Schistosomes are the causative agents of schistosomiasis, a neglected tropical disease affecting over 230 million people worldwide. Additionally to their major impact on human health, they are also models of choice in evolutionary biology. These parasitic ﬂatworms are unique among the common hermaphroditic trematodes as they have separate sexes. This so-called “evolutionary scandal” displays a female heterogametic genetic sex-determination system (ZZ males and ZW females), as well as a pronounced adult sexual dimorphism. These phenotypic differences are determined by a shared set of genes in both sexes, potentially leading to intralocus sexual conﬂicts. To resolve these conﬂicts in sexually selected traits, molecular mechanisms such as sex-biased gene expression could occur, but parent-of-origin gene expression also provides an alternative. In this work we investigated the latter mechanism, that is, genes expressed preferentially from either the maternal or the paternal allele, in Schistosoma mansoni species. To this end, tran- scriptomes from male and female hybrid adults obtained by strain crosses were sequenced. Strain-speciﬁc single nucleotide poly- morphism (SNP) markers allowed us to discriminate the parental origin, while reciprocal crosses helped to differentiate parental expression from strain-speciﬁc expression. We identiﬁed genes containing SNPs expressed in a parent-of-origin manner consistent with paternal and maternal imprints. Although the majority of the SNPs was identiﬁed in mitochondrial and Z-speciﬁc loci, the remaining SNPs found in male and female transcriptomes were situated in genes that have the potential to explain sexual differences in schistosome parasites. Furthermore, we identiﬁed and validated four new Z-speciﬁc scaffolds. Key words: parent-of-origin gene expression, sexual dimorphism, intralocus sexual conﬂict, male–female coevolution, Schistosoma mansoni. Introduction and sexually reproduce (Beltran and Boissier 2008). The pro- Schistosomiasis, also known as bilharzia, is a highly prevalent duced eggs are released out of the host via the feces and, in tropical disease affecting over 200 million people worldwide contact with freshwater, hatch to liberate free-swimming lar- (Engels et al. 2002). It mainly occurs in developing countries vae called miracidium. These miracidia actively search and in- and ranks second in term of parasite morbidity and mortality fect their intermediate hosts, transform into two successive after malaria (King 2010). This chronic infection is caused by intramolluskan stages (i.e., sporocysts), which produce by different species of the Schistosoma genus, blood ﬂukes, with asexual multiplication thousands of vertebrate-infecting larvae a complex lifecycle involving two obligatory hosts. (i.e., cercaria). Schistosoma mansoni responsible of intestinal schistosomiasis Besides the medical importance of this parasite for human uses Biomphalaria genus as mollusk intermediate host, and health, it is also a very interesting model in terms of evolu- humans or rodents as vertebrate deﬁnitive hosts. In the ver- tionary biology due to its original sexual features. Among the tebrate host, males and females form monogamous couples, 18,000–24,000 hermaphroditic species recorded in The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non- commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org 840 Genome Biol. Evol. 10(3):840–856. doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Parent-of-Origin-Dependent Gene Expression GBE Trematodes, only the Schistosomatidae family (100 species) restricted to one of the parental alleles leading to paternally has evolved separate sexes and has thus been qualiﬁed as an versus maternally preferential or exclusive gene expression “evolutionary scandal” by Claude Combes in 1991 (Combes (aka parent-of-origin gene expression, or genomic imprinting) 1991). This gonochorism is accompanied by a pronounced (Reik and Walter 2001). Different theories have been pro- dimorphism between sexes (Basch 1990; Loker and Brant posed to explain the evolution of genomic imprinting and 2006). These differences between males and females exist the effects of such gene expression in the offspring. The sex- on different scales ranging from a molecular, to a behavioral ual antagonism (Day and Bonduriansky 2004; Bonduriansky level, and have been documented in several studies (see Mone 2007; Patten et al. 2014) or maternal–offspring coadaptation and Boissier 2004 for review). Larval stages are morphologi- (Wolf and Hager 2006) theories suggest that genomic im- cally indistinguishable and one striking difference at the adult printing has the potential to modify resemblance of an indi- stage is the worm musculature (muscular adult male vs. a thin vidual to its parents (Patten et al. 2014). The sexual adult female), as well as the presence of a gynaecophorous antagonism theory proposes that as fathers and mothers canal, and a large oral sucker in males allowing them to carry have passed the ﬁlter of sex-speciﬁc selection (alleles success- and shelter their female mating partner (Beltran and Boissier fully transmitted to progeny), it is thus more likely that male 2008, 2009). Sexes are genetically determined during egg offspring will beneﬁt from paternally expressed alleles for fertilization with a ZZ/ZW chromosomal system where females male traits, whereas female offspring will beneﬁt from mater- are heterogametic (Grossman et al. 1981). In S. mansoni, nally expressed alleles for female traits (Day and Bonduriansky females have a W chromosome characterized by large pseu- 2004). It is therefore predicted that phenotypic traits beneﬁt- doautosomal regions as well as W-speciﬁc sequences almost ing males or females, as for example muscular bodies for entirely composed of heterochromatic satellite-type repeats males, would be preferentially expressed from the paternal (Lepesant et al. 2012; Protasio et al. 2012). No W-speciﬁc alleles. In schistosomes we therefore expect paternal genes protein-coding genes were identiﬁed so far (Criscione et al. coding for growth enhancers or muscle development to be 2009). Thus, in spite of the observed sexual dimorphism at the preferentially expressed in males, and maternal expression in adult stage, males and females share a common set of protein- females for traits under sexually antagonistic selection. coding genes. There are 782 genes speciﬁc to the Z chromo- On the basis of the maternal–offspring coadaptation the- some and are thus found twice in the ZZ males and only once in ory, maternal alleles may be selected for imprinted expression the ZW females. Studies on ZW female heterogametic systems to provide the greatest combined ﬁtness between the mother frequently report a lack of dosage compensation that equalizes and progeny (Wolf and Hager 2006). Therefore in organisms Z-linked transcript levels in males and females (Graves 2016). It providing maternal investment (e.g., most mammals) we ex- is also thecasein S. mansoni adults where Z-linked expression is pect maternal expression in both sexes for genes involved in reduced relative to autosomal expression in females but not in maternal–offspring interactions and more speciﬁcally mater- males (Vicoso and Bachtrog 2011). However, it is not known nal care. In schistosome species, it has been proposed that whether speciﬁc genes may be escaping the dosage compen- prezygotic paternal investment (transport of the female to the sation mechanism in schistosome males, therefore being po- oviposition site, female maturation, and feeding) is higher tentially responsible for male-biased gene expression and than maternal investment (Beltran and Boissier 2008). accentuating phenotypic differences between sexes. If selec- Therefore, paternal expression in both sexes may be expected. tion for a particular trait at a particular locus favors different The evolutionary roles of parent-of-origin expression nev- alleles in males versus females (i.e., sexual antagonism), one ertheless remain controversial and the patterns predicted by would expect to ﬁnd a mechanism to resolve these intralocus those models may be very contrasted but also non-exclusive. sexual conﬂicts. Gonochoric species usually “solve” these con- Indeed, most of the studies have been carried in plants, mam- ﬂicts through sex-biased expression of the sexually antagonistic mals and some insects such as coccids and bees (da Rocha genes (Ingleby et al. 2015; Lipinska et al. 2015). In schisto- and Ferguson-Smith 2004; Ferguson-Smith 2011; Macdonald somes, sex-biased gene expression was