article: Among more than 20,000 species of hermaphroditic trematodes, Schistosomatidae are unusual since they have evolved gonochorism. In schistosomes, sex is determined by a female heterogametic system, but phenotypic sexual dimorphism appears only after infection of the vertebrate definitive host. The completion of gonad maturation occurs even later, after pairing. To date, the molecular mechanisms that trigger the sexual differentiation in these species remain unknown, and in vivo studies on the developing schistosomulum stages are lacking. To study the molecular basis of sex determination and sexual differentiation in schistosomes, we investigated the whole transcriptome of the human parasite Schistosoma mansoni in a stage- and sex-comparative manner. We performed a RNA-seq on males and females for five developmental stages: cercariae larvae, three in vivo schistosomulum stages and adults. We detected 7,168 genes differentially expressed between sexes in at least one of the developmental stages, and 4,065 of them were functionally annotated. Transcriptome data were completed with H3K27me3 histone modification analysis using ChIP-Seq before (in cercariae) and after (in adults) the phenotypic sexual dimorphism appearance. In this paper we present (i) candidate determinants of the sexual differentiation, (ii) sex-biased players of the interaction with the vertebrate host, and (iii) different dynamic of the H3K27me3 histone mark between sexes as an illustration of sex-biased epigenetic landscapes. Our work presents evidence that sexual differentiation in S. mansoni is accompanied by distinct male and female transcriptional landscapes of known players of the host-parasite crosstalk, genetic determinants and epigenetic regulators. Our results suggest that such combination could lead to the optimized sexual dimorphism of this parasitic species. As S. mansoni is pathogenic for humans, this study represents a promising source of therapeutic targets, providing not only data on the parasite development in interaction with its vertebrate host, but also new insights on its reproductive function.
  Among the hundred species of Schistosomatidae, the Schistosoma genus is of particular medical importance as it is pathogenic in humans. Seven Schistosoma species are responsible for schistosomiasis (or bilharziasis) [1,2], which represents the second most important parasitic disease after malaria and affects at least 240 million people worldwide [3]. Schistosoma mansoni, responsible for the intestinal schistosomiasis, is endemic in Africa and South America [1] and has been a study model for the Schistosomatidae. S. mansoni has a genome of 364. 5MB (genome version 5. 2), containing 10,852 genes, seven pairs of autosomal chromosomes and one pair of ZZ/ZW sex chromosomes [4,5]. S. mansoni has a complex life cycle in which a freshwater snail from Biomphalaria genus serves as intermediate host and primates or rodent species as definitive host. By definition, sexual reproduction occurs in the vertebrate host. The parasite’s eggs are released in freshwater via the feces. Free-living larvae (miracidia) hatch out, and infect the mollusc intermediate host where they transform into sporocysts that release human infecting cercariae after asexual multiplication. These larvae actively seek definitive host skin contact and penetrate the epidermis. During skin penetration, the cercariae lose their tail, and their head undergo drastic morphological and physiological transformations. Within two hours, the free-living larvae become obligatory endoparasitic schistosomula. They leave the dermis to reach the bloodstream and migrate to the liver via the lungs [6,7]. At the first steps of the life cycle, some life-history traits have been shown to be different between males and females [8], but no apparent phenotypic sexual dimorphism exists from the eggs to the early stages of schistosomula, in spite of the fact that the parasites possess genetically different sexes. After two to five weeks within the definitive host, and once they reach the hepatic portal system [9], the schistosomula develop from 150 μm juvenile sexually undifferentiated individuals into one centimeter differentiated male or female adult worms. This developmental step could be defined as “sexual differentiation”. Then, dimorphic males and females mate and migrate together to mesenteric venules where they intensely reproduce. Mating is critical for completing gonadal maturation of both sexes [10]. In the adults, the dimorphic phenotype is crucial for the fitness of the parasite: (i) the muscular male clasps the female into his gynaecophoric groove to resist the high blood pressure environment, (ii) the filiform female