Activity of NAD-sorbitol dehydrogenase is caused by biosynthesis of enzyme protein in diapause and non-diapause eggs of the silkworm,Bombyx mori

• 1.1. Using SDS-PAGE and immunoblotting analyses with anti-sorbitol dehydeogenase (EC 1.1.1.14, SDH) serum, changes in amount of SDH protein were examined in diapause and non-diapause eggs of the silkworm, Bombyx mori.
• 2.2. When diapause eggs were exposed to 5°C from 2 days after oviposition to break the diapause gradually, SDH protein appeared after 50-day chilling, and then the amount increased along with chilling period. This changing pattern paralleled that in SDH activity.
• 3.3. In diapause eggs treated with HCl after chilling at 5°C for 30 days to break the diapause quickly, and non-diapause eggs, changing patterns in amount of SDH protein also paralleled those in SDH activity.
• 4.4. These results showed that SDH activity was caused by biosynthesis of SDH protein, independent of diapause or non-diapause eggs.
• 5.5. Occurrence of SDH correlates with the three developmental phases: diapause termination, embryonic growth, and larval differentiation. In larva, SDH was mainly localized in the fat-body.

     

Studies on the embryonic diapause of thepnd mutant of the silkworm,Bombyx mori

Summary The changing pattern in free amino acids following the embryonic development in the non-diapause and diapause eggs of thepnd mutant of theBombyx silkworm was studied. In the diapause eggs, heterozygous for thepnd gene, the levels of most of the amino acids increased concomitantly with the substantial decrease in oxygen consumption. Among the amino acids, alanine was the only amino acid that showed a large accumulation. The accumulation could be induced experimentally in the non-diapause eggs, homozygous for thepnd gene, by reducing the oxygen supply. In contrast, it was prevented in the diapause eggs by increasing the oxygen supply. From these results, it is suggested that the alanine accumulation is the consequence of anaerobic metabolism in the eggs during diapause. The possible significance of the alanine accumulation is discussed in relation to the anaerobic carbohydrate metabolism associated with the embryonic diapause in thepnd mutant.     

Molecular characterization and functional analyses of a diapause hormone receptor-like gene in parthenogenetic Artemia

In arthropods, mature females under certain conditions produce and release encysted gastrula embryos that enter diapause, a state of obligate dormancy. The process is presumably regulated by diapause hormone (DH) and diapause hormone receptor (DHR) that were identified in the silkworm, Bombyx mori and other insects. However, the molecular structure and function of DHR in crustaceans remains unknown. Here, a DHR-like gene from parthenogenetic Artemia (Ar-DHR) was isolated and sequenced. The cDNA sequence consists of 1410 bp with a 1260-bp open reading frame encoding a protein consisting of 420 amino acid residues. The results of real-time PCR (qRT-PCR) and Western blot analysis showed that the mRNA and protein of Ar-DHR were mainly expressed at the diapause stage. Furthermore, we found that Ar-DHR was located on the cell membrane of the pre-diapause cyst but in the cytoplasm of the diapause cyst by analysis of immunofluorescence. In vivo knockdown of Ar-DHR by RNA interference (RNAi) and antiserum neutralization consistently inhibited diapause cysts formation. The results indicated that Ar-DHR plays an important role in the induction and maintenance of embryonic diapause in Artemia. Thus, our findings provide an insight into the regulation of diapause formation in Artemia and the function of Ar-DHR.     

Effects of transgenic overexpression of diapause hormone and diapause hormone receptor genes on non-diapause silkworm

Diapause is a state of developmental arrest that is most often observed in arthropods, especially insects. The domesticated silkworm, Bombyx mori, is a typical insect that enters diapause at an early embryonic stage. Previous studies have revealed that the diapause hormone (DH) signaling molecules, especially the core members DH and DH receptor 1 (DHR1), are crucial for the determination of embryonic diapause in diapause silkworm strains. However, whether they function in non-diapause silkworm strains remains largely unknown. Here, we generated two transgenic lines overexpressing DH or DHR1 genes in a non-diapause silkworm strain, Nistari. Our results showed that developmental expression patterns of DH and DHR1 are quite similar in transgenic silkworms: both genes are highly expressed in the mid to late stages of pupae and are most highly expressed in day-6 pupae but are expressed at very low levels in other developmental stages. Moreover, the overexpression of DH or DHR1 can affect the expression of diapause-related genes but is not sufficient to induce embryonic diapause in their offspring. This study provides new insights into the function of DH and DHR1 in a non-diapause silkworm strain.     

