Fig 1.
MA plots of expressed genes in Aag2 cells.
(A) MA plot showing differentially expressed genes in Aag2 cells under FAC (100 μM) vs DFO (50 μM). (B) MA plot for the genes in Aag2 cells under DFO (50 μM) vs L15. Log2FC: log2-transformed fold change values; FDR: false discovery rate; grey dots: FDR ≥ 0.05; yellow dots: FDR < 0.05 and Log2FC < |1|; red dots: FDR < 0.05 and Log2FC ≥ |1|.
Table 1.
Candidate iron transporter genes selected from RNAseq data.
GeneID: VectorBase gene ID. Log2FC: log2 fold change between indicated comparison groups. in/ex: predicted iron transporting function; in: importer, ex: exporter. Cell line: cell line used for dual-luciferase reporter assay; AAEL013277 was used for both cell lines as the expression was high in both cell lines. Normalized count: normalized CPM (counts per million) for the two cell lines. There was only one dsRNA available for AAEL014972 and AAEL020524 (red fonts) due to high identity between these genes. AAEL008624 and AAEL012698 (grey-shaded) could not be analyzed downstream due to failure of synthesizing dsRNA. Genes in bold fonts are present in both the candidate tables from AaegL3 and AaegL5 assemblies. TMD: number of predicted transmembrane domains. Blue fonts in Log2FC indicates values lower than threshold in AaegL5 mapping, but higher in AaegL3 mapping. Superfamily/Domain: Predicted superfamily/domain by blastp. SLC: solute carrier. TMEM: transmembrane protein. MFS: major facilitator superfamily. ABC: ATP binding cassette. CLC: chloride channel. DUF: domain of unknown function.
Fig 2.
Graphical representation of dual-luciferase reporter plasmid and assay procedure.
(A) Two luciferase reporter genes in a single plasmid in tail-to-tail orientation. FerLCH: Ae. aegypti ferritin light chain (AAEL007383). polyUb: Ae. aegypti poly-ubiquitin (AAEL003888). (B) Timeline for gene silencing and dual-luciferase assay procedure. FAC: ferric ammonium citrate.
Fig 3.
RNAi knockdown of candidate iron transporters changes iron-responsive reporter expression in mosquito cells.
(A) Dual-luciferase assay in Aag2 cells and (B) in A20 cells. Normalized firefly luciferase luminescence values (to EGFP control) are plotted on a Log2 scale with mean ± SD. Different dot colors indicate individual experiments. Neutral level is indicated by a black dotted line; sky-blue line indicates manually set cutoff (−0.8log2) for downstream analysis. The targets of dsRNA (either VectorBase gene ID [AAEL number] or gene name) are on the x axis. The predicted exporters are indicated by red fonts and the five genes selected for downstream analyses are in bold fonts. Statistical significance by mixed effect model (see methods) between EGFP control and each sample are shown in the figure.
Fig 4.
Ferritin light chain mRNA expression.
(A) qRT-PCR quantification of transcript expression pre- and post-bloodmeal in the midgut (Mg), Malpighian tubules (MT), Ovaries (Ov) and remaining body parts (C) (mean±SD normalized to rpS7), (B) ferritin LCH expression in Mg 24 hPBM in dsRNA-injected mosquitoes (n = 8–12) (mean±SD of ratio to dsEGFP of rpS7 normalized expression). **: p < 0.01 by Kruskal-Wallis test followed by multiple comparison.
Fig 5.
Fecundity and fertility of candidate iron transporter RNAi.
(A) Egg number (Fecundity) and hatch rate (Fertility) for female mosquitoes injected with dsRNA against each candidate iron transporter gene. Results are representative of three independent experiments (n = 24). (B) Second dsRNA against AAEL000471 (471b) showed comparable results to the first dsRNA (471a) (n = 12–17). Lines in the graphs show mean ± SD. ANOVA with multiple comparison between EGFP control and each sample was performed for statistical analysis as significant levels shown in the figure.
Fig 6.
AAEL000471-silenced mosquitoes exhibited small, pale-colored eggs and delayed excretion.
Representative EAgaL assay wells after individual females were placed in each well for 48 h (72–120 hPBM). Females injected with dsRNA against AAEL000471 laid small (short), pale-colored eggs and exhibited delayed excretion (magenta arrowheads in the top middle panel).
Fig 7.
Organ-specific transcript expression of dysp (AAEL000471) pre- and post-bloodfeeding.
qRT-PCR quantification of mRNA expression in the midgut (Mg), Malpighian tubules (MT), Ovaries (Ov) and remaining body parts (C). Transcript abundance is normalized to the housekeeping gene (rpS7). PBM: post-bloodmeal. The graph shows mean ± SD.
Fig 8.
Phylogenetic analysis of SLC16 proteins between vertebrates, dipterans and yeast.
Predicted amino acid sequences of 86 SLC16 members were aligned using Muscle as implemented in MEGA7 under the default parameters. The resulting alignment was used to generate a tree using the Neighbor-joining method, with the pairwise deletion option and bootstrapping (n = 1000). The bootstrap consensus tree is displayed, with percent bootstrap support indicated on each node when greater than 50%. SLC16 members with known substrates are indicated; dipteran-specific clade containing dysp is highlighted orange.