No pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: From mating interactions to differential gene expression.

Species usually develop reproductive isolation mechanisms allowing them to avoid interbreeding. These preventive barriers can act before reproduction, "pre-zygotic barriers", or after reproduction, "post-zygotic barriers". Pre-zygotic barriers prevent unfavourable mating, while post-zygotic barriers determine the viability and selective success of the hybrid offspring. Hybridization in parasites and the underlying reproductive isolation mechanisms maintaining their genetic integrity have been overlooked. Using an integrated approach this work aims to quantify the relative importance of pre-zygotic barriers in Schistosoma haematobium x S. bovis crosses. These two co-endemic species cause schistosomiasis, one of the major debilitating parasitic diseases worldwide, and can hybridize naturally. Using mate choice experiments we first tested if a specific mate recognition system exists between both species. Second, using RNA-sequencing we analysed differential gene expression between homo- and hetero-specific pairing in male and female adult parasites. We show that homo- and hetero-specific pairing occurs randomly between these two species, and few genes in both sexes are affected by hetero-specific pairing. This suggests that i) mate choice is not a reproductive isolating factor, and that ii) no pre-zygotic barrier except spatial isolation "by the final vertebrate host" seems to limit interbreeding between these two species. Interestingly, among the few genes affected by the pairing status of the worms, some can be related to pathways affected during male and female interactions and may also present interesting candidates for species isolation mechanisms and hybridization in schistosome parasites.

Introduction

A subset of obstacles evolved in the course of speciation in order to limit gene flow via hybridization and maintain species boundaries. These obstacles are traditionally classified as pre- and post-zygotic barriers (also known as pre- or post-mating barriers) and can be defined as any mechanism preventing or reducing gene flow between groups of potentially interbreeding individuals [1]. Pre-zygotic barriers include spatial isolation (e.g., two species live in different habitats), behavioural isolation (e.g., individuals can choose to mate with individuals of their own species), temporal isolation (reproduction does not occur at the same time e.g., different seasons), mechanical isolation (sex organs are not compatible) and gametic isolation (sperm and eggs mix but fertilization does not occur). When the first barrier is crossed, post-zygotic isolation mechanisms can arise to prevent gene flow. Post-zygotic barriers include hybrid unviability (hybrids die prematurely), reduced fitness with low fertility (hybrids are less fertile, infertile or non-viable) or hybrid breakdown (a longer process where the hybrid lines are counter-selected compared to their parental forms). The strength and/or the order of each reproductive barrier vary among species. This makes difficult to predict the outcome of inter-species mating, and the evolution of reproductive isolation mechanisms [2]. Moreover, reproductive isolation is often the result of an accumulation and interaction of multiple pre- and post-zygotic mechanisms restricting most gene flow [3]. However, it is generally recognized that pre-zygotic isolation barriers are enhanced in sympatric species [4], and are the most effective because they act early to prevent the production of hybrid progeny.

Despite their importance in terms of biodiversity [5], but also animal and human health, parasite species have received less attention than other free-living organisms regarding both hybridization and the role of reproductive isolation mechanisms [6]. Pre-zygotic barriers in parasites usually include additional and stronger obstacles to overcome compared to those of free-living organisms. For instance, the "habitat barrier" includes the geographic area, the host species and the tropism within the host. For parasites, hosts are dynamic habitats imposing strong selective pressures (co-evolutionary arms race) requiring constant adaptation of parasites for the completion of their life cycle. The specialisation of parasite species to a particular host is thus expected to be a strong pre-zygotic isolation mechanism preventing hybridization and favouring speciation. However, some closely related species do manage to retain their genetic identity whilst parasitizing the same host, meaning that they have acquired selective mechanisms for reproductive isolation. Hybridization and pre-zygotic reproductive barriers have been studied on very few parasite models such as plasmodium species, cestodes and schistosomes [6-8]. Partial pre-zygotic barriers have been evidenced between Plasmodium berghei and P. yoeli [7]. It was not the case between Schistocephalus solidus and S. pungitii [6], suggesting in the latter that post-zygotic selection against hybridization is presumably the most important driving force limiting gene flow between these two parasitic sister species [6].

Schistosomes are parasitic agents that cause schistosomiasis, a debilitating disease affecting over 240 million people worldwide, mainly in tropical and subtropical areas [9]. There are currently 23 know species in the genus Schistosoma, including six species that infect humans and 20 species that infect animals [8]. These parasites have a two-host life cycle, which includes a mammalian definitive host, in which sexual reproduction occurs and a mollusc intermediate host in which asexual multiplication takes place. Schistosomes have the particularity of having separate sexes, a feature not observed in other trematodes that are hermaphroditic [10,11]. Schistosomes have therefore been intensively studied for their sexual features including male-female interactions [12,13], sex-ratios [14,15], mating systems [16,17] and mating behaviour [18]. One direct consequence of dioecism in these species is the necessity of individuals of both sexes to infect the same definitive host. This constraint can lead to interactions between species infecting the same host, and in the case of porous reproductive pre-zygotic barriers this can lead to hybridization.

To conserve their genetic identity, schistosomes that inhabit the same definitive host are expected to present pre-zygotic isolation mechanisms. Among these barriers, habitat and behavioural isolation have a great influence in schistosome’s sexual interactions. First, habitat isolation is a three-level constraint that initially has to be overcome (i.e., same geographic area, same host individual, and same localisation in the host). Indeed, schistosomes species are distributed worldwide (the majority in Africa), the vertebrate host specificity depends on the parasite species, and while the majority of species live in the mesenteric vein system, one species (S. haematobium) lives in the veins surrounding the bladder of humans. Second, behavioural isolation is more complex in schistosomes than in other species because mating is followed by a pairing-dependent differentiation of the female’s sexual organs [12,13]. Studies have clearly established that the presence of the male (independently of the species paired) is necessary not only for the female’s sexual development, but also for the maintenance of a sexually mature and active state [19-21]. It was also demonstrated that female schistosomes stimulate males through changes in levels of glutathione and lipids, and stimulate tyrosine uptake in the male worms [12]. Hence, while males transfer glucose and lipid secretions to females, females also release factors affecting the physiology of male worms [22-25]. Thus, male and female schistosomes are strongly co-dependent, in terms of behaviour (i.e., they have complementary roles in the hosts), but also physiologically [10] with an intimate and permanent association between sexes necessary for reproduction to occur.

Nevertheless, several hybrid schistosomes have been evidenced [8,22,26,27]. Similarly to other groups, isolation mechanisms increase with divergence time between taxa [4,8]. The success of inter-species interactions on the viability of hybrid offspring also depends on the direction of the cross and thus which parental species provides the maternal and paternal genome [27-29]). Studies on schistosome mate choices have revealed that depending on the parasite species interacting, some combinations may readily pair with no preference (S. haematobium x S. intercalatum (referred to as S. guineensis since 2003 based on their mitochondrial divergence [30,31]) S. bovis x S. curassoni and S. mansoni x S haematobium), whereas when involved in other combinations, species may present a mate recognition system favoring or not interspecies pairing (S. mansoni x S. intercalatum (now S. guineensis), S. haematobium x S. mattheeii, and S. mansoni x S. margrebowiei crosses) [28,32-34]. However, competition between schistosome species can also explain the frequency of some interspecific crosses [28,29,32]. For instance it has been shown that S. haematobium males can take away females from other species when competing with male S. intercalatum (now S. guineensis) [35], S. mattheei [29] or S. mansoni [28] hence promoting or favouring hetero-specific pairing. For schistosome species that randomly pair with no mate preference and for many related parasitic species capable of hybridizing, final host specificity may be the sole barrier preventing interbreeding [35]. This isolation mechanism “by the host” may be so efficient that species may lack any post-zygotic or other pre-zygotic mechanisms ultimately allowing them to hybridize when the opportunity arises. Therefore, the lack of reproductive incompatibility (i.e., isolation by behaviour and physiology) between schistosome species infecting humans and animals may facilitate gene flow if the host isolation barriers are broken down.

Schistosoma haematobium x S. bovis hybrids are today the most studied hybrid system of schistosomes. These hybrids were first identified in Niger by Brémont [36] and more recently in Senegal [26] but appeared widely distributed in West Africa [26,27,36-39]. Moreover, these hybrids have recently been involved in a large-scale outbreak in Europe (Corsica, France), where transmission of the disease is persistent [37,40]. Schistosoma haematobium and S. bovis are co-endemic in Africa, but their host specificity and tropism within their definitive hosts are different (urogenital and human vs. intestinal and cattle, respectively). S. haematobium is mainly a parasite of humans, however, sporadic studies have shown that non-human primates, Cetartiodactyla members or rodents could be naturally infected by this parasite species (although these accounts were based on egg morphology and could thus involve other species) [41-43]. Conversely, S. bovis is mainly a parasite of ruminants with sporadic cases of rodent infection [41,44]. Interestingly, although data are scarce, recent studies showed that S. haematobium x S. bovis hybrids may naturally infect rodents or cattle [39,44].

Hybridization between these two species is particularly worrying because it raises the eventuality for a human parasite to have animal reservoirs of infection and the animal parasite to be zoonotic [45]. Likewise, hybridization may lead to changes in the parasites life history traits, including host range expansion, increased virulence and host morbidity, but also response to chemotherapeutic treatment [46]. Indeed, in experimental infections, these hybrids often display heterosis, in which their fitness outperforms the fitness of parental species [8,26,27]. Importantly the existence of a mate recognition system between the two species would prevent natural occurrences of hybridization in sympatric areas. In contrary a lack of reproductive isolation could indicate that occurrences of hybridization may be more frequent.

