Metabolome of diapause-destined Megachile rotundata prepupae differs from those undergoing direct development

Diapause in many insects is a non-feeding state, yet energetic demands persist. This imbalance between energy in and energy out can be met by two main mechanisms: reduced metabolism, facilitated by decreasing temperatures, and storage of nutrients. In bivoltine insects, some individuals may continue direct development while others may enter diapause. Few studies have compared these two groups prior to the decision to diapause, even though biochemical differences are likely due to accumulation of nutrient stores and molecules involved in metabolic suppression. In Megachile rotundata, offspring oviposited in early summer develop directly to adults, while those oviposited later that summer enter diapause as prepupae. Here we test the hypothesis that prepupae between the two groups have distinct metabolomic signatures that will indicate key differences in energetic status. We collected 12 non-diapause and 15 diapause-destined nests. Two prepupae from each nest were flash frozen and kept in -80°C until extracted. Diapause state of frozen bees was verified by observing whether nestmates developed or underwent diapause. We used an untargeted GC-MS-based approach that identified 57 significant metabolites with the potential to differentiate between diapause-destined and non-diapause prepupae. Altered metabolites included sugars, polyols, intermediates of the tricarboxylic acid (TCA) cycle, and amino acids. Seventeen metabolites (60% carbohydrates) were more abundant in diapause destined prepupae. Forty metabolites (30% amino acids) were more abundant in non-diapausing prepupae. Several metabolites were previously implicated in diapausing insects. Some metabolites, e.g., putrescine, could be used as a biomarker to identify diapause status of nests, benefitting bee growers and farmers.