intensively studied at 2012; Kocher et al. 2015) and support an alternative, but not the whole transcriptome level (Fitzpatrick et al. 2005, 2008; exclusive conjecture: The kinship theory (Moore and Haig Anderson et al. 2015; Lu et al. 2016; Picard et al. 2016), but 1991; Haig 2000), which links parent-of-origin gene expres- other molecular mechanisms such as parent-of-origin gene ex- sion not to sexual, but more generally to parental conﬂicts. It pression have never been investigated. assumes that parental genomes are not functionally equiv- In diploid gonochoric organisms, sexual reproduction leads alent and do not have the same reproductive interests. In a to the presence of two copies of each gene in each somatic polyandrous mating system, maternal alleles tend to preserve cell of an individual, one inherited from the mother (i.e., mother and progeny by a restriction of resources, while the matrigenes) and the other from the father (i.e., patrigenes). paternal genome favors growth of offspring. An interesting Usually both copies of a gene cooperate and are expressed at example for such a “battle of sexes” was shown by the pa- equal levels allowing compensation of the function in case of ternal expression of the insulin-like growth factor 2 (IGF2) deleterious mutation, but in some cases gene expression is inducing growth, while the maternal expression of its Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 841 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Kincaid-Smith et al. GBE antagonist receptor (IGF2R) tends to reduce growth in mam- The resulting hybrid miracidiae were individually used to infect mals (Wilkins and Haig 2003). The kinship model therefore mollusks, and adult worms were recovered after mouse uni- predicts that maternal expression will be favored if a gene has sexual infestation (ﬁg. 1a). Total RNA was extracted from the a positive effect when maternally expressed but a negative eight samples separately (ﬁg. 1b) and global sequencing was effect when paternally derived and vice versa for paternal performed on four lanes (double multiplexing) using paired- expression. Therefore, we would expect paternal expression end (2 125 nt) Hiseq Illumina technology. in both sexes for genes involved in extraction of maternal resources by offspring. Schistosomes are oviparous organisms that shed hundreds of eggs daily per female, and resource is Total RNA Isolation already present in the egg before fertilization (maternal allo- For each sex and cross, experiments were performed in two cation of energy) but once released the subsequent develop- biological replicates. RNA extractions were performed alter- ment is independent of parental resources. The expression natively from 20 adult males or 100 adult females. Brieﬂy, patterns will therefore depend on the phenotypic effect of parasites were ground in liquid nitrogen and solubilized in the genes. TRIzol (Thermo Fisher Scientiﬁc). Total RNA was then Differences in gene expression according to parental origin extracted by adding chloroform. PureLink RNA Mini kit in schistosomes may be important to identify candidates for (Ambion) was used for further puriﬁcation following the man- sex-speciﬁc phenotypes. To this end, we screened male and ufacturer’s protocol. Total RNA was eluted in 30 ml RNAsecure female transcriptomes in reciprocal hybrids of S. mansoni for (Ambion) and incubated at 65 C for 10 min. Samples were parent-of-origin expressed genes, considering both known then treated with TURBO DNase (TURBO DNA-free, Ambion) sex-linked and autosomal genes. This approach relied on and the reaction was stopped by cooling down on ice for strain-speciﬁc single nucleotide polymorphic (SNP) markers 2 min. RNA was ﬁnally puriﬁed on a column (RNeasy mini allowing us to identify the potential allelic expression proﬁle kit, QIAGEN) and eluted in 30 ml RNase-free water. Quality of each transcript, while reciprocal crosses allowed us to dis- and concentration were assessed by spectrophotometry criminate parent-of-origin effects from strain-of-origin effects. with the Agilent 2100 Bioanalyzer system. Further details We show that most SNPs detected in genes with monoallelic are available at Environmental and Evolutionary Epigenetics expression are located in mitochondrial and Z-speciﬁc loci, thus Webpage (http://methdb.univ-perp.fr/epievo/; last accessed validating our experimental design and analysis pipeline. We February 16, 2018). also identiﬁed a number of SNPs with preferential expression from one parental allele in the transcriptome of male and fe- male schistosomes, suggesting paternal and maternal imprints. Illumina Libraries Construction and High-Throughput As the genes concerned by these parent-of-origin type SNPs Sequencing are closely related to development and sex-speciﬁc functions, we suggest that parent-of-origin gene expression needs to be cDNA library construction and sequencing were performed at explored in depth because it may be used to resolve intralocus the sequencing facility of Montpellier GenomiX (MGX, France). sexual conﬂicts underlying sexual dimorphism in this species. The TruSeq stranded mRNA library construction kit (Illumina Finally, we discuss our results in regard to current knowledge Inc., USA) was used according to the manufacturer’s recom- on schistosome parasites life-history traits and the evolutionary mendations on 300 ng of total RNA per condition. Brieﬂy, poly- theories supporting imprinting in other organisms. A RNAs were puriﬁed using oligo-d(T) magnetic beads. The poly-Aþ RNAs were fragmented and reverse transcribed using random hexamers, Super Script II (Life Technologies, ref. 18064-014) and Actinomycin D. During the second-strand Materials and Methods generation step, dUTP substitued dTTP to prevent the second Parasite Origin, Crossing Protocol, and Sequencing strand to be used as a matrix during the ﬁnal PCR ampliﬁcation. Hybrid adult parasites were obtained by reciprocally crossing Double-stranded cDNAs were adenylated at their 3 -ends be- two different geographic isolates of the S. mansoni species: fore ligation was performed using Illumina’s indexed adapters. The Guadeloupian strain GH2 and the Brazilian strain BRE. We LigatedcDNAswere ampliﬁed following15cycles PCRand performed biological duplicates of two independent crosses, PCR products were puriﬁed using AMPure XP Beads F1 male and female hybrids (GH2 mother BRE father) and (Beckman Coulter Genomics, ref.A63881). The quantitative the reciprocal male and female crosses (BRE mother GH2 and qualitative analysis of the library was carried on father) as shown in ﬁgure 1 and table 1.Each cross was Agilent_DNA 1000 chip and qPCR (Applied Biosystems 7500, performed in mouse (Mus musculus) deﬁnitive hosts, by using SYBR Green). The sequencing was performed on a HiSeq2500 Biomphalaria mollusks that were individually exposed to a in paired-end 2x125nt mode. RNA-Seq reads are available at single miracidium of the BRE or GH2 strain (releasing either the NCBI-SRA under the BioProject accession number male or female clonal populations of BRE or GH2 cercariae). PRJNA378178. 842 Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Parent-of-Origin-Dependent Gene Expression GBE FIG.1.—(a) Crossing protocol example for Cross 1 and (b) samples used for RNA-sequencing. (a) Mollusks releasing clonal populations of male BRE (ZZ) and female GH2 (ZW) cercariae were used to infect M. musculus deﬁnitive hosts and produce F1 eggs releasing hybrid miracidiae. These male or female miracidiae were used to infect mollusks and produce male of female cercariae. Unisexual infection of M. musculus allowed us to recover after 7 weeks, male and female hybrid adult worms. Notice that Cross 2 (male GH2 x female BRE), which is not presented in this ﬁgure, was performed the same way. Table 1 F1 and Mirror Individuals from Two Reciprocal Crosses F1 Male F1 Female F1-Mirror Male F1-Mirror Female Biological duplicates $ GH2 # BRE $ GH2 # BRE $ BRE # GH2 $ BRE # GH2 $ GH2 # BRE $ GH2 # BRE $ BRE # GH2 $ BRE # GH2 RNA-Seq Data Processing performed in single reads with intron length parameter set be- tween 20 and 50,00 bp. We authorized one mismatch in the Quality Control anchor region of spliced alignment, applied a microexon search Quality control and initial cleaning of the reads was performed because of the presence of microexon genes in schistosome with the ﬁlter by quality program (version 1.0.0) based on genomes (DeMarco et al. 2010), and used the very sensitive FASTX-toolkit (Blankenberg et al. 2010). Reads with less Bowtie2 option (Langmead et al. 2009). The GTF annotation ﬁle than 90% of bases with Phred quality score inferior or equal (S. mansoni sex-speciﬁc transcriptome) produced in a previous to 30 were discarded (probability of 1 incorrect base call out work (Picard et al. 2016) served as the reference (http://ihpe.u- of 1,000, and a base call accuracy of 99, 9%). Adaptors used niv-perp.fr/acces-aux-donnees/; last accessed February 16, for sequencing were removed using the cutadapt program 2018). In order to avoid false positives in SNP calling, we ﬁltered version 1.6 (Martin 2011). PCR duplicates by eliminating exact reads found more than eight times (option “Tolerated Duplicates” ¼ four) using “Remduplicates” (Althammer et al. 2011). Mapping of the Reads against the Reference Genome Each sample’s paired-end sequencing reads were indepen- Parent-of-Origin Gene Identiﬁcation dently mapped (i.e., unique reads) to the S. mansoni’s reference genome version 5.2 (Berriman et al. 2009; Protasio et al. 2012) The approach is based on the analysis of the frequency of SNP using TopHat2 software (Kim et al. 2013). The alignment was markers presenting allelic imbalance within the transcriptomic Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 843 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Kincaid-Smith et al. GBE FIG.2.