Although experimental crosses in hamsters have demonstrated their capacity to pair and the viability of S. haematobium x S. bovis hybrids [27], their pairing frequency and underlying molecular mechanisms need to be assessed. This study hence uses an integrated approach, from mating behaviour to male and female gene expression, in order to quantify the importance of pre-zygotic barriers involved in the interactions between S. haematobium x S. bovis. First, using a mate choice experiment we tested whether specific mate recognition or competition exists by quantifying the frequency of hetero-specific and homo-specific pairs compared to random mating expectations. Second, given the strong co-dependence between male and female schistosomes, we also analysed the influence of pairing (homo- vs hetero-specific) on the transcriptomic profile of male and female parasites using RNA sequence analysis. We hypothesize that since these hybrids are frequently encountered in the field [37,37,39] and since parental species are able to pair in the laboratory [27] mate recognition should not constitute a strong barrier to reproduction. However, depending on species dominance in mating, the direction of pairing could be affected. Since females undergo strong developmental changes upon pairing [13,47,48] we would expect finding strong transcriptomic changes associated with inter-species interactions for females but not for males. The molecular determinants of the very first step towards hybridization may give further insight into the permeability of the two species and reveal some important genes linked to male and female interaction, species isolation and hybridization.

Materials and methods

Ethics statement

Experiments were carried out according to national ethical standards established in the writ of February 1st, 2013 (NOR: AGRG1238753A), setting the conditions for approval, planning and operation of establishments, breeders and suppliers of animals used for scientific purposes and controls. The French Ministry of Agriculture and Fishery (Ministère de l’Agriculture et de la Pêche), and the French Ministry for Higher Education, Research and Technology (Ministère de l’Education Nationale de la Recherche et de la Technologie) approved the experiments carried out for this study and provided permit A66040 for animal experimentation. The investigator possesses the official certificate for animal experimentation delivered by both ministries (Décret n° 87–848 du 19 octobre 1987; number of the authorization 007083).

Origin and maintenance of schistosome strains

Schistosoma haematobium and S. bovis were maintained in the laboratory using Bulinus truncatus snails as intermediate hosts and Mesocricetus auratus as definitive hosts. The parasite strains originated from Cameroon and Spain for S. haematobium and S. bovis, respectively [49]. The S. haematobium strain was initially recovered from the urine of infected patients in 2015 (Barombi Kotto lake; 4°28’04"N, 9°15’02"W). Eggs from positive samples were hatched, miracidia were harvested, and sympatric B. truncatus molluscs, bred from snails collected from the same location as the parasites, were individually exposed to five miracidia before being transferred to the IHPE laboratory for their maintenance. The S. bovis, strain isolated in the early 80’s [49,50] was kindly provided by Ana Oleaga from the Spanish laboratory of parasitology of the Institute of Natural Resources and Agrobiology in Salamanca, and originates from Villar de la Yegua-Salamanca.

Experimental infections

Protocols of experimental infections were set for two objectives, i) quantifying the frequency of hetero-specific and homo-specific pairings and, ii) forcing hybridization and then assessing the transcriptomic changes between homo-specific and hetero-specific paired males and females. The successive steps of our experimental infection procedure are presented in Fig 1. Detailed procedures for mollusc and rodent infections have been previously described [51-53]. Step 1: 3–5 mm B. truncatus were placed in 24-well plates containing 1ml of spring water per well. Each mollusc was exposed overnight to a single miracidium (i.e., a single male or female genotype) of either S. haematobium or S. bovis. The following morning molluscs were placed in breeding tanks and fed ad libitum for the duration of the experiment. After a minimum period of 55 days, corresponding to the development time of the parasites in their intermediate host, molluscs were stimulated under light for cercariae shedding. Step 2: cercariae from each infected mollusc were recovered for molecular sexing as previously described [49]. Step 3: molluscs were gathered into four distinct tanks according to the species and the sex of the infecting parasite. Step 4: hamsters were individually exposed to cercariae using the surface application method for one hour [51-53]. The sex and the species of the cercariae used for each experiment are presented in Table 1 and are described further below (Mate choice analysis, Force pairing and underlying molecular analysis).

Fig 1

Schematic representation of experimental infection procedure.

Table 1

Number of cercariae used for each experiment according to the species and the sex of the parasite.

Experiments	S. haematobium		S. bovis		Number of hamsters
	Males	Females	Males	Females
Objective 1: Quantification of homo- and hetero-specific pairs frequency
Limited Choice Experiment
Exp. 1 (limiting sex: S. haematobium males)	150	225	-	225	5
Exp. 2 (limiting sex: S. haematobium females)	225	150	225	-	5
Exp. 3 (limiting sex: S. bovis males)	-	225	150	225	5
Exp. 4 (limiting sex: S. bovis females)	225	-	225	150	5
Full Choice Experiment
Exp. 5	150	150	150	150	5
Objective 2: Assess the transcriptomic profiles of homo- and hetero-specific paired worms
Homo-specific forced pairing 1	300	300	-	-	6
Homo-specific forced pairing 2	-	-	300	300	6
Hetero-specific forced pairing 1	300	-	-	300	6
Hetero-specific forced pairing 2	-	300	300	-	6

Hamsters were euthanized at three months after cercarial exposition and adult worms were recovered by hepatic perfusion [53]. Hamsters were autopsied and specific organs such as the mesenteric and portal veins were carefully checked to identify potential remaining worms. We recorded each worm’s sex inferred by their strong sexual dimorphism [11] and their paring status (paired or single). Paired worms were manually separated under a light microscope. All worms collected were individualized in 96-well plates and were subjected to DNA extraction using the method described previously in Beltran et al. (2008) [54]. The species of each worm was identified using the rapid diagnostic procedure based on multiplex PCR reaction described by Webster and colleagues [55,56].

Mate choice analysis

Experimental design

The experimental procedure to quantify the frequency of homo- and hetero-specific pairs between S. bovis and S. haematobium consisted of five experiments (i.e., Exp. 1 to Exp. 5, see Table 1). The first four experiments aimed to test individually the choice of each species and sex (Exp. 1 and Exp. 3 for male choice—Exp. 2 and Exp. 4 for female choice for S. haematobium and S. bovis, respectively). In each experiment, five hamsters (used as biological replicates) were infected with mixed combinations of cercariae (Table 1). These four experiments represented a limited choice of mate where excess of one sex (of both species competing for mating) ensuring that all individuals of the other sex (that had the choice for homo- or hetero-specific pairings) will be mated (Table 1). Finally, the last experiment (Exp. 5, Table 1) represented full choice of mate. Hamsters were infected with equal numbers of cercariae of both sexes and both species so that all combination of mating can be assessed at the same time.

Statistical analysis

After counting the total number of adult worms recovered for each species (e.g., homo-specific pairs, hetero-specific pairs and single worms), we calculated the expected number of single and paired worms according to the null hypothesis of random pairing (e.g., in the Exp. 1, the expected number of homo-specifically paired S. haematobium males equals the total number of S. haematobium males, times the total number of S. haematobium females over the total number of females). Expected and observed numbers of homo- and hetero-specific pairs were then compared using Chi-square tests.

Forced pairing and underlying molecular analysis

Hamsters were infected with four combinations of parasites (Table 1). Homo-specific pairing consisted of infections with single species of cercariae, while hetero-specific pairing consisted of infections with male and female cercariae of the opposite species. Hamsters were euthanized three months after their exposition to cercariae and adult worms were recovered by hepatic perfusion. Paired worms were separated under a magnifier using a small paintbrush (to avoid causing any damage to the worms) and pooled according to their sex (male or female) and the species of their sexual partner (same or opposite species). Pools of 10–12 female or male worms were placed in 2ml microtubes and immediately frozen in liquid nitrogen and stored at -80°C. Three biological replicates were constituted for each combination representing a total of 24 samples (2 sexes x 4 combinations x 3 replicates) for subsequent RNA extraction and transcriptome sequencing (see Fig 2 for a schematic view of the procedure).

Fig 2

Schematic representation of the procedure used to obtain the reciprocal homo- and hetero-specific pairs of S.

haematobium and S. bovis.

RNA extraction and transcriptome sequencing of homo- and hetero-specific S. haematobium and S. bovis male and female pairs

Trizol RNA extraction and subsequent paired-end Illumina HiSeq 4000 PE100 sequencing technology was performed on the 24 samples. Briefly, pools of adult worms were ground with two steel balls using a Retsch MM400 cryobrush (2 pulses at 300Hz for 15s). Total RNA was extracted using the Trizol Thermo Fisher Scientific protocol (ref: 15596018) slightly modified as the volume of each reagent was halved. Total RNA was eluted in 44 μl of ultrapure water before undergoing a DNase treatment using Thermofisher Scientific Turbo DNA-free kit. RNA was then purified using the Qiagen RNeasy mini kit and eluted in 42μl of ultrapure water. Quality and concentration of the RNA was assessed by spectrophotometry with the Agilent 2100 Bioanalyzer system and using the Agilent RNA 6000 nano kit. Further details are available at Environmental and Evolutionary Epigenetics Webpage (http://methdb.univ-perp.fr/epievo).

