Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus.

Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the Ace-1 target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan Culex quinquefasciatus display CNV for the voltage-gated sodium channel gene (Vgsc), target-site of pyrethroid and organochlorine insecticides. In order to develop field-applicable diagnostics for Vgsc CN, and as a prelude to investigating the possible association of CN with insecticide resistance, three assays were compared for their accuracy in CN estimation in this species. The gold standard method is droplet digital PCR (ddPCR), however, the hardware is prohibitively expensive for widespread utility. Here, ddPCR was compared to quantitative PCR (qPCR) and pyrosequencing. Across all platforms, CNV was detected in ≈10% of mosquitoes, corresponding to three or four copies (per diploid genome). ddPCR and qPCR-Std-curve yielded similar predictions for Vgsc CN, indicating that the qPCR protocol developed here can be applied as a diagnostic assay, facilitating monitoring of Vgsc CN in wild populations and the elucidation of association between the Vgsc CN and insecticide resistance.

Genetic variability and spatial distribution in small geographic scale of Aedes aegypti (Diptera: Culicidae) under different climatic conditions in Northeastern Brazil.

BACKGROUND: The study of the genetic structure of Aedes aegypti is essential to understanding their population dynamics as well as for the analysis of factors responsible for their resistance and ecological adaptation. The use of molecular markers in identifying differences amongst populations of Ae. aegypti in different geographical areas as well as the temporal variation of the vector populations has contributed to the improvement of vector control strategies. The present study aims to determine the genetic variability of Ae. aegypti populations in a small geographical area (state of Sergipe, Northeastern Brazil) by means of inter-simple sequence repeat (ISSR) and single nucleotide polymorphism (SNP) molecular markers. RESULTS: ISSR markers revealed a more heterogeneous pattern of genetic diversity among the populations with an expected heterozygosity (H E) ranging from 0.261 ± 0.03 to 0.120 ± 0.032, while a similar trend was detected through SNPs across populations with an H E between 0.375 ± 0.054 and 0.269 ± 0.042. The population's genetic differentiation assessed with ISSR and SNP markers indicated a very low structuring among the populations with the highest diversity observed within the populations 72 % (ISSR) and 92 % (SNP). Clustering analysis also suggested little variation among populations: the seven populations were grouped into only three ISSR clusters and a single panmictic group based on SNP markers. The present study identified a close relationship between the populations, which probably results mainly from passive gene flow between mosquitoes from distinct geographic regions, influenced by humans commuting along roads. CONCLUSIONS: There was an intense migration of mosquitos across municipalities, leading to a potential increase in risk of arbovirus and insecticide resistance associated-alleles spreading between mosquito populations.

Spatial distribution and esterase activity in populations of Aedes (Stegomyia) aegypti (Linnaeus) (Diptera: Culicidae) resistant to temephos.

INTRODUCTION: The need for studies that describe the resistance patterns in populations of Aedes aegypti (Linnaeus) in function of their region of origin justified this research, which aimed to characterize the resistance to temephos and to obtain information on esterase activity in populations of Aedes aegypti collected in municipalities of the State of Paraíba. METHODS: Resistance to temephos was evaluated and characterized from the diagnostic dose of 0.352mg i.a./L and multiple concentrations that caused mortalities between 5% and 99%. Electrophoresis of isoenzymes was used to verify the patterns of esterase activity among populations of the vector. RESULTS: All populations of Aedes aegypti were resistant to temephos, presenting a resistance rate (RR) greater than 20. The greatest lethal dose 50% of the sample (CL50) was found for the municipality of Lagoa Seca, approximately forty-one times the value of CL50 for the Rockefeller population. The populations characterized as resistant showed two to six regions of α and ß-esterase, called EST-1 to EST-6, while the susceptible population was only seen in one region of activity. CONCLUSIONS: Aedes aegypti is widely distributed and shows a high degree of resistance to temephos in all municipalities studied. In all cases, esterases are involved in the metabolism and, consequently, in the resistance to temephos.

Spatial distribution and esterase activity in populations of Aedes (Stegomyia) aegypti (Linnaeus) (Diptera: Culicidae) resistant to temephos

INTRODUCTION: The need for studies that describe the resistance patterns in populations of Aedes aegypti (Linnaeus) in function of their region of origin justified this research, which aimed to characterize the resistance to temephos and to obtain information on esterase activity in populations of Aedes aegypti collected in municipalities of the State of Paraíba. METHODS: Resistance to temephos was evaluated and characterized from the diagnostic dose of 0.352mg i.a./L and multiple concentrations that caused mortalities between 5% and 99%. Electrophoresis of isoenzymes was used to verify the patterns of esterase activity among populations of the vector. RESULTS: All populations of Aedes aegypti were resistant to temephos, presenting a resistance rate (RR) greater than 20. The greatest lethal dose 50% of the sample (CL50) was found for the municipality of Lagoa Seca, approximately forty-one times the value of CL50 for the Rockefeller population. The populations characterized as resistant showed two to six regions of α and β-esterase, called EST-1 to EST-6, while the susceptible population was only seen in one region of activity. CONCLUSIONS: Aedes aegypti is widely distributed and shows a high degree of resistance to temephos in all municipalities studied. In all cases, esterases are involved in the metabolism and, consequently, in the resistance to temephos. .