Effect of fumigation and juvenile hormone on oxygen consumption of the larvae of Corcyra cephalonica (Stainton)

Reduced effect of JH-I, JH-II and JH-III on oxygen consumption of II-V instars and increased effect on the oxygen consumption of VI instar larvae suggested that control of corcyra with juvenile hormones could be brought about only when applied to just emerged VI instar larvae. Similarly fumigation of juvenile hormone treated larvae could prove beneficial only at VI instar stage. The possibility of enhancing the effect of fumigant with pretreatment of JH will be futile as even only JH treated VI instar larvae develop into abnormal individuals which die later.     

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.     

Activite de l'hormone juvenile en C17 (JH-II) chez Locusta migratoria

The effects of C₁₇ juvenile hormone (JH-II) have been investigated in Locusta on morphogenesis, ovarial development, and pigmentation, by means of injections in oil. These effects have been compared with those of injecting C₁₈ juvenile hormone (JH-I) and of implanting corpora allata into Locusta. JH-I and JH-II are similar in their effects upon morphogenesis and pigmentation, and also on ovarial development in which JH-III has been found to be more effective in other insects. Injections of JH-I and JH-II have similar effects to those seen after implanting corpora allata. However in experiments on heart beat (in which the corpora allata have been shown to be involved) JH-I is the only substance to increase the rate of heart beat in the same way as active corpora allata. These observations are discussed, and it is concluded that JH-I is the hormone with effects nearest to those of the corpus allatum hormone itself.     

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.     

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.     

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.     

Developmental regulation of juvenile hormone esterase in Trichoplusia ni: Its multiple electrophoretic forms occur during each larval ecdysis

Juvenile hormone esterase activity has been found during the intramoult period of each larval stadium in Trichoplusia ni. The activity is indistinguishable from that occurring during the final larval stadium, on the basis of its four isoelectric forms and kinetic data. The 4th- to 5th-instar intramoult peak in activity also occurs in other Lepidoptera (Heliothis virescens, Spodoptera exigua, Manduca sexta and Hyphantria cunea). Further, the final species also possessed a peak of activity during the intramoult period to the penultimate larval instar. The findings have important implications for the current concept that the function of juvenile hormone esterase is, by reason of its anti-juvenile hormone action, an enzyme of the last larval instar which enables metamorphosis to begin.     

Effects of juvenile hormone on the secretion of prothoracicotropic hormone in the last- and penultimate-instar larvae of the silkworm Bombyx mori

The effect of juvenile hormone (JH) on the secretion of the prothoracicotropic hormone (PTTH) was investigated, by examining the changes in hemolymph PTTH titer after the topical application of JH-I on the larvae of the silkworm, Bombyx mori. The titer of PTTH was determined by the time-resolved fluoroimmunoassay. JH-I application at very early stages of development in the fifth (last) instar resulted in a significant increase in the PTTH titer, but this effect became less evident thereafter. After the onset of wandering (day 6 of the fifth instar), JH-I did not affect the hemolymph PTTH titer. JH-I application on day 5 resulted in the delay of spinneret pigmentation on day 6, which is induced by an increase in the ecdysteroid titer on day 5 and is the first visible indication of larval-pupal transformation. However, the JH-I application did not suppress the increase in either PTTH or ecdysteroid titer on day 5, suggesting that JH-I acts on the spinneret to inhibit the response of the tissue to ecdysteroids. JH-I also exhibited a PTTH titer-elevating effect in the fourth instar. These results suggest that JH has a role as a potent stimulator of PTTH secretion in both the penultimate and last instar of the silkworm.     

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.     

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.     

Juvenile hormone and DNA synthesis in imaginal disks of Calliphora erythrocephala: results of a new incubation technique.

An in vitro incubation technique in which imaginal disks are exposed to juvenile hormone and some of its analogues is presented. These substances were shown to have an inhibitory effect on the incorporation of tritiated thymidine (³HTdR) during the post-feeding period of the last larval instar of Calliphora. The technique makes it possible to investigate the nature of the effects of ecdysterone and juvenile hormones on the DNA synthesis in imaginal disks of exo- and endopterygote insects.     

Purification,characterization and developmental changes in the titer of a new larval serum protein of the silkworm,Bombyx mori

A new protein was found as a major component of hemolymph proteins up to day 1 of the last larval instar of Bombyx mori, and was named Bombyx mori larval serum protein (BmLSP). The BmLSP was purified to homogeneity by ammonium sulfate precipitation, CM-cellulose column chromatography and gel permeation chromatography. The molecular weight of BmLSP was estimated to be 30,000 by SDS-PAGE and 25,000 by gel permeation chromatography. The amino acid composition of BmLSP was similar to that of 30 kDa proteins which are the major serum proteins in the older last (fifth) instar larvae. The 20 NH₂-terminal amino acids were sequenced and found to be quite different from those of the 30 kDa proteins. Developmental changes in BmLSP titer were followed throughout post-embryonic life by Western blotting using a specific antiserum against BmLSP. Within 1 day after larval hatching, BmLSP appeared in the hemolymph and remained at an almost constant level until day 1 of the last instar. On day 2 of the last instar, the BmLSP level suddenly fell and then gradually decreased toward larval-pupal metamorphosis. Thus, BmLSP is a true larval serum protein and is different from proteins stored for metamorphosis.     