Illumina library construction and high-throughput sequencing

cDNA library construction and sequencing were performed at the Génome Québec platform. The TruSeq stranded mRNA library construction kit (Illumina Inc., USA) was used following the manufacturer’s protocol on 300 ng of total RNA per sample. Sequencing of the 24 samples was performed in 2x100 bp paired-end on a Illumina HiSeq 4000 (S1 Table). Sequencing data are available at the NCBI-SRA under the BioProject PRJNA491632.

Transcriptomic analysis of hetero-specific pairing versus homo-specific pairing

Raw sequencing reads were analysed on the Galaxy instance of the IHPE laboratory [57,58] First, raw reads were subjected to quality assessment and sequence adaptor trimming. We used the set of tools based on the FASTX-toolkit [59], as well as Cutadapt program (Galaxy Version 1.16.1) to remove adapter sequences from Fastq files [60]. Finally, paired end reads were joined in a single fastq file using the FASTQ interlacer/de-interlace programs (Galaxy Version 1.1). Processed reads were mapped using RNA-star Galaxy Version 2.6.0b-1 [61] to the S. haematobium reference genome [62] downloaded from the Schistosoma Genomic Resources website SchistoDB (http://schistodb.net/common/downloads/Current_Release/ShaematobiumEgypt/fasta/data/). Exon-intron structure was thereafter reconstructed for each mapping BAM file using Cufflinks transcript assembly Galaxy Version 2.2.1.2, by setting the max intron length at 50000, but without any correction parameters [63]. Finally, in order to create a reference transcriptome representative of S. haematobium and S. bovis male and female reads, we merged all cufflinks data with Cuffmerge Galaxy Version 2.2.1.2 [63] without using any guide or reference. This enabled us to create a representative reference transcriptome of both species and both sexes using the same reference genome. The Genomic DNA intervals of all newly assembled genes of this reference transcriptome were extracted from the S. haematobium reference genome and converted into a Fasta file.

The number of reads per transcript for each sample (i.e., the read abundance representative of each gene) was quantified using HTseq-count Galaxy Version 0.9.1 on the reference transcriptome, setting the overlap resolution mode on “union” [64]. Finally, we evaluated the differential gene expression levels between homo-specifically and hetero-specifically paired worms for each species and each sex separately using DESeq2 Version 1.28.1 [65] run on R version 4.0.0 [66]. We carried out four types of comparisons which respectively focused on S. haematobium males, S. haematobium females, S. bovis males and S. bovis females and contrasted gene expression profiles between hetero-specifically paired individuals and homo-specifically paired individuals. Differential gene expression results were filtered on the adjusted P-value (Benjamini-Hochberg multiple testing based False Discovery Rate (FDR)) and considered significant when ≤ 5%.

Functional annotation

Using our BLAST local server, we annotated the entire de novo assembled transcriptome by Blastx search against the non-redundant database of the NCBI. We conserved only the longest unique transcript (TCONS) of each representative gene (XLOC) for Blastx search and subsequent analysis. Output XML files were used for gene ontologies (GO) mapping and annotation using Blast2Go version 4.1.9 [67]. Finally, enrichment Fisher’s exact tests were performed on up and down regulated sets of genes focusing on biological process (BP) ontology terms. The P-value for significance was set to 5% False Discovery Rate (FDR).

Results

Mating choice experiments

Limited choice: Experiments 1 to 4

Details on the number of worms recovered from each hamster and whether they were paired or single are summarized in Table 2. For each mate choice experiment both homo-specific and hetero-specific pairs were observed (Table 2, Exp. 1–4). Also, in each limited choice of mate experiment (Exp. 1–4) we consistently obtained an excess of single worms of both species competing for pairing (i.e., male or female depending on the experiment) whereas all worms of the limiting sex (i.e., choosing partners, such as female choice or male competition) were paired (Table 2). This indicates that the choosing partners in each experiment were not limited in their choice by the number of potential homo- or hetero-specific partners. Specifically, in the experiment 1, the number of homo- and hetero-specific pairs of male S. haematobium was significantly different from those expected under the random mating hypothesis (χ2 = 11.10; d.f. = 4; P-value = 0.049, Table 2). This was due to the deviation from the random mating hypothesis in one hamster (hamster number 3, see Table 2). Regarding S. haematobium females’ choice (Table 2, Exp. 2) at the contrary, the numbers of homo-specific pairs and hetero-specific pairs were not significantly different from expectations under the random mating hypothesis (χ2 = 3.118; d.f. = 4; P-value = 0.682, Table 2). In the experiment 3 that focused on S. bovis males’ choice, the total number of paired worms recovered was extremely low, due to premature death of two hamsters and only two hamsters had enough worms to be analysed (Table 2, Exp. 3). Although in this case, statistics should be interpreted with caution, the numbers of homo-specific and hetero-specific pairs were once again not significantly different from expectations under a random mating hypothesis (χ2 = 4.522; d.f. = 1; P-value = 0.104, Table 3). Finally, regarding S. bovis females’ choice (Table 2, Exp. 4) similarly we did not find a significant difference between the numbers of observed and expected homo-specific pairs and hetero-specific pairs under random mating hypothesis (χ2 = 3.246; d.f. = 4; P-value = 0.662, Table 2). Overall, when analysing all limited choice experiments together (i.e., Exp. 1 to 4) no significant difference was recorded between the number of observed homo- and hetero-specific pairs and those expected under a random mating scenario (χ2 = 21.71, d.f. = 16, P-value = 0.152, Table 2).

Table 2

Summarized information of experiments 1 to 4 (limited choice).

For each experiment are displayed the sex and the species of the choosing partner (such as female choice or male competition), the number of observed homo- and hetero-specific pairs and the number of worms that remained single. Sh = S. haematobium and Sb = S. bovis. Expected number of pairs under random mating hypothesis is shown in brackets (see the statistics section in Materials and Methods for details). Chi-square statistic, degree of freedom and P-value are given for each hamster, for each experiment and for all experiments combined. * indicates significant results at 5% level. In Exp. 3, worms from only two hamsters could be analysed, while others died prematurely (two hamsters) or presented too few numbers of paired worm (one hamster).

Exp.	Host	Choosing partner	Homo-specific pairs	Hetero-specific pairs	Single worms		χ2-statistic	d.f.	P-value
Exp. 1			♂ Sh x♀ Sh	♂ Sh x♀ Sb	♀ Sh	♀ Sb	11.104	4	0.049*
1	1	♂ Sh	11 (14)	14 (11)	20	9	1.838	1	0.175
1	2	♂ Sh	11 (15)	22 (18)	15	11	1.543	1	0.214
1	3	♂ Sh	14 (20)	16 (10)	25	4	5.057	1	0.025*
1	4	♂ Sh	9 (9)	6 (6)	36	26	0.015	1	0.903
1	5	♂ Sh	10 (13)	10 (7)	41	15	2.651	1	0.103
Exp. 2			♀ Sh x ♂ Sh	♀ Sh x ♂ Sb	♂ Sh	♂ Sb	3.118	4	0.682
2	1	♀ Sh	10 (9)	2 (4)	7	5	0.908	1	0.341
2	2	♀ Sh	6 (5)	1 (2)	0	1	0.429	1	0.513
2	3	♀ Sh	12 (10)	3 (5)	11	10	1.688	1	0.194
2	4	♀ Sh	16 (15)	3 (4)	6	2	0.094	1	0.759
2	5	♀ Sh	12 (12)	13 (13)	11	12	0.001	1	0.993
Exp. 3			♂ Sb x♀ Sb	♂ Sb x♀ Sh	♀ Sb	♀ Sh	4.522	1	0.104
3	2	♂ Sb	4 (2)	0 (2)	46	55	4.400	1	0.036
3	3	♂ Sb	2 (1)	1 (2)	22	32	0.742	1	0.389
Exp. 4			♀ Sb x ♂ Sb	♀ Sb x ♂ Sh	♂ Sb	♂ Sh	3.246	4	0.662
4	1	♀ Sb	15 (12)	17 (20)	4	14	1.070	1	0.301
4	2	♀ Sb	10 (8)	25 (28)	2	19	1.061	1	0.303
4	3	♀ Sb	3 (3)	8 (8)	4	13	0.030	1	0.862
4	4	♀ Sb	9 (8)	15 (16)	8	19	0.188	1	0.665
4	5	♀ Sb	49 (49)	15 (15)	18	5	0.007	1	0.932
All Exp.							21.719	16	0.152

Table 3

Summarized information of experiment 5 (full choice).

For each combination (i.e., sex and species) are given the number of observed pairs and the number of single partners that remained single. Sh = S. haematobium and Sb = S. bovis. Expected number of pairs under random mating is shown in brackets (see the statistics section in Materials and Methods for details). Chi squared statistics, degree of freedom and P-value are given per hamster and for the whole experiment.

