Juvenile hormone-mediated termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

Larvae of the bamboo borer, Omphisa fuscidentalis are in diapause for more than nine months (Singtripop, T., Wanichaneewa, S., Tsuzuki, S., Sakurai, S. 1999. Larval growth and diapause in a tropical moth, Omphisa fuscidentalis Hampson. Zool. Sci. 16, 725-733). To examine the endocrine mechanisms underlying this larval diapause, we assayed the responsiveness of the diapausing larvae to 20-hydroxyecdysone (20E) and a juvenile hormone analogue (JHA: S-methoprene). 20E injection caused the larvae to halt movement, followed by deposition of a pupal cuticle. Topical application of JHA induced pupation in a dose-dependent manner. JHA also induced pupation of the larvae whose brains were removed before JHA application. In those larvae, the prothoracic glands became active and competent to respond to brain extracts within seven days after JHA treatment, and the hemolymph ecdysteroid concentration began to increase 12 days after JHA application. These results indicate that JHA stimulates the prothoracic glands of diapausing Omphisa larvae, terminating larval diapause, in contrast with previous findings that JH inhibits the brain-prothoracic gland axis and thus maintains the larval diapause. Current results therefore suggest a novel regulatory mechanism for larval diapause in this species.     

Programmed cell death of larval tissues induced by juvenile hormone in the bamboo borer, Omphisa fuscidentalis

Programmed cell death (PCD) plays a critical role during animal development through the destruction of unneeded cells and tissues. In some insects, the prothoracic glands (PGs) and anterior silk glands (ASGs) are larval-specific tissues that are normally eliminated by PCD after pupation. Previous studies report that juvenile hormone analog (JHA) terminates the larval diapause of Omphisa fuscidentalis by increasing the hemolymph ecdysteroids that trigger PCD. Because JHA may indirectly induce the PCD of the PGs and ASGs of Omphisa diapausing larvae, the effects of JHA on the induction of PCD were determined. The application of 1μg JHA induced PCD in the PGs and ASGs of larvae identified as stage G0 (prior to pupation). The injection of 1μg 20E triggered the PCD of the ASGs when the larvae expressed a G0-G1 morphology, whereas PCD occurred in the PGs on day 1 post-injection. Histological studies revealed similar patterns of morphological changes during the PG and ASG PCD in the JHA- and 20E-treated larvae. Furthermore, to confirm that PCD was induced by a high ecdysteroid level that increases after JHA application, the expression profiles of EcR-A and EcR-B1 in the PGs and ASGs from the JHA-treated larvae were examined, and the results showed that the expression levels of EcR-A and EcR-B1 mRNA increased during the G0 stage. These results suggest that JHA may be involved in PCD by increasing the ecdysteroid titer, leading to termination of the larval diapause period in Omphisa fuscidentalis.     

Dual control of midgut trehalase activity by 20-hydroxyecdysone and an inhibitory factor in the bamboo borer Omphisa fuscidentalis Hampson

During larval diapause lasting 9 months from September to May, trehalase activity in the midgut of the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae) was low from December to April, followed by a fourfold increase in May that remained high during the pupal stage in July. An application of juvenile hormone analog (JHA) produced increases in the ecdysteroid titer, while trehalase activity was increased by both JHA and 20-hydroxyecdysone (20E) injection. The trehalase activity in the midgut of diapausing larvae was doubled by incubating the midgut with 20E for 48h. During diapause as well as after JHA application, expression of two ecdysone receptor isoform genes (EcR-A and EcR-B1) in the midgut increased simultaneously with the increase in hemolymph ecdysteroid titer, followed by an increase in trehalase activity. The hemolymph of diapausing larvae contained a trehalase inhibitor and inhibitory activity was high during diapause. After 20E injection, trehalase inhibition decreased as midgut trehalase activity increased. Taken together, at least two factors may participate in the change in midgut trehalase activity: increase in trehalase activity and decrease in trehalase inhibitor activity, both of which may be induced by 20E.     

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.     

