# Morphological and genomic characterisation of the Schistosoma hybrid infecting humans in Europe reveals admixture between Schistosoma haematobium and Schistosoma bovis

Julien Kincaid-Smith, Alan Tracey, Ronaldo de Carvalho Augusto, Ingo Bulla, Nancy Holroyd, Anne Rognon, Olivier REY, Cristian Chaparro, Ana Oleaga, Santiago Mas-Coma, et al.

► To cite this version:

Julien Kincaid-Smith, Alan Tracey, Ronaldo de Carvalho Augusto, Ingo Bulla, Nancy Holroyd, et al. Morphological and genomic characterisation of the Schistosoma hybrid infecting humans in Europe reveals admixture between Schistosoma haematobium and Schistosoma bovis. PLoS Neglected Tropical Diseases, 2021, 15 (12), pp.e0010062. 10.1371/journal.pntd.0010062. hal-03508340

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# Morphological and genomic characterisation of the Schistosoma hybrid infecting humans in Europe reveals admixture between Schistosoma haematobium and Schistosoma bovis

Julien Kincaid-Smith, Alan Tracey, Ronaldo de Carvalho Augusto, Ingo Bulla, Nancy Holroyd, Anne Rognon, Olivier REY, Cristian Chaparro, Ana Oleaga, Santiago Mas-Coma, et al.

► To cite this version:

Julien Kincaid-Smith, Alan Tracey, Ronaldo de Carvalho Augusto, Ingo Bulla, Nancy Holroyd, et al. Morphological and genomic characterisation of the Schistosoma hybrid infecting humans in Europe reveals admixture between Schistosoma haematobium and Schistosoma bovis. PLoS Neglected Tropical Diseases, 2021, 15 (12), pp.e0010062. 10.1371/journal.pntd.0010062. hal-03508340v2

HAL Id: hal-03508340

https://hal.science/hal-03508340v2

Submitted on 25 Apr 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH ARTICLE

# Morphological and genomic characterisation of the *Schistosoma* hybrid infecting humans in Europe reveals admixture between *Schistosoma haematobium* and *Schistosoma bovis*

<figure><img src="image_2.png" /></figure>

Julien Kincaid-Smith<sup>1,2,●</sup>, Alan Tracey<sup>3,●</sup>, Ronaldo de Carvalho Augusto<sup>1,†</sup>, Ingo Bulla<sup>1,4,§</sup>, Nancy Holroyd<sup>3</sup>, Anne Rognon<sup>1</sup>, Olivier Rey<sup>1</sup>, Cristian Chaparro<sup>1,†</sup>, Ana Oleaga<sup>6</sup>, Santiago Mas-Coma<sup>7</sup>, Jean-François Allienne<sup>1,†</sup>, Christoph Grunau<sup>1,†</sup>, Matthew Berriman<sup>3</sup>, Jérôme Boissier<sup>1</sup>, Eve Toulza<sup>1,†</sup>

1 IHPE, Univ Perpignan Via Domitia, CNRS, IFREMER, Univ Montpellier, Perpignan, France, 2 CBGP, IRD, CIRAD, INRAE, Institut Agro, Univ Montpellier, Montpellier, France, 3 Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom, 4 Institute for Mathematics and Informatics, University of Greifswald, Greifswald, Germany, 5 Department of Computer Science, ETH Zürich, Zürich, Switzerland, 6 Parasitology Laboratory, Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA, CSIC), Cordel de Merinas, Spain, 7 Departamento de Parasitologia, Facultad de Farmacia, Universidad de Valencia, Burjassot, Valencia, Spain

● These authors contributed equally to this work.

* kincaid-smith.julien@ird.fr

<figure><img src="image_3.png" /></figure>

OPEN ACCESS

**Citation:** Kincaid-Smith J, Tracey A, de Carvalho Augusto R, Bulla I, Holroyd N, Rognon A, et al. (2021) Morphological and genomic characterisation of the *Schistosoma* hybrid infecting humans in Europe reveals admixture between *Schistosoma haematobium* and *Schistosoma bovis*. PLoS Negl Trop Dis 15(12): e0010062. https://doi.org/10.1371/journal.pntd.0010062

**Editor:** Bonnie L. Webster, Natural History Museum, UNITED KINGDOM

**Received:** August 22, 2021

**Accepted:** December 6, 2021

**Published:** December 23, 2021

**Copyright:** © 2021 Kincaid-Smith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** The Illumina datasets for the Corsican hybrid strain are available in the SRA repository under submission number: PRJNA486714. The S. bovis PacBio data are available under accession number ERS2549235. (https://www.ncbi.nlm.nih.gov/sra/?term=ERS2549235) The *Schistosoma bovis* assembly SBOS_v1.1 is available at https://doi.org/10.5281/zenodo.5779218. Annotations are available as

## Abstract

Schistosomes cause schistosomiasis, the world's second most important parasitic disease after malaria in terms of public health and social-economic impacts. A peculiar feature of these dioecious parasites is their ability to produce viable and fertile hybrid offspring. Originally only present in the tropics, schistosomiasis is now also endemic in southern Europe. Based on the analysis of two genetic markers the European schistosomes had previously been identified as hybrids between the livestock- and the human-infective species *Schistosoma bovis* and *Schistosoma haematobium*, respectively. Here, using PacBio long-read sequencing technology we performed genome assembly improvement and annotation of *S. bovis*, one of the parental species for which no satisfactory genome assembly was available. We then describe the whole genome introgression levels of the hybrid schistosomes, their morphometric parameters (eggs and adult worms) and their compatibility with two European snail strains used as vectors (*Bulinus truncatus* and *Planorbarius metidjensis*). Schistosome-snail compatibility is a key parameter for the parasites life cycle progression, and thus the capability of the parasite to establish in a given area. Our results show that this *Schistosoma* hybrid is strongly introgressed genetically, composed of 77% *S. haematobium* and 23% *S. bovis* origin. This genomic admixture suggests an ancient hybridization event and subsequent backcrosses with the human-specific species, *S. haematobium*, before its introduction in Corsica. We also show that egg morphology (commonly used as a species diagnostic) does not allow for accurate hybrid identification while genetic tests do.
<figure><img src="image_2.png" /></figure>

RESEARCH ARTICLE

# Morphological and genomic characterisation of the *Schistosoma* hybrid infecting humans in Europe reveals admixture between *Schistosoma haematobium* and *Schistosoma bovis*

Julien Kincaid-Smith<sup>1,2,*</sup>, Alan Tracey<sup>3,*</sup>, Ronaldo de Carvalho Augusto<sup>1</sup>, Ingo Bulla<sup>1,4,5</sup>, Nancy Holroyd<sup>3</sup>, Anne Rognon<sup>1</sup>, Olivier Rey<sup>1</sup>, Cristian Chaparro<sup>1</sup>, Ana Oleaga<sup>1,6</sup>, Santiago Mas-Coma<sup>7</sup>, Jean-François Allienne<sup>1</sup>, Christoph Grunau<sup>1</sup>, Matthew Berriman<sup>3</sup>, Jérôme Boissier<sup>1</sup>, Eve Toulza<sup>1</sup>

1 IHPE, Univ Perpignan Via Domitia, CNRS, IFREMER, Univ Montpellier, Perpignan, France, 2 CBGP, IRD, CIRAD, INRAE, Institut Agro, Univ Montpellier, Montpellier, France, 3 Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom, 4 Institute for Mathematics and Informatics, University of Greifswald, Greifswald, Germany, 5 Department of Computer Science, ETH Zürich, Zürich, Switzerland, 6 Parasitology Laboratory, Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA, CSIC), Cordel de Merinas, Spain, 7 Departamento de Parasitologia, Facultad de Farmacia, Universidad de Valencia, Burjassot, Valencia, Spain

* These authors contributed equally to this work.

* kincaid-smith.julien@ird.fr

<figure><img src="image_3.png" /></figure>

OPEN ACCESS

**Citation:** Kincaid-Smith J, Tracey A, de Carvalho Augusto R, Bulla I, Holroyd N, Rognon A, et al. (2021) Morphological and genomic characterisation of the *Schistosoma* hybrid infecting humans in Europe reveals admixture between *Schistosoma haematobium* and *Schistosoma bovis*. PLoS Negl Trop Dis 15(12): e0010062. https://doi.org/10.1371/journal.pntd.0010062

**Editor:** Bonnie L. Webster, Natural History Museum, UNITED KINGDOM

**Received:** August 22, 2021

**Accepted:** December 6, 2021

**Published:** December 23, 2021

**Copyright:** © 2021 Kincaid-Smith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** The Illumina datasets for the Corsican hybrid strain are available in the SRA repository under submission number: PRJNA486714. The S. bovis PacBio data are available under accession number ERS2549235. (https://www.ncbi.nlm.nih.gov/sra/?term=ERS2549235) The *Schistosoma bovis* assembly SBOS_v1.1 is available at https://doi.org/10.5281/zenodo.5779218. Annotations are available as

## Abstract

Schistosomes cause schistosomiasis, the world's second most important parasitic disease after malaria in terms of public health and social-economic impacts. A peculiar feature of these dioecious parasites is their ability to produce viable and fertile hybrid offspring. Originally only present in the tropics, schistosomiasis is now also endemic in southern Europe. Based on the analysis of two genetic markers the European schistosomes had previously been identified as hybrids between the livestock- and the human-infective species *Schistosoma bovis* and *Schistosoma haematobium*, respectively. Here, using PacBio long-read sequencing technology we performed genome assembly improvement and annotation of *S. bovis*, one of the parental species for which no satisfactory genome assembly was available. We then describe the whole genome introgression levels of the hybrid schistosomes, their morphometric parameters (eggs and adult worms) and their compatibility with two European snail strains used as vectors (*Bulinus truncatus* and *Planorbarius metidjensis*). Schistosome-snail compatibility is a key parameter for the parasites life cycle progression, and thus the capability of the parasite to establish in a given area. Our results show that this *Schistosoma* hybrid is strongly introgressed genetically, composed of 77% *S. haematobium* and 23% *S. bovis* origin. This genomic admixture suggests an ancient hybridization event and subsequent backcrosses with the human-specific species, *S. haematobium*, before its introduction in Corsica. We also show that egg morphology (commonly used as a species diagnostic) does not allow for accurate hybrid identification while genetic tests do.
trackhub at http://ihpe.univ-perp.fr/IHPE tracks/S.
bovi/hub.txt.

**Funding:** CG and RA were supported by the Wellcome Trust [grant number 107475/Z/15/Z], and Deutsche Forschungsgemeinschaft Fellowship BU 2685/5-1. MB was supported by the Wellcome Trust [grant number 098051]. SMC was funded by Health Research Project No. PI16/00520, Plan Estatal de Investigación Científica y Técnica y de Innovación, ISCIII-MINECO, Madrid, Spain; Red de Investigación de Centros de Enfermedades Tropicales - RICET (Project No. RD16/0027/0023 of the PN de I+D+, ISCIII-RETICS), Ministry of Health and Consumption, Madrid, Spain. JB, ET, OR were funded by the French National Research Agency ANR project HySWARM ANR-18-CE35-0001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing interests:** The authors have declared that no competing interests exist.

## Author summary

In 2013, schistosomiasis reached Southern Europe. Since then, endemic infections were recurrently identified clearly indicating that the parasite has settled and established locally. Using two molecular markers, we had previously demonstrated that the parasite was a hybrid between *Schistosoma haematobium* and *Schistosoma bovis*, two species known to infect humans and livestock, respectively. Nevertheless, this method has very low resolution and is not informative for determining the origins and the mechanisms of hybridization events, e.g. if the hybrid had been recently generated in Corsica or long before it's introduction. The genome-wide sequencing approach, used in this study, allowed us to reveal genomic admixture suggesting that these hybrids are the result of ancient crossing events between *S. haematobium* and *S. bovis*, with subsequent backcrossing with *S. haematobium*. This introgression has prevented compatibility with *P. metidjensis* snail vectors, which are specific to *S. bovis*. Additionally, whether in Africa or in Europe, a clear discrepancy exists between the egg shape usually used for species identification and the genomic composition of schistosomes. Therefore, egg shape cannot be used as a good indicator for hybrid detection. Knowing the phenotypic traits and the genetic features of such hybrids may have important implications in terms of diagnostics and disease management either through vector control strategies or treatment of patients.

## Introduction

Schistosomes are dioecious parasitic flatworms, responsible for the major Neglected Tropical Disease (NTD) schistosomiasis. The epidemiological statistics associated with this disease are sobering: 800 million people are at risk in 78 countries, mostly concentrated in sub-Saharan Africa; 230 million are infected and the disease causes more than 200,000 deaths each year as well as between 1.7 and 4.5 million Disability Adjusted Life Years (DALYs) [1]. The most exposed groups are children and young adults who have activities linked to contaminated freshwater environments. In addition to humans, schistosomiasis severely impacts livestock in Africa and Asia with over 165 million animals estimated to be infected [2].

The parasites have a complex life cycle that includes passage through specific freshwater snail intermediate hosts (hereafter termed vectors) in which the parasites undergo clonal multiplication, and a final vertebrate definitive host in which, adult worms sexually reproduce. The liberation of *Schistosoma* eggs into the host tissues is the principal cause of chronic and acute morbidity [3].

Global changes, including both anthropogenic and environmental modifications, may contribute to modifications in the geographical distribution of species and expand their potential ecological niches [4,5]. Distinct species may thus acquire a new capacity to interact, hybridize and subsequently introgress their genomes by backcrossing with parental species or other hybrids, a phenomenon called “hybrid swarm” [6]. Hybridization between individuals from two previously reproductively isolated species is generally expected to result in the production of offspring less fit than the parents, sometimes non-viable or sterile. In some cases however, hybridization may lead to viable progeny that can even have a greater fitness than parental species, a genetic effect known as hybrid vigour or heterosis that is generally observed during early generations [7]. These advantageous combinations of parental genes in offspring may enable progeny to adapt to new environments as potentially exemplified by the recent outbreak of schistosomiasis in Southern Europe (Corsica, France) [8].
trackhub at http://ihpe.univ-perp.fr/IHPE tracks/S.
bovi/hub.txt.

**Funding:** CG and RA were supported by the Wellcome Trust [grant number 107475/Z/15/Z], and Deutsche Forschungsgemeinschaft Fellowship BU 2685/5-1. MB was supported by the Wellcome Trust [grant number 098051]. SMC was funded by Health Research Project No. PI16/00520, Plan Estatal de Investigación Científica y Técnica y de Innovación, ISCIII-MINECO, Madrid, Spain; Red de Investigación de Centros de Enfermedades Tropicales - RICET (Project No. RD16/0027/0023 of the PN de I+D+I, ISCIII-RETICS), Ministry of Health and Consumption, Madrid, Spain. JB, ET, OR were funded by the French National Research Agency ANR project HySWARM ANR-18-CE35-0001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing interests:** The authors have declared
that no competing interests exist.

Author summary

In 2013, schistosomiasis reached Southern Europe. Since then, endemic infections were recurrently identified clearly indicating that the parasite has settled and established locally. Using two molecular markers, we had previously demonstrated that the parasite was a hybrid between *Schistosoma haematobium* and *Schistosoma bovis*, two species known to infect humans and livestock, respectively. Nevertheless, this method has very low resolution and is not informative for determining the origins and the mechanisms of hybridization events, e.g. if the hybrid had been recently generated in Corsica or long before it's introduction. The genome-wide sequencing approach, used in this study, allowed us to reveal genomic admixture suggesting that these hybrids are the result of ancient crossing events between *S. haematobium* and *S. bovis*, with subsequent backcrossing with *S. haematobium*. This introgression has prevented compatibility with *P. metidjensis* snail vectors, which are specific to *S. bovis*. Additionally, whether in Africa or in Europe, a clear discrepancy exists between the egg shape usually used for species identification and the genomic composition of schistosomes. Therefore, egg shape cannot be used as a good indicator for hybrid detection. Knowing the phenotypic traits and the genetic features of such hybrids may have important implications in terms of diagnostics and disease management either through vector control strategies or treatment of patients.

