Insulin Pathway Gene Expression in a Diapausing Solitary Bee Megachile rotundata
Authors: Cambron Liz D , Yocum George D , Greenlee Kendra J 
Published: June 2021

Insulin signaling pathways are conserved across taxa, but interestingly insects have insulin‐like peptides (ILP) that are encoded by multigene families. These genes can be differentially expressed across tissues, developmental stages, and species. Although studies have shown that ILP can function as hormones, neurotransmitters, and growth factors, their direct functions are still unclear. In other animals, insulin signaling is involved in many other physiological processes such as reproduction, development, and metabolism, but further studies are needed to investigate its role in insects. One area that needs further investigation is the role of insulin signaling in regulating diapause. Diapause is a hibernation‐like stage that many insects undergo to survive winter. Diapausing insects are exposed to environmental stressors, such as fluctuating temperatures, changes in oxygen concentrations, and pathogens. For some insects, including the alfalfa leafcutting bee, Megachile rotundata, this can also be a non‐feeding period. With fixed resources, overall metabolism and insulin signaling are maintained at low levels, but it is unclear if these change in response to stressors. Our hypothesis is that insulin signaling is responsible for allocating energy in response to stressors. To test this hypothesis, M. rotundata were overwintered in either a lab setting at constant 4°C or in the field in naturally fluctuating temperatures. Samples from both lab and field were collected monthly, and RNA was extracted. November, December, January, and March samples were used to span the entire overwintering period. Previous Illumina sequence data was analyzed to find sequences for potential reference and target genes in the insulin pathway, and then sequences were confirmed by aligning to the genome for M. rotundata using BLAST. The three most stable reference genes were chosen using geNorm software and used for subsequent qPCR on target genes. qPCR data was normalized using reference genes and analyzed using the ΔΔCt method. Two‐way ANOVA was used to test for differences between lab and field groups and across months. We predicted that bees in the field would have more changes in gene expression in response to the stress of fluctuating temperatures. Relative to November, gene expression between lab and field groups differed for the insulin receptor and mTOR. However, an overall trend of down‐regulation from November to March was seen for both groups. This trend was expected, because rising temperatures are usually a cue for bees to end overwintering and resume development in the spring. Future studies will focus on investigating more genes in this pathway to develop a full picture of how insulin signaling changes in diapausing bees. Studying insulin signaling in diapausing bees is important for understanding how stress affects energy allocation in insects and may provide insight into how insulin signaling evolved. With many conserved pathways, including the insulin pathway, the use of insect models in metabolic studies is increasing. By better understanding insect physiology, the possibilities of research avenues and translational research are vastly expanded.Support or Funding InformationNSF GRFP/GRIPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.