The two duplicated insecticyanin genes, ins-a and ins-b are differentially expressed in the tobacco hornworm, Manduca sexta.

Two gene-specific probes were generated from the unique sequences in the 3' non-coding regions of the two insecticyanin genes, ins-a and ins-b to study the developmental expression of these genes in Manduca sexta. Both genes were initially transcribed in the freshly hatched first instar larvae and then expressed in the epidermis and to a lesser degree in the fat body during every larval feeding stage. In the epidermis of the 4th and 5th instar larvae, both mRNAs appeared shortly before ecdysis and accumulated to maximal levels within a day. As the larval epidermis became pupally committed on day 3 of the 5th (final) instar, INS-a mRNA quickly decreased, whereas INS-b mRNA showed a second peak of accumulation. In the fat body, both genes showed a similar expression pattern within the 4th instar to that of the epidermis except that levels were lower and ins-b mRNA dominated. In the final instar, only ins-b mRNA was present in significant amount. These findings not only reveal that the two duplicated insecticyanin genes have diverged in their expression pattern but also demonstrate, for the first time, that fat body also expresses insecticyanin genes.     

Cloning and structural characterization of juvenile hormone diol kinase in Spodoptera litura

Juvenile hormone (JH) is one of the key insect hormones that regulate metamorphosis. Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH metabolism and catalyzes JH diol to form a polar end product, JH diol phosphate that has no JH activity. In this study, a JHDK complementary DNA (cDNA) was cloned from Spodoptera litura and the structure and expression of the gene was characterized. The cDNA was 714 base pairs in length and encoded a protein of 183 amino acids with a molecular mass of 21 kDa and an isoelectric point of 4.55. Based on the structure, three putative calcium binding motifs and guanosine triphosphate‐binding motifs were predicted in the protein. Modeling of the 3‐D structure showed that the protein consisted of eight α‐helixes linked with loops, with no β‐sheets. The gene was expressed in the epidermis, fat body and midgut of fifth and sixth instar larvae. The expression level in the epidermis was lower than in the fat body and midgut. The gene was expressed at higher levels at the early stages than in the later stages of fifth and sixth instar midgut and fat body. The results suggest that this gene may be involved in the regulation of the JH titer in larvae of S. litura.     

Requirements for molting of the crochet epidermis of the tobacco hornworm larva in vivo and in vitro

Summary In the tobacco hornworm,Manduca sexta, the epidermis which underlies the larval crochets is the first tissue to become independent of the prothoracic glands (PG) in a larval molt. In each successive larval molt, crochet forming cells increase in size, form hooks at their distal ends and, finally, secrete cuticle. This paper examines the endocrine requirements for competence to molt and describes parallel cultures in vivo and in vitro to define the hormonal control of crochet molting. When implanted into a fourth instar host larva prior to initiation of the last larval molt, competent crochet epidermis molted, forming crochets synchronously with its host. In the fourth instar, competence to form crochets is attained slowly during the first two days following ecdysis from the third instar. During the feeding phase of the fifth (last) instar, the crochet epidermis remains competent to molt (to form an extra sixth instar set of crochets) until the larva attains a weight of about 4.5 gm. Then, concurrent with the decline in the titer of juvenile hormone (JH) in the hemolymph, competence to form crochets declines. A similar loss of competence did not occur when fourth instar crochet epidermis was exposed to a declining JH titer by culture in either fourth instar isolated abdomens for 72 h or in fifth instar host larvae between 4 and 7 gm. Responses of crochet epidermis cultured in vitro also were examined. Competent fourth instar crochet epidermis formed crochets following 3–6 h exposure to ecdysone in vitro. Six ×10^–7M -ecdysone was required for 50% response, whereas a 10–50-fold higher concentration of -ecdysone was necessary. Although formation of morphologically complete crochets in vitro proceeded with similar time course to that in situ, no molt-induced growth occurred in vitro. When crochet epidermis was exposed to ecdysone in vitro immediately after explantation, exogenous JH was not required for molting. But when tissue was first cultured for 72 h without hormones, subsequent molting in vitro could not be elicited, although molting still could occur when the tissue subsequently was implanted into a fourth instar host. Exposure to corpora allata or to JH during the 72 h of culture in vivo partially prevented the loss in capacity to respond to ecdysone in vitro, suggesting that JH may be one factor involved directly or indirectly in maintenance of tissue responsiveness.Preliminary presentation of some of this work given at the December, 1973 Meeting of the American Society of Zoologists (Fain and Riddiford, 1973)     

Upregulation of the immune protein gene hemolin in the epidermis during the wandering larval stage of the Indian meal moth, Plodia interpunctella

