Govindan Bhaskaran

On the Identity of the Juvenile Hormone in Insects

The secretion of the corpora allata, the juvenile hormone (JH), has a morphogenetic function during the larval life of an insect and may act as a gonadotropin in the adult. On the basis of transplantation experiments, it had been concluded that the hormone of the corpora allata is stage and order unspecific (Wigglesworth, 1970; Novak, 1966). Consequently, the name “juvenile hormone” was retained irrespective of its endocrinological function during a specific postembryonic stage. Piepho (1950) even had suggested that the juvenile hormones of all insects be chemically related, if not identical.

The transfer of juvenile hormone from male to female during mating in the cecropia silkmoth.

The juvenile hormone (JH) stored in the accessory sex glands (ASG) of adult maleHyalophora cecropia (L.) originates both from sequestration of circulating hormone and from JH synthesized de novo in the ASG from JH acid taken up from the hemolymph. The secretions present in the lumina of the ASG contain most of the accumulated JH. During mating, endogenous JH, labeled biosynthetically via injected [3H-methyl]-methionine, is transferred along with the other seminal material to the bursa copulatrix of the female. The physiological significance of the JH transfer remains unknown.

Neuroendocrine regulation of corpus allatum activity in Manduca sexta: The endocrine basis for starvation-induced supernumerary larval moult

Abstract When newly-ecdysed 5th instar larvae of Manduca sexta were starved for 3 days and thereafter fed on standard diet the majority (90%) of the surviving larvae moulted into 6th instars. Allatectomy prior to starvation abolished the supernumerary moult, while denervation of the corpora allata (CA) had no effect. Cautery of medial neurosecretory cells, but not of the lateral cells, prevented supernumerary moulting and pupation ensued. Transplantation of brains from young 5th instar donors into larvae, whose medial neurosecretory cells were cauterized prior to starvation, restored the extra larval moult. Neither CA nor corpora cardiaca (CC) could be substituted for the medial neurosecretory cells. For induction of the supernumerary moult the medial neurosecretory cells are required only until day 1 after refeeding whereas the CA are required until day 3 after refeeding. Allatectomy on day 3 after refeeding resulted in the production of black 6th instar larvae. We conclude that starvation-induced supernumerary moulting is due to activation of the CA by allatotropin produced by medial neurosecretory cells in the brain. The anteromedial cells (group II) appear to be the source of allatotropin.

Juvenile hormone acid: Evidence for a hormonal function in induction of vitellogenin in larvae of Manduca sexta†

In the tobacco hornworm (Manduca sexta), vitellogenin (Vg), the major yolk protein precursor, and its mRNA are first detectable in the prepupal stage; and production of both can be enhanced by methoprene, a juvenile hormone (JH) analog. Competence to respond to methoprene is acquired after ecdysteroid-initiated commitment for metamorphosis. Here we show that acquisition of competence requires prior exposure to JH-II acid in addition to ecdysteroid. Application of 20-hydroxyecdysone or RH5992, an ecdysteroid analog, to isolated abdomens from feeding larvae (precommitment) results in exposure of the dorsal vessel (EDV), a sign of metamorphic commitment--but such abdomens do not make Vg in response to methoprene. However, injection of JH-II acid along with 20-hydroxyecdysone into isolated abdomens causes Vg production in response to methoprene. Methoprene acid similarly induces competence to respond to methoprene. Northern blot analysis confirmed that Vg transcripts are present in fat body only if isolated abdomens were pretreated with both ecdysteroid, and JH-II acid or methoprene acid. The latter two can induce competence even in precocious prepupae resulting from removal of the corpora allata (the glands that produce JH) from early penultimate larvae. JH-III acid and related metabolites such as farnesol, farnesoic acid, and methyl farnesoate do not induce competence. Hitherto, JH acids have been regarded as precursors or catabolites of JHs. Here we show for the first time that JH acid has a hormonal function that cannot be performed by JH itself.

Allatinhibin, a neurohormonal inhibitor of juvenile hormone biosynthesis in Manduca sexta

The corpora allata (CA) of Manduca sexta larvae become incapable of synthesizing juvenile hormone (JH) early in the wandering stage of the last larval stadium. They then switch to the synthesis and release of JH acids. This change in CA activity is induced by an inhibitory factor--allatinhibin (AI)--from the brain. AI activity is present in the fifth (Vth) instar hemolymph from about Day 4 (day of wandering) until Day 7 (early prepupa). CA of early fifth instar larvae (uninhibited) incubated in vitro with brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes or brain alone from wandering larvae are inhibited as demonstrated by bioassay. On the basis of these observations, an in vitro-in vivo assay for AI was developed. Br-CC-CA or Br alone were first incubated in tissue culture medium overnight. Day 0 (0d) Vth instar CA incubated for 16 hr in such medium will lose the ability to induce a larval molt in allatectomized 0d IVth instar larvae if the medium contained AI activity. The highest AI activity was exhibited by the medium obtained from incubations of brain from wandering larvae whereas the medium from incubation of 0d Vth and 0d pupal brains showed no AI activity. Dose-response data show that AI is active at 0.03 brain equivalents/200 microliters medium. CA must be exposed to AI for 12-16 hr for manifestation of inhibition. AI causes a stable inhibition of CA. AI is heat-labile, protease sensitive, has a molecular size between 1.0 and 3.5 kDa, and is clearly distinct from the allatostatins described by others.