Identification and purification of a Bombyx mori homologue of FTZ-F1.

Extracts from embryos and from posterior and middle silk glands of the silkworm, Bombyx mori contain a sequence specific DNA binding factor termed BmFTZ-F1. The factor binds to the recognition site of FTZ-F1, a positive regulator of the fushi tarazu gene in Drosophila melanogaster. BmFTZ-F1 and FTZ-F1 share the same methylation interference patterns, the same chromatographic behaviors and similar protease digestion profiles. Anti-FTZ-F1 cross reacts with BmFTZ-F1. These results indicate that BmFTZ-F1 is a B. mori homologue of FTZ-F1. The mobility of the factor-DNA complex formed in the silk gland extract changes depending on the developmental stages. Purification of BmFTZF1 to an almost homogeneous state reveals that the factor is a 73 kd protein.     

Studies on embryonic diapause in thepnd mutant of the silkworm,Bombyx mori

Summary Two-dimensional gel electrophoresis has been used to analyse patterns of proteins synthesized in the eggs from theBombyx mutantpnd, whose homozygous embryo never enters diapause owing to a genetic defect. At the middle to late stage of gastrulation the diapause type of the heterozygous embryo, derived from a homozygouspnd female mated to a wild-type male, synthesizes eight proteins which are not detected in the homozygouspnd embryo. To examine the relationship between embryonic diapause and the appearance of the heterozygote-specific proteins, the pattern of proteins synthesized in the heterozygotes of the diapause type was compared with that in heterozygotes which were artificially altered so that they would continue development. Only one of the eight heterozygote-specific proteins was constitutively synthesized according to the embryonic genome, irrespective of their developmental state, whereas appearance of the remaining seven proteins was exclusively dependent on their developmental nature. This finding strongly suggests that the unique protein might result from the expression of thepnd ⁺ gene, and the other proteins might be synthesized along with diapause initiation in the heterozygotes. The possible role of the putativepnd ⁺ gene-specific protein at the onset of embryonic diapause is discussed in relation to the action of the diapause factor, which predetermines embryonic diapause by affecting the developing oocytes.     

Agonist-mediated activation of Bombyx mori diapause hormone receptor signals to extracellular signal-regulated kinases 1 and 2 through Gq-PLC-PKC-dependent cascade

Diapause is a developmental strategy adopted by insects to survive in challenging environments such as the low temperatures of a winter. This unique process is regulated by diapause hormone (DH), which is a neuropeptide hormone that induces egg diapause in Bombyx mori and is involved in terminating pupal diapause in heliothis moths. An G protein-coupled receptor from the silkworm, B. mori, has been identified as a specific cell surface receptor for DH. However, the detailed information on the DH-DHR system and its mechanism(s) involved in the induction of embryonic diapause remains unknown. Here, we combined functional assays with various specific inhibitors to elucidate the DHR-mediated signaling pathways. Upon activation by DH, B. mori DHR is coupled to the Gq protein, leading to a significant increase of intracellular Ca²⁺ and cAMP response element-driven luciferase activity in an UBO-QIC, a specific Gq inhibitor, sensitive manner. B. mori DHR elicited ERK1/2 phosphorylation in a dose- and time-dependent manner in response to DH. This effect was almost completely inhibited by co-incubation with UBO-QIC and was also significantly suppressed by PLC inhibitor U73122, PKC inhibitors Gö6983 and the Ca²⁺ chelator EGTA. Moreover, DHR-induced activation of ERK1/2 was significantly attenuated by treatment with the Gβγ specific inhibitors gallein and M119K and the PI3K specific inhibitor Wortmannin, but not by the Src specific inhibitor PP2. Our data also demonstrates that the EGFR-transactivation pathway is not involved in the DHR-mediated ERK1/2 phosphorylation. Future efforts are needed to clarify the role of the ERK1/2 signaling pathway in the DH-mediated induction of B. mori embryonic diapause.     

