Establishment of a method for Lutzomyia longipalpis sand fly egg microinjection: The first step towards potential novel control strategies for leishmaniasis

Leishmaniases is a group of vector-borne parasitic diseases transmitted by sand flies that affects 1.3 million people across 98 countries, with limited control strategies due to the lack of an available vaccine and the emergence of insecticide resistance. Novel control strategies that are being explored for mosquito-borne diseases, such as Wolbachia bacterial inhibition of pathogens and genetically modified insects (e.g. using CRISPR-Cas9 editing), rely on the ability to consistently inject eggs of the target species. Here we present a novel method to obtain and inject preblastoderm sand fly eggs of the genus Lutzomyia (Lu.) longipalpis, the principle vector of zoonotic visceral leishmaniasis in South America. The procedures required to obtain sufficiently young Lu. longipalpis colony eggs are described alongside a microinjection technique that permits rapid injection and minimal handling of small sand fly eggs post-injection. Using a strain of Wolbachia as a ‘marker’ for successful injection, our protocol produced early generation Wolbachia transinfected Lu. longipalpis lines, demonstrating its potential as the first step for use in novel applied strategies for sand fly control.