Esterases e resistência a acaricidas no carrapato bovino Boophilus microplus (Acari, Ixodidae)

Abstract

The cattle tick Boophilus microplus is one of the most important ectoparasites for the Brazilian farming, and it is responsible, according to Agriculture Ministry, for direct and indirect damages to livestock of, approximately, two billions dollars a year. The traditional method of control is based on the intensive use of pyrethroids and organophosphate pesticides, whose systematic use has been promoting the selection of resistant genotypes in tick populations. Point mutations in esterase genes and the increase of production of these enzymes represent important strategies of resistance, conferring insensivity to neurotoxic effects of these organic compounds in this specie. In this work, esterase patterns were compared among the stages of the life cycle of B. microplus, in non-denaturing polyacrilamide gel electrophoresis (PAGE), using specific staining for esterases. Electrophoretical results indicate the presence of nine regions displaying esterase activity. These esterases were considered as products from nine alleles distributed in seven gene loci. Esterases that were detected in all the stages of development were considered as products of housekeeping genes. Some stage-specific and sex-specific esterases were also detected. Strains with different levels of resistance were selected after the resistance test with the malation organophosphate and with the deltametrin pyrethroid in engorged females from a farm of Uberlândia region (MG), with description of resistance. Electrophoretical profiles of esterase pattern using non-denaturing polyacrilamide gel electrophoresis among malathion and deltametrin-susceptible, tolerant and resistant tick strains revealed four bands displaying esterase activity, named EST-1 to EST-4. The esterases EST-3 and EST-4, classified as carboxylesterases, were observed in all strains. The esterase EST-2, classified as an acetylcholinesterase in inhibitor tests, was detected in all strains, but it has presented an increasing degree of staining, from susceptible to resistant strains, indicating an increase in the enzyme production according to resistance level, probably as a result of gene amplification or post-traditional alterations. The enzyme EST-1, classified as an acetylcholinesterase, was detected exclusively in tolerant and resistant strains to both acaricides, however, with higher activity in resistant strains. These data indicate that the detected acetylcholinesterases could xviii be representing important detoxification strategy developed to overcome the effects of the acaricides. These strains were also analyzed by the techniques Low Ionic Strength Single Stranded Conformation Polymorphism (LIS-SSCP) and Polimerase Chain Reaction-Restriction Fragment Length Polymorphism (PCRRFLP) to investigate mutations in a putative carboxylesterase fragment. The digestion of the amplified fragment of 372bp with the restriction enzyme Eco RI showed three band patterns, associated to three distinct genotypes: W, H and M, which were observed in different frequencies in the analyzed strains. The homozygous wild genotype (W) was detected only in the susceptible strains, in high frequency. The heterozygous genotype (H) was observed in all strains, however, in higher frequency in tolerant strains, and the homozygous mutant genotype (M) was observed only in the resistant and tolerant strains, with higher frequency in the resistant strains, suggesting that the resistance can be associated to the presence of the m mutant allele (EcoRI polymorphism). The genotyping by LIS-SSCP showed four haplotypes, named 1, 2, 3 and 4. The haplotype 1 was observed only in the susceptible strains and in high frequency. The haplotype 4 was observed in the resistant and tolerant strains with higher frequency in tolerant strains. The haplotype 3 was observed in resistant and tolerant strains, and was detected in higher frequency in the resistant strains. The haplotype 2, was also detected in resistant and tolerant strains, however, in very low frequency in population. The comparison among the sequences demonstrated the presence of mutations, beyond the EcoRI polymorphism in resistant and tolerant strains. The presence of stop codons was also observed, creating truncated proteins in the susceptible and tolerant strains. The domains analysis showed additional motifs in resistant strain. The obtained data suggest mechanisms mediated by point mutations, gene amplification and post-traditional modifications, which can be altering the activity of the translated proteins and acting together in the expression of resistance in the analyzed population.