Simple Summary Diapause is a programmed developmental arrest coupled with an evident reduction in metabolic rate and a dramatic increase in stress tolerance. Sitodiplosis mosellana, a periodic but devastating wheat pest, spends the hot summer and cold winter as diapausing larvae. However, little is known about the metabolic changes underlying this obligatory diapause. The objective of this study was to identify significantly altered metabolites and pathways in diapausing S. mosellana at stages of pre-diapause, diapause, post-diapause quiescence and post-diapause development using gas chromatography/time-of-flight mass spectrometry and the orthogonal partial least squares discriminant analysis. Pairwise comparisons of the four groups showed that 54 metabolites significantly changed. Of which, 37 decreased in response to diapause, including four TCA cycle intermediates and most amino acids, whereas 12 increased. Three metabolites were significantly higher in the cold quiescence stage than in other stages. The elevated metabolites included the well-known cryoprotectants trehalose, glycerol, proline and alanine. In conclusion, the low metabolic rate and cold tolerance S. mosellana displayed during diapause may be closely correlated with its reduced TCA cycle activity or/and the increased biosynthesis of cryoprotectants. The results have contributed to our understanding of the biochemical mechanism underlying diapause and the related stress tolerance in this key pest. Sitodiplosis mosellana, a notorious pest of wheat worldwide, copes with temperature extremes during harsh summers and winters by entering obligatory diapause as larvae. However, the metabolic adaptive mechanism underlying this process is largely unknown. In this study, we performed a comparative metabolomics analysis on S. mosellana larvae at four programmed developmental stages, i.e., pre-diapause, diapause, low temperature quiescence and post-diapause development. In total, we identified 54 differential metabolites based on pairwise comparisons of the four groups. Of these metabolites, 37 decreased in response to diapause, including 4 TCA cycle intermediates (malic acid, citric acid, fumaric acid, alpha-ketoglutaric acid), 2 saturated fatty acids (palmitic acid, stearic acid) and most amino acids. In contrast, nine metabolites, including trehalose, glycerol, mannitol, proline, alanine, oleic acid and linoleic acid were significantly higher in both the diapause and quiescent stages than the other two stages. In addition to two of them (trehalose, proline), glutamine was also significantly highest in the cold quiescence stage. These elevated metabolites could function as cryoprotectants and/or energy reserves. These findings suggest that the reduced TCA cycle activity and elevated biosynthesis of functional metabolites are most likely responsible for maintaining low metabolic activity and cold tolerance during diapause, which is crucial for the survival and post-diapause development of this pest.
