HRM Analysis of Spermathecal Contents to Determine the Origin of Drones that Inseminated Honey Bee Queens

Europe, Africa and the Middle East have several original subspecies of the western honey bee (Apis mellifera L.), each with distinctive characteristics. These subspecies are the product of natural selection in their native range. Nevertheless, anthropogenic impacts such as migratory beekeeping and use of non-native queens result in an admixture of these subspecies and their ecotypes. I aimed to develop a SNP-based method to detect whether queen honey bees were mated with drones from foreign subspecies. For this purpose, Caucasian and Italian queens and drones were reared. Each queen was instrumentally inseminated with mixed semen collected from Caucasian (4 μl) and Italian drones (4 μl). The spermathecae of queens were dissected out after the onset of oviposition. The DNA was extracted from each spermatheca and from the thoraces of Caucasian and Italian drones. Seven regions on mtDNA that were isolated from drones were sequenced to determine the SNPs, enabling the discrimination of Caucasian sperm from Italian in spermathecal contents. Based on one SNP (11606. bp, T/C) residing on the Cytb gene, a specific primer was designed to be used in High Resolution Melting (HRM) analysis. HRM analysis indicated that heteroduplex peak profiles were present in all spermathecal contents of instrumentally inseminated queens. The results provide proof of the concept that the presence of likely non-native mitochondrial lineages can be detected by HRM analysis based on the SNP genotyping of spermathecal contents.