—Detection strategy of parent-of-origin expression: example for Cross 1. Cross 1 was performed using males from the S. mansoni Brazilian strain (BRE) and females from the Guadeloupian strain (GH2). Each hybrid contains a chromosome from the mother (GH2) and from the father (BRE). The trancriptomic analysis of hybrid offspring allowed identifying genes 1) expressed in equal proportions between the mother’s (GH2) and father’s (BRE) allele (i.e., biallelic expression), 2) expressed exclusively from the mother’s (GH2) or father’s (BRE) allele (i.e., monoallelic expression), or 3) expressed preferentially from the mother’s (GH2) or the father’s (BRE) allele (i.e., biased expression). data of the hybrids. The identity of the base can then be used Parental Origin of the Transcripts Analysis to distinguish allelic origin, and the reciprocal cross helps to In order to discriminate parent-of-origin from strain-of- discriminate parent-of origin from strain-of-origin biases in origin SNP expression, each sample’s transcriptome vari- allelic expression (ﬁg. 1). ant ﬁles were compared with the previous selected strain- speciﬁc SNPs (BRE or GH2) and that according to variant positions. We only kept SNPs in the transcriptome for Selecting Strain-Speciﬁc SNPs in the Genomic Data of BRE which we had parental information in order to identify and GH2 the origin from which the SNP is expressed. Three catego- SNPs in the genome of the BRE and GH2 strains were previ- ries were deﬁned based on the expression proﬁles of the ously described (Clementetal. 2013). We used the Freebayes SNPs. Those with alternative allele frequency between 0% generated BAM ﬁles available at http://methdb.univ-perp.fr/ and 10% or 90% and 100% were deﬁned as monoallelic downloads/, last accessed February 16, 2018 to assess strain- (or imprinted). Those with alternative allele frequency be- speciﬁc SNPs. We considered informative, any SNP showing tween 10% and 35% or 65% and 90% were deﬁned as ﬁxed alternative allele frequency (AF 0.9) in one strain, and biased (preferential expression), and those with alterna- absent or having an alternative allele frequency inferior or tive allele frequency between 35% and 65% were con- equal to 0.1 in the other (AF 0.1) compared with the reference sidered as biallelic (ﬁg. 2). Among these, SNPs were genome (NMRI strain, Puerto Rican Origin). conserved only if found in the same category in both rep- licates. We further added mpileup coverage information SNPs Calling in the Transcriptomic Data to assess whether the SNPs were not detected, or whether the regions were not transcribed. When coverage infor- Using SAMtools (Li et al. 2009), we produced mpileup ﬁles mation indicated reads in the region we considered the with read coverage information and call quality from the allele similar to the reference genome and set allele fre- TopHat2 alignment BAM ﬁles. We then performed SNPs call- quency to 0% (minimum coverage of ten reads). If the ingby using VarScansoftware (version0.1) onthe mpileup region was not covered and therefore not transcribed, ﬁles (Koboldt et al. 2012). The minimum depth at position to we considered position as noninformative. make a call was set to ten reads, the minimum supporting reads for alternative allele at position were set to two. The minimum base quality to count a read was set to 15, mini- Comparisons between the Reciprocal Crosses mum variant allele frequency threshold was set to 1%, and the p-value threshold for calling variants was set to 5%. The SNPs with biased and monoallelic expression proﬁles were number of reads, the number of variant allele, and the alter- compared between reciprocal crosses. This allowed us to native base identity were visually checked for ten randomly identify genes potentially expressed in a parent-of-origin man- selected SNPs in each sample with the Integrative Genomics ner when reciprocal crosses agreed with each other on Viewer (IGV) software (Thorvaldsdottiretal. 2013). parent-of-origin bias. 844 Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Parent-of-Origin-Dependent Gene Expression GBE Table 2 Number of Sequences Remaining after Each Bioinformatic Steps Quality Filtering Q30 TopHat2 Mapping PCR Duplicate Removal Crosses Sex Input Discarded Input Mapped Output $ GH2 # BRE M 203,732,076 43,590,513 160,141,563 144,201,576 64,795,806 (90%) $ GH2 # BRE M 278,553,568 62,102,488 216,451,080 197,048,330 84,039,096 (91.0%) $ BRE # GH2 M 249,198,708 54,496,632 194,702,076 179,194,594 81,242,489 (92.0%) $ BRE # GH2 M 237,406,392 52,577,939 184,828,453 169,678,849 79,231,052 (91.8%) $ GH2 # BRE F 197,048,594 34,868,591 162,180,003 147,618,963 76,799,603 (91.0%) $ GH2 # BRE F 226,038,046 42,007,698 184,030,348 167,729,763 86,451,322 (91.1%) $ BRE # GH2 F 231,467,876 57,694,047 173,773,829 159,153,208 80,775,575 (91.6%) $ BRE # GH2 F 320,963,252 81,771,733 239,191,519 220,417,355 103,756,971 (92.2%) efﬁciency¼1þ 10 (1/slope). Quantitative PCR reactions Annotation of the Transcriptome were carried out in a total volume of 10 ml, containing 5 mlof The transcriptome ﬁle (extracted from the GTF reference the Takyon 2X reaction mix (Eurogentec), 2 mlof 10 mM pri- [Picard et al. 2016]) was de novo reannotated on the local mers (forward and reverse), 2 mlof DNA template, and 1 mlof server of the laboratory by searching protein databases using nuclease free water. The qPCR program was set for 40 cycles a translated nucleotide query (BlastX) against the nonredun- containing a 10-s denaturation phase at 95 C, annealing– dant protein sequence database (nr). The 25 best hits were extension phase at 59 C for 20 s and 72 C for 25 s. then used to search for gene ontology terms using the Blast2Go program (Conesa et al. 2005). Correspondence be- tween transcript and current version of genome annotation Results was manually checked using IGV software (Thorvaldsdottir RNA-Seq Data Processing et al. 2013). Between 197 and 320 million reads were produced after RNA sequencing for each of the eight samples, representing male Quantitative PCR Validation of Z-Speciﬁc Scaffolds and female adult progenies of biological duplicate crosses and Simple or double copy of genomic DNA was assessed with mirror crosses of a Brazilian (BRE) and Guadeloupian (GH2) male (ZZ) and female (ZW) DNA extracted from adult worms strain of S. mansoni (table 2 and ﬁg. 1). After ﬁltering reads to verify the Z-speciﬁc status of unplaced scaffolds containing according to their quality Phred score, between 160 and 239 genes with monoallelic paternal expression. DNA of single million (80%) of them were kept for subsequent read map- adult worms of each sex was extracted as recommended ping against S. mansoni’s reference genome v5.2 (Berriman from the QIAGEN QIAamp DNA Micro Kit protocol for isolation et al. 2009; Protasio et al. 2012). At least 90% of the ﬁltered of genomic DNA from tissues. Final elution was performed us- reads were successfully mapped to the reference genome. ing 40 ml of buffer AE (10 mM TrisCl, 0.5 mM ethylenedi- Because SNPs frequency reliability was essential to detect aminetetraacetic acid, pH 9.0). Primers were designed using allele-speciﬁc transcripts, potential PCR duplicates (50% of the online Primer3plus program (Rozen and Skaletsky 2000) reads) were removed. and aligning the primers against S. mansoni’s reference ge- nome v5.2 to verify for high speciﬁcity. We used a known Z- Identiﬁcation of Parent-of-Origin Expressed Genes speciﬁc (double copy in ZZ males and single in ZW females) and an autosomal (double copy in each sex) genomic region as SNPs Discriminating Parents internal control. Primer efﬁciencies were assessed with serial In this work, we beneﬁted from 708,898 SNPs identiﬁed in the dilutions of genomic DNA in DNAse free water (dilutions = , Brazilian (BRE) and Guadeloupian (GH2) strains (Clementetal. 