Characterization of Boophilus microplus esterases and their role in acaricide resistance.

摘要

Several esterases were detected from reference strains of acaricide resistant and susceptible Boophilus microplus ticks. Esterase activity was detected by histochemical staining and an SDS-PAGE assays. An 87 kDa esterase was detected in the saliva of organophosphate (OP) resistant adult ticks. A 180 kDa esterase was found in larval extracts of OP resistant ticks, and a 47 kDa esterase was abundant in larval extracts of both OP and pyrethroid resistant ticks.;The esterases were inhibited in vitro by the synergist S,S,S-tributiryl phosphorotrithioate (DEF). Both OP resistant ticks (OP resistance factor (RF) of 6.3) as well as field collected ticks (RF 0.5 to 5.4) were compared in a synergist bioassay using DEF. The RF values were reduced, suggesting that some of the esterases found were involved in the acaricide resistance mechanism of the analyzed ticks. Esterase detection by SDS-PAGE and the measurement of specific enzyme activity was found to complement the standard acaricide resistance bloassay.;An esterase gene coding for 409 amino acids was isolated and identified as an esterase B. The nucleotide sequence of the gene encoding the isolated enzyme showed homology with esterase sequences from Culex pipiens and B. microplus. The esterase gene was cloned in the vector Pbacgus2cp in the correct open reading frame for the polh promoter. The recombination of the vector with baculovirus generated a recombinant virus capable of infecting the insect cell line, Sf9.;A 65 kDa protein band was detected by SDS-PAGE esterase assays as active in extracts of Sf9 cells infected with the recombinant virus. No similar esterase activity was detected in wild type virus infected Sf9 cells, or in uninfected cells. Specific esterase activity measured for the cell pellet obtained at 72 hours post recombinant virus infection showed increased activity when compared to controls, however, western immunoblots showed a reaction with a 50 kDa protein. No direct confirmation of expression was obtained from the experimental results.