Microbiome composition shapes temperature tolerance in a Hawaiian picture-winged Drosophila

Hawaiian picture-winged Drosophila are undergoing rapid biodiversity loss, with 12 species listed as endangered and others in decline. Microbiota are increasingly recognized as contributors to host adaptation and influence stress tolerance, reproduction and survival. We investigated the role of microbial communities in two Drosophila basisetae populations from Hawaiian rainforests at 900 m and 1200 m elevation. Microbiome profiling of wild flies by high throughput amplicon sequencing revealed distinct bacterial and fungal communities between sites. To test microbiome effects on host physiology, we conducted a fully factorial research design with microbiome inoculations in laboratory-reared flies acclimated at 18°C (control) or 24°C (stressful). Flies receiving low-elevation microbiota exhibited higher survival across temperatures, whereas those given high-elevation microbiota produced more eggs, indicating microbiome-mediated differences in survival and reproductive investment. Activity levels were higher when flies received microbiota from their native population, whereas critical thermal maximum and male accessory gland size revealed complex interactions among microbiome, temperature and population. These population effects are consistent with whole-genome resequencing of wild-caught flies that identified outlier SNPs in genes associated with immunity, heat tolerance and reproduction, despite signs of admixture and gene flow across populations. Overall, these results indicate that microbiome-host-environment interactions may modulate thermal tolerance and reproduction in population- and environment-specific ways and contribute to both phenotypic plasticity and evolutionary adaptation. These insights inform conservation strategies for Hawaiian Drosophila that incorporate microbial management to bolster adaptive potential and resilience in Hawaiian ecosystems.