1/16, 1/64, and 1/256). Primer efﬁciency was calculated 2013). These two inbred strains display low nucleotide diver- using the slope of the standard curve with sity within each strain, but a high differentiation level between Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 845 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Kincaid-Smith et al. GBE Table 3 Summary Information of SNPs Recovered in the Transcriptomic Data and Associated SNPs Expression Patterns Parental Discriminating Categorized Number of Biallelic Strain-of- Parent-of- Other SNPs in Transcriptome SNPs between SNPs in SNPs Origin Origin SNPs SNPs Replicates Reciprocal SNPs Crosses M: $ GH2 # BRE 29,796 20,273 13,333 10,665 5,422 4,422 128 693 M: $ GH2 # BRE 23,509 (70%) (51%) (41%) (1%) (6%) M: $ BRE # GH2 24,731 16,455 M: $ BRE #GH2 24,966 (72%) F: $ GH2 # BRE 25,092 17,595 14,412 7,095 5,455 1,063 799 F: $ GH2 #BRE 26,286 (74%) (49%) (38%) (7%) (6%) F: $ BRE # GH2 27,349 21,014 F: $ BRE # GH2 28,885 (77%) them (mean F ¼ 0.73) (Clementetal. 2013). We selected SNPs were located in the exons of 15 genes (in addition to one st 230,425 of those SNPs (32%) that were ﬁxed and discriminant mitochondrial) and expressed from the same parent in both between the two strains (“private” SNPs) (genome browser reciprocal crosses. Six different chromosomes were con- available at http://genome.univ-perp.fr; last accessed February cerned, with three genes on chromosomes 1, 2, and 4, and 16, 2018). Among these, 29,796 SNPs (13%) were found one gene on the unplaced scaffold_0193 of chromosome 2, across the transcriptome of the hybrid progenies. The majority the unplaced scaffold_0083 of chromosome 3, chromosome (56%) of these SNPs was situated in exons of known genes 5, ZW linkage group (Z-speciﬁc region), unplaced scaf- (16,587 exons from 6,612 genes), while 13,209 (44%) were fold_0138 and unplaced scaffold_0164. Most SNPs (n¼ 86) situated in introns or nonannotated regions. were located in the same gene (cytochrome c oxidase I) of the mitochondrial genome of maternal inheritance and thus pro- SNPs Expression between Replicates and Reciprocal viding an internal control as they had a strictly maternal ex- Crosses pression pattern (table 4). Among the 15 genes in males, one displayed monoallelic expressed SNPs from the maternal al- We compared SNP expression patterns between replicates. lele, while 14 genes showed parentally biased SNPs in equal Between 70% and 77% of the SNPs had the same expression proportion between the mother’s (n¼ 7) and the father’s al- patterns in biological replicates when attributed to one of the lele (n¼ 7) (table 5). three deﬁned categories (i.e., biallelic, biased or monoallelic, In females, SNPs showing consistent parent-of-origin ex- see description in Materials and Methods) (table 3). We thus pression were found in exons of a total of 378 different genes. analyzed 10,665 SNPs for males and 14,412 SNPs for females Among these, 278 genes had SNPs with a strict paternal ex- and compared their allele expression proﬁles between recipro- pression and were known to be located in Z-speciﬁc regions cal crosses to distinguish strain-of-origin effects from parent- (i.e., ZW linkage group: position 3550000–13340000, of-origin effects (table 3). Among them, 5, 422 (51%) and 13860000–19650000, and 23230000–30820000 of the 7,095 (49%) of the SNPs had a biallelic expression pattern for v5.2 assembly [Protasio et al. 2012]) (supplementary table S1, males and females, respectively. In total, 128 (1%) and 1,063 Supplementary Material online), which is consistent with the (7%) SNPs showed a parent-of-origin expression pattern for heterogametic status of female schistosomes. Indeed, hetero- males and females, respectively. Others were either expressed gametic female schistosomes inherit their W chromosome in a strain-of-origin manner or had a nonconcordant expression from the mother and the Z from the father: Such patterns pattern between reciprocal crosses, representing 6% of all provided again an internal control validating the accuracy of SNPs (e.g., biallelic in one and biased in the other [table 3]), our method for the detection of parent-of-origin genes. Z-spe- which could be due to mitonuclear incompatibilities in the ciﬁc genes were therefore not considered in further analysis. hybrid crosses (Wolf 2009; Wolf et al. 2014). Indeed, this Additionally, 92 genes locatedon22individualunplaced type of incompatibilities is expected to be manifested and scaffolds (including Chr_1.unplaced. SC_0034 and may lead to aberrant gene expression patterns and/or loss of Chr_3.unplaced. SC_0192) contained SNPs with a paternal imprinting in one direction of the cross (Wolff et al. 2014). expression pattern (supplementary table S1, Supplementary Material online). We thus considered them as potentially Parent-of-Origin Expressed Genes Z-speciﬁc and veriﬁed their status in the four biggest unplaced We investigated whether SNPs with parent-of-origin patterns scaffolds containing the larger amount of genes, for unique were situated in known genes of S. mansoni. In males, 108 (ZW) or double copy (ZZ) of genomic DNA in males and 846 Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Parent-of-Origin-Dependent Gene Expression GBE Table 4 Functional Annotation of Genes Containing Parent-of-Origin Expressed SNPs Chromosomes Number of Genes or Number of SNPs Origin SNPs Genes Genes with Position Discriminant Positions Expression Function Parental SNPs with a Pattern SNPs Parent-of-Origin Expression Males Chr_1 3 Smp_151660 1 2817123 $ Biased Putative uncharacterized protein Smp_128980 7 21888575 $ Monoallelic Aminomethyltransferase Smp_083130 2 39963130 $ Biased Beta1, 3-glucuronyltransferase I Chr_2 3 XLOC_009689 1 10177131 # Biased Endonuclease-reverse transcriptase (10176395–10178030) Smp_169030 1 17479792 # Biased Probable asparagine–tRNA mitochondrial Smp_147330 1 19943769 # Biased Probable ATP-dependent RNA heli- case dhx34 Chr_2.SC_0193 1 Smp_171530 1 63337 # Biased Beta Parvin related Chr_3.SC_0083 1 Smp_168560 1 661857 $ Biased Steroid dehydrogenase Chr_4 3 Smp_149950 1 3351765 $ Biased Bifunctional coenzyme a synthase XLOC_015537 1 18631541 # Biased Gag-pol polyprotein (18626181–18631701) Smp_131150 1 28344103 $ Biased Exosome component 10 Chr_5 1 XLOC_017885 1 538919 $ Biased Tpa: endonuclease-reverse (537474–540461) transcriptase ZW linkage 1 Smp_171960 1 15939680 # Biased Dehydrogenase: reductase SDR fam- group ily 1 SC_0138 1 Smp_125620 1 248617 # Biased Coiled-coil domain-containing pro- tein 60 SC_0164 1 Smp_094930 1 60425 $ Biased Early growth response protein 1 Mitochondria 1 XLOC_034755 86 Not shown $ Monoallelic Cytochrome c oxidase subunit i (1–14415) (mitochondrial) Females Chr_1 5 Smp_034860 1 15501570 $ Biased Nuclear receptor 2dbd gamma Smp_128970 1 21880758 # Biased Endonuclease-reverse transcriptase/ Inhibitor of growth protein 3 Smp_173620 1 30834128 $ Biased Transmembrane protein C9orf5/ Strawberry notch related XLOC_001902 2 39435074 $ Biased Gag-pol polyprotein (39431891–39435207) 39435138 Smp_154960 1 44621620 # Biased Putative cop-coated vesicle mem- brane protein P24 Emp24/gp25l family Chr_2 2 Smp_142400 1 4664882 # Biased Bhlhzip transcription factor Bigmax Smp_122810 2 31605145 # Biased Mechanosensory protein 2/ 31605184 MEChanosensory abnormality family member Mitochondria 1 XLOC_034755 24 Not shown $ Monoallelic Cytochrome c oxidase subunit i (1–14415) (mitochondrial) NOTE.—The localization of the genes in known chromosomes or unassembled portion of S. mansoni’s genome for male and female adults is represented in this table. Number of SNPs, parental origin, expression pattern, and functional annotation are also presented. Expressed genes detected in this study that were not in the current annotated regions of S. mansoni’s genome version (v5.2) are mentioned as XLOCs as identiﬁed in the GTF transcriptome reference (Picard et al. 2016). Notice that for those genes, the genomic position has been provided. Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 847 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Kincaid-Smith et al. GBE Table 5 Number of Genes Containing Parent-of-Origin SNPs SNP Patterns in Reciprocal Crosses Male Progeny Female Progeny Total Genes with paternal monoallelic SNPs 0 0 0 Genes with paternal biased SNPs 7 4 11 Genes with maternal monoallelic SNPs 1 (þ1 mitochondrial) 0 (þ1 mitochondrial) 1 Genes with maternal biased SNPs 7 3 10 Total monoallelic 1 0 1 Total biased 14 7 21 Total genes with parent-of-origin expressed SNPs 15 7 22 NOTE.—Genes with parent-of-origin SNPs are categorized 1) according to their expression in male and female progenies and 2) according to their expression pattern (i.e., maternal or paternal, and monoallelic or biased). Table 6 Quantitative PCR Results: Validation of Four New Z-Speciﬁc Scaffolds 0 0 Tested Scaffolds or Controls Target Positions Primer Sequences (5 –3 ) Expected Product Primer Fold Changes Sizes (bp) Efﬁciencies (female/male) Chr_ZW (Z-speciﬁc control) 28071666–28072040 TGTTATCAAACGCCCAGTGA- 375 1.8 0.47 CGTTGAAAAGCCGAGTTTGT- Chr_1 (autosome) 32740790–32741111 CCTCACGAGGTACTCGAAGC- 322 1.8 1 TATGGGACCTGCAACCTTTC- Chr_ZW.unplaced.SC_0115 23140–23512 CCTGCTTAGACCGCCTGTAG- 373 2 0.45 ACTGTTTCGGCCGTAATGTC- 198332–198645 TCGGTTGGTGTCTGATGGTA- 314 2 0.48 CCACTGACCAATTTCCTCAAA- 729203–729462 TCATCTGTCTCCCAGGCATT- 260 2 0.42 GGCAAGAACATGACCGAGAT- SC_0111 155978–156340 GCTCCTCCATGTCCAACTCT- 363 1.8 0.44 ACGCATTCGTAGCCGAGATA- 626009–626295 GGCACCCTGTAAATTCATCC- 287 1.8 0.55 CCTGCTTTTAGTTGCCCTGA- 1059389–1059655 TGGATCCGAAAATTGTTTGTC- 267 1.8 0.46 GTACCGCTTTCAAAACATGC- SC_0129 99114–99482 GATGTCAATGTGAGGCCAAA- 369 1.8 0.52 GGCTACTCGTGTCCCGTAAG- 320296–320629 GCTTAGGAATAAGCGGTTCG- 334 1.8 0.42 AACGGCATAAATGGGTGAAT- SC_0136 134667–134949 TCGATAATCCCATGCACTCA- 283 1.8 0.41 CCTTCATGAAAAACAGGGAAA- 23063–23394 AAAAGAACGCTTCACCGAAA- 332 2 0.45 TGAATCGTGCTGATTCTCCA- NOTE.—Two control regions were used with one in a known Z-speciﬁc region (see Protasio et al. 2012) and one in an autosomal region. Three primers were taken arbitrary in the unplaced scaffolds to be tested. The target positions, primer sequences, as well as the expected product sizes, primer efﬁciencies and female/male fold changes are presented in the table. females using a quantitative PCR approach (table 6). The remaining seven genes in females (in addition to one Unplaced scaffold_0115 of the ZW linkage group mitochondrial) presented biased SNPs consistent with parent- (Chr_ZW.unplaced.SC_0115), as well as the unplaced of-origin expression. Two distinct regions in the genome were scaffolds number 0111 (SC_0111), 0129 (SC_0129), and concerned, with SNPs located in ﬁve genes on chromosome 0136 (SC_0136), were thereafter classed as Z-speciﬁc 1, and two genes on chromosome 2. These genes contained (table 6). The other scaffolds were not validated by biased SNPs expressed from the maternal (n¼ 3) or paternal qPCR but were considered as potentially Z-speciﬁc and (n¼ 4) alleles. And last, the “cytochrome c oxidase” was excluded from further analysis (supplementary table S1, identiﬁed again with 24 SNPs located in the gene and with Supplementary Material online). a maternal expression (tables 4 and 5). 848 Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Parent-of-Origin-Dependent Gene Expression GBE Function of the Parent-of-Origin Expressed Genes the ligand-binding domain of C. elegans nuclear hormone receptor Sex-1 protein. This transcription factor plays pivotal The majority of genes identiﬁed with a parent-of-origin SNP role in sex fate of C. elegans by regulating the transcription of expression in male and female schistosomes (table 4)had bi- the sex-determination gene xol-1, which speciﬁes male fate ological processes involved in metabolism, growth, and devel- when active and hermaphrodite fate when inactive (Carmi opment. We furthermore identiﬁed putative genetic mobile et al. 1998; Farboud and Meyer 2006). Other genes related elements of unknown functions. Two types of expression to developmental pathways were identiﬁed such as the patterns were observed with 1) either the expression of pa- “Strawberry Notch related” gene (Smp_173620). Notch ternal SNPs in males and maternal SNPs in females, or 2) the genes encode for transmembrane proteins and have con- opposite feature with paternal SNPs expressed in females or served functions in developmental pathways. They are re- maternal SNPs expressed in males. Interestingly, most of the quired during embryogenesis and oogenesis in Drosophila genes concerned with these SNPs expression patterns were and zebraﬁsh (Coyle-Thompson and Banerjee 1993; related to developmental pathways and therefore potentially Majumdar et al. 1997; Takano et al. 2010). related to sex-speciﬁc and dimorphic phenotypes. At last, a genetic mobile element (XLOC_001902), identi- Is ﬁrst described in this section the function of genes pre- ﬁed as a gag-pol polyprotein, was maternally expressed in senting SNPs expressed from the parent of the same sex in schistosome females. progeny. In males, seven genes with paternally expressed The analysis of SNPs with opposite expression patterns in SNPs were identiﬁed. Among those, we pinpointed the puta- males and females (i.e., from the parent of opposite sex) also tive ATP-dependent RNA helicase dhx34 (Smp_147330) pro- revealed genes potentially involved in male and female devel- tein-coding gene, which is part of the DEAD box proteins opmental pathways but may also be relevant in a coevolution family, conserved in metazoan they are known for their im- context between sexes. Concerning maternally expressed plication in embryogenesis, spermatogenesis, cellular growth, SNPs in males, eight genes were identiﬁed including one and division (Godbout and Squire 1993; Johnstone et al. with monoallelic expressed SNPs (Smp_128980). This amino- 2005; Matsumoto et al. 2005). Its ortholog in methyltransferase gene is nuclear-encoded but conﬁned to Caenorhabditis elegans (smgl-2) is critical for muscle develop- the mitochondria and is involved in glycine metabolism. ment (Williams and Waterston 1994). Furthermore, such hel- Also, mainly present in the mitochondrial matrix, we identiﬁed icases are part of a complex called the “compensasome” the Bifunctional coenzyme A synthase gene (Smp_149950). because they mediate dosage compensation mechanism This last gene predicted to be involved in ATP-binding activity (Sanjuan and Marın 2001) in other organisms. The Beta- and dephospho-CoA kinase activity has an ortholog in Parvin related gene (Smp_171530) is essential during embryo- C. elegans (Y65B4A.8), which seems involved in embryo de- genesis in other organisms (Zhang et al. 2004; Montanez velopment, feminization of hermaphroditic germ-line, germ et al. 2009) and its ortholog in C. elegans is required for mus- cell development, nematode larval development, regulation cle assembly and function, while mutation of the gene leads of cell proliferation, and regulation of meiotic nuclear division to embryonic lethality. The coiled-coil domain-containing pro- (Kerins et al. 2010; Waters et al. 2010). tein 60 (Smp_125620) potentially involved in protein demeth- Other genes with maternally biased SNPs in males were ylation and protein–protein interactions is also thought to be identiﬁed. The “putative Beta1-3 glucuronyltransferase” important for embryonic development in vertebrates, with an (Smp_083130) which is involved in carbohydrate metabolic identiﬁed role in the dorsoventral axial establishment of zebra- process is mainly expressed in the brain of Drosophila and is ﬁsh (Wei et al. 2016). A dehydrogenase reductase sdr family involved in the growth of peripheral nerves during larval member gene (Smp_171960) has also been identiﬁed and development (Pandey et al. 2011). In C. elegans sqv-8 (homol- interestingly a previous association study has shown a pater- ogous to three distinct glucuronyl transferases [GlcAT-I, nal parent-of-origin effect of that gene on language impair- GlcAT-P, and GlcAT-D]), which encodes a glucuronyl transfer- ment (Nudel et al. 2014). At last three genes were identiﬁed ase, is required for one-cell embryos and for vulval morpho- with paternally expressed SNPs, one being a probable aspar- genesis. Sqv mutants have a vulval defect, but also an oocyte agine tRNA mitochondrial coding gene (Smp_169030) and and somatic gonad defect, resulting in hermaphrodite sterility. has been related to developmental diseases in humans Moreover, some sqv mutations cause maternal-effect lethality (Sofou et al. 2015; Vanlander et al. 2015), while two are (Herman et al. 