Optimising Lutzomyia longipalpis Processing for Gut Microbiome Analysis: Sex and Dietary Impacts

Lutzomyia longipalpis (Lu. longipalpis) is the primary vector for the protozoan parasite Leishmania, the causative agent of Visceral Leishmaniasis in Latin America. Species-specific DNA extraction kits for Lu. longipalpis analysis is not currently available. Therefore, the optimisation of generic insect kits that are instead used is needed to ensure maximum DNA yield of such a small organism. Here, we used the QIAGEN Blood and Tissue Kit as a base protocol. We found that increasing the exoskeleton degradation time and repeating purification steps enhanced DNA concentrations without compromising purity. This improved technique allowed us to explore intrinsic and extrinsic factors that could potentially alter the sandfly midgut microbiome. Whilst there are hormonal and physiological differences between the sexes of Lu. longipalpis, we found no distinction in midgut microbiome diversity and composition between male and female sandflies. Female Lu. longipalpis feed on mammalian blood, which can have a profound effect on microbiome populations. However, smaller time resolutions to identify precise changes have yet to be utilised, owing to the difficulty of controlling the sandfly feeding cycle in the wild. By using lab-reared Lu. longipalpis, were able to observe bacterial populations 2, 4, 7 and 9 days post blood feed. We found the species number to stay the same across samples, with a small but significant difference in species type occurring by day 4. This delayed reaction points to questions regarding the wider role of the peritrophic matrix (PM), which could initially shield the microbiome from blood meals. Further research is required to determine the time points of the PM formation alongside microbiome restructuring.