Hot and Bothered, Bees’ Gut Microbiome Shifts Under Thermal Stress and Pathogen Infection

Abstract Understanding bumble bee gut health is imperative as these vital pollinators are subjected to pathogenic infections and thermal stress from climate change. The gut microbiome serves as an indicator for health and fitness and indicates the types of stress. To investigate the combined effects of thermal stress and pathogenic infection on bee guts, we performed a two-by-two crossed design where Bombus impatiens workers were subjected to hot conditions, pathogenic infection, or both. Incubation groups were given 2-weeks of stress conditions, with infected bees initially inoculated with Crithidia bombi, a common bee gut parasite. We measured body size, quantified the infection intensity of C. bombi using qPCR, and defined the composition of the gut microbiome using full-length 16S rRNA gene amplicon sequencing on an Oxford Nanopore Technologies Mk1D. While the core gut microbiome thrived with genera such as Bombilactobacillus and Snodgrassila which were not impacted by treatment; there were notable changes in other key organisms. Asaia bogorensis spiked in control temperature infected organisms, while species of Lactobacillus were overtaken in hot temperatures by significant increases in Apilactobacillus kunkeei. Species such as Citrobacter freundii dominated in hot infected bees suggesting an increased immunocompromised state from the combined stressors impacts on bee gut health. Our novel combined effects from thermal stress and pathogenic infection strengthen existing literature and provide new directions on how to quantify the health-state of wild bees based on their gut microbiome composition. These insights enable us to better understand how bees will be further impacted in changing landscapes. Importance We find significant changes in bees’ gut microbiome especially with an increased abundance of lactic acid bacteria. These lactic acid bacteria are often specialized: based on infection status, temperature, and the combined effects. These insights are vital to researchers, especially those studying wild bee gut health where they have an uncontrolled system and need to make assumptions about bee stress based on fitness and microbiome. Our detailed outline of relevant species will provide wild bee researchers with a baseline to determine thermal stress and recent infection status based on microbiome communities. Graphical Abstract Overview of the experimental design where bees were either given control temperatures or thermal stress and/or an infection by Crithidia bombi. The bees microcolonies were incubated for 2-weeks before sacrificing the bees, extracting DNA, and performing downstream analysis. Full length 16S rRNA gene amplicon sequencing was performed on an Oxford Nanopore MK1D sequencer while Crithidia bombi infections were quantified using quantitative polymerase chain reaction.