A species-specific lncRNA modulates the reproductive ability of the asian tiger mosquito

Abstract

Long non-coding RNA (lncRNA) research has emerged as an independent scientific field in recent years. Despite their association with critical cellular and metabolic processes in plenty of organisms, lncRNAs are still a largely unexplored area in mosquito research. We propose that they could serve as exceptional tools for pest management due to unique features they possess. These include low inter-species sequence conservation and high tissue specificity. In the present study, we investigated the role of ovary-specific lncRNAs in the reproductive ability of the Asian tiger mosquito, Aedes albopictus. Through the analysis of transcriptomic data, we identified several lncRNAs that were differentially expressed upon blood feeding; we called these genes Norma (NOn-coding RNA in Mosquito ovAries). We observed that silencing some of these Normas resulted in significant impact on mosquito fecundity and fertility. We further focused on Norma3 whose silencing resulted in 43% oviposition reduction, in smaller ovaries and 53% hatching reduction of the laid eggs, compared to anti-GFP controls. Moreover, a significant downregulation of 2 mucins withing a neighboring (∼100 Kb) mucin cluster was observed in smaller anti-Norma3 ovaries, indicating a potential mechanism of in-cis regulation between Norma3 and the mucins. Our work constitutes the first experimental proof-of-evidence connecting lncRNAs with mosquito reproduction and opens a novel path for pest management.

Keywords: Aedes albopictus; RNAi pest control; lncRNAs (long non-coding RNAs); species-specific control; tiger mosquito.

FIGURE 1

Computational analysis of RNAs predicted to be non-coding via automated NCBI analysis. (A) A coding potential prediction model (FEELnc Random Forests) was trained specifically on Ae. albopictus sequences. The optimal cutoff to discriminate coding from non-coding sequences was set at the point where sensitivity and specificity was maximized (10-fold cross validation). (B) Overlap of coding potential predictions between FEELnc and CPC2 models. (C) Depiction of number of transcripts presenting hits to other Hexapoda species. Transcripts are tallied by the number of species in which they presented BLASTn hits (x-axis). (D) Genomic localization of lncRNAs, per type (genic/intergenic), subcategory (exonic/intronic/upstream/downstream/distal (i.e., >100 kb afar from other transcripts) and strand relative to close/overlapping elements (sense/antisense). (E) Heatmap of per-context fractions for all lncRNAs presenting tau > 0.5.

FIGURE 2

Expression of Norma transcripts across mosquito developmental contexts and oviposition rate change after dsRNA-treatment against them. (A) Heatmap of Norma expression levels in carcass, ovary, testis, diapause, larva, pupa and embryo samples from a publicly available transcriptomic dataset by Gamez et al. Expression is depicted as log₂-transformed TMM-normalized counts. (B) Impact of dsRNA-treatment, against each Norma separately, in oviposition rate. Each dot corresponds to the number of eggs that were laid individually by each female mosquito treated with dsRNA against 10 Norma or GFP dsRNA-treated mosquitoes and untreated control. Each sample contained at least 25 mosquitoes (biological replicates) and an unpaired two-tailed student’s t-test was conducted to assess the statistical significance of the results, by comparing the anti-GFP sample (control) with each anti-Norma sample. Treatment with dsRNA against Norma3, 9, 16, 17, 18, and 19 displayed statistically significant differences that may represent the influence of those genes on oviposition. Results are presented as mean ± SD. *:p-value≤0.05, **:p-value ≤0.01.

FIGURE 3

Spatiotemporal expression of Norma3 and its silencing efficacy. (A) Expression profile of Norma3 among non-blood fed (NBF) ovaries, post-blood meal (PBM) ovaries and other tissues collected 60 h PBM. Ovaries of blood-fed mosquitoes were collected every 12 h upon a blood meal, ranging from 12 to 72 h PBM when egg development is completed. Ovary samples (NBF & PBM) contained ovaries collected from 5-6 individual mosquitoes (biological replicates). Norma3 exhibits a basal expression in NBF, 12, 24, 36, and 48 h PBM ovaries, which abruptly increases at 60 h PBM leading to a fold change of >2,000 at 60 h PBM (x̄ = 2,387 ± 455.8, n = 5) which drops to 200-fold in 72 h-PBM (x̄ = 199.6 ± 46.6, n = 6), compared to NBF ovaries (x̄ = 1 ± 0.27, n = 6). The other tissues that are presented were collected 60 h PBM. OV = Ovaries, HD = Head, MG = Midgut, CR = carcass, MT = Malpighian tubes. Each sample contained tissues collected from three individual mosquitoes (biological replicates). Fold change is presented relatively to NBF ovaries. All values were normalized with ribosomal genes RpL32 & RpS17 and are presented as mean ± SEM. (B) Relative quantification of Norma3 expression in replicates of the anti-Norma3 dsRNA vs. anti-GFP dsRNA samples. Ovaries were collected 60 h PBM, the time point when Norma3 peaks its expression. Each sample contains 8-9 biological replicates. Average expression of Norma3 in anti-GFP replicates was set as 1 (x̄ = 100 ± 11.4, n = 9) and the overall expression drop of anti-Norma3 replicates, compared to anti-GFP, was measured to 50% (x̄ = 49.5 ± 6.2, n = 8, p = 0.0031). All values were normalized with ribosomal genes RpL32 & RpS17 and are presented as mean ± SEM, **:p-value ≤0.01.

