Single-cell transcriptional landscapes of Aedes aegypti midgut and fat body after a bloodmeal

Abstract

Aedes aegypti mosquitoes transmit arboviruses that pose a growing global health threat. After a bloodmeal, mosquitoes experience complex physiological changes orchestrated by the midgut and fat body, beginning with digestion and culminating in egg production. Our study provides comprehensive midgut and fat-body cell atlases using single-cell RNA sequencing and metabolomics. Our analyses reveal highly diverse cell populations specialized in digestion, metabolism, immunity, and reproduction. The midgut primarily comprises enterocytes, enteroendocrine, and intestinal stem cells, while the fat body features trophocytes and oenocytes but also a substantial hemocyte population and a newly found fat-body-yolk cell population. Additionally, Phasi Charoen-like virus was detected in midgut cells 7 days post bloodmeal. These findings highlight the complexity of mosquito abdominal tissues and inform the development of refined vector-control strategies, focusing on specific cell populations and metabolic pathways essential for mosquito reproductive success.

Keywords: Aedes aegypti; atlas; bloodmeal; immunity; insect-specific virus; metabolism; metabolomics; mosquito; single cell.

Graphical abstract

Figure 1

Cellular landscape of Ae. aegypti abdominal tissues after a bloodmeal (A) Schematic experimental workflow. Female Ae. aegypti mosquitoes were blood-fed, and pools of seven midgut and fat-body tissues were collected at 2 and 7 days post bloodmeal (dpbm), for subsequent cell isolation and scRNA-seq. (B and C) Integrated uniform manifold approximation and projection (UMAP) of midgut (B) and fat body (C) at 2 and 7 dpbm, with dots representing single cells colored by cluster. EC, enterocytes; ISC/EB, intestinal stem cells/enteroblasts; EE, enteroendocrine cells; VM, visceral muscle cells; HC, hemocytes; CA, cardia cells; TP, trophocytes; FYC, fat-body-yolk cell; HC-G, hemocytes-granulocytes; HC-O, hemocytes-oenocytoids; TB, tracheoblast-like; EP, epidermal cells; MU, muscle cells; NE, neuroendocrine cells; OE, oenocytes. (D) Schematic of midgut- and fat-body-associated cell types. See also Figures S1 and S2; Tables S1–S3.

Figure 2

Dynamic midgut cell composition after a bloodmeal (A) Expression of top two marker genes per midgut cell cluster at 2 and 7 dpbm. (B) Gene ontology (GO) of top 20 cluster marker genes, curated from Aedes aegypti, Drosophila, or Anopheles GO terms, and functional annotations from Hixson et al. Transcr, transcription; Transl, translation; Cell diff, cell differentiation. (C) nubbin (AAEL017445) expression in enterocytes (EC) and EC-like clusters at 2 and 7 dpbm. EC clusters are outlined in brown, with cell proportions relative to total cell number. (D) Dotplot of EC and EC-like marker gene expression at 2 and 7 dpbm. Ser, serine; CPA, carboxypeptidase; Oxy. Phos, oxidative phosphorylation; TF, transcription factor. (E–H) Specific cell clusters visualized on UMAP associated with gene markers and relative proportions of (E) cardia cells (CA), (F) intestinal stem cells and enteroblasts (ISC/EB), (G) enteroendocrine cells (EE), and (H) visceral muscle cells (VM). (I) Hemocyte (HC) cluster visualized by UMAP and co-expression of Nimrod B2 (AAEL019650) and SPARC (AAEL013656) markers. Dot color shows expression intensity at 2 dpbm (red) and 7 dpbm (blue). Dot size reflects the percentage of expressing cells per cluster. Each gene is indicated by AAEL0 Vectorbase ID and abbreviation if available. Drosophila or Anopheles ortholog abbreviations are indicated in italics. See also Figure S3; Tables S4, S6, and S7.

