) alone causes a reduction in host ecdysteroid titres at the pupal cell formation stage and prevents pupation. Here we report three new findings. (1) We show that parasitization causes a reduction in haemolymph ecdysteroid titre immediately after the moult to the 5th instar; similarly low values were seen in nonparasitized larvae after the moult to the 6th instar. These data along with parasitoid removal experiments indicate that the low ecdysteroid titre after the moult is a very early sign of the upcoming metamorphosis. (2) In vitro experiments with prothoracic glands and brain extracts showed that CiBV affects both prothoracic glands and prothoracicotropic hormone after the stage of pupal cell formation. (3) In the haemolymph of parasitized larvae the ecdysteroid titre increased in the late cell formation stage, i.e. immediately before egression of the parasitoid. In vitro experiments showed that late 2nd instar parasitoids release ecdysteroids and are thus very likely responsible for the rise in host ecdysteroids.     

Regulation and significance of ecdysteroid titre fluctuations in lepidopterous larvae and pupae

The last larval moult of Galleria mellonella is induced by an elevation of ecdysteroid titre to more than 200 ng/g. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr, is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 ng/g) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500–2500 ng/g) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 ng/g) in the middle of pupal instar.Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 30–40 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing the prothoracicotropic neurohormone (PTTH). In either case, the pupation-inducing increase of ecdysteroids is 3 times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed.Neither in G. mellonella larvae, nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid production or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5–15 times lower than usual ecdysteroid concentration when the last-instar larvae are exposed to JH.     

Haemolymph ecdysteroid levels and cellular events in the intermoult/moult sequence of Calpodes ethlius

The haemolymph ecdysteroid titre of the last larval and pupal stadia of Calpodes ethlius was determined by radioimmunoassay. During the last larval stadium, four significant ecdysteroid peaks are present, two of which have been reported for other Lepidoptera. The first peak occurs 12 hr after ecdysis and correlates temporally with nucleolar activity, RNA synthesis and organelle formation in the fat body and epidermis. It correlates also with fat body DNA synthesis, polyploidy and the initiation of a low rate of lipid synthesis. Another peak, at 78 hr, starts its increase when the prothoracic glands no longer require the influence of the brain to produce ecdysone for pupation, and marks the first critical period. It correlates with the initiation of epidermal DNA synthesis and mitosis, and with the progressive determination of pupal characteristics (change in commitment, reprogramming). This ecdysteroid peak may also be involved in the massive intermoult syntheses in the epidermis (lamellate cuticle, wax) and the fat body (lipid, protein). The largest ecdysteroid peak is seen at 162 hr, 6 hr after the tissues no longer require the prothoracic glands for pupation (second critical period). It correlates temporally with the cessation of massive synthetic activity in both epidermis and fat body and initiates preparation for pupal synthesis in both tissues. At this time the ratio of ecdysone: 20-hydroxyecdysone is ～ 1 : 6.6.In common with other Lepidoptera, a single large ecdysteroid peak occurs during the first half of the pupal stadium. Comparisons between these events and the ecdysteroid titre are made between Calpodes and other insects.     

Activation of the prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mamestra brassicae

The effects of JHA (ZR-515) application or brain implantation on metamorphosis and adult development were examined in the last instar larvae and pupae of Mamestra brassicae. When JHA was applied to neck-ligated 4- or 5-day-old larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands taken from 5-day-old larvae, the insects pupated. Dauer pupae and diapausing pupae treated with JHA showed adult development. By contrast, pupation could not be induced by the application of JHA to 2- or 3-day-old neck-ligated larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands from 0-day-old larvae. Implantation of a brain into neck-ligated 3- or 5-day-old larvae (at the beginning of gut emptying and wandering) caused pupation of the host. A similar result was obtained when both a brain and the prothoracic glands from 0- or 5-day-old larvae were implanted into the isolated abdomens of 5-day-old larvae. These results indicate that activation of the prothoracic glands by application of JHA is temporally restricted to the last part of the last larval instar and to the pupal stage, while the activation by prothoracicotropic hormone (PTTH) can occur throughout the last larval instar and the pupal stage. In addition, the implantation of brains or application of JHA to neck-ligated 5-day-old larvae 25 days after ligation seldom induced pupation of the hosts, a result which suggests that larval prothoracic glands maintained under juvenile hormone (JH) or PTTH-free conditions for long periods of time may become insensitive to reactivation by both hormones.     

Ecdysteroids in adult males and females of Drosophila melanogaster

Ecdysteroid titres in whole flies and different tissues of adult male and female Drosophila were determined at various times after eclosion using a radioimmunoassay. The ecdysteroid titre decreased as the flies matured after eclosion. The differences in titre between males and females can be accounted for by their difference in body weight. The ecdysteroids were found to be distributed throughout several tissues. At eclosion not all of the ecdysteroid complement present could be accounted for by that found localised in tissues. After maturation of the flies the ecdysteroids in various tissues can account for the majority of that detected in whole-fly extracts. Ecdysteroids were produced during in vitro culture of various tissues, but the quantities detected were low by comparison with ring glands of wandering 3rd-instar larvae. Neither the ovaries nor the abdominal body walls (fat body) seem to be a major source of hormone, and they are only able to convert minute quantities of ecdysone to the biologically active form, 20-hydroxyecdysone, in vitro. The amounts of 20-hydroxyecdysone present were measured using high performance liquid chromatography and radioimmunoassay. We tentatively suggest that the differential experession of the yolk-protein-genes in the fat bodies of males and females does not result from differences in hormone titres between them.     

