Infection, Genetics and Evolution 10 (2010) 1218–1227

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Infection, Genetics and Evolution

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Molecular diversity of franki in two intermediate hosts ( auricularia and ): A complex of species

D. Jouet a,*, K. Skı´rnisson b, L. Kola´rˇova´ c, H. Ferte´ a a JE 2533 – USC AFSSA «VECPAR» UFR de Pharmacie, Universite´ de Reims Champagne – Ardenne, 51 rue Cognacq-Jay, 51096 Reims, b Institute for Experimental Pathology, University of , Keldur, Reykjavı´k, Iceland c Institute of Immunology and Microbiology of the First Faculty of Medicine, Charles University in Prague and General Teaching Hospital, Studnicˇkova 7, 128 00 Prague 2, Czech Republic

ARTICLE INFO ABSTRACT

Article history: Recently, the systematic use of the molecular approach as a complement to the other approaches Received 23 March 2010 (morphology, biology, life cycle) has brought help for the identification of species considered as different Received in revised form 19 May 2010 in the past to be regrouped and synonymised, and distinctions to be drawn between species similar at Accepted 4 August 2010 the morphological level. Among these species, we tried to clarify the situation of Trichobilharzia franki Available online 11 August 2010 Mu¨ ller and Kimmig, 1994, species that today include more than 50 haplotypes notably coming from larval stages isolated from intermediate hosts belonging to gastropods of the Radix genus. Cercariae were Keywords: isolated in France and Iceland from various molluscs, before being analyzed, with their hosts, by Trichobilharzia franki molecular analysis of various fields such as the D2 and ITS of the ribosomal DNA and the COX1 of Radix peregra mitochondrial DNA. We thus show the presence of two clades depending on the specificity of their Molecular analyses intermediate in which they were isolated (Radix auricularia or Radix peregra), thus allowing Cercariae separation of the species T. franki that had been described in the past as a probable new species. Snails ß 2010 Elsevier B.V. All rights reserved.

1. Introduction mitochondrial DNA (Littlewood and Johnston, 1995; Mollaret et al., 1997; Snyder and Loker, 2000; Picard and Jousson, 2001; Lockyer Among schistosomatids, representatives of the genus Tricho- et al., 2003; Olson et al., 2003; Snyder, 2004; Brant et al., 2006; bilharzia, with more than 40 described species, are probably the Littlewood et al., 2006; Webster et al., 2007) finally allowed most frequent in the field. The parasite life cycle comprises various definitive and precise taxonomical identification of the parasites. (mainly waterfowl) and water snails as definitive and On the basis of morphological and molecular characteristics, intermediate hosts, respectively. Due to a complicated life cycle including intermediate-host specificity, three Trichobilharzia spe- and the difficulty of isolation and morphological identification of cies maturing predominantly in waterfowl (for a review see Hora´k the adult worms, the within this genus is difficult. In the et al., 2002) are currently recognized in Europe: Trichobilharzia past, characterization of the species was based mainly on szidati Neuhaus, 1952 developing in Lymnaea stagnalis, Trichobil- morphology of the larval (cercariae) and adult stages. Unfortu- harzia franki Mu¨ ller and Kimmig, 1994 developing in Radix nately, in many cases cercariae and adults were described auricularia and Hora´k Kola´rˇova´ and Dvorˇa´k, separately with regard to their further development in definitive 1998 developing in Radix peregra. Four species that had been or intermediate hosts, respectively, meaning that the morphology discovered some time ago were recently characterized in North of all developmental stadia of such species remained unknown. America – Trichobilharzia physellae (Talbot, 1936) McMullen and Valuable data were obtained by studies on schistosomatids Beaver, 1945 developing in Physa parkeri, Trichobilharzia querque- developing in experimentally infected hosts, but numerous dulae McLeod, 1937 developing in Physa acuta (experimentally), problems such as unavailability of compatible hosts or isolation Trichobilharzia stagnicolae (Talbot, 1936) McMullen and Beaver, of adult flukes from the birds (Kola´rˇova´ et al., 2010) limited the 1945 developing in Stagnicola emarginata and Trichobilharzia wide use of these methods. The use of recent molecular tools such brantae Farr and Blankemeyer, 1956 developing in Gyraulus parvus. as the 28S and ITS of the ribosomal DNA and the COX1 of the For the latter, phylogenetic analysis of DNA suggests that this species probably does not belong in the genus Trichobilharzia (Brant and Loker, 2009).

