Analysis of genetic variability and haplotype correlation between honey bee Apis mellifera and honey bee mite Varroa destructor

Abstract

In this study, genetic variability of honey bee Apis mellifera and honey bee mite Varroa destructor populations was analyzed, monitoring haplotype-based distribution pattern between the host and the parasite. For the identification of honey bee haplotypes, samples from 48 honey bee colonies were used, while 245 Varroa mite samples originating from 29 colonies were analyzed for haplotype identification and monitoring correlation between A. mellifera and V. destructor haplotypes. Sequencing of tRNAleu-cox2 region revealed six A. mellifera haplotypes, with C2aa haplotype observed for the first time. Different frequency of identifed bee haplotypes was detected in total sample, with C2d haplotype being the most frequent, and C2aa and C2i haplotypes with the lowest frequency. Distribution of A. mellifera haplotypes was found to be dependent on the locality, with three sites represented by only one haplotype, one locality represented by two haplotypes and four localities represented by three honey bee haplotypes. Sequencing results of V. destructor mitochondrial fragments revealed the point mutations at position 1932 and 10133 mtDNA within cox1 and cytb sequences, respectively. Based on these polymorphic sites, two novel Varroa haplotypes were defined and named as Serbia 1 (S1) and Peshter 1 (P1). Both detected mutations were the silent ones and did not affect the protein sequence. At the sequence chromatograms in some of the analyzed samples, double peaks were detected at haplotype-defining sites within cox1 and cytb, observing the phenomenon of nucleotide heteroplasmy for the first time in V. destructor. After the polymorphic sites had been observed using the sequencing method, ARMS and RFLP assays were performed for identification of Varroa haplotypes and detection of heteroplasmic individuals. Results of the cox1 sequence analysis showed that 48,2% of individuals belonged to the K haplotype, 31,8% belonged to the S1 haplotype and 20,0% Varroa samples was found to be heteroplasmic...

U ovom radu ispitivana je genetička varijabilnost medonosne pčele Apis mellifera i pčelinjeg krpelja Varroa destructor, uz praćenje korelacije u distribuciji haplotipova domaćina i parazita. Za identifikaciju haplotipova pčela korišćeni su uzorci iz 48 pčelinjih društava sa osam lokaliteta na teritoriji Srbije, dok je za tipizaciju varoa i praćenje korelacije haplotipova domaćina i parazita analizirano 245 uzoraka varoe iz 29 košnica. Sekvencioniranjem tRNAleu-cox2 regiona definisano je šest haplotipova A. mellifera, pri čemu je haplotip C2aa opisan po prvi put. Zastupljenost identifikovanih haplotipova pčela razlikovala se u ukupnom uzorku, pri čemu je najzastupljeniji bio haplotip C2d, dok su najmanje prisutni bili haplotipovi C2aa i C2i. Distribucija haplotipova A. mellifera razlikovala se u zavisnosti od lokaliteta, pri čemu je na tri posmatrana lokaliteta utvrđen po jedan haplotip, na jednom lokalitetu dva i na četiri lokaliteta utvrđeno je prisustvo tri haplotipa pčela. Rezultati sekvencioniranja delova mitohondrijalnih gena V. destructor otkrili su prisustvo tačkastih mutacija na pozicijama 1932 i 10133 mtDNK unutar cox1 i cytb sekvenci. Na osnovu tih polimorfnih mesta, definisana su dva nova haplotipa varoe koji su nazvani Srbija 1 (S1) i Pešter 1 (P1). Obe otkrivene mutacije po svojoj prirodi spadaju u „tihe‟ mutacije koje ne dovode do promene proteinskog lanca. Na hromatogramima pojedinih uzoraka otkriveni su dupli pikovi na haplotip definišućim mestima unutar cox1 i cytb, čime je po prvi put utvrđena nukleotidna heteroplazmija kod vrste V. destructor. Nakon utvrđivanja polimorfnih mesta metodom sekvencioniranja, za identifikaciju haplotipova varoe i otkrivanje jedinki sa heteroplazmijom korišćene su ARMS i RFLP metode. Analizom cox1 sekvence ARMS metodom utvrđeno je 48,2% jedinki K haplotipa, 31,8% jedinki S1 haplotipa i 20,0% jedinki sa heteroplazmijom...