Fig 1.
The lincRNA identification pipeline flowchart.
Fig 2.
Aedes aegypti lincRNA characterization.
A) Comparison of the GC content in protein-coding genes and the putative lincRNA genes. B) Sequence length distribution of Ae. aegypti lincRNA candidates. C) Comparison of gene length in protein-coding genes and putative lincRNA genes. D) lincRNA distribution among different Ae. aegypti genome scaffolds. The majority of scaffolds (~77%) only contain 1–4 lincRNAs, while only 23 Ae. aegypti genome scaffolds contain more than 10 lincRNAs (~2%).
Table 1.
Distribution of potential Ae. aegypti lincRNAs in different genome scaffolds with more than 10 lincRNAs and their comparison with the number of protein-coding genes.
Fig 3.
Ae. aegypti lincRNAs share some conserved areas with other closely related species.
A) The similarity bit score showed more similar sequences with high-degree of similarity were present in Ae. albopictus. B) The Venn diagram displays the number of Ae. aegypti lincRNAs with similarity scores above the cut off (E-value above 10−50) in other species using the BLAST algorithm.
Fig 4.
Volcano plot of differentially expressed Ae. aegypti lincRNAs in DENV-2 infected samples (midgut and carcass) compared with their corresponding controls.
Dots with red color represent lincRNAs with more than 4-fold changes due to DENV-2 infection.
Fig 5.
DENV infection leads to changes in the abundance of Ae. aegypti lincRNAs.
The relative transcript levels of selected numbers of Ae. aegypti lincRNAs were measured by RT-qPCR analysis of Aa20 cells infected with 1 MOI of DENV-2 for three days. Three biological replicates were used for each treatment with three technical replicates each. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
Fig 6.
The transcript levels of Ae. aegypti lincRNAs were altered in Wolbachia-infected mosquitoes.
RT-qPCR was used to analyze the relative transcript levels of selected numbers of Ae. aegypti lincRNAs in response to Wolbachia infection. For this, RNA from 4-day-old female mosquitoes from wMelPop (Pop)-infected and their tetracycline-cured line (Tet) mosquitoes were used in three biological replicates, each with three technical replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
Fig 7.
Possible involvement of lincRNA_1317 in DENV-2 replication in Ae. aegypti Aa20 cells.
A) Aa20 cells were double transfected with the transfection reagent only (Mock), dsRNA to GFP (dsCont) or dsRNA to lincRNA_1317 (dslncRNA) for three days followed by 1 MOI infection of the cells with DENV-2. Primers to the NS2A region (S1 Table) were used for measuring the relative DENV-2 genomic RNA levels. B) RNAi silencing of lincRNA_1317 using dsRNA was confirmed by RT-qPCR. C) Changes in DENV-2 genomic RNA levels during the course of infection analyzed by RT-qPCR on RNA extracted from Aa20 cells at 1, 3 and 5 days post-infection (dpi). D) DENV-2 infection increased the transcript levels of Ae. aegypti lincRNA_1317 in 3rd and 5th days post-infection when RNA from cells were analyzed by RT-qPCR. In all the experiments shown in this figure, three biological replicates, each with three technical replicates were used. **, p < 0.01; ***, p < 0.001.
Table 2.
Ae. aegypti miRNA recognition site distribution on lincRNA_1317.