FIGURE 4

Anti-Norma3 treatment leads to abnormal maturation of ovaries and reduced oviposition. (A) Comparison of the length (nm) of the long axis on the ovoid follicles from ovaries obtained from mosquitoes injected with anti-GFP and anti-Norma3 dsRNA. Follicles of the anti-Norma3 sample have a smaller size (x̄ = 240 ± 65.1, n = 40) (p = 0.0003), compared to the anti-GFP (x̄ = 293.6 ± 42, n = 29). Smaller cohort is a subgroup of the anti-Norma3 sample that includes highlighted smaller replicates (x̄ = 153.4 ± 37.8, n = 9). (B) Representative ovaries dissected 60 h PBM from the anti-GFP and the anti-Norma3 samples. Smaller follicle size and nurse cells are evident in smaller anti-Norma3 ovaries. (C) Number of deposited eggs per individual mosquito of untreated (x̄ = 44.6 ± 16.3, n = 106), anti-GFP (x̄ = 41.5 ± 16.4, n = 104) and anti-Norma3 (x̄ = 24.1 ± 16.7, n = 104) samples. Each dot corresponds to the number of eggs that were laid individually by each female mosquito treated with dsRNA against Norma3 or GFP and untreated control. Each sample contained more than 100 mosquitoes (biological replicates) and an unpaired two-tailed student’s t-test was conducted to assess the statistical significance of the results, by comparing the anti-GFP (control) with anti-Norma3 sample. Anti-Norma3 exhibits statistically significant reduced oviposition. All values are presented as mean ± SD. Error bars include values from min to max. ***: p-value ≤0.001, ****: p-value ≤0.0001.

FIGURE 5

anti-Norma3 treatment reduces hatch rate and disrupts regular embryonic development. (A) Comparison of hatchability of eggs laid by individual females of untreated (x̄ = 78.9 ± 19.9, n = 28), anti-GFP (x̄ = 77.9 ± 20.2, n = 30), and anti-Norma3 (x̄ = 36.9 ± 28.9, n = 26) samples. No statistical significance was observed between the untreated and anti-GFP samples, while high significance was detected between anti-GFP and anti-Norma3 samples (p < 0.0001, Mann-Whitney test). All values are presented as mean ± SD. ****: p-value ≤0.0001 (B) Effect of anti-Norma3 treatment in eggs that were dechorionated with Trpiš solution. Two representative embryos are displayed. Anti-GFP embryo presents regular development as based on the presence of respiratory siphon (Rs), eight abdominal segments (As), thoracic segments (Ts), head (H) and ocelli (Oc). On the contrary, anti-Norma3 does not present any of those structures.

FIGURE 6

Expression patterns through Post-blood meal timepoints (A,B) and in relation to Norma3 silencing (C). (A) Based on its expression across all timepoints, Norma3 was grouped in Cluster 34, which gradually peaks at 48 h Post-blood meal. (B) Three mucins neighboring Norma3 (mucin1-3) belong to Cluster 5 which presents an acute peak at 48 h. (C) Expression of Norma3-neighboring proteins in replicates of the anti-Norma3 vs. anti-GFP sample. All mucins exhibit a statistically significant expression drop. Mucin1 61% (x̄ = 39.1 ± 19.3, n = 9), mucin2 41% (x̄ = 59.2 ± 19.3, n = 9), mucin3 80% (x̄ = 20 ± 12.5, n = 9) while other neighboring proteins (chymo1, venom1) do not (Mann-Whitney test). Both anti-GFP and anti-Norma3 samples contain 9 biological replicates. Results were normalized with ribosomal genes RpL32 and RpS17. All values are presented as mean ± SEM. *: p-value ≤0.05, **: p-value ≤0.01.