Immunoreactive intensity of FXPRL amide neuropeptides in response to environmental conditions in the silkworm, Bombyx mori.

In the silkworm Bombyx mori, the diapause hormone-pheromone biosynthesis activating neuropeptide gene, DH-PBAN, is a neuropeptide gene that encodes a polypeptide precursor consisting in five Phe-X-Pro-Arg-Leu-NH(2) (FXPRL) amide (FXPRLa) neuropeptides; DH (diapause hormone), PBAN (pheromone-biosynthesis-activating neuropeptide) and α-, ß- and γ-SGNPs (subesophageal ganglion neuropeptides). These neuropeptides are synthesized in DH-PBAN-producing neurosecretory cells contained within three neuromeres, four mandibular cells, six maxillary cells, two labial cells (SLb) and four lateral cells of the subesophageal ganglion. DH is solely responsible, among the FXPRLa peptide family, for embryonic diapause. Functional differentiation has been previously suggested to occur at each neuromere, with the SLb cells releasing DH through brain innervation in order to induce embryonic diapause. We have investigated the immunoreactive intensity of DH in the SLb when thermal (25°C or 15°C) and light (continuous illumination or darkness) conditions are altered and following brain surgery that induces diapause or non-diapause eggs in the progeny. We have also examined the immunoreactivity of the other FXPRLa peptides by using anti-ß-SGNP and anti-PBAN antibodies. Pupal SLb somata immunoreactivities seem to be affected by both thermal and light conditions during embryogenesis. Thus, we have been able to identify a close correlation between the immunoreactive intensity of neuropeptides and environmental conditions relating to the determination of embryonic diapause in B. mori.

Changes in the expression of soluble and integral-membrane trehalases in the midgut during metamorphosis in Bombyx mori.

To elucidate the relationship between soluble trehalase (Treh1) and integral-membrane trehalase (Treh2) in the Bombyx mori midgut, expression profiles for both proteins and mRNAs were examined during metamorphosis by using Western-blotting and quantitative real-time PCR analyses. Two bands of Treh2 (about 74 kDa) were detected in the midgut of 0-day-old 5th (last) instar larvae. Levels of Treh2 decreased as the developing larvae approached spinning (8 days old). In contrast, towards the onset of the spinning stage, Treh1 (68 kDa) was clearly observed, and levels increased until the middle of the pupal stage. Treh2 mRNA expression relative to Bmrp49 mRNA expression was almost constant, although fluctuations were detected. Treh1 mRNA expression relative to Bmrp49 mRNA increased sharply just after spinning. To further examine the expression mechanism of the Treh1 gene in midgut, actively feeding larvae (4 days old) were starved or ligated between the 4th and 5th segments. Injection of a molting hormone into the larval-isolated abdomen led to activation of Treh1, demonstrating that molting hormone acts on the midgut and activates this gene.

ERK/MAPK regulates ecdysteroid and sorbitol metabolism for embryonic diapause termination in the silkworm, Bombyx mori.

The eggs of the silkworm Bombyx mori undergo a state of suspended overt development and diminished metabolism called "diapause" to escape adverse environmental conditions. Termination of Bombyx embryonic diapause requires 2-3 months of low temperature (5 degrees C), but the molecular mechanisms underlying diapause termination are unknown. Diapause termination requires a decrease in the sorbitol concentration, which arrests embryonic development, and the secretion factors from yolk cells that promote embryonic development. In the present study, we report that 20-hydroxyecdysone promoted the development of denuded embryos and that ecdysteroid-phosphate phosphatase (EPPase), which is a key enzyme for active ecdysteroid production, was induced by incubation of diapausing eggs at 5 degrees C. In dechorionated egg cultures, extracellular signal-regulated kinase (ERK), which is activated by incubating diapausing eggs at 5 degrees C, regulated sorbitol-glycogen conversion, ecdysteroid secretion via gene transcription of key enzymes, sorbitol dehydrogenase-2, and EPPase, suggesting that ERK has a key role in diapause termination.

