Fig 1.
Schematic representation of the pairing assays performed by Lu et al. 2016 and in the present work.
(A) Lu et al. [14] exposed hamsters to a S. mansoni mixed-sex cercariae infection (left) or to single-sex infections (middle); 46 days after infection (left) or 67 days after infection (middle), hamsters were perfused, and the adult worms recovered. For the mixed-sex infection, parasites were manually separated. Gonads were isolated from a fraction of the recovered worms, and RNA was extracted from mixed-sex infection whole males (bM) and testes (bT), from mixed-sex whole females (bF) and ovaries (bO), as well as from single-sex whole males (sM) and testes (sT) and single-sex whole females (sF) and ovaries (sO). Samples were submitted to RNA-Seq; in the present work, we re-analyzed the RNA-Seq data to search for differentially expressed lncRNAs. (B) Here, hamsters were exposed to a S. mansoni mixed-sex cercariae infection; adult worms were retrieved by perfusion 42 days post-infection. Worms retrieved in the perfusion as paired couples were collected separate from adult males and females that were retrieved as naturally separated worms. Worm pairs or separated worms were cultured in vitro for 2, 4 and 8 days in ABC media; separated worms are known to experience regression of the reproductive organs. At the end of the incubation times, the paired worm couples were manually separated. Male and female worms that were either paired or unpaired during in vitro culturing were submitted to RNA extraction, and RT-qPCR measurements were performed. Images reproduced with permission from commons.wikimedia.org (trematode lifecycle stages), from stock.adobe.com (hamsters), and from Elsevier B.V. through PLSclear ([68], immature and mature females).
Fig 2.
Heatmap of Schistosoma mansoni long non-coding RNAs (lncRNAs) or protein-coding genes differentially expressed (DE) between parasites from mixed-sex and single-sex infections.
Non-supervised hierarchical clustering of (A) 3681 DE lncRNA genes (lines) or (B) 11,109 DE protein-coding gene isoforms (lines) in each of the biological replicates (columns) of S. mansoni parasites retrieved from mixed-sex (b) or single-sex (s) infections, as indicated in the sample labels at the bottom of the heatmaps. These results were obtained through a re-analysis of the RNA-Seq data from Lu et al., 2016 [14], now using as reference the lncRNA transcriptome previously published by Maciel et al., 2019 [41]. Statistically significant DE genes were determined with DESeq2 (FDR < 0.05). Gene expression levels are shown as Z-scores, which are the number of standard deviations below (blue lines, downregulated) or above (red lines, up-regulated) the mean expression value of each gene among all samples, as indicated by the color scale on the right. Females from mixed-sex or single-sex infections are identified by bF or sF; males from mixed-sex or single-sex infections by bM or sM; ovaries from mixed-sex or single-sex infection females by bO or sO; testes from mixed-sex or single-sex infection males by bT or sT.
Fig 3.
Expression of lincRNAs enriched in females from mixed-sex infections, and in vitro differential expression validation by RT-qPCR.
Three lincRNAs detected as enriched in females from mixed-sex infections (bF) in the re-analyses of the RNA-Seq dataset of Lu et al., 2016 [14], were selected for in vitro RT-qPCR assays, namely (A) SmLINC142881, (B) SmLINC175062, and (C) SmLINC110998. Male related results are shown on the left (green) and female results on the right (orange). Paired couples (P) or unpaired (U) parasites were obtained by perfusion of hamsters infected for 42 days with S. mansoni cercariae. After perfusion, males and females were cultured in vitro for 2, 4 or 8 days as paired (P) couples or unpaired (U) male and female worms. RT-qPCR results (solid-colored graphs) are normalized to the geometric mean of reference genes Smp_099690 and Smp_023150. Expression values from 4 different biological replicates are shown. Standard error of the mean (SEM) is shown in the error bars. (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001, Student t test. N.D.: Not detected. ns: p-value > 0.05. For comparison, RNA-Seq data from the re-analysis of Lu et al., 2016 [14] is shown (plaid-colored graphs) and the expression is measured in TPM (transcripts per million); RNA-Seq data is retrieved from males (M), females (F), testes (T) or ovaries (O) from either a mixed-sex (b) or a single-sex (s) infection; (#) = FDR<0.005. The fold-change differences between the compared groups are represented under the brackets.