Dopamine is a key factor for the induction of egg diapause of the silkworm, Bombyx mori.

The silkworm, Bombyx mori, enters diapause in the early embryonic stage. Embryonic diapause is induced by incubating eggs of the maternal generation at high temperature (diapause type), whereas incubation at low temperature results in non-diapausing progeny (non-diapause type). Measurement of catecholamine concentrations in haemolymph and brain-subesophageal ganglia (Br-SGs) showed that only dopamine concentrations in both tissues are consistently higher in diapause-type than non-diapause-type larvae and pupae. In particular, the difference in dopamine concentrations in both tissues increases around pupal ecdysis. During the early pupal stage, Dopa decarboxylase activities and mRNA concentrations in Br-SGs were also much higher in diapause-type than non-diapause-type insects. Elevation of dopamine levels induced by feeding Dopa to penultimate-instar and last-instar larvae, and by injecting Dopa or dopamine into pupae 2 days after pupation made the non-diapause-destined insects lay diapause-destined eggs at 59% and approximately 70% frequencies, respectively. Furthermore, injection of Dopa or dopamine elevated mRNA levels of the diapause hormone in the Br-SGs of non-diapause-type pupae 1 day after injection. Incubation of Br-SGs isolated from non-diapause-type day-2 pupae with Dopa or dopamine also stimulated the expression of diapause hormone mRNA. These data indicate that environmental stimuli during embryonic development increase dopamine levels in both hemolymph and Br-SGs from the larval stage to early pupal stage, which results in laying of diapause-destined eggs by female adults through enhanced expression of the diapause hormone gene.     

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.     

Samui, a novel cold-inducible gene, encoding a protein with a BAG domain similar to silencer of death domains (SODD/BAG-4), isolated from Bombyx diapause eggs.

Cellular responses to cold-acclimation have not yet been studied in depth. To explore this field, we focussed on insect diapause development. Although embryonic diapause of Bombyx mori is sustained at 25 degrees C, chilling at 5 degrees C for 2 months causes diapause termination, a transition that is marked when the sorbitol dehydrogenase gene (SDH) is activated. To clarify the relationship between this activation and incubation at 5 degrees C, we isolated a novel cold-inducible gene, Samui. Expression of Samui mRNA and protein was activated after incubation at 5 degrees C for 5-6 days, lasted for another 30 days and then weakened. Exposure to 25 degrees C suppressed both mRNA and protein expression. In nondiapause eggs incubated at 5 degrees C, Samui was also up-regulated, although the expression was weaker. Samui contained nuclear localization-signals, a ssDNA-binding motif and a BAG domain similar to that of SODD/BAG-4. Because Samui could bind to HSP70, it is a member of BAG protein family. It is proposed that Samui serves to transmit the '5 degrees C signal' for SDH expression in diapause eggs, while also protecting against cold-injures in nondiapause eggs, through binding to respective partners. This is the first report that a member of BAG protein family is up-regulated by cold.     

Control of diapause-factor secretion from the suboesophageal ganglion in the silkworm,Bombyx mori: The roles of the protocerebrum and tritocerebrum

To localize any functional areas in the brain which control the secretion of the diapause factor from the suboesophageal ganglion in the Bombyx silkworm, various parts of the brain were excised or transected surgically. It was demonstrated that cortices slightly lateral to the median line on the dorsal side of the protocerebrum (Lmdp) are closely related to the inhibitory mechanism in the pupa producing non-diapause eggs. On the other hand, cortices of anterolateral areas on the ventral side of the protocerebrum (Alvp) participate in the stimulatory mechanism in the pupa producing diapause eggs. Moreover, it is suggested that the tritocerebrum is competent to stimulate secretion of the diapause factor from the suboesophageal ganglion not only in the pupae producing diapause eggs but also in the pupae producing non-diapause eggs. From these results, we propose a possible relationship between the Alvp, Lmdp and tritocerebrum concerning regulation of diapause-factor secretion from the suboesophageal ganglion.     