Introduction

Schistosomes are dioecious parasitic flatworms, responsible for the major Neglected Tropical Disease (NTD) schistosomiasis. The epidemiological statistics associated with this disease are sobering: 800 million people are at risk in 78 countries, mostly concentrated in sub-Saharan Africa; 230 million are infected and the disease causes more than 200,000 deaths each year as well as between 1.7 and 4.5 million Disability Adjusted Life Years (DALYs) [1]. The most exposed groups are children and young adults who have activities linked to contaminated freshwater environments. In addition to humans, schistosomiasis severely impacts livestock in Africa and Asia with over 165 million animals estimated to be infected [2].

The parasites have a complex life cycle that includes passage through specific freshwater snail intermediate hosts (hereafter termed vectors) in which the parasites undergo clonal multiplication, and a final vertebrate definitive host in which, adult worms sexually reproduce. The liberation of *Schistosoma* eggs into the host tissues is the principal cause of chronic and acute morbidity [3].

Global changes, including both anthropogenic and environmental modifications, may con-
tribute to modifications in the geographical distribution of species and expand their potential
ecological niches [4,5]. Distinct species may thus acquire a new capacity to interact, hybridize
and subsequently introgress their genomes by backcrossing with parental species or other
hybrids, a phenomenon called “hybrid swarm” [6]. Hybridization between individuals from
two previously reproductively isolated species is generally expected to result in the production
of offspring less fit than the parents, sometimes non-viable or sterile. In some cases however,
hybridization may lead to viable progeny that can even have a greater fitness than parental spe-
cies, a genetic effect known as hybrid vigour or heterosis that is generally observed during
early generations [7]. These advantageous combinations of parental genes in offspring may
enable progeny to adapt to new environments as potentially exemplified by the recent out-
break of schistosomiasis in Southern Europe (Corsica, France) [8].
<figure><img src="image_4.png" /><figcaption>**Fig 1.** Egg morphologies of the pure parental species and the European *S. haematobium-bovis* hybrids. Eggs 1 and 2 show typical morphologies of *S. haematobium* (elliptical with a terminal spine) and *S. bovis* (spindle shape with a terminal spine), respectively from laboratory isolates. Eggs 3–9 show the egg morphology of the European *S. haematobium-bovis* hybrid schistosome. While most eggs were of a typical *S. haematobium* morphotype (3–7), there was a high variability in the morphologies and near 10% (4/44) of eggs were non-typical (8–9).</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0010062.g001

supports the initial conclusion that the hybrid originates from a cross between *S. bovis* and *S. haematobium*.

As with all other *Schistosoma* species strong sexual dimorphism was observed for the *S. haematobium-bovis* hybrids with females being held within the gynaecophoric canal of the males. The detailed morphological characteristics and morphometric measurements of the adult worms are presented in Table 1.

The adult *S. haematobium-bovis* hybrid males had an elongated body measuring, on average, 5.3 (±1.0) mm in length and 0.3 (±0.07) mm in width at largest part of the body (middle). The male body was dorso-ventrally flattened with folds in the ventral axis forming the gynaecophoric canal (Fig 2A). The anterior region was narrower than the rest of the body and presented a sub-terminal mouth formed into the oral sucker (length: 0.2±0.01 mm; width: 0.2 ±0.006 mm) and a robust ventral sucker or acetabulum (length: 0.3±0.008 mm; width: 0.3 ±0.01 mm) near the top of the gynaecophoric canal (Fig 2B). The oesophagus began at the oral sucker and was extended posteriorly to the acetabulum where it bifurcates into the gut caeca until it reunites at the posterior end of the body.

Four to five testes were observed per male, situated dorsally posterior to the ventral sucker and were round to ovoid (4.2±0.5). Posterior to the acetabulum, in the dorsal region, tubercles with a round extremity began to appear at the level of the gynaecophoric canal and occurred to all the posterior region of the body (Fig 2C). The presence of tegument projections on the

<table><caption>Table 1. Morphological measurements of the European male and female hybrid adult worms.</caption><thead><tr><th rowspan="3"></th><th colspan="4">Hybrid schistosomes from Corsica</th></tr><tr><th colspan="2">Male</th><th colspan="2">Female</th></tr><tr><th>Mean (±SD)</th><th>Range</th><th>Mean (±SD)</th><th>Range</th></tr></thead><tbody><tr><td>Total length (mm)</td><td>5.3 (±1.0)</td><td>3.3–6.6</td><td>9.6 (±1.2)</td><td>8.6–14.1</td></tr><tr><td>Largest body width (mm)</td><td>0.3 (±0.07)</td><td>0.2–0.5</td><td>0.2 (±0.04)</td><td>0.1–0.3</td></tr><tr><td>Oral sucker length (mm)</td><td>0.2 (±0.01)</td><td>0.2–0.2</td><td>0.05 (±0.004)</td><td>0.04–0.06</td></tr><tr><td>Oral sucker width (mm)</td><td>0.2 (±0.006)</td><td>0.1–0.2</td><td>0.05 (±0.002)</td><td>0.05–0.06</td></tr><tr><td>Ventral sucker length (mm)</td><td>0.3 (±0.008)</td><td>0.2–0.3</td><td>0.06 (±0.001)</td><td>0.06–0.07</td></tr><tr><td>Ventral sucker width (mm)</td><td>0.3 (±0.01)</td><td>0.2–0.3</td><td>0.06 (±0.001)</td><td>0.06–0.07</td></tr><tr><td>Testes number</td><td>4.2 (±0.5)</td><td>4–5</td><td>-</td><td>-</td></tr><tr><td>Ovary length (mm)</td><td>-</td><td>-</td><td>0.3 (±0.01)</td><td>0.3–0.3</td></tr><tr><td>Ovary width (mm)</td><td>-</td><td>-</td><td>0.09 (±0.01)</td><td>0.08–0.1</td></tr><tr><td>Extension of the vitellaria (mm)</td><td>-</td><td>-</td><td>5.0 (±0.7)</td><td>4–5.9</td></tr></tbody></table>

https://doi.org/10.1371/journal.pntd.0010062.t001
<figure><img src="image_4.png" /><figcaption>**Fig 1.** Egg morphologies of the pure parental species and the European *S. haematobium-bovis* hybrids. Eggs 1 and 2 show typical morphologies of *S. haematobium* (elliptical with a terminal spine) and *S. bovis* (spindle shape with a terminal spine), respectively from laboratory isolates. Eggs 3–9 show the egg morphology of the European *S. haematobium-bovis* hybrid schistosome. While most eggs were of a typical *S. haematobium* morphotype (3–7), there was a high variability in the morphologies and near 10% (4/44) of eggs were non-typical (8–9).</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0010062.g001

supports the initial conclusion that the hybrid originates from a cross between *S. bovis* and *S. haematobium*.

As with all other *Schistosoma* species strong sexual dimorphism was observed for the *S. haematobium-bovis* hybrids with females being held within the gynaecophoric canal of the males. The detailed morphological characteristics and morphometric measurements of the adult worms are presented in Table 1.

The adult *S. haematobium-bovis* hybrid males had an elongated body measuring, on average, 5.3 (±1.0) mm in length and 0.3 (±0.07) mm in width at largest part of the body (middle). The male body was dorso-ventrally flattened with folds in the ventral axis forming the gynaecophoric canal (Fig 2A). The anterior region was narrower than the rest of the body and presented a sub-terminal mouth formed into the oral sucker (length: 0.2±0.01 mm; width: 0.2 ±0.006 mm) and a robust ventral sucker or acetabulum (length: 0.3±0.008 mm; width: 0.3 ±0.01 mm) near the top of the gynaecophoric canal (Fig 2B). The oesophagus began at the oral sucker and was extended posteriorly to the acetabulum where it bifurcates into the gut caeca until it reunites at the posterior end of the body.

Four to five testes were observed per male, situated dorsally posterior to the ventral sucker and were round to ovoid (4.2±0.5). Posterior to the acetabulum, in the dorsal region, tubercles with a round extremity began to appear at the level of the gynaecophoric canal and occurred to all the posterior region of the body (Fig 2C). The presence of tegument projections on the

<table><caption>Table 1. Morphological measurements of the European male and female hybrid adult worms.</caption><thead><tr><th rowspan="3"></th><th colspan="4">Hybrid schistosomes from Corsica</th></tr><tr><th colspan="2">Male</th><th colspan="2">Female</th></tr><tr><th>Mean (±SD)</th><th>Range</th><th>Mean (±SD)</th><th>Range</th></tr></thead><tbody><tr><td>Total length (mm)</td><td>5.3 (±1.0)</td><td>3.3–6.6</td><td>9.6 (±1.2)</td><td>8.6–14.1</td></tr><tr><td>Largest body width (mm)</td><td>0.3 (±0.07)</td><td>0.2–0.5</td><td>0.2 (±0.04)</td><td>0.1–0.3</td></tr><tr><td>Oral sucker length (mm)</td><td>0.2 (±0.01)</td><td>0.2–0.2</td><td>0.05 (±0.004)</td><td>0.04–0.06</td></tr><tr><td>Oral sucker width (mm)</td><td>0.2 (±0.006)</td><td>0.1–0.2</td><td>0.05 (±0.002)</td><td>0.05–0.06</td></tr><tr><td>Ventral sucker length (mm)</td><td>0.3 (±0.008)</td><td>0.2–0.3</td><td>0.06 (±0.001)</td><td>0.06–0.07</td></tr><tr><td>Ventral sucker width (mm)</td><td>0.3 (±0.01)</td><td>0.2–0.3</td><td>0.06 (±0.001)</td><td>0.06–0.07</td></tr><tr><td>Testes number</td><td>4.2 (±0.5)</td><td>4–5</td><td>-</td><td>-</td></tr><tr><td>Ovary length (mm)</td><td>-</td><td>-</td><td>0.3 (±0.01)</td><td>0.3–0.3</td></tr><tr><td>Ovary width (mm)</td><td>-</td><td>-</td><td>0.09 (±0.01)</td><td>0.08–0.1</td></tr><tr><td>Extension of the vitellaria (mm)</td><td>-</td><td>-</td><td>5.0 (±0.7)</td><td>4–5.9</td></tr></tbody></table>

https://doi.org/10.1371/journal.pntd.0010062.t001
<figure><img src="image_5.png" /><figcaption>**Fig 2.** Morphological observations of the European male hybrid adult worms. (A) Whole parasite body showing the oral sucker, acetabulum (ventral sucker) and gynaecophoral canal; (B) Frontal view of the anterior region showing the oral sucker and acetabulum (ventral sucker) in detail; (C) Dorso-lateral view of the spines and tubercles on the parasite surface.</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0010062.g002

tubercles where identified with several apical spines which decreased in distribution and size
towards the back and sides of the male's bodies (Fig 2C). The females were elongated and fili-
form measuring 9.6 (±1.2) mm in length and 0.2 (±0.04) mm in width, with the posterior half
of body expanded (Fig 3A). The anterior regions of the females were smaller compared to
males, had a small oral sucker (length: 0.05±0.004 mm; width: 0.05±0.002 mm) and acetabu-
lum (length: 0.06±0.001 mm; width: 0.06±0.001 mm) (Fig 3B).

The oesophagus started near to the oral sucker and bifurcated immediately after the acetabu-
lum. The genital pore was situated at the posterior end of the ventral sucker (dorsally) and some
eggs could be observed in the uterus (Fig 3C). A single ovary measuring 0.3 (±0.01) long and 0.09
(±0.01) wide was situated in the posterior third of the female's body (Fig 3A). The vitelline glands,
also called vitellaria were extensive, occupying roughly 50% of the posterior part of the worm and
extending further posteriorly than the intestine. The females' tegument was smooth and uniform
without significant projections. The posterior extremity was tapered and rounded.

**Whole genome sequencing shows introgression of S. bovis into S. haematobium**

Unfortunately, we were not able to produce enough high molecular weight genomic DNA to
directly proceed to long-read sequencing of individual hybrid adult worms. To better
<figure><img src="image_5.png" /><figcaption>**Fig 2.** Morphological observations of the European male hybrid adult worms. (A) Whole parasite body showing the oral sucker, acetabulum (ventral sucker) and gynaecophoral canal; (B) Frontal view of the anterior region showing the oral sucker and acetabulum (ventral sucker) in detail; (C) Dorso-lateral view of the spines and tubercles on the parasite surface.</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0010062.g002

tubercles where identified with several apical spines which decreased in distribution and size towards the back and sides of the male's bodies (Fig 2C). The females were elongated and filiform measuring 9.6 (±1.2) mm in length and 0.2 (±0.04) mm in width, with the posterior half of body expanded (Fig 3A). The anterior regions of the females were smaller compared to males, had a small oral sucker (length: 0.05±0.004 mm; width: 0.05±0.002 mm) and acetabulum (length: 0.06±0.001 mm; width: 0.06±0.001 mm) (Fig 3B).

The oesophagus started near to the oral sucker and bifurcated immediately after the acetabulum. The genital pore was situated at the posterior end of the ventral sucker (dorsally) and some eggs could be observed in the uterus (Fig 3C). A single ovary measuring 0.3 (±0.01) long and 0.09 (±0.01) wide was situated in the posterior third of the female's body (Fig 3A). The vitelline glands, also called vitellaria were extensive, occupying roughly 50% of the posterior part of the worm and extending further posteriorly than the intestine. The females' tegument was smooth and uniform without significant projections. The posterior extremity was tapered and rounded.

**Whole genome sequencing shows introgression of *S. bovis* into *S. haematobium***

Unfortunately, we were not able to produce enough high molecular weight genomic DNA to directly proceed to long-read sequencing of individual hybrid adult worms. To better
<figure><img src="image_6.png" /><figcaption>**Fig 3.** Morphological observations of the European female hybrid adult worms. (A) Lateral view of whole parasite body showing oral sucker, acetabulum (ventral sucker), ovary and vitellaria; (B) Frontal view of anterior region showing oral sucker, acetabulum (ventral sucker) in detail; (C) Dorso-lateral view of two eggs inside female uterus.</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0010062.g003

characterize the level of hybridization we therefore used Illumina short read high-throughput sequencing and alignment to parental genomes. As the previous *S. bovis* assembly was highly fragmented (111 328 scaffolds, N50 7kb), and another *S. bovis* strain from Spain was used to produce the experimental hybrids examined in this study (as below) we resequenced the *S. bovis* (Villar de la Yegua, Salamanca, Spain) genome using 200 worms with PacBio long reads. This produced 4,102,584 filtered subreads (48,987,175,429 bases). The genome assembly led to a new genome version of 486 scaffolds (N50: 3.1Mb) from which 14,104 protein-coding genes were identified with an average length of 18,725 bp (5.3% of genome length) (Table 2), consistent with the known characteristics of *Schistosoma* genomes [17,18].

Using the Benchmarking Universal Single-Copy Orthologs (BUSCO) [19] to assess the genome assembly and annotation completeness, we identified 66.9% of complete BUSCOs for

<table><caption>**Table 2.** Assembly and gene prediction metrics of *S. bovis* from Spain.</caption><tr><td>Total assembly length (bp)</td><td>446,282,422</td></tr><tr><td>Number of scaffolds</td><td>486</td></tr><tr><td>N50 (bp)</td><td>3,109,635</td></tr><tr><td>Number of scaffolds ≥ N50 (L50)</td><td>38</td></tr><tr><td>Proportion of the genome that is coding (%)</td><td>5.27</td></tr><tr><td>Number of genes</td><td>14,104</td></tr><tr><td>Average gene length (bp)</td><td>18,725 ± 21,625</td></tr><tr><td>Average number of exons per gene</td><td>7.9</td></tr><tr><td>Average exon length (bp)</td><td>299</td></tr><tr><td>Average intron length (bp)</td><td>2,389</td></tr><tr><td>Repeat rate (%)</td><td>49.5%</td></tr></table>

https://doi.org/10.1371/journal.pntd.0010062.t002
<figure><img src="image_6.png" /><figcaption>**Fig 3.** Morphological observations of the European female hybrid adult worms. (A) Lateral view of whole parasite body showing oral sucker, acetabulum (ventral sucker), ovary and vitellaria; (B) Frontal view of anterior region showing oral sucker, acetabulum (ventral sucker) in detail; (C) Dorso-lateral view of two eggs inside female uterus.</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0010062.g003

characterize the level of hybridization we therefore used Illumina short read high-throughput sequencing and alignment to parental genomes. As the previous *S. bovis* assembly was highly fragmented (111 328 scaffolds, N50 7kb), and another *S. bovis* strain from Spain was used to produce the experimental hybrids examined in this study (as below) we resequenced the *S. bovis* (Villar de la Yegua, Salamanca, Spain) genome using 200 worms with PacBio long reads. This produced 4,102,584 filtered subreads (48,987,175,429 bases). The genome assembly led to a new genome version of 486 scaffolds (N50: 3.1Mb) from which 14,104 protein-coding genes were identified with an average length of 18,725 bp (5.3% of genome length) (Table 2), consistent with the known characteristics of *Schistosoma* genomes [17,18].