Expression of hemolin, which generates an immune protein, was up-regulated in wandering fifth instar larval stage of Plodia interpunctella. The mRNA level peaked in the middle of the wandering stage. Major expression was in the epidermis, rather than in the fat body or gut. To test a possible ecdysteroid effect on hemolin induction we treated with RH-5992, an ecdysteroid agonist, and KK-42, which inhibits ecdysteroid biosynthesis in both feeding and wandering fifth instar larvae. When feeding larvae were treated with RH-5992 the hemolin mRNA level was increased. When wandering larvae were treated with KK-42 its level was reduced. In addition, when KK-42-treated larvae were subsequently treated with RH-5992 the hemolin mRNA level was recovered. These results strongly suggest that ecdysteroid up-regulates the expression of hemolin mRNA. Hormonal and bacterial effects on hemolin induction were further analyzed at the tissue level. Major induction of hemolin mRNA was detected following both RH-5992 treatment and bacterial injection in the epidermis of both feeding and wandering larvae. Minor induction of hemolin was detected in the fat body following a bacterial injection, but not RH-5992 treatment. We infer that in P. interpunctella larvae, the epidermis is the major tissue for hemolin induction in na?ve insects and in insects manipulated with bacterial and hormonal treatments.     

Tissue-specific regulation of juvenile hormone esterase gene expression by 20-hydroxyecdysone and juvenile hormone in Bombyx mori

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.     

Hydrolysis of juvenile hormone in diapausing and non-diapausing larvae of the southwestern corn borer,Diatraea grandiosella

Summary The role of juvenile hormone (JH) esterases in relation to the diapause state of the southwestern corn borer,Diatraea grandiosella, was examined. The facultative larval diapause of this insect is dependent upon the presence of JH. Plasma, fat body, midgut, and body wall extracts metabolized [³H]JH I and [³H]JH III to JH-acid in vitro. JH-diol, JH-acid-diol, or conjugated polar metabolites were not detected. A longer half life of [³H]JH I was found in vitro in the plasma of diapausing larvae than in that of non-diapausing larvae. Although JH hydrolytic activity was relatively low in the plasma of pre-diapausing and diapausing larvae, systematic changes were observed suggesting that JH esterases may be involved in regulating the JH titer during this period. The JH hydrolytic activity found in the plasma of diapausing larvae was 3 to 5 times lower than that found in the plasma of mid-last instar non-diapausing larvae. Gel filtration profiles obtained from the plasma of diapausing and non-diapausing larvae suggested that JH esterases and -naphthyl-acetate esterases are different enzymes. Multiple overlapping peaks of JH hydrolytic activity with an apparent molecular weight range of 43,000 to 75,000 were detected, whereas 2 separate peaks of -naphthyl-acetate hydrolytic activity (apparent mol. wt. ca. 54,000, and 120,000) were detected. Gel filtration of supernatants of fat body indicated that JH was hydrolyzed at a lower rate by the fat body of pre-diapausing larvae than by that of non-diapausing larvae.     

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED₅₀=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED₅₀ = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Juvenile hormone modulates 20-hydroxyecdysone-inducible ecdysone receptor and ultraspiracle gene expression in the tobacco hornworm, Manduca sexta

 Insect molting and metamorphosis are orchestrated by ecdysteroids with juvenile hormone (JH) preventing the actions of ecdysteroids necessary for metamorphosis. During the molt and metamorphosis of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, the isoforms involved in the ecdysone receptor (EcR)/Ultraspiracle (USP) complex change with the most dramatic switch being the loss of USP-1 and the appearance of USP-2 during the larval and pupal molts. We show here that this switch in USP isoforms is mediated by high 20-hydroxyecdysone (20E) and that the presence of JH is necessary for the down-regulation of USP-1 mRNA. The decrease of USP-1 mRNA in day 2 fourth instar larval epidermis in vitro required exposure to a high concentration (10^–5 M) of 20E equivalent to the peak ecdysteroid concentration in vivo, whereas the increase of USP-2 mRNA occurred at lower concentrations (effective concentrations, EC₅₀=6.3×10^–7 M). During the pupal molt of allatectomized larvae which lack JH, USP-2 mRNA increased normally with the increasing ecdysteroid titer, whereas USP-1 mRNA remained high until pupation. When day 2 fifth instar larval epidermis was exposed to 500 ng/ml 20E in the absence of JH to cause pupal commitment of the cells by 24 h, USP-1 RNA remained at its high preculture level for 12 h, then increased two- to threefold by 24 h. The increase was prevented by the presence of 1 μg/ml JH I which also prevents the pupal commitment of the cells. By contrast, USP-2 mRNA increased steadily with the same EC₅₀ as in fourth stage epidermis, irrespective of the presence or absence of JH. Under the same conditions, mRNAs for both EcR-B1 and EcR-A isoforms were up-regulated by 20E, each in its own time-dependent manner, similar to that seen in vivo. These initial mRNA increases were unaffected by the presence of JH I, but those seen after 12 h exposure to 20E were prevented by JH, indicating a difference in response between larvally and pupally committed cells. The presence of JH which maintained larval commitment of the cells also prolonged the half-life of the EcR proteins in these cells. These results indicate that both EcR and USP RNAs are regulated by 20E and can be modulated by JH in a complex manner with only that of USP-2 apparently unaffected. Received: 16 July 1998 / Accepted: 5 August 1998     