Host no.	♂Shx♀ Sh	♂Sbx♀ Sb	♂Shx♀ Sb	♂Sbx♀ Sh	♂Sh	♂Sb	♀ Sh	♀ Sb	χ2-statistic	d.f.	P-value
1	1 (2)	(24)	5 (9)	4 (5)	8	5	4	9	3.358	3	0.340
2	8 (8)	2 (1)	5 (4)	1 (2)	8	3	23	8	1.786	3	0.618
3	7 (5)	2 (2)	1 (2)	2 (3)	6	5	19	11	2.307	3	0.511
4	4 (4)	6 (3)	3 (4)	1 (3)	10	4	13	11	4.806	3	0.187
5	5 (6)	1 (1)	6 (6)	2 (1)	20	3	17	16	0.796	3	0.850
Total									13.053	12	0.365

Full choice: Experiment 5

Details on the number of worms recovered from each hamster and whether they were paired or single are summarized in Table 3. When all mating combinations were allowed between S. haematobium and S. bovis, four types of pairing combination were obtained: two being homo-specific (♂ Sh x ♀ Sh and ♂ Sb x ♀ Sb, Table 3) and two being hetero-specific (♂ Sh x ♀ Sb and ♂ Sb x ♀ Sh, Table 3). There was also an excess of males and females of both species remaining single, suggesting that all possible pairings were not limited by partner availability (Table 3). Regarding the number of homo-specific and hetero-specific pairs observed between S. haematobium and S. bovis, Chi-square tests did not reveal significant departure from random mating hypothesis, when the number of each pairing combination was analysed in each hamster separately and also when analysing all replicate together (Table 3).

Transcriptomic response in homo- vs. hetero-specific pairs

RNA sequencing, transcriptome assembly and gene annotation of the homo- and hetero-specific pairs

We have separately analysed 24 samples, corresponding to biological triplicates of males and females of the four forced pairing combinations described in Table 1 and Fig 2 (i.e., homo- and hetero-specifically paired males and females). Between ~24.7 and ~42.3 million high quality Illumina HiSeq 4000 PE100 RNA-seq reads were obtained after sequencing of the 24 samples. After quality control and adaptor trimming, between ~19.2 and ~33,1 million reads were uniquely mapped to the S. haematobium reference genome and used for gene expression analysis [62]. On average ~78% of raw reads were mapped to the reference genome, with 51% of which corresponded to S. haematobium and 49% to S. bovis (S1 Table).

The reference transcriptome assembly on which tests were carried out, was composed of 73,171 putative isoform sequences identified as TCONS, and 18,648 unique genes identified as XLOCS. We conserved the longest isoform (TCONS) for each gene (XLOC) for subsequent annotation. The GTF and Fasta file of this transcriptome are available in a Figshare repository (https://doi.org/10.6084/m9.figshare.12581156, [68]). Blast annotations and Gene Ontology terms of the complete reference transcriptome are available in Sheet A S1 File. On the 18,648 genes, 14,414 found at least one hit following Blastx analysis, and 12,332 of them were mapped to at least one GO term using Blast2GO [67].

Differential gene expression

Quantification of read abundance as well as differential gene expression analysis were performed on the 18,648 genes for each homo- and hetero-specific conditions (Sheet B, Sheet C, Sheet D, Sheet E and Sheet G in S1 File). The heatmap of the sample-to-sample distances as well as the principal component analysis plot are presented in Fig 3. A total of 1,277 genes (~7% of the 18,648 genes present in the reference transcriptome) were differentially expressed in at least one of the four homo- versus hetero-specific comparisons with a FDR <5% (Sheet G in S1 File). Of these, 1,234 (97%) had a match using Blastx against the non-redundant database of the NCBI and 1,088 (85%) were mapped and successfully annotated with at least one GO term using Blast2GO [67] (Sheet G in S1 File).

Fig 3

Differential gene expression profiles.

a) Principal component plot of the samples and b) Heatmap of the sample-to-sample distances.

Most of the differentially expressed genes (DEGs) were identified in S. haematobium males, with 1,166 DEGs between the hetero-specific and homo-specific pairing combinations (734 over-expressed and 432 under-expressed in hetero-specific paired males compared to homo-specific ones). Log2-Fold changes were quite low with only one of these 1,166 DEGs having a Log2-Fold Change higher than 1.5 and none had Log2-Fold change lower than -1.5 (Fig 4, Sheet C and Sheet G in S1 File). In S. haematobium females, 47 genes were differentially expressed between hetero- vs. homo-specific conditions (22 over-expressed and 25 under-expressed in hetero-specific females). Among these 47 DEGs, six had Log2-Fold changes higher than 1.5 and one had a Log2-Fold change lower than -1.5 (Fig 4, Sheet D and Sheet G in S1 File). In S. bovis females, 88 genes were differentially expressed between hetero- vs. homo-specific conditions (58 over-expressed and 30 under-expressed in hetero-specific females). Among these 88 DEGs, 48 had Log2-Fold changes higher than 1.5 and 11 had Log2-Fold changes lower than -1.5 (Fig 4, Sheet E and Sheet G in S1 File). Finally, no DEGs were identified in S. bovis males (Sheet F and Sheet G in S1 File). Significantly (p<5%) over- and under-expressed genes (XLOC) for each comparison as well as their annotation are shown in Sheet G in S1 File.

Fig 4

Genes expression profiles in hetero-specifically compared to homo-specifically paired worms.

Volcano plots showing the log transformed adjusted P-values (i.e., FDR) and the log fold changes for the 18,648 unique genes of the reference transcriptome assembly for S. haematobium males a), S. haematobium females b), S. bovis females c) and S. bovis males d). Black dots refer to non-significant genes regarding their expression profile (over an FDR of 5%). Red dots refer to differentially expressed genes at a FDR of 5%, green dots refer to differentially expressed genes at a FDR between 5% and 1% and blue dots refer to DEGs at a FDR between 1% and 1 ‰.

Gene Ontology and enrichment analysis of the differentially expressed genes

Gene ontology categories significantly enriched in either over- or under-expressed genes were found in S. haematobium males (Fig 5, Sheet H in S1 File) whereas in S. haematobium females, S. bovis males and females (in which fewer DEG were detected), no GO terms were significantly enriched.

Fig 5

Biological processes impacted by hetero-specific pairing in male S. haematobium.

Barplot showing the biological processes significantly enriched in DEGs (at a FDR threshold of 5%), either over-expressed or under-expressed in hetero-specific condition compared to homo-specific condition, in S. haematobium males.

In S. haematobium males, biological processes enriched in under-expressed genes (in hetero-specific paired males compared to homo-specific ones) were related to signal transduction, notably through neuronal processes (synaptic transmission, cholinergic, chemical synaptic transmission, postsynaptic, G protein−coupled receptor signalling pathway), development (anatomical structure development), metabolism (glycogen biosynthetic process, negative regulation of endopeptidase activity), transmembrane transport (potassium ion transmembrane transport), response to stimuli (response to drug, peptidyl−proline hydroxylation, cell redox homeostasis) and cell adhesion (homophilic cell adhesion via plasma membrane adhesion molecules) (Fig 5, Sheet H in S1 File).

On the other hand, biological processes enriched in over-expressed genes (in hetero-specific males) were related to signal transduction including again some neuronal processes (e.g., transmembrane receptor protein tyrosine kinase signalling pathway, regulation of Ras protein signal transduction, regulation of axon extension), metabolism (e.g., proteolysis involved in cellular protein catabolic process, phosphatidylcholine metabolic process, long−chain fatty acid metabolic process, lipid droplet organization), response to stimuli (e.g., response to other organism, phagocytosis, cellular response to chemical stimulus), transmembrane transport (e.g., anion transmembrane transport, vesicle fusion, regulation of vesicle−mediated transport, inorganic cation import across plasma membrane, exocytosis, positive regulation of Notch signaling pathway), localization (e.g., establishment of localization in cell), locomotion (e.g., regulation of locomotion, microtubule−based process, actin filament organization) and also cell adhesion (e.g., cell junction assembly) (Fig 5, Sheet H in S1 File).

No GO terms were found enriched neither in over- nor under-expressed genes in S. bovis and S. haematobium hetero- vs. homo-specifically paired females. However, based on annotations, in S. haematobium females, we found differentially expressed genes that corresponded to genetic mobile elements (e.g., XLOC_014282: integrase core domain, XLOC_014741: TPA: endonuclease-reverse transcriptase, XLOC_009783: endonuclease-reverse transcriptase), genes involved in transmembrane transport (e.g., XLOC_009318: phosphatase methylesterase 1 (S33 family) and XLOC_010891: Calcium-binding mitochondrial carrier S -1), stress response including oxidation-reduction processes (e.g., XLOC_017856: heat shock, XLOC_012518: epidermal retil dehydrogese 2 and XLOC_018492: iron-dependent peroxidase) and other functions such as reproduction, or development (e.g., XLOC_015776: egg CP391S, XLOC_007823: Craniofacial development 2) (Sheet G in S1 File). Similarly, in S. bovis females, we found differentially expressed genes that correspond to genetic mobile elements as well (e.g., XLOC_017328: R-directed D polymerase from transposon X-element, XLOC_018050: R-directed D polymerase from mobile element jockey-like or XLOC_018156: gag-pol poly), genes involved in ion transport (e.g., XLOC_008268: Bile salt export pump, XLOC_003851: sodium-coupled neutral amino acid transporter 9 isoform X2 and XLOC_005754: Y+L amino acid transporter), response to stress (e.g., XLOC_017856: heat shock and XLOC_009339: Universal stress) as well as other functions such as reproduction, growth or metabolism (e.g., XLOC_014939: early growth response, XLOC_015393: Syptotagmin-1, XLOC_016728: egg CP391S-like and XLOC_012591: Cathepsin B-like cysteine proteinase precursor) (Sheet G in S1 File). Hence, for S. haematobium and S. bovis females, DEGs were quite similar in term of function, regardless of their expression profile (under- or over-expression in hetero-specific pairs) and regardless of the schistosome species.

