Figure 1.
Microarray gene expression analysis of the naïve and DENV-infected salivary gland transcriptomes.
(A) Numbers of genes that displayed differential (red and green circles) and similar (yellow circle) transcript abundance between the naïve salivary gland and carcass. The superimposed gray triangle shows the numbers of genes displaying significant differences in transcript abundance in the DENV-infected salivary gland, and their overlap with genes expressed in the naïve gland. (B) Salivary gland-expressed genes were classified into low, medium and high abundance categories according to their spot intensities on the array. The pie chart shows the number of genes in each category and its corresponding percentage of the total number of expressed genes. (C) Functional classification of differentially expressed genes in the naïve salivary gland (relative to the carcass), the DENV-infected salivary gland, and the DENV infected carcass. Functional group abbreviations: CS, cytoskeletal and structural; CSR, chemosensory reception; DIV, diverse functions; DIG, blood and sugar food digestive; IMM, immunity; MET, metabolism; PROT, proteolysis; RSM, redox, stress and mitochondrion; RTT, replication, transcription, and translation; TRP, transport; UKN, unknown functions. (D) Venn diagram showing numbers of uniquely and commonly regulated genes in DENV-infected salivary glands and carcasses. Arrows represent the direction of gene regulation.
Table 1.
Candidate genes selected for functional testing via RNAi-mediated gene knockdown.
Figure 2.
Effect of candidate gene knockdown on salivary gland DENV titers.
Candidate genes were silenced in DENV-infected mosquitoes, and salivary gland virus titers at 14 dpbm were determined by plaque assay. A pool of three biological replicates is shown, and p values were determined using the Mann-Whitney U test (*, p<0.05). Gene name abbreviations: AnkP, ankyrin repeat-containing protein; Cyst, cystatin; SSP, salivary secreted peptide; CatB, cathepsin B.
Figure 3.
Effect of OBP gene knockdown on mosquito blood-feeding behavior.
A behavioral feeding assay was performed 4 days post-silencing of OBPs 10 and 22. Mosquitoes were offered an anesthetized mouse and observed individually for 400 seconds. The following parameters were measured and are represented here: (A) Probing propensity: the percentage of mosquitoes that probed within the observation period; (B) Probing initiation time: the time from the introduction of the mouse until the mosquito starts to probe; (C) Probing time: the time from the initial insertion of the mouthparts in the skin to the initial engorgement of blood. The data are a pool of six biological replicates, and p values were determined with the Mann-Whitney U test (*, p<0.05).
Figure 4.
OBP silencing efficiency and gene expression in the chemosensory organs; detection of DENV by RT-PCR in the chemosensory organs.
(A) Silencing efficiencies for OBPs 10 and 22 in the salivary gland and chemosensory organs (antennae and palps) 4 days post-injection of 2 ug dsRNA, relative to dsGFP-injected controls. (B) Detection of DENV by RT-PCR in the antennae and palps of DENV-infected mosquitoes at 10 and 14 dpbm. *, p<0.05 in Student's t-test. (C) OBP and Aaeg\Orco gene expression in the antennae and palps of DENV-infected mosquitoes at 10 and 14 dpbm, relative to mock-infected mosquitoes. p values were determined with the Student's t-test.
Figure 5.
Detection of DENV infection in the chemosensory organs by immunofluorescence staining.
Head squashes from mosquitoes at 14 dpbm were stained with mouse hyperimmune ascitic fluid to DENV and an AlexaFluor568-conjugated anti-mouse antibody. (A–C) Uninfected antennae, (D–F) Infected antennae at segments 2–4, (G–I) Infected antennae at segments 9–11, (J–L) Infected maxillary palp, (M–O) Infected proboscis. Red, DENV antigen.
Figure 6.
Effect of DENV infection on mosquito blood-feeding behavior.
A behavioral feeding assay was performed on DENV- and mock-infected mosquitoes at 14 dpbm. Mosquitoes were offered an anesthetized mouse and observed for 400 seconds. The following parameters were measured and are represented here: (A) Probing initiation time; (B) Probing time; and (C) Probing propensity. The data are a pool of three biological replicates, and p values were determined with the Mann-Whitney U test.