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Culex mosquitoes, as well as being vectors of filariasis and Japanese encephalitis, are a world wide biting nuisance. Organophosphorus insecticides (OPs) have been widely used to control Culex populations. Resistance to OPs has occurred and is typically mediated by the increase in non-specific esterase activity. The two esterases involved are classified as 'A' and 'B' esterases with respect to their preference for the substrates α- or β- naphthyl acetate. The commonest phenotype involves two elevated esterases, A2 and B2, which occur in complete linkage disequilibrium. The over expression of esterase B1 is due to gene amplification. Initially, in order to further study the molecular biology of OP resistance, full length cDNAs coding for both A2 and B2 esterases were isolated and sequenced from an OP resistant Sri Lankan strain of Culex quinquefasciatus, PelRR. The B2 esterase cDNA was isolated with PCR using primers sharing homology with the B1 esterase cDNA and has 97.4% homology with esterase B1 at the amino acid level. This confirmed that the B esterases belong to an allelic series. Partial genomic sequences of B2 esterase from PelRR and four other OP resistant Culex strains were identical. This suggests that the initial B2 esterase amplification has occurred only once. However, the cDNA sequence of a B1 esterase cDNA isolated from an OP resistant Cuban strain of Culex quinquefasciatus, MRES, was different to that of the previously published B1 esterase gene sequence. At the genomic level, the haplotype of the Cuban B1 esterase gene, based on EcoRI endonuclease analysis, was also different, suggesting that the initial B1 esterase gene amplification event has occurred at least twice. AB esterase cDNA from an OP susceptible strain, PelSS, has also been partially sequenced. PelSS was derived from the same origins as PelRR but its B esterase cDNA sequence and haplotype of the gene are different. Thus, the B2 esterase gene conferring OP resistance, as well as being amplified, is only found in the resistant strain, PelRR. The A2 esterase cDNA was isolated by screening a PelRR cDNA expression library with an anti-A2 antiserum. The cDNA coded for a protein of 540 amino acids (the same as B2 esterase) and shared 47% amino acid homology with B2 esterase. This strongly suggests that the two genes arose from a duplication of an ancestral counterpart. Furthermore, screening of a PelRR genomic library with A2 and B2 esterase gene probes suggests that the two esterase genes, A2 and B2 are situated in tandem within the genome. PCR was used to amplify the coding region of the PelRR A2 esterase cDNA and this was co-transfected into the baculovirus expression system. The recombinant virus expressed an active A esterase.

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Thesis (M.E.S.)--Evergreen State College, 2008. / "June 2008." Title from title screen (viewed 4/8/2010). Includes bibliographical references (leaves 69-71).

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisors: Charles E. Mason and J. Lindsey Flexner, Dept. of Entomology & Wildlife Ecology. Includes bibliographical references.

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The prevalence of insecticide resistance in vector species around the world is a continuous threat for any success at mitigating the spread of vector-borne diseases. With a limited arsenal of new insecticides, it is crucial for public health programs to understand the geographic range and the genetic mechanisms of resistance to best approach controlling insect vectors. Insecticide resistance is being increasingly observed in phlebotomine sand fly (Diptera: Psychodidae) populations in both the Old World and New World. Sand flies transmit the protozoans that cause leishmaniasis, a disfiguring disease that kills tens of thousands of people each year. The goal of this dissertation was to have both an applied and basic research focus towards understanding resistance in phlebotomines. I began by comparing in vivo and in vitro methods for blood-feeding two species of sand flies, Phlebotomus papatasi and Lutzomyia longipalpis, in the laboratory, both of which are important leishmaniasis vectors. I investigated the susceptibility of both species to ten different insecticides by calculating lethal concentrations that caused varying levels of mortality. Based on these results, I determined diagnostic doses and diagnostic times for both species to the same ten insecticides using an accepted, but novel, assay for sand flies. Finally, I tested for known mechanisms of insecticide resistance in four artificially resistant-selected colonies of sand flies, as well as tested for novel resistance mechanisms. Through applied research, I developed methods for efficient sand fly rearing and for determination of population resistance to insecticides, tools that have worldwide applicability. Through basic research, I determined that laboratory populations of sand flies have sufficient standing genetic variation needed to survive sublethal doses of insecticides; however, I was unable to develop artificially-selected colonies resistant to these insecticides. My research has generated information to provide new insights into the evolution of insecticide resistance in natural sand fly populations. My results support that resistance development may be possible, but evolutionary challenging, an encouraging finding that may be exploited by vector biologists and public health officials to prevent or slow the development of resistance in sand flies to insecticides

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