Fig 1.
Conservation, coding probability, and genomic localization information for NCBI automatically annotated lncRNAs in Aedes aegypti.
(A) The number of lncRNA sequences vs. the number of species against which they were found to exhibit Blast hits (top 20 hits shown). (B) Density plot of the coding potential model scores for all lncRNAs. The vertical dotted line indicates the cutoff point (0.432), below which transcripts were classified as non-coding. (C) Sankey plot displaying the overall annotation process and metrics for all RefSeq ncRNA transcripts (denoted by the “XR” prefix).
Fig 2.
Volcano plot depicting differentially expressed transcripts in different time-points post Zika virus infection.
(A) 2 days post-infection (dpi) with ZIKV, (B) at 7dpi, and (C) at 14dpi. The y-axis shows the posterior probability that each transcript was differentially expressed. Vertical lines indicate the applied log2FC threshold (|log2(FC)| ≥ 1), and the horizontal line indicates the statistical significance threshold, set at probability ≥95% (corresponding to a 5% false-discovery rate).
Fig 3.
Expression of six Zinc lncRNAs in Ae. aegypti mosquitoes after a Zika virus-infected vs. non-infected blood meal.
Data were collected at three different time points (2, 7, and 14 days post-blood-feeding). Comparisons were made between Zika virus-infected and non-infected samples at the same time point. On average, in infected samples, 7dpi Zinc1 was upregulated 6.6-fold, Zinc2 was up-regulated 15.4-fold, Zinc3 8.6-fold, Zinc9 17.1-fold, and Zinc10 10.9-fold, whereas Zinc22 was down-regulated 7.0-fold at 2dpi. Each sample contained three biological replicates. Expression values are transcript-level read counts, normalized for library size using the median-by-ratio method. Bars indicate mean value ± SEM. The significance was determined by Student’s t-test. *:P<0.05, **:P<0.01, ***:P<0.001
Fig 4.
Zinc gene silencing efficiency.
Relative quantification of Zinc1, Zinc2, Zinc3, Zinc9, Zinc10 and Zinc22 RNA abundance in Zinc-silenced vs GFP dsRNA-treated control mosquitoes. Whole mosquitoes were collected and pooled at three time-points (3-, 7- and 10-days) post-dsRNA injection. 3–5 biological replicates were performed for each Zinc and the average expression of GFP dsRNA treated replicates was set as 1 and the decreased dsZinc replicates, compared to GFP dsRNA, was measured. Samples were normalized using RNA abundance of the ribosomal RpS7 gene and are presented as the mean ± SEM. The significance was determined by Student’s t-test. *: P<0.05, **:P<0.01.
Fig 5.
Effects of Ae. aegypti Zinc lncRNA silencing on ZIKV infection.
Viral titers of Zika virus in midguts and carcasses obtained from dsRNA-treated mosquitoes at 7- or 10-days post-infection, respectively. Each panel corresponds to the comparison between Zinc-silenced mosquitoes and GFP dsRNA-treated controls: A) Zinc1-silenced, B) Zinc2-silenced, C) Zinc3-silenced, D) Zinc9-silenced, E) Zinc10-silenced, F) Zinc22-silenced. Each dot corresponds to the logarithmic number of plaque-forming units (pfu) that were counted per tissue and represents data from individual mosquitoes. The numbers in parentheses represent the number of virus-infected mosquitoes plotted against the number of mosquitoes checked. Data are presented as box plots in which the horizontal lines indicate the median value and whiskers show the minimum to maximum values. The significance was determined by Mann-Whitney U-test. **:P<0.01
Fig 6.
Impact of silencing of Zinc22 lncRNAs on fecundity (oviposition), fertility (hatching) and progeny of Ae. aegypti mosquitoes.
(A) Each dot corresponds to the number of eggs that were laid by each individual Zinc22-silenced or GFP dsRNA-treated female mosquito. (B) The fertility was defined as the percentage (%) of hatched larvae in relation to the total number of laid eggs per mosquito. (C) The number of offspring produced by each Zinc-silenced or GFP dsRNA-treated mosquito. Data are presented as box plots in which the horizontal lines indicate the median value and whiskers the minimum to maximum values. The significance was determined by Mann-Whitney U-test. **P<0.001.
Fig 7.
Impact of Zinc1, Zinc2, Zinc3, Zinc9 and Zinc10 lncRNA silencing on fecundity, fertility and progeny.
(A) Each dot corresponds to the number of eggs that were laid by each individual Zinc-silenced or GFP dsRNA-treated female mosquito. (B) The fertility was defined as the percentage (%) of hatched larvae in relation to the total number of laid eggs per mosquito. (C) The number of offspring produced by each Zinc-silenced or GFP dsRNA-treated mosquito. Data are presented as box plots in which the horizontal lines indicate the median value and whiskers the minimum to maximum values. The significance was determined by Mann-Whitney U-test. **P<0.001.