Mutation Report for: G137D_colho-E3aest7

BACKGROUND: The emergence of insecticide resistance is a fast-paced example of the evolutionary process of natural selection. In this study, we investigated the molecular basis of resistance in the myiasis-causing fly Cochliomyia hominivorax (Diptera: Calliphoridae) to dimethyl-organophosphate (OP) insecticides. METHODS: By sequencing the RNA from surviving larvae treated with dimethyl-OP (resistant condition) and non-treated larvae (control condition), we identified genes displaying condition-specific polymorphisms, as well as those differentially expressed. RESULTS: Both analyses revealed that resistant individuals have altered expression and allele-specific expression of genes involved in proteolysis (specifically serine-endopeptidase), olfactory perception and cuticle metabolism, among others. We also confirmed that resistant individuals carry almost invariably the Trp251Ser mutation in the esterase E3, known to confer OP and Pyrethroid resistance. Interestingly, genes involved in metabolic and detoxifying processes (notably cytochrome P450s) were found under-expressed in resistant individuals. An exception to this were esterases, which were found up-regulated. CONCLUSIONS: These observations suggest that reduced penetration and aversion to dimethyl-OP contaminated food may be important complementary strategies of resistant individuals. The specific genes and processes found are an important starting point for future functional studies. Their role in insecticide resistance merits consideration to better the current pest management strategies.

Livestock production is an important economic activity in Brazil, which has been suffering significant losses due to the impact of parasites. The New World screwworm (NWS) fly, Cochliomyia hominivorax, is an ectoparasite and one of the most important myiasis-causing flies endemic to the Americas. The geographic distribution of NWS has been reduced after the implementation of the Sterile Insect Technique (SIT), being eradicated in North America and part of Central America. In South America, C. hominivorax is controlled by chemical insecticides, although indiscriminate use can cause selection of resistant individuals. Previous studies have associated the Gly137Asp and Trp251Leu mutations in the active site of carboxylesterase E3 to resistance of diethyl and dimethyl-organophosphates insecticides, respectively. Here, we have sequenced a fragment of the carboxylesterase E3 gene (ChalphaE7), comprising part of intron iII, exon eIII, intron iIII and part of exon eIV, and three mitochondrial gene sequences (CR, COI and COII), of NWS flies from 21 locations in South America. These markers were used for population structure analyses and the ChalphaE7 gene was also investigated to gain insight into the selective pressures that have shaped its evolution. Analysis of molecular variance (AMOVA) and pairwise FST analysis indicated an increased genetic structure between locations in the ChalphaE7 compared to the concatenated mitochondrial genes. Discriminant analysis of principal components (DAPC) and spatial analysis of molecular variance (SAMOVA) indicated different degrees of genetic structure for all markers, in agreement with the AMOVA results, but with low correlation to geographic data. The NWS fly is considered a panmitic species based on mitochondrial data, while it is structured into three groups considering the ChalphaE7 gene. A negative association between the two mutations related to organophosphate resistance and Fay & Wu's H significant negative values for the exons, suggest that these mutations evolved under positive selection.

Altered acetylcholinesterase (AChE) has been identified in numerous arthropod species resistant to organophosphate (OP) and carbamate insecticides. The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel), one of the most important myiasis-causing flies in the Neotropics, has been controlled mainly by the application of OP insecticides in its current geographical distribution. However, few studies have investigated insecticide resistance in this species. Based on previous studies about mutations conferring OP resistance in related dipteran species, AChE cDNA was sequenced allowing a survey for mutations (I298V, G401A, F466Y) in NWS populations. In addition, the G137D mutation in the carboxylesterase E3 gene, also associated with OP resistance, was analyzed in the same NWS populations. Only 2/135 individuals presented an altered AChE gene (F466Y). In contrast, a high frequency of the G137D mutation in the E3 gene was found in some localities of Brazil and Uruguay, while the mutant allele was not found in Cuba, Venezuela or Colombia. These findings suggest that the alteration in the carboxylesterase E3 gene may be one of the main resistance mechanisms selected in this ectoparasite. The knowledge of the frequency of these resistance-associated mutations in the NWS natural populations may contribute to the selection of appropriate chemicals for control as part of pest management strategies.

The New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), is one of the most important myiasis-causing flies in South America. It is responsible for severe economic losses to livestock producers, mainly because it causes mortality in newborn calves and reductions in the quality of leather and in the production of milk and meat. The economic losses caused by myiasis, along with those caused by other internal and external parasites, are the main factors limiting meat production. In Brazil, 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. This, associated with the fast development of OP resistance in other myiasis-causing flies, shows the importance of investigating resistance in C. hominivorax. Based on the findings of previous studies, the objective of the current work was to isolate and sequence the E3 gene in C. hominivorax. Mutations at the positions (Gly137 and Trp251) responsible for conferring OP resistance in Lucilia cuprina and Musca domestica L. (Muscidae) were identified in C. hominivorax. In addition, the orthologous region in C. hominivorax contained motifs that are highly conserved among carboxyl/cholinesterases and contribute to the catalytic mechanism of the active site. The characterization of this gene in natural populations of New World screwworm can be an important tool for monitoring resistance to insecticides throughout its current geographic distribution. This will provide information for the selection and implementation of more effective pest management programmes.

Cochliomyia hominivorax (Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. In Brazil, C. hominivorax has been controlled mainly with organophosphate (OP) insecticides, although the inappropriate use of these chemicals can result in the selection of resistant flies. Changes in carboxylesterase activity have been associated with OP insecticides in some arthopodan species. In this work, we isolated and characterized part of the E3 gene in C. hominivorax (ChalphaE7), which contained the same substitutions responsible for the acquisition of OP hydrolase activity in Lucilia cuprina (Calliphoridae). Digestion of the polymerase chain reaction products with a restriction enzyme that specifically recognized the mutation site unambiguously differentiated wild and mutated esterase alleles. The PCR-RFLP assay therefore provided a fast, reliable DNA-based method for identifying C. hominivorax individuals with a mutation in the esterase gene. Further bioassays to determine the association of this mutation with OP resistance in C. hominivorax should allow the development of more effective strategies for managing this species.