A rapid luminescent assay for measuring cytochrome P450 activity in individual larval Culex pipiens complex mosquitoes (Diptera: Culicidae).

Multiple assays are available to measure P450 activity in insects, including mosquitoes; however, each of these assays has drawbacks in terms of the number of mosquitoes required, specificity, sensitivity, cost, and/or time required to prepare active enzyme homogenates. In this study, a commercially available luminescent assay, P450-Glo, was modified and evaluated to measure P450 activity from the gut of a single larva after removal of the gut contents. We also compared this assay to an earlier developed fluorescent assay. After optimization of assay conditions, the P450-Glo assay held considerable promise to be used as an effective, inexpensive, high-throughput, and sensitive screening assay to measure P450 activities in single mosquitoes. Furthermore, we tested the utility of the single gut assay using the pyrethroid resistant Marin strain of Culex pipiens pipiens form molestus and the pyrethroid sensitive CQ-1 strain of Cx. pipiens quinqefasciatus. We observed on average 1.8-fold higher levels of P450 activity in the resistant mosquitoes in comparison to the sensitive mosquitoes. Additionally, consistent with our previous findings, distribution plots of P450 activity showed 33% of individual Marin mosquitoes had higher P450 activities than the highest activity displayed by a CQ-1 mosquito. The assay platform is highly flexible in terms of choice of tissue, method of preparation, isozyme specificity, and sample quantity and thus could easily be adapted to be used for other arthropod species.

A set of broadly applicable microsatellite markers for analyzing the structure of Culex pipiens (Diptera: Culicidae) populations.

Microsatellite markers were isolated and developed from Culex pipiens quinquefasciatus Say (Diptera: Culicidae) sampled in Johannesburg, South Africa, to identify those that are broadly useful for analyzing Cx. pipiens complex populations between continents. Suitable loci should be 1) inherited in a codominant Mendelian manner, 2) polymorphic, 3) selectively neutral, 4) randomly associated, 5) without null alleles, and 6) applicable across broad regions and between diverse biotypes. Loci in Cx. p. quinquefasciatus from Johannesburg ranged from two to 17 alleles per locus and expected heterozygosities (H(e)) were 0.02-0.87. Loci in Cx. p. pipiens L. from Johannesburg had five to 19 alleles per locus and H(e) values ranging from 0.57 to 0.93, whereas those from George, South Africa, had five to 17 alleles per locus and H(e) values ranging from 0.54 to 0.88. Loci in North American mosquitoes were more variable. Cx. p. quinquefasciatus from South Carolina had five to 19 alleles per locus and H(e) values ranging from 0.64 to 0.90, whereas Cx. p. pipiens from Massachusetts had six to 28 alleles per locus and with H(e) values ranging from 0.65 to 0.94. All loci were associated randomly. Overall, four of nine of these new loci satisfied all six criteria for broad utility for analyzing the genetic structure of Cx. pipiens populations.