Structure of the VAP-peptide (BmACP-6.7) gene in the silkworm, Bombyx mori and a possible regulation of its expression by BmFTZ-F1

The VAP-peptide (BmACP-6.7) is a hydrophobic peptide localized in adult cuticle of the silkworm, Bombyx mori. We isolated and characterized the VAP-peptide gene as a useful marker gene to analyze molecular mechanisms of terminal differentiation processes in the adult. The gene is composed of two exons interrupted by one intron. The 5' upstream promoter region is shown to bear a nucleotide sequence similar to the cis-element that is recognized and bound by the Bombyx mori FTZ-F1 protein (BmFTZ-F1). Expression of the BmFTZ-F1 gene preceded expression of the VAP-peptide gene and injection of 20-hydroxyecdysone suppressed the expression of both genes. An in vitro binding assay indicated direct interaction of BmFTZ-F1 with the VAP-peptide gene promoter sequence. Therefore, BmFTZ-F1 is proposed to be a possible factor regulating the stage-specific expression of the VAP-peptide gene towards adult life.     

Recent advances in insect neuropeptides

1. The number of insect neuropeptides identified chemically grows rapidly and most important neuropeptides have already been characterized. After multi-year efforts Bombyx diapause hormone has recently been isolated and sequenced.2. New approaches to search for new insect neuropeptides have been carried out by two groups of workers, which have succeeded in identifying several unique peptides.3. cDNAs for more than 10 insect neuropeptides have been cloned and sequenced. It was found that two functionally distinct neuropeptides. Bombyx diapause hormone and pheromone biosynthesis activating neuropeptide, are encoded in a single gene.     

Genome‐wide microarray screening for Bombyx mori genes related to transmitting the determination outcome of whether to produce diapause or nondiapause eggs

The bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae) exhibits a maternally controlled embryonic diapause. Maternal silkworms decide whether to lay diapause or nondiapause eggs depending on environmental factors such as the temperature and photoperiod during the egg and larval stages, and then induce diapause eggs during the pupal stage. However, little is known about the molecular mechanism that conveys the outcome of whether to produce diapause or nondiapause eggs from the egg or larval stages to the pupal stage. This study used microarray analysis to investigate differentially expressed genes in the larval brains of diapause‐ and nondiapause‐egg producers, to which bivoltine silkworms were destined by thermal or photic stimulation during the egg stage. The cytochrome P450 18a1 and Krüppel homolog 1 genes were upregulated in producers of diapause eggs compared with those of nondiapause eggs under both experimental conditions. Cytochrome P450 18a1 encodes a key enzyme for steroid hormone inactivation and Krüppel homolog 1 is an early juvenile hormone‐inducible gene that mediates the repression of metamorphosis. The upregulation of these genes during the larval stage might be involved in the signaling pathway that transmits information about the diapause program from the egg stage to the pupal stage in the silkworm.     

An active principle from the corpora cardiaca,corpora allata and suboesophageal ganglion of the locust,inducing egg diapause in Bombyx mori

The activity of the substance(s) which are contained in the cephalic endocrine organs of the locust which induce egg diapause in Bombyx mori was examined by implantation and injection of saline extracts of these organs. Extracts from the median and lateral neurosecretory parts of the locust brain were not effective in inducing egg diapause. Extracts of the corpora cardiaca, corpora allata, and suboesophageal ganglion of the locust induced diapause eggs in Bombyx pharate adults from which the suboesophageal ganglion had been removed. The first two extracts could induce egg diapause even in isolated abdomens of pharate adults of Bombyx. In the locust corpora cardiaca, the activity was present only in the glandular lobe and not in the nervous region. This activity decreased when the nervi corporis cardiaci I and II and of nervi corporis allati I were cut. Allatectomy also brought about a decrease in the activity in the glandular lobe which could not be restored by the injection of juvenile hormone. The activity in the corpora allata was enhanced slightly by the disconnection though not significantly.From these results, it is assumed that the corpora cardiaca, corpora allata and suboesophageal ganglion of the locust contain and active principle(s) capable of inducing egg diapause in Bombyx mori. The nervous connections between the brain, corpora cardiaca, and corpora allata are essential for the accumulation of the active substance(s) in the glandular lobes of the corpora cardiaca.     

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 °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 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °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 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.