A Biochemical Evidence of the Inhibitory Effect of Diflubenzuron on the Metamorphosis of the Silkworm,Bombyx mori

Diflubenzuron (DFB) has been known to prevent metamorphosis of silkworm, Bombyx mori, from larval to pupal stage at low dose exposure. To explain this inhibitory action of DFB, a hypothesis was raised that DFB acts like juvenile hormone (JH) or DFB inhibits JH esterase to increase endogenous JH titer. A JH bioassay using isolated abdomen clearly indicates that DFB does not act as JH analog because DFB did not induce vitellogenesis in the isolated female abdomen, while endogenous JHs did significantly. General esterase activities in hemolymph were lower in DFB-treated fifth instar larvae than in the control larvae, but there was no difference between fat body esterase activities in both groups. Two hemolymph esterases (‘E1’ and ‘E2’) of the fifth instar larvae were separated and visualized by α-and β-naphthyl acetate. From in vitro incubation experiment, the cathodal esterase (‘E1’) was sensitive to DFB at its nanomolar range. Considering the fact that early fifth instar larvae have high level of JH esterase in the hemolymph, these results suggest that DFB inhibit larval to pupal metamorphosis by blocking JH degradation, which increases endogenous JH titer especially at the critical period when the larvae determine metamorphic development at the following molt.     

Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

Application of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female-specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.     

Short neuropeptide F (sNPF) is a stage-specific suppressor for juvenile hormone biosynthesis by corpora allata,and a critical factor for the initiation of insect metamorphosis

Molting and metamorphosis are essential events for arthropod development, and juvenile hormone (JH) and its precursors play critical roles for these events. We examined the regulation of JH biosynthesis by the corpora allata (CA) in Bombyx mori, and found that intact brain-corpora cardiaca (CC)–CA complexes produced a smaller amount of JH than that in CC–CA complexes and CA alone throughout the 4th and 5th (last) instar stadium. The smaller amount of synthesis was due to allatostatin-C (AST-C) produced by the brain. The CC synthesized short neuropeptide F (sNPF) that also suppressed the JH synthesis, but only in day 3 4th stadium and after the last larval ecdysis. For the suppression, both peptides prevented the expression of some of the distinct JH biosynthetic enzymes in the mevalonate pathway. Allatotropin (AT) stimulated sNPF expression in the CC of day 1 5th instar stadium, not of day 3 4th; therefore the stage-specific inhibition of JH synthesis by sNPF was partly due to the stimulative action of AT on the sNPF expression besides the stage-specific expression of the sNPF receptors in the CA, the level of which was high in day 2 4th and day 0 5th instar larvae. The cessation of JH biosynthesis in the last instar larvae is a key event to initiate pupal metamorphosis, and both sNPF and AST-C are key factors in shutting down JH synthesis, along with the decline of ecdysone titer and dopamine.     

Parasite regulation of host insect metamorphosis: a new form of regulation in pseudoparasitized larvae ofTrichoplusia ni

Summary When eggs ofTrichoplusia ni (Lepidoptera) are stung by a parasitic wasp,Chelonus sp., the developing host larvae precociously initiate metamorphosis ten days later. Precocious initiation of metamorphosis occurs even in ‘pseudoparasitized’ stung hosts which contain no living parasites at the time of symptoms of host regulation by the parasite. In feeding, penultimate instar, pseudoparasitized hosts, the corpora allata activity, hemolymph juvenile hormone esterase activity, in vivo rates of juvenile hormone metabolism and changes in hemolymph protein composition all follow the pattern of the normal last instar. This and other evidence suggests the entire developmental pattern of the last larval instar is precociously expressed in penultimate instar, pseudoparasitized hosts. The cause of precocious expression of the developmental program leading to metamorphosis is a significant decrease in the critical size parameter that, in normal larvae, signals attainment of the last instar. The induction, in preultimate instar larvae, of the entire feeding stage developmental program leading to metamorphic commitment, using either biochemical, surgical or parasitic experimental probes, has not been previously reported. The results have important implications for the study of host-parasite endocrine interaction, of normal insect metamorphosis and even of human puberty.     

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.     

Analysis of precocious metamorphosis induced by application of an imidazole derivative to early third instar larvae of the silkworm,Bombyx mori

When an imidazole derivative (KK-42) was applied to day 1 third instar larvae of the silkworm, Bombyx mori, 100% underwent precocious metamorphosis at the end of the fourth instar. Thus, the fourth instar becomes the last instar in these KK-42–treated larvae. The endocrine systems underlying the precocious metamorphosis were analyzed in the present study. Hydroprene application during the prolonged third instar after KK-42 treatment can prevent precocious metamorphosis, and the results showed dose-dependent and stage-specific effects. From analysis of the developmental changes in ecdysteroid levels in both KK-42–treated larvae and KK-42– and hydroprene-treated larvae, we conclude that changes in JH levels during the third larval instar can modify the secretion pattern of prothoracic glands and that during the next larval instar, very low ecdysteroid levels during the early stages of the presumptive last (fourth) larval instar are directly related to precocious metamorphosis. Arch. Insect Biochem. Physiol. 36:349–361, 1997. © 1997 Wiley-Liss, Inc.