Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 nanograms of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

Detection of Anopheles stephensi Mosquitoes by Molecular Surveillance, Kenya.

The Anopheles stephensi mosquito is an invasive malaria vector recently reported in Djibouti, Ethiopia, Sudan, Somalia, Nigeria, and Ghana. The World Health Organization has called on countries in Africa to increase surveillance efforts to detect and report this vector and institute appropriate and effective control mechanisms. In Kenya, the Division of National Malaria Program conducted entomological surveillance in counties at risk for An. stephensi mosquito invasion. In addition, the Kenya Medical Research Institute conducted molecular surveillance of all sampled Anopheles mosquitoes from other studies to identify An. stephensi mosquitoes. We report the detection and confirmation of An. stephensi mosquitoes in Marsabit and Turkana Counties by using endpoint PCR and morphological and sequence identification. We demonstrate the urgent need for intensified entomological surveillance in all areas at risk for An. stephensi mosquito invasion, to clarify its occurrence and distribution and develop tailored approaches to prevent further spread.

Polymerase chain reaction-based identification and genotyping of Anopheles mosquitoes with a 96-pin bacterial replicator.

A simple method for rapid identification of large numbers of Anopheles mosquitoes was developed based on polymerase chain reaction (PCR) amplification of the rDNA intergenic spacer and internal transcribed spacer 2. By means of previously described primers for the Anopheles gambiae and An. quadrimaculatus species complexes, rDNA was amplified simultaneously from 96 whole mosquitoes or parts. No homogenization or individual DNA preparation was necessary, and transfer of 96 samples to PCR reactions was performed simultaneously with a bacterial replicator. Control reactions indicate that the level of cross-contamination is negligible, and false-negative findings are rare. The method was tested on larvae, pupae, adult heads, whole adult males and females, and single tarsi. All parts except tarsi provided satisfactory template. Fresh, ethanol-preserved, dried, and frozen adults were also tested with similar results. The method was also tested for amplification of a single-copy gene, white. Results were generally positive, although some false-negative findings were observed. This method allows rapid analysis of large numbers of mosquitoes without robotic equipment and should enable rapid and extensive PCR analysis of field-collected samples and laboratory specimens.

Unassisted isolated-pair mating of Anopheles gambiae (Diptera: Culicidae) mosquitoes.

Female Anopheles mosquitoes usually mate only once, but mating is seldom seen in small containers containing only one female and male. Therefore, matings are often performed among many adults in large cages or by forced copulation. Isolated-pair mating of Anopheles gambiae G3 strain-derived mosquitoes without forced copulation in small vials is described. We observed that the experimental variables eye color and male number were significant factors in the mating frequency. Females mated more frequently when three males were present over only one male. White-eyed females were more likely to be mated than wild-eyed females, but wild males mated more frequently than did white-eyed males. Experiments were also conducted to determine when mating was occurring by using wild-eye-color mosquitoes in isolated pairs. Almost no matings were observed before day 6 rather than the frequencies typically observed after 1-2 d in standard large-cage matings among large numbers of adults.