QIIME2 pipeline.

<div><p>West Nile virus (WNV) is a flavivirus naturally circulating between mosquito vectors and birds, occasionally infecting horses and humans and causing epidemiologically relevant outbreaks. In Spain, the first big WNV outbreak was recorded in 2020, resulting in 77 people infected and 8 fatalities, most of them in southern Spain. <i>Culex perexiguus</i> was identified as the primary vector of WNV maintaining its enzootic circulation of the virus. Growing evidence highlights the role of mosquito microbiota as a key component determining the vectorial capacity of mosquitoes, largely contributing to disease epidemiology. Here, we develop, to our knowledge, the first identification of the microbiota composition of this mosquito vector under natural conditions and test for the potential relationship between mosquito microbiota composition and WNV infection. To do so, we collected mosquitoes in a natural area of southern Spain during the 2020 WNV outbreak and identified the microbiota composition of mosquitoes using a 16S rRNA gene metabarcoding approach. The microbiota of <i>Cx</i>. <i>perexiguus</i> was dominated by the phylum <i>Proteobacteria</i>. The most abundant families were <i>Burkholderiaceae</i> and <i>Erwiniaceae</i>, including the genera <i>Burkholderia</i>, <i>Erwinia</i>, and <i>Pantoea</i>. The genus <i>Wolbachia</i>, which use to dominate the microbiota of <i>Cx</i>. <i>pipiens</i> and negatively interact with WNV according to the literature, had a low prevalence and relative abundance in <i>Cx</i>. <i>perexiguus</i> and its abundance did not differ between WNV-positive and WNV-negative mosquito pools. The microbiota diversity and composition of <i>Cx</i>. <i>perexiguus</i> were not significantly related to the WNV infection status. These results provide the first identification of the mosquito microbiota in an endemic area of WNV circulation in Spain.</p></div>