Intrinsic and Extrinsic Factors Shape Cane Toad Gut Bacteria Across an Expanding Invasive Range

Abstract

The cane toad (Rhinella marina) is one of the most successful invasive species worldwide. Since their introduction to Queensland in the 1930’s, Australian cane toads have expanded westward and now are present in Western Australia. My thesis examines the gut bacteria in Australian cane toads to determine how environmental factors (e.g., diet, climate) and intrinsic factors of hosts (e.g., genetics, body size, parasite infection) interact to maintain and influence the composition and stability of intestinal bacteria. I first investigated sampling methodologies to determine whether non-lethal (cloacal and faecal) sampling accurately represent gut bacteria. I found that cloacal swabs are better proxies for large intestinal bacteria than faeces in toads. I then tested whether behaviours associated with invasion are correlated with intestinal bacterial community assemblage and function. Behaviours thought to be linked to invasion ability differ in toads from the extreme ends of this range. Although behaviour has been linked to gut bacteria in other taxa, cane toad gut bacteria has not been investigated. I characterised gut bacteria composition and behaviour of wild-sampled cane toads across their northern Australian range and found significant difference in bacterial community and predicted functions between Western Australia and Queensland cane toads, based on 16S rRNA sequencing. Environmental factors including Isothermality, Annual Mean Temperature and the presence of co-introduced lungworms (Rhabdias pheudosphaerocephala) best explained bacterial community assemblage. These same factors, in addition to certain behaviours linked to invasion ability (righting reflex time and the presence of righting reflex movements) best explained bacterial function. I then used Next Generation Sequencing to characterize and compare cane toad genetic (single nucleotide polymorphism), epigenetic (DNA methylation), and gut bacteria differences across populations. I found no significant association between host heterozygosity and gut bacterial diversity within individuals. However, I did find that pairwise genetic diversity was positively associated with pairwise epigenetic diversity. Interestingly, the positive correlation between pairwise epigenetic diversity and bacteria diversity was greater in pairs with lower genetic diversity. Finally, I examined cane toad diet (taxonomy of stomach contents) and found that the presence of plant matter in cane toads’ stomachs was associated with gut bacteria variation, but that gut bacteria was not significantly associated with the main component of cane toad diet (insects). My thesis provides important methodological advances in the study of amphibian gut bacteria and suggests that in cane toads, gut bacteria variation is strongly linked to lungworm infection and to DNA methylation. These results highlight possible mechanisms through which cane toads could increase the plasticity of their response to novel environments encountered during invasion.