is able to insinuate into the tiniest venules of the intestine to lay the eggs one by one [11] up to 300 eggs per day. Approximately 50% of these eggs remain trapped in the vertebrate host, causing inflammatory chronic disease [12]. The paired adult parasites can remain for decades in their host, showing their highly adapted interaction with the vertebrate host and the success of the gonochoristic strategy. In this context, the understanding of developmental and reproductive biology of schistosomes is crucial to fight schistosomiasis. Many studies have addressed the question of the development of the parasite within its vertebrate host and the establishment of the highly efficient reproductive system [13]. Particularly, numerous works have highlighted the responsiveness of the developing S. mansoni to the host blood microenvironment and shown that the parasite might exploit endocrine and host immune signals to accomplish its development [14–18]. The mating status (i. e. paired vs unpaired) has also been shown to play an essential role for the maturation of both male and female [19–26]. Other molecular studies have highlighted male- or female-biased pathways essential for the development and the reproduction of the parasite [27–35]. Global transcriptomic analyses were carried out on diverse developmental stages [4,36–38] but sex-biased expressions were only explored in adult [38,39] or cercariae [40]. Epigenetic control for gene expression regulation has also been investigated and highlighted sex-specific epigenetic processes with chromatin structural changes occurring on female-specific microsatellite repeats of the W-chromosome during the development of the parasite [41]. Moreover sex-biased and stage-specific microRNA (miRNA) precursor expression suggests that non-coding RNAs (ncRNAs) participate in the S. mansoni sexual differentiation process [42–44]. In the present report, we propose to correlate molecular mechanisms to phenotypic sexual dimorphism appearance. With this in mind, we present the global transcriptome of S. mansoni in a stage- and sex-comparative manner. Here, we employed an exhaustive RNA-sequencing analysis in five stages of in vivo development of S. mansoni, from the undifferentiated cercariae to the phenotypically very distinctive male and female worms. The in vivo schistosomulum differentiating stages, critical in the host/parasite interaction, were sub-categorized in three morphologically consistent groups of individuals [45–47] and are described here for the first time. In addition to the transcriptomic study, we present male and female genome-wide chromatin structure profiles before (in cercariae) and after (in adults) the somatic sexual differentiation, using ChIP-Seq against H3K27me3 as a proxy.
  The bioinformatic pipeline and the quality of the metrics are described in S1 supporting information. All data treatment was carried out under a local galaxy instance [52]. After grooming (i. e. Fastq sanger format checking) (Fastq galaxy tool v1. 0. 4, [53]) and quality assessment of the reads (FASTX-Toolkit v0. 0. 13, [54]) we determined Phred quality scores over 25 for all the nucleotides. Consequently, neither quality filtering nor trimming was applied and all the reads were mapped to the S. mansoni reference genome (assembly version 5. 2) [4] using TopHat (v2. 0. 9) [55], with the “very sensitive” option for Bowtie2 settings (v2. 1. 0. 0, [56]). The resulting BAM files were converted to SAM format (SAM tools v0. 1. 18. 0, [57]) and the unmapped reads were removed. In order to be consistent in the further differential analyses between the different samples, all datasets were adjusted to the smallest by randomly picking 65,844,021 reads from each file. Exon-intron structure was then reconstructed with Cufflinks (v2. 1. 1) [58] without any correction parameter: neither quartile normalization, nor bias corrections were applied. All the obtained data were joined with Cuffmerge (v1. 0. 0) [58] without any genome reference nor guide, in order to create a S. mansoni de novo reference transcriptome containing the exon-intron structures of the five developmental stages. The GTF file of this transcriptome is available at the IHPE laboratory webpage [59] and the sequences of all the assembled unique transcripts (TCONS) in the S5 Table (sheet1). We quantified each sample read abundance by mapping each condition to this de novo reference transcriptome with HTseq (v0. 6. 1p1) with the overlap resolution mode union [60]. Finally, the differential gene expression levels between sexes were analyzed with the DESeq package (v1. 12. 