Functional characterization of heat shock proteins during stress response in pacific white shrimp Penaeus vannamei and brine shrimp Artemia franciscana

Abstract

Acute hepatopancreatic necrosis disease (AHPND) caused by the bacterium Vibrio parahaemolyticus carrying toxin produced plasmid (VPAHPND), has led to severe mortalities in farmed penaeid shrimp throughout Asia. Our studies demonstrated that a chronic non-lethal heat shock (chronic-NLHS) could enhance resistance of the Pacific white shrimp Penaeus vannamei to VPAHPND infection. Shrimp exposed to chronic-NLHS (28 °C to 38 °C, 5 min for 7 days) had higher survival rate (>50%) than that of the control non-heated shrimp (20%) when they were challenged with VPAHPND. The qRT-PCR analysis revealed that the expression of heat shock proteins, LvHSP70 and LvHSP90, as well as other immune-related genes, LvproPO1 and LvCrustin1, were induced upon exposure of shrimp to chronic NLHS. Moreover, LvHSP70 and LvHSP90 gene silencing eradicated the VPAHPND resistance in the chronic-NLHS shrimp and decreased PO activity. Further investigation showed that chronic-NLHS immediately activated the production of LvHSP70 in shrimp hemocytes. Injection of 1.0 nmol recombinant LvHSP70 (rLvHSP70) of injection induced the expression of several immune-related genes (LvMyD88, LvIKKε, LvIKKβ, LvCrustin1, LvPEN2, LvPEN3, LvproPO1, LvproPO2, and LvTG1) in the shrimp immune system. Interestingly, rLvHSP70 enhanced P. vannamei resistance to VPAHPND by increasing survival rate from 20% (control group) to >75%. These results suggested that LvHSP70 plays crucial roles in bacterial defense by activating shrimp immune system. In addition, four novel HSP70 family genes designated as HSC70, HSC70-5, BIP, and HYOU1 were identified from the draft Artemia transcriptome database by sequence analysis that contained the specific conserved domain of HSP70 family gene. Expression analysis revealed that in the juvenile state, the transcript level of HSP70 and HSC70 was significantly higher in the selective breeding for induced thermotolerance population (TF12) of Artemia relative to a control population (CF12). Moreover, after a NLHS treatment of TF12 at the nauplii state, HSP70, HSC70, and HSC70-5 transcrips were significantly (P<0.05) up-regulated. In contrast, the expression of the other members of Artemia franciscan HSP70 family showed no significant (P>0.05) induction. Furthermore, SNP polymorphisms of HSP70 and HSC70 genes were investigated and the result identified a SNP at position 171 in HSC70 (C171A; N57K) located at ATP-binding domain which might be potentially associated with the increased thermotolerance. The genotype analysis result showed significant (P<0.01) difference of this SNP between CF12 and TF12. Moreover, the phenotypic analysis by yeast (Saccharomyces cerevisiae) system confirmed that yeast contained the HSC70-N57K plasmid could tolerate high temperature than yeast containing the WT-HSC70 plasmid. As the N57K genotype was more prevalent in TF12 population than that in CF12 suggesting that it can be used in selective breeding for induced thermotolerance in A. franciscana.