Identification and expression analysis of the heat shock proteins Hsp70, Hsp90, and Hsp90b in Litopenaeus vannamei under low-temperature stress

Litopenaeus vannamei is a tropical and subtropical species of shrimp, and its growth, physiology, and immune functions are affected by low temperatures. This study investigated the tissue-specific expression patterns of the heat shock protein genes hsp70, hsp90, and hsp90b in L. vannamei under low-temperature stress, and their correlation with cold tolerance. Fluorescence in situ hybridization revealed widespread expression of hsp70, hsp90, and hsp90b in the nucleus and cytoplasm of hepatopancreatic tubular cells. Notably, the expression of hsp70 was significantly higher in a cold-tolerant family of L. vannamei compared to a common family at 28 degrees C. The expression level of hsp90 was considerably elevated in the cold-tolerant family following exposure to low temperatures (18 degrees C and 10 degrees C) compared to that of the common family. During low-temperature stress, hsp70 and hsp90 expression levels increased gradually in both families, while hsp90b expression decreased. Downstream genes regulated by hsp90, such as akt, were initially upregulated, but then became downregulated, under low- temperature stress. RNA interference-mediated knockdown of hsp90 affected PI3K/AKT pathway signaling, which downregulated the downstream genes akt and apoptosis-inhibiting bcl-xl, bcl-2, and creb, and upregulated the apoptosis-promoting genes casp1 and casp9. Additionally, the survival rate of L. vannamei significantly decreased following hsp90 knockdown. DNA sequence alignment identified 12 single nucleotide polymorphisms (SNPs) in the hsp70 gene and 17 SNPs in the hsp90 gene. Linkage disequilibrium analysis revealed that eight SNPs in hsp70 were significantly associated with low-temperature tolerance (P < 0.01), and 4 SNPs were moderately associated with low-temperature tolerance (P < 0.05). All 17 SNPs in hsp90 were significantly associated with low-temperature tolerance (P < 0.01). These findings highlight the critical role of heat shock proteins in the low- temperature response of L. vannamei and provide valuable insights into understanding the mechanisms of shrimp cold tolerance and the development of cold-tolerant varieties.