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

## TL;DR

This study identifies two PEPCK genes in a wheat pest insect that are linked to diapause and temperature stress, revealing their role in larval development and stress tolerance.

## Contribution

The study characterizes two PEPCK genes in Sitodiplosis mosellana and shows their involvement in diapause and temperature stress adaptation.

## Key findings

- SmPEPCK1-1 and SmPEPCK1-2 are expressed in fat bodies of diapausing larvae and are regulated by diapause and environmental stress.
- Knockdown of SmPEPCK1-2 prolongs larval development but does not affect cocoon-breaking rates.
- Both genes show increased expression under specific heat or cold stress conditions.

## Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) is a crucial metabolic enzyme involved in gluconeogenesis. However, little is known of its role in the diapause response. In this study, two cytoplasmic genes encoding PEPCK (SmPEPCK1-1 and SmPEPCK1-2) in Sitodiplosis mosellana Géhin (Diptera: Cecidomyiidae), a significant wheat pest that undergoes an obligatory diapause during its third-instar larval stage, were cloned, and their expression during diapause and temperature stress was analyzed, together with the effects of SmPEPCK1-2 knockdown on larval development. The results suggested that the expression of both SmPEPCK1-1 and SmPEPCK1-2 is regulated by diapause and the environment, with SmPEPCK1-2 also involved in the regulation of larval development.

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 (SmPEPCK1-1 and SmPEPCK1-2) in Sitodiplosis mosellana, 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 SmPEPCK1-2 knockdown on larval development. Both SmPEPCK1-1 and SmPEPCK1-2 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 SmPEPCK1-2 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 SmPEPCK genes are closely involved in tolerance to diapause-related stress, with SmPEPCK1-2 also contributing to the regulation of larval development.