1999; Bulik et al. 2000) It has also been shown genetic mobile elements with an endonuclease or endopep- that glct-6 (another Beta1-3 glucuronyltransferase ortholog in tidase activity (XLOC_009689 and XLOC_015537). C. elegans) is involved in determination of adult lifespan (Kim In females, the genes with maternally expressed SNPs iden- and Sun 2007). The “exosome component 10” gene tiﬁed had functions linked to development and female specif- (Smp_131150), also known as “polymyositis scleroderma icities. The most relevant gene that can directly be related to autoantigen” in humans, is a cell death-related nuclease, female-speciﬁc phenotypes was the nuclear receptor 2dbd that is required for DNA degradation during apoptosis. It is gamma gene (Smp_034860). It contains a domain similar to also thought to participate in dosage compensation Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 849 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Kincaid-Smith et al. GBE mechanism by inactivation of the X chromosome. Indeed, in Ferguson-Smith 2004; Arico et al. 2011; Ferguson-Smith mouse, a speciﬁc nuclear component of the exosome 2011; Macdonald 2012; Kocher et al. 2015), and aberrant (Exosc10) gene involved in mRNA degradation pathways leads expression in gene subject to imprinting has severe conse- to downregulation of spliced Xist transcript production and quences, mainly on development and growth (Tycko and blocks the onset of the X-inactivation process (Ciaudo et al. Morison 2002). The term imprinting was ﬁrst used to describe 2006). The transcription factor gene “Early growth response the elimination of an entire paternal chromosome in sciara protein 1” (Smp_094930) targeting important genes for nor- ﬂies (Crouse 1960) and refers to the entire heterochromatiza- mal development and differentiation (Silverman et al. 1998; tion (silencing) of the paternal genome in the soccid mealy- Pagel and Deindl 2011) may also play an important role in bug, which is involved in maleness determination (Khosla Schistosoma blood ﬂukes development. Finally, a Steroid de- et al. 2006). In addition to the kinship theory of genomic hydrogenase gene (Smp_168560) was detected with a ma- imprinting (Haig 2000), another interesting alternative expla- ternal SNPs expression in males. Interestingly, steroid nation proposes that imprinting may be related to a mecha- dehydrogenase pathway was also found overrepresented in nism for the resolution of intralocus sexual conﬂict and may male schistosomes from cercariae to adult stages (Picard et al. be important for traits under sex-speciﬁc selection (Day and 2016). In C. elegans, some ortholog genes to Smp_168560 Bonduriansky 2004; Bonduriansky 2007). This sexual antago- were found (stdh-4, stdh-1,and let-767) they encode for a nism theory involving male and female coevolution has the putative steroid dehydrogenase and expressed not only in potential to act differently for each sex (sex-speciﬁc imprint- larval and adult pharynx (stdh-4) but also in larval intestine, ing) and predicts that imprinting may affect organisms with and in both larval and adult body wall muscle and neurons sexual dimorphism (Bonduriansky 2007) asit isthe case in (stdh-1) and is required for normally short lifespan. The schistosomes adult parasites. For that reason, we have inves- ortholog gene let-767 is particularly important for C. elegans tigated S. mansoni transcriptome for parent-of-origin gene development, as it is required for embryogenesis, and female expression considering intralocus sexual conﬂicts and sexual reproduction. This gene is zygotically expressed in the intes- antagonism. We have identiﬁed genes containing SNPs tine, but a maternal-effect lethal allele (let-767 [s2464]) also expressed in a parent-of-origin manner and even if larger scale exists, indicating that LET-767 is probably provided maternally parent-of-origin gene expression in this species could not be which is consistent with our maternal expressed SNPs identi- addressed here we argue that regarding to the function of the ﬁed. Mutations in this gene cause abnormal embryonic devel- genes concerned by these pattern, they have the potential to opment, slow growth and small adult body size as well as a explain sexual differences in schistosome parasites. failure to mature gonads and last an hypersensitivity to low A total of 1,191 SNPs were identiﬁed as expressed in a cholesterol (Kuervers et al. 2003). Other genes with opposite parent-of origin manner in male (128 SNPs) and female expression patterns in males and females could not be asso- (1,063 SNPs) adult schistosomes (table 3). These parental ciated to any known functions (Smp_151660) or were pre- SNPs were located in 15 genes in males (in addition to the dicted as a genetic mobile element with an endonuclease cytochrome c oxidase) and in 378 genes in females. As activity (XLOC_017885). expected, the mitochondrial cytochrome c oxidase gene In females, ﬁve genes were found containing SNPs was also detected with a maternal expression in both sexes, expressed from the paternal allele. Among them, one gene thus providing validation of our methodology. In females, exhibited a monoallelic expressed SNP, while the four others these parental expressed SNPs also revealed as expected contained biased SNPs. In this last situation we identiﬁed an 278 known Z-speciﬁc genes expressed exclusively from the uncharacterized protein-coding gene (Smp_128970), con- paternal allele (monoallelic SNPs expression), which is consis- taining PHD ﬁnger domains, related to the “inhibitor of tent with the schistosomes female heterogametic situation. growth protein family” which has been implicated in Furthermore, in females, 92 genes situated on 22 unplaced chromatin-mediated transcriptional regulation in C. elegans scaffolds were found with a paternal expression pattern. We embryo and is therefore potentially important for schistosome hypothesized that they might be related to Z-speciﬁc loci and development (Luo et al. 2009). Other genes were involved in tested the four biggest unplaced scaffolds containing the ma- not only protein and vesicle-mediated transport jority of paternal expressed genes using a quantitative PCR (Smp_154960) but also transcriptional regulation of develop- approach. This allowed us to identify four new Z-speciﬁc ge- mental pathways (Smp_142400) (Steingrımsson et al. 1998; nomic regions (unplaced scaffold 0115 of the ZW linkage Hallsson et al. 2004; Hsu et al. 2004) and mechanosensory group, unplaced scaffold 0111, 0129 and 0136, table 6). response (Smp_122810) (Huang et al. 1995). We believe that other unplaced scaffolds are potentially Z- speciﬁc and need further validation (supplementary table S1, Supplementary Material online). Discussion A total of 22 genes showing expressed SNPs consistent Parent-of-origin gene expression has been studied in with parent-of-origin expression were identiﬁed in reciprocal mammals, plants, and some invertebrates (da Rocha and cross duplicates of female (seven genes) and male (15 genes) 850 Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Parent-of-Origin-Dependent Gene Expression GBE adult schistosomes. These genes only contained few SNPs identiﬁed genes containing SNPs expressed from the paternal consistent with parental expression and the majority of alleles that seem important for muscle development them was biased rather than monoallelic. Notwithstanding, (Smp_147330, Smp_171530, Smp_125620), which is clearly in males gene “Smp_128980” contained seven monoallelic predicted by the sexual antagonism theory. In some case, SNPs expressed from the maternal allele and gene mutations of orthologous genes in C. elegans lead to embry- “Smp_083130” contained two biased SNPs expressed from onic lethality (Smp_171530). Moreover, some of those genes the maternal allele, while in females two biased SNPs were have been related to paternal parent-of-origin effects identiﬁed in XLOC_001902 and Smp_122810 expressed from (Smp_171960) (Nudel et al. 2014) and developmental dis- the maternal and paternal alleles, respectively (table 4). This is eases in humans (Smp_169030) (Sofou et al. 2015; probably the consequence of the poor amount of discrimi- Vanlander et al. 2015), which often ﬁnd their cause in im- nant SNPs from which we have based our analysis and there- printing defaults. Another interesting point may concern the fore hampers our ability to conﬁrm any existing imprinting at relationship with dosage compensation mechanism the whole gene level in this species. Another consideration (Smp_147330) as such helicases genes mediate