Myocyte enhancer factor 2 (MEF2) is a key modulator of the expression of the prothoracicotropic hormone gene in the silkworm, Bombyx mori.

Prothoracicotropic hormone (PTTH) plays a central role in controlling molting, metamorphosis, and diapause termination in insects by stimulating the prothoracic glands to synthesize and release the molting hormone, ecdysone. Using Autographa californica nucleopolyhedrovirus (AcNPV)-mediated transient gene transfer into the central nervous sytem (CNS) of the silkworm, Bombyx mori, we identified two cis-regulatory elements that participate in the decision and the enhancement of PTTH gene expression in PTTH-producing neurosecretory cells (PTPCs). The cis-element mediating the enhancement of PTTH gene expression binds the transcription factor Bombyx myocyte enhancer factor 2 (BmMEF2). The BmMEF2 gene was expressed in various tissues including the CNS. In brain, the BmMEF2 gene was expressed at elevated levels in two types of lateral neurosecretory cells, namely PTPCs and corazonin-like immunoreactive lateral neurosecretory cells. Overexpression of BmMEF2 cDNA caused an increase in the transcription of PTTH. Therefore, BmMEF2 appears to be particularly important in the brain where it is responsible for the differentiation of lateral neurosecretory cells, including the enhancement of PTTH gene expression. This is the first report to identify a target gene of MEF2 in the invertebrate nervous system.

Establishment of a sandwich ELISA system to detect diapause hormone, and developmental profile of hormone levels in egg and subesophageal ganglion of the silkworm, Bombyx mori.

In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.

Temperature-dependent activation of ERK/MAPK in yolk cells and its role in embryonic diapause termination in the silkworm Bombyx mori.

The silkworm Bombyx mori requires 2-3 months of low temperature (5 degrees C) to terminate embryonic diapause. The molecular mechanisms, however, are unknown. Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) is temperature-dependently activated in the yolk cells of diapausing eggs after 45 days at 5 degrees C, coincident with the acquisition of developmental competence of the embryos at 25 degrees C. Yolk cell granulation and dissociation also begin in diapause eggs incubated at 5 degrees C for 45 days. We used dechorionated egg culture as a model system of diapause termination and observed that both yolk cell dissociation and embryonic development are inhibited by MAPK-ERK kinase (MEK) inhibitor U0126. Therefore, we suggest that ERK in yolk cells has a role in regulating changes in yolk morphology and termination of embryonic diapause in B. mori.

The profiles of mRNA levels for BHR39, a Bombyx homolog of Drosophila hormone receptor 39, and Bombyx FTZ-F1 in the course of embryonic development and diapause.

In the silkworm, Bombyx mori, the 5'-upstream region of the sorbitol dehydrogenase (SDH) gene contains nucleotide sequences that might be recognized and bound by steroid hormone receptors such as Drosophila hormone receptor 39 (DHR39) and Fushi tarazu transcription factor 1 (FTZ-F1). As a first trial to find out regulator(s) involved in gene expression for SDH in Bombyx eggs, we have cloned a cDNA encoding a Bombyx homolog (BHR39) of DHR39, and examined levels of mRNA expressions for BHR39 and Bombyx FTZ-F1 (BmFTZ-F1) during the entire embryonic development and diapause. During embryonic development of non-diapause eggs, mRNA expression for BmFTZ-F1, but not for BHR39, was shown to occur in parallel with that for SDH, although such a parallelism was not found at the termination of diapause, when SDH mRNA was expressed.

Inhibitory Action of an Imidazole Compound on Ecdysone Synthesis in Prothoracic Glands of the Silkworm, Bombyx mori: (imidazole compound/ecdysteroids/prothoracic gland/precocious pupation/Bombyx mori).