* Corresponding author. Tel.: +33 326 913 597; fax: +33 326 913 569. T. franki has been reported in many European countries: Czech E-mail address: [email protected] (D. Jouet). Republic (Kola´rˇova´ et al., 1997; Rudolfova´ et al., 2005),

1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.08.001 D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) 1218–1227 1219

(Brant and Loker, 2009), (Picard and Jousson, 2001), SchistoCox1-30 (50-TAA TGC ATM GGA AAA AAA CA-30)(Webster France (Ferte´ et al., 2005; Jouet et al., 2008, 2009), Iceland et al., 2007). (Skı´rnisson and Kola´rˇova´, 2008; Skı´rnisson et al., 2009; Aldhoun ITS-2 was also used for snail identification: Amplification of the et al., 2009a), Russia (Semyenova et al., 2005), Belarus (Chrisanfova positive snails was made by using the specific primers of Radix spp. et al., 2009), (Rudolfova´ et al., 2005) and Finland (Aldhoun DIX1 (50-CGC GCT CTG GWC CKT CGC GGC-30) and DIX2 (50-ATY et al., 2009b). At present, many haplotypes of cercariae and/or TYG TYC GAT TTG AGG TTG-30)(Jouet et al., 2008). adults originating from naturally infected snails and aquatic birds, PCR products were directly sequenced in both directions with respectively, are available in GenBank. Except for one record of T. the primers used for DNA amplification (QIAGEN, Germany). The franki isolated from L. stagnalis by Rudolfova´ et al. (2005), the larval sequences are deposited in GenBank under the accession numbers stages were found only in snails of genus Radix: mainly R. HM131131–HM131205 and HQ003220–HQ003235. auricularia but also R. peregra were then identified as the Sequences were aligned using the ClustalW routine included in intermediate hosts. the MEGA version 3.1 software (Kumar et al., 2004) and checked by Since T. franki has been found in a variety of geographical areas eye. The D2 domain (554 bp) and ITS region (1637 bp) of the in a large number of hosts and considering the increasing number ribosomal DNA and the COX1 domain (822 bp) of the mitochon- of sequences available in the databases (in particular the recent drial DNA for the parasite and ITS-2 (440 bp) of the rDNA for the description of new American species), we studied the possible snails were used for tree construction and rooted with the variations of the parasite within the species and revised the outgroup taxon (Bilharziella polonica (D2), T. szidati (ITS and COX1) complex of parasites with regard to species-type described by and L. stagnalis (ITS-2)). Mu¨ ller and Kimmig (1994). We attempted to clarify the position of Phylogenetic analyses were performed using haplotypes the haplotypes of parasites found in various Radix snails, using obtained in this study (Table 1) and sequences available in molecular analysis. GenBank: T. regenti, T. szidati, T. physellae, T. querquedulae, Trichobilharzia sp., T. stagnicolae, T. brantae, Allobilharzia visceralis, 2. Materials and methods Dendritobilharzia pulverulenta, Gigantobilharzia huronensis, B. polo- nica for ocellate furcocercariae; R. auricularia, Radix balthica This study was carried out for an international project (EGIDE) (=peregra, ovata), Radix ampla, Radix lagotis, Radix labiata and in the aim of studying the common parasites between France and Radix sp. for snails (Tables 2 and 3). Phylogenetic reconstruction Iceland. Snails of the genus Radix were collected between 2000 and using Neighbour-Joining (NJ) with the Kimura-2 parameter and 2009 in France and during summer and autumn 2009 in Iceland. uniform rates among sites was performed using the MEGA version Cercarial emergence was stimulated according to Kola´rˇova´ et al. 3.1 software (Kumar et al., 2004). Maximum Likelihood (ML) (2010). Ocellate furcocercariae were preserved in 95% ethanol and analysis was performed in Phyml online (Guindon et al., 2005). The frozen (20 8C) until the DNA analysis. Positive snail hosts were model and the parameters were chosen using the hierarchical