Combined effect of a neonicotinoid insecticide and a fungicide on honeybee gut epithelium and microbiota, adult survival, colony strength and foraging preferences.

Fungicides, insecticides and herbicides are widely used in agriculture to counteract pathogens and pests. Several of these molecules are toxic to non-target organisms such as pollinators and their lethal dose can be lowered if applied as a mixture. They can cause large and unpredictable problems, spanning from behavioural changes to alterations in the gut. The present work aimed at understanding the synergistic effects on honeybees of a combined in-hive exposure to sub-lethal doses of the insecticide thiacloprid and the fungicide penconazole. A multidisciplinary approach was used: honeybee mortality upon exposure was initially tested in cage, and the colonies development monitored. Morphological and ultrastructural analyses via light and transmission electron microscopy were carried out on the gut of larvae and forager honeybees. Moreover, the main pollen foraging sources and the fungal gut microbiota were studied using Next Generation Sequencing; the gut core bacterial taxa were quantified via qPCR. The mortality test showed a negative effect on honeybee survival when exposed to agrochemicals and their mixture in cage but not confirmed at colony level. Microscopy analyses on the gut epithelium indicated no appreciable morphological changes in larvae, newly emerged and forager honeybees exposed in field to the agrochemicals. Nevertheless, the gut microbial profile showed a reduction of Bombilactobacillus and an increase of Lactobacillus and total fungi upon mixture application. Finally, we highlighted for the first time a significant honeybee diet change after pesticide exposure: penconazole, alone or in mixture, significantly altered the pollen foraging preference, with honeybees preferring Hedera pollen. Overall, our in-hive results showed no severe effects upon administration of sublethal doses of thiacloprid and penconazole but indicate a change in honeybees foraging preference. A possible explanation can be that the different nutritional profile of the pollen may offer better recovery chances to honeybees.

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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.