The precocious pupation was induced either by allatectomy at the time of third ecdysis or by topical application of an imidazole compound (KK-42; 1-benzyl-5-[(E)-2, 6-dimethyl-1, 5-heptadienyl] imidazole) to the fourth (penultimate) instar larvae of the silkworm, Bombyx mori. However, the critical period for KK-42 treatment in induction of precocious pupation was longer than that for allatectomy. The effects of KK-42 depended on the doses applied and a half-maximum dose was estimated to be approx. 10 µg/larva. KK-42 suppressed the increase in hemolymph ecdysteroid titres leading to larval ecdysis in controls. Ecdysteroid levels remained at low levels for about 6 days after the treatment, followed by an increase toward precocious pupation. When the prothoracic glands from the mature fifth instar larvac were incubated in vitro in Grace's medium containing various concentrations of KK-42, secretion of ecdysone into the medium was suppressed depending upon the doses of KK-42 added and a half-inhibition concentration was estimated to be approx. 1 nM. Thus, KK-42 was shown to be an inhibitory agent to ecdysteroid secretion in silkworm larvae.

Physiological differentiation of DH-PBAN-producing neurosecretory cells in the silkworm embryo.

Embryonic diapause of the silkworm, Bombyx mori, is induced by a neuropeptide hormone, the diapause hormone (DH), which is secreted from a limited number of neurosecretory cells in the subesophageal ganglion (SG) at the maternal generation. We examined the developmental fate of the hormone-producing cell (DH-pheromone biosynthesis activating neuropeptide [PBAN]-producing cell) in the embryonic stage at the level of gene expression and cell biology. The DH-PBAN gene expression started at the histogenesis stage and gradually increased toward hatching. DH is an amidated peptide belonging to FXPRLamide family. The immunoreactive somata against anti FXPRLamide antiserum were found in the SG from blastokinesis. Immunoreactive neural processes with varicosites were also found on the corpus cardiacum and the corpus allatum. The implantation of a part of a developing embryo including the SG into the pupae with the SG removed induced diapause eggs in the progeny. These results were obtained from eggs incubated under diapause-averting conditions as well as diapause-inducing conditions. Thus, a neurosecretory system responsible for biosynthesis of FXPRLamide neuropeptides is established as early as histogenesis, although the system to regulate the secretion of neuropeptide hormones has not been fully formed by that time.

Expression of the Gene Encoding Diapause Hormone and Pheromone Biosynthesis Activating Neuropeptide During Post-embryonic Development in the Silkworm, Bombyx mori.

Embryonic diapause and sex pheromone biosynthesis in the silkworm, Bombyx mori, are induced by the diapause hormone (DH) and the pheromone biosynthesis activating neuropeptide (PBAN), respectively. These are produced in the suboesophageal ganglion from a common polyprotein precursor (DH-PBAN precursor) encoded in a single gene (DH-PBAN gene). Using RT-PCR and Southern hybridization methods, we quantitatively measured DH-PBAN mRNA content throughout the post-embryonic development. The results show that DH-PBAN gene not only expresses during pupal stage, but also during larval and adult stages. The high level of expression during pupal stage suggests that DH and PBAN are respectively synthesized.

Cloning of cDNA encoding a Bombyx mori homolog of human oxidation resistance 1 (OXR1) protein from diapause eggs, and analyses of its expression and function.