The homeodomain protein PBX participates in JH-related suppressive regulation on the expression of major plasma protein genes in the silkworm, Bombyx mori

In the silkworm, Bombyx mori, major plasma proteins referred to as 30K proteins are the most abundant proteins in the hemolymph of final (fifth) instar larvae. Surgical extirpation of corpora allata, the source of a juvenile hormone (JH), causes rapid accumulation of 30K proteins in the hemolymph of fourth instar larvae. The 30K protein 6G1 (30K6G1) gene was repressed in primary cultured fat body cells treated with a JH analog (JHA), methoprene. To identify the JH response element present in the promoter region of the 30K6G1 gene, we performed transfection analyses of the 5'-deletion mutants of the 30K6G1 gene using primary cultured fat body cells, gel retardation assays and in vivo footprinting analysis. The results from those analyses revealed that a JH response element exists in the sequence between positions -147 and -140. When the promoter construct mutated at positions -143, -142, and -141 was transfected to fat body primary cultured cells, the suppression effect on the reporter gene expression caused by JHA was reduced. Gel retardation assay using specific antibody revealed that a PBX protein binds to the JH response element. Northern blot analysis revealed that the gene expression of Bombyx PBX is enhanced in the fat body cells by JHA treatment. These results indicate that PBX proteins are involved in the JH signaling pathway and play an important role in suppressing 30K protein gene expression in the fat body of B. mori.     

Nonshivering thermogenesis and cold resistance during seasonal acclimatization in the Djungarian hamster

Summary The absence of juvenile hormone (JH) at the time of head capsule slippage during the molt to the fifth (final) instar of the tobacco hornworm was found to cause ommochrome (primarily dihydroxanthommatin) synthesis in the epidermis during the first two days after ecdysis. Then synthesis decreased until its transient reappearance during the wandering stage. Either JH-I (ED₅₀=8x10^–4 g) or methoprene (ED₅₀=1.4x10^–2 g) applied at this critical time during the molt prevented the first synthesis. A comparison of developmental profiles of tryptophan and its metabolites, kynurenine and 3-hydroxykynurenine, in normal and allatectomized wild type larvae showed that JH at this critical time prevented both the conversion of kynurenine to 3-hydroxykynurenine and 3-hydroxykynurenine to ommochromes. A similar study in normal and methoprene-treatedblack mutant larvae showed that only the latter conversion was inhibited by JH. The accumulation of 3-hydroxykynurenine in the epidermis of the JH-treatedblack mutant is thought to be due to the altered tryptophan metabolism in these mutants in previous instars due to lower JH levels. Neither starvation of theblack mutant nor injection of 3-hydroxykynurenine significantly affected ommochrome synthesis by the epidermis. Preliminary studies of the enzymes involved showed that JH at the critical period suppressed the later activity and/or production of kynurenine 3-hydroxylase in the wild type larva, but had little effect on the particulate ommochrome synthetase activity of the epidermis.Abbreviations CA corpora allata - JH juvenile hormone - PTTH prothoracicotropic hormone     

Juvenile hormone changes associated with diapause induction, maintenance, and termination in the beet webworm, Loxostege sticticalis (Lepidoptera: Pyralidae)

At 22°C and under a long-day photoperiod of L:D 16:8, all the last fifth instar Loxostege sticticalis larvae undergo prepupal stage and pupate without diapause. Under a short-day photoperiod of L:D 12:12, in contrast, they all enter diapause with approximately 36 days diapause maintenance and then terminate diapause spontaneously, although only 44% of the larvae terminated diapause successfully. Changes in hemolymph juvenile hormone (JH I) titers of diapause-destined larvae across diapause induction, maintenance and termination were examined using HPLC, and were compared with those of non-diapause-destined larvae from the fifth instar through pupation. JH I titer of the earliest fifth instar diapause-destined larvae remained at a high level with a peak of 220.4 ng/ml, though it decreased continuously to a minimum of 69.0 ng/ml on day 5 in the fifth instar when the larvae stopped feeding to enter diapause. During the diapause maintenance, JH I titer of the mature larvae increased significantly and maintained a high level until day 31 in prepupae. JH I titer declined and fluctuated at low level from 5 days before pupation. In contrast, JH I titer of both the fifth instar non-diapause-destined larvae and prepupae remained and fluctuated at low level consistently, as well as decreased before pupation. These results indicate that diapause induction and maintenance in this species might be a consequence of high JH, whereas diapause termination can be attributed to low JH titer, which was in agreement with the hormonal regulation observed in many other larval-diapausing insects.