Positive feedback regulation of prothoracicotropic hormone secretion by ecdysteroid – A mechanism that determines the timing of metamorphosis

When insect larvae have fully grown, prothoracicotropic hormone (PTTH) is released from the brain, triggering the initiation of metamorphic development through stimulation of ecdysteroid secretion by the prothoracic glands. The present study analyzes the mechanism that regulates the occurrence of this PTTH surge. In the silkworm Bombyx mori, the PTTH surge occurs on day 6 of the fifth instar and is preceded by a small rise in hemolymph ecdysteroid titer, which occurs late on day 5. We therefore hypothesized that this rise of ecdysteroid titer is involved in the induction of the PTTH surge. To test this hypothesis, two experiments were conducted. First, a small amount of 20-hydroxyecdysone was injected on day 4, two days before the expected day of the PTTH surge, to simulate the small rise in hemolymph ecdysteroid titer on day 5. This injection led to a precocious surge of PTTH the next day. Next, the hemolymph ecdysteroid titer on day 5 was artificially lowered by injecting ecdysteroid-22-oxidase, which inactivates 20-hydroxyecdysone. After this treatment, the PTTH surge did not occur on day 6 in 80% of the animals. These results indicate that a small rise of the hemolymph ecdysteroid titer plays a critical role in the induction of the PTTH surge. Since basal ecdysteroidogenic activity of the prothoracic glands increases with larval growth, a circulating level of ecdysteroids may convey information about larval maturity to the brain, to coordinate larval growth and metamorphosis. This is the first report in invertebrates to demonstrate positive feedback regulation of the surge of a tropic hormone by a downstream steroid hormone.     

Hormonal mechanisms underlying termination of larval diapause by juvenile hormone in the bamboo borer, Omphisa fuscidentalis

Topical application of methoprene, a juvenile hormone analogue (JHA), induces pupation by activating the prothoracic glands (PGs) in diapausing larvae of the bamboo borer, Omphisa fuscidentalis. To determine the minimum stimulation period for PG activation, we transplanted PGs of JHA-treated larvae (donors) into non-treated larvae (recipients) on successive days after JHA treatment and observed the recipients for pupation. JHA stimulation for 1 day was sufficient to induce pupation. In recipient larvae, the hemolymph ecdysteroid titer increased transiently on day 18 after transplantation and significantly on days 24-28, prior to pupation. Secretory activity of recipient PGs increased transiently on day 16 and days 22-28. Because the recipient PG activity was too low to account for an increased ecdysteroid titer, the JHA-stimulated donor PGs must produce the major part of hemolymph ecdysteroids. In addition, the ecdysteroid produced by the donor PGs might have stimulated the recipient PGs. We examined the possible involvement of two ecdysone receptor (EcR) isoforms, OfEcR-A and OfEcR-B1, in PG activation by JHA, and found that although both isoforms were up-regulated, accompanied by an increased ecdysteroid titer in the hemolymph, the isoform mRNA levels were not altered at all before the increase in PG secretory activity. Thus, EcR expression might not be involved in feedback activation of PGs.     

Effects of juvenile hormone analogs and 20-hydroxyecdysone on diapause termination in eggs of Locusta migratoria and Oxya yezoensis

To understand the hormonal control of embryonic diapause, juvenile hormone analogs (JHAs), methoprene and hydroprene, and 20-hydroxyecdysone (20E) were applied onto diapause eggs of Locusta migratoria and Oxya yezoensis. These insects enter diapause at the mid-stage of embryogenesis prior to blastokinesis. Topical application of JHAs significantly facilitated diapause termination in both species but JHA-treated embryos underwent abnormal morphogenesis, pigmentation and sclerotization without dorsal closure. The Locusta eggs immersed in the 20E solution for 24h terminated diapause in a dose-dependent manner. We also investigated phosphorylation of extracellular signal-regulated kinase (ERK), a member of mitogen-activated protein kinase (MAPK), during diapause-terminating process of Locusta migratoria and found that ERK was activated either by cold exposure or JHA treatment. The possible involvement of the hormones and ERK in embryonic diapause and the possibility of ecdysteroids synthesis by prothoracic glands of diapause embryo were proposed.     