To better understand the molecular mechanisms of diapause initiation, we used the sensitive cDNA subtraction (selective amplification via biotin- and restriction-mediated enrichment) method and isolated a novel gene expressed abundantly in diapause eggs of the silkworm, Bombyx mori, which encodes a homolog of the human oxidation resistance 1 (OXR1) protein. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analyses confirmed that BmOXR1 mRNA and its 140-kDa protein were differentially expressed in diapause eggs compared to non-diapause eggs. OXR1 double-stranded RNA (dsRNA) was injected into diapause-destined eggs before the cellular blastoderm stage, and 4days later, when untreated eggs reached the diapause stage, the OXR1 protein disappeared; however, these eggs remained in diapause, suggesting that BmOXR1 is not essential for diapause initiation and/or maintenance. To further investigate the in vivo function of BmOXR1 apart from its role in diapause, we overexpressed BmOXR1 in Drosophila melanogaster. The fruit fly male adult life-span was significantly extended in the 50%-survival time when adults were reared on diets both with and without H2O2 solution under 25°C incubation. These results suggest that BmOXR1 functions in D. melanogaster via a possible antioxidant effect. As BmOXR1 was expressed mainly in the nuclei of D. melanogaster cells, the mechanism underlying its antioxidation effect appears to be different from that in humans where it is expressed mainly in the mitochondria. Taken together, these results suggest that BmOXR1 might serve as an antioxidant regulator during the early diapause stage.

Heat shock factor binds to heat shock elements upstream of heat shock protein 70a and Samui genes to confer transcriptional activity in Bombyx mori diapause eggs exposed to 5°C.

To understand the molecular mechanisms of how 5°C-incubation activates mRNA expression of Hsp70a and Samui genes in Bombyx mori diapause eggs, we first searched the 5'-upstream regions of the Hsp70a and Samui genes for heat shock elements (HSEs) and found two regions [Hsp70aHSE-1 (-95 to -58) and -2 (-145 to -121), and SamuiHSE-1 (-84 to -55) and -2 (-304 to -290)] corresponding to HSEs (repeats of nGAAn and/or nTTCn). We cloned four cDNAs encoding heat shock factor (HSF)-a2 (627 amino acids), -b (685 aa), -c (682 aa) and -d (705 aa), which were produced by alternative splicing. When we exposed diapause eggs to 5°C beginning at 2 day post-oviposition to break diapause, HSFd mRNA only increased after chilling for 6-8 days, a pattern very similar to those of Hsp70a and Samui mRNAs. To examine further whether HSFd binds to the respective HSEs, we carried out a gel shift assay using HSFd protein expressed in a cell-free system and the isolated HSEs; migration of the respective digoxigenin(DIG)-labeled HSE-1 and -2 of Hsp70a and Samui was retarded by addition of HSFd; the retarded bands disappeared after addition of the corresponding unlabeled HSE-1 and -2 as competitors, but were not affected by addition of the respective mutated unlabeled HSE-1 and -2. These results indicated that HSFd protein binds to the respective HSEs and may activate mRNA expression of Hsp70a and Samui genes upon exposure of diapause eggs to 5°C.

Trehalase-2 protein contributes to trehalase activity enhanced by diapause hormone in developing ovaries of the silkworm, Bombyx mori.

Diapause hormone (DH) targets developing ovaries in female pupae to induce embryonic diapause immediately after completion of mesoderm segregation of the silkworm, Bombyx mori. At the same time, DH enhances trehalase activity on the oolemma, which leads to higher concentrations of glycogen in oocytes through the stimulated incorporation of hemolymph trehalose. In B. mori, the treh-1 and -2 genes encoding soluble trehalase (68 kDa) and integral-membrane trehalase (74kDa) have been isolated. DH stimulates mRNA expression of both of these genes. In this study, we aimed to clarify whether ovarian trehalase originates from Treh-1 or Treh-2. Western blotting of the developing ovaries showed positive bands in the membrane-bound fraction, containing trehalase activity, only with antibodies against Treh-1&2 and Treh-2, but not Treh-1, irrespective of nondiapause or diapause egg-producers. The intensities of the positively stained 74 kDa bands were increased approximately 4-fold in ovaries from pupae with intact subesophageal ganglion (SG, a unique DH-biosynthesizing organ), and from pupae that were injected with DH at the middle pupal stage after their SGs were removed on the day of pupation. Furthermore, quantitative real-time PCR data showed that in developing ovaries, copy number of treh-2 mRNA per one copy of rp49 mRNA was approximately 1000-fold higher than that of treh-1 mRNA. These results demonstrate that trehalase activities enhanced by DH originate mainly from treh-2 protein regulated at the transcriptional level.

