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Thermal adaptation plays a fundamental role in the expansion and distribution of insects, and heat shock proteins (Hsps) play important roles in the temperature adaptation of various organisms. To determine the roles of Hsp genes ( Hsp70 ,  Hsp21 , and  sHsp21 ) on the high temperature tolerance of  Agasicles hygrophila , we obtained complete cDNA (complementary DNA) sequences for  Hsp70 ,  Hsp21 , and  sHsp21  by rapid amplification of cDNA ends (RACE), analyzed their expression profiles under different high temperature treatments by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and performed functional verification by RNA interference (RNAi). The open reading frames of  Hsp70 ,  Hsp21 , and  sHsp21  were 1940, 543, and 567 bp, encoding 650, 180, and 188 amino acids, respectively. Their molecular weights (MWs) were 71.757, 20.879, and 21.510 kDa, and the isoelectric points were 5.63, 6.45, and 6.24, respectively. Phylogenetic tree analysis showed that the  Hsp70 ,  Hsp21 , and  sHsp21  genes of  A. hygrophila  were relatively conserved in evolution. The  Hsp70  and  Hsp21  genes in  A. hygrophila  were homologous to those in  Leptinotarsa decemlineata  (87 and 79% similarity, respectively), and the  sHsp21  gene in  A. hygrophila  was homologous to that in  Lissorhoptrus oryzophilus  (74% similarity). The amino acid polypeptide chain had highly conserved sequences of DLGGGTFD, VLVGGSTR, and GPTIEEVD. The sequence of EEVD was the characteristic motif of cytoplasmic  Hsp70 , and the highly conserved sequences of MALFR and MSLLP were characteristic sequences of  Hsp2  and  sHsp21 , respectively. Relative quantitative real time PCR showed that the three  Hsps  could be induced by 4-h treatment at high temperatures. Significant upregulation of these  Hsps  was observed when the temperature was further increased. The RNAi results showed that the injection of the three  Hsps ’ dsRNA could suppress the expression at the gene level significantly. Compared with the control group, high temperature heat shock reduced the fecundity of  A. hygrophila  significantly, and the fecundity decreased with the increase in temperature. Our results suggest that  Hsp70 ,  Hsp21 , and  sHsp21  might play key roles in high temperature adaptation of  A. hygrophila  and help improve our understanding of their mechanism of thermotolerance.

frontiers in physiology

Induced Thermotolerance and Expression of Three Key Hsp Genes (Hsp70, Hsp21, and sHsp21) and Their Roles in the High Temperature Tolerance of Agasicles hygrophila