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Simple Summary  Reference genes are key to normalizing expression data across samples of organisms collected after different treatments are applied, but often, reference genes are not properly validated for this purpose. In this report, we screened several genes for a solitary bee,  Megachile rotundata , and identified two (RPS18, and RPL8) with very stable expression levels across all life stages of the bee, and under a variety of environmental conditions, including during and after diapause. These genes should make good reference genes. We also identified other genes with stable expression, even if used only for a limited number of developmental stages. This information is important for future gene expression studies on these bees, but it also demonstrates the importance of validating reference genes in general.    Abstract  The alfalfa leafcutting bee,  Megachile rotundata  is widely used in the western United States as a pollinator for alfalfa seed production. Unfortunately, immatures experience high mortality in agriculturally managed populations. Quantified gene expression could be used to identify how this bee responds during different life stages to pathogens, environmental toxins, and other stresses, but stably expressed reference genes are needed to normalize transcription data. We evaluated twelve candidate genes for their transcription stability across different life stages, including during and after diapause. RPS18 and RPL8 were the two most stably expressed genes, followed by RPS5 and RPL27A. These genes were also very stable even during and after diapause, while the most variable genes being APN, PMIIM, NPC2, and Cr-PII had increased expression levels during larval growth and were also variable during and after diapause. The four reference genes we identified in  M. rotundata  may prove useful for transcriptomic studies in other bees as well, such as honey bees.

Variation in Expression of Reference Genes across Life Stages of a Bee, Megachile rotundata