Characterization of Two Novel Heat Shock Protein 70 Transcripts from Sitodiplosis mosellana and Their Response to Larval Diapause and Thermal Stress

The heat shock protein 70 (Hsp70) family mediates responses to environmental stress in insects. The wheat midge <i>Sitodiplosis mosellana</i>, a worldwide pest, avoids summer and winter temperature extremes by diapause of the third-instar larvae in the soil. To explore the functions of Hsp70s in this process, we characterized two cytoplasmic Hsp70 genes (<i>SmHsp70A1-1</i> and <i>SmHsp70A1-2</i>) from this insect. Both SmHsp70s contained three signature motifs of the family and lacked introns. Developmental expression profiling revealed maximal <i>SmHsp70A1-1</i> expression during early larval stages, while the expression of <i>SmHsp70A1-2</i> was highest in the pupal stages. The expression of <i>SmHsp70A1-1</i> was significantly upregulated during diapause, particularly during summer and winter, whereas <i>SmHsp70A1-2</i> showed marked downregulation and dose-dependent induction by 20-hydroxyecdysone (20E). Furthermore, both genes exhibited similar expression patterns in over-summering and over-wintering larvae under thermal stress, with maximal expression at 40 °C and -10 °C, respectively, but were not significantly induced at prolonged extreme temperatures (50 °C or -15 °C). Knockdown of the two <i>SmHsp70</i> genes by RNA interference (RNAi) significantly increased the susceptibility of the larvae to cold stress. These results suggest the important role of both <i>SmHsp70</i> genes in diapause-associated stress tolerance and provide crucial insights into the mechanisms underlying thermal adaptation in <i>S. mosellana</i>.