Stress Test: Effects of Endosymbiotic Bacteria on Thermal Tolerance of a Montane Leaf Beetle

Insects are a diverse group of organisms found in most terrestrial and aquatic ecosystems. Because insects are ectotherms, they are frequently exposed to variation in environmental temperature and thus variation in body temperature, the latter of which may cause physiological stress. Insects host a variety of microbes, including intracellular and extracellular bacteria, endosymbiotic or parasitic, which may alter how the host responds to environmental temperature variation. Physiological, biochemical and molecular responses to thermal variation have been well characterized for many species of insects, yet few studies have examined how microbes alter their response to environmental stress. In this thesis, I examined features of the microbiome of a montane insect, the leaf beetle Chrysomela aeneicollis. Prior studies of beetle populations living at high elevation in the Eastern Sierra Nevada mountains of California have shown that adult and larval beetles are exposed to both elevated and sub-zero temperatures during the summer growing season, and that exposure to these thermal extremes impacts survival, performance, and reproductive success. Like most insects, this beetle is host to endosymbiotic bacteria; however, little is known about the composition of this microbiome, or the role endosymbionts play in how beetles respond to thermal extremes. My study pursued three aims: 1) describe the beetle microbiome and determine which microbes are most common; 2) assess the evolutionary relationships between willow beetle microbes and those of other known insect symbionts; and 3) quantify the interacting effects of host genetic variation and microbe composition and abundance on the beetles’ response to an environmentally realistic cold stress. To examine the beetle microbiome, I used the metagenomic application Metaphlan, a computational tool used for profiling the composition of microbiome communities. The results of this analysis indicated that Wolbachia was the most abundant endosymbiont in Sierra willow beetles, making up 99% of the microbiome. I therefore focused the remainder of my work on that common endosymbiont. To determine the number of Wolbachia strains infecting C. aeneicollis, I used Multi Locus Sequence Typing (MLST), which is based on five genetic loci known to have appropriate properties for use in distinguishing Wolbachia types. To quantify the relationship between Wolbachia density and recovery from cold exposure, I performed quantitative PCR (qPCR) on beetles exposed to either control conditions, or an ecologically relevant sub-lethal cold exposure. I used restriction digestion and SNP genotyping to determine the haplotype of cytochrome oxidase II (COII) and genotype of phosphoglucose isomerase (PGI), two genes coding for proteins of central metabolism, for each beetle. MLST analysis determined the presence of at least three strains of Wolbachia; two of these belonged to Wolbachia Supergroup A and one belonged to Wolbachia Supergroup B. Results from qPCR analysis showed that Wolbachia density was greater in non-stressed individuals than those exposed to cold stress, and that Wolbachia density was related to both PGI and COII genotype. I found that running speed measured directly after field collection of beetle adults was related to nuclear and mitochondrial genotype, and negatively related to the density of Wolbachia B. Running speed after cold exposure was also related to nuclear and mitochondrial genotype, and was negatively correlated with the density of Wolbachia A. These results implicate both host genetics and pathogen density in mediating a response to thermal stress. Thus, the microbiome of Sierra willow beetles may influence the ability of these insects to live in a rapidly changing thermal environment.