Using the Benchmarking Universal Single-Copy Orthologs (BUSCO) [19] to assess the genome assembly and annotation completeness, we identified 66.9% of complete BUSCOs for

<table><caption>**Table 2.** Assembly and gene prediction metrics of *S. bovis* from Spain.</caption><tr><td>Total assembly length (bp)</td><td>446,282,422</td></tr><tr><td>Number of scaffolds</td><td>486</td></tr><tr><td>N50 (bp)</td><td>3,109,635</td></tr><tr><td>Number of scaffolds ≥ N50 (L50)</td><td>38</td></tr><tr><td>Proportion of the genome that is coding (%)</td><td>5.27</td></tr><tr><td>Number of genes</td><td>14,104</td></tr><tr><td>Average gene length (bp)</td><td>18,725 ± 21,625</td></tr><tr><td>Average number of exons per gene</td><td>7.9</td></tr><tr><td>Average exon length (bp)</td><td>299</td></tr><tr><td>Average intron length (bp)</td><td>2,389</td></tr><tr><td>Repeat rate (%)</td><td>49.5%</td></tr></table>

https://doi.org/10.1371/journal.pntd.0010062.t002
<table><caption>Table 3. Genome assembly and annotation completeness using BUSCO.</caption><thead><tr><th></th><th>S. bovis (Spanish strain, this study)</th><th>S. bovis (Tanzanian strain [14])</th><th>S. haematobium v1 (Egyptian strain [17])</th><th>S. haematobium v2 (Egyptian strain [18])</th></tr></thead><tbody><tr><td>Complete BUSCOs (C)</td><td>654</td><td>619</td><td>584</td><td>613</td></tr><tr><td>Complete and single-copy BUSCOs (S)</td><td>586</td><td>590</td><td>577</td><td>604</td></tr><tr><td>Complete and duplicated BUSCOs (D)</td><td>68</td><td>29</td><td>7</td><td>9</td></tr><tr><td>Fragmented BUSCOs (F)</td><td>86</td><td>94</td><td>103</td><td>111</td></tr><tr><td>Missing BUSCOs (M)</td><td>238</td><td>265</td><td>291</td><td>254</td></tr><tr><td>Total BUSCO groups searched</td><td>978</td><td>978</td><td>978</td><td>978</td></tr><tr><td>Completeness</td><td>66.90%</td><td>63.30%</td><td>59.70%</td><td>62.70%</td></tr></tbody></table>

https://doi.org/10.1371/journal.pntd.0010062.t003

our *S. bovis* genome (Spanish strain), compared to 63.3%, 59.7% and 62.7% for the previously sequenced *S. bovis* strain from Tanzania [14] and the *S. haematobium* v1 [17] and v2 [18] assembly, respectively using the same pipeline (Table 3). Due to the divergence of the Platyhelminth lineage and the very limited representation of Lophotrochozoa in the BUSCO protein set, it has already been shown that BUSCO underestimate completeness, particularly using its genome mode [20,21].

For the European *S. haematobium-bovis* hybrid, a total of 289,873,531 reads (76.5%) were mapped against the 681.2 Mb concatenated genomes of *S. haematobium* [17] and *S. bovis* (this study), representing 42X mean coverage (Table 4). We also sequenced F1 males from experimental first generation cross between male *S. haematobium* (Cameroon) x female *S. bovis* (Spain) as a control. A total of 4,910,354 reads (92.3%) were mapped against a concatenate of the *S. haematobium* and *S. bovis* genomes (Table 4).

The mapping of the F1 reads to the *S. haematobium* and *S. bovis* genomes was of ~50% on each of the parental species genomes (Table 4). This control was crucial to validate our analytical pipeline and thus validate the results obtained for the natural European hybrid schistosomes. Moreover, 98.3% of reads of mitochondrial origin, aligned to the mitochondrial genome of *S. bovis*, which is consistent with the maternal inheritance of mitochondrial genome within *Schistosoma* species (Table 4).

Interestingly, the mapping of the European hybrid reads against the *S. haematobium* and *S. bovis* reference genomes revealed admixture between the parental genomes with a proportion of 76.9% of sequences mapping to the *S. haematobium*, and 23.1% mapping to the *S. bovis* genomes for both male and female parasites (Table 4). Alignment to the mitochondrial genomes of both parents also showed results concordant with the previous Sanger sequencing data for this marker, with 97.8% of reads being mapped to the *S. bovis* mitochondrial genome, and 2.2% to the *S. haematobium* mitochondrial genome [8]. To assess the divergence level between the nuclear genomes of the two “pure” species, we identified homologous regions

<table><caption>Table 4. Summary of the introgression level analysis of the experimental F1 hybrids (control) and the natural European hybrid strain recovered in Corsica.</caption><thead><tr><th rowspan="2"></th><th rowspan="2">Experimental F1 genome (males)</th><th colspan="2">European hybrid genome</th></tr><tr><th>Males</th><th>Females</th></tr></thead><tbody><tr><td> mapped reads (% of total number)</td><td>4,910,354 (92.3%)</td><td>150,994,161 (80.8%)</td><td>138,879,370 (72.2%)</td></tr><tr><td> Reads mapped against S. haematobium genome</td><td>2,417,196 (49.2%)</td><td>116,052,836 (76.9%)</td><td>106,969,327 (77%)</td></tr><tr><td> Reads mapped against S. bovis genome</td><td>2,493,158 (50.8%)</td><td>34,941,325 (23.1%)</td><td>31,910,043 (23%)</td></tr><tr><td> Reads mapped against S. haematobium mitochondrion</td><td>168 (1.7%)</td><td>50,436 (2.2%)</td><td>33,454 (2.2%)</td></tr><tr><td> Reads mapped against S. bovis mitochondrion</td><td>9,825 (98.3%)</td><td>2,229,096 (97.8%)</td><td>1,455,815 (97.8%)</td></tr></tbody></table>

https://doi.org/10.1371/journal.pntd.0010062.t004
<table><caption>Table 3. Genome assembly and annotation completeness using BUSCO.</caption><thead><tr><th></th><th>S. bovis (Spanish strain, this study)</th><th>S. bovis (Tanzanian strain [14])</th><th>S. haematobium v1 (Egyptian strain [17])</th><th>S. haematobium v2 (Egyptian strain [18])</th></tr></thead><tbody><tr><td>Complete BUSCOs (C)</td><td>654</td><td>619</td><td>584</td><td>613</td></tr><tr><td>Complete and single-copy BUSCOs (S)</td><td>586</td><td>590</td><td>577</td><td>604</td></tr><tr><td>Complete and duplicated BUSCOs (D)</td><td>68</td><td>29</td><td>7</td><td>9</td></tr><tr><td>Fragmented BUSCOs (F)</td><td>86</td><td>94</td><td>103</td><td>111</td></tr><tr><td>Missing BUSCOs (M)</td><td>238</td><td>265</td><td>291</td><td>254</td></tr><tr><td>Total BUSCO groups searched</td><td>978</td><td>978</td><td>978</td><td>978</td></tr><tr><td>Completeness</td><td>66.90%</td><td>63.30%</td><td>59.70%</td><td>62.70%</td></tr></tbody></table>

https://doi.org/10.1371/journal.pntd.0010062.t003

our *S. bovis* genome (Spanish strain), compared to 63.3%, 59.7% and 62.7% for the previously sequenced *S. bovis* strain from Tanzania [14] and the *S. haematobium* v1 [17] and v2 [18] assembly, respectively using the same pipeline (Table 3). Due to the divergence of the Platyhelminth lineage and the very limited representation of Lophotrochozoa in the BUSCO protein set, it has already been shown that BUSCO underestimate completeness, particularly using its genome mode [20,21].

For the European *S. haematobium-bovis* hybrid, a total of 289,873,531 reads (76.5%) were mapped against the 681.2 Mb concatenated genomes of *S. haematobium* [17] and *S. bovis* (this study), representing 42X mean coverage (Table 4). We also sequenced F1 males from experimental first generation cross between male *S. haematobium* (Cameroon) x female *S. bovis* (Spain) as a control. A total of 4,910,354 reads (92.3%) were mapped against a concatenate of the *S. haematobium* and *S. bovis* genomes (Table 4).

The mapping of the F1 reads to the *S. haematobium* and *S. bovis* genomes was of ~50% on each of the parental species genomes (Table 4). This control was crucial to validate our analytical pipeline and thus validate the results obtained for the natural European hybrid schistosomes. Moreover, 98.3% of reads of mitochondrial origin, aligned to the mitochondrial genome of *S. bovis*, which is consistent with the maternal inheritance of mitochondrial genome within *Schistosoma* species (Table 4).

Interestingly, the mapping of the European hybrid reads against the *S. haematobium* and *S. bovis* reference genomes revealed admixture between the parental genomes with a proportion of 76.9% of sequences mapping to the *S. haematobium*, and 23.1% mapping to the *S. bovis* genomes for both male and female parasites (Table 4). Alignment to the mitochondrial genomes of both parents also showed results concordant with the previous Sanger sequencing data for this marker, with 97.8% of reads being mapped to the *S. bovis* mitochondrial genome, and 2.2% to the *S. haematobium* mitochondrial genome [8]. To assess the divergence level between the nuclear genomes of the two “pure” species, we identified homologous regions

<table><caption>Table 4. Summary of the introgression level analysis of the experimental F1 hybrids (control) and the natural European hybrid strain recovered in Corsica.</caption><thead><tr><th rowspan="2"></th><th rowspan="2">Experimental F1 genome (males)</th><th colspan="2">European hybrid genome</th></tr><tr><th>Males</th><th>Females</th></tr></thead><tbody><tr><td> mapped reads (% of total number)</td><td>4,910,354 (92.3%)</td><td>150,994,161 (80.8%)</td><td>138,879,370 (72.2%)</td></tr><tr><td> Reads mapped against S. haematobium genome</td><td>2,417,196 (49.2%)</td><td>116,052,836 (76.9%)</td><td>106,969,327 (77%)</td></tr><tr><td> Reads mapped against S. bovis genome</td><td>2,493,158 (50.8%)</td><td>34,941,325 (23.1%)</td><td>31,910,043 (23%)</td></tr><tr><td> Reads mapped against S. haematobium mitochondrion</td><td>168 (1.7%)</td><td>50,436 (2.2%)</td><td>33,454 (2.2%)</td></tr><tr><td> Reads mapped against S. bovis mitochondrion</td><td>9,825 (98.3%)</td><td>2,229,096 (97.8%)</td><td>1,455,815 (97.8%)</td></tr></tbody></table>

https://doi.org/10.1371/journal.pntd.0010062.t004
between *S. haematobium* and *S. bovis* using CACTUS [22]. A total of 234.5 Mb sequences aligned between the parental species, representing 64% of the *S. haematobium* genome length. The mean similarity of these shared sequences was 95.9% compared to ~99% and ~90% for commonly used phylogenetic markers such as ITS and cox1, respectively. The mean similarity between *S. haematobium* mitochondria (GenBank accession number DQ157222.2) and *S. bovis* mitochondria (Contig 00439F of our assembly) was 82.1%.

Besides being of fundamental interest for the evolutionary biology of the parasite, this finding could also have immediate consequences for parasite control. One of the few phenotypic features that are of relevance for infection success and for which the genetic basis is known is resistance to Oxamniquine (OXA). In *S. mansoni* the mutations that confer resistance occur in the SmSULT-OR gene (*Smp_089320*), encoding a sulfotransferase that is required for drug activation, are p.E142del and p.C35R. [23,24]. In *S. haematobium* the drug is not efficient due to a F39 Sm > Y54 Sh substitution [24]. We reasoned that if the corresponding *S. bovis* allele would have introgressed into the hybrid it would follow the *S. mansoni* trait making the hybrid more sensitive to OXA. We have undertaken reciprocal homology searches with *Smp_089320* against the *S. haematobium* and the new *S. bovis* genome to identify orthologues. Orthologues exist in both genomes and both possess the F->Y Sh54 and no mutations in C Sm35 or L Sm256 or deletion in E Sm142.

**The European hybrid is compatible with the natural intermediate snail hosts of *S. haematobium*, *B. truncatus* but not with *P. metidjensis* which is also a host of *S. bovis***

The presence of 23% of *S. bovis* genetic material in the hybrid genome raises the possibility that the hybrid is capable of infecting specific vector snails uniquely compatible with *S. bovis*. To test this hypothesis we exposed *P. metidjensis*, to miracidia of the European hybrid found in Corsica. The prevalence in its natural host, *B. truncatus* from Corsica was 24% (9 infected snails out of 37) whereas, no *P. metidjensis* became infected (0 infected snails out of 29 alive).

**Discussion**

The emergence of infectious diseases are currently among the greatest concerns of our chang-
ing world and has strong outreach effects for society. Besides the important impacts that global
changes may have on the spread and transmission of tropical infectious diseases in higher lati-
tudes, other phenomenon may combine and act as driving forces promoting the emergence of
novel disease in unsuspected areas. The importance and the frequency of hybridization in
infectious agents are certainly underestimated, and very little attention has been given so far to
the role of genetic introgression on infectious disease emergence, spread and control [25]. In
the genus *Schistosoma*, several reports have revealed that inter-species hybrids are frequent
and are a real concern for human health [9,10]. In particular hybridization between *S. haema-
tobium* and *S. bovis* have now been identified with molecular tools in Senegal, Niger, Benin,
Mali, Cote D'Ivoire and also Malawi [26-30]. To date genomic evidence indicates that
although only a small proportion of *S. bovis* seems to have introgressed in the genomic back-
ground of *S. haematobium* (i.e. 3-8%), introgression from *S. bovis* is widespread across *S. hae-
matobium* populations in endemic areas. The signatures of introgression observed indicate
ancient and unidirectional events with a potential adaptive significance related to host-patho-
gen interactions [11,14,15]. Nevertheless, it is the first time that a hybrid schistosome has been
involved in a large-scale outbreak in Europe [8,31,32]. Although usually restricted to tropical
areas, schistosomiasis transmission is now persisting in Corsica and the hybrid status of the
parasite might have increased its invasive and adaptive capacities. Indeed, our study revealed
between *S. haematobium* and *S. bovis* using CACTUS [22]. A total of 234.5 Mb sequences aligned between the parental species, representing 64% of the *S. haematobium* genome length. The mean similarity of these shared sequences was 95.9% compared to ~99% and ~90% for commonly used phylogenetic markers such as ITS and cox1, respectively. The mean similarity between *S. haematobium* mitochondria (GenBank accession number DQ157222.2) and *S. bovis* mitochondria (Contig 00439F of our assembly) was 82.1%.

Besides being of fundamental interest for the evolutionary biology of the parasite, this finding could also have immediate consequences for parasite control. One of the few phenotypic features that are of relevance for infection success and for which the genetic basis is known is resistance to Oxamniquine (OXA). In *S. mansoni* the mutations that confer resistance occur in the SmSULT-OR gene (*Smp_089320*), encoding a sulfotransferase that is required for drug activation, are p.E142del and p.C35R. [23,24]. In *S. haematobium* the drug is not efficient due to a F39 Sm > Y54 Sh substitution [24]. We reasoned that if the corresponding *S. bovis* allele would have introgressed into the hybrid it would follow the *S. mansoni* trait making the hybrid more sensitive to OXA. We have undertaken reciprocal homology searches with *Smp_089320* against the *S. haematobium* and the new *S. bovis* genome to identify orthologues. Orthologues exist in both genomes and both possess the F->Y Sh54 and no mutations in C Sm35 or L Sm256 or deletion in E Sm142.

