Stress response in black tiger prawn penaeus monodon by detecting heat shock proteins and blood glucose level

Abstract

The investigation on the stress response of p. monodon to various temperature revealed that the proper temperature for non-lethal thermal shock on 20 g shrimps ranged from 15 to 35℃ for 6 h where shrimps survived for more than 2 days. Exposing to higher or lower temperatures of this range resulted in complete mortality within 10 and 15 min. After induction with non-lethal thermal shock, protein levels of haemolymph from induced shrimps raised significantly in corresponding to the level of the increase temperature in heat shock and decreased when exposed to cold shock. Similar results from the increase or decrease levels of plasma glucose concentration were obtained from thermal shock shrimps. The results on protein analysis of thermal-shock shrimps with and without vibrio infection using SDS-PAGE revealed several different protein bands between the unstressed and stressed samples. However, the results were not conclusive due to the un-consistency of the protein profiles between replications and the interference of haemocyanin in the samples. The Western blotting analysis of the haemocyte lysates from the shrimps from control, cold and heat shock experiments showed a considerably clear signal of cross reaction of anti-HSP70 monoclonal antibody and the proteins at 76 kDa. The increasing intensity of the band correlated to the level of thermal induction and time indicated that HSP70 was inducible by heat and cold condition and could be detected by cross-reaction with antibody raised with HSP70 from other organisms. The changes of HSP60 and HSP90 in thermal shock shrimps, however, were not detected by this method. The differential expressed genes in the haemocytes of the shrimps in response to heat shock were detected by RAP-PCR technique. From the result of amplification with 10 primer combinations, 7 DNA fragments were detected to display differentially between control and heat shock shrimps, 10 fragments were up regulated and 3 fragments were down regulated in corresponding to the heat shock temperatures. From the results of sequence comparison of those markers, 3 fragments showed significant similarity to some known genes in the GenBank. The expression levels of 8 stress-related genes including RAP-PCR markers (RAP 12, 16, 22, and 58), PO, HSP60, HSP70 and HSP90 from heat-induced shrimps exposed to Vibrio were determined. The results showed that the transcription levels of RAP 12, RAP 16, RAP21, RAP58, HSP70, and HSP90 increased in responding to heat induction. The response of HSP60 gene to any of the inductions in this experiment was not detected. Vibrio treatment did not affect the expression level of those genes. The expression of PO gene was up-regulated by the Vibrio treatment but not by heat shock.