Fig 1.
Schematic representation of the experimental design and biological samples used for RNA-seq.
The diagram illustrates key mosquito tissues sampled for RNA sequencing. Depicted within the mosquito body are: the gut (midgut shown in dark red at T1 and light red at T5), the crop (dark grey diverticulum connected to the foregut), the salivary glands (light blue structures in the thorax), hemocytes and fat body cells (represented by black dots in the grey hemolymph), and virions (blue hexagons). The term carcass refers to the entire body after the midgut has been removed.
Table 1.
Summary of samples and RNA-Seq Data.
Table 2.
Summary of Differential Expression (DE) Analysis. The table reports the number of upregulated (UP) and downregulated (DOWN) genes at three significance thresholds (FDR < 0.05, FDR < 0.01, FDR < 0.001). Comparisons shown are: Mi T1 V vs C (midguts at 1 dpi, CHIKV-infected vs control) and Ca T5 V vs C (carcasses at 5 dpi, CHIKV-infected vs control).
Fig 2.
Volcano plots displaying the results of the differential expression (DE) analysis.
(A) DE contigs in midguts at T1. (B) DE contigs in carcasses at T5. The x-axis represents Log10 fold change (FC), while the y-axis represents -Log10 false discovery rate (FDR). DE genes are highlighted in red. Transcripts differentially upregulated in the infected groups compared to control groups are shown on the left side of each graph, whereas those downregulated in the infected groups compared to control groups are displayed on the right side.
Fig 3.
Selection of enriched terms in infected midguts at 1 dpi, i.e., upregulated in response to CHIKV infection.
(A) List of enriched Gene Ontology (GO) terms according to the three categories, Biological process (BP, red bars), Molecular Function (MF, blue bars) and Cellular Component (CC, green bars); (B) list of enriched Pfam domains, families and repeats: Pfam IDs and descriptions are reported as the adjusted p value (adjp); (C) list of differentially expressed (DE) contigs: transcript IDs, according to AalbF5 genome assembly, are reported with relative annotation; enrichment parameters are logFC (Fold Change) logCPM (Counts Per Million) and FDR, False Discovery Rate.
Fig 4.
Selection of enriched terms in control midguts at 1 dpi, i.e., downregulated in response to CHIKV infection.
(A) List of enriched Gene Ontology (GO) terms according to the three categories, Biological process (BP, red bars), Molecular Function (MF, blue bars) and Cellular Component (CC, green bars); (B) list of enriched Pfam domains, families and repeats: Pfam IDs and descriptions are reported as the adjusted p value (adjp); (C) list of differentially expressed (DE) contigs: transcript IDs, according to AalbF5 genome assembly, are reported with relative annotation; enrichment parameters are logFC (Fold Change) logCPM (Counts Per Million) and FDR, False Discovery Rate. The asterisk indicates XM_019688536.3, whose expression is analysed through RT-qPCR validation (Fig 9).
Fig 5.
Selection of enriched terms in infected carcasses at 5 dpi, indicating upregulation in response to CHIKV infection.
(A) List of enriched Gene Ontology (GO) terms according to the three categories, Biological process (BP, red bars), Molecular Function (MF, blue bars) and Cellular Component (CC, green bars); (B) list of enriched Pfam domains, families and repeats: Pfam IDs and descriptions are reported as the adjusted p value (adjp); (C) list of differentially expressed (DE) contigs: transcript IDs, according to AalbF5 genome assembly, are reported with relative annotation; enrichment parameters are logFC (Fold Change) logCPM (Counts Per Million) and FDR, False Discovery Rate.
Fig 6.
Selection of enriched terms in control carcasses at 5 dpi, indicating downregulation in response to CHIKV infection.
(A) List of enriched Gene Ontology (GO) terms according to the three categories, Biological process (BP, red bars), Molecular Function (MF, blue bars) and Cellular Component (CC, green bars); (B) list of enriched Pfam domains, families and repeats: Pfam IDs and descriptions are reported as the adjusted p value (adjp); (C) list of differentially expressed (DE) contigs: transcript IDs, according to AalbF5 genome assembly, are reported with relative annotation; enrichment parameters are logFC (Fold Change) logCPM (Counts Per Million) and FDR, False Discovery Rate.
Fig 7.
Transcriptional modulation of immune gene family members following viral challenge.
The mirrored bar chart displays on the x-axis the percentage of transcripts that were at least twice as abundant in infected versus control samples from each immune group as listed on the y-axis (with the total number of family members in brackets). Red bars refer to data from midguts at 1 dpi, while grey bars represent data from carcasses at 5 dpi.
Fig 8.
Heatmaps illustrating the transcriptional profiles of members of the (A) Autophagy, (B) RNAi and (C) JAK/STAT families.
Each heatmap includes: the identifier of each contig (including possible isoforms) in the TRANCRIPT_ID column; the log2 values of the ratio between the FPKM values (+1 FPKM) of infected samples (V) and control samples (C), presented for midgut samples at 1 dpi (T1-Mi V/C) and carcass samples at 5 dpi (T5-Ca V/C), with a corresponding color legend below; the gene description or annotation provided in the GENE_NAME column.
Fig 9.
Transcriptional Modulation of Digestive Factors Upon CHIKV Infection.
(A) Heatmap displaying transcriptional changes. (B) Pie chart illustrating the percentage of genes with a control/infected ratio >1 (blue) or <1 (red). (C) Validation of the trypsin gene XM_019688536.3 by RT-qPCR across different tissues, including whole females, dissected heads, ovaries, midguts, and carcasses (whole body excluding head, ovaries, and midgut). Statistical analysis was conducted using ordinary one-way ANOVA followed by Tukey’s multiple comparison test. (D) Summary of differentially expressed (DE) contigs within the digestive factor list.
Fig 10.
Heatmaps of Transcriptional Profiles Across Immune Categories.
(A) AMPs (Antimicrobial Peptides); (B) LRR (Leucine-Rich Repeat proteins); (C) CTL-PAFs (C-type Lectins and Pathogen-Associated Factors); (D) TEP (Thioester-Containing Proteins); (E) PPO (Prophenoloxidase). Validation by RT-qPCR (contigs marked with asterisk): (F) Holotricin gene (XM_029879658.2); (G) LRR gene (XM_019686715.3); H. PAF2 gene (XM_029857423.2). RT-qPCR was performed across different tissues, including whole females, dissected heads, ovaries, midguts, and carcasses (whole body excluding head, ovaries, and midgut). Statistical analysis was conducted using ordinary one-way ANOVA followed by Tukey’s multiple comparison test.