**The European hybrid is compatible with the natural intermediate snail hosts of *S. haematobium*, *B. truncatus* but not with *P. metidjensis* which is also a host of *S. bovis***

The presence of 23% of *S. bovis* genetic material in the hybrid genome raises the possibility that the hybrid is capable of infecting specific vector snails uniquely compatible with *S. bovis*. To test this hypothesis we exposed *P. metidjensis*, to miracidia of the European hybrid found in Corsica. The prevalence in its natural host, *B. truncatus* from Corsica was 24% (9 infected snails out of 37) whereas, no *P. metidjensis* became infected (0 infected snails out of 29 alive).

**Discussion**

The emergence of infectious diseases are currently among the greatest concerns of our chang-
ing world and has strong outreach effects for society. Besides the important impacts that global
changes may have on the spread and transmission of tropical infectious diseases in higher lati-
tudes, other phenomenon may combine and act as driving forces promoting the emergence of
novel disease in unsuspected areas. The importance and the frequency of hybridization in
infectious agents are certainly underestimated, and very little attention has been given so far to
the role of genetic introgression on infectious disease emergence, spread and control [25]. In
the genus *Schistosoma*, several reports have revealed that inter-species hybrids are frequent
and are a real concern for human health [9,10]. In particular hybridization between *S. haema-
tobium* and *S. bovis* have now been identified with molecular tools in Senegal, Niger, Benin,
Mali, Cote D'Ivoire and also Malawi [26-30]. To date genomic evidence indicates that
although only a small proportion of *S. bovis* seems to have introgressed in the genomic back-
ground of *S. haematobium* (i.e. 3-8%), introgression from *S. bovis* is widespread across *S. hae-
matobium* populations in endemic areas. The signatures of introgression observed indicate
ancient and unidirectional events with a potential adaptive significance related to host-patho-
gen interactions [11,14,15]. Nevertheless, it is the first time that a hybrid schistosome has been
involved in a large-scale outbreak in Europe [8,31,32]. Although usually restricted to tropical
areas, schistosomiasis transmission is now persisting in Corsica and the hybrid status of the
parasite might have increased its invasive and adaptive capacities. Indeed, our study revealed
that the European hybrids established in Corsica are highly introgressed, 77% *S. haematobium*
origin and 23% *S. bovis* origin. Together with the maternal inheritance of the mitochondria
this suggested that these hybrids were generated by an initial cross between a male *S. haematobium*
and a female *S. bovis* with successive backcrosses with *S. haematobium*. This seems to
confirm an ancient and mostly unidirectional introgression event(s) that could potentially be
advantageous for the parasites. The hybrid status of the parasite may thus have important
implication for disease control in term of host spectrum, diagnostics, and treatment in
endemic areas but also Europe.

Implications for host spectrum and parasite distribution

*S. haematobium* and *S. bovis* have different intermediate host specificities. *S. haematobium*
only infects snails within the genus *Bulinus* while *S. bovis* can also infect *Planorbarius* snails
(widely present in the Iberian Peninsula), together with *Bulinus* species. The potential distribu-
tion range of the disease may thus be enhanced if the hybrid is able to infect the intermediate
hosts of both parental species. Interestingly, the natural European hybrid schistosomes, that
we recovered in Corsica, were not able to infect our laboratory strain of *P. metidjensis*, but dis-
played high levels of compatibility with Corsican *B. truncatus* (24% infection prevalence)
which is consistent with previous schistosome-snail compatibility assessments for the hybrid
parasite recovered directly from an infected patient in 2014 [8]. This is consistent with the pre-
dominance of *S. haematobium* within the genome of this hybrid, and may open the door to
vector based control, e.g. by control strategies targeting these snails. However, although our
findings indicate that *P. metidjensis* is not a host for this European hybrid we cannot exclude
the possibility that other strains of *P. metidjensis* from Europe or Africa could be compatible
and act as a host. Moreover, as the tested miracidia were collected after passage through a labo-
ratory host, potentially inducing a population bottleneck, further testing on various hybrid
field isolates is warranted. One other fundamental concern is the capacity of such introgressed
schistosomes to infect livestock or other animal reservoir hosts. The zoonotic potential of the
hybrids would strongly impact the parasite transmission in the field, in and out of endemic
areas, and may hamper our capacity to maintain adequate control strategies as schistosomiasis
treatment focuses almost exclusively on humans. Recent studies are now showing the presence
of not only *S. bovis*, *S. haematobium*, and *S. mansoni* in rodents (hosts in which hybridization
may occur), but also the occurrence of *S. haematobium x bovis* hybrids in such hosts in Senegal
[33,34] and Benin [35] although the importance of rodents in transmission dynamics needs
further exploration. Moreover, beside widespread investigation of animal reservoir in Senegal
[26] to date only one study in Benin suggests that other animals such as cattle may be natural
hosts for such hybrid parasites [27]. The situation in Corsica and the role of animal reservoir
needs to be precisely investigated as despite ongoing transmission and developing endemicity
on the island [36], no infection has been detected in livestock in the region, and the only
infected animals found were two rats that do not seem to play a significant role in the transmis-
sion for this particular foci [37]. However, we cannot rule out the influence of an undetected
animal reservoir such as *Ovis aries musimon*, a wild sheep native to Corsica, that have never
been tested for infections [36].

**Implications for diagnostics**

The hybrid status of the parasite may impair parasitological, serological and molecular diag-
nostics used to diagnose infections. In endemic countries, parasitological diagnosis (egg detec-
tion) is the gold standard, whereas serological tests are commonly used for imported cases of
schistosomiasis in non-endemic, developed countries. In humans, schistosome eggs that are
that the European hybrids established in Corsica are highly introgressed, 77% *S. haematobium*
origin and 23% *S. bovis* origin. Together with the maternal inheritance of the mitochondria
this suggested that these hybrids were generated by an initial cross between a male *S. haemato-
bium* and a female *S. bovis* with successive backcrosses with *S. haematobium*. This seems to
confirm an ancient and mostly unidirectional introgression event(s) that could potentially be
advantageous for the parasites. The hybrid status of the parasite may thus have important
implication for disease control in term of host spectrum, diagnostics, and treatment in
endemic areas but also Europe.

Implications for host spectrum and parasite distribution

*S. haematobium* and *S. bovis* have different intermediate host specificities. *S. haematobium*
only infects snails within the genus *Bulinus* while *S. bovis* can also infect *Planorbarius* snails
(widely present in the Iberian Peninsula), together with *Bulinus* species. The potential distribu-
tion range of the disease may thus be enhanced if the hybrid is able to infect the intermediate
hosts of both parental species. Interestingly, the natural European hybrid schistosomes, that
we recovered in Corsica, were not able to infect our laboratory strain of *P. metidjensis*, but dis-
played high levels of compatibility with Corsican *B. truncatus* (24% infection prevalence)
which is consistent with previous schistosome-snail compatibility assessments for the hybrid
parasite recovered directly from an infected patient in 2014 [8]. This is consistent with the pre-
dominance of *S. haematobium* within the genome of this hybrid, and may open the door to
vector based control, e.g. by control strategies targeting these snails. However, although our
findings indicate that *P. metidjensis* is not a host for this European hybrid we cannot exclude
the possibility that other strains of *P. metidjensis* from Europe or Africa could be compatible
and act as a host. Moreover, as the tested miracidia were collected after passage through a labo-
ratory host, potentially inducing a population bottleneck, further testing on various hybrid
field isolates is warranted. One other fundamental concern is the capacity of such introgressed
schistosomes to infect livestock or other animal reservoir hosts. The zoonotic potential of the
hybrids would strongly impact the parasite transmission in the field, in and out of endemic
areas, and may hamper our capacity to maintain adequate control strategies as schistosomiasis
treatment focuses almost exclusively on humans. Recent studies are now showing the presence
of not only *S. bovis*, *S. haematobium*, and *S. mansoni* in rodents (hosts in which hybridization
may occur), but also the occurrence of *S. haematobium x bovis* hybrids in such hosts in Senegal
[33,34] and Benin [35] although the importance of rodents in transmission dynamics needs
further exploration. Moreover, beside widespread investigation of animal reservoir in Senegal
[26] to date only one study in Benin suggests that other animals such as cattle may be natural
hosts for such hybrid parasites [27]. The situation in Corsica and the role of animal reservoir
needs to be precisely investigated as despite ongoing transmission and developing endemicity
on the island [36], no infection has been detected in livestock in the region, and the only
infected animals found were two rats that do not seem to play a significant role in the transmis-
sion for this particular foci [37]. However, we cannot rule out the influence of an undetected
animal reservoir such as *Ovis aries musimon*, a wild sheep native to Corsica, that have never
been tested for infections [36].

**Implications for diagnostics**

The hybrid status of the parasite may impair parasitological, serological and molecular diag-
nostics used to diagnose infections. In endemic countries, parasitological diagnosis (egg detec-
tion) is the gold standard, whereas serological tests are commonly used for imported cases of
schistosomiasis in non-endemic, developed countries. In humans, schistosome eggs that are
partly retained in the tissues are the cause for the disease and host-induced pathology, but are
also classical tools for diagnosis and species identification. At first sight, egg morphology and
their localization in the urine of infected patients in Corsica strongly suggested an *S. haemato-
bium* infection [8]. Indeed *S. haematobium* eggs that are usually voided by the urine have a typ-
ical round to oval shape (elliptical or elongated) with a terminal spine. According to previous
studies, *S. haematobium* eggs measure between 100–156 µm long and 40–50 µm wide with
usual length between 115–135 µm long [38–40]. A previous analysis of the European hybrid
ggs revealed smaller eggs (n = 15) with a mean length of 106.5 µm, a width of 42.8 µm with a
spine length of 10.4 µm [32]. According to our results the eggs generally show an ovoid shape
measuring 126.4 x 60.8 µm, more similar to *S. haematobium* eggs. This is also consistent with
the introgression levels that show a predominance of *S. haematobium*-type genetic material
(Table 4). However, sometimes eggs appeared intermediate with spindle or diamond shapes,
which are characteristic of *S. bovis* eggs (usually bigger and measuring between 170–223.9 µm
long and 55–66.0 µm wide) [38,41,42] (Fig 1). In addition, *S. bovis* eggs are released in the
feces of infected animals, due to *S. bovis* locating around the mesenteric vessels. Thus, we
could expect that hybrid parasite eggs may also be released in part in the feces of human hosts.
This could explain, together with the low parasite intensity, why only 30% of patients infected
in Corsica had eggs that were able to be detected in their urine [43]. The route of excretion
associated with egg shape is the current standard for diagnostic and species determination,
however our results confirm earlier publications showing that it is impossible to detect hybrid-
ization in schistosome species using egg morphology alone [44]. Although adult worm mor-
phology has a limited interest for diagnostics in humans, principally because worms are not
accessible, they may be useful for a taxonomic purpose. Males and females of the European
hybrids were generally smaller (in length and width) when compared to both parental species
[45–49], but most interestingly, the presence of spines on the tubercles of the males, a trait not
found in *S. bovis* male, [50–52], was concordant with the predominance of *S. haematobium* in
the genetic make up of these hybrids.

Concerning serological diagnosis, the majority of commercial tests, ELISA or IHA (indirect
hemagglutination) use *S. mansoni* antigens. A discrepancy between those antigens and the
infecting species may induce false negative results [53]. The efficiency of these commercial
diagnostic kits thus needs to be reevaluated in a context of different species and hybrid forms.
Finally, molecular diagnostic for urogenital schistosomiasis using PCR has already been used
in urine or serum, targeting a highly repeated sequence (*Dra*I), which is restricted to the *S. haem-
matobium* group of schistosomes (including both *S. haematobium* and *S. bovis*) [54,55]. We
expect that this test would be efficient to detect such infection but not to identify the hybrid
status of the parasite.

**Implications for treatment**

Praziquantel (PZQ) is currently the main drug used to treat schistosomiasis and the applica-
tion of mass chemotherapy programs is the prevailing strategy for schistosomiasis control
[56]. PZQ is also efficient for treating bovine schistosomiasis, but the dose needed is quite high
(60 mg/kg for 95% deworming efficacy in goats [57,58]) and cannot be considered in endemic
areas where treatment capacities are primarily focused on human schistosomiasis. Moreover,
it has been shown in humans that a dose of 40 mg/kg of PZQ is only 63.5% efficient for mixed
infections, compared to 76.7% and 77.1%, for mono infections of *S. mansoni* and *S. haemato-
bium*, respectively [59]. To date, neither experimental nor field trials have tested the sensitivity
of hybrid parasites to PZQ. Thus, there is no current evidence that there is any difference in
drug response in natural infections and changes associated to PZQ response in hybrids is still
partly retained in the tissues are the cause for the disease and host-induced pathology, but are
also classical tools for diagnosis and species identification. At first sight, egg morphology and
their localization in the urine of infected patients in Corsica strongly suggested an *S. haemato-
bium* infection [8]. Indeed *S. haematobium* eggs that are usually voided by the urine have a typ-
ical round to oval shape (elliptical or elongated) with a terminal spine. According to previous
studies, *S. haematobium* eggs measure between 100–156 µm long and 40–50 µm wide with
usual length between 115–135 µm long [38–40]. A previous analysis of the European hybrid
ggs revealed smaller eggs (n = 15) with a mean length of 106.5 µm, a width of 42.8 µm with a
spine length of 10.4 µm [32]. According to our results the eggs generally show an ovoid shape
measuring 126.4 x 60.8 µm, more similar to *S. haematobium* eggs. This is also consistent with
the introgression levels that show a predominance of *S. haematobium*-type genetic material
(Table 4). However, sometimes eggs appeared intermediate with spindle or diamond shapes,
which are characteristic of *S. bovis* eggs (usually bigger and measuring between 170–223.9 µm
long and 55–66.0 µm wide) [38,41,42] (Fig 1). In addition, *S. bovis* eggs are released in the
feces of infected animals, due to *S. bovis* locating around the mesenteric vessels. Thus, we
could expect that hybrid parasite eggs may also be released in part in the feces of human hosts.
This could explain, together with the low parasite intensity, why only 30% of patients infected
in Corsica had eggs that were able to be detected in their urine [43]. The route of excretion
associated with egg shape is the current standard for diagnostic and species determination,
however our results confirm earlier publications showing that it is impossible to detect hybrid-
ization in schistosome species using egg morphology alone [44]. Although adult worm mor-
phology has a limited interest for diagnostics in humans, principally because worms are not
accessible, they may be useful for a taxonomic purpose. Males and females of the European
hybrids were generally smaller (in length and width) when compared to both parental species
[45–49], but most interestingly, the presence of spines on the tubercles of the males, a trait not
found in *S. bovis* male, [50–52], was concordant with the predominance of *S. haematobium* in
the genetic make up of these hybrids.

Concerning serological diagnosis, the majority of commercial tests, ELISA or IHA (indirect
hemagglutination) use *S. mansoni* antigens. A discrepancy between those antigens and the
infecting species may induce false negative results [53]. The efficiency of these commercial
diagnostic kits thus needs to be reevaluated in a context of different species and hybrid forms.
Finally, molecular diagnostic for urogenital schistosomiasis using PCR has already been used
in urine or serum, targeting a highly repeated sequence (*Dra*I), which is restricted to the *S. hae-
matobium* group of schistosomes (including both *S. haematobium* and *S. bovis*) [54,55]. We
expect that this test would be efficient to detect such infection but not to identify the hybrid
status of the parasite.

**Implications for treatment**

Praziquantel (PZQ) is currently the main drug used to treat schistosomiasis and the applica-
tion of mass chemotherapy programs is the prevailing strategy for schistosomiasis control
[56]. PZQ is also efficient for treating bovine schistosomiasis, but the dose needed is quite high
(60 mg/kg for 95% deworming efficacy in goats [57,58]) and cannot be considered in endemic
areas where treatment capacities are primarily focused on human schistosomiasis. Moreover,
it has been shown in humans that a dose of 40 mg/kg of PZQ is only 63.5% efficient for mixed
infections, compared to 76.7% and 77.1%, for mono infections of *S. mansoni* and *S. haemato-
bium*, respectively [59]. To date, neither experimental nor field trials have tested the sensitivity
of hybrid parasites to PZQ. Thus, there is no current evidence that there is any difference in
drug response in natural infections and changes associated to PZQ response in hybrids is still
theoretical. However, a lower sensitivity to PZQ of *S. bovis* x *S. haematobium* hybrid schistosomes compared to pure *S. haematobium* parasites, although not tested, has been proposed to be at the origin of the spread of the hybrid forms in Senegal [60] and as discussed earlier hybridization may also affect Oxamniquine efficiency [61]. Since the genetic basis of Oxamniquine residence is known, our data suggests that OXA is not a treatment option.

