Midgut cell atlas and virome of four important mosquito species and immune landscapes of dengue virus infection
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Mosquitoes are the most important arthropod vectors of human diseases globally, with their midgut serving as both a digestive organ and the primary site of viral infection. Single-cell RNA sequencing (scRNA-seq) studies in insects, including mosquitoes, remain limited due to the absence of established protocols. Here, we fill this gap by developing a compatible microwell-based scRNA-seq workflow, and generated midgut cell atlas and virome for four important mosquito species: Aedes aegypti, Aedes albopictus, Culex pipiens pallens and Culex tritaeniorhynchus. Eight distinct cell types were identified, and the cell composition was inter-generic conserved in types but divergent in proportions. Culex mosquitoes exhibited higher proportions of intestinal stem cells/enteroblasts (ISC/EBs) than Aedes mosquitoes. In response to dengue virus 2 (DENV-2) infection, stress-associated genes were broadly downregulated, whereas endoenterocytes (EEs) and ISC/EBs showed marked upregulation of diverse immune related genes. Notably, we identified a novel midgut cell cluster (termed SV2/UNC-93) characterized by high expression of synaptic vesicle protein-2 and UNC-93, along with broad upregulation of apical-basal polarity-related genes linked to viral infection. The midguts of Aedes and Culex mosquitoes harbored diverse virome belonging to 5 viral families and one unclassified group. Hanko iflavirus 1 (HKIFV1) was the most common virus, distributed in all four mosquito species, while Tiger mosquito bi-segmented tombus-like virus (TMBSTLV) was the most abundant virus in Ae. albopictus and exhibited strong tropism in hemocytes. DENV-2 only detected in experimental infected Ae. aegypti, with no obvious midgut cell tropism but highest viral reads occurred in enterocytes (ECs) and EEs.Our study provides fundamental insights into the cellular composition of these four medically important mosquitoes and facilitate future research on virus-midgut interactions.
