Effects of juvenile hormone on some phase characteristics in the common cutworm, Spodoptera litura

Phase characters of the common cutworm, Spodoptera litura, were influenced by different rearing densities from the 4th-larval instar. Primarily the final feeding period of isolated larvae was 1 day longer than that of crowded larvae causing an increase in pupal weight. Applications of juvenile hormone I, II, or methoprene to crowded larvae caused an increased feeding period similar to that of isolated larvae when the juvenile hormones were applied within 1 day after the last-larval ecdysis. Allatectomy of isolated Spodoptera during the moult to the final-larval instar decreased the duration of the final feeding period to that of intact crowded larvae. These results suggested that one of the characters of phase variation, pupal weight, is influenced by the differences in the regulation and activity of the corpora allata during the last-larval instar. Other characteristics of phase variation such as behaviour (feigned death) and colour were not affected by alteration in juvenile hormone levels after the last larva ecdysis.     

Influence of a juvenile hormone analogue on vitellogenin synthesis and oögenesis in larvae of Nauphoeta cinerea

In experiments on the synthesis of the vitellogenic protein, farnesylmethylester, a juvenile hormone (JH) analogue, was injected into female Nauphoeta cinerea larvae at various stages during their development. Two and 4 days after injection, 2 μl of haemolymph were assayed in a vitellogenin immunodiffusion test. In second last and last instar larvae less than 6 days before adult ecdysis, high doses (100 μg) of farnesylmethylester are necessary to induce vitellogenin synthesis, whereas older last stage larvae and decapitated adults respond to small doses (1 μg) with the synthesis of vitellogenin. It seems that the competence to synthesize the vitellogenic protein changes at the time of induction of the moulting process. If farnesylmethylester is injected into last instar larvae with a supposedly high titre of ecdysone, the vitellogenic protein can be detected in the haemolymph of a small percentage of animals only.Oöcyte maturation can be observed in last instar larvae injected after the fifth to ninth day with farnesylmethylester. The observed volume changes of the corpora allata suggest that an absence of JH for a short time is necessary for the oöcytes to become competent to grow. Last instar larvae treated with farnesylmethylester become larval-adult intermediates with partly developed oöcytes, demonstrating a simultaneous juvenilizing and gonadotropic influence of the JH analogue. In last instar larvae injected with farnesylmethylester a partial degeneration of already maturing oöcytes is induced at the time when the ecdysone titre is supposedly high and the possible reasons for this are discussed.     

Factors influencing juvenile hormone esterase activity in the wax moth, Galleria mellonella

The effects of juvenile hormone, antiallatotropins, selected surgical procedures and starvation on the juvenile hormone esterase levels in Galleria larvae and pupae were investigated. JH reduced JH esterase activity in larvae but induced the enzyme in 1-day-old pupae. In vitro studies confirmed that the peak of synthesis and/or release of JH esterase from the fat body of last instar larvae occurred 4 days after ecdysis. These studies also showed that fat body from JH-treated larvae released much less enzyme than controls. Antiallatotropins, precocene 2 and ZR 2646 also reduced JH esterase levels in larvae, but ZR 2646 induced JH esterase in pupae. In starved larvae, JH esterase did not increase during the first five days. A minimum of 36 hr of feeding was necessary for the larval esterase activity to increase on schedule on day 4 of the last larval stadium. When day-l larvae were ligated behind the head or the prothorax, they had lower JH esterase levels and yet showed a slight increase in the enzyme when the larvae reached the age of 4 days. The significance of these results is discussed in relation to the possible control of esterase activity during metamorphosis.     

Bursicon and the metabolism of tyrosine in the moulting cycle of Pieris larvae

In fourth instar larvae of Pieris brassicae the haemolymph tyrosine level begins to rise about 1 day before apolysis to reach a level about treble that in the middle of the instar. Between apolysis and ecdysis the haemolymph tyrosine level appears to decline, until just before ecdysis another steep rise occurs. About 30 min after ecdysis a steep decline starts, levelling off gradually until the level in the middle of the instar is restored.Bursicon assays show that this hormone operates in the haemolymph both during apolysis and after ecdysis; but during the actual ecdysis no bursicon activity can be demonstrated in the haemolymph.Indications have been found that the bursicon activity can restore itself spontaneously in the haemolymph of newly ecdysed larvae. This would suggest that during ecdysis a bursicon inhibitor of restricted life is operating.     

