Methodological insight into mosquito microbiome studies: dissection and preservation

Symbiotic bacteria affect competence for pathogen transmission in insect vectors, including mosquitoes. However, knowledge on mosquito-microbiome-pathogen interactions remains limited, largely due to methodological reasons. The current, cost-effective practice of sample pooling used in mosquito wild populations surveillance and epidemiology prevents correlation of individual traits (i.e. microbiome profile) and infection status. As a consequence, epidemiological and microbiome studies in mosquitoes are to some extent uncoupled, and the interactions among pathogens, microbiomes, and natural mosquito populations remain poorly understood. This study tests different approaches to find an optimized low-cost methodology for extensive sampling while allowing for accurate, individual-level microbiome studies. We tested two methodological aspects that directly affect the cost and feasibility of broad-scale molecular studies: tissue dissection and sample preservation. We compared the microbiome of freshly extracted whole specimens and dissected guts. We also tested different preservation reagents such as AllProtect (Qiagen), NAP (nucleic acid preservation) buffer and absolute ethanol, with or without freezing for short-term storage (at -20 or 4 ºC, respectively).