The characterisation of the Maltese honey bee using morphometric and mitochondrial DNA analysis.

Abstract

This study aimed to determine the status of the Maltese honey bee using morphometric and mitochondrial DNA analysis. In addition, the presence of introgression from neighbouring honey bee subspecies was investigated. Between October and December 2013, Apis mellifera samples were collected from 52 colonies across the Maltese Islands. Eight to ten bees per colony from a total of 35 colonies were dissected and examined for the 35 standard morphometric parameters proposed by Ruttner et al. (1978). Colony means were then calculated and compared to a number of reference subspecies (obtained from the Institut fϋr Bienenkunde, Oberursel) using Principal Components Analysis, Factor analysis and K-means cluster analysis. The DNA from two samples for each of the 52 colonies were subjected to mitochondrial DNA analysis. The latter involved the amplification of the intergenic region between the Cytochrome oxidase C sub-unit I (COl) and Cytochrome oxidase C sub-unit ii (COII) genes, using the primer pair E2-H2. The latter intergenic region spans repeats of non-coding sequences, known as the P and Q sequences. Samples of each amplified fragment were subjected to RFLP analysis using the restriction endonuclease Oral, as well as to Sanger sequencing. Multiple alignment as well as manual investigation for specific nucleotide changes, with the aim of designating a specific mitochondrial haplotype, was then carried out. A neighbour-joining phylogenetic dendrogram was also constructed to observe clustering patterns. PCA and K-means cluster analysis confirmed the identity of the Maltese honey bee, A. m. ruttneri, as a separate subspecies, as well as showing the potential presence of a subpopulation. Introgression was minimally observed with the Sicilian subspecies and was absent with respect to the Italian subspecies. 83% of the samples were found to display the African lineage mitochondrial haplotypes A8, A9 and A4, whilst 17% of the samples were found to pertain to the European haplotypes Cl, C2 and M7. The sequences of the different COl-COll intergenic regions for the native A. m. ruttneri were established for the first time in the Maltese Islands. Some samples displayed a novel Po structure, that was never reported in previous studies. The use of both morphometry and mitochondrial DNA analysis provided a more complete picture of the status of the Maltese honey bee. Due to shared common North African haplotypes (A8 and A9), the Maltese honey bee could not be distinguished from the Sicilian subspecies based on mitochondrial DNA analysis. However, morphometry offered better resolution in completely distinguishing between these two subspecies. It was postulated that the origin of both the Maltese and Sicilian subspecies took place during the Messinian Crisis, through a northern migration of the North African A. mellifera subspecies. Aside from supporting the A. mellifera dispersion routes proposed by Garnery et al. (1992), this study gave further insight to the gaps present in the latter routes, showing possible evidence for the migration of North African bees towards Europe. The distinct nature of the Maltese honey bee was surprising after the infestation by the Varroa mite and subsequence importations of non-native bees and although only a small percent of colonies were found to pertain to non-native European haplotypes, proper regulation of imported honey bee nucs must be carried out to safeguard the native Maltese subspecies.