## Conclusion

This work provides new insight into the *S. haematobium-bovis* hybrids that emerged, in Europe, revealing admixture between *S. haematobium* and *S. bovis* parasites. Such levels of genomic introgression appear to be the result of several ancient inter species crosses and subsequent backcrosses with parental species, as found in other genomic studies [11,15]. However, interestingly, it is the first time that such a high proportion of *S. bovis* has been identified within the genomic background of *S. haematobium*. As the European hybrid strain, analysed here, was originally recovered from a single infected patient infected in Corsica in 2014, it is now necessary to extend our conclusions to further infections in Corsica, and also to investigate the dynamics of these hybridization events in their original endemic areas of Africa, particularly Senegal which is thought to be the origin of the Corsican outbreak [8]. It is now also essential to precisely characterize the impact of hybridization and introgression on the parasites' life history traits, including sensitivity to current treatments and assess the molecular mechanisms underlying these phenotypic changes. These hybrids may indeed have the capacities to dominate the geographical distribution of the parental species, but also become established in new areas fueled by ongoing climate/habitat change, together with the increased movement of people.

## Materials and methods

### Ethics statement

Housing, feeding, animal care and experiments were carried out according to the national ethical standards established in the writ of 1 February 2013 (NOR: AGRG1238753A) and accordingly to the directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. The Direction Départementale de la Cohésion Sociale et de la Protection des Populations (DDSCPP) provided the permit N°C66-136-01 to our laboratory and approved experiments on animals. The investigator possesses the certificate for animal experimentation (Decree n° 87-848 du 19 octobre 1987; authorization 007083). Samples were collected within previous studies [8]. Informed consent was collected by the InVS (National institute for Public Health Surveillance) for each patient taking part to this outbreak investigation. Serological diagnostic test conducted for each patient were part of the (standard) diagnostic work-up for schistosomiasis. Data and specimens collected were transferred anonymously to Perpignan for egg detection. The study was approved by the French Commission for Data Protection (Commission Nationale de l'Informatique et des Libertés).

### Parasite / Snail strains and experimental infections

The European *S. haematobium-bovis* hybrid isolate originated from eggs isolated from the urine of a locally infected tourist from Corsica and maintained in laboratory passage in Corsican *B. truncatus* snails and the experimental vertebrate host, the golden hamster, *Mesocricetus auratus* [8]. Briefly, eggs from the urine sample were hatched in drilling water, and the resulting larvae (miracidia and then cercariae) were used to infect intermediate host snails and
theoretical. However, a lower sensitivity to PZQ of *S. bovis* x *S. haematobium* hybrid schistosomes compared to pure *S. haematobium* parasites, although not tested, has been proposed to be at the origin of the spread of the hybrid forms in Senegal [60] and as discussed earlier hybridization may also affect Oxamniquine efficiency [61]. Since the genetic basis of Oxamniquine residence is known, our data suggests that OXA is not a treatment option.

## Conclusion

This work provides new insight into the *S. haematobium-bovis* hybrids that emerged, in Europe, revealing admixture between *S. haematobium* and *S. bovis* parasites. Such levels of genomic introgression appear to be the result of several ancient inter species crosses and subsequent backcrosses with parental species, as found in other genomic studies [11,15]. However, interestingly, it is the first time that such a high proportion of *S. bovis* has been identified within the genomic background of *S. haematobium*. As the European hybrid strain, analysed here, was originally recovered from a single infected patient infected in Corsica in 2014, it is now necessary to extend our conclusions to further infections in Corsica, and also to investigate the dynamics of these hybridization events in their original endemic areas of Africa, particularly Senegal which is thought to be the origin of the Corsican outbreak [8]. It is now also essential to precisely characterize the impact of hybridization and introgression on the parasites' life history traits, including sensitivity to current treatments and assess the molecular mechanisms underlying these phenotypic changes. These hybrids may indeed have the capacities to dominate the geographical distribution of the parental species, but also become established in new areas fueled by ongoing climate/habitat change, together with the increased movement of people.

## Materials and methods

### Ethics statement

Housing, feeding, animal care and experiments were carried out according to the national ethical standards established in the writ of 1 February 2013 (NOR: AGRG1238753A) and accordingly to the directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. The Direction Départementale de la Cohésion Sociale et de la Protection des Populations (DDSCPP) provided the permit N°C66-136-01 to our laboratory and approved experiments on animals. The investigator possesses the certificate for animal experimentation (Decree n° 87-848 du 19 octobre 1987; authorization 007083). Samples were collected within previous studies [8]. Informed consent was collected by the InVS (National institute for Public Health Surveillance) for each patient taking part to this outbreak investigation. Serological diagnostic test conducted for each patient were part of the (standard) diagnostic work-up for schistosomiasis. Data and specimens collected were transferred anonymously to Perpignan for egg detection. The study was approved by the French Commission for Data Protection (Commission Nationale de l'Informatique et des Libertés).

### Parasite / Snail strains and experimental infections

The European *S. haematobium-bovis* hybrid isolate originated from eggs isolated from the urine of a locally infected tourist from Corsica and maintained in laboratory passage in Corsican *B. truncatus* snails and the experimental vertebrate host, the golden hamster, *Mesocricetus auratus* [8]. Briefly, eggs from the urine sample were hatched in drilling water, and the resulting larvae (miracidia and then cercariae) were used to infect intermediate host snails and
subsequently laboratory hamsters. Hamsters were euthanized by intraperitoneal injection of sodium pentobarbital (100 mg/kg). Adult worms were recovered from the hamsters, after partial perfusion and male/female couples were manually separated. Detailed methods are described previously [62]. *Schistosoma bovis* (isolated in 1970 in Villar de la Yegua, Salamanca, Spain) [63] and *Schistosoma haematobium* (isolated in 2015 in Barombi Kotto Lake, Cameroon) [64] were also maintained in the laboratory using *P. metidjensis* and *B. truncatus* as the intermediate hosts, respectively, and *M. auratus* hamsters as the definitive hosts [64]. F1 hybrids were produced after experimental cross between male *S. haematobium* from Cameroon and female *S. bovis* from Spain. Molluscs were infected with a single miracidium of the parental species to obtain male or female clonal cercariae, which were molecularly sexed as described in [64]. We simultaneously exposed hamsters to 300 cercariae of male *S. haematobium* and 300 cercariae of female *S. bovis*. Three months after infection, hamsters were euthanized, eggs were collected from the liver using a series of metal sieves (425, 180, 106, and 45 µm pore size) and F1 miracidia hatched in drilling water to infect molluscs. At latency infected snails were stimulated to produce cercariae which were used to infect hamsters (pools of 600 cercariae per hamster), with F1 hybrid adult male worms collected three months post infection.

## Hybrid parasite compatibility with snail hosts

*Planorbarius metidjensis* (n = 40) and *B. truncatus* (n = 40) snails were individually exposed overnight to five miracidia of the European *S. haematobium-bovis* hybrid strain, maintained in the laboratory, in 24-well plates containing 1ml of drilling water per well. The following morning molluscs were placed in the same tank at 26°C with a 12:12 light cycle and fed *ad libitum* for the duration of the experiment. At 35 days after infection, corresponding to the development time of the parasites in their intermediate host, snails were individually checked for parasite emission of the cercariae after light stimulation for 4 hours.

## Morphological analysis of the European *S. haematobium-bovis* hybrid eggs and adult worms

Morphometric analysis of *Schistosoma* eggs is a classical way to identify species. Since eggs are easily accessible in the field, excreted within stool or urine, they are commonly used to diagnose the infecting *Schistosoma* species. Twenty adult worm pairs (20 males and 20 females) were collected three months post infection after hepatic perfusion together with encysted eggs from the hamster livers. Adult worms and eggs were washed in 8.5% w/v Tris-NaCl solution for subsequent morphological analysis. Male and female worms were manually separated and stored at -80°C before subsequent DNA or RNA extraction.

After being whole-mounted on glass slides [65] the adult worms and eggs were viewed under a light microscopy and photographed using a Wild Heerbrugg M400 ZOOM Makroskop (Leica, Germany) or Dialux20 (Leitz, Germany) coupled to Nikon digital sight DS-F11 digital camera. All measurements were produced with ImageJ version 1.51 [66] using a real graduated scale to set up pixel numbers vs. mm correspondance, and drawings were done by image overlay in Adobe Photoshop CS2 version 9.0.1.

For the adult worms, the following characters were measured: worm length and width, orientation of the oral and ventral suckers, the sucker's ratio, sucker ratio per worm length and distance from the genital opening to the anterior region. Additionally, in female worms the area of the ovary and extension of the vitellarium were measured. In males we also recorded the number of testes and the presence or absence of tegumental tubercles and characteristic
subsequently laboratory hamsters. Hamsters were euthanized by intraperitoneal injection of sodium pentobarbital (100 mg/kg). Adult worms were recovered from the hamsters, after partial perfusion and male/female couples were manually separated. Detailed methods are described previously [62]. *Schistosoma bovis* (isolated in 1970 in Villar de la Yegua, Salamanca, Spain) [63] and *Schistosoma haematobium* (isolated in 2015 in Barombi Kotto Lake, Cameroon) [64] were also maintained in the laboratory using *P. metidjensis* and *B. truncatus* as the intermediate hosts, respectively, and *M. auratus* hamsters as the definitive hosts [64]. F1 hybrids were produced after experimental cross between male *S. haematobium* from Cameroon and female *S. bovis* from Spain. Molluscs were infected with a single miracidium of the parental species to obtain male or female clonal cercariae, which were molecularly sexed as described in [64]. We simultaneously exposed hamsters to 300 cercariae of male *S. haematobium* and 300 cercariae of female *S. bovis*. Three months after infection, hamsters were euthanized, eggs were collected from the liver using a series of metal sieves (425, 180, 106, and 45 µm pore size) and F1 miracidia hatched in drilling water to infect molluscs. At latency infected snails were stimulated to produce cercariae which were used to infect hamsters (pools of 600 cercariae per hamster), with F1 hybrid adult male worms collected three months post infection.

## Hybrid parasite compatibility with snail hosts

*Planorbarius metidjensis* (n = 40) and *B. truncatus* (n = 40) snails were individually exposed overnight to five miracidia of the European *S. haematobium-bovis* hybrid strain, maintained in the laboratory, in 24-well plates containing 1ml of drilling water per well. The following morning molluscs were placed in the same tank at 26°C with a 12:12 light cycle and fed *ad libitum* for the duration of the experiment. At 35 days after infection, corresponding to the development time of the parasites in their intermediate host, snails were individually checked for parasite emission of the cercariae after light stimulation for 4 hours.

## Morphological analysis of the European *S. haematobium-bovis* hybrid eggs and adult worms

Morphometric analysis of *Schistosoma* eggs is a classical way to identify species. Since eggs are easily accessible in the field, excreted within stool or urine, they are commonly used to diagnose the infecting *Schistosoma* species. Twenty adult worm pairs (20 males and 20 females) were collected three months post infection after hepatic perfusion together with encysted eggs from the hamster livers. Adult worms and eggs were washed in 8.5% w/v Tris-NaCl solution for subsequent morphological analysis. Male and female worms were manually separated and stored at -80°C before subsequent DNA or RNA extraction.

After being whole-mounted on glass slides [65] the adult worms and eggs were viewed under a light microscopy and photographed using a Wild Heerbrugg M400 ZOOM Makroskop (Leica, Germany) or Dialux20 (Leitz, Germany) coupled to Nikon digital sight DS-F11 digital camera. All measurements were produced with ImageJ version 1.51 [66] using a real graduated scale to set up pixel numbers vs. mm correspondance, and drawings were done by image overlay in Adobe Photoshop CS2 version 9.0.1.

For the adult worms, the following characters were measured: worm length and width, orientation of the oral and ventral suckers, the sucker's ratio, sucker ratio per worm length and distance from the genital opening to the anterior region. Additionally, in female worms the area of the ovary and extension of the vitellarium were measured. In males we also recorded the number of testes and the presence or absence of tegumental tubercles and characteristic
tubercle spines. For eggs, the objective lens magnification was set to x10 and we measured the length (including the spine), the width (at its largest point) and the size of the terminal spine.

## DNA extraction and sequencing for *Schistosoma bovis* assembly

Two hundred clonal adult male worms of *Schistosoma bovis* from Spain were produced after monomiracidial infection of snails, sexing and hamster portal perfusion as described above. High molecular weight genomic DNA was prepared using CHEF Genomic DNA Plug Kits (BioRad). The DNA was quantified on a FEMTO Pulse, qubit and nanodrop. A total of 8.1 µg genomic DNA was used to generate a size selected PacBio library. First the DNA was sheared to an average fragment size of 45 kb by gently passing the DNA sample through a 2" long, 26 gauge needle, four times and then concentrated using Ampure PB (Pacific Biosciences 100-265-900) before the library was prepared following the standard PacBio size selected library preparation protocol using the BluePippin Size-Selection System. The library was size selected at 15kb, and run on 6 SMRT cells on the Sequel platform, generating 47.9 Gb of data.

## RNA extraction and sequencing for *Schistosoma bovis* annotation

Pools of 10–12 adult male or female worms of *Schistosoma bovis* from Spain isolated from infected hamsters were frozen with liquid nitrogen and ground using a Retsch MM400 cryo-brush (2 pulses at 300Hz for 15s). Total RNA was extracted using TRIzol Thermo (Fisher Scientific) followed by DNase treatment with Turbo DNA-free kit. RNA was then purified using the RNeasy mini kit (Qiagen). The TruSeq stranded mRNA library construction kit (Illumina) was used on 300 ng of total RNA per condition. Library preparation and sequencing was performed at the McGill University in the Génome Québec Innovation Centre, Montréal, Canada on a Illumina HiSeq 4000 (100 bp paired-end reads).

### *Schistosoma bovis* genome assembly and annotation

PacBio reads were assembled using the Hierarchical Genome Assembly Process (HGAP4) de novo assembly analysis application, followed by polishing with Pilon using consensus PacBio reads and illumina short reads. Gene prediction was carried out with AUGUSTUS 3.3.1. No new training was performed for our data but the parameter set “schistosoma2” of the AUGUSTUS distribution was used. RepeatMasker 4.0.7 and RepeatScout 1.0.5 were used for repeat prediction and masking. RNAseq data from male and female adult worms was employed as external hints. This RNA-seq data was aligned with STAR version 020201. The Benchmarking Universal Single-Copy Orthologs (BUSCO) version 3.0.2 [19] was used in—genome mode and with the metazoa_odb9 dataset to assess genome assembly and annotation completeness.

## DNA extraction and sequencing for the European *S. haematobium-bovis* hybrid and experimental *S. haematobium* x *S. bovis* F1 adult worms

Genomic DNA of the European schistosome hybrid was recovered from one pool of 10 adult males and one pool of 40 adult females separately, while DNA of the experimental F1 progeny from the inter-species cross was recovered from a pool of 10 adult males. DNA was extracted using the Qiamp DNA Micro Kit tissue kit (Qiagen) followed by RNase A treatment. Genomic DNA of the European hybrid worms was then sent to Genome Quebec (https://www.genomequebec.com/) for library construction using the Illumina TruSeq kit starting from 200 ng of genomic DNA (for both males and females), and sequencing was performed on a Illumina HiSeq 2000 (100 bp paired-end reads). For the experimental F1 hybrid males, library construction was performed using the Nextera XT kit starting from 1 ng and sequenced on a
tubercle spines. For eggs, the objective lens magnification was set to x10 and we measured the length (including the spine), the width (at its largest point) and the size of the terminal spine.

## DNA extraction and sequencing for *Schistosoma bovis* assembly

Two hundred clonal adult male worms of *Schistosoma bovis* from Spain were produced after monomiracidial infection of snails, sexing and hamster portal perfusion as described above. High molecular weight genomic DNA was prepared using CHEF Genomic DNA Plug Kits (BioRad). The DNA was quantified on a FEMTO Pulse, qubit and nanodrop. A total of 8.1 µg genomic DNA was used to generate a size selected PacBio library. First the DNA was sheared to an average fragment size of 45 kb by gently passing the DNA sample through a 2" long, 26 gauge needle, four times and then concentrated using Ampure PB (Pacific Biosciences 100-265-900) before the library was prepared following the standard PacBio size selected library preparation protocol using the BluePippin Size-Selection System. The library was size selected at 15kb, and run on 6 SMRT cells on the Sequel platform, generating 47.9 Gb of data.

