Fig 1.
Single-cell sequencing of Cx. tarsalis midguts and cell-typing of midgut cell populations.
Uniform Manifold Approximation and Projection (UMAP) reduction was used to visualize midgut cell populations. (A) 20 distinct clusters were identified at 4dpi and (B) 17 distinct clusters were identified at 12dpi. (C) Expression of canonical marker genes that were conserved between infection condition (mock and WNV-infected) were used to determine cell type. Dot size denotes percent of cells expressing each gene, color denotes scaled gene expression. Panel C was derived from the total population of cells for each subtype.
Fig 2.
Further characterization of enteroendocrine cells, hemocytes and progenitor cells.
Expression of characterizing genes visualized for EEs (A), HCs (B), and ISC/EBs (A). UMAP limits were set to include only clusters of interest, orientation of clusters in the total population can be found in panel C. (D) Expression of canonical enteroendocrine/neuroendocrine, neuropeptide, and secretory genes. (E) Expression of canonical HC, and granulocyte and oenocytoid genes (F) Expression of canonical ISC and EB genes as well as gene markers for proliferation and mitosis. Panels A and B were created in BioRender. Gallichotte, E. (2024) https://BioRender.com/f66n058. Gene counts for the total population (timepoints and conditions combined) were converted to binary (blue = 1, grey = 0) and visualized via UMAP.
Fig 3.
WNV vRNA is detected in most midgut cell populations.
(A) Levels of WNV vRNA across midgut cell populations at 4 and 12dpi. Purple to grey gradient denotes high to low levels of vRNA. (B) percent of cells in the total population containing WNV vRNA (percent expressing) and average WNV vRNA level in the total population (average expression) calculated for each WNV-infected replicate and compared between timepoints. Significance was determined by unpaired t-test, * = p<0.05. (C) WNV vRNA level in individual cells of distinct clusters. Black asterisks denote significant enrichment of vRNA as a cluster marker, pink asterisks denote significant absence of vRNA as a cluster marker. The percentage of cells containing WNV vRNA in each epithelial cell population at (D) 4dpi and (E) 12dpi. Average WNV vRNA level in epithelial cell populations at (F) 4dpi and (G) 12dpi. Significance determined by one-way ANOVA, * = p<0.05, ** = p<0.005, *** = p<0.0005, **** = p<0.0001. Points = replicate values, bars = mean of replicate values, error bars = SD. (E) Trajectory inference for ISC/EB and ISC/EB-prol populations identified two lineages. (F) vRNA levels visualized along lineage progression. Green = lineage 1 (EC), blue = lineage 2 (EE). Panels B-G were derived from only WNV-infected samples. Panels D-G exclude values derived from clusters of ≤5 cells. Panels A, and H-I were derived from the total population.
Fig 4.
The impact of WNV infection on midgut cell populations.
(A) Number of significant (adjusted p-value<0.05) differentially expressed genes (DEGs) associated with WNV infection in each midgut cell type. Green = 12dpi, purple = 4dpi, black = upregulated, pink = downregulated. (B) Total significant DEGs identified in midgut cell populations. Green = 12dpi, purple = 4dpi, black = upregulated, pink = downregulated. (C) Visualizing WNV-infection associated DEGs in cell populations of interest (ISC/EBs, EEs). Negative log2FC indicates downregulation and positive log2FC indicates upregulation associated with WNV infection. In the interest of data density, the bar representing the log2FC of the WNV 5’ UTR is not shown. WNV 5’ UTR log2FC values can be found in S4 Table. (D) Gene ontology enrichment analysis (GOEA) for all downregulated DEGs (timepoints and cell populations combined). (E) GOEA for all upregulated DEGs (timepoints and cell populations combined). Color denotes -log10(FDR). FDR cutoff<0.05 was used for GOEA.
Fig 5.
Identifying genes upregulated in response to WNV infection with differential expression and correlation analyses.
DEGs associated with WNV-infected condition when compared to the mock condition at 4dpi (A) and 12dpi (B) were identified with DESeq2. Negative log2FC indicates downregulation and positive log2FC indicates upregulation associated with WNV infection. (C) Genes upregulated during WNV infection at 4dpi (negative log2FC) and 12dpi (positive log2FC). (D) Characterized genes that are significantly correlated with WNV vRNA at 4dpi (purple) and 12dpi (teal), with correlation values of >0.65. Only significant (p<0.05) relationships shown. (E) Correlation of enteroendocrine specific genes and WNV vRNA at 4dpi (purple) and 12dpi (teal). Solid line represents the mean correlation of WNV vRNA with 1000 randomly selected genes at the specified timepoint (dotted lines represent the upper and lower 95% confidence intervals for this value). Labeled dotted lines denote vRNA correlation with RPL8 and RPL32 housekeeping reference genes. Panels C-E were derived from only WNV-infected replicates.
Fig 6.
Lack of key immune gene upregulation in response to WNV infection.
(A-B) Percent of the total population of each replicate comprised by hemocytes compared between mock and WNV-infected conditions at each timepoint. Significance was determined by unpaired t-test. Points = replicate values, bars = mean of replicate values, error bars = SD. (C-D) Average expression of, and percent of cells expressing, mosquito innate immune genes compared between mock and WNV-infected replicates for each timepoint. Significance was determined by multiple unpaired t-tests. Points = replicate values, bars = mean of replicate values, error bars = SD. (E) Correlation of immune genes and WNV vRNA at 4dpi (purple) and 12dpi (teal). Solid line represents the mean correlation of WNV vRNA with 1000 randomly selected genes at the specified timepoint (dotted lines represent the upper and lower 95% confidence intervals for this value). Labeled dotted lines denote vRNA correlation with RPL8 and RPL32 housekeeping reference genes. (F) Feature scatter of IMD vs. WNV 5’ UTR for both timepoints combined. (G) Expression level of IMD in mock and WNV-infected conditions for both timepoints combined. Panels E-F were derived from only WNV-infected replicates.