Fig 1.
Experimental strategy to compare molecular events occurring through male and female S. mansoni development.
(A) A monomiracidial infection of mollusks was first performed in order to obtain either ZZ male or ZW female clonal cercariae. (B) The sex of the cercarial clones was determined by PCR of female-specific sequences and unisexual infection of mice was then performed. For the molecular study, we recovered one free-living undifferentiated stage of the parasite: cercariae; and four intra-vertebrate stages: (C) the three differentiating schistosomulum stages and (D) the adult stage. The schistosomulum stages are characterized by the onset of sexual dimorphism between s#2 and s#3, and are described more in details in the S1 protocol. For these five stages, we compared the male and female transcriptomes using RNAseq (biological duplicate). For the non-differentiated cercariae and the sexually differentiated adults we also performed a ChIPseq in duplicate for males and triplicate for females.
Fig 2.
Venn diagrams of sex-biased genes.
For each of the three developmental stages (a) cercariae, (b) schistosomula s#2 and (c) adults, significant differences (Padj<0.05) between sexes were detected by DEseq analyses. Here are represented the number of sex-biased genes by stage and those that are shared between different stages: (A) when pooling male- and female-biased genes, or considering only (B) female-biased genes or (C) male-biased genes. (D) The table represents the number of sex-switching biased genes that are more expressed alternatively in females or in males depending on the developmental stage.
Fig 3.
GO term analysis of sex-biased genes.
(A) Distribution of total sex-biased GO categories (i.e. both sexes and considering the three stages). (B) Percentage of male (in blue) and female (in pink) sex-biased GO terms for each category within each stage: cercariae, schistosomula s#2 and adults. (C) Percentage of sex-biased GO terms related to chromatin structure in schistosomula and adults. (D) Sex-biased GO terms related to chromatin structure in schistosomula and adults.
Fig 4.
General clustering of sex-biased gene expression depending on their “general developmental pathway” functional category.
Male-biases are represented in blue, female-biases in pink and non-significant differences (Padj>0.05) in black. Three stages are considered: (a) cercariae, (b) schistosomula s#2 and (c) adults. (A) 39 homeotic genes are mainly female-biased in cercariae. (B) 58 genes linked to growth factor pathways are mainly male-biased in cercariae and adults. (C) 40 genes are related with the steroid pathway and are mainly male-biased through the three stages. The functional annotation and details on gene expression are provided in S6 Table.
Fig 5.
Overview of the 12 sex-biased miRNAs through three stages of the schistosome lifecycle (a: cercariae, b: schistosomula s#2, c: adults). (A) General clustering of sex-biased miRNA expression: seven male-biased miRNAs were detected (in blue), five were female-biased (in pink). (B) Table of sex-biased miRNA precursors IDs, chromosomal location and expression bias. Four miRNAs were located on sex chromosomes: three on the Z-specific regions (as defined by Protasio et al. [4]) were overexpressed in males. Key: PSA = PseudoAutosomalRegion, Z = Z-specific gene, N.A. = no sex-bias of expression.
Fig 6.
General clustering of sex-biased gene expression depending on their “regulation of gene expression” functional category.
male-biaises are represented in blue, female-biaises in pink and non-significant differences (Padj>0.05) in black. Three stages are considered: (a) cercariae, (b) schistosomula s#2 and (c) adults. (A) 873 mobile genetic elements were detected as sex-biased, mainly in female adult worms. (B) 45 splicing-linked genes are mainly male-biased in schistosomula s#2. (C) 59 genes involved in chromatin modification are mainly male-biased in schistosomula s#2. The functional annotation and details on gene expression are provided in S6 Table.
Fig 7.
Average H3K27me3 enrichment profile of the chromosome 1 genes.
X axis represents the position in bases relative to the transcriptional start site (TSS, position 0), Y axis represents the normalized mean enrichment of reads obtained after a ChIP targeting the H3K27me3 mark on (A) cercariae and (B) adults. The EpiChIP enrichment has been calculated around the TSS of the chromosome 1 transcripts obtained in our RNA-Seq experiment. It has been normalized with the same mean enrichment of reads obtained after a ChIP without antibody. The mean profile for two male biological replicates (blue) and three female biological replicates (red) are represented. The profile for each replicate is provided in S1 supporting information (Slide 8). The same profile was obtained for the ZW linkage group.
Fig 8.
Male-biased (in blue) and female-biased (in pink) players accompanying Schistosoma mansoni sexual differentiation, and hypothetical links with known life-history traits (dotted line).
(A) The cercarial stage is marked by gene expression differences in environment detection: the male-response to more specific chemical stimuli can facilitate the host detection and thus could allow a larger dispersion of the male cercariae explaining explain the higher genetic diversity observed later in male adults (1) [130]; and (ii) be responsible of the male-biased sex ratio (3) [130]. (B) Molecular events preparing parasite development start before the host penetration: while intrinsic pathways (homeogenes) are female-biased, growth factor and hormonal pathways are male-biased. These pathways could not only be intrinsic to the parasite, but also linked to the host microenvironment. These differences in developmental strategy could lead to the previously hypothesized faster male development (2) [128] and thus indirectly to the male-biased sex ratio (3) [130]. Several important candidates to sex determination and/or differentiation were identified both in males and females through the three stages of development. (C) Different putative players of the host/parasite interaction were detected as sex-biased trough the different stages of development. These differences could lead to both higher male immunogenicity and pathogenicity (4) [131, 142, 143]. Furthermore, if they facilitate male-resistance against the host immune system, they could indirectly be responsible of the male-biased sex-ratio (3) [130]. (D) Different putative regulators of gene expression were detected as sex-biased. Particularly, the depletion in H3K27me3 histone mark could lead to an optimized male development (2). Lines and dots link subcategories to more general biological process. “Black” life-history traits were previously published and “grey” ones are hypothesized. Abbreviations: VALs = Venom Allergen Like proteins, MEGs = Micro Exon Genes