## RNA extraction and sequencing for *Schistosoma bovis* annotation

Pools of 10–12 adult male or female worms of *Schistosoma bovis* from Spain isolated from infected hamsters were frozen with liquid nitrogen and ground using a Retsch MM400 cryo-brush (2 pulses at 300Hz for 15s). Total RNA was extracted using TRIzol Thermo (Fisher Scientific) followed by DNase treatment with Turbo DNA-free kit. RNA was then purified using the RNeasy mini kit (Qiagen). The TruSeq stranded mRNA library construction kit (Illumina) was used on 300 ng of total RNA per condition. Library preparation and sequencing was performed at the McGill University in the Génome Québec Innovation Centre, Montréal, Canada on a Illumina HiSeq 4000 (100 bp paired-end reads).

## *Schistosoma bovis* genome assembly and annotation

PacBio reads were assembled using the Hierarchical Genome Assembly Process (HGAP4) de novo assembly analysis application, followed by polishing with Pilon using consensus PacBio reads and illumina short reads. Gene prediction was carried out with AUGUSTUS 3.3.1. No new training was performed for our data but the parameter set “schistosoma2” of the AUGUSTUS distribution was used. RepeatMasker 4.0.7 and RepeatScout 1.0.5 were used for repeat prediction and masking. RNAseq data from male and female adult worms was employed as external hints. This RNA-seq data was aligned with STAR version 020201. The Benchmarking Universal Single-Copy Orthologs (BUSCO) version 3.0.2 [19] was used in—genome mode and with the metazoa_odb9 dataset to assess genome assembly and annotation completeness.

## DNA extraction and sequencing for the European *S. haematobium-bovis* hybrid and experimental *S. haematobium* x *S. bovis* F1 adult worms

Genomic DNA of the European schistosome hybrid was recovered from one pool of 10 adult males and one pool of 40 adult females separately, while DNA of the experimental F1 progeny from the inter-species cross was recovered from a pool of 10 adult males. DNA was extracted using the Qiamp DNA Micro Kit tissue kit (Qiagen) followed by RNase A treatment. Genomic DNA of the European hybrid worms was then sent to Genome Quebec (https://www.genomequebec.com/) for library construction using the Illumina TruSeq kit starting from 200 ng of genomic DNA (for both males and females), and sequencing was performed on a Illumina HiSeq 2000 (100 bp paired-end reads). For the experimental F1 hybrid males, library construction was performed using the Nextera XT kit starting from 1 ng and sequenced on a
Illumina NextSeq 550 (150 bp paired-end reads) on the Bio-Environment NGS platform at
University of Perpignan.

Estimation of the genomic introgression levels for the hybrid strain

The sequencing reads, with PHRED quality scores over 30, with no adapter contamination
were retained for further analysis and aligned to a chimeric concatenate of the *S. haematobium*
and *S. bovis* genomes using Bowtie v2.3.3.31 [67]. We used the SchistoDB *S. haematobium*
genome v1 [17] and the *S. bovis* SBOS_v1.1 assembly genome produced for this study. To
avoid mapping bias due to differences in assembly size between the two genomes, only scaf-
folds >1Mb were retained for further analyses. The genome size after concatenation of *S. hae-
matobium* and *S. bovis* was 681.2 Mb. Mapping was thus performed by allowing each read,
depending on its origin, to map against the more similar location in either the *S. haematobium*
or *S. bovis* genome. We then counted the proportion of best locations of aligned reads (*S. bovis*
or *S. haematobium* genomes) in the SAM files. The same procedure was applied for the mito-
chondrial genomes using a concatenate of the scaffold 000439F that contained the mitochon-
drial genomes of *S. bovis* (this study) and *S. haematobium* (GenBank accession NC_008074).

Similarity analysis between the *S. haematobium* and *S. bovis* genomes

The two genomes were aligned using CACTUS [22]. We then processed the output, identify-
ing all alignments blocks composed of exactly one genome part *S. bovis* and exactly one part *S.
haematobium* (S1 Table). Orthologous region for SmSULT-OR (Smp_089320) was retrieved
undertaking reciprocal homology searches (BlastX) against *S. haematobium* and the new *S.
bovis* genome, generated as part of this study.

**Supporting information**

S1 Table. Global alignment summary between *Schistosoma haematobium* and *Schistosoma bovis* genomes. For each scaffold of the *S. haematobium* genome, the scaffold length and the number of nucleotides, which were uniquely aligned to the *S. bovis* genome, are reported. (XLSX)

Acknowledgments

We would like to acknowledge the NGS facility at the bio-environment platform (University of Perpignan) and DNA Pipelines at Wellcome Sanger Institute, in particular Michelle Smith and Craig Corton. This study is set within the framework of the « Laboratoire d'Excellence (LabEx) » TULIP (ANR-10-LABX-41).

Author Contributions

Conceptualization: Jérôme Boissier, Eve Toulza.

**Formal analysis:** Julien Kincaid-Smith, Alan Tracey, Ronaldo de Carvalho Augusto, Ingo Bulla, Nancy Holroyd, Anne Rognon, Olivier Rey, Cristian Chaparro, Jean-François Allienne, Christoph Grunau, Matthew Berriman, Jérôme Boissier, Eve Toulza.

**Project administration:** Nancy Holroyd.

**Resources:** Ana Oleaga, Santiago Mas-Coma.

**Supervision:** Jérôme Boissier, Eve Toulza.

**Writing – original draft:** Julien Kincaid-Smith, Eve Toulza.
Illumina NextSeq 550 (150 bp paired-end reads) on the Bio-Environment NGS platform at University of Perpignan.

**Estimation of the genomic introgression levels for the hybrid strain**

The sequencing reads, with PHRED quality scores over 30, with no adapter contamination were retained for further analysis and aligned to a chimeric concatenate of the *S. haematobium* and *S. bovis* genomes using Bowtie v2.3.3.31 [67]. We used the SchistoDB *S. haematobium* genome v1 [17] and the *S. bovis* SBOS_v1.1 assembly genome produced for this study. To avoid mapping bias due to differences in assembly size between the two genomes, only scaffolds >1Mb were retained for further analyses. The genome size after concatenation of *S. haematobium* and *S. bovis* was 681.2 Mb. Mapping was thus performed by allowing each read, depending on its origin, to map against the more similar location in either the *S. haematobium* or *S. bovis* genome. We then counted the proportion of best locations of aligned reads (*S. bovis* or *S. haematobium* genomes) in the SAM files. The same procedure was applied for the mitochondrial genomes using a concatenate of the scaffold 000439F that contained the mitochondrial genomes of *S. bovis* (this study) and *S. haematobium* (GenBank accession NC_008074).

**Similarity analysis between the *S. haematobium* and *S. bovis* genomes**

The two genomes were aligned using CACTUS [22]. We then processed the output, identifying all alignments blocks composed of exactly one genome part *S. bovis* and exactly one part *S. haematobium* (S1 Table). Orthologous region for SmSULT-OR (Smp_089320) was retrieved undertaking reciprocal homology searches (BlastX) against *S. haematobium* and the new *S. bovis* genome, generated as part of this study.

**Supporting information**

S1 Table. Global alignment summary between *Schistosoma haematobium* and *Schistosoma bovis* genomes. For each scaffold of the *S. haematobium* genome, the scaffold length and the number of nucleotides, which were uniquely aligned to the *S. bovis* genome, are reported. (XLSX)

**Acknowledgments**

We would like to acknowledge the NGS facility at the bio-environment platform (University of Perpignan) and DNA Pipelines at Wellcome Sanger Institute, in particular Michelle Smith and Craig Corton. This study is set within the framework of the « Laboratoire d'Excellence (LabEx) » TULIP (ANR-10-LABX-41).

**Author Contributions**

**Conceptualization:** Jérôme Boissier, Eve Toulza.

**Formal analysis:** Julien Kincaid-Smith, Alan Tracey, Ronaldo de Carvalho Augusto, Ingo Bulla, Nancy Holroyd, Anne Rognon, Olivier Rey, Cristian Chaparro, Jean-François Allienne, Christoph Grunau, Matthew Berriman, Jérôme Boissier, Eve Toulza.

**Project administration:** Nancy Holroyd.

**Resources:** Ana Oleaga, Santiago Mas-Coma.

**Supervision:** Jérôme Boissier, Eve Toulza.

**Writing – original draft:** Julien Kincaid-Smith, Eve Toulza.
**Writing – review & editing:** Julien Kincaid-Smith, Matthew Berriman, Eve Toulza.

References

1. Chitsulo L, Engels D, Montresor A, Savioli L. The global status of schistosomiasis and its control. Acta Trop. 2000; 77: 41–51. https://doi.org/10.1016/s0001-706x(00)00122-4 PMID: 10996119

2. De Bont J, Vercruysse J. The epidemiology and control of cattle schistosomiasis. Parasitology Today. 1997. pp. 255–262. https://doi.org/10.1016/s0169-4758(97)01057-0 PMID: 15275062

3. Mawa PA, Kincaid-Smith J, Tukahebwa EM, Webster JP, Wilson S. Schistosomiasis Morbidity Hot-spots: Roles of the Human Host, the Parasite and Their Interface in the Development of Severe Morbidity. Front Immunol. 2021; 12: 1–21. https://doi.org/10.3389/fimmu.2021.635869 PMID: 33790908

4. Wu X, Lu Y, Zhou S, Chen L, Xu B. Impact of climate change on human infectious diseases: Empirical evidence and human adaptation. Environ Int. 2016; 86: 14–23. https://doi.org/10.1016/j.envint.2015.09.007 PMID: 26479830

5. Kim K-H, Kabir E, Ara Jahan S. A review of the consequences of global climate change on human health. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2014; 32: 299–318. https://doi.org/10.1080/10590501.2014.941279 PMID: 25226222

6. Cockayne L, Allan HH. The naming of wild hybrid swarms. Nature. Nature Publishing Group; 1926. pp. 623–624. https://doi.org/10.1038/118623a0

7. Bar-Zvi D, Lupo O, Levy AA, Barkai N. Hybrid vigor: The best of both parents, or a genomic clash? Curr Opin Syst Biol. 2017; 6: 22–27. https://doi.org/10.1016/j.coisb.2017.08.004

8. Boissier J, Grech-Angelini S, Webster BLBLBL, Allienne J-FJ-FFJ-F, Huyse T, Mas-Coma S, et al. Out-break of urogenital schistosomiasis in Corsica (France): an epidemiological case study. Lancet Infect Dis. 2016; 16: 1–9. https://doi.org/10.1016/S1473-3099(15)00504-6 PMID: 26738818

9. King KC, Stelkens RB, Webster JP, Smith DF, Brockhurst MA. Hybridization in Parasites: Conse-quences for Adaptive Evolution, Pathogenesis, and Public Health in a Changing World. PLoS Pathog. 2015; 11: 1–12. https://doi.org/10.1371/journal.ppat.1005098 PMID: 26336070

10. Leger E, Webster JP. Hybridizations within the Genus Schistosoma: Implications for evolution, epidemi-ology and control. Parasitology. 2017. pp. 65–80. https://doi.org/10.1017/S0031182016001190 PMID: 27572906

11. Platt RN, McDew-White M, Le Clec'H W, Chevalier FD, Allan F, Emery AM, et al. Ancient Hybridization and Adaptive Introgression of an Invadolsin Gene in Schistosome Parasites. Mol Biol Evol. 2019; 36: 2127–2142. https://doi.org/10.1093/molbev/msz154 PMID: 31251352

12. Van den Broeck F, Savill NJ, Imamura H, Sanders M, Maes I, Cooper S, et al. Ecological divergence and hybridization of Neotropical Leishmania parasites. Proc Natl Acad Sci U S A. 2020; 117: 25159–25168. https://doi.org/10.1073/pnas.1920136117 PMID: 32958676

13. Tihon E, Imamura H, Dujardin J-C, Van Den Abbeele J, Van den Broeck F. Discovery and genomic analyses of hybridization between divergent lineages of Trypanosoma congolense, causative agent of Animal African Trypanosomiasis. Mol Ecol. 2017; 26: 6524–6538. https://doi.org/10.1111/mec.14271 PMID: 28752916

14. Oey H, Zakrzewski M, Gravermann K, Young ND, Korhonen PK, Gobert GN, et al. Whole-genome sequence of the bovine blood fluke Schistosoma bovis supports interspecific hybridization with S. Hae-matobium. PLoS Pathog. 2019; 15: 1–16. https://doi.org/10.1371/journal.ppat.1007513 PMID: 30673782

15. Rey O, Toulza E, Chaparro C, Allienne JF, Kincaid-Smith J, Mathieu-Bégné E, et al. Diverging patterns of introgression from Schistosoma bovis across S. haematobium African lineages. PLoS Pathog. 2021; 17: 1–20. https://doi.org/10.1371/journal.ppat.1009313 PMID: 33544762

16. Kincaid-Smith J, Mathieu-Bégné E, Chaparro C, Reguera-Gomez M, Mulero S, Allienne JF, et al. No pre-zygotic isolation mechanisms between schistosoma haematobium and schistosoma bovis para-sites: From mating interactions to differential gene expression. PLoS Negl Trop Dis. 2021; 15: 1–24. https://doi.org/10.1371/journal.pntd.0009363 PMID: 33945524

17. Young ND, Jex AR, Li B, Liu S, Yang L, Xiong Z, et al. Whole-genome sequence of Schistosoma hae-matobium. Nat Genet. 2012; 44: 221–225. https://doi.org/10.1038/ng.1065 PMID: 22246508

18. Stroehlein AJ, Korhonen PK, Chong TM, Lim YL, Chan KG, Webster B, et al. High-quality Schistosoma haematobium genome achieved by single-molecule and long-range sequencing. Gigascience. 2019; 8: 1–12. https://doi.org/10.1093/gigscience/giz108 PMID: 31494670
**Writing – review & editing:** Julien Kincaid-Smith, Matthew Berriman, Eve Toulza.

