Characterization and Expression of Two Cytoplasmic Phosphoenolpyruvate Carboxykinase Genes Associated with Larval Diapause and Temperature Stress in the Wheat Blossom Midge, Sitodiplosis mosellana

Phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, is associated with adaptation to environmental stress. However, its potential role in diapause is not known. Here, two cytoplasmic genes encoding PEPCK (<i>SmPEPCK1-1</i> and <i>SmPEPCK1-2</i>) in <i>Sitodiplosis mosellana</i>, a significant wheat pest undergoing obligatory larval diapause during the third instar, were cloned, and their expression patterns during diapause and thermal stress were assessed, together with the effects of <i>SmPEPCK1-2</i> knockdown on larval development. Both <i>SmPEPCK1-1</i> and <i>SmPEPCK1-2</i> were evolutionarily conserved and contained canonical functional domains and motifs. Their expression was induced by diapause, and was found to be tissue-specific, with the highest levels observed in the fat bodies of diapausing larvae. Furthermore, exposure to heat stress in oversummering larvae or cold stress in overwintering larvae enhanced the expression of both genes within specific temperature ranges (35-40 °C and -10-0 °C, respectively). RNA interference-mediated knockdown of <i>SmPEPCK1-2</i> did not affect cocoon-breaking rates and timing but significantly prolonged the duration of larval development from cocoon-breaking to pupation. These findings indicate that both <i>SmPEPCK</i> genes are closely involved in tolerance to diapause-related stress, with <i>SmPEPCK1-2</i> also contributing to the regulation of larval development.