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.