Effects of RH-5992 on ecdysteroidogenesis of the prothoracic glands during the fourth larval instar of the silkworm, Bombyx mori

Stage-dependent effects of RH-5992 on ecdysteroidogenesis of the prothoracic glands during the fourth larval instar of the silkworm, Bombyx mori, were studied in the present report. When larvae were treated with RH-5992 during the early stages of the fourth larval instar (between day 0 and day 1), initially ecdysteroid levels in the hemolymph were inhibited. However, 24 h after RH-5992 application, ecdysteroid levels were greatly increased as compared with those treated with acetone. The examination of the in vitro prothoracic gland activity upon RH-5992 application during the early stages of the fourth larval instar confirmed a short-term inhibitory effect. When RH-5992 was applied to the later stages of the fourth larval instar, no effects on both hemolymph ecdysteroid levels and prothoracic gland activity were observed. Addition of RH-5992 to incubation medium strongly inhibited ecdysteroid secretion by the prothoracic glands from the early fourth instar, indicating direct action of RH-5992 on ecdysteroidogenesis by prothoracic glands. Four hours after application with RH-5992 on day 1.5, prothoracic glands still showed an activated response to PTTH in both PTTH-cAMP signaling and the extracellular signal-regulated kinase (ERK) signaling. Moreover, addition of RH-5992 to incubation medium did not interfere with the stimulatory effect of the glands to PTTH in ecdysteroidogenesis. These results indicated that both PTTH-cAMP signaling and PTTH-ERK signaling may not be involved in short-term inhibitory regulation by RH-5992.     

Endocrine Mechanisms Regulating Post-Diapause Development in the Cabbage Armyworm,Mamestra brassicae

Diapause, a programmed developmental arrest at a specific stage, is common in insects and is regulated by hormones. It is well established that in pupal diapause, cessation of ecdysteroid secretion from the prothoracic glands (PGs) after pupal ecdysis leads to diapause initiation, while resumption of its secretion induces post-diapause development. However, what regulates the activity of the glands is poorly understood, especially for the glands of diapause-terminated pupae. In the present study, we investigate the mechanisms by which post-diapause development is regulated in the cabbage armyworm Mamestra brassicae. We demonstrate that the brain is necessary for the initiation of post-diapause development and that the factor in the brain responsible for the activation of the PGs is the prothoracicotropic hormone (PTTH). Further, through measuring the hemolymph PTTH titers by time-resolved fluoroimmunoassay, we show that PTTH is actually released into the hemolymph prior to the activation of the PGs. Although its peak titer is much lower than expected, this low concentration of PTTH is most likely still effective to activate the PGs of post-diapause pupae, because the responsiveness to PTTH of the glands at this stage is very high compared to that of nondiapause pupal PGs. These results strongly suggest that in M. brassicae, PTTH serves as a trigger to initiate pupa-adult development after diapause termination by stimulating the PGs to secrete ecdysteroid.     

Juvenile hormone regulation of the larval diapause of the Southwestern corn borer,Diatraea grandiosella

The hormonal mechanism which controls the larval diapause of the southwestern corn borer was examined. The onset of this facultative mature larval diapause is marked by a transition from a spotted to an immaculate larval form, and during diapause individuals may undergo one or more stationary larval ecdyses. Experiments were designed to uncover the nature of the humoral mechanism regulating this diapause state. The finding that injecting diapause larvae with 20-hydroxyecdysone only brought about a stationary larval ecdysis suggests that diapause was not maintained by the lack of ecdysone. Neck ligations performed on larvae which had just entered diapause resulted in a premature termination of diapause, and larval-pupal ecdysis occurred in the thoraco-abdominal section, suggesting that a cephalic factor was necessary for the maintenance of diapause. This finding was further supported by the discovery that injecting 20-hydroxyecdysone into the thoraco-abdominal section of previously ligated diapause larvae also resulted in a premature termination of diapause and larval-pupal ecdysis, indicating that ecdysone only initiated the pupal moulting cycle when the cephalic factor was absent.Further experiments led to the conclusion that the juvenile hormone is the cephalic factor. Topical treatment with a juvenile hormone mimic caused non-diapause mature larvae to become immaculate and enter diapause. Periodical topical application of this mimic to diapause larvae prolonged diapause and increased the number of stationary larval ecdyses. These findings suggest that the initiation and maintenance of diapause are regulated by juvenile hormone titre. Results indicate that larvae retain a high titre of juvenile hormone until the last stages of diapause. Injection of 20-hydroxyecdysone into early or middiapause larvae only caused stationary larval ecdyses, while the same injection into larvae in the late stages of diapause caused some of them to pupate. Histological studies of the neurosecretory cells, corpus cardiacum-allatum complex, and prothoracic glands showed that the endocrine system was not inactive during diapause. A new hypothesis is therefore proposed which recognizes the existence of hormonal activity during larval diapause and emphasizes the principal regulatory rôle of juvenile hormone.     