1) [61]. We carried out five comparisons: male vs female cercariae, male vs female schistosomula s#1, male vs female schistosomula s#2, male vs female schistosomula s#3, male vs female adult worms (Fig 1). Considering one stage and one transcript, significant difference in expression between sexes was evaluated according to the adjusted P-value (Padj) for multiple testing with the Benjamini-Hochberg procedure which controls false discovery rate (FDR). Genes that were significantly (Padj <0. 05) overexpressed in one sex compared to the opposite sex, were defined as “sex-biased genes”. The de novo assembled transcriptome was entirely and automatically annotated (S5, sheet2). Blastx searches against the non-redundant database of the NCBI (14-oct-2013) were performed on a local server using BLAST 2. 2. 26+ version [62]. XML files were loaded onto Blast2GO for gene ontologies (GO), mapping and annotation with version b2g_sep13 of the Blast2GO database [63]. InterProScan 5–44. 0 version [64] was then used and Interproscan GO were merged to Blast2GO. Using BLAT (v34) [65] we aligned the de novo transcriptome to the S. mansoni coding sequences of the reference genome v5. 2 (ASM23792v2. 30) got at the Ensemble Genomes resource [66], setting the minimum score to 50. In the case of alignment of a Cufflinks gene (XLOC_ID) to multiple reference genes (Smp_IDs), the hit with the higher identity was considered as the correct alignment. Reciprocally, only the higher coverage for a reference transcript was conserved. We then use Cuffcompare, an associate utility program of Cufflinks (v2. 2. 1) in order to characterize the type of matches between the Cufflinks transcripts and the reference transcripts (v5. 2). Functional analysis introduced in this paper mainly consider the three stages cercariae, schistosomula s#2 and adults because these three stages display the highest number of significant sex-biased genes, due to the quality of the biological replicates [61]. Concerning the two other schistosomulum stages s#1 and s#3, we detected less than 100 significant sex-biased genes (Padj<0. 05). Thus, only the 100 best adjusted P-values were analyzed (S2 Table) and heatmaps revealed the consistence of replicates in these stages (S1 supporting information, slide5). For each developmental stage and sex of the parasite, first strand cDNA synthesis and qPCR validation experiments were achieved on two different biological replicates. 500ng of the purified total RNA were reverse transcribed using identical concentration (250 nM) of random and oligo-dT primers of Maxima H Minus Reverse Transcriptase kit (ThermoSCIENTIFIC). qPCRs were performed using a LightCycler 480 System (Roche Diagnostics) with the LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics). Single product amplification was checked by analysis of the amplicon melting curve and capillary migration on a Labchip GX DNA assay system (PerkinElmer). For each reaction, the crossing point (Cp) was determined using the second derivative maximum method using Light Cycler Software version 3. 3 (Roche Diagnostics). For each studied stage, sex, and replicate, the level of transcription was normalized using the mean geometric transcription rate of three reference sequences Smp_093230 (Sm-arp 10, actin protein 10), Smp_197220 (Sm-RPL35, subunit of the oligosaccharyltransferase) and Smp_089880 (Sm-fad oxidoreductase, FAD dependent oxidoreductase domain containing protein) previously described [72]. The stability indexes of those reference genes were calculated using NormFinder (v20) [73] to assess if they were stable (i) during all the developmental stages and (ii) between males and females. Forward and reverse primers (Eurogentec) were designed for 43 genes (in addition to the three housekeeping genes) with the Primer3plus web based interface [74], the lack of putative primer dimer was checked with Perlprimer (v1. 1. 21) and the uniqueness of the target was verified using blast on the S. mansoni genome v5. 2. Primer efficiencies were >1. 8 for each couple. Primer sequences and expected PCR product sizes are listed in S1 Table. Correlation between RNAseq and qPCR was tested both globally and individually for each gene with a Spearman Rank test (S2 supporting information). Protein coding genes mentioned in the text are accessible on GeneDB ([70], http: //www. genedb. org/) under the following accession numbers: Smp_093230: actin protein 10 (Sm-arp10) / Smp_197220: subunit of the oligosaccharyltransferase (Sm-RLP35) / Smp_089880: FAD dependent oxidoreductase domain containing protein (Sm-fad oxidoreductase) / Smp_196410: dachshund / Smp_000530: zinc finger transcription factor gli2 / Smp_141030: EGF receptor kinase substrate 8-like / Smp_035260: EGF receptor kinase substrate 8-like / Smp_134550: Neuropeptide (Sma-npp-27) / Smp_212730: tyrosine kinase, TK group, Src family (SmTK3) / Smp_174880: FOG / Smp_103470: protein mago nashi / Smp_045950: transformer 2 protein / Smp_009600: serine: threonine protein kinase PLK1 (polo-like kinase 1) / Smp_159800: MEG-2 (ESP15) family / Smp_159810: MEG-2 (ESP15) family / Smp_010550: MEG 15 / Smp_163630: MEG-4 (10. 