Impact of chemical pollutants on the gut microbiome of pollinators

1. Introduction Exposure to chemical pollutants, particularly agrochemicals, has been recognized as one of the leading factors contributing to the decline of pollinator populations recorded in recent years (Goulson et al., 2015). Exposure to agrochemicals may indirectly compromise insect health by disrupting the gut microbiome, which plays crucial roles for insect physiology (Motta and Moran, 2020; Engel and Moran, 2013). Most studies assessing the effects of chemical pollutants on pollinators have focused on honeybees (Apis mellifera) and bumblebees and have evaluated a limited number of substances, mainly insecticides, at typical field application levels (Motta et al., 2018). Thus, there is a significant knowledge gap regarding the effects of sub-lethal concentrations of various chemical pollutants on different pollinator species. In this context, in accordance with the PhD thesis project previously presented, this poster concerns the main results of activity A1: study of the influence of chemical pollutants on the structure of the gut microbial community of the red mason bee Osmia bicornis and of the honeybee Apis mellifera. Experiments are ongoing also on the hoverfly Eristalis tenax. These species were selected because they are among the most important crop pollinators, with social (A. mellifera) or solitary (O. bicornis, E. tenax) behaviour. The chemicals studied are boscalid, copper (II) chloride dihydrate, glyphosate and ivermectin. Boscalid and copper (II) chloride dihydrate act as fungicides, while glyphosate is used as herbicide and ivermectin is a veterinary drug. These compounds have been previously detected as residues in nectar and pollen, making them relevant for studying the potential impacts on the gut microbiome of pollinators (Wallner, 2009; Lewis et al., 2016; Thompson et al., 2014; Sands and Noll, 2022). 2. Materials and Methods A. mellifera and O. bicornis adults were subjected to chronic oral toxicity tests using sublethal doses of boscalid, copper (II) chloride dihydrate, glyphosate and ivermectin, administered singularly and in combination. The tested concentrations were 2 mg/kg for boscalid (Wallner, 2009), 7,2 mg/kg for copper (II) chloride dihydrate (Nikolić et al., 2016), 30 mg/kg for glyphosate (Thompson et al., 2014) and 0,16 μg/ml for ivermectin (Guseman et al., 2016). For O. bicornis, individuals were subjected to a period of three days of pollen-feeding for microbiome acquisition, while for A. mellifera microbiome acquisition was achieved by feeding the specimens with a gut homogenate preparation for five days. In both species, exposure to chemicals lasted four days. To account for potential genetic variability in susceptibility to chemicals, three distinct A. mellifera colonies (F3, F5, and L1) were selected for exposure to boscalid and copper (II) chloride dihydrate. After surface sterilization, insects were dissected and their guts were extracted under sterile conditions (Callegari et al., 2021). Total genomic DNA was extracted from guts with QIAamp PowerFecal Pro DNA Kit (Qiagen). The gut bacterial community of O. bicornis and A. mellifera was investigated by molecular analyses in terms of bacterial abundance using quantitative PCR targeting the phylogenetic marker 16S rRNA gene (Callegari et al., 2021). Additionally, the gut microbiome of A. mellifera was characterized by studying the abundance of fungal species, through quantitative PCR targeting ITS1 region, a reference taxonomic marker for fungi (Callegari et al., 2021). 3. Results and Discussion 3.1 Quantification of gut bacterial abundance in O. bicornis and A. mellifera In O. bicornis, qPCR results targeting the 16S rRNA gene showed that gut bacterial abundance was significantly reduced when specimens were exposed to boscalid, copper (II) chloride dihydrate, and their combination (50:50 mixture), suggesting that fungicides are detrimental for the gut bacterial community. Gut bacterial abundance is not affected by treatments with glyphosate, ivermectin and the mixture of the two compounds. A metataxonomic analysis targeting the 16S rRNA gene is ongoing to evaluate the effect of the chemicals on the structure of the bacterial community of O. bicornis (Callegari et al., 2021). For A. mellifera, no differences among the different treatments in terms of gut bacterial abundance were recorded, apart from colony L1, which was susceptible to treatment with copper (II) chloride dihydrate. This result indicates that the response to chemicals may vary between colonies. qPCR analyses targeting the main bacterial taxa of A. mellifera are ongoing to assess whether exposure to the agrochemicals has affected the core microbiome (Callegari et al., 2021). 3.2 Quantification of fungal load in the gut of A. mellifera Considering individuals from F3 colony, qPCR results targeting ITS1 region showed that exposure to boscalid and to 50:50 boscalid:copper mixture led to a reduction in fungal abundance present in the gut. In F5 and L1 colonies, a significant reduction in fungal load was observed when specimens were fed with boscalid. These findings suggest a potential negative impact of boscalid on the gut-associated fungal community of A. mellifera, confirming that the response to chemicals can differ between colonies. Exposure to glyphosate, ivermectin and to the mixture of the two compounds had no effect on the abundance of fungal species. A metataxonomic analysis targeting the ITS2 region will be carried out to assess the effect of the chemicals on the composition of the fungal community of A. mellifera (Callegari et al., 2021).