frozen directly at 20 8C in individual sterile bags for storage. Only likelihood ratio test implemented in Modeltest 3.7 (Posada and the samples from which cercariae and their corresponding snails Crandall, 2001). Maximum Parsimony (MP) analysis was per- were sequenced were used for molecular analyses. The sequenced formed in MEGA version 3.1 software (Kumar et al., 2004) using the samples are listed in Table 1. Photographs and measurements of Maxi-mini Branch and Bound method. For all NJ, ML and MP cercariae were made on fresh material or preserved in formalin, analyses, gaps were treated as missing data and internal node with a digital camera (Leica DC 300) attached to a microscope support was assessed by bootstrapping over 500 replicates. (Leica DMLB or Olympus BX50) equipped for differential interfer- ence contrast or Nomarski system. 3. Results After removing all ethanol from samples, DNA was extracted using the Qiamp DNA Mini Kit (Qiagen, Germany) following 3.1. Morphological comparison of cercariae manufacturer’s instructions. During the first step (tissue lysis), cercariae or a small part of the foot of each positive snail were Measurements of the cercariae isolated from R. auricularia and crushed using a piston pellet (Treff, Switzerland). The DNA was R. peregra show variations between them and relative to the eluted in 50 ml of the buffer provided. Polymerase Chain Reaction cercariae of T. franki, T. querquedulae and T. physellae, particularly in was performed in a 50 ml volume using 5 ml of DNA, and 50 pmol the overall length of the cercariae (Table 4). However, cercariae of of each of the primers. The PCR mix contained (final concentra- avian schistosomes are contractile, which may explain such tions) 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.01% variability. It is thus impossible to determine to which species Triton X-100, 200 mM dNTP each base, and 1.25 units of Taq the parasites belong on the basis of morphological criteria alone. polymerase (Eppendorf, Germany). Sequencing of the D2 domain of the 28S subunit, internal 3.2. Molecular analyses of cercariae transcribed spacers (ITS-2 and ITS-1) of ribosomal DNA and COX1 domain of the mitochondrial DNA were used for the identification The molecular analysis of the D2, ITS and COX1 domains are of avian schistosomes obtained from naturally infected snails. PCR congruent (Figs. 1–3): they show that the cercariae isolated from was performed under conditions that had been published Radix belong to Trichobilharzia. Our analyses clearly show that the previously (Jouet et al., 2009). haplotypes T. franki ‘‘auricularia’’ and T. franki ‘‘peregra’’ belong to Ribosomal DNA: ITS-2 and ITS-1 of furcocercariae were two distinct clades, separated by the haplotypes corresponding to amplified using primers ITS3Trem (50-GCG TCG ATG AAG AGT the recently described American species: T. querquedulae and T. GCA GC-30), ITS4Trem (50-TCC TCC GCT TAT TGA TAT GC-30), physellae. ITS2Trem (50-GCT GCA CTC TTC ATC GAC GC-30) and ITS5Trem (50- For the D2 domain, each clade (‘‘auricularia’’ and ‘‘peregra’’)is GGA AGT AAA AGT CGT AAC AAG G-30)(Dvorˇa´k et al., 2002). The D2 composed of 100% homologous haplotypes. In spite of the domain was amplified using the primers C20B(50-GAA AAG TAC conserved character of this domain (specific level of the D2), TTT GRA RAG AGA-30) and D2 (50-TCC GTG TTT CAA GAC GGG-30) three variations are present between these two haplotypes. In according to Mollaret et al. (1997). comparison, T. franki ‘‘auricularia’’ and T. physellae are also Mitochondrial DNA: The domain COX1 was amplified using the separated by three variations, two variations between T. franki primers SchistoCox1-50 (50-TCT TTR GAT CAT AAG CG-30) and ‘‘peregra’’ and T. physellae, and also between T. franki ‘‘peregra’’ and 1220 D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) 1218–1227

Table 1 Isolates of schistosomes used for molecular analysis.