3) family / Smp_033600: Dicer 2 / Smp_118190: staphylococcal nuclease domain-containing protein / Smp_165220: polycomb protein EED / Smp_006250: polycomb protein Scm1 (Sex comb on midleg homolog, Scm1). miRNA precursors mentioned in the text are accessible on miRBase ([71,145], http: //www. mirbase. org/index. shtml) under the following accession numbers: MI0027256: sma-mir-1a / MI0027255: sma-mir-8458 / MI0027196: sma-mir-125c / MI0027285: sma-mir-8483 / MI0027247: sma-mir-8451 / MI0027258: sma-mir-8459 / MI0027222: sma-mir-8429.
  We generated the transcriptomes of cercariae, three developmental stages of schistosomula (s#1, s#2 and s#3) and adult worms from unisexual infection. Details and illustrations for the sorting criteria of the three classes of schistosomula are shown in S1 protocol. For each developmental stage, males and females were analyzed separately with two biological replicates (Fig 1). In total, the transcriptome sequencing of these 20 samples yielded 1,080,386,261 Illumina single reads of 50bp and 981,363,482 mapped to the S. mansoni reference genome (v5. 2). These mapped reads could be assembled in 54,956 unique transcripts identified as “TCONS”, (S5 Table, sheet1) representing putative splice variants of 34,755 genes identified as “XLOC” (S5 Table, sheet1). Among these expressed genes, 9,581 annotated genes of the S. mansoni reference genome (v5. 2) could be identified with the blat aligner (S5 Table, sheet3). Notably, we found that 6. 95% of the transcripts could correspond to novel isoforms of known transcripts and that 45. 39% were located in intergenic region of the reference genome (S1 supporting information, slide6). Quantification of read abundance and DEseq analysis of differential gene expression between sexes were performed for each stage. We present here significant transcriptomic differences (adjusted P-value < 0. 05) observed in cercariae, schistosomula s#2 and adults. These three stages taken together, 7,168 genes appeared as significantly differentially expressed between sexes (i. e. either more expressed in males or more expressed in females) in at least one of the classes (Fig 2A). They were defined as “sex-biased genes” and represent 20. 62% of the total number of expressed genes. For cercariae, schistosomula s#2 and adults, we identified respectively 5,264 (2,353 male-biased and 2,911 female-biased); 1,534 (1,040 male-biased and 494 female-biased) and 1,645 (1,043 male-biased and 602 female-biased) sex-biased genes (Fig 2). Ninety-eight male-biased and 26 female-biased genes were consistent through all the stages (Fig 2). Interestingly, several genes are alternatively more expressed in females or in males, they were qualified as “sex-switching biased genes” (Fig 2D). To further support our transcriptomic analysis, qPCR experiments were performed to validate the expression patterns obtained from the DEseq analysis. Forty-three transcripts were randomly tested and a Spearman Rank test determined that RNAseq and qPCR experiments were significantly (p<0. 0001) and positively correlated, with a coefficient R = 0. 65 (S2 supporting information). Among the 7,168 “sex-biased” genes, 2,468 corresponded to known genes (S4 Table, sheet20) and 1,598 other could be de novo functionally annotated by the blast2GO analysis (S4 Table, Sheet19), totalizing 4,065 functionally annotated sex-biased genes. To gain insight into major biological processes that would be enriched during development in either male or female individuals, we performed an exact Fisher test using the blast2GO software, focusing on the “biological processes” ontology. Enriched GO terms are presented in the S3 Table. To obtain a more synthetic overview, we manually sorted the GO terms into more general categories and considered the GO term enrichment for each of them (Fig 3A). Of particular interest in the context of host-parasite interaction, we detected that the response to “environmental stimulus” occurred in cercariae at the same level for both males and females (Fig 3B). Nonetheless, GO terms were different and indicated a distinct perception of environmental factors (S3 Table). Female cercariae displayed a better “response to light” and “mechanical stimulus” than males. Male cercariae seemed better responsive to “chemical stimuli” and this capacity was maintained in schistosomula and adult worms. Among the GO terms involved