Discussion

In this study we aimed to investigate potential reproductive isolation mechanisms between two major African schistosome species that cause major debilitating parasitic disease and show evidence of extensive hybridization in nature [37,69]. Specifically, we tested whether hybridization between S. haematobium and S. bovis could be constrained or promoted by mate choices and whether these mate choices were associated with specific transcriptomic profiles in hetero- and homo-specifically paired individuals. Overall, the data shows that S. haematobium and S. bovis mate in a random fashion and depend only on the presence and the relative abundance of each species in the definitive host. Likewise, we did not detect any major transcriptomic changes associated with hetero-specific pairing in male and female S. haematobium and S. bovis.

First, we showed that the two frequently co-endemic sister species S. haematobium and S. bovis readily pair with no preferences for neither homo-specific nor hetero-specific associations in simultaneous infections. The only exception was found for male S. haematobium mate choice. Indeed, we found a significantly higher number of hetero-specific pairs compared to that expected under the assumption of random mating. However, as we cannot differentiate mate recognition initiated by males from female competition, our results suggest that either male S. haematobium prefer mating with female S. bovis or alternatively, that female S. bovis may be more competitive than female S. haematobium. Interestingly, although female competition is possible, it is assumed that male schistosomes are the competitive sex and in particular male S. haematobium are usually better at pairing when compared to males from other species including S. intercalatum (now S. guineensis) [35], S. mattheei [29] or S. mansoni [28]. However, since the bias toward hetero-specific pairing was observed in a unique hamster, this result should be considered with caution. Indeed, this bias was not retrieved in our full mate choice experiments and future studies are warranted to confirm if this observation is repeatable as it may have important epidemiological consequences regarding pairing directionality and hybrid representation in the field. Similarly, premature death of some hamsters in the experiment focusing on the mate choice of male S. bovis limited our ability to draw specific conclusions. Consequently, our mate choice experiments overall rather indicate no differences in species mate choice or competitiveness and that S. haematobium and S. bovis males and females mate randomly. Such a result is in line with Webster and colleagues [27]. Altogether, this highlights that there are no behavioural barriers preventing hetero-specific pairing once both species encounter each other in the same definitive host.

The second part of this study aimed to assess the transcriptomic profiles associated with hetero-specific pairings between S haematobum and S. bovis. Since different species might constitute a different stimulus for the other partner, we expected at first to find an impact of the hetero-specific pairing, and especially on female transcriptomes compared to male transcriptomes since they respond to male stimuli for their sexual maturation [20]. However, only few DEGs were observed in both males and females. Biological processes enriched in DEGs were identified only for male S. haematobium pairings. Likewise, most of the genes detected presented low Log2-Fold changes (notably in S. haematobium males where only one DEG exceeded a Log2-Fold change of 1.5). Thus, the influence of hetero-specific pairing on male and female adult worms of both species in terms of numbers of DEGs, related biological processes and gene expression level was not striking. Such results suggest that both species may be highly receptive to each other since no major transcriptomic adjustments are induced by hetero-specific pairings. This observation is hence consistent with our previous mating experiments that suggest random pairing between both species and further show that there are no major physiological nor molecular barriers making hetero-specific pairings and thus hybridization less prone to occur.

Although hetero-specific pairings did not result in many DEGs, it is worth noting that most of the DEGs were found in the comparison between homo- and hetero-specifically paired male S. haematobium. So far transcriptomic studies on Schistosoma pairing tended to show large molecular reprograming of female genes rather than male genes, in part due to the initiation of their sexual maturation [13,47,48]. The biological explanations for our results are thus not straightforward. First, we cannot rule out the possibility of an artefact induced by extrinsic factors or other technical issues such as a lower variability in the transcriptomic profiles of the different biological replicates of male S. haematobium in comparison to other samples. However, our results also show that male S. haematobium displayed more DEGs than females but DEG identified in females presented overall higher log2 Fold Changes. Hence, another hypothesis could be that females may differentially express fewer genes, but at higher levels. Finally, we could also hypothesize that the molecular plasticity in expression of genes is a mechanism by which male S. haematobium manage to be more competitive (compared to females from both species and S. bovis males) in hetero-specific pairing, for instance by properly initiating female maturation depending on their species. This latter hypothesis is particularly appealing since male S. haematobium are thought to be dominant over several other Schistosoma species [28,29,35]. This is also congruent with the potential bias toward hetero-specific pairing of male S. haematobium found in our mating experiments and also with field studies that show that the majority of the hybrids in the field appear to be a result of a cross between male S. haematobium and female S. bovis [37,70]. Nevertheless, since we did not identify any DEG in S. bovis males, and also because the log2-Fold change of the DEG identified in S. haematobium males were low, it seems difficult to conclude that one or the other sex is preferentially impacted during hetero-specific pairing, or that one species is more prone to initiate the sexual maturation of females. However, we are confident that the small number of DEGs identified when comparing homo- and hetero-specific parings together with their low log2 Fold Change reflect the relatedness between S. bovis and S. haematobium that undergo only few transcriptomic adjustments following hetero-specific pairing. Moreover, the molecular changes that we identified here at the very first step in the hybridization process may reveal some important genes linked to male and female interactions, species isolation and hybridization.

Indeed, some of the DEGs identified in our work show functions that can be linked to sexual interactions, notably to reproductive functions suggested by other studies. Notably, among female schistosomes we found three genes encoding egg proteins that were differentially expressed in S. haematobium and/or S. bovis females, and that are well-known female-associated gene products [71]. Similarly, a transcript matching the Syptotagmin-1 gene was under-expressed in hetero-specifically paired female S. bovis. This gene was previously shown to have a female-specific expression and to be regulated during pairing [47]. Moreover, two DEGs that encode digestive enzymes, specifically expressed by paired females (i.e., cathepsin B and L) were found in female S. bovis [71]. Similarly, in S. haematobium DEGs were related to biological processes known to be involved in male-female interactions. Previous studies looking at the molecular basis of Schistosoma male-female interaction, with a particular interest in the pairing process, proliferation, differentiation and maturation of female gonads, have underlined the major role of signal transduction cascades and particularly signalling pathways such as the TGF-beta and Ras (e.g., receptor tyrosine kinase coupled pathway) signalling pathways [72-78]. These pathways, notably the TGF-beta signaling pathway are known to induce the production of the gynecophoric canal protein by males during pairing which is a trigger for maturation of females [73]. Interestingly, in this work, among genes whose expression was affected by homo- and hetero-specific pairing in male S. haematobium, we notably found the TGF-beta signal transducer gene, and two gynecophoral canal protein genes. Moreover, both transmembrane receptor protein tyrosine kinase signaling pathway and regulation of Ras protein signal transduction processes were enriched in over-expressed genes in hetero-specific pairs. Also echoing more recent studies on the gonad-specific and pairing-dependent transcriptomes of male schistosomes, we found several biological processes enriched either in over- or under-expressed genes in S. haematobium males that were involved in neuronal processes which are associated with male-female interaction patterns [13,48]. We consequently found that genes and processes impacted between homo- and hetero-specific pairing in S. haematobium and S. bovis at least partly overlapped those generally affected in other male-female interaction studies. These results suggest that both species may have maintained similar patterns of interactions between males and females allowing them to reproduce. A moderate regulation of these genes during pairing with another species may thus allow the two parasite species to overcome their divergence resulting in successful hetero-specific mating. Finally, it is worth noting that among the DEGs identified, the majority of them were also related to processes that were not particularly documented to be impacted during male-female interactions (e.g., genetic mobile elements, response to drug and stimuli, oxidation-reduction). Several DEGs were related to stress response and stimuli responses (e.g., oxidation-reduction processes as well as the genetic mobile elements [79,80]), indicating that at least at the molecular level schistosome species may perceive hetero-specific pairing as a stress, although this does not seem to impede hetero-specific pairing. Alternatively, the pairing status (i.e., homo-specific or hetero-specific) could impact the worms’ responses to external stimuli including host and/or environmental stimuli. In particular hetero-specific male S. haematobium under-expressed a fair amount of genes involved in response to drugs compared to homo-specific ones (e.g., Multidrug and toxin extrusion, Multidrug and toxin extrusion 2, Multidrug resistance or Multidrug resistance-associated). These observations may raise important questions regarding schistosomes’ drug response in the context of co-infection and hybridization especially since a lower sensitivity to PZQ of S. bovis x S. haematobium hybrids compared to pure S. haematobium parasites has been proposed to be at the origin of the spread of the hybrid form in Senegal [27]. However, it is important to pinpoint that any changes associated to PZQ response in hybrids is still theoretical and there is not current evidence that there is any difference in drug response in natural infections. Here we found a differential expression of genes involved in response to drugs in male S. haematobium only, which call for future clarification to assess if this is a peculiarity of our study and/or of male S. haematobium. More generally, several other genes identified in this work may be of potential significance for the encounter, interaction, and communication between these two species. Further attention is thus required to decipher the role of each of them in the context of hybridization or at the contrary in the context of speciation.

