Study On The Antimicrobial Peptide And Heat Shock Protein In The Shrimp Fenneropenaeus Chinensis

Antimicrobial peptides are important for non-specific host defence in many animals. Here we report the presence of a novel antimicrobial peptide, active against both gram-positive and gram-negative bacterial, in the shrimp Fenneropenaeus chinensis. CZE (capillary zone electrophoresis) was applied to analyse hemolymph samples from shrimp before and after an immune challenge with the bacteria strains Vibrio anguillarum. The analysis of these spectra led to the following observation: the peptides relative concentration was found to increase by the first 3 hours post-challenge; and reached a maximum at 6 hours; after 24 hours, the plasma content of peptides appeared to be similar to that observed in unchallenged animals. On the basis of CZE and antimicrobial assay, the antimicrobial peptide (AP) was purified to homogeneity by Sep-Pak C18 extraction and reverse-phase HPLC. The partial N-terminal amino acid sequence obtained via Edman degradation revealed that the AP was proline rich and shared 61% identity in a 15-amino-acid overlap with the penaeidins, a family of APs from shrimp Litopenaeus vannamei. The classification and differential count of circulating hemocytes have also been studied at different time after microbial challenge. The mean number of total hemocytes per ml showed a distinct decrease at 6 hours post-challenge. The proportion of agranular hemocytes was found to increase after bacterial challenge, and reach a maximum at 6 hours later. Simultaneously, the proportions of large-granule hemocytes and small-granule hemocytes were found to decrease after bacterial challenge. The former reached a minimum at 6 hours later, and the latter did at 24 hours post-challenge. After 48 hours, the numbers of three types of hemocytes appeared to be similar to that observed in unchallenged animals. The results showed that bacterial challenge triggered a plasmatic increase of the antimicrobial peptide concentration and gave implication of the simultaneous release of the peptide from the shrimp granulocytes.RT-PCR assays of total RNA from the shrimp Fenneropenaeus chinensis of different ages provide the first evidence of developmental expression of antimicrobial peptide in Crustacean and indicate that the antimicrobial peptide gene is constitutively expressed at every stage examined in the shrimp Fenneropenaeus chinensis. Using western immunoblotting, and monoclonal anti-HSP70 and HSP70, we obtained stress protein induction data from the shrimp Fenneropenaeus chinensis during thermal and immune-challenged stresses. This work is the first report of the effects of various stressors on the expression of HSP70 in various tissues such as muscle, hepatopancreas, eyestalk, hemolymph, and gills. HSP70 was prominently induced in hepatopancreas and gills, but not in muscle, eyestalk and hemolymph, during the shrimps were exposed to heat shock and Vibrio anguillavium-challenged stresses. Cold shock and WSSV treatment had no significant effects on the levels of HSP70 expression in all tissues examined. HSP70 induction was greatest after 2 h exposure to heat shock stress, which was elevated after acute heat shock exposure of 10℃ above ambient temperature.