Putrescine is more abundant in larval bees destined to diapause compared to those that develop directly

Univoltine species spend winter months in a state of arrested development called diapause, whereas in multivoltine species, some generations develop directly to adults and some diapause. In many cases, the signal to enter diapause is the change in photoperiod during the late summer months. However, in the alfalfa leafcutting bee, Megachile rotundata, the signal to enter diapause is not clear. The alfalfa leafcutting bee is a solitary, cavity-nesting bee that readily builds nests in commercial structures, made from Styrofoam blocks with pre-drilled cavities. Females construct individual brood cells using leaf pieces that they cut with their mandibles. Brood cells are provisioned with a mixture of nectar and pollen that the female collects. She then lays an egg in the cell, caps the cell, and begins constructing the next one. A nest typically has 5-8 brood cells in series. After eggs hatch, larvae consume the entire provision over a period of about two weeks. After the provision is gone, larvae will either begin metamorphosis or enter diapause. Some data suggest that maternal effects determine whether offspring will enter diapause or develop directly to adults. To identify possible signals, we collected eggs from the field that were likely to be direct-developers (July-long days) and diapause-destined (September-short days) and reared them in the lab until they had consumed their entire provision. At that point, we froze the first and last offspring from several nests and kept them at -80°C, but we could not tell which bees would enter diapause or develop. To verify the diapause state of the frozen bees, we observed development of nestmates. Frozen samples were omitted from the study if nests were mixed in voltinism (i.e., some siblings in the nest entered diapause and some continued development to adults). Frozen prepupae (n=22 diapause-destined and n=26 direct developers) were shipped to University of Wyoming on dry ice for untargeted metabolomics analysis. Samples were subjected to biphasic extraction using MTBE, methanol, and water to produce an organic phase for lipidomics and an aqueous phase for metabolomics. Aqueous and organic layers were stored at -80°C prior to derivatization. Derivatization of the aqueous phase was conducted using methoxyamine and MSTFA, while the organic layer was derivatized using methanolic acetyl chloride and MSTFA (for fatty-acid and cholesterol analysis). GC-MS analysis was performed, and data were analyzed using MS-DIAL and MetaboAnalyst softwares. We identified 21 metabolites that were more abundant in diapause-destined larvae and 52 that were more abundant in those that would develop directly. No differences in the lipidomes were detected. One metabolite, putrescine, was nearly seven times higher in diapause-destined larvae compared to those that would develop directly. In a previous study, putrescine fed to caterpillars, Helicoverpa armigera, increased the likelihood that they would diapause, despite being exposed to long days. To determine whether putrescine could alter diapause status in M. rotundata, we added 0.01% or 0.1% putrescine to provisions of M. rotundata eggs and observed development. Putrescine had no effect on the likelihood of entering diapause, but higher levels increased mortality. It is unclear if putrescine levels in the diet vary with season. We plan to analyze provisions from early and late in the season to determine if putrescine levels vary. Knowing what factors contribute to diapause is especially important for commercially used insects. Understanding the environmental signals that contribute to diapause is important as environmental changes occur. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.