Altogether, the integrative assessment of lack of pre-zygotic reproductive mechanisms we present here may have profound implications regarding what we could expect in term of hybridization dynamics in the field. In particular it suggests that both species have retained similar processes allowing them to find their partner in the host, pair and produce viable offspring. This result is in line with several recent studies that have presented evidences of introgression between S. haematobium and S. bovis and that suggest that their relatively recent divergence compared to other schistosomes and thus the genetic distance between both species is not sufficient to limit hybridization [39,40,81]. This relies in part in the fact that they have retained the same karyotype with n = 8 chromosome pairs, including sex chromosomes that are morphologically similar [82], hence allowing the species’ genomes to be highly permeable to each other’s alleles [83]. In that case, the most significant reproductive isolation mechanisms preserving the genetic integrity between these species would be habitat isolation, including geographical location and definitive host specificity. Also, it is worth noting however that the two species used in this study have been isolated from distinct geographical zones and this could contribute to explain the absence of pre-zygotic isolation. Indeed, sympatric African schistosome species are likely to respond differentially as sympatric species tend to have enhanced pre-zygotic isolation barriers [4]. Nevertheless, the lack of pre-zygotic barriers does imply that in areas where S. haematobium and S. bovis are sympatric and infect the same definitive hosts, hybrids and introgressed individuals should be more likely to be found. This may be particularly relevant for parasite species that are brought together by global changes (enhanced human migration for S. haematobium, and animal transhumance for S. bovis) and may have porous reproductive isolation mechanisms.

While our study opens new avenues regarding the understanding of the mechanisms allowing or preventing hybridization between schistosome species, it also calls for future experimental and field work to fully understand hybridization patterns observed in natura. First, our observation of random mating between the two species suggests that first-generation hybrids may be frequent in endemic areas. A recent study in Senegal found hybrids with mixed genetic profiles between parental species suggesting that they may be of early generation [45]. However, current genomic analyses of parasites recovered in the field indicate that introgression between S. haematobium and S. bovis is the result of an ancient event rather than an ongoing process [40,81,84]. This is also supported by the genetic differentiation between hybrids and parental species populations in Senegal and Niger [85,86]. Second, although a broader view of the hybridization dynamics is warranted by increasing the number of samples collected across the African continent, the current data suggests that at least in the field S. haematobium could be dominant over S. bovis and that hybridization patterns may differ between foci. Indeed, several studies report unidirectional introgression of S. bovis genes into S. haematobium [36,40] and a predominance for an initial cross between a male S. haematobium and a female S. bovis, (leading to the introgression of mitochondrial DNA of the latter in the genomic background of the former [26,37]). Such biases in the direction of the crosses and introgression patterns are frequent in the hybridization landscape. For instance while some species hybridize in both directions and over multiple generations (S. bovis and S. curassoni; [27,36]; S. mansoni and S. rodhaini; [69]), others may produce offspring with strong asymmetries in their fitness (S. haematobium and S. mattheei;[87], S. haematobium and S. intercalatum (now S. guineensis) [29]) and sometimes in the directionality of introgression (S. rodhaini and S. mansoni [28]). However, since our analysis of the pre-zygotic isolation mechanisms does not support any type of asymmetry in the direction of the crosses it is most likely that if any, post-zygotic barriers may be at the origin of such biased patterns in the field and also potentially the relatively rare encounter in early generation hybrids. Consequently, the genomic landscape of introgression and the transmission patterns of hybrids may not be uniform, are highly complex and potentially dynamic. In this context it would be necessary as a next step to assess the importance of post-zygotic isolation mechanisms in terms of snail compatibility, hybrid life history traits and potential heterosis, which are important biological features that may shape hybridization outcomes by potentially reducing or promoting inter-species interaction and admixture. This may have strong implications as hybridization in schistosomes is a major concern and since heterosis in offspring may increase the parasite virulence compared to their parental species [88,89]. Such changes in the parasites life history traits may have important outcomes in terms of epidemiological dynamics (hybrids may take over parental species range [90], but also threaten the transmission, control and ultimate elimination of schistosomiaisis). In this context, a better understanding of the consequences of hybridization in parasites is a necessary next step to anticipate its effect in terms of disease dynamics and spread.

In conclusion, in this integrative study of S. haematobium and S. bovis behavioural and physiological isolation mechanisms we showed that natural hybridization between S. haematobium and S. bovis lack strong pre-zygotic barriers apart from their host specificity. Our data suggest that no mate recognition system mitigates hybridization between these two species and that no major transcriptomic adjustments are associated with hetero-specific pairings. This highlights that the two species remain sufficiently coadapted to each other to allow an efficient reproduction once they are in contact. Besides the current evidence of ancient introgression and biases in hybrid profiles, this weak pre-zygotic isolation exemplified raises the risk that in the absence of other reproduction isolation mechanisms, hybridization between these two species may be common. This also implies that contact zones may need further consideration to assess if hybridization is ongoing. Finally, our results may also partly explain the high prevalence of these hybrids in the field. Because such inter-species interaction may increase the offspring’s virulence compared to parental species, one could expect to find increased prevalence and intensities of the disease in areas where hybridization occurs. Understanding the modifications in the parasite life history traits, including their zoonotic potential and epidemiological outcomes are warranted to control human and animal morbidity, reduce transmission and ultimately eliminate schistosomiasis.

Metrics of the RNA-sequencing reads processing

(XLSX)

Click here for additional data file.

Transcriptome analysis related information: Sheet A in S1 File: Table showing the full annotation of the reference transcriptome representative of S. haematobium and S. bovis assembled for this study. Sheet B in S1 File: Table of the transcript counts (i.e., HTseq) in each triplicate of each condition. Sheet C in S1 File: DESeq2 results for male S. haematobium. Sheet D in S1 File: DESeq2 results for female S. haematobium. Sheet E in S1 File: DESeq2 results for female S. bovis. Sheet F in S1 File: DESeq2 results for male S. bovis. Sheet G in S1 File: Annotations associated to each differentially expressed genes that have been identified. Sheet H in S1 File: Gene Ontologies enriched in differentially expressed genes in male S. haematobium.

(XLSX)

Click here for additional data file.

13 Oct 2020

Dear Mrs Mathieu-Bégné,

Thank you very much for submitting your manuscript "Pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: from mating interactions to differential gene expression" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

All three reviewers have commented on the level of English and grammar within this paper, that makes it hard to properly review this paper. I would recommend that in addition to all that scientific comments and specific language comments included that all authors really help rewrite this manuscript and if needed that you ask a native English speaker to help with the next version. I will send this out to reviewers again upon resubmission if the level of English has improved, but that really does need to be addressed.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Sincerely,

Poppy Lamberton

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

All three reviewers have commented on the level of English and grammar within this paper, that makes it hard to properly review this paper. I would recommend that in addition to all that scientific comments and specific language comments included that all authors really help rewrite this manuscript and if needed that you ask a native English speaker to help with the next version. I will send this out to reviewers again upon resubmission if the level of English has improved, but that really does need to be addressed.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

Yes

-Is the study design appropriate to address the stated objectives?

Yes but more discussion is needed on the limitations of the study design

-Is the population clearly described and appropriate for the hypothesis being tested?

More information is needed on the strains used and the details of the experiments

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

Yes

-Were correct statistical analysis used to support conclusions?

I would like this checked by a statistician

-Are there concerns about ethical or regulatory requirements being met?

No

Reviewer #2: - The overall objective is clear but the hypotheses could be more clearly formulated in the introduction. For example, somewhere in the paper it was stated that the authors expected the opposite outcome (more transcriptomic changes in females than in males), so this hypothesis could have been included at the end of the introduction, instead of the vague kind of conclusion in line 194-197. Additional testable hypotheses can be formulated.

- The study design is appropriate although some issues arise regarding statistical power. The results discussed in line 362 (premature death of two hamsters) does raise the question whether 5 replica’s (5 hamsters) is enough to make robust conclusions as in this particular case no reliable statistics can be done for S. bovis males’ choice.

- No concerns about ethical issues.

For more comments on the Methods section see General Comments.

Reviewer #3: Methods seem appropriate

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: -Does the analysis presented match the analysis plan?

Yes

-Are the results clearly and completely presented?

Improvement is needed for clarity of the results to allow interpretation

-Are the figures (Tables, Images) of sufficient quality for clarity?

More information and clarity is needed as detailed in the attached document

Reviewer #2: The analysis is sound and the results are well presented.

Table 2 is redundant I would say.

For more comments on the Results section see General Comments.

Reviewer #3: Analysis matches described goals of the work.

REsults are clearly presented.

Figures are sufficient quality

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: -Are the conclusions supported by the data presented?

Yes

-Are the limitations of analysis clearly described?

No

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

Ye

-Is public health relevance addressed?

Yes

Further comments on the discussion are available in the attached

Reviewer #2: As put in my General comments, the Discussion needs reworking and should be more substantiated. The conclusions are not always clear or strong, I miss more references to similar studies, but also a proper discussion on the limitations and recommendations for future research are missing. The public health relevance is not really thoroughly discussed.

Reviewer #3: Conclusions are supported by the data. They speculate quite a bit in the discussion about the potential importance of various DE genes. But this type of speculation is rampant in gene expression papers and they don't make any hard conclusions.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: The main modidications are needed in the clarity of the data and how it is presented. Comments are in the attached document.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This is an important and interesting study that shows the lack of pre zygotic isolation between S. haematobium and S. bovis supporting inter-species hyrbidisation an important scenario for human and animal health in Africa. Although, I consider the study of vlaue and there is a substantial body of high quality of work, which is not easy to do (particularly the generation of the isolates and the mating experiments) the paper needed considerable improvements before it can be reviewed further. The authors should consider that the readers will not be familiar with these types of experiments and there is a need to add the detail that allows the readers to understand the experimental procedures and the data produced. There are also english and gramma errors many of which can be improved with careful reading. In the attached these are highlighted in the text showing the errors and where changes are needed. I have tried to cover the whole document but due to time some places may have been missed and careful reading and revision is needed. There are also comments on the attached to highlight where more clarity is needed and also where further information or discuss is warranted. The discussion is also very long and could be condensed by not repeating what is in the results.

