Bees under interactive stressors: the novel insecticides flupyradifurone and sulfoxaflor along with the fungicide azoxystrobin disrupt the gut microbiota of honey bees and increase opportunistic bacterial pathogens

The gut microbiome plays an important role in bee health and disease. But it can be disrupted by pesticides and in-hive chemicals, putting honey bee health in danger. We used a controlled and fully crossed laboratory experimental design to test the effects of a 10-day period of chronic exposure to field-realistic sublethal concentrations of two nicotinic acetylcholine receptor agonist insecticides (nACHRs), namely flupyradifurone (FPF) and sulfoxaflor (Sulf), and a fungicide, azoxystrobin (Azoxy), individually and in combination, on the survival of individual honey bee workers and the composition of their gut microbiota (fungal and bacterial diversity). Metabarcoding was used to examine the gut microbiota on days 0, 5, and 10 of pesticide exposure to determine how the microbial response varies over time; to do so, the fungal ITS2 fragment and the V4 region of the bacterial 16S rRNA were targeted. We found that FPF has a negative impact on honey bee survival, but interactive (additive or synergistic) effects between either insecticide and the fungicide on honey bee survival were not statistically significant. Pesticide treatments significantly impacted the microbial community composition. The fungicide Azoxy substantially reduced the Shannon diversity of fungi after chronic exposure for 10 days. The relative abundance of the top 10 genera of the bee gut microbiota was also differentially affected by the fungicide, insecticides, and fungicide-insecticide combinations. Gut microbiota dysbiosis was associated with an increase in the relative abundance of opportunistic pathogens such as Serratia spp. (e.g. S. marcescens), which can have devastating consequences for host health such as increased susceptibility to infection and reduced lifespan. Our findings raise concerns about the long-term impact of novel nACHR insecticides, particularly FPF, on pollinator health and recommend a novel methodology for a refined risk assessment that includes the potential effects of agrochemicals on the gut microbiome of bees.