Clonagem e sequenciamento parcial do gene co-i de varroa destructor haplótipo J: um método de apoio à caracterização molecular

Abstract

Honey bees are fundamental pollinator insects ecosystems and economically relevant. Through its relatively simple and inexpensive handling, the exploitation of its products, mainly honey, makes beekeeping quite profitable for the rural producer. Reduction of populations of Apis mellifera has been registered in many countries. Most of the reports have been associated with the phenomenon called Colony Collapse Disorder (CCD), a sum of adverse conditions including pathogens, parasites and sub-lethal doses of agrochemicals, culminating in the abandonment of hives. In Brazil, CCD is less observed and the reports of losses of colonies in the country have been more associated with accidental poisoning. However, is necessary evidence the presence of such pathogens and parasites in Brazilian hives. One of factors related to the disappearance of bees is the Varroa destructor ectoparasite. The parasite, besides being a vector of pathogens, such as bacteria and viruses, also sucks the hemolymph of adult bees and larvae, affecting its development and shortening life span. Because of this, the cited mite is considered a threat to worldwide beekeeping. In this way, identification of the different haplotypes, directly related to the reproductive vigor of species, from a given region is fundamental for implementation of control programs. Within this context, this work aimed to establish, within APIPAMPA Research Group, the method for identification of V. destructor J haplotype. To this end, cloning and partial sequencing of a 458 bp fragment of the mitochondrial gene Cytochrome C Oxidase subunit I (co-I) from V. destructor was performed. The fragment was amplified by PCR, using the COXF primers[5´GG(A/G)GG(A/T)GA(C/T)CC(A/T)ATT(C/T)T(A/T)TATCAAC3´] and COXR [5´GG(A/T)GACCTGT(A/TA(A/T)AATAGCAAATAC3´], purified and cloned. Subsequent to cloning and transformation, the fragment was sequenced. Sequence obtained was deposited in the database of the National Center for Biotechnology Information (NCBI) under accession number KX458253. Sequencing results enabled the confirmation of the identity of V. destructor J haplotype by comparing the alignment of nucleotide sequence obtained with sequences already published by other authors. The data obtained in the present work allow next researchers to use a suitable method to identify the V. destructor J haplotype in APIPAMPA Research Group laboratories with greater accuracy, allowing the monitoring of the fluctuation of this haplotype in infested apiaries.