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

Authors: MONDAL, BANANI - University Of Wyoming; WALTER, RIKKI - North Dakota State University; Rinehart, Joseph; Yocum, George; BASILE, FRANCO - University Of Wyoming; GREENLEE, KENDRA - North Dakota State University

Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2026
Publication Date: 2/4/2026
Citation: Mondal, B., Walter, R., Rinehart, J.P., Yocum, G.D., Basile, F., Greenlee, K.J. 2026. Metabolome of diapause-destined Megachile rotundata prepupae differs from those undergoing direct development. Journal of Insect Physiology. https://doi.org/10.1016/j.jinsphys.2026.104949.
DOI: https://doi.org/10.1016/j.jinsphys.2026.104949

Interpretive Summary: The alfalfa leafcutting bee is a critically important pollinator for alfalfa seed production and other specialty crops. This bee spends a majority of its life cycle in diapause, which is an overwintering stage during which the insect lowers its metabolism, suspends development, and increases its ability to survive cold stress. When managed for pollination, this bee can spend as much as 9 months of the year as a cold-stored diapauser. While much is known about how this insect survives this extended period of time, little is known about the nutritional stores that they must develop to survive. In this study, we compared the lipids and other compounds associated with metabolism in diapause-destined insects with those destined to continue their development without delay. Ou results show significant differences between the two types of bees, with diapause-destined bees exhibiting higher levels of specific amino acids and carbohydrates, including several sugar alcohols. Diapause destined insects also had much high levels of putrescine, a compound linked to diapause in other species. These data contribute to a growing knowledge base that will help inform pollination managers how to improve the health of their bees during winter storage.

Technical Abstract: Diapause in many insects is a non-feeding state, yet energetic demands persist due to maintenance, some development, and possible production of key cryoprotectant molecules. This imbalance between energy in and energy out is met by two main mechanisms: reduced metabolism and nutritional stores. Insects reduce their metabolic rates, facilitated by their exposure to low temperatures. Numerous studies have measured changes in gene and protein expression in an effort to test the hypothesis that there is a core set of genes involved in regulating diapause. During diapause, metabolites also vary, including amino acids, sugars and polyols, metabolic intermediates, and fatty acids. However, few studies have looked at insects prior to overwintering. We tested the hypothesis that bees destined to overwinter, diapause, and those that will develop directly have distinct metabolomic signatures that are indicative of key differences in energetic status. We collected nests of alfalfa leafcutting bees, Megachile rotundata, oviposited in the field in early July (nondiapause) and mid-September (diapause). Two prepupae from each nest were frozen on liquid nitrogen and kept in -80°C until sample extractions. The diapause state of the frozen bee was verified by observing whether nestmates developed or underwent diapause. Prepupae were subjected to biphasic extraction to produce an organic phase for lipidomics and an aqueous phase for metabolomics. We used an untargeted GC-MS-based metabolomics approach. We found 31 significant metabolites with the potential to differentiate between diapause-destined and direct developing prepupae. These altered metabolites primarily encompass sugars, polyols, intermediates of the tricarboxylic acid (TCA) cycle, and amino acids. In diapausing prepupae, 22 metabolites were upregulated, and 48 were upregulated in non-diapausing prepupae. Metabolites upregulated in diapause-destined prepupae were mostly classified as amino acids and carbohydrates, including several sugar alcohols. Metabolites upregulated in non-diapausing bees included mostly amino acids and carbohydrates. In diapause-destined bees, more than 50% of the upregulated metabolites were carbohydrates. In non-diapausing bees, only about 20% of the upregulated metabolites were carbohydrates. Surprisingly, the GC-MS analysis of the lipid acyl groups (as their fatty acid methyl esters) did not reveal differentiation between diapause-destined and non-diapausing prepupae. Several of the identified metabolites have been previously implicated in diapausing insects. Some of these metabolites could be used as biomarkers to identify which nests will diapause, which could benefit bee growers and farmers.