Antijuvenile influence of the precocene on the development of adult antennae in the large fruit-tree tortrix Archips podana Scop. (Lepidoptera: Tortricidae)

The influence of precocene II, an antijuvenile agent, on the development of adult antennae in the large fruit-tree tortrix A. podana Scop. was demonstrated. Treatment of the fifth instar larvae and prepupae with different doses of precocene proved to cause different sensitivity of the specimens to the juvenile hormone deficit. Treatment with 450 and 600 ??g precocene per specimen during the first days after ecdysis to the fifth instar caused the death of larvae. Treatment with 300, 450, and 600 ??g per specimen on the third day of the fifth instar larvae and prepupae caused a delay in the development of adult antennae. The results are discussed with respect to the role of the juvenile hormone in the development of imaginal structures during metamorphosis.     

Composition,synthetic and cytolytic activities of Galleria mellonella silk glands during the last-larval instar under the action of juvenile hormone

Within the first 48 hr of the last-larval instar of Galleria mellonella the silk glands grow but silk production is restrained. This ‘preparatory phase’ of the glands is probably maintained by juvenile hormone. Silk production and accumulation are stimulated in the ‘accumulation phase’ between 60 and 132 hr by unknown factors in the absence of juvenile hormone. The rate of RNA synthesis culminates at 84 hr but the RNA content increases until the end of cocoon spinning at 144 hr. In the following ‘regression phase’ (144–160 hr), when the glands exhibit high activities of acid and alkaline DN-ases and of acid phosphatase, the RNA and protein contents rapidly decrease, but that of DNA remains high. This phase is typical of moulting insects, is independent of juvenile hormone, and seems to be caused either by an increase in ecdysteroids or by lack of nutrients. The following ‘degeneration phase’ occurs when the surge of ecdysteroids terminates the larval-pupal transformation. Disintegration of silk glands by autolysis and phagocytosis is completed after pupal ecdysis (180 hr). Treatment of larvae with a juvenoid (ZR 512) at 48 or 132 hr in the last instar dramatically alter the composition, synthetic and cytolytic activities of silk glands. At the next ecdysis the glands attain a state very similar to that of the preparatory phase. They are capable of intensive silk production and completion of developmental cycle when the supernumerary larvae prepare for pupation. The results indicate that juvenile hormone can reverse the development of the silk glands.     

Action of two juvenile hormone antagonists on lepidopteran epidermis

The juvenile hormone antagonist ETB (ethyl-4-2(t-butylcarbonyloxy)-butoxybenzoate) caused formation of precocious larval-pupal intermediates after the 4th (penultimate)-larval instar of the tobacco hornworm, Manduca sexta, when 50 μg were applied to any 3rd stage larvae or to 4th stage larvae within 12 hr after ecdysis. This dose was most effective within 12 hr after ecdysis to the 3rd stage. In the black mutant larval assay for juvenile hormone, ETB had activity, 0.75 μg per larva giving half-maximal score. In vitro ETB acted as a juvenile hormone to prevent the ecdysteroid-induced change in commitment at concentrations above 0.1 μg/ml with an ED₅₀ at 2.8 μg/ml and as a partial juvenile hormone antagonist to 0.1 μg/ml juvenile hormone I at concentrations between 10^?3 and 10^?2 μg/ml. By contrast, EMD (ethyl-E-3-methyl-2-dodecenoate) had little juvenile hormone-like activity in vitro up to its limits of solubility (100 μg/ml) and exhibited sporadic partial juvenile hormone antagonistic activity in vitro at concentrations between 1 and 100 μg/ml. Since these concentrations were 10–1000 times that of juvenile hormone I in the medium, EMD apparently is not an efficient competitor.     

Reduced titres of ecdysone following juvenile hormone treatment in the German cockroach, Blattella germanica

Changes in ecdysone titre of the larvae of the German cockroach, Blattella germanica, exposed continuously to the juvenile hormone (JH), or to the insect growth regulator (IGR) with JH activity, can be correlated with the nature of the substance applied, its dose, and the time of application. The younger larvae exposed to the high dose of the IGR die in the next ecdysis, whereas the same treatment induces a diapause-like stage of developmental arrest in the last larval stage. The affected larvae have very little or no ecdysone, the synthesis of which takes place in the second part of the instar. The same treatment after this period has a lesser effect. The extent of the effect is correlated to the amount of ecdysone synthesized before the application of IGR. Last instar larvae exposed to the lower dose of the IGR or JH lack the peak of ecdysone normally found in the controls at the end of the second third of the instar when metamorphosis takes place. In these insects the first rise of the ecdysone titre begins towards the end of the instar, and ecdysis into the supernumerary larval stage is initiated when the ecdysone titre reached a level permitting ecdysis.A direct or indirect antagonism between these hormones, both fundamental to insect development, can explain the morphogenetic, inhibitory, and lethal effects observed in insects treated with JH or IGR with JH activity.     