Cloning of cDNAs encoding sorbitol dehydrogenase-2a and b, enzymatic characterization, and up-regulated expression of the genes in Bombyx mori diapause eggs exposed to 5 °C.

We previously cloned a cDNA for sorbitol dehydrogenase (SDH1) from Bombyx mori. In the present study we cloned two additional cDNAs encoding SDHs (designated as SDH2a and SDH2b). The amino acid sequences of SDH2ab were almost the same and had higher similarity to the SDHs of other organisms than to B. mori SDH1. The SDH2ab and SDH1genes were located in tandem within about 40 kbp on chromosome 21. SDH2ab mRNAs increased after exposing diapause eggs to 5 °C for 40 days, beginning at 2 days post-oviposition, to break diapause. However, they were at very low levels in diapausing eggs incubated at 25 °C continuously from oviposition. These changes in expression pattern of SDH2ab mRNA were almost the same as that of SDH1 mRNA. To understand whether SDH1 and SDH2 were responsible for the SDH activity seen in diapause eggs exposed to 5 °C for more than 60 days, we expressed a His-tagged SDH2a fusion protein in Escherichia coli and examined its enzymatic parameters. The maximum activity of SDH2a observed at pH 8.4∼9.0, and the Km value for sorbitol was 12.6 mM, similar to the kinetic properties of other SDHs. Due to the significantly higher similarity between SDH2a and b, they were thought to have similar kinetic properties. Therefore, we purified SDH from B. mori diapause-terminated eggs exposed to 5 °C for 300 days which showed higher SDH activity using two-step affinity chromatography. The highly purified SDH showed a higher Km value (125 mM) for sorbitol, being similar to the value (136 mM) determined previously from Eadie-Hofstee plots using egg crude extract as an enzyme source; additionally, the plots showed one slope indicating one Km value. Moreover, in silico analysis indicated that no SDH genes other than SDH1 and 2ab are present in B. mori genomic DNA. These results suggest that SDH1 activity may be responsible for the majority of the increased SDH activity seen in diapause eggs after acclimation to 5 °C rather than SDH2ab. Further, the relative sequence divergence among these genes is consistent with the idea/hypothesis that the original SDH gene was first duplicated into SDH1 and SDH2, and then SDH2 was duplicated into the SDH2a and SDH2b genes.

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs.

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 degrees C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 degrees C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 degrees C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 degrees C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 degrees C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.

Disappearance of chorion proteins from Bombyx mori eggs treated with HCl solution to prevent diapause.

Bombyx mori eggs enter diapause immediately after completion of mesoderm segregation. HCl treatment of approximately 24-hour-old eggs (germband formation stage) is well known to be the most effective procedure to prevent entry into diapause, although the molecular mechanism remains unclear. In this study, we examined the protein composition of diapausing and nondiapausing eggs after various HCl treatments known to prevent or break diapause and found that proteins of approximately 11 and 8 kDa disappeared immediately after HCl treatment. Partial amino acid sequences of these proteins indicated that they were members of the chorion class A protein L12 family synthesized in follicle cells. Under the hypothesis that the chorion provides a barrier to oxygen, dechorionation of diapausing eggs induces resumption of embryonic development. Hence, to test this and other hypotheses about the function of these proteins, we used 20% SDS-PAGE with Coomassie Brilliant Blue staining to trace their disappearance from embryos and eggshells after treatment with HCl under different conditions and on polyvoltine, univoltine, and bivoltine silkworm races. Even when 10-day-old diapausing eggs were treated with HCl, which did not break diapause, the 11 and 8 kDa proteins disappeared. Our results suggest that disappearance of these proteins is not directly associated with preventing entry into or breaking a diapause state. Nevertheless, our results cannot completely rule out the possibility that the 11 and 8 kDa proteins function to block permeability of O(2) during the period when HCl treatment is physiologically effective to prevent diapause so that after the diapause system is established within the egg, even removing the 11 and 8 kDa proteins may not affect to prevent diapause. We also discuss the role of these proteins in choriogenesis.

