Microscopic visualization of Wuchereria and Brugia larval stages in intact cleared mosquitoes

Over the past several decades, epidemiologic data from filarial vectors typically has been obtained by mass dissection or by dissection of individual specimens. The former is quick and easy to do on large numbers of insects but provides no information on the frequency distribution of infection, presence of early developmental stages, or larval location; the latter is labor-intensive and tedious. We describe a new technique that can provide data comparable to those obtained by individual dissection, including calculation of infection and infective rates, and this technique is easy enough to accommodate large numbers of insects. Brief treatment of ethanol-fixed, intact mosquitoes in sodium hypochlorite, followed by treatments in increasing concentrations of ethanol and an organic solvent allowed microscopic visualization of filarial larvae within the abdomen, thorax, head, and proboscis of Brugia malayi-infected Aedes aegypti and Wuchereria bancrofti-infected Anopheles punctulatus. We compared the classic techniques to our technique using Ae. aegypti infected by feeding on jirds with B. malayi microfilaremias. Comparisons of the infective rate, total number of infective stage larvae (L3s) observed, and locations of L3s showed that this new technique was comparable to the established methods, while being faster and more precise in determining the location of larvae.