Warming restructures the Drosophila melanogaster microbiome and constrains genotype-dependent responses to agrochemical contamination

Insects are increasingly exposed to concurrent environmental stressors, including climate warming and chronic agrochemical contamination. Although both temperature and insecticides can reshape insect-associated microbiomes, their combined effects, particularly in the context of host genetic variation, remain poorly understood. Here, we used a fully factorial design in Drosophila melanogaster to test how thermal regime, sublethal insecticide exposure, and detoxification genotype influence early-life performance and bacterial community structure. Flies carrying either the insecticide-resistant Cyp6g1-BA haplotype or the susceptible Cyp6g1-M haplotype were reared under a cool temperature cycle (averaging 18 °C) or a warm temperature cycle (averaging 28 °C), with or without insecticide treatment. Warming was the dominant driver of microbiome restructuring, significantly reducing observed richness and Shannon diversity and promoting community simplification characterised by increased dominance of a limited number of genera, notably Acetobacter. Under cool conditions, insecticide treatment elicited genotype-dependent microbiome responses across approximately 27% of retained genera. In contrast, under warming this interaction-responsive fraction declined sharply to ~5%, despite comparable effect sizes among responsive taxa, indicating a contraction of microbiome plasticity under thermal stress. Parallel to these microbiome patterns, genotype-specific host survival costs emerged under combined warm and insecticide exposure. Together, our results show that environmental temperature acts as the strongest overarching factor governing gut community diversity and stability; warming radically restructures microbiome composition while constraining genotype-dependent responses to agrochemical stress. This suggests that rising temperatures may reduce microbiome-mediated buffering and narrow the ecological resilience of insect populations facing layered environmental challenges.