Fig 4.
Expression of lincRNAs enriched in samples other than females from mixed-sex infections and in vitro differential expression validation by RT-qPCR.
Three lincRNAs detected as enriched in samples other than females from mixed-sex infections in the re-analyses of the RNA-Seq dataset of Lu et al., 2016 [14], were selected for in vitro RT-qPCR assays, namely (A) SmLINC133371, enriched in sT, sF and sO; (B) SmLINC101519, enriched in bM and sF; and (C) SmLINC141426, enriched in bT. Male related results are shown on the left (green) and female results on the right (orange). Paired couples (P) or unpaired (U) parasites were obtained by perfusion of hamsters infected for 42 days with S. mansoni cercariae. After perfusion, males and females were cultured in vitro for 2, 4 or 8 days as paired (P) couples or unpaired (U) male and female worms. RT-qPCR results (solid-colored graphs) are normalized to the geometric mean of reference genes Smp_099690 and Smp_023150. Expression values from 4 different biological replicates are shown. Standard error of the mean (SEM) is shown in the error bars. (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001, Student t test. N.D.: Not detected. ns: p-value > 0.05. For comparison, RNA-Seq data from the re-analysis of Lu et al., 2016 [14] is shown (plaid-colored graphs) and the expression is measured in TPM (transcripts per million); RNA-Seq data is retrieved from males (M), females (F), testes (T) or ovaries (O) from either a single-sex (s) or mixed-sex (b) infection; (#) = FDR<0.005. The fold-change differences between the compared groups are represented under the brackets.
Fig 5.
Phenotypic changes in Schistosoma mansoni adult worm couples upon in vitro silencing (RNAi) of each of three pairing-dependent lincRNAs.
Paired couples were obtained by perfusion of hamsters infected for 42 days with S. mansoni cercariae. Couples were cultured in vitro for 8 days, in ABC media supplemented with 30 μg/mL of dsRNA targeting each of the lincRNAs indicated by the different colors, namely SmLINC101519 (pink), SmLINC110998 (blue), or SmLINC175062 (orange). Medium was exchanged every other day while dsRNA was added every day. dsRNA targeting mCherry (a gene that is not present in S. mansoni) was assayed in parallel as a negative control (light gray). Results for parasites cultured with no dsRNA are also shown (dark gray). (A) RT-qPCR results for each lincRNA expression level are normalized to the geometric mean of reference genes Smp_099690 and Smp_023150. (B-D) Pairing status, adhesion to the plate and motility of worm couples were traced along the 8 days of the experiment. (E) Viability of adult worms (males+females) was monitored using the ATP-Glo Assay. (F) At the end of the experiment (8 days), eggs were collected and counted. (G-J) Collected eggs were monitored for their size (area) (G), integrity of their eggshell (autofluorescence) (H), proliferation status of the embryos (Egg Edu+/DAPI+ cell ratio) (I), and the percentage of egg hatching was measured by keeping the eggs in culture for another 7 days in ABC media for synchronization of their development, then assessing egg hatching as described in Methods, with the percentage of hatched eggs being shown (J). Violin plot representation at Figs (G-I) with the median indicated by the red line and the quartiles represented by the dashed lines. Results from 4 different biological replicates are shown. Standard error of the mean (SEM) is shown in the error bars. Student t test (panels A to D) or One-Way ANOVA test with multiple comparisons to dsmCherry group (panels E to J) were used. (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001; (****) = p < 0.0001.
Fig 6.
In vitro silencing of pairing-dependent lincRNAs in S. mansoni adult worm couples leads to an impaired cell proliferation status.
Paired couples were obtained by perfusion of hamsters infected for 42 days with S. mansoni cercariae. Couples were cultured in vitro for 8 days in ABC media supplemented with 30 μg/mL of dsRNA targeting each of the indicated lincRNAs, namely SmLINC101519, SmLINC110998, or SmLINC175062, with a negative control dsRNA targeting mCherry (a gene that is not present in S. mansoni) or with no dsRNA. Medium was exchanged every other day while dsRNA was added every day. Cell proliferation was assayed by labeling with thymidine analog 5-ethynyl-2′-deoxyuridine (EdU), which was added to the cultures on the 7th day of culture at a final concentration of 10 μM and incubating for 24 h. EdU labeling detection in (A) adult worms, and in (B) the gonads was performed as described [64]. DAPI stained cells nuclei are shown in gray and EdU+ cells (proliferating cells) are stained in red. Scale bars: 250 μm for the adult worm images in (A), and 25 μm for the adult worm gonad images in (B). Representative images from 3 experiments with n > 10 parasites. The red rectangles define zoomed-in insets of interest that correspond to the regions within green rectangles.
