Abstract 1701 Single-cell RNA Sequencing of Culex tarsalis Ovaries

Rift Valley Fever Virus (RVFV) causes a mosquito-borne viral disease that affects livestock and humans, primarily in Africa, occasionally resulting in mass animal die-offs. These outbreaks can lead to devastating economic and food security losses for the farming communities. Over 40 mosquito species can potentially act as transmission vectors. RVFV is thought to be maintained in nature in part by transovarial transmission during mosquito reproduction. Transovarial transmission begins with a disseminated viral infection of the adult female and moves to the developing eggs. Previous reports indicated that about 30% of mosquito's total transcriptomic response is in the ovaries. We chose Culex tarsalis, which is a major vector of West Nile Virus and highly competent for transmission of RVFV as a subject for single cell sequencing analysis. The overall objective of this project is to identify expressed marker genes that can be used to identify specific ovarian cell types. However, the genome annotation is highly provisional. Methods: Ovarian single cell suspensions were prepared from pools of 20 sets of ovaries. The Chromium Next GEM Single Cell 3' Reagent kit was used to create libraries for high throughput sequencing of RNA from single cells (scRNA-Seq) of adult ovaries that had already laid one round of eggs. Initial scRNA-Seq was performed, then Cell Ranger and cluster analyses were performed. Results: Improved gene annotation of the Culex tarsalis genome has quickly yielded informative marker genes for further analysis. Putative marker genes were confirmed by cross-checking with orthologs from Aedes Aegypti and Culex quinquefasciatus mosquitoes. Genes of interest from scRNA-seq clusters include the vitellogenin receptor (VgR) and vitellogenin, a crucial lipoprotein for egg development. Another gene cluster appears to represent circulating immune cells, due to expression of cecropin-A1 (CeCA1), an important anti-microbial effector. We have also identified a number of representative genes from the RNA Interference (RNAi) pathway in our datasets. The RNAi pathway is a major immune response in mosquitoes that triggers induction of a silencing complex that eliminates gene expression of critical proteins, protecting cells against viral infection. Validation of marker genes using in situ hybridization methods is currently underway. Conclusions: We have identified putative target gene markers that will aid in the colocalization of RVFV and those marker genes in ovarian cells. These results provide foundation for future work to investigate transovarial transmission of RVFV in Culex tarsalis mosquitoes. This research was supported by the United States Department of Agriculture - Agricultural Research Service.