Figure 3

Cellular mosaic of abdominal fat-body tissues after a bloodmeal (A) Expression of top two marker genes per fat-body cell cluster at 2 and 7 dpbm. (B) GO of top 20 cluster marker genes, curated from Aedes aegypti, Drosophila, or Anopheles GO terms, and functional annotations from Hixson et al. Egg dev, egg development; Transcr, transcription; Transl, translation; Cell diff, cell differentiation. (C–F) Specific cell clusters visualized on UMAP associated with gene markers and relative proportions of (C) trophocytes, (D) oenocytes, (E) hemocytes, and (F) fat-body-yolk cell. Dot color depicts expression intensity at 2 dpbm (red) and 7 dpbm (blue). Dot size reflects the percentage of expressing cells per cluster. Each gene is indicated by AAEL0 Vectorbase ID and abbreviation if available. Drosophila or Anopheles ortholog abbreviations are indicated in italics. Vittelo., vitellogenin genes; Vit-R, vitellogenin membrane receptor; Treh, trehalose metabolic genes; Glyc, glycogen metabolic genes; Detox, detoxification genes; FAS, fatty acid synthase; Elon, elongase; FAR, fatty acyl-CoA reductase; TCA, tricarboxylic acid cycle genes; EcR, nuclear ecdysone receptor; Cho, chorion; LDL-R, low-density lipoprotein receptor; Atg, autophagy; SC, stem cell gene markers; Histone, histone protein genes; HMT, histone methyltransferase. See also Figures S3 and S4; Tables S5, S7, and S8.

Figure 4

Immune and metabolic pathways are expressed mainly by fat-body cells and disparately in midgut cells Heatmap displays proportion of cells expressing genes in key immune and metabolic pathways per cluster, with at least 15% expression in a minimum of one cluster across the tissues. Each gene is indicated by AAEL0 Vectorbase ID and abbreviation if available. Drosophila or Anopheles ortholog abbreviations are indicated in italics. PGRP, peptidoglycan recognition protein; LYS, lysozyme; Glyco, glycolysis; Pent, pentose phosphate pathway; TCA, tricarboxylic acid cycle; Oxy. Phos, oxidative phosphorylation; CYP450, cytochrome P450. See also Figure S5 and Table S9.

Figure 5

Tissue metabolomics highlight the physiological differences and the metabolic expertise of the fat body (A) Overview of midgut and fat-body collection from blood-fed mosquitoes for metabolite extraction and UHPLC-MS/MS analysis. (B) Principal-component (PC) analysis of metabolomic dataset from dissected midgut and fat-body tissues. Each dot represents a pool of five tissues from the same individual. Samples are normalized by the total ion chromatogram. The dataset is log₁₀ transformed and scaled by autoscaling. (C) Venn diagram of significantly regulated metabolites between tissues or time points. Significant metabolites were selected with a false discovery rate (FDR)-adjusted p value of <0.05 and at least |2| fold-change difference. (D) Pathway analysis of top ten differential metabolic pathways between midgut and fat body at 2 and 7 dpbm. Pathway analysis used HMDB annotated metabolites and global test enrichment method, based on Aedes aegypti KEGG pathway library. Significant pathways were selected with −log₁₀(p) > 1.5 and FDR-adjusted p value of <0.05. (E) Heatmap of selected metabolites differential abundance between midgut and fat body at 2 and 7 dpbm. Significantly regulated metabolites in bold correspond to a fold change >|2| and adjusted p value of <0.05 between tissues. Metabolites are grouped by chemical classes. Carbo, carbohydrates; CA, carboxylic acids; FA, fatty acids; GL, glycerolipids; LysoPL, lysophospholipids; PL, phospholipids. (F) Selected metabolite intensity repartition in midgut and fat body at 2 and 7 dpbm. Significant differences were based on a two-way ANOVA Tukey’s multiple comparison.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Error bars represent standard error of the mean. See also Table S10.

Figure 6

Insect-specific virus PCLV is highly expressed in midgut and fat-body cells at later stage post blood meal (A) Proportion of cells expressing PCLV, CFAV, and HTV in midgut and fat body at 2 and 7 dpbm. Data were analyzed with a Fisher's exact test, ∗∗∗∗p < 0.00001. nd, not detected. (B) Dotplot showing PCLV expression for midgut (top) and fat-body (bottom) clusters. Dot color shows relative average expression intensity at 2 dpbm (red) and 7 dpbm (blue). Dot size reflects the percentage of cells expressing PCLV in each cell cluster. (C) Proportion of PCLV-positive cells in each cluster at 7 dpbm. Higher proportions significant by chi-squared test with Bonferroni correction, ∗p < 0.05. (D) Dotplot showing top ten genes differentially expressed between PCLV-positive and PCLV-negative cells at 7 dpbm. Dot size reflects the percentage of cells expressing corresponding genes in each cell cluster. (E) Violin plot showing top five gene markers differentially expressed between PCLV-positive and PCLV-negative cells within EC-like2 and merged OE and OE-like clusters at 7 dpbm.