in response to “chemical stimuli”, we noticed the particular biological processes “response to vitamin D” and “growth epidermal factor” (both enriched in male cercariae, S3 Table sheet1), and “response to cortico/gluco-steroid stimulus” (enriched in male schistosomula, see S3 Table sheet3). Along with these differences in environment perception we detected distinct representation of terms related to metabolic functions between sexes. Particularly, some categories were more enriched in males whatever the developmental stage such as “protein process”, “heme related process”, “energy” and “carbon metabolism” (Fig 3B). Besides, and of special interest for gene expression regulation, we detected a particular enrichment of the category “chromatin structure” in male schistosomula and adults. This category did not display any differences between sexes in the larval stage (Fig 3C). The corresponding GO terms are represented in the Fig 3D. They all converged toward a strong chromatin re-organization during the vertebrate stages of the parasite, emphasized in males. Finally, considering processes putatively involved in the sexual dimorphism appearance, our GO term analysis indicated that the very general “development” category was over-represented in female cercariae and adults, but more in male schistosomula (Fig 3B). More specifically, “sexual differentiation” was a category over-represented in female cercariae while the “reproduction” category was over-represented in male cercariae and schistosomula and then reversely over-represented in adult females (Fig 3B). The 4,065 functionally annotated sex-biased genes were manually classified into 16 classes related to (i) the general developmental pathways, (ii) the sex determination and/or sexual differentiation pathways and (iii) the detection of the environment and/or the interaction with the vertebrate host (S4 Table). Players of transcription mechanisms, genetic mobile elements, chromatin remodeling and other DNA modifications together with RNA processing were examined by a combination of the transcriptomic approach and epigenetic analysis.
  Despite the large attention given to S. mansoni reproduction biology, only few molecular candidates have been shown to be key players of sex determination and differentiation and most are related to sexual maturation after pairing. They mainly belong to the “kinome” [27,29–32,103,104], particularly the TGFβ pathway [17,25,105,106], and were identified as new potential target for therapies [107]. Our transcriptional study allows the identification of several promising candidates with a sex-biased expression (Fig 8B), and we discuss here four of them that, in our opinion, need further attention. The “mago nashi protein homolog” (Smp_103470) is male-biased in schistosomula. Its gene product is a nuclear factor highly conserved from plants to animals [85,108] and is a sex-determined protein in some model organisms. For instance, it is necessary for oocyte organization in Drosophila melanogaster [108,109] and for maintaining oogenesis in Caenorhabditis elegans hermaphrodite individuals [110]. Moreover, previous experiment performed in S. japonicum showed its importance for male teste organization [87]. However, no data was provided for females. Another important candidate is the female-biased “dachshund” gene (Smp_196410) which by contrast is involved in male development of D. melanogaster [79]. It could be an example of downstream actor of the sex-differentiation cascade. The zinc finger transcription factor “gli2” could also be a candidate of particular interest because it is related to the TGFβ pathway intensely studied for its involvement in sexual differentiation of S. mansoni [17,25,105,106]. Moreover, “gli2” is alternatively male or female biased depending on the parasite developmental stage and thus confirms its “bipotentiality” [111] which could play a pivotal role in “sex-orientation” of the development. For instance, in the sexual differentiation context it could switch to shape the sperm/oocyte decision. This kind of mechanism is known to be involved in germ-line cell decision in hermaphroditic nematodes [112]. In our study, several sex-switching genes appear alternatively overexpressed in males or females depending on the developmental stage. We for instance hypothesize that these genes could be particularly sensitive to host environment, and molecular dysfunction on these candidates or environment variation could consequently lead to abnormal hermaphroditism occasionally observed in S. mansoni [113,114]. Besides, we describe here for the first time the male-biased expression of the neuropeptide Sma-npp-27 (Smp_134550) in adults. In acoelomate, like schistosomes, the