Microbiota diversity and hygienic behavior in a honey bee breeding population: Insights into Varroa resistance

Hygienic behavior is a key trait in the western honey bee (Apis mellifera) associated with resistance to diseases and tolerance to the parasitic mite Varroa destructor. Worker bees expressing this behavior are able to detect and remove unhealthy or infested brood, thereby limiting pathogen transmission and mite reproduction. In recent years, the gut microbiota has emerged as an important factor in honey bee health, influencing immunity and disease resistance. Hygienic behavior is a form of social immune defense; however, whether gut microbiota composition contributes to variation in this disease-resistance mechanism remains poorly understood. This study investigated the relationship between gut microbiota composition and hygienic behavior in a honey bee breeding population selected for docility, honey production, and hygiene since 2015. Seventy-seven colonies were sampled at three time points between June and October 2021 for gut microbiota analysis using high-throughput sequencing. Hygienic behavior was evaluated using the average pin test score, calculated from tests performed in March and July. Significant association were identified between gut microbiota diversity and hygienic behavior, with marked seasonal pattern. Colonies with higher pin test scores showed increased alpha diversity in October, while beta-diversity analyses indicated significant difference in July. In particular, LAB genera such as Lactobacillus, Bifidobacterium, and Bombilactobacillus, were more abundant in highly hygienic colonies in July, suggesting a microbiota configurations potentially linked enhanced immune capacity and resilience to brood diseases and Varroa infestations. Overall, these findings support a seasonal association between gut microbiota composition and hygienic behavior in honey bees and highlight specific microbial taxa that may contribute to colony robustness. This study provides a basis for future research exploring the functional role of microbiota in social immunity and its potential integration into selective breeding strategies aimed at improving colony health.