Reviewer #2: This is a very interesting study, with interesting results. Experimental infections in hamsters show that there are no pre-zygotic barriers to mating between the human Schistosoma haematobium and the animal S. bovis parasite, neither are there any major transcriptomic responses following hetero-specific pairings. Even though a previous study by Webster and colleagues already showed that S. bovis and S. haematobium readily paired in laboratory hamsters (to which the present authors not refer, which I think is an omission), this is the first time that any transcriptomic study is done on hybrid crosses.

I was frustrated by the sloppy grammar and spelling throughout the entire manuscript, which gave the impression that the authors were in a hurry to submit this manuscript or they didn’t really care about this.

I also lack a discussion on the asymmetry in the direction of hybridisation and introgression between schistosomes, which is sometimes even unidirectional, but nothing is mentioned on this. Also, it is repeatedly said that isolation ‘by the host’ is apparently the only barrier to hybridisation between these two schistosome species, but it is never specified which host they mean with that, the final or the intermediate one. Since mate choice and reproduction takes place in the final host, this one is of particular interest of course, but if the different intermediate hosts are not sympatric, then hybridisation will also be less frequent (if they final host does not move around too much). For S. bovis and S. haematobium this is more complicated, as intermediate host specificity of the latter varies with geography, but still this should be properly discussed as it could also explain why we see such regional differences in the distribution of hybrids. Then finally, I also have some problems with parts of the discussion: some questions remain unanswered (e.g. why would only S. haematobium males in heterospecific pairing have this transcriptomic response?), the statistical power, and with the fact that I miss the broader picture, the reference to all the previous work on schistosome mating experiments. So therefore I think that these concerns should be addressed first before it can be accepted for publication.

Abstract & author summary

- line 44: make two sentences out of this long sentence

- line 47: delete ‘allowing them to maintain…’ this is repetition from above

- line 48: what do you mean with misunderstood? Not understood? Or are there really mistakes and / or misconceptions out there in the literature?

- line 57: ‘by the host’: replace with ‘final host choice’ or something like that, because at the snail host level there is not always spatial isolation

- line 67-68: evolutionary biology?

- Line 68: replace ‘If’ by ‘While’

- Line 73: S. haematobium (species names always in full when first mentioning) and parasitize

- Line 70-74: sentence too long, split in two and rephrase ‘including out of endemic areas’

- Line 75: ‘…rather than having a homo-specific mate preference’

- Line 78: mechanisms

- Line 78: ‘but the one imposed by host specificity’? what do you mean? Please rephrase ‘except the one imposed by final host specificity’

- Line 81: ‘encounter each other’

Especially the author summary does not read very fluently, and there are quite some grammar mistakes.

Introduction

- line 88= mechanism

- line 90: this sounds more like mate choice rather than behavioural isolation

- line 91: individuals

- line 91: I would replace copulation by reproduction

- line 93: encounter

- line 96: less fertile

- line 96: hybrid lines

- line 98: making it difficult to predict

- line 104: terms

- line 105 and others: free-living

- line 108: compared to those of free-living…

- line 109: hostile rather than inimical?

- Line 112-113: rephrase this sentence, it is not really shaped through the host alone, it is shaped through the host – parasite interactions, and you first call this a strong isolation mechanism and a few words later you say ‘potentially’ preventing hybridization… the sounds less convinced. Also, you write ‘its hosts’, so plural, I would make the distinction already here between intermediate and final hosts, because in case of schistosomes sexual reproduction only takes place in the final host, so host choice at this level is more important than at the other level. You should discuss it at least somewhere, because in the abstract you only talk about ‘host’ choice.

- Line 113: you mean ‘closely related’ species? Because all species are related somehow…

- Line 116: plasmodium species (or you provide the genus names for the other two parasites you add here)

- Line 123: rephrase ‘schistosomiasis debilitating diseases’, this is not an official term (also, the disease is of great concern, rather than the parasites themselves I would say)

- Line 128: rephrase ‘among other trematodes’, you want to say here that they are an exception within the Trematoda

- Line 133: ‘this can lead to hybridisation’

- Line 136: influence rather than interest

- Line 136: First,

- Line 138: same host individual and schistosome species

- Line 144: female’s

- Line 145: of a sexually …

- Line 145: what does not depend upon species-specific pairing? Discuss this in a separate sentence in order to avoid too long sentences

- Line 147: stimulate

- Line 149: male worms’ physiology or the physiology of male worms

- Line 153: groups instead of clades?

- Line 155: schistosome

- Line 157: others, you mean other combinations?

- Line 158: S. mattheei

- Line 156-159: in all these cases it should be mentioned that the viability of these crosses depend on the type of crosses, which parental species provides the male and which the female in the hybrid cross. Also, the way you write this you suggest that the first group of species can readily pair because there is less divergence between them (because this is what you write in the preceding sentence), while others are more selective because they are more divergent… but the divergence between S. haematobium and S. intercalatum is similar to the divergence between S. haematobium and S. mattheei. Also how can a combination be more selective or readily pair? It is the species that forms this combination that can be selective I would say. The grammar is quite sloppy in many cases, please take care of this.

- Line 174: non-human; also Cetartiodactyla is a superorder, and a superorder or a genus cannot be infected by parasites, but their members can

- Line 175: ruminants

- Line 177: rodents

- Line 191: outperforms the fitness of parental species

- Line 194: the sentence starting with ‘Relying on such an integrative…’ is redundant as it is mainly repetition

Material and methods

- Line 214: in or with Bulinus

- Line 219: B. truncatus

- Line 223: were performed

- Line 242: versus

- Line 244: each worm and its

- Figure 1: I am a bit confused why you use the same color red for S. haematobium and S. bovis females?

- Table 1: line 248: ‘and’ should not be in italic. Line 250: the number of male and female S. haematobium and S. bovis worms

- Line 254: rephrase this sentence, grammatically incorrect

- Table 2: I think this table can be left out as everything is already explained in the text

- Line 278-279: parasite species names not in italic since the title is in italic

- Line 289: rephrase: as the volume of each reagent was halved

- Line 296: library construction

- Line 302: vs in full and not italic

- Line 307: all sample reads

- Line 318: each newly assembled gene or all newly assembled genes

- Line 319: were extracted from the S. haematobium

- Line 321: transcript

- Line 324: hetero-specific paired worms (or elsewhere you write hetero-specifically paired worms)

- Line 327: gene expression

- Line 338: sets of genes

Results

- line 349: experiment

- line 353: partners

- line 362: these are the risks of experimental research of course, and this cannot be avoided, but it does raise the question whether 5 replica’s (5 hamsters) is enough to make robust conclusions as in this particular case no reliable statistics can be done for S. bovis males’ choice

- line 367: find instead of found

- Table 3, line 376: remaining single or that remained single

- Line 379: elsewhere you write P-value

- Line 380: experiments

- line 385: male and female S. haematobium

- line 412: providing from?

- Line 426: does this 7% means 7% of all schistosome genes? Please specify this

- line 427: vs. in full as elsewhere

- line 462: GO terms

- line 463: male S. haematobium

- line 468: biological

- line 489: and not in italic

- line 500: correspond (in present tense)

- line 503: involved in ion

- line 506: functions

Discussion

- line 513: reproductive isolation mechanisms?

- Line 521: male and female

- Line 523: definitive host. This sentence is actually a final conclusion before starting the Discussion itself

- Line 532: S. mattheei

- Line 534: so this calls for more replica’s in future experiments to verify this possibility!

- Line 539: there are no barriers or there is no barrier

- Line 557: previous instead of precedent

- Line 558: there are no

- Line 560: what do you mean with ‘male and female S. haematobium and S. bovis’? Between male S. haematobium and female S. bovis?

- Line 561: male S. haematobium

- Line 571: display a more

- Line 576: ‘females species sexual maturation’?

- Line 587: female S. bovis

- Line 602: gonad-specific

- Line 579 – 612: this lengthy discussion is confusing and less convincing because right before this discussion you conclude that only few transcriptomic adjustments are associated with hetero-specific pairing and that the log2-Fold change in males were low, and that it seems difficult to conclude that one or the other sex is preferentially impacted, suggesting that your results are not so convincing. This is not so motivating for the reader then to follow this subsequent discussion

- Line 627-628: these observations could have indeed important consequences with respect to drug treatment, but again, how serious do we have to take this, and why would only S. haematobium males in heterospecific pairing have this response, and not S. bovis males?

- Line 632: avenues

- Line 634: but these report unidirectional introgression of a few bovis genes into the haematobium genome, so a dominance of haematobium genomic DNA, how can you reconcile or link this with your results? Wouldn’t you expect more differences between the different crosses?

It has been proven in co-infection experiments that crosses are not always reciprocal, i.e. one cross performing better than the reverse cross. This could also be expected in these crosses as in Senegal many hybrids that were found appeared to have arisen of a female S. bovis x male S. haematobium pairing (although backcrossing in nature obscures these patterns, and in other areas, like Niger, the reverse crosses are more frequently found). I do not see any reference to this or to the topic of unidirectional hybridisation and unidirectional introgression, which I think is missing.