nervous system occupies a particular role: as they lack both body cavity and circulatory system, the nervous system is not only involved in sensory and neuromuscular signalization, but can also transmit developmental and hormonal signals [115]. Accordingly, the study of Collins et al. [80] defined a role for peptide hormones in controlling reproductive physiology, particularly in males, in the planaria Schmidtea mediterranea. Our results could fit into their hypothesis of shared molecular mechanisms of reproduction among flatworms, independently of their hermaphroditic or gonochoric status. During the cercarial stage, homeogenes are more expressed in females while growth factor and hormonal pathways are over expressed in males successively in cercariae and vertebrate stages (Fig 8B). The obligate implication of the host system regarding the parasite development has received much attention [15,116–124] because interfering with the perception of the host environment might be a possible therapeutic target [107,122]. Previous studies showed that the physiological and reproductive status of the worm is strongly influenced by the host [125] and hermaphroditic-like structures can occasionally appear under the environmental pressure of this host [113]. In this context, growth factor and hormonal pathway over-expression in males could not only result from intrinsic developmental pathways but also could reflect a better receptivity to the host micro-environment (Fig 8A). Cercariae actively seek vertebrate skin contact and prepare host invasion [126,127]. We have identified here female biased genes involved in response to light and mechanical stimuli in cercariae while males biased genes encodes function involved in response to chemical stimuli (Fig 8A). We hypothesize that males may be consequently more efficient to encounter their host, which is consistent with the male-biased sex ratio in adults [128,129] (Fig 8. 3). Furthermore, it could account for the higher genetic diversity in male adult worms [129,130] (Fig 8. 1), via a better capacity of dispersion. After penetrating the vertebrate host, parasites have to overcome its immune system. In this context, we detected sex-biased expression of secreted and/or tegumental molecules known or supposed to be involved in host invasion (Fig 8C) among which, known and new micro-exon genes encoding for variant secreted proteins [92], proteases and protease inhibitors [131,132], “tetraspanins” [90]. In addition, we show for the first time sex-biased expression of “venom-allergen like proteins” [92,133]. We thus further hypothesize that the sex-biased expression of these candidates could explain the known differences between male and female immunogenicity and pathogenicity (Fig 8. 4) [134]. Our analysis highlights different putative mechanisms of gene expression regulation between males and females: miRNA (microRNA) precursors, mobile genetic elements, genes encoding proteins involved in chromatin reorganization, and post-transcriptional events (splicing, mRNA editing) (Fig 8D). Previous studies have highlighted miRNAs stage- and sex-biased expression [42–44,98] as important factors for female maturation [44,135]. In addition, other non-coding RNAs (e. i. W-specific long non-coding RNAs) are involved in sex chromosome evolution [41]. Besides, schistosome miRNAs have been detected in the blood of the definitive host that underlines their possible implication in the pathological processes of schistosomiasis [136,137]. Although our experiment was not fully designed to identify mature miRNAs, we found 12 transcripts encoding miRNA hairpin precursors with a sex-bias expression in favor of either male or female individuals. The sex-biased pattern of their expression may be another argument to explain the differences of pathogenicity between males and females (Fig 8. 4). Our result suggest that, non-coding RNAs certainly deserve fully dedicated experiments for both their implication in the sexual biology of schistosomes and their role in the interaction with the human host. Another interesting molecular mechanism highlighted in our study is the sex-biased expression of mobile genetic elements. This aspect undoubtedly needs further investigation because: (i) of their high bias of expression (great proportion among the 100 best P-values (S2 Table) ), (ii) of their particular structural features (intronic localization of genes), and (iii) previous works in model organisms showing that they could be essential in the reproduction by playing a role in sex chromosome inactivation [138] and in the dosage compensation mechanism [139]. Another interesting aspect of schistosome developmental biology is the intense chromatin