Diapause duration of Paratlanticus ussuriensis is prolonged as an egg that enter both initial and final diapause stgaes. Environmental conditions, such as temperature, can modify the duration of initial diapause. Eggs enter initial diapause at 20℃, but continued early embryonic development at 30℃. Final diapause at a fully developed embryonic stage is obligatory regardless of temperature conditions. To determine temperature effects on initial diapause mechanism of P. ussuriensis eggs, we compared weights, DNA and RNA amounts of eggs incubated at either 20℃ or 30℃ for 50 days after oviposition. We identified small heat shock protein (shsp), heat shock protein 90 (hsp90) and three heat shock protein 70 (hsp70a, hap70b, hsp70c) genes of P. ussuriensis and determined those expression levels at different temperature conditions. The levels of shsp, hsp70a, hsp70b and hsp90 was not detectable until 20 days after oviposition at both temperature conditions, but highly increased at 50 and 60 days when incubated at 30℃. In contrast, hsp70c level was rapidly peaked at 20 days after oviposition, which is the time of initial diapause entrance. We analysis of temperature sensitivity of P. ussuriensis eggs. Hsp70a is expressed after the first cold treatment of mature eggs. Hsp70b is highly expressed just before hatching. Both shsp and hsp70c was highly expressed at the heat shock condition into immature egg stage. Our results suggest that high temperature breakdown initial diapause and one hsp gene, such as hsp70c, may be involved into the mechanism of initial diapause of P. ussuriensis eggs.

To find some antibacterial peptides responsible for bacterial resistance, we performed differential hybridization with total cDNA probes which synthesized from normal and immunized larvae. Thirteen individual cDNA transcripts were expressed differentially in a total 1,862 random cDNA clones. One of upregulated genes is a novel member of the insect defensin-like peptide(Coprisin), a family of antibacterial peptide. Northern blot analysis showed that Coprisin was up-regulated at 4h and reached the highest point level at 16h after injection of E.coli. The deduced amino acid sequence of Coprisin was composed of 80 amino acids with predicted molecular weight of 8.6 kDa and PI of 8.72. Comparison of the deduced amino acid mature portion of Coprisin with defensin-like peptide of other insect indicated that it has 79.1% and 67.4% identity with Anomala cuprea and Allomyrina dichotoma, respectively. To find antibacterial active region of Coprisin, we synthesized four peptides corresponding to amino acid residues 1V-43N-NH2(CopN1), 5-16(CopN2), 19-30(CopN3) and 31-43(CopN4) of coprisin having amidated amino acid residues at their Cterminal. A 12-mer amidated at its C-terminus, ACALHCIALRKK-NH2 (Ala19-Lys30-NH2) was synthesized based on the deduced amino acid sequence, assumed to be an active site sequence. This peptides showed antibacterial activity against E.coli, Staphylococcus aureus, MRSA, Psedomonas syringae, and Pectobacterium carotovorium. Modified 9-mer peptide, LRCIALRKK-NH2, showed strong antibacterial activity than mellitin peptide used as a positive control against gram-negative and gram-positive bacteria. This peptide showed no haemolytic activity and quite stable at 100℃ for several hours of incubation and in a wide pH range(pH2-12). Therefore, this peptide may be a good candidate for the development of new drug with potent antibacterial activity without cytotoxicity.