Host: snail Geographical data

Taxa Species Taxa Life cycle stage Locality GPS coordinates

RAN79 Radix pergeraa EAN79 Cercariae Annecy Lake, France 4585104100N/681000200E RAN80 Radix pergeraa EAN80 Cercariae Radix pergera ICR1 Cercariae Raudavatn, Iceland 6480602800N/2184600800O Radix pergera ICR21 Cercariae Helgavogur-Myvatn, Iceland 6583500800N/1685903800O Radix pergera ICR22 Cercariae Radix pergera ICR23 Cercariae Radix pergera ICR24 Cercariae Radix pergera ICR25 Cercariae IS1F Radix pergeraa F1IS Cercariae Botnsvatn, Iceland 6680200000N/1780000000O IS2F Radix pergeraa F2IS Cercariae IS3F Radix pergeraa F3IS Cercariae Raudavatn, Iceland 6480602800N/2184600800O IS4F Radix pergeraa F4IS Cercariae IS5F Radix pergeraa F5ISA Cercariae IS5F Radix pergeraa F5ISB Cercariae IS6F Radix pergeraa F6IS Cercariae RAN77 Radix auriculariaa EAN77 Cercariae Annecy Lake, France 4585104100N/681000200E RSFO1 Radix auriculariaa FORS1 Cercariae Der-Chantecoq Lake, France 4883401600N/484500800E AY795574 Radix auriculariaa FORS3 Cercariae RSFO4 Radix auriculariaa FORS4 Cercariae RSBE1 Radix auriculariaa BERS1 Cercariae Beauvais, France 4982702100N/280301700E RSBE2 Radix auriculariaa BERS2 Cercariae RSBE13 Radix auriculariaa BERS13 Cercariae RSBE67 Radix auriculariaa BERS67 Cercariae Radix auricularia STRS2 Cercariae Strasbourg, France 4883405800N/784403700E Radix auricularia STRS4 Cercariae Radix auricularia STRS5 Cercariae Radix auricularia STRS6 Cercariae

a Molecular identification.

Table 2 GenBank sequences of bird schistosomes used for molecular analysis.

Species Host Locality GenBank accession numbers

D2 ITS2 ITS1 COX1

Trichobilharzia franki ‘‘peregra’’ Radix peregra France EU413962 Radix peregra France EU413963 Radix peregra France EU413965 Radix peregra France EU413966 Trichobilharzia franki ‘‘auricularia’’ Radix auricularia France AY795572 Radix auricularia France AY795573 Trichobilharzia physellae Aythya affinis USA FJ174473 FJ174518 Bucephala alveola USA FJ174474 FJ174561 FJ174561 FJ174514 Physa gyrina USA FJ174562 FJ174562 FJ174523 Aythya affinis USA FJ174563 FJ174563 FJ174512 Aythya affinis USA FJ174564 FJ174564 FJ174515 Aythya valisineria USA FJ174565 FJ174565 Aythya collaris USA FJ174566 FJ174566 FJ174517 Mergus merganser USA FJ174567 FJ174567 FJ174521 Physa gyrina USA FJ174568 FJ174568 FJ174513 Mergus merganser USA FJ174569 FJ174569 FJ174519 Aythya affinis USA FJ174575 FJ174575 FJ174522 Clangula hyemalis USA FJ174516 Trichobilharzia querquedulae Anas discors USA FJ174468 FJ174558 FJ174558 FJ174498 Anas discors USA FJ174469 FJ174555 FJ174555 FJ174510 Anas cyanoptera USA FJ174470 FJ174556 FJ174556 FJ174505 Anas clypeata USA FJ174547 FJ174547 FJ174509 Anas clypeata USA FJ174548 FJ174548 Anas clypeata USA FJ174549 FJ174549 FJ174503 Anas discors USA FJ174550 FJ174550 FJ174507 Anas clypeata USA FJ174551 FJ174551 FJ174504 Anas clypeata USA FJ174552 FJ174552 FJ174508 Anas cyanoptera USA FJ174553 FJ174553 FJ174501 Anas discors USA FJ174554 FJ174554 FJ174502 Anas clypeata USA FJ174557 FJ174557 FJ174497 Anas cyanoptera USA FJ174559 FJ174559 FJ174499 Anas clypeata USA FJ174560 FJ174560 FJ174506 Anas cyanoptera USA FJ174500 Anas discors USA FJ174511 Trichobilharzia sp. Radix peregra France EU413961 Radix peregra France EU413964 D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) 1218–1227 1221