A Role for Ecdysteroids in the Induction and Maintenance of the Pharate First Instar Diapause of the Gypsy Moth,Lymantria dispar

Several lines of evidence suggest a novel regulatory mechanism for diapause regulation in the gypsy moth. We propose that ecdysteroids play a role in the induction and maintenance of the pharate first instar larval diapause in this species. A 55 kDa gut protein that is indicative of diapause is expressed in intact and neck-ligated pharate larvae but is not expressed when a ligature is placed posterior to the prothorax, site of the prothoracic gland. Guts cultured in vitro for 12 h cease to synthesize the 55 kDa protein, but synthesis of the protein resumes if the culture medium is enriched with a prothorax extract from pharate larvae or a prothoracic gland extract from fifth instar larvae. Injection of 20-hydroxyecdysone or the ecdysteroid agonist, RH-5992, into isolated abdomens stimulates synthesis of the diapause-specific 55 kDa protein, suggesting that the essential factor from the prothorax is an ecdysteroid. KK-42, an imidazole derivative known to inhibit ecdysteroid biosynthesis, averts diapause when applied to prediapausing pharate first instar larvae, but this effect can be countered by application of 20-hydroxyecdysone or RH-5992, i.e. KK-42 treated pharate larvae that are exposed to an ecdysteroid or RH-5992 readily enter diapause. A chilling period (120 days at 5 degrees C) is normally adequate to prompt an immediate termination of diapause when pharate larvae are transferred to 25 degrees C, but if such larvae are held in hanging drop cultures with ecdysteroids they fail to terminate diapause. Together, these results suggest that ecdysteroids are essential for the induction and maintenance of diapause and imply that a drop in the ecdysteroid titer is essential for diapause termination. Copyright 1997 Elsevier Science Ltd. All rights reserved     

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.     

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.     

Hemolymph ecdysteroid titer and ecdysteroid-dependent developmental events in the last-larval stadium of the silkworm,Bombyx mori: role of low ecdysteroid titer in larval-pupal metamorphosis and a reappraisal of the head critical period

The endocrine regulation of larval-pupal metamorphosis was studied in the silkworm, Bombyx mori, by measuring the following changes: hemolymph ecdysteroid titer, the secretory activity of prothoracic glands and the responsiveness of larvae to ecdysteroids and prothoracicotropic hormone (PTTH), with regard to developmental events such as the occurrence of spinneret pigmentation, initiation of cocoon spinning and onset of wandering stage as indicated by gut purge. These measurements were concentrated especially on the time before and after the head critical period (HCP) which falls 3-4 days before the gut purge ([Sakurai, 1984]). A small increase in the hemolymph ecdysteroid titer was first found during the HCP, and then the titer increased with daily fluctuations. Small but significant titer peaks were found prior to the occurrence of both spinneret pigmentation and gut purge, indicating that an individual titer peak could possess a specific role in development. Responsiveness of larvae to exogenous 20-hydroxyecdysone (20E) after the HCP was markedly higher than that before the HCP. The sensitivity of the prothoracic gland to PTTH also changed during the HCP. The results thus showed that the HCP is not the period after which an additional PTTH release is not required for the developmental events occurring on schedule, but rather it is the period during which complex events occur not only in the endocrine glands but also in the peripheral tissues. In addition, various developmental phenomena before gut purge are brought about by the hemolymph ecdysteroid whose concentration gradually increased with daily fluctuations, and these precise changes in the titer appeared to be important for the sequential occurrence of developmental events in the larval-pupal metamorphosis.     

Stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis by the prothoracic glands during the last larval instar of the silkworm, Bombyx mori

The stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori, were studied in the present study. When last instar larvae were starved beginning on day 1 of that instar, all larvae died between days 5 and 7 of the instar. Although the prothoracicotropic hormone (PTTH) release from the brain-corpus cardiacum-corpus allatum (BR-CC-CA) did not significantly change during starvation, a deficiency in PTTH signal transduction was maintained, which led to very low levels of hemolymph ecdysteroids after the beginning of starvation. However, when starvation began on day 3 of the last larval instar, the major hemolymph ecdysteroid peak, preceding larval-pupal transformation, occurred 1 day earlier than that in control larvae. Protein content of the prothoracic glands in day 3-starved larvae was maintained at a low level as compared to that of control larvae. The secretory activity of the prothoracic glands in day 3-starved larvae was maintained at a level similar to that of control larvae. However, the rate of ecdysteroidogenesis, expressed per microgram of glandular protein, was greatly enhanced in these starved larvae, indicating that upon starvation, larvae increased the ecdysteroid production rate to enhance the rate of survival.     

Temperature-dependent regulatory mechanism of larval development of the wax moth (Galleria mellonella)

The mechanisms underlying larval diapause in the wax moth (Galleria mellonella) is one of the most throughly studied aspects. At the low temperature of 18 degrees C, the last instar larvae did not pupate but transferred to 30 degrees C they initiated development and pupation in a circadian manner. Different types of surgical manipulations including head-ligation, nerve cord-severance, implantation of the brain, prothoracic glands, accompanied with ecdysteroid titre measurements indicated that diapausing arrest of larval development at 18 degrees C might be due to the nervous inhibition of their prothoracic glands.     

Decreased JH biosynthesis is related to precocious metamorphosis in recessive trimolter (rt) mutants of the silkworm, Bombyx mori

In recessive trimolter (rt) mutants of the silkworm, Bombyx mori, that have four larval instars rather than five larval instars of normal B. mori, a decrease after a small increase in the hemolymph ecdysteroid titer during the early stages of the last (fourth) larval instar appeared to be a prerequisite for larvae to undergo precocious metamorphosis. The present study was carried out to investigate the possible mechanism underlying this decrease in the ecdysteroid titer. It was found that juvenile hormone (JH) biosynthetic activity of the corpora allata (CA) increased during the first day of the last larval instar, but its absolute JH biosynthesis activity was relatively lower compared to that of normal fourth-instar larvae in tetramolters. This lowered JH biosynthetic activity appeared to be related to a decrease in prothoracic gland ecdysteroidogenesis during the second day of the last instar, because hydroprene application prevented this decrease in prothoracic gland ecdysteroidogenesis, leading to the induction of a supernumerary larval molt. The in vitro incubation of prothoracic glands with hydroprene showed that hydroprene did not directly exert its action on prothoracicotropic hormone (PTTH) release. Further study showed that the application of hydroprene enhanced the competency of the glands to respond to PTTH. From these results, it was supposed that the lowered JH biosynthesis of the CA during the first day of last instar in rt mutants was related to decreased ecdysteroidogenesis in the prothoracic glands during the second day, thus playing a role in leading to precocious metamorphosis.     

Identification, characterization, and developmental regulation of two storage proteins in the bamboo borer Omphisa fuscidentalis

Two insect storage proteins, OfSP1 (75 kDa) and OfSP2 (72 kDa), were purified using three different chromatographies from the hemolymph of Omphisa fuscidentalis larvae during diapause, and their genes were cloned. OfSP1 and OfSP2 concentrations in the hemolymph were high during diapause. During pupation, OfSP1 levels decreased in the male hemolymph and disappeared from the female hemolymph. OfSP1 and OfSP2 mRNA levels in the fat bodies were low during the third instar, but increased greatly during the fourth and fifth larval instars. During diapause, mRNA expression continued at a lower level than during the feeding period. The injection of 20-hydroxyecdysone (20E) into diapausing larvae caused an increase in OfSP1 and OfSP2 mRNA levels 2-3 days post-injection, followed by a decrease in expression until pupation, which occurred 2-4 days thereafter. When larvae were treated with juvenile-hormone analog (JHA), OfSP1 and OfSP2 mRNA levels gradually decreased until the onset of pupation. In Omphisa, OfSP1 and OfSP2 proteins are produced and released by the larval fat bodies in the fourth and fifth-instar larvae, and the proteins accumulate in the hemolymph until the insects enter diapause. OfSP1 may be reabsorbed by the fat bodies at the end of diapause for subsequent re-use during pupation.