dosage com- comes from the fact that imprinting may be tissue speciﬁc and pensation mechanism in other organisms (Sanjuan and Marın that working with whole adult worms may mitigate the 2001). In schistosomes, heterogametic (ZW) females need to resulting expression patterns found in this work. Moreover, balance Z-linked transcript levels relative to homogametic (ZZ) in the present experimental design the adult worms recovered males (Vicoso andBachtrog2011; Graves 2016). These pre- from single-sex infections present an immature phenotype vious observations are consistent with the sexual antagonism (especially for female worms that rely on males for their mat- theory proposed by Day and Bonduriansky (Day and uration), which may have biased the expression and hamper Bonduriansky 2004; Bonduriansky 2007). the detection of further imprinted genes. Nevertheless, the Maternal expressed SNPs found in male genes were in- genes identiﬁed with these parent-of-origin expressed SNPs in volved in development (Smp_149950, Smp_094930), with males and females had interesting functions related to sex- embryogenesis and reproduction (Smp_168560) but also speciﬁc phenotypes and sexual dimorphism in this species and growth (Smp_083130). In C. elegans let-767 (orthologous share interesting common relations with parent-of-origin to Smp_168560) is zygotically expressed in the intestines, genes expression theories. and mutations in this gene cause abnormal embryonic devel- If we base our reﬂection on the kinship theory (Haig 2000) opment, slow growth and small adult body size as well as a deﬁned by parental conﬂict over resource allocation and off- failure to mature gonads and last a hypersensitivity to low spring ﬁtness, we would of expected to ﬁnd an impact of cholesterol (Kuervers et al. 2003). Other mutations of C. ele- gene dosage on the ﬁtness of matrilineal or patrilineal rela- gans orthologous genes (sqv genes orthologous to tives. Moreover, usually schistosome couples are monoga- Smp_083130; let-767 orthologous to SMP_168560) cause mous, but mate change can occur among these parasites maternal-effect lethality (Herman et al. 1999; Bulik et al. (Tchuem Tchuente et al. 1996; Beltran and Boissier 2008, 2000) indicating that they are probably provided maternally 2009) potentially causing a source of relatedness asymmetry in C. elegans, which is similar to our observations in schisto- for matrigenes and patrigenes in offspring. For example, ma- somes. This may suggest that the maternal genome largely ternally expressed alleles would be selected to reduce the ex- contributes to male phenotypes and is probably also indis- traction of resources and therefore reduce organism size, pensable for proper schistosome development. Moreover, while paternally expressed genes should favor taking more we identiﬁed a probable candidate gene (Smp_131150, exo- resources from the mother and therefore induce growth. some component 10) known to be involved in dosage com- Indeed males are much more muscular than females (i.e., pensation mechanism in mouse (Exosc10)as it leads to sexual dimorphism), and sexual selection may favor large downregulation of spliced Xist transcript production and body in males and the opposite in females, thus paternal blocks the onset of the X-inactivation process (Ciaudo et al. genes should be selected in males to extract resources at a 2006). This seems to indicate a strong coadaptation between greater rate than in females. Schistosomes are oviparous sexes in schistosomes. Moreover, consistent with such coad- organisms therefore neither the paternal or maternal genome aptation we identiﬁed in males two genes (Smp_128980, can directly inﬂuence resource allocation after spawning. As aminomethyltransferase, containing maternal monoallelic observed in this study for schistosomes, it is difﬁcult to recon- SNPs together with the Bifunctional coenzyme A synthase cile these predictions to the function of the genes identiﬁed in gene [Smp_149950] containing maternal biased SNPs) that this work except for genes that may inﬂuence growth and are mainly conﬁned to the mitochondria. This may underlie development of offspring, but this may overlap with the func- coevolved complexes between the maternal inherited mito- tions predicted by other models and in particular the sexual chondrial genome and nuclear maternally expressed genes in antagonism theory. males, and therefore male and female coadaptation. A clear dimorphic trait found in adult males is their strong Cytonuclear interacting genes seem important in the evolu- musculature compared with the thin females. Interestingly we tion of genomic imprinting (Wolf 2009). Such genes are Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 851 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Kincaid-Smith et al. GBE important in a coevolution context between sexes because it that female cannot reach sexual maturity without pairing with may allow stronger coadaptation between the maternal amale(Popiel 1986; Beltran and Boissier 2008). In order to inherited mitochondrial genome and the expression of mater- explain the monogamous mating system of schistosomes it nal nuclear alleles in males (Beekman et al. 2014). has been proposed that prezygotic paternal investment (trans- In females, we identiﬁed genes involved in developmental port of the female to the oviposition site, female maturation, pathways (Smp_173620), embryogenesis and oogenesis and and feeding) is higher than maternal investment (Beltran and also in transcriptional regulation of embryo development and Boissier 2008) in contrast with other models on which imprint- animal reproduction (Smp_142400). Others were related not ing has been studied. Second, it is well established that the sex only to protein and vesicle-mediated transport (Smp_154960) ratio is male-based making the male the more competitive sex but also to mechanosensory response (Smp_122810) (Huang (Boissier and Mone 2000; Mone and Boissier 2004; Beltran et al. 1995). Interestingly, we identiﬁed an uncharacterized and Boissier 2010). protein-coding gene (Smp_128970), which seems to play a At last in females the genes expressed from the maternal role in chromatin-mediated transcriptional regulation, and allele once more provide interesting elements concordant also contain a PHD ﬁnger domain, related to the “inhibitor with the sexual antagonism theory. Indeed, some had partic- of growth protein family.” This gene found in females has ular conserved functions in developmental pathway SNPs expressed from the paternal allele and together with (Smp_173620; Strawberry Notch related) and required during other genes expressed from the paternal allele in females embryogenesis and oogenesis in other organisms (Coyle- may once more allow a better coadaptation between sexes. Thompson and Banerjee 1993; Majumdar et al. 1997; Indeed, the paternal genome may inﬂuence the transcrip- Takano et al. 2010). Moreover, we identiﬁed a gene with a tomic proﬁle of females in order to increase the adaptive in- maternal biased SNP in females (Smp_034860) containing a tegration of offspring and the paternal genome leading to domain similar to the ligand-binding domain of C. elegans higher female’s ﬁtness. This observation is also consistent nuclear hormone receptor Sex-1 protein. It is used as an with the sexual antagonism theory, but rather than expressing X-chromosome signal that determines nematode sex and male- or female-speciﬁc coding genes in the same sexes, it may also play a role in dosage compensation mechanisms shows that the opposite parent may inﬂuence the phenotype (Carmi et al. 1998; Farboud and Meyer 2006). As the molec- of the progeny in order to stick with its own sexual features ular determinants of sex determination are not yet known in (e.g., Smp_128970; inhibitor of growth protein family schistosomes besides that it is genetically determined during expressed from the paternal allele in females potentially tends egg fertilization, this gene seems to be an interesting candi- to reduce females size which is one important dimorphic trait date to explore. representative of schistosome sexual features). This again shows how difﬁcult the patterns observed in our organism Why Is Parent-of-Origin Expression an Interesting can be reconciled with any particular model on the evolution Phenomenon to Explain Gonochorism and Sexual of parent-of-origin gene expression and be nonexclusive. Dimorphism in Schistosomes and May Need Further Importantly, this also suggests that parent-of-origin gene Consideration? expression or imprinting may occur in