Table 2 (Continued )

Species Host Locality GenBank accession numbers

D2 ITS2 ITS1 COX1

Trichobilharzia regenti Radix peregra Czech Republic AF263829 AF263829 Radix peregra Czech Republic AY157244 AY157190 Radix peregra France EU413960 Anas clypeata Poland EF094533 EF094533 Aythya fuligula Poland EF094534 EF094534 Anas platyrhynchos Poland EF094535 EF094535 Anas platyrhynchos Poland EF094537 EF094537 Anas platyrhynchos Poland EF094538 EF094538 Aans clypeata Czech Republic EF094540 EF094540 Radix peregra France EU413967 EU413967 Radix peregra UK DQ859919 Radix peregra UK NC009680 Trichobilharzia szidati Lymnaea stagnalis Czech Republic AF263828 AF263828 AY157191 Lymnaea stagnalis Czech Republic AY157245 Lymnaea stagnalis Czech Republic AY713972 AY713972 Lymnaea stagnalis Germany AY713971 AY713971 Lymnaea stagnalis Netherlands AY713970 AY713970 Lymnaea stagnalis Czech Republic AY713968 AY713968 Lymnaea stagnalis Poland AY713965 AY713965 Lymnaea stagnalis Czech Republic AY713961 AY713961 Anas platyrhynchos Poland EF094536 EF094536 Anas platyrhynchos Czech Republic EF094541 EF094541 Lymnaea stagnalis Finland FJ609409 FJ609409 Lymnaea stagnalis Finland FJ609410 FJ609410 Lymnaea stagnalis France AY795570 AY795570 Lymnaea stagnalis France AY795571 AY795571 Lymnaea stagnalis USA FJ174476 FJ174588 FJ174588 FJ174496 Stagnicola elrodi USA FJ174495 Trichobilharzia stagnicolae Stagnicola sp. USA FJ174477 Mergus merganser USA FJ174478 Stagnicola emarginata USA FJ174479 Allobilharzia visceralis Cygnus columbianus USA EF114222 Cygnus columbianus USA EF114223 Trichobilharzia brantae Gyraulus parvus USA FJ174466 Chen caerules USA FJ174467 Dendritobilharzia pulverulenta Gallus gallus USA AY157241 Chicken USA AF167090 Gigantobilharzia huronensis Agelaius phoeniceus USA AY157242 Agelaius phoeniceus USA AF167091 Bilharziella polonica Anas platyrhynchos Ukraine AY157240 Planorbis planorbis Bohemia AF167088

T. querquedulae, two variations between T. querquedulae and T. comparison with distances separating them to the haplotypes of T. physellae and a single variation between T. franki ‘‘auricularia’’ and regenti and T. szidati. The results obtained for these two domains T. querquedulae. confirm those obtained for the D2, with the separation of the For the total ITS and the COX1, only sequences corresponding haplotypes T. franki ‘‘auricularia’’ and T. franki ‘‘peregra’’ into two to the species T. szidati, T. regenti, T. querquedulae, T. physellae and distinct clades. haplotypes T. franki ‘‘auricularia’’ and T. franki ‘‘peregra’’ were compared. Taking into account the considerable variability of 3.3. Prevalence and molecular identification of snails these fields, the sequences were compared using the pairwise distance between each group, whose results appear in Table 5. Among snails of the Radix genus isolated, the prevalence of These results show very small distances for these four groups in ocellate furcocercariae pertaining to the species T. franki varied

Table 3 GenBank sequences of snails used for molecular analysis.