Fig 7.
In vitro silencing of pairing-dependent lincRNAs in S. mansoni adult worm couples causes female vitellaria impairment.
Paired couples were obtained by perfusion of hamsters infected for 42 days with S. mansoni cercariae. Couples were cultured in vitro for 8 days in ABC media supplemented with 30 μg/mL of dsRNA targeting each of the indicated lincRNAs, namely SmLINC101519, SmLINC110998, or SmLINC175062. Medium was exchanged every other day while dsRNA was added every day. dsRNA targeting mCherry (a gene that is not present in S. mansoni) was assayed in parallel as a negative control. Results for parasites cultured with no dsRNA are also reported. Female vitellaria were stained with Fast Blue BB (pink) and BODIPY (green), which labeled vitelline and lipid droplets in the vitellaria, respectively. DAPI staining of cells nuclei is shown in gray. Scale bars: 25 μm. Representative images from 3 experiments with n > 10 parasites. The red squares define zoomed-in insets of interest that correspond to the regions within green squares.
Fig 8.
Localization of the selected pairing-dependent lincRNAs in adult worm tissues by whole mount in situ hybridization.
Whole mount in situ hybridization (WISH) of each lincRNA is shown as the blue color stains in the male (left) or female (right) adult worm images of the heads and bodies. Scale bars are 100 μm. Results for (A) SmLINC101519, (B) SmLINC110998, and (C) SmLINC175062. For comparison purposes, single-cell RNA-Seq data from Morales-Vicente et al., 2022 [28] was retrieved from (http://verjolab.usp.br:8081/). LincRNA expression patterns across the single-cell clusters are shown with UMAP plots, colored by gene expression levels (blue = low, red = high), and the scale represents log10(UMIs+1). The regions enclosed by red dashed lines indicate the relevant cell clusters: Region 1, parenchyma 1 cells cluster; Region 2, different muscle cell clusters; Region 3, different neuron cell clusters; Region 4, late vitellocyte cells cluster.
Fig 9.
In vivo silencing (RNAi) of pairing-dependent lincRNAs decreases the number of S. mansoni adult worms recovered from infected mice.
Five weeks old C57BL/6 female mice were infected with an average of 145 cercariae. At weeks 1, 3, 5 and 6 post infection, mice were injected in the retro orbital vein with 30 μg of dsRNA targeting the specified lincRNA (in a 100 μL solution with non-pyrogenic saline), namely SmLINC101519 (pink), SmLINC110998 (blue), or SmLINC175062 (orange). A dsRNA targeting mCherry (a gene that is not present in S. mansoni and mice) was assayed in parallel as a negative control. At week 7, the mice were perfused, and the worms retrieved. The liver from each mouse was processed for measuring eggs. (A) RT-qPCR results for each lincRNA expression level are normalized to the geometric mean of reference genes Smp_099690, Smp_023150, Smp_196510, and Smp_101310. (B-E) Worm burden results are reported, where the number of paired worm couples (B), number of unpaired male worms (C), number of unpaired female worms (D) retrieved from perfusion, and total number of worms (E) are shown. The number of eggs retrieved from the livers of infected mice was measured after liver processing as mentioned in Methods. Those eggs were counted and the number of eggs per gram of liver is reported (F). Egg health parameters such as size (area) (G) and eggshell integrity (autofluorescence) (H) were measured. The eggs were kept in culture for another 7 days in ABC media for synchronization of their development. Then egg hatching assay was performed as described in Methods and the percentage of hatched eggs is shown (I). Standard error of the mean (SEM) is shown in the error bars. Student t test (panel A) or One-Way ANOVA test with multiple comparisons to dsmCherry group (panels B to I) were used. (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001; (****) = p < 0.0001.