Dissection of Mosquito Ovaries, Midgut, and Salivary Glands for Microbiome Analyses at the Organ Level.

The global burden of mosquito-transmitted diseases, including malaria, dengue, West Nile, Zika, Usutu, and yellow fever, continues to increase, posing a significant public health threat. With the rise of insecticide resistance and the absence of effective vaccines, new strategies are emerging that focus on the mosquito's microbiota. Nevertheless, the majority of symbionts remain resistant to cultivation. Characterizing the diversity and function of bacterial genomes in mosquito specimens, therefore, relies on metagenomics and subsequent assembly and binning strategies. The obtention and analysis of Metagenome-Assembled Genomes (MAGs) from separated organs can notably provide key information about the specific role of mosquito-associated microbes in the ovaries (the reproductive organs), the midgut (key for food digestion and immunity), or the salivary glands (essential for the transmission of vector-borne diseases as pathogens must colonize them to enter the saliva and reach the bloodstream during a blood meal). These newly reconstructed genomes can then pave the way for the development of novel vector biocontrol strategies. To this aim, it is required to isolate mosquito organs while avoiding cross-contamination between them or with microorganisms present in other mosquito organs. Here, we describe an optimized and contamination-free dissection protocol for studying mosquito microbiome at the organ level.