Scientific Reports
Honeybee colonies are under the threat of many stressors, biotic and abiotic factors that strongly affect their survival. Recently, great attention has been directed at chemical pesticides, including their effects at sub-lethal doses on bee behaviour and colony success; whereas the potential side effects of natural biocides largely used in agriculture, such as entomopathogenic fungi, have received only marginal attention. Here, we report the impact of the fungus Beauveria bassiana on honeybee nestmate recognition ability, a crucial feature at the basis of colony integrity. We performed both behavioural assays by recording bee guards' response towards foragers (nestmate or non-nestmate) either exposed to B. bassiana or unexposed presented at the hive entrance, and GC-MS analyses of the cuticular hydrocarbons (CHCs) of fungus-exposed versus unexposed bees. Our results demonstrated that exposed bees have altered cuticular hydrocarbons and are more easily accepted into foreign colonies than controls. Since CHCs are the main recognition cues in social insects, changes in their composition appear to affect nestmate recognition ability at the colony level. The acceptance of chemically unrecognizable fungus-exposed foragers could therefore favour forager drift and disease spread across colonies. Bees are declining worldwide with considerable consequences on the pollination services they provide for crop production and the integrity of terrestrial ecosystems 1-3. Agriculture intensification, pesticide exposure, and increased pressure of native and invasive parasites/pathogens have largely contributed to such decline 1,4-10. As the adoption of intensive farming models aimed at feeding the world's ever-growing population, cannot be separated from the massive use of agrochemicals 11,12 , in the last decades, an increasing effort has been dedicated to biological control strategies (biocontrol) to develop a more eco-friendly pest management approach in agriculture 13-15. Nowadays, the use of microbial pathogens, parasites/parasitoids or predators to cope with insect pests, has partially replaced conventional synthetic plant-protection products in many countries 12,15-17. Among microbial pathogens, entomopathogenic fungi are extensively used as natural biocides in organic agriculture 18-23. The worldwide-distributed Beauveria bassiana is a natural biocide widely used since the '80s 19,21,22. This fungus attacks insects percutaneously: hydrophobic spores adhere to the cuticular hydrocarbons (CHCs) layer, germinate and penetrate the insect cuticle, killing the host a few days after the infection 20,21. The frequent use of B. bassiana as a biocontrol agent is justified by its proved efficacy on target pests and by the low cost compared to conventional chemical insecticides; notably, potential side effects on humans and other non-target organisms have so far been considered as negligible 21. Honeybees often forage on crops biologically controlled with B. baussiana 24,25 therefore, spores of the fungus are likely to adhere to the insect body during the repeated foraging flights 21,24,25 but such B. baussiana spore contamination does not seem to represent a threat for bee survival 25,26. The apparent low sensitivity of insect pollinators, such as honeybees and bumblebees, to this entomopathogenic agent has even encouraged the possible use of bee foragers as vectors to disseminate fungal spores on crops against target insect pests 24,25,27 or the direct spread of B. bassiana spores inside hives to control Varroa mite populations 28,29. In recent years a strong debate has developed about potential side effects of chemical biocides on non-target species, especially insect pollinators 30-33. Sub-lethal doses of insecticides and pesticides have been reported to influence behavioural traits, such as flight capacity, orientation ability and memory in honeybees and bumblebees, which in turn affect foraging performance or the return of foragers to their colony 31-40. In this framework, beside the importance of toxicological tests to evaluate the survival of target and not-target insects, it is also crucial to assess the potential effects of biocides on the behaviour of non-target species as well as on the complex interactions at the colony level of non-target social species.