Determination Accession number Origin

Radix auricularia AJ319628 Czech Republic, Austria, UK AJ319629 Czech Republic AJ319630, AJ319631, AJ319632, AY795574 France Radix peregra (=R. ovata;=R. balthica) AJ319633 Iceland AJ319634, AJ319635, EU413980–EU413986 France Radix labiata AJ319636 Turkey AJ319637 Germany Radix lagotis AJ319638 Czech Republic AJ319639 Austria Radix ampla AJ319640 Austria Radix sp. AJ319641 Turkey Lymnaea stagnalis AJ319614 Germany AJ319615, AJ319616 France AJ319617 Italy, France, Germany 1222 D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) 1218–1227

Table 4 Dimensions and comparison of cercariae of Trichobilharzia franki, T. querquedulae, T. physellae and cercariae isolated on Radix auricularia and Radix peregra (in mm).

T. franki T. franki ‘‘auricularia’’ T. franki ‘‘peregra’’ T. querquedulae T. physellae

Reference Mu¨ ller and This paper This paper Brant and Loker McMullen and Brant and Kimmig (1994) (2009) Beaver (1945) Loker (2009) Snail host R. auricularia R. auricularia R. peregra Physa gyrina P. parkeri, P. gyrina P. gyrina Number 55 31 5 Total length 960 1018 864 958–961 835 810 Ant. end-eye 145 134 Ant. end-acetabulum 244 182 Head organ length 307 318 257 327 265 270 Head organ width 81 73 Acetabulum 37 28 Tail stem length 419 427 379 410 374 352 Tail stem width 56 50 Furca length 234 273 227 221–224 196 188 Furca width 26 24

from 0.07 to 1.6%, according to the different sites. The phylogenetic auricularia and R. peregra) were included in the same complex trees based on ITS-2 sequences of snail isolates (Fig. 4), rooted on L. ‘‘T. franki’’. stagnalis, show four different clades, as reported by Bargues et al. Today, the recent morphological and molecular description of (2001, 2003): the first is formed by R. labiata, the second by R. new species, particularly in North America (Brant and Loker, 2009), ampla and R. lagotis, the third by Radix sp. and R. auricularia and the more precise characterization of the intermediate hosts by (including six haplotypes from France with the following molecular biology have allowed us to question the haplotypes sequences RSFO1, RSFO4, RSBE1, RSBE2, RSBE13, RSBE67 and described in the past. In our study, we showed that the parasites RAN77) and the fourth by R. balthica (=R. peregra, ovata) with three isolated from R. auricularia and R. peregra seem not to belong to the haplotypes (including isolates RAN 79 and RAN80 from France, and single species ‘‘T. franki’’, but to two different clades that we IS1F, IS2F, IS3F, IS4F, IS5F and IS6F, from Iceland). Although the consider to represent distinct species. The molecular variations haplotypes of ITS-2 from snails showed diversity, all snails observed between these two haplotypes thus do not correspond to emitting the furcocercariae we had found were included in the intraspecific variations within the same species, but rather to R. auricularia or the R. balthica (=peregra, ovata) branches which are interspecific variations between two distinct species. According to well supported by the data. the principle of priority, we need to take into account that the species T. franki was described by Mu¨ ller and Kimmig (1994) from 4. Discussion R. auricularia. It thus appears that the haplotypes isolated from R. auricularia in the past should still be considered as belonging to As previously shown, morphological identification of snails this species. However, it is important to note that, in the future, the of the genus Radix based on the external features (shell size identification of a snail host belonging