Ecdysteroids and juvenile hormones play a key role in the regulation of development and reproduction of insects. The search for adenotropic factors that regulate the synthesis of these hormones led to the isolation and identification of a large number of neuropeptides with ecdysio- or allatoregulating functions from various insect orders. Many of these peptides are multifunctional and widespread among insects. This review mainly deals with the isolation, identification and physiological actions in vitro and in vivo of ecdysiostatic and allatostatic peptides isolated from the Mediterranean field cricket, Gryllus bimaculatus, and presents some previously unpublished results. The identified peptides and their physiological actions are compared with those isolated from other insect orders. It seems likely that in addition to the ecdysio- and allatoregulating factors, peptides of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family might also be involved in the regulation of insect development and reproduction. These latter peptides are well known for their energy-mobilizing function in flying insects, but they also regulate other energy-demanding events such as egg-production. The possible roles of these neuropeptides and their interactions with other hormones are discussed.

View full abstract

We purified vitellin (ApVn) and vitellogenin (ApVg) from Antheraea pernyi by a combination of gel permeation chromatography, anion-exchange chromatography, and hydrophobic chromatography. Our results showed that the molecular size of the ApVg heavy chain determined by SDS-PAGE was approximately 210 kDa. ApVn and ApVg each consisted of only a large subunit, suggesting that ApVg should be classified in group 2 of the insect vitellogenin family. We analyzed the N-terminal amino acid sequence of ApVg and the terminal amino acid sequences of four lysyl endopeptidase-degraded fragments of ApVg. The nucleotide sequence was determined using overlapping cDNA fragments generated from reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) reactions. The ApVg cDNA was 5720 nucleotides long and coded for the entire subunit, which consisted of 1778 amino acids. The molecular weight of the predicted polypeptide was 200, 000. There is no RXRR motif, which is the cleavage site between the small and large subunits in the previtellogenins of the silkworm, Bombyx mori, the mosquito, Aedes aegypti, the sawfly, Athalia rosae, and the boll weevil, Anthonomous grandis. Two polyserine regions were found in the deduced amino acid sequence.

View full abstract

We isolated a protein from pupal brain extract of the silkworm, Bombyx mori, using two chromatographic steps, and identified its N-terminal amino acid sequence. Based on the amino acid sequence, degenerate oligodeoxynucleotide was synthesized and used as a hybridization probe to screen a pupal brain cDNA library. The sequence analysis of the identified cDNA clones indicated that a 603bp open reading frame encoded a 201-amino-acid protein containing a 15-amino-acid leader peptide. This protein has a high similarity to the Galleria mellonella Gallerin as a member of the lipocalin family. Northern blot analysis clearly showed a ca 1.4kb transcript in the central nervous system, but not in the fat body, salivary glands and Malpighian tubules of 1-day old female pupae. We named this novel protein from the Bombyx central nervous system, Bombyrin, and discussed possible functions of Bombyrin in the central nervous system, although members of lipocalin family are well known to serve as transport proteins.

View full abstract

Our previous experiment using a trehalase inhibitor, trehazolin, indicated the possibility that the diapause hormone (DH) could induce embryonic diapause through a regulating expression of some genes other than the trehalase gene in developing ovaries of the silkworm, Bombyx mori. To examine this possibility, we employed the differential display after a DH challenge. We identified three cDNA fragments (ID1, ID3, UP3) in which expression was up-regulated by DH. ID1, ID3, UP3 and trehalase mRNA were detected at different levels in all tissues examined, but only the developing ovary responded positively to a DH challenge. DH induced the gene expression at a high level in the vitellogenic or younger follicles. ID1, ID3 and UP3 gene expression was significantly stimulated as early as 30min after DH administration and reached a maximum level at 1h. By contrast, the trehalase gene was slowly up-regulated. Actinomycin D completely inhibited DH-dependent stimulation of the expression, whereas cycloheximide did not inhibit the expression. Thus, DH is conceived to act on one organ to regulate the expression of different genes through different mechanisms.