Haemolymph juvenile hormone esterase activity in synchronous last instar larvae of the cabbage looper, Trichoplusia ni

Weight and time of moult during the last instar of the cabbage looper (Trichoplusia ni) were examined and used to select last instar larvae that had similar rates of development. Haemolymph protein content and titres of haemolymph esterases hydrolyzing juvenile hormone I, juvenile hormone III, and α-naphthyl acetate were monitored during the last instar using these closely timed larvae. Juvenile hormone I and juvenile hormone III esterase profiles were very similar and differed markedly from the α-naphthyl acetate esterase and protein content profiles. Two major peaks of juvenile hormone esterase activity were observed, one before ecdysone release and the other just prior to pupal ecdysis. Juvenile hormone I was hydrolyzed 15 times faster than juvenile hormone III when assayed at 5 × 10^?6 M.     

Inhibition of the larval ecdysis and emergence behavior of the parasitoid Cotesia congregata by methoprene

Parasitism of the tobacco hornworm, Manducasexta, by the braconid wasp Cotesiacongregata, induces developmental arrest of the host in the larval stage. During the final instar of the host, its juvenile hormone (JH) titer is elevated, preventing host metamorphosis. This study investigated the effects of hormonal manipulation of the host on the parasitoid’s emergence behavior. The second larval ecdysis of the wasps coincides with their emergence from the host, and application of the juvenile hormone analogue methoprene to day 4 fifth instar hosts either delayed or totally suppressed the subsequent emergence of the wasps. Effects of methoprene were dose-dependent and no parasitoids emerged following treatment of host larvae with doses >50 μg. Parasitoids which failed to emerge eventually succumbed as unecydsed pharate third instar larvae in the hemocoel of the host. Effects of host methoprene treatment on parasitoid metamorphosis were also assessed, and metamorphic disruption occurred at much lower dosages compared with doses necessary to suppress parasitoid emergence behavior. The inhibitory effect of methoprene on parasitoid emergence behavior appears to be mediated by effects of this hormone on the synthesis or release of ecdysis-triggering hormone (ETH) in the parasitoid, the proximate endocrine cue which triggers ecdysis behavior in free-living insects. ETH accumulated in the epitracheal Inka cells of parasitoids developing in methoprene-treated hosts, suggestive of a lack of hormone release. Thus, the hormonal modulation of parasitoid emergence behavior appears to be complex, involving a suite of hormones including JH, ecdysteroid, and peptide hormones.     

Juvenile hormone titres and metabolism during starvation-induced supernumerary larval moulting of the tobacco hornworm, Manduca sexta L.

When tobacco hornworm (manduca sexta) larvae are starved for 5 days immediately after ecdysis to the 5th instar, then fed normal diet, they undergo a supernumerary moult instead of metamorphosis. During starvation the titre of juvenile hormone in the haemolymph increased to a maximum of 3 ng juvenile hormone I equivalents/ml (determined by the black Manduca larval bioassay) on the fourth day of starvation, then began a decline which continued through the subsequent feeding period. The changes in juvenile hormone titre were not attributable to changes in haemolymph volume during starvation (only a 5% decrease) and subsequent feeding. During starvation the esterase activity of the haemolymph declined 4-fold with a 2-fold larger decrease in the DFP-insensitive, presumably juvenile hormone specific, esterase activity. Both the total and the juvenile hormone-specific esterase activity then increased as a function of larval weight during the subsequent feeding period. As growth was slow in the prolongedly starved larvae, sufficient juvenile hormone was present at the time of prothoracicotropic hormone (PTTH) and ecdysteroid release at the beginning of the fourth day of feeding to prevent metamorphosis.     

The hormonal regulation of the larval diapause in the codling moth, Laspeyresia pomonella (Lep. Tortricidae)

The hormonal control of the facultative diapause of the codling moth has been investigated. The diapause can be divided into 4 phases or periods: (1) diapause induction by short-day conditions (SD) in young larvae, (2) initiation of the diapause in the early last larval instar by a high titre of juvenile hormone, (3) onset and maintenance of diapause with inactivity of the neuroendocrine system, as evidenced by the results of neck-ligation experiments, (4)termination of diapause by the production of ecdysteroid.Diapause-induced larvae pupated after spinning the cocoon, if the state of induction was changed by injection with the anti-juvenile hormone precocene II at the beginning of the last larval instar and subsequent results of neck-ligation experiments, (4) termination of diapause by the production of ecdysteroid. treated with juvenile hormone during the first 1.5 days after the last larval moult and subsequently reared under SD. Under LD, continuous application of juvenile hormone during the last larval instar and after spinning did not prevent the insects from moulting to either a supernumerary larva, a pupa or a larval-pupal intermediate. Termination of diapause, i.e. pupation, was achieved by injecting diapausing larvae with 20-hydroxyecdysone. Although juvenile hormone was found to have a prothoractropic effect in diapausing larvae, no pupal moult could be induced by the application of the hormone. Contrary to the hormonal situation before pupation of nondiapausing larvae, no juvenile hormone could be detected before or during the pupation of larvae after diapause.     

