The development of resistance to insecticides has become a classic exemplar of evolution occurring within human time scales. In this study we demonstrate how resistance to DDT in the major African malaria vector Anopheles gambiae is a result of both target-site resistance mechanisms that have introgressed between incipient species (the M- and S-molecular forms) and allelic variants in a DDT-detoxifying enzyme. Sequencing of the detoxification enzyme, Gste2, from DDT resistant and susceptible strains of An. gambiae, revealed a non-synonymous polymorphism (I114T), proximal to the DDT binding domain, which segregated with strain phenotype. Recombinant protein expression and DDT metabolism analysis revealed that the proteins from the susceptible strain lost activity at higher DDT concentrations, characteristic of substrate inhibition. The effect of I114T on GSTE2 protein structure was explored through X-ray crystallography. The amino acid exchange in the DDT-resistant strain introduced a hydroxyl group nearby the hydrophobic DDT-binding region. The exchange does not result in structural alterations but is predicted to facilitate local dynamics and enzyme activity. Expression of both wild-type and 114T alleles the allele in Drosophila conferred an increase in DDT tolerance. The 114T mutation was significantly associated with DDT resistance in wild caught M-form populations and acts in concert with target-site mutations in the voltage gated sodium channel (Vgsc-1575Y and Vgsc-1014F) to confer extreme levels of DDT resistance in wild caught An. gambiae.

Our laboratory recently developed a diagnostic test (ELISA) for human fascioliasis based on the detection of serum IgG4 antibodies reactive with Fasciola hepatica cathepsin L1 (CL1). In the present study, we have used recombinant CL1, generated by functional expression of the cDNA in Saccharomyces cerevisiae, in this immunodiagnostic test and compared its performance with native CL1. Sera obtained from 64 individuals living in Cutusuma village in the northern Altiplano of Bolivia, a region with a high prevalence of human fascioliasis, were analyzed by the IgG4-ELISA. A highly statistically significant correlation (r2 = 0.751, P < 0.001) was demonstrated between the absorbances obtained using the recombinant and native proteins. These assays showed that 38 (59%) of the individuals tested were seropositive for fascioliasis, whereas only 26 of them were coprologically positive for F. hepatica eggs. All seronegative patients were also coprologically negative. Serum from individuals infected with schistosomiasis mansoni, cysticercosis, hydatidosis, and Chagas disease did not contain antibodies reactive with the recombinant or native CL1. Therefore, recombinant CL1 shows excellent potential for the development of the first standardized assay for the sensitive and specific diagnosis of human fascioliasis. Finally, our data supports earlier reports on the high prevalence of human fascioliasis in the Bolivian Altiplano, which collectively suggest that the disease has been endemic there for more than a decade.