Regulation of vitellogenin synthesis by juvenile hormone in the corn earworm, Helicoverpa zea

Summary In the corn earworm Helicoverpa zea yolk deposition in oocytes begins 8–10 h after adult eclosion and is controlled by juvenile hormone (JH). Hemolymph from females at the time of yolk formation, analyzed by SDS-PAGE, contains polypeptides apparently identical to polypeptides from eggs; and these have been tentatively designated as apovitello-genins (apoVgs). ApoVgs were not found in male hemolymph. The hormonal control of production of the apoVg with an apparent molecular weight of 165 kDa (apoVg-I) was studied by SDS-PAGE. Decapitation of females immediately after eclosion did not prevent the appearance of apoVg-I whereas the same operation done 5–7 h prior to eclosion abolished its production. Treatment of the latter group of females with methoprene, a JH analogue, restored production of apoVg-I whereas injection of 20-hydroxyecdysone (20-HE) did not. When pharate adults were kept at 10°C from 10–12 h before the time of their anticipated eclosion, eclosion was delayed for 4–7 d. In such females...

INDUCTION OF SUPERNUMERARY MOLTING BY STARVATION IN MANDUCA SEXTA LARVAE

Studies were carried out on the effects of prolonged starvation on the development of fifth‐ (last‐) instar larvae of the tobacco hornworm Manduca sexta. Following ecdysis, larvae were starved for varying lengths of time and subsequently fed normal diet. The percent of starved larvae molting to sixth instars increased, while the percent survival decreased with increasing length of the starvation period. When larvae were provided with agar as a source of water during the starvation period, the percent survival increased, but the percent undergoing supernumerary molting decreased. The optimal condition for maximum survival and supernumerary molting appeared to be 3 days of starvation with 0.5–1.0 g of agar provided on day 0. The endocrine basis for the supernumerary larval molt induced by starvation is briefly discussed.

Dietary Sugars, Hemolymph Trehalose Levels, and Supernumerary Molting of Manduca sexta Larvae

The influence of selected dietary factors on the prevention of starvation-induced supernumerary larval molt was studied. The incidence of supernumerary molting was greatly reduced when larvae were provided with sucrose during the starvation period. Neither casein nor a diet deficient only in sugar had such an effect. Among the various sugars tested, sucrose and fructose were the most effective in suppressing the supernumerary molt. Mannose and sorbose reduced supernumerary molting to a lesser extent. The percentage of larvae undergoing a supernumerary molt was inversely related to the concentration of sucrose, with 100% of those fed on 1% sucrose and 10% of those fed on 7% sucrose molting into sixth instars. The incidence of supernumerary molting was inversely correlated with hemolymph trehalose concentration but was not correlated with protein concentration. A hypothesis relating low hemolymph trehalose titers with activation of the brain-corpora allata system is proposed to explain the JH titer changes after starvation and during molting cycles.

Regulation of Corpus Allatum Activity in Last Instar Manduca Sexta Larvae

The corpora allata (CA) , like other endocrine glands, go through phases of activity and inactivity which correspond to well defined physiological changes occurring during post-embryonic development. This cyclic activity is not intrinsically programmed but is instead regulated by factors external to the gland. It is only logical that the secretory activity of the CA which produce juvenile hormone (JH) , a hormone with profound effects on morphogenesis and reproduction, is finely tuned by environmental factors and the internal milieu via the central nervous system. Evidence suggests that the brain (and perhaps other ganglia) controls the activity of the CA by both humoral and nervous mechanisms during the stages preceding metamorphosis (Wigglesworth, 1954; 1970; Scharrer, 1958; Highnam, 1967; Doane, 1973).

Juvenile hormone acid and ecdysteroid together induce competence for metamorphosis of the Verson's gland in Manduca sexta.

In the last larval instar of Lepidoptera, ecdysteroid in the absence of juvenile hormone (JH) is believed to cause the shift from larval to pupal development. In Manduca sexta, tissues such as the Versons gland and crochet epidermis become pupally committed before the earliest pulse of ecdysteroid that occurs on day 2. What causes the change in commitment in these tissues? First it was necessary to determine at what stage these tissues become competent to express the pupal program. Last instar larvae of different ages were induced to molt prematurely by feeding the ecdysteroid analog RH5992 and Versons gland proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Glands became competent to make pupal proteins between 24 and 32 h after the last larval ecdysis. Next, hormonal regulation of competence was examined in ligated abdomens of 12h last instar larvae. Treatment with JH II acid or methoprene acid plus a low dose (1/50th of the molt inducing dose) of RH5992 induced competence, whereas RH5992 alone, methoprene acid alone or methoprene plus RH5992 did not. Versons glands maintained in vitro produced pupal proteins in response to methoprene acid together with RH5992 but not with RH5992 alone. Likewise, crochet epidermis lost the ability to make crochets (metamorphic change) only in isolated abdomens treated with JH II acid or methoprene acid and low doses of RH5992. In conclusion, JH acid in the presence of basal levels of ecdysteroid induces tissue competence for metamorphosis. Metamorphic competence is followed by commitment, induced by a small pulse of ecdysteroid in the absence of JH, and finally by expression caused by a high titer of ecdysteroid. It is proposed that JH acid is an essential metamorphic hormone.