remodeling that occurs after penetration into the definitive host [102]. We show here that the male schistosomulum development is characterized by an over-expression of genes involved in nucleosome assembly, structural maintenance of chromatin and structural genes encoding for histones compared to female' s development. These transcriptomic observations are reinforced by a depletion of the H3K27me3 histone mark, emphasized in males, from cercariae to adult. H3K27me3 is known to be widely involved in developmental gene silencing from invertebrates to vertebrates [101,102,140]. We hypothesize that the observed accentuated depletion of this histone mark in males could be a strategy for enhancing male development (Fig 8D and 8D. 2). Finally all our observations seem to converge to an optimized male survival and development in a host-parasite context. Considering the particular sexual biology of S. mansoni [141] this strategy could serve to an enhanced pre-zygotic paternal investment and thus benefit not only the males but also to the females which need males to achieve their maturation. This ability is essential for the survival and thus the reproductive success of the couple. In our study we present sex-biased pathways, related to development and host-parasite interaction, which accompany sexual differentiation in S. mansoni. We further propose putative gene determinants of sex determination/differentiation in this organism and candidate epigenetic mechanisms involved in its regulation. Our analysis represents a first step towards the identification of sex-pivotal genes and now further studies are required to validate the candidate functions and to clarify sexual differentiation pathways. These coming works could involve tissue-specific expression, knock-down and/or over-expression of the candidate genes. In a larger context, we observed an important proportion of newly transcribed regions: further works leading to their deep characterization could participate to the improvement of the actual reference genome v5. 2. Since schistosomiasis represents the second most important parasitic disease worldwide and affects millions of people, providing new therapeutic targets is a substantial issue for the scientific community [144]. We describe here a new in vivo transcriptome of free-living as well as parasitic developmental stages of both sexes of S. mansoni. Our work paves a new way toward understanding the complex molecular interplay that occurs between the host and S. mansoni through the sexual differentiation of the parasite, thereby suggesting new potential drug targets and vaccine candidates.
summary: Parasitic flatworms include more than 20,000 species that are classically hermaphrodites. Among them, the roughly hundred species of Schistosomatidae are intriguing because they are gonochoric. Schistosomes are responsible of the second most important parasitic disease worldwide, and eggs are the main cause of the inflammatory symptoms. Thus, studying the sexual reproduction mechanisms of schistosomes is of particular interest for drug development. Schistosome’s sex is genetically determined by the presence of sex chromosomes: ZZ in males or ZW in females. There is, however, no phenotypic dimorphism in the larval stages: sexual dimorphism appears only in the vertebrate host. In order to understand the molecular mechanisms underlying phenotypic sexual dimorphism, we performed a transcriptome analysis (RNA-Seq) in five different stages of the parasite lifecycle as well as a chromatin status analysis (ChIP-Seq) in the non-differentiated stage cercariae and in the adult differentiated stage, for males and females separately. Our work presents evidence that sexual differentiation in S. mansoni is accompanied by distinct male and female transcriptional landscapes of known players of the host-parasite crosstalk, developmental pathways and epigenetic regulators. Our sex-comparative approach provides therefore new potential therapeutic targets to affect development and sexual reproduction of parasite.
keywords: schistosoma
  invertebrates
  schistosoma mansoni
  medicine and health sciences
  pathology and laboratory medicine
  helminths
  gene regulation
  vertebrates
  animals
  developmental biology
  micrornas
  genome analysis
  morphogenesis
  genomics
  gene expression
  pathogenesis
  biochemistry
  rna
  nucleic acids
  host-pathogen interactions
  sexual differentiation
  genetics
  transcriptome analysis
  biology and life sciences
  computational biology
  non-coding rna
  organisms
title: Sex-Biased Transcriptome of Schistosoma mansoni: Host-Parasite Interaction, Genetic Determinants and Epigenetic Regulators Are Associated with Sexual Differentiation