A survey of mutations in the Cochliomyia hominivorax (Diptera: Calliphoridae) esterase E3 gene associated with organophosphate resistance and the molecular identification of mutant alleles
Introduction
The infestation of live vertebrate hosts by dipteran larvae, a condition known as myiasis (Zumpt, 1965), remains an unsolved problem for animal production in Neotropical regions. Cochliomyia hominivorax (Coquerel) is one of the most important myiasis-causing flies in the Neotropics (Hall and Wall, 1995) and is responsible for severe economic losses to the livestock industry, mainly by reducing the quality of leather and the production of milk and meat. In South American countries, C. hominivorax has been controlled by applying insecticides, particularly organophosphate (OP)-based compounds. However, the improper and continuous use of these chemicals can lead to the selection of OP-resistant strains.
The major mechanisms of insecticide resistance described so far involve either metabolic detoxification of the insecticide before it reaches its target site, or changes in the sensitivity of the target site that abolish its susceptibility to the insecticide (Hemingway, 2000). The carboxylesterase-mediated detoxification of insecticides has been reported for more than 30 medical, veterinary and agricultural insect pests (reviewed in Hemingway and Karunaratne, 1998). A decrease in carboxylesterase activity has been observed in OP-resistant strains of Lucilia cuprina (Hughes and Raftos, 1985), Musca domestica (Van Asperen and Oppenoorth, 1969) and Chrysomya putoria (Townsend and Busvine, 1969). These findings have been hypothesized to involve a mutant ali-esterase in which a structural mutation in a carboxylesterase results in a reduced ability to hydrolyze aliphatic ester substrates, such as methyl butyrate and naphthyl acetate, but an acquired ability to hydrolyze OP substrates (Claudianos et al., 1999).
The LcαE7 gene (Newcomb et al., 1997b) encodes the major ali-esterase of L. cuprina, also known as esterase isozyme E3. L. cuprina αE7 alleles from three strains (an OP-susceptible, a diazinon and a malathion-resistant) have been isolated and expressed in vitro (Campbell et al., 1998). Biochemical assays using these expressed proteins showed that a single amino acid substitution at position 137 (Gly137Asp) in the diazinon-resistant strain simultaneously abolished the ali-esterase activity and increased the rate of dephosphorylation to yield OP insecticide hydrolase activity, especially of diethyl OPs. Diethyl OP insecticides contain two ethoxy groups attached to their central phosphorus atom and are commonly used to control C. hominivorax. In contrast, malathion-resistant strain contained a different amino acid substitution (Trp251Leu) that conferred specificity towards dimethyl OPs (Campbell et al., 1998), which are not commonly used to control C. hominivorax. In the putative E3 model based on the solved tertiary structure of the related acetylcholinesterase from Torpedo californica (Sussman et al., 1991), the Gly137Asp mutation is located in the motif known as the oxyanion hole and Trp251Leu is part of the acyl binding site within the active site of the insect esterases (Newcomb et al., 1997b, Campbell et al., 1998).
Campbell et al. (1997) showed that there is substantial functional equivalence in the mutant enzymes associated with esterase-mediated resistance across the species M. domestica (MdαE7), Drosophila melanogaster (DmαE7) and L. cuprina (LcαE7). There are many similarities among these systems, including amino acid sequence, electrophoretic mobilities and inhibitor sensitivities, which strongly suggests that they are orthologous (Bigley and Plapp, 1961, Hughes and Raftos, 1985, Spackman et al., 1994, Parker et al., 1996).
Based on the foregoing studies, we have isolated and sequenced a region of the E3 gene in C. hominivorax, which is orthologous to that previously described for L. cuprina and contains the Gly137Asp and Trp251Leu mutations. The characterization of this sequence allowed the development of a rapid and efficient method for identifying Gly137Asp mutants of C. hominivorax.
Section snippets
Amplification and sequencing
Larvae of C. hominivorax were collected from the wounds of infested animals from several locations and stored at −70 °C. Genomic DNA from eight individuals of C. hominivorax was extracted according to Infante and Azeredo-Espin (1995). Four primers previously described for L. cuprina, 7F1, 7F1a, 7R2 and 7R4 (Newcomb et al., 1997a, Newcomb et al., 1997b), were used to amplify the orthologous region in C. hominivorax. A novel primer, 7R3a (5′-ATCCTTATCATTATTTTCACCC-3′), was designed to specifically
Amplification and sequencing
The primer pairs 7F1/7R2 and 7F1/7R4 did not amplify the expected fragments, probably because of the low specificity between these fragments and the C. hominivorax sequences. The 7F1a/7R4 primer pair amplified a fragment of approximately 700 bp, as expected from L. cuprina sequences (Newcomb et al., 1997b), and this fragment was used to design a C. hominivorax-specific reverse primer (7R3a). The 7F1a/7R3a primer pair amplified a fragment of 536 bp and was used for all subsequent amplifications,
Discussion
The primers 7F1a, previously described for L cuprina, and 7R3a, designed specifically for C. hominivorax, amplified a 536 bp fragment that included intron III (63 bp) and the exon region coresponding to positions 359–831 in the L. cuprina αE7 nucleotide sequence, as well as both amino acid positions (137 and 251) related to OP resistance. Like LcαE7, DmαE7 and MdαE7, the orthologous region in C. hominivorax (ChαE7) contained motifs that are highly conserved among carboxyl/cholinesterases and
Acknowledgements
The authors thank N.M. da Silva for comments on the manuscript, R.A. Rodrigues for technical assistance and M.S. Couto for maintaining the screw-worm colonies. We are also grateful to M.L. Lyra for help with the sample collections. This research was supported by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo to A.M.L.A.E (FAPESP, grant no. 03/01458-9). R.A.C. and T.T.T. were supported by fellowships from FAPESP (grant nos. 04/12532-8 and 02/00035-4).
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