Disruptive developmental effects of benzyl-1,3-benzodioxole derivatives on unparasitized and parasitized Manduca sexta larvae

Treatment of tobacco hornworm larvae with the benzyl-1,3-benzodioxole derivative J-2710 immediately after ecdysis to the fourth instar disrupted development either during the moult to the fifth instar or shortly thereafter. Larvae given topical applications of 100 μg J-2710 in 1 μl acetone suffered 100% mortality, often after secreting moulting fluid in large pockets between the epidermis and the cuticle later in the fourth instar. Larvae that successfully ecdysed had abnormalities of the mouthparts and cervix that interfered with normal feeding, inhibiting growth in the fifth instar. Larvae of the gregarious endoparasitic wasp Cotesia congregata (=Apanteles congregatus) frequently failed to emerge from host Manduca sexta larvae treated with high doses of J-2710, particularly when the host failed to feed normally. Less potent disruptive effects on Manduca and Cotesia were seen after treatment of larvae with the derivatives J-3370 and J-2581.No anti-juvenile hormone action of J-2710 was observed. J-2710-treated M. sexta larvae showed no precocious metamorphosis and the developmental effects of J-2710 were not prevented by co-application of the juvenile hormone analogue methoprene in doses ranging from 1 to 100 μg/larva. Moreover, J-2710 had no effect on the action of methoprene in the black larval assay for juvenile hormone-like activity, unlike results reported to occur using the Galleria wax wound assay.     

Pupal commitment and its hormonal control in wing imaginal discs

The timing of pupal commitment of the forewing imaginal discs of the silkworm, Bombyx mori, was determined by a transplantation assay using fourth instar larvae. The wing discs were not pupally committed at the time of ecdysis to the fifth instar. Pupal commitment began shortly after the ecdysis and was completed in 14 h. When the discs of newly molted larvae (0-h discs) were cultured in medium containing no hormone, they were pupally committed in 26 h. In vitro exposure of 0-h discs to 20-hydroxyecdysone accelerated the progression of pupal commitment. Methoprene, a juvenile hormone analog (JHA), did not suppress the change in commitment in vitro at physiological concentrations. Thus the wing discs at the time of the molt have lost their sensitivity to JH, and 20E is not a prerequisite for completion of pupal commitment. These results suggest that the change in commitment in the forewing discs may begin before the last larval molt.     

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.     

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.     

Hormonal regulation of phase polymorphism and storage-protein fluctuation in the common cutworm, Spodoptera litura

Three storage proteins are synthesised by Spodoptera litura last-instar larvae as detected by an antiserum against pupal fat body proteins. The putative pupal storage proteins 1 and 2, appear in the haemolymph of the last-instar larvae 36 h after ecdysis under crowded rearing conditions: they appear 1 day later in isolated conditions. The appearance of these proteins in the haemolymph is prevented by juvenile hormone treatment and enhanced by allatectomy. Injection of 20-hydroxyecdysone into ligatured larvae does not induce appearance of these 2 proteins. Accumulation of protein 3 that reacts with Bombyx mori arylphorin antiserum is not blocked by juvenile hormone and is similar in both phases. It also accumulates to a small extent in the haemolymph during the moult to the final-larval instar and then disappears at ecdysis. One-hundred ng/ml ecdysteroid caused the sequestration of these proteins by the fat body, but a higher concentration of ecdysteroid (200 ng/ml) produced pupal cuticle in the isolated abdomens, suggesting that different ecdysteroid concentrations are necessary for these two events.     

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.     

Effect of low rearing temperature on development of Galleria mellonella larvae and their sensitivity to juvenilizing treatment

• 1.1. The development of Gallena mellonella is strongly affected by a low temperature of 18°C (the last instar persists for more than one year, instead of about 9 days at 30°C). At 18°C the last instar Galleria mellonella larvae respond to juvenilizing treatment—chilling stress or juvenile hormone analogue—with a very low percentage or no supernumerary moults, respectively.
• 2.3. Experiments in which larvae subjected to such treatments were transferred from 18°C to 30°C and vice versa showed that for the realization of the larval programme after chilling stress application the higher (30°C) temperature is needed.
• 3.4. In last instar larvae reared at 18°C there coexist very high juvenile hormone titre and high juvenile hormone esterase activity.
• 4.5. This phenomenon which is found in both, chilled and unchilled larvae, is discussed.