- line 648: I don’t completely agree, because the different ‘compartments’ (strange term) do not prevent them from meeting each other in the liver, where mating takes place, only after that stage the couple moves through the hepatic portal vein to the egg-laying site. So there is plenty of opportunity in the liver, irrespective of the difference in tropism

- line 653: how can these parasites be co-occurring (I would use the word sympatric) of their hosts are not? Please adapt

- line 657: rephrase ‘parental species individuals’

- line 655-659: what do the authors want to say here exactly? Does ‘such hybrids’ refer to those hybrids resulting from an ancient hybridisation event? And do only ‘those hybrids’ present heterosis, in contrast to ‘other, more recent hybrids’? Please rephrase. Also, how do your results explain the fact that previous studies show that mainly ‘ancient hybrids’ are found in nature (Platt et al, 2019), while your experiments show that hybridisation is so ‘easy’? In line 668 you write that your result ‘echoes recent evidence of introgression’. I am not sure what you mean with this. Do you mean ‘evidence of recent introgression’ or ‘recent evidence of (ancient) introgression’? I would say the opposite, the high prevalence of hybrids is an echo or a reflection of the weak pre-zygotic barrier that you observed. But still, I don’t see how your results can be matched to the outcome of Platt et all, suggestion ancient hybridisation.

- Line 669: a higher

- Line 670: if the hybrids always have higher fitness, why do you still see ‘pure S. haematobium’ and ‘pure’ S. bovis in places where they overlap? Wouldn’t the hybrids take over?

- Line 672: ‘new issues in the disease control’ and ‘alteration in the efficacy’ sound rather vague as a closing sentence, please be more specific.

Reviewer #3: Summary

The goal of this work appeared to be to test for evidence of species-specific mate choice between S. haemotobium and S. bovis, and also to see whether there are differentially expressed genes in adults engaged in homo vs. hetero-specific pairings. The found minimal evidence for mate choice and few strongly DE genes. In the discussion they speculate on possible roles of the few DE genes, but make no strong conclusions about any of them.

Comments

The authors did a mate choice experiment using cercariae of known sex in hamsters. They found no strong evidence that species-specific mate choice occurs by males or females of either species. The statistical analysis of the mate choice experiments seemed appropriate to me.

If they have the data, it would be interesting if the authors could comment on the physical location of the hetero vs homo-specific pairings within the hamster (urogenital vs. mesenteric). I wondered whether different behavior might be observed if the definitive host was a larger mammal such as a bovine or human. Perhaps the authors would care to speculate or at least mention the possibility.

The authors also looked for evidence of differential gene expression in individuals of each sex and species when engaged in hetero vs. homo-specific pairings. They observed only a few dozen DE genes in females of either species. Oddly, they found zero DE genes in male S. bovis, but over 1000 in male S. haemotobium (although I wonder if figure 2a suggests the difference in S. haemotobium might be driven by one individual).

The almost complete lack of even minimally DE genes in S. bovis males shown in figure 3d is puzzling. I have never seen a volcano plot like this one. I would have expected more by chance alone with only n = 3 per treatment. Analysis of gene expression data is not my expertise, so I defer to other reviewers to comment on this. Or perhaps the authors could head off puzzled readers by explaining why this pattern obtains.

Minor comments:

The manuscript could use some editing to fix various small grammatical errors and instances of odd English usage. Perhaps asking a native English speaker to read it through once for them would be helpful.

It would help Figure 3 if the authors would label, on the figure, which combination of sex and species is represented by each panel. Going back and forth between the legend and the figure is tedious for the reader.

Figure 4. Is there some measure of statistical significance associated with the difference between blue and red bars that could be indicated on the figure?

Supplementary table S1 would be helped by species identifications.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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29 Dec 2020

Submitted filename: 1_Letter_responses to reviewer comments_def.docx

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2 Feb 2021

Dear Mrs Mathieu-Bégné,

Thank you very much for submitting your manuscript "No pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: from mating interactions to differential gene expression" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic and we all agree that the manuscript is now greatly improved. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations. I look forward to seeing an updated paper resubmitted very soon.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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Poppy Lamberton

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The manuscript is now greatly improved and I look forward to seeing these minor changes suggested by two of the reviewers amended and an updated paper resubmitted very soon.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The methods are now clear and easy to follow

Reviewer #2: yes

Reviewer #3: Acceptable

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results are clearly presented.

Reviewer #2: yes

Reviewer #3: Acceptable

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The discussion supported the data presented and is informative. The limitations are clearly presented and there relevance to public health is addressed.

Reviewer #2: yes

Reviewer #3: Acceptable

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: See general comments

Reviewer #2: The authors did a good job in addressing all the comments of the referees, they adapted the text and figures/tables where needed or suggested, and this resulted in a much-improved version of the manuscript. I think this version can be accepted for publication.

A few minor comments:

Line 43-44: before reproduction is also ‘during the life cycle’, so why not simply saying ‘These preventive barriers can act before reproduction, “pre-zygotic barriers”, or after reproduction/fertilisation, “post-zygotic barriers”?

Line 561; draw

Line 569: male transcriptomes

Line 640; to overcome their divergence only I would say, not to overcome their relatedness

Line 654: a lower sensitivity to PZQ of the hybrid being at the origin of the spread was not at all proposed by Huyse et al., 2009 so please adapt.

Line 708: S. mansoni [28]).

Line 711; I think the biased patterns in the field might indeed by due to post-zygotic isolation but not the rare encounter of first generation hybrids (or not only because of that). The latter is probably (also) due geographical variation in the level of sympatry between parental species, no?

Reviewer #3: They adequately addressed the issues of language usage.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The paper is much improved, and the authors have taken the time and effort to amend the manuscript according to the reviewer’s comments. This is a highly informative paper that adds to our understand on the interactions between S. haematobium and S. bovis. It will also open several research avenues for future work.

I have the following minor comments.

Gramma punctuation is needed in several places so a good go over will help

Some further modifications of the English is needed. Areas that I managed to pick up are highlighted.

Line 120 – there are 23 species not 25

Line 121 – to be precise 20 infect animals if you include S. mansoni that is found in rodents and monkeys.

Line 188-189 – make it clear that the heterosis has been observed in experimental infections not in the natural setting.

Line 220-222- the animal license information should be checked with the editors regarding how it should be written.

Line 229 – what do you mean by sympatric B. truncatus – do you mean local natural hosts?

Line 250 – add “below” to the end of the sentence.

Figure 2 – S. h = S. bovis needs correcting. Also, the symbols are both in the figure and legend. Only one is needed.

Table 1: in Exp. 2 and 4 you say that this is a female choice experiment. Is this not more competition of the two male species?

Line 290 – make it clear that this section is moving onto the molecular work and not the analysis of pairings.

Line 296 – make it clear how the worms were separated. It is important here that there is not contamination between males and females, so the detail is needed.

Line 327 – “effect” not needed

Line 328 – reference the Galaxy instance. E.g., is it a software or a machine?

Table 2 column 6 and 7 remove the words or change homo specific or hetero specific as they are not paired. Thy can just be called unpaired worms. It would also be better to have the exp. row above the data rather and below.

For figure 4 is it important to distinguish between hetero and homo paired worms ?

Line 481 – show what GO means,

Line 553 – is there any evidence for female selecting their partners. They are very underdeveloped until they are paired?

Line 555 – this S. intercalatum strain is actually now named S. guineensis – maybe add a note on this and also where it is referenced in the intro.

Line 565 – reference the study Webster et al., 2012 that also suggested that there was no mate choice

Line 599 - suggest you add here that the majority of the hybrids found in the field appear to be a result of a cross between S. h male and S. b female based on cox1 and ITS sequencing. It is better to say this than state that they are as more work is needed to clarify that.

Line 655 – I think it is important to point out that any changes in PZQ response by hybrids is very theoretical and there is not current evidence that there is any difference in drug response in natural infections.

Line 707- check if the S. intercalatum should be called S. guineensis. S. intercalatum is the Zaire/DRC strain. Cameroon and other areas is S. guineensis.

Reviewer #2: (No Response)

Reviewer #3: I believe the authors have adequately addressed my comments on their original submission. The main points of the paper, that there is random mating and few differentially expressed genes in interspecies pairings, are sufficiently supported and worth putting into the literature

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Reviewer #1: Yes: Dr Bonnie Webster

Reviewer #2: No

Reviewer #3: No

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30 Mar 2021

Submitted filename: response reviewers.docx

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6 Apr 2021

Dear Mrs Mathieu-Bégné,

We are pleased to inform you that your manuscript 'No pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: from mating interactions to differential gene expression' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Deputy Editor

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Poppy Lamberton

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PLOS Neglected Tropical Diseases

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There is some minor type editing required, such as:

Line 160: remove full stop and close bracket.

Line 231: remove space at start

Line 233: I think you can revert to sympatric here, I think it is clear, but maybe if clarification is needed write 'and sympatric B. truncatus molluscs, bred from snails collected from the same location as the parasites, were individually .....' and please calrify here also, or if these were collected straight from the field and exposed, then remove the 'bred from'

Line 376: Please add 'worms of the limiting sex (i.e., choosing partners, such as female choice or male competition) and please include this in the table legend as well to explain it when it is first used.

Several references need the Latin names in italics and the titles having the full capitalisation removed

29 Apr 2021

Dear Mrs Mathieu-Bégné,

We are delighted to inform you that your manuscript, "No pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: from mating interactions to differential gene expression," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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