systems that have pre- viously not been the focus of research because of the absence As seen, most of the expression patterns observed in this work of conﬂict over maternal investment, but that imprinting shall are difﬁcult to reconcile with all predictions made by the dif- be present in organisms where coadapted traits may have ferent models of the evolution of genomic imprinting, namely ﬁtness effects for progeny (Wolf and Hager 2006). This theory the kinship theory (Moore and Haig 1991; Haig 2000), the described in the maternal–offspring coadaptation theory maternal–offspring coadaptation theory (Wolf and Hager (Wolf and Hager 2006), in any case does not reject the pos- 2006), and the sexual antagonism theory (Day and sibility of having coadaptation between paternal and offspring Bonduriansky 2004; Bonduriansky 2007). Nevertheless re- traits (Wolf and Hager 2006). Therefore, we may have stron- garding sexual dimorphism, we have mainly focused here ger coadaptation between the paternal genome and off- on patterns predicted by the sexual antagonism theory. Day spring in schistosomes rather than relative to the maternal and Bonduriansky predicted in their sexual antagonism theory genome. Thus, the maternal–offspring coadaptation theory (Day and Bonduriansky 2004) that sex-speciﬁc imprinting may may not be obvious in schistosomes and this may be a reason occur, and in combination with sexually antagonistic selection why we do not observe any particular features predicted by result in sexual dimorphism. Studies have clearly shown that this model. Furthermore, this is plausible considering the some parentally expressed genes are expressed in a sex- strong inﬂuence that male schistosomes have on their speciﬁc manner and might be at the base of sexual dimor- female’s ﬁtness (Popiel 1986; Boissier and Mone2001; Lu phism and differences in behavior like, for example, maternal et al. 2016). An example of such coevolution at the pheno- care (Gregg et al. 2010), but also have effects on complex typic level can be observed from the fact that the male schis- traits (Hager et al. 2008). Sex-speciﬁc parent-of-origin expres- tosomes continuously feed the female via the tegument and sion in adults can thus contribute to transcriptional differences 852 Genome Biol. Evol. 10(3):840–856 doi:10.1093/gbe/evy037 Advance Access publication February 12, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/840/4852791 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Parent-of-Origin-Dependent Gene Expression GBE leading to morphological and or physiological changes at the It allowed us to not only identify new Z-Speciﬁc loci but also base of male and female adult phenotypes in contrast with detect genes containing SNPs consistent with parent-of-origin the schistosomes morphologically indistinguishable larval patterns. Because of the poor amount of SNP markers avail- stages. Therefore, sex-speciﬁc imprinting would allow males able, we cannot conclusively afﬁrm that imprinting exists at a and females to approach their phenotypic optima and may larger scale in this species, but suggest that it needs further contribute to the evolution of sexual dimorphism considerations. Interestingly, in males and females an impor- (Bonduriansky 2007). In this work, we highlighted interesting tant number of genes identiﬁed with parent-of-origin SNPs elements concordant with such predictions. are related to developmental processes such as embryogene- First, most of the genes identiﬁed have functions related to sis, spermatogenesis, and oogenesis and may act as funda- development and sex-speciﬁc functions. As we know that mental contributors to male and female phenotypes and schistosomes have evolved from hermaphroditic ancestors, more speciﬁcally to sexual dimorphism. Other genes related both sexes have therefore obligatorily coevolved and to dosage compensation mechanisms also seem important parent-of-origin gene expression may allow stronger coadap- for sex-speciﬁc phenotypes, especially in males, and the iden- tation between sexes as seen previously. Second, gonochor- tiﬁcation of the Sex-1 protein gene ortholog found in females ism is thought to have appeared with the colonization of is an important candidate to explore the molecular bases of warm-blooded species (Loker and Brant 2006). The diversity sexual determination in schistosomes. We propose that generated by sexual reproduction was proposed to allow the parent-of-origin gene expression may be a mechanism allow- parasite to counter vertebrate’s immune system. Moreover, ing to mitigate conﬂict linked to parenthood and potentially sexual dimorphism is essential for schistosomes and allows solve intralocus sexual conﬂict in this species as it has been male and female schistosomes to have complementary roles proposed from Day and Bonduriansky’s sexual conﬂict theory, in the deﬁnitive hosts (Basch 1990). Indeed, males are much deﬁning imprinting as a mechanism for the evolution of sex more muscular than females (i.e., sexual dimorphism), and speciﬁc traits. In schistosomes, it is appealing to think that if sexual selection may favor large body in males and the oppo- imprinting exists in this species, it may coincide with the shift site in females. It is interesting to consider that parent-of- from hermaphroditism to gonochorism and thus may be at origin expression may have allowed sexual reproduction main- the base sexual reproduction and sexual dimorphism mainte- tenance in order to generate diversity in these species, as nance. Importantly, future studies on parent-of-origin expres- imprinting defaults can have detrimental effects in other sion in schistosomes should address the question of the organisms (Swales and Spears 2005) and cause embryonic underlying molecular mechanisms. Indeed, the imprinting by lethality. Furthermore, it might have contributed to the evo- the usual mechanism of DNA methylation could be contro- lution of sexual dimorphism as proposed by Day and versial in this species for which such mechanism is still under Bonduriansky’s predictions (Day and Bonduriansky 2004). At debate (Raddatz et al. 2013); therefore other epigenetic last, the identiﬁcation of genetic mobile elements with actors will have to be explored, such as histone modiﬁcations. parent-of-origin expressed SNPs is also an interesting point Growing evidences show that chromatin modiﬁcations are as it has been shown that imprinted regions tend to show indeed involved in schistosomes development (Roquis et al. signiﬁcantly more transposon insertions or more generally, 2015), moreover in a sex-speciﬁc manner (Picard et al. 2016). retroviral repeats, than other regions (Pignatta et al. 2014). Another interesting perspective would be to look for parental Thus, imprinting in schistosomes could also be the side effect biased expression in the nondifferentiated cercarial stage. of host defense mechanism against foreign DNA (McDonald Finally, by detecting genes with strain-speciﬁc expressions, et al. 2005) that was thereafter selected because of its adap- our unprecedented analysis opens new perspectives to under- tive signiﬁcance. It has recently been shown that “captured” stand hybridization mechanisms in schistosomes and thus on genes of retroviral origin (Syncytins) have sex-dependent the disease dynamics, including interspeciﬁc interactions in effects in mice, and that they are involved in the development the context of the modiﬁcation of species geographical distri- of the placenta in females, and have a role in muscle devel- bution due to climate change (Boissier et al. 2016). opment in males (Redelsperger et al. 2016). This again under- lies a possible contribution of imprinted genes to sexual Ethic Statement dimorphism in schistosome parasites. All experiments were carried out according to national ethical standards established in the writ of February 1, 2013 (NOR: Conclusion AGRG1238753A), setting the conditions for approval, plan- Schistosomes are diploid metazoan parasites with separate ning, and operation of establishments, breeders and suppliers sexes, thus the maternal and paternal genomes are thought of animals used for scientiﬁc purposes and controls. The to contribute equally to the ﬁtness of their offspring. In this French Ministe ` re de l’Agriculture et de la P^ eche and work, we explored the transcriptome of male and female the French Ministe ` re de l’Education Nationale de la adult worms to search for parent-of-origin expressed genes. Recherche et de la Technologie provided permit A66040 to Genome Biol. 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