Dataset Information

Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping.

ABSTRACT: Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA) from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This report describes development and evaluation of an automated, quantitative PCR method based upon TaqMan single nucleotide polymorphism (SNP) genotyping.Standard PCR, and TaqMan SNP genotyping with newly designed primers and fluorophore-labeled probes hybridizing to sequences of complementary rDNA specific for either An. gambiae s.s. or An. arabiensis, were conducted in three experiments involving field-collected An. gambiae s.l. from western Kenya, and defined laboratory strains. DNA extraction was from a single leg, sonicated for five minutes in buffer in wells of 96-well PCR plates.TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR. In an extensive field study, only 29 of 3,041 (0.95%) were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species), however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1%) error rate for TaqMan genotyping in mistakenly identifying species hybrids.TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.

SUBMITTER: Walker ED

PROVIDER: S-EPMC1808465 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping.

Walker Edward D ED Thibault Alisha R AR Thelen Annette P AP Bullard Blair A BA Huang Juan J Odiere Maurice R MR Bayoh Nabie M NM Wilkins Elizabeth E EE Vulule John M JM

Malaria journal 20070227

<h4>Background</h4>Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA) from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This r ...[more]

PMID: 17326831

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Project description:BackgroundWhilst significant progress has been made in the fight against malaria, vector control continues to rely on just two insecticidal methods, i.e., indoor residual spraying and insecticidal bed nets. House improvement shows great potential to complement these methods and may further reduce indoor mosquito biting and disease transmission. Open eaves serve as important mosquito house entry points and provide a suitable location for intercepting host-seeking anophelines. This study describes semi-field experiments in western Kenya with eave tubes, a household protection product that leverages the natural behaviour of host-seeking malaria mosquitoes.MethodsSemi-field experiments were conducted in two screen-houses. In both of these a typical western Kenyan house, with mud walls and corrugated iron sheet roofing, was built. Eave tubes with bendiocarb- or deltamethrin-treated eave tube inserts were installed in the houses, and the impact on house entry of local strains of Anopheles gambiae and Anopheles arabiensis was determined. Experiments with open eave tubes (no netting) were conducted as a control and to determine house entry through eave tubes. Insecticidal activity of the inserts treated with insecticide was examined using standard 3-min exposure bioassays.ResultsExperiments with open eave tubes showed that a high percentage of released mosquitoes entered the house through tubes during experimental nights. When tubes were fitted with bendiocarb- or deltamethrin-treated inserts, on average 21% [95% CI 18-25%] and 39% [CI 26-51%] of An. gambiae s.s. were recaptured the following morning, respectively. This contrasts with 71% [CI 60-81%] in the treatment with open eaves and 54% [CI 47-61%] in the treatment where inserts were treated with fluorescent dye powder. For An. arabiensis recapture was 21% [CI 14-27%] and 22% [CI 18-25%], respectively, compared to 46% [CI 40-52%] and 25% [CI 15-35%] in the treatments with open tubes and fluorescent dye.ConclusionsInsecticide-treated eave tubes resulted in significant reductions in recapture rates for both malaria vector species, representing the first and promising results with this novel control tool against Kenyan malaria vectors. Further field evaluation of eave tubes under more realistic field conditions, as well as their comparison with existing approaches in terms of cost-effectiveness and community acceptance, is called for.

Project description:In areas where the morphologically indistinguishable malaria mosquitoes Anopheles gambiae Giles and An. arabiensis Patton are sympatric, hybrids are detected occasionally via species-diagnostic molecular assays. An. gambiae and An. arabiensis exhibit both pre- and post-reproductive mating barriers, with swarms largely species-specific and male F1 (first-generation) hybrids sterile. Consequently advanced-stage hybrids (back-crosses to parental species), which would represent a route for potentially-adaptive introgression, are expected to be very rare in natural populations. Yet the use of one or two physically linked single-locus diagnostic assays renders them indistinguishable from F1 hybrids and levels of interspecific gene flow are unknown.We used data from over 350 polymorphic autosomal SNPs to investigate post F1 gene flow via patterns of genomic admixture between An. gambiae and An. arabiensis from eastern Uganda. Simulations were used to investigate the statistical power to detect hybrids with different levels of crossing and to identify the hybrid category significantly admixed genotypes could represent.A range of admixture proportions were detected for 11 field-collected hybrids identified via single-locus species-diagnostic PCRs. Comparison of admixture data with simulations indicated that at least seven of these hybrids were advanced generation crosses, with backcrosses to each species identified. In addition, of 36 individuals typing as An. gambiae or An. arabiensis that exhibited outlying admixture proportions, ten were identified as significantly mixed backcrosses, and at least four of these were second or third generation crosses.Our results show that hybrids detected using standard diagnostics will often be hybrid generations beyond F1, and that in our study area around 5% (95% confidence intervals 3%-9%) of apparently 'pure' species samples may also be backcrosses. This is likely an underestimate because of rapidly-declining detection power beyond the first two backcross generations. Post-F1 gene flow occurs at a far from inconsequential rate between An. gambiae and An. arabiensis, and, especially for traits under strong selection, could readily lead to adaptive introgression of genetic variants relevant for vector control.

Dataset Information

Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping.

Publications

Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping.

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