Glycerol kinase activity and glycerol kinase-3 gene are up-regulated by acclimation to 5 degrees C in diapause eggs of the silkworm, Bombyx mori.

With initiation of diapause, glycogen is converted into sorbitol and glycerol in eggs of the silkworm Bombyx mori. At diapause termination promoted by incubation at 5 degrees C, sorbitol and glycerol are utilized. Although sorbitol utilization is triggered by NAD-sorbitol dehydrogenase induced by acclimation to 5 degrees C, the initial enzyme utilizing glycerol remains unclear. In this study, we detected glycerol kinase activity in diapause-terminated eggs and then characterized its properties; maximal activity was seen at pH 8.5-9.0, and Km values for glycerol and ATP were 0.32 and 0.095 mM, respectively. In diapause eggs continuously kept at 25 degrees C, the activity was almost negligible. However, activity was seen after chilling for 60 days and thereafter increased when the eggs were exposed to 5 degrees C after 2 days post-oviposition, indicating that glycerol kinase is a rate-limiting enzyme in glycerol utilization. We then cloned cDNAs encoding glycerol kinase-1, -2 and -3 from B. mori. Only gene expression of glycerol kinase-3 was up-regulated in diapause eggs exposed to 5 degrees C, indicating that glycerol kinase activity is regulated via transcription of the glycerol kinase-3 gene.

Novel gene encoding precursor protein consisting of possible several neuropeptides expressed in brain and frontal ganglion of the silkworm, Bombyx mori.

A novel gene (BmK5) expressed in the central nervous system of the silkworm, Bombyx mori, was isolated using a cDNA subtraction method. BmK5 was first cloned as a candidate regulator of diapause hormone release from subesophageal ganglion via corpus cardiacum-corpus allatum into the hemolymph; however, subsequent analyses revealed that the gene expression patterns in brain-subesophageal ganglion complexes did not differ between diapause and nondiapause egg producers. The deduced amino acid sequence showed the characteristics of secretory protein precursor or nuclear localization protein. Immunohistochemical experiments with an anti-BmK5 antibody revealed that BmK5 precursor protein exists in the cytoplasm of specific cells of brain and frontal ganglion, but not in the nuclei. In addition, a peptide (GSGTKVGGAGAATKVVTKSGS-NH(2)) possibly processed from the BmK5 precursor protein was immunohistochemically detected in the axons connecting the anti-BmK5 antibody-positive cells to the neurohemal organ, corpus cardiacum-corpus allatum. These results suggest that BmK5 encodes a precursor of the novel neurosecretory protein and that several mature peptides are released into the hemolymph via the corpus cardiacum-corpus allatum, although the functions of these peptides are yet unclear.

G protein-coupled receptor for diapause hormone, an inducer of Bombyx embryonic diapause.

Bombyx diapause hormone was the first chemical substance identified as a maternal control factor that arrests offspring development. However, the molecular mechanisms by which the hormone transduces the signal to the oocyte that induces embryonic diapause immediately after mesoderm segmentation are not fully understood. Here, we describe a cDNA for a G protein-coupled diapause hormone receptor with seven transmembrane domains. Its amino-acid sequence shows a high level of similarity to the receptors of mammalian neuromedin U and insect regulatory peptide, an FXPRL-amide C-terminus. When expressed in a Xenopus oocyte system, the receptor exhibited the highest affinity (EC(50), approximately 70nM) for diapause hormone, when compared with other Bombyx FXPR/KL-amide peptides. Diapause hormone without amidation at the C-terminus, which never induces embryonic diapause in vivo, had no effect in this heterologous expression system. The mRNA is expressed in the ovaries during Bombyx pupal-adult development. These results strongly indicate that the cDNA encodes the diapause hormone receptor.