Pollen Exposed to Aerial Pesticide Spray Is a Major Exposure Pathway for Stingless Bees

Testing possible exposure routes of pesticides is essential for assessing the risks to pollinators. In Australia, the pesticide Bacillus thuringiensis var. israelensis (Bti) is widely used in mosquito control programs and applied aerially to mangrove and saltmarsh habitats. Bti formulations contain both bacterial spores and toxins, allowing detection using DNA-based methods, whereas bee foraging behaviour can be quantified by pollen DNA metabarcoding. We combined these approaches to investigate both the foraging behaviour of stingless bees and their potential exposure to Bti in coastal mosquito habitats. Sentinel hives of the stingless bee Tetragonula carbonaria (Meliponini) were placed at two field sites subjected to routine aerial Bti treatments. Stingless bee foraging behaviour was assessed by ITS2 metabarcoding of pollen collected from returning foragers, and a Bti-specific qPCR assay was applied to the same pollen samples, hive stores and the bodies of foraging bees after pollen removal. ITS2 metabarcoding revealed that foragers predominantly visited Avicennia marina (grey mangrove; Acanthaceae) flowers during the early exposure period, a period of peak flowering in A. marina, shifting to Schinus terebinthifolia (Brazilian peppertree; Anacardiaceae) and other non-mangrove species later in the study. Bti was detected in pollen loads from 27% of pollen samples (n = 184) and 4% of adults (n = 287) after pollen removal, identifying contaminated A. marina pollen as the primary exposure pathway in the studied environments. A single pollen pot in one of the eight tested was positive, suggesting potential accumulation of Bti in hive stores. Detected Bti concentrations matched the expected concentrations from field applications and likely pose little risk to stingless bees, but our findings highlight exposure mechanisms that are likely to be significant for pesticides with greater toxicity. The Bti qPCR system developed here provides a way to experimentally manipulate and further investigate insecticide exposure pathways for stingless bees.