View full abstract

A cDNA clone encoding an ATPase inhibitor-like protein was found in a Bombyx mori EST (expressed sequence tag) database and designated as BmAl-a. Also a novel cDNA clone encoding a different ATPase inhibitor-like protein was isolated from a testis library of B. mori after mRNA subtraction, and named BmAl-b. Both BmAl-a and BmAl-b cDNA clones were determined for their nucleotide sequences; the deduced amino acid sequences showed that the relevant proteins were composed of 127 and 107 amino acid residues, respectively. The BmAl-a and BmAl-b proteins have the highest homologies of 41% and 37%, respectively, with the Caenorhabditis elegance ATPase inhibitor-like protein CelF, among the animal homologs so far reported. Expression analysis by reverse transcription polymerase chain reaction demonstrated that the BmAl-a mRNA was transcribed in all tissues examined, while the BmAl-b mRNA was expressed exclusively in the testis. A computational analysis of amino acid residues by a method available from a web-server, suggested that BmAl-a is located in the mitochondria, whereas BmAl-b is allocated in organelles other than the mitochondria.

View full abstract

In cultured cells, karyotypes and genomic dose are variable, especially in ones passaged for a long time. We have improved a method for the genome confirmation of cultured cells of the silkworm, Bombyx mori. A rapid, simple procedure was developed for direct extraction of genomic DNA from cultured cells which has two main steps of proteinase K treatment and heating, without phenol extraction. Genomic characterization is based on PCR amplification of three known genes which vary in copy number and chromosome location. The retrotransposon, BMC1, is dispersed in the genome; rDNA is clustered in one locus; and the fibroin gene is single copy. These genes were sufficient to identify cells as Bombyx mori for comparing among the other species. It was shown that this method could also be used for genotyping and strain identification using larval hemolymph samples.

View full abstract

To characterize the infection of Bombyx mori nucleopolyhedrovirus (BmNPV) in Spodoptera frugiperda (Sf9) cells, several relevant parameters to viral replication were examined. Sf9 cells showed no clear overall cytopathology and proliferated continuously following inoculation with BmNPV. However, careful examination under a microscope revealed a small number of cells with polyhedra. Also the immunoblot analysis showed significant but low levels of BmNPV polyhedrin expression in BmNPV-inoculated Sf9 cells. Examination with a recombinant BmNPV, BmΔpolh-lacZ that possessed lacZ driven by polyhedrin gene (polh) promoter, further indicated that the number of Sf9 cells expressing lacZ increased with the increase in viral multiplicities of inoculation. Slot-blot hybridization analysis detected low levels of viral DNA replication and northern blot analysis showed that transcription of an immediate early viral gene, ie-1, was restricted strikingly in BmNPV-inoculated Sf9 cell cultures, suggesting that early events leading to productive viral infection occurred in only a limited number of BmNPV-inoculated Sf9 cells. Comparative infectivity assay between inoculation of BmNPV budded virions (BVs) and transfection of BmNPV DNA revealed that infection efficiency was higher in the transfection than in the inoculation. Viral adsorption assay showed that BmNPV BVs were able to be adsorbed efficiently by Sf9 cells. These results suggest that BmNPV replication in majority of Sf9 cells is restricted mainly at the step prior to the expression of immediate early viral genes, that includes viral entry into the cells through endocytotic pathway and nucleocapsid uncoating in the nuclei.

View full abstract

A sericin hydrogel membrane with a good dynamic property was prepared by chemical cross-linking, and the relationship between the structure and the physical properties of the membrane was studied. The chemical cross-link increases with increasing dimethylolurea (DMU) as a cross-linking agent even after the extraction of the excess DMU. The increase in the cross-link strains the segmental motion, but that motion becomes easier as a mole ratio of 0.5 increases because of the extraction of the excess DMU and the turbulence of the crystal structure of sericin. Water content is lowered to a 0.25 mole ratio of DMU, and then increases to over 0.5 again. Young's modulus shows a tendency that is reversing the result for water content.

View full abstract