Variability and Number of Circulating csd Alleles in a Honey Bee Breeding Population After Four Years of Single-Drone Insemination

Background: Varroa destructor is the major threat to honey bee health, and selective breeding for resistance traits such as Varroa-sensitive hygiene represents a promising long-term strategy for controlling mite populations. However, breeding programs that rely on highly controlled mating schemes, including single-drone instrumental insemination, may reduce allelic diversity at the complementary sex determiner (csd) locus, potentially increasing the production of non-viable diploid males and compromising colony fitness. Methods: To evaluate whether csd diversity can be maintained under these conditions, we characterized the hypervariable region of csd in a selectively bred Apis mellifera population subjected to four years of selection. Using a validated de novo assembly pipeline, we reconstructed 43 amino-acid sequences from 33 diploid worker pupae sampled across 13 colonies. Results: Seven distinct alleles were identified, five of which were shared among multiple colonies and corresponded to variants already described in the literature, while two were private to individual colonies and novel in the literature. Colony-level frequency data revealed a moderate diversity: the most common allele was detected in nine colonies, with an allelic frequency of 31%. Moreover, the expected heterozygosity of the population was estimated at 0.79. Conclusions: Overall, these findings show that csd diversity can be partially maintained even under strong selective pressure when multiple maternal lines are retained, and they underscore the importance of incorporating genetic information into breeding decisions to support the long-term sustainability of selective breeding programs.