**References**

1. Chitsulo L, Engels D, Montresor A, Savioli L. The global status of schistosomiasis and its control. *Acta Trop.* 2000; 77: 41–51. https://doi.org/10.1016/s0001-706x(00)00122-4 PMID: 10996119

2. De Bont J, Vercruysse J. The epidemiology and control of cattle schistosomiasis. *Parasitology Today*. 1997. pp. 255–262. https://doi.org/10.1016/s0169-4758(97)01057-0 PMID: 15275062

3. Mawa PA, Kincaid-Smith J, Tukahebwa EM, Webster JP, Wilson S. Schistosomiasis Morbidity Hot-spots: Roles of the Human Host, the Parasite and Their Interface in the Development of Severe Morbidity. *Front Immunol.* 2021; 12: 1–21. https://doi.org/10.3389/fimmu.2021.635869 PMID: 33790908

4. Wu X, Lu Y, Zhou S, Chen L, Xu B. Impact of climate change on human infectious diseases: Empirical evidence and human adaptation. *Environ Int.* 2016; 86: 14–23. https://doi.org/10.1016/j.envint.2015.09.007 PMID: 26479830

5. Kim K-H, Kabir E, Ara Jahan S. A review of the consequences of global climate change on human health. *J Environ Sci Health C Environ Carcinog Ecotoxicol Rev.* 2014; 32: 299–318. https://doi.org/10.1080/10590501.2014.941279 PMID: 25226222

6. Cockayne L, Allan HH. The naming of wild hybrid swarms. *Nature*. *Nature Publishing Group*; 1926. pp. 623–624. https://doi.org/10.1038/118623a0

7. Bar-Zvi D, Lupo O, Levy AA, Barkai N. Hybrid vigor: The best of both parents, or a genomic clash? *Curr Opin Syst Biol.* 2017; 6: 22–27. https://doi.org/10.1016/j.coisb.2017.08.004

8. Boissier J, Grech-Angelini S, Webster BLLBL, Allienne J-FJ-FFJ-F, Huyse T, Mas-Coma S, et al. Out-break of urogenital schistosomiasis in Corsica (France): an epidemiological case study. *Lancet Infect Dis.* 2016; 16: 1–9. https://doi.org/10.1016/S1473-3099(15)00504-6 PMID: 26738818

9. King KC, Stelkens RB, Webster JP, Smith DF, Brockhurst MA. Hybridization in Parasites: Conse-quences for Adaptive Evolution, Pathogenesis, and Public Health in a Changing World. *PLoS Pathog.* 2015; 11: 1–12. https://doi.org/10.1371/journal.ppat.1005098 PMID: 26336070

10. Leger E, Webster JP. Hybridizations within the Genus Schistosoma: Implications for evolution, epidemi-ology and control. *Parasitology*. 2017. pp. 65–80. https://doi.org/10.1017/S0031182016001190 PMID: 27572906

11. Platt RN, McDew-White M, Le Clec'H W, Chevalier FD, Allan F, Emery AM, et al. Ancient Hybridization and Adaptive Introgression of an Invadolsin Gene in Schistosome Parasites. *Mol Biol Evol.* 2019; 36: 2127–2142. https://doi.org/10.1093/molbev/msz154 PMID: 31251352

12. Van den Broeck F, Savill NJ, Imamura H, Sanders M, Maes I, Cooper S, et al. Ecological divergence and hybridization of Neotropical Leishmania parasites. *Proc Natl Acad Sci U S A.* 2020; 117: 25159–25168. https://doi.org/10.1073/pnas.1920136117 PMID: 32958676

13. Tihon E, Imamura H, Dujardin J-C, Van Den Abbeele J, Van den Broeck F. Discovery and genomic analyses of hybridization between divergent lineages of Trypanosoma congolense, causative agent of Animal African Trypanosomiasis. *Mol Ecol.* 2017; 26: 6524–6538. https://doi.org/10.1111/mec.14271 PMID: 28752916

14. Oey H, Zakrzewski M, Gravermann K, Young ND, Korhonen PK, Gobert GN, et al. Whole-genome sequence of the bovine blood fluke Schistosoma bovis supports interspecific hybridization with S. Haematobium. *PLoS Pathog.* 2019; 15: 1–16. https://doi.org/10.1371/journal.ppat.1007513 PMID: 30673782

15. Rey O, Toulza E, Chaparro C, Allienne JF, Kincaid-Smith J, Mathieu-Begné E, et al. Diverging patterns of introgression from Schistosoma bovis across S. haematobium African lineages. *PLoS Pathog.* 2021; 17: 1–20. https://doi.org/10.1371/journal.ppat.1009313 PMID: 33544762

16. Kincaid-Smith J, Mathieu-Bégné E, Chaparro C, Reguera-Gomez M, Mulero S, Allienne JF, et al. No pre-zygotic isolation mechanisms between schistosoma haematobium and schistosoma bovis para-sites: From mating interactions to differential gene expression. *PLoS Negl Trop Dis.* 2021; 15: 1–24. https://doi.org/10.1371/journal.pntd.0009363 PMID: 33945524

17. Young ND, Jex AR, Li B, Liu S, Yang L, Xiong Z, et al. Whole-genome sequence of Schistosoma hae-matobium. *Nat Genet.* 2012; 44: 221–225. https://doi.org/10.1038/ng.1065 PMID: 22246508

18. Stroehlein AJ, Korhonen PK, Chong TM, Lim YL, Chan KG, Webster B, et al. High-quality Schistosoma haematobium genome achieved by single-molecule and long-range sequencing. *Gigascience*. 2019; 8: 1–12. https://doi.org/10.1093/gigascience/giz108 PMID: 31494670
19. Simão FA, Waterhouse RM, Ioannidis P, Kriventseva E V, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31: 3210–3212. https://doi.org/10.1093/bioinformatics/btv351 PMID: 26059717

20. Buddenberg SK, Tracey A, Berger DJ, Lu Z, Doyle SR, Fu B, et al. Assembled chromosomes of the blood fluke Schistosoma mansoni provide insight into the evolution of its ZW sex-determination system. bioRxiv. 2021; 2021.08.13.456314. https://doi.org/10.1101/2021.08.13.456314

21. Olson PD, Tracey A, Baillie A, James K, Doyle SR, Buddenberg SK, et al. Complete representation of a tapeworm genome reveals chromosomes capped by centromeres, necessitating a dual role in segregation and protection. BMC Biol. 2020; 18: 165. https://doi.org/10.1186/s12915-020-00899-w PMID: 33167983

22. Paten B, Earl D, Nguyen N, Diekhans M, Zerbino D, Haussler D. Cactus: Algorithms for genome multiple sequence alignment. Genome Res. 2011; 21: 1512–1528. https://doi.org/10.1101/gr.123356.111 PMID: 21665927

23. Chevalier FD, Le Clec'h W, McDew-White M, Menon V, Guzman MA, Holloway SP, et al. Oxamniquine resistance alleles are widespread in Old World Schistosoma mansoni and predate drug deployment. PLoS Pathog. 2019; 15: 1–25. https://doi.org/10.1371/journal.ppat.1007881 PMID: 31652296

24. Valentim CLL, Cioli D, Chevalier FD, Cao X, Taylor AB, Holloway SP, et al. Genetic and molecular basis of drug resistance and species-specific drug action in schistosome parasites. Science. 2013; 342: 1385–1389. https://doi.org/10.1126/science.1243106 PMID: 24263136

25. Detwiler JT, Criscione CD. An infectious topic in reticulate evolution: Introgression and hybridization in animal parasites. Genes. 2010. pp. 102–123. https://doi.org/10.3390/genes1010102 PMID: 24710013

26. Léger E, Borlase A, Fall CB, Diouf ND, Diop SD, Yasenev L, et al. Prevalence and distribution of schistosomiasis in human, livestock, and snail populations in northern Senegal: a One Health epidemiological study of a multi-host system. Lancet Planet Heal. 2020; 4: e330–e342. https://doi.org/10.1016/S2542-5196(20)30129-7 PMID: 32800151

27. Savassi BAES, Mouahid G, Lasica, Mahaman SDK, Garcia A, Courtin D, et al. Cattle as natural host for Schistosoma haematobium (Bilharz, 1852) Weinland, 1858 x Schistosoma bovis Sonsino, 1876 interactions, with new cercarial emergence and genetic patterns. Parasitol Res. 2020; 119: 2189–2205. https://doi.org/10.1007/s00436-020-06709-0 PMID: 32468189

28. Soentjens P, Cnops L, Huyse T, Yansouni C, De Vos D, Bottieau E, et al. Diagnosis and Clinical Management of Schistosoma haematobium-Schistosoma bovis Hybrid Infection in a Cluster of Travelers Returning From Mali. Clin Infect Dis. 2016; 63: 1626–1629. https://doi.org/10.1093/cid/ciw493 PMID: 27941144

29. Pennance T, Allan F, Emery A, Rabone M, Cable J, Garba AD, et al. Interactions between Schistosoma haematobium group species and their Bulinus spp. intermediate hosts along the Niger River Valley. Parasites and Vectors. 2020; 13: 1–15. https://doi.org/10.1186/s13071-019-3862-4 PMID: 31900233

30. Tian-Bi YNT, Webster B, Konan CK, Allan F, Diakité NR, Ouattara M, et al. Molecular characterization and distribution of Schistosoma cercariae collected from naturally infected bulinid snails in northern and central Côte d'Ivoire. Parasites and Vectors. 2019; 12: 1–10. https://doi.org/10.1186/s13071-018-3256-z PMID: 30606222

31. Holtfreter MC, Moné H, Müller-Stöver I, Mouahid G, Richter J. Schistosoma haematobium infections acquired in Corsica, France, August 2013. Eurosurveillance. 2014; 19.

32. Moné H, Holtfreter MC, Allienne JF, Mintsa-Nguéma R, Ibikounlé M, Boissier J, et al. Introgressive hybridizations of Schistosoma haematobium by Schistosoma bovis at the origin of the first case report of schistosomiasis in Corsica (France, Europe). Parasitol Res. 2015; 114: 4127–4133. https://doi.org/10.1007/s00436-015-4643-4 PMID: 26268566

33. Catalano S, Sène M, Diouf ND, Fall CB, Borlase A, Léger E, et al. Rodents as Natural Hosts of Zoonotic Schistosoma Species and Hybrids: An Epidemiological and Evolutionary Perspective From West Africa. J Infect Dis. 2018; jiy029. https://doi.org/10.1093/infdis/jiy029 PMID: 29365139

34. Catalano S, Léger E, Fall CB, Borlase A, Diop SD, Berger D, et al. Multihost Transmission of Schistosoma mansoni in Senegal, 2015–2018. Emerg Infect Dis J. 2020; 26: 1234. https://doi.org/10.3201/eid2606.200107 PMID: 32441625

35. Savassi BAES, Dobigny G, Etougbétché JR, Avocegan TT, Quinsou FT, Gauthier P, et al. Mastomys natalensis (Smith, 1834) as a natural host for Schistosoma haematobium (Bilharz, 1852) Weinland, 1858 x Schistosoma bovis Sonsino, 1876 introgressive hybrids. Parasitol Res. 2021; 120: 1755–1770. https://doi.org/10.1007/s00436-021-07099-7 PMID: 33687566

36. Rothe C, Zimmer T, Schunk M, Wallrauch C, Helfrich K, Gültekin F, et al. Developing endemicity of schistosomiasis, Corsica, France. Emerg Infect Dis. 2021; 27: 319–321. https://doi.org/10.3201/EID2701.204391 PMID: 33264582
19. Simão FA, Waterhouse RM, Ioannidis P, Kriventseva E V, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31: 3210–3212. https://doi.org/10.1093/bioinformatics/btv351 PMID: 26059717

20. Buddenberg SK, Tracey A, Berger DJ, Lu Z, Doyle SR, Fu B, et al. Assembled chromosomes of the blood fluke Schistosoma mansoni provide insight into the evolution of its ZW sex-determination system. bioRxiv. 2021; 2021.08.13.456314. https://doi.org/10.1101/2021.08.13.456314

21. Olson PD, Tracey A, Baillie A, James K, Doyle SR, Buddenberg SK, et al. Complete representation of a tapeworm genome reveals chromosomes capped by centromeres, necessitating a dual role in segregation and protection. BMC Biol. 2020; 18: 165. https://doi.org/10.1186/s12915-020-00899-w PMID: 33167983

22. Paten B, Earl D, Nguyen N, Diekhans M, Zerbino D, Haussler D. Cactus: Algorithms for genome multiple sequence alignment. Genome Res. 2011; 21: 1512–1528. https://doi.org/10.1101/gr.123356.111 PMID: 21665927

23. Chevalier FD, Le Clec'h W, McDew-White M, Menon V, Guzman MA, Holloway SP, et al. Oxamniquine resistance alleles are widespread in Old World Schistosoma mansoni and predate drug deployment. PLoS Pathog. 2019; 15: 1–25. https://doi.org/10.1371/journal.ppat.1007881 PMID: 31652296

24. Valentim CLL, Cioli D, Chevalier FD, Cao X, Taylor AB, Holloway SP, et al. Genetic and molecular basis of drug resistance and species-specific drug action in schistosome parasites. Science. 2013; 342: 1385–1389. https://doi.org/10.1126/science.1243106 PMID: 24263136

25. Detwiler JT, Criscione CD. An infectious topic in reticulate evolution: Introgression and hybridization in animal parasites. Genes. 2010. pp. 102–123. https://doi.org/10.3390/genes1010102 PMID: 24710013

26. Léger E, Borlase A, Fall CB, Diouf ND, Diop SD, Yasenev L, et al. Prevalence and distribution of schistosomiasis in human, livestock, and snail populations in northern Senegal: a One Health epidemiological study of a multi-host system. Lancet Planet Heal. 2020; 4: e330–e342. https://doi.org/10.1016/S2542-5196(20)30129-7 PMID: 32800151

27. Savassi BAES, Mouahid G, Lasica, Mahaman SDK, Garcia A, Courtin D, et al. Cattle as natural host for Schistosoma haematobium (Bilharz, 1852) Weinland, 1858 x Schistosoma bovis Sonsino, 1876 interactions, with new cercarial emergence and genetic patterns. Parasitol Res. 2020; 119: 2189–2205. https://doi.org/10.1007/s00436-020-06709-0 PMID: 32468189

28. Soentjens P, Cnops L, Huyse T, Yansouni C, De Vos D, Bottieau E, et al. Diagnosis and Clinical Management of Schistosoma haematobium-Schistosoma bovis Hybrid Infection in a Cluster of Travelers Returning From Mali. Clin Infect Dis. 2016; 63: 1626–1629. https://doi.org/10.1093/cid/ciw493 PMID: 27941144

29. Pennance T, Allan F, Emery A, Rabone M, Cable J, Garba AD, et al. Interactions between Schistosoma haematobium group species and their Bulinus spp. intermediate hosts along the Niger River Valley. Parasites and Vectors. 2020; 13: 1–15. https://doi.org/10.1186/s13071-019-3862-4 PMID: 31900233

30. Tian-Bi YNT, Webster B, Konan CK, Allan F, Diakité NR, Ouattara M, et al. Molecular characterization and distribution of Schistosoma cercariae collected from naturally infected bulinid snails in northern and central Côte d'Ivoire. Parasites and Vectors. 2019; 12: 1–10. https://doi.org/10.1186/s13071-018-3256-z PMID: 30606222

31. Holtfreter MC, Moné H, Müller-Stöver I, Mouahid G, Richter J. Schistosoma haematobium infections acquired in Corsica, France, August 2013. Eurosurveillance. 2014; 19.

32. Moné H, Holtfreter MC, Allienne JF, Mintsa-Nguéma R, Ibikounlé M, Boissier J, et al. Introgressive hybridizations of Schistosoma haematobium by Schistosoma bovis at the origin of the first case report of schistosomiasis in Corsica (France, Europe). Parasitol Res. 2015; 114: 4127–4133. https://doi.org/10.1007/s00436-015-4643-4 PMID: 26268566

33. Catalano S, Sène M, Diouf ND, Fall CB, Borlase A, Léger E, et al. Rodents as Natural Hosts of Zoonotic Schistosoma Species and Hybrids: An Epidemiological and Evolutionary Perspective From West Africa. J Infect Dis. 2018; jiy029. https://doi.org/10.1093/infdis/jiy029 PMID: 29365139

34. Catalano S, Léger E, Fall CB, Borlase A, Diop SD, Berger D, et al. Multihost Transmission of Schistosoma mansoni in Senegal, 2015–2018. Emerg Infect Dis J. 2020; 26: 1234. https://doi.org/10.3201/eid2606.200107 PMID: 32441625

35. Savassi BAES, Dobigny G, Etougbétché JR, Avocegan TT, Quinsou FT, Gauthier P, et al. Mastomys natalensis (Smith, 1834) as a natural host for Schistosoma haematobium (Bilharz, 1852) Weinland, 1858 x Schistosoma bovis Sonsino, 1876 introgressive hybrids. Parasitol Res. 2021; 120: 1755–1770. https://doi.org/10.1007/s00436-021-07099-7 PMID: 33687566

36. Rothe C, Zimmer T, Schunk M, Wallrauch C, Helfrich K, Gültekin F, et al. Developing endemicity of schistosomiasis, Corsica, France. Emerg Infect Dis. 2021; 27: 319–321. https://doi.org/10.3201/EID2701.204391 PMID: 33264582
37. Oleaga A, Rey O, Polack B, Grech-Angelini S, Quilichini Y, Pérez-Sánchez R, et al. Epidemiological surveillance of schistosomiasis outbreak in Corsica (France): Are animal reservoir hosts implicated in local transmission? PLoS Negl Trop Dis. 2019; 13: e0007543. https://doi.org/10.1371/journal.pntd.0007543 PMID: 31233502

38. Pitchford RJ. Differences in the Egg Morphology and Certain Biological Characteristics of Some African and Middle Eastern Schistosomes, Genus Schistosoma, with Terminal-Spined Eggs. Bull World Health Organ. 1965; 32: 105–120. PMID: 14292062

39. Moné H, Minguez S, Ibikounlé M, Allienne JF, Massougbodji A, Mouahid G. Natural interactions between S. haematobium and S. guineensis in the Republic of Benin. Sci World J. 2012; 2012: 793420. https://doi.org/10.1100/2012/793420 PMID: 22645454

40. Loker ES. A comparative study of the life-histories of mammalian schistosomes. Parasitology. 1983; 87: 343–369. https://doi.org/10.1017/s0031182000052689 PMID: 6359028

41. Alves W. The Eggs of Schistosoma bovis, S. mattheei and S. haematobium. J Helminthol. 2009/11/18. 1948; 23: 127–134. https://doi.org/10.1017/S0022149X00032466 PMID: 15402061

42. Rollinson D, Southgate VR, Vercruysse J, Moore PJ. Observations on natural and experimental interactions between Schistosoma bovis and S. curassoni from West Africa. Acta Trop. 1990; 47: 101–114. https://doi.org/10.1016/0001-706x(90)90072-8 PMID: 1969699

43. Noël H, Ruello M, Maccary A, Pelat C, Sommen C, Boissier J, et al. Large outbreak of urogenital schistosomiasis acquired in Southern Corsica, France: monitoring early signs of endemicization? Clin Microbiol Infect. 2018; 24: 295–300. https://doi.org/10.1016/j.cmi.2017.06.026 PMID: 28669843

44. Boon NAM, Fannes W, Rombouts S, Polman K, Volckaert FAM, Huyse T. Detecting hybridization in African schistosome species: Does egg morphology complement molecular species identification? Parasitology. 2017/02/20. 2017; 144: 954–964. https://doi.org/10.1017/S0031182017000087 PMID: 28215207

45. Sahba GH, Malek EA. Unisexual infections with Schistosoma haematobium in the white mouse. Am J Trop Med Hyg. 1977; 26: 331–333. https://doi.org/10.4269/ajtmh.1977.26.331 PMID: 848652

46. Brumpt E. Cycle évolutif complet de Schistosoma bovis-Infection naturelle en Corse et infection expérimentale de Bullinus contortus. Ann Parasitol Hum Comp. 1930; 8: 17–50.