Developmental arrest induced by juvenile hormone in larvae of Bombyx mori

Injection of the juvenile hormone analog (JHA) methoprene into day 3, fifthinstar larvae of Bombyx mori induced developmental arrest. Feeding activity declined, and the larvae remained as larvae for more than 2 weeks, after which they died. After JHA injection, the hemolymph ecdysteroid titer was low, and the prothoracic glands were almost inactive for 7 days. During this period, prothoracic glands were stimulated by prothoracicotropic hormone (PTTH) in vitro, indicating that JHA did not inhibit the competence of the glands to respond to PTTH. When brain-corpora cardiaca-corpora allata complexes were removed from intact fifth-instar larvae on day 4, the prothoracic glands became autonomously active and produced enough ecdysone for pupation. When PTTH injections were given to larvae previously injected with JHA (7 days before), the larvae recovered feeding activity, purged their guts, and pupated. Injections of 20-hydroxyecdysone into larvae that had been injected with JHA 7 days earlier induced larval molting. These results suggest that JHA affects both the brain and the prothoracic gland.     

Conversion of 3-dehydroecdysone by a ketoreductase in post-diapause, pre-hatch eggs of the gypsy moth Lymantria dispar

The prothoracic glands (PGs) of Lymantria dispar (day-5 female, last-stage larvae) produce both ecdysone and an ecdysteroid which has the same retention time on reverse-phase liquid chromatography (RPLC) as a known standard of 3-dehydroecdysone. The latter ecdysteroid can be converted by a heat-labile factor in extracts of post-diapause, pre-hatch L. dispar eggs to an ecdysteroid which has the same retention time on RPLC as ecdysone. Purified 3-dehydroecdysone, similarly treated with egg extract, also gives the same retention time on RPLC as ecdysone. Taken together, these data suggest that, like Manduca sexta, a major product of the PGs in L. dispar is 3-dehydroecdysone. Furthermore, these data suggest that L. dispar eggs, which contain mature embryos, possess ecdysteroid ketoreductase activity capable of converting 3-dehydroecdysone to ecdysone. This is the first report of ecdysteroid ketoreductase activity in embryonated eggs.     

Growth and respiratory enzyme activity in the prothoracic glands of Galleria

In the penultimate-larval instar, the total volume of the prothoracic gland and the activities of some oxidative mitochondrial enzymes (cytochrome oxidase, NADH: cytochrome c oxidoreductase, succinate: cytochrome c oxidoreductase) undergo cyclic variations associated with larval growth. These specifically larval-larval growth cycles are absent in the prothoracic glands of normal last-instar larvae. Here the cycles can be induced artificially by implantation of brain or corpora cardiaca-allata complexes or, by exogenous application of juvenile hormone. The smallest size of the prothoracic gland in relation to the size of the body, as well as the minimal activity of all the three mitochondrial enzymes in the gland, have been found exactly at the moment of the pre-pupal peak of ecdysteroid in the body. The possibility that the prothoracic glands alone can synthetize ecdysteroid during the peak is questioned.     

Neurotropic effect of juvenile hormone III in larvae of the cockroach,Periplaneta americana

Juvenile hormone III and juvenile hormone analogs (methoprene, farnesyl methyl ether) cause a short-term depression of spike activity in last instar larvae of the cockroach Periplaneta americana, specifically in the prothoracic gland nerves, both in vivo and in vitro. The decreased nervous activity in the prothoracic gland nerves leads to decreased ecdysteroid secretion by the prothoracic gland.     

Effects of juvenile hormone analogue on ecdysis prevention induced by precocene in Rhodnius prolixus (Hemiptera:Reduviidae)

Precocene II, added to the meal of fourth-instar larvae of Rhodnius prolixus (25 micrograms/ml of blood), induced an increase in the duration of the molting cycle. This effect was related to the decrease of both the nuclear area of the prothoracic gland cells and the mitotic activity in epidermal cells. Juvenile hormone analogue applied topically (60 micrograms/insect) together with Precocene II treatment avoided atrophy of the prothoracic glands and induced a higher number of epidermal mitosis accelerating the time of subsequent ecdysis. A possible relationship between juvenile hormone and production of ecdysone is discussed.     

Activity of the prothoracic gland and its sensitivity to prothoracicotropic hormone in the penultimate and last-larval instar of Bombyx mori

The time course of secretion of ecdysone in vitro by the prothoracic glands of Bombyx mori was studied through the penultimate and last-larval instars. Ecdysone was produced by the glands in high amounts by the penultimate instar at 72 and 84 h while the glands in the last instar exhibited a high activity over 4 days around the time of gut purge and thereafter. The glands in the penultimate instar produced ecdysone at a low level throughout the instar before the sharp peak of activity, when they became inactive and remained so for the first 3 days of the last instar after when they regained secretory activity. Sensitivity of the glands to prothoracicotropic hormone varied in accord with the changes in their secretory activity. Inactive glands were not stimulated by 22K-prothoracicotropic hormone. In addition, glands with maximal activity in the penultimate instar were insensitive to 22K-prothoracicotropic hormone. These results suggest that the prothoracic glands in the penultimate and last-instar larvae are physiologically different.