47. James C, Webbe G. A comparison of Sudanese and South African strains of Schistosoma haematobium. J Helminthol. 2009/06/01. 1975; 49: 191–197. https://doi.org/10.1017/s0022149x00023634 PMID: 1184947

48. Basch PF. Schistosomes: development, reproduction, and host relations. 1991.

49. Leroux PL. Abnormal adaptations and development of schistosomes in experimental animals. Trans R Soc Trop Med Hyg. 1949; 43: 2–3.

50. Southgate VR, Rollinson D, Vercruysse J. Scanning electron microscopy of the tegument of adult Schistosoma curassoni, and comparison with male S. bovis and S. haematobium from Senegal. Parasitology. 2009/04/01. 1986; 93: 433–442. https://doi.org/10.1017/s0031182000081154; 10.1017/S0031182000081154 PMID: 3797058

51. Leitch B, Probert AJ, Runham NW. The ultrastructure of the tegument of adult Schistosoma haematobium. 1984; 71–78. https://doi.org/10.1017/s0031182000001141 PMID: 6472886

52. Fournier A, Pages JR, Touassem R, Mouahid A. Can tegumental morphology be used as a taxonomic criterion between Schistosoma haematobium, S. intercalatum, and S. bovis? Parasitol Res. 1989; 75: 375–380. https://doi.org/10.1007/BF00931133 PMID: 2726719

53. Kinkel HF, Dittrich S, Baümer B, Weitzel T. Evaluation of eight serological tests for diagnosis of imported schistosomiasis. Clin Vaccine Immunol. 2012; 19: 948–953. https://doi.org/10.1128/CVI.05680-11 PMID: 22441394

54. Hamburger J, He-Na, Abbasi I, Ramzy RM, Jourdane J, Ruppel A. Polymerase chain reaction assay based on a highly repeated sequence of Schistosoma haematobium: a potential tool for monitoring schistosome-infested water. Am J Trop Med Hyg. 2001; 65: 907–911. https://doi.org/10.4269/ajtmh.2001.65.907 PMID: 11791997

55. Cnops L, Soentjens P, Clerinx J, van Esbroeck M. A Schistosoma haematobium-Specific Real-Time PCR for Diagnosis of Urogenital Schistosomiasis in Serum Samples of International Travelers and Migrants. PLoS Negl Trop Dis. 2013; 7. https://doi.org/10.1371/journal.pntd.0002413 PMID: 24009791

56. Colley DG, Bustinduy AL, Secor WE, King CH. Human schistosomiasis. Lancet (London, England). 2014; 383: 2253–2264. https://doi.org/10.1016/S0140-6736(13)61949-2 PMID: 24698483

57. Monrad J, Sörén K, Johansen M V., Lindberg R, Ørnbjerg N. Treatment efficacy and regulatory host responses in chronic experimental Schistosoma bovis infections in goats. Parasitology. 2006/04/01. 2006; 133: 151–158. https://doi.org/10.1017/S0031182006000102 PMID: 16623963
37. Oleaga A, Rey O, Polack B, Grech-Angelini S, Quilichini Y, Pérez-Sánchez R, et al. Epidemiological surveillance of schistosomiasis outbreak in Corsica (France): Are animal reservoir hosts implicated in local transmission? PLoS Negl Trop Dis. 2019; 13: e007543. https://doi.org/10.1371/journal.pntd.0007543 PMID: 31233502

38. Pitchford RJ. Differences in the Egg Morphology and Certain Biological Characteristics of Some African and Middle Eastern Schistosomes, Genus Schistosoma, with Terminal-Spined Eggs. Bull World Health Organ. 1965; 32: 105–120. PMID: 14292062

39. Moné H, Minguez S, Ibikounlé M, Allienne JF, Massougbodji A, Mouahid G. Natural interactions between S. haematobium and S. guineensis in the Republic of Benin. Sci World J. 2012; 2012: 793420. https://doi.org/10.1100/2012/793420 PMID: 22645454

40. Loker ES. A comparative study of the life-histories of mammalian schistosomes. Parasitology. 1983; 87: 343–369. https://doi.org/10.1017/s0031182000052689 PMID: 6359028

41. Alves W. The Eggs of Schistosoma bovis, S. mattheei and S. haematobium. J Helminthol. 2009/11/18. 1948; 23: 127–134. https://doi.org/10.1017/S0022149X00032466 PMID: 15402061

42. Rollinson D, Southgate VR, Vercruysse J, Moore PJ. Observations on natural and experimental interactions between Schistosoma bovis and S. curassoni from West Africa. Acta Trop. 1990; 47: 101–114. https://doi.org/10.1016/0001-706x(90)90072-8 PMID: 1969699

43. Noël H, Ruello M, Maccary A, Pelat C, Sommen C, Boissier J, et al. Large outbreak of urogenital schistosomiasis acquired in Southern Corsica, France: monitoring early signs of endemicization? Clin Microbiol Infect. 2018; 24: 295–300. https://doi.org/10.1016/j.cmi.2017.06.026 PMID: 28669843

44. Boon NAM, Fannes W, Rombouts S, Polman K, Volckaert FAM, Huyse T. Detecting hybridization in African schistosome species: Does egg morphology complement molecular species identification? Parasitology. 2017/02/20. 2017; 144: 954–964. https://doi.org/10.1017/S0031182017000087 PMID: 28215207

45. Sahba GH, Malek EA. Unisexual infections with Schistosoma haematobium in the white mouse. Am J Trop Med Hyg. 1977; 26: 331–333. https://doi.org/10.4269/ajtmh.1977.26.331 PMID: 848652

46. Brumpt E. Cycle évolutif complet de Schistosoma bovis-Infection naturelle en Corse et infection expérimentale de Bullinus contortus. Ann Parasitol Hum Comp. 1930; 8: 17–50.

47. James C, Webbe G. A comparison of Sudanese and South African strains of Schistosoma haematobium. J Helminthol. 2009/06/01. 1975; 49: 191–197. https://doi.org/10.1017/s0022149x00023634 PMID: 1184947

48. Basch PF. Schistosomes: development, reproduction, and host relations. 1991.

49. Leroux PL. Abnormal adaptations and development of schistosomes in experimental animals. Trans R Soc Trop Med Hyg. 1949; 43: 2–3.

50. Southgate VR, Rollinson D, Vercruysse J. Scanning electron microscopy of the tegument of adult Schistosoma curassoni, and comparison with male S. bovis and S. haematobium from Senegal. Parasitology. 2009/04/01. 1986; 93: 433–442. https://doi.org/10.1017/s0031182000081154; 10.1017/S0031182000081154 PMID: 3797058

51. Leitch B, Probert AJ, Runham NW. The ultrastructure of the tegument of adult Schistosoma haematobium. 1984; 71–78. https://doi.org/10.1017/s0031182000001141 PMID: 6472886

52. Fournier A, Pages JR, Touassem R, Mouahid A. Can tegumental morphology be used as a taxonomic criterion between Schistosoma haematobium, S. intercalatum, and S. bovis? Parasitol Res. 1989; 75: 375–380. https://doi.org/10.1007/BF00931133 PMID: 2726719

53. Kinkel HF, Dittrich S, Baümer B, Weitzel T. Evaluation of eight serological tests for diagnosis of imported schistosomiasis. Clin Vaccine Immunol. 2012; 19: 948–953. https://doi.org/10.1128/CVI.05680-11 PMID: 22441394

54. Hamburger J, He-Na, Abbasi I, Ramzy RM, Jourdane J, Ruppel A. Polymerase chain reaction assay based on a highly repeated sequence of Schistosoma haematobium: a potential tool for monitoring schistosome-infested water. Am J Trop Med Hyg. 2001; 65: 907–911. https://doi.org/10.4269/ajtmh.2001.65.907 PMID: 11791997

55. Cnops L, Soentjens P, Clerinx J, van Esbroeck M. A Schistosoma haematobium-Specific Real-Time PCR for Diagnosis of Urogenital Schistosomiasis in Serum Samples of International Travelers and Migrants. PLoS Negl Trop Dis. 2013; 7. https://doi.org/10.1371/journal.pntd.0002413 PMID: 24009791

56. Colley DG, Bustinduy AL, Secor WE, King CH. Human schistosomiasis. Lancet (London, England). 2014; 383: 2253–2264. https://doi.org/10.1016/S0140-6736(13)61949-2 PMID: 24698483

57. Monrad J, Sörén K, Johansen M V., Lindberg R, Ørnbjerg N. Treatment efficacy and regulatory host responses in chronic experimental Schistosoma bovis infections in goats. Parasitology. 2006/04/01. 2006; 133: 151–158. https://doi.org/10.1017/S0031182006000102 PMID: 16623963
58. Johansen M V, Monrad J, Ø. Christensen N. Effects of praziquantel on experimental Schistosoma bovis infection in goats. Vet Parasitol. 1996; 62: 83–91. https://doi.org/10.1016/0304-4017(95)00849-7 PMID: 8638396

59. Zwang J, Olliaro PL. Clinical Efficacy and Tolerability of Praziquantel for Intestinal and Urinary Schistosomiasis—A Meta-analysis of Comparative and Non-comparative Clinical Trials. PLoS Negl Trop Dis. 2014; 8. https://doi.org/10.1371/journal.pntd.0003286 PMID: 25412105

60. Webster BL, Diaw OT, Seye MM, Webster JP, Rollinson D. Introgressive Hybridization of Schistosoma haematobium Group Species in Senegal: Species Barrier Break Down between Ruminant and Human Schistosomes. PLoS Negl Trop Dis. 2013; 7. https://doi.org/10.1371/journal.pntd.0002110 PMID: 23593513

61. Pitchford RJ, Lewis M. Oxamniquine in the treatment of various schistosome infections in South Africa. S Afr Med J. 1978; 53: 677–680. PMID: 354047

62. Boissier J, Chlichlia K, Digon Y, Ruppel A, Moné H. Preliminary study on sex-related inflammatory reactions in mice infected with Schistosoma mansoni. Parasitol Res. 2003; 91: 144–150. https://doi.org/10.1007/s00436-003-0943-1 PMID: 12910415

63. Silva ML, Vicente FS, Avelino IC, Martin VR. Susceptibility of Planorbarius metidjensis from Portugal and Spain to Schistosoma bovis from Salamanca, Spain. Malacologia. 1977; 16: 251–254. PMID: 904368

64. Kincaid-Smith J, Boissier J, Allienne JFJ-F, Oleaga A, Djuikwo-Teukeng F, Toulza E. A Genome Wide Comparison to Identify Markers to Differentiate the Sex of Larval Stages of Schistosoma haematobium, Schistosoma bovis and their Respective Hybrids. PLoS Negl Trop Dis. 2016; 10: e0005138. https://doi.org/10.1371/journal.pntd.0005138 PMID: 27861520

65. Hulstijn M, Barros LA, Neves RH, Moura EG, Machado-Silva JR. Morphological changes in the reproductive organs of male and female Schistosoma mansoni worms caused by streptozotocin, a drug used to induce diabetes mellitus. Parasitology. 2003; 126: 53–61. https://doi.org/10.1017/s0031182002002512 PMID: 12613763

66. Prodanov D, Verstreke K. Automated Segmentation and Morphometry of Cell and Tissue Structures. Selected Algorithms in ImageJ. Molecular Imaging. 2012. https://doi.org/10.5772/36729

67. Langmead B, Trapnell C, Pop M, Salzberg S. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10: R25. https://doi.org/10.1186/gb-2009-10-3-r25 PMID: 19261174
58. Johansen M V, Monrad J, Ø. Christensen N. Effects of praziquantel on experimental Schistosoma bovis infection in goats. Vet Parasitol. 1996; 62: 83–91. https://doi.org/10.1016/0304-4017(95)00849-7 PMID: 8638396

59. Zwang J, Olliaro PL. Clinical Efficacy and Tolerability of Praziquantel for Intestinal and Urinary Schistosomiasis—A Meta-analysis of Comparative and Non-comparative Clinical Trials. PLoS Negl Trop Dis. 2014; 8. https://doi.org/10.1371/journal.pntd.0003286 PMID: 25412105

60. Webster BL, Diaw OT, Seye MM, Webster JP, Rollinson D. Introgressive Hybridization of Schistosoma haematobium Group Species in Senegal: Species Barrier Break Down between Ruminant and Human Schistosomes. PLoS Negl Trop Dis. 2013; 7. https://doi.org/10.1371/journal.pntd.0002110 PMID: 23593513

61. Pitchford RJ, Lewis M. Oxamniquine in the treatment of various schistosome infections in South Africa. S Afr Med J. 1978; 53: 677–680. PMID: 354047

62. Boissier J, Chlichlia K, Digon Y, Ruppel A, Moné H. Preliminary study on sex-related inflammatory reactions in mice infected with Schistosoma mansoni. Parasitol Res. 2003; 91: 144–150. https://doi.org/10.1007/s00436-003-0943-1 PMID: 12910415

63. Silva ML, Vicente FS, Avelino IC, Martin VR. Susceptibility of Planorbarius metidjensis from Portugal and Spain to Schistosoma bovis from Salamanca, Spain. Malacologia. 1977; 16: 251–254. PMID: 904368

64. Kincaid-Smith J, Boissier J, Allienne JFJ-F, Oleaga A, Djuikwo-Teukeng F, Toulza E. A Genome Wide Comparison to Identify Markers to Differentiate the Sex of Larval Stages of Schistosoma haematobium, Schistosoma bovis and their Respective Hybrids. PLoS Negl Trop Dis. 2016; 10: e0005138. https://doi.org/10.1371/journal.pntd.0005138 PMID: 27861520

65. Hulstijn M, Barros LA, Neves RH, Moura EG, Machado-Silva JR. Morphological changes in the reproductive organs of male and female Schistosoma mansoni worms caused by streptozotocin, a drug used to induce diabetes mellitus. Parasitology. 2003; 126: 53–61. https://doi.org/10.1017/s0031182002002512 PMID: 12613763

66. Prodanov D, Verstreke K. Automated Segmentation and Morphometry of Cell and Tissue Structures. Selected Algorithms in ImageJ. Molecular Imaging. 2012. https://doi.org/10.5772/36729

67. Langmead B, Trapnell C, Pop M, Salzberg S. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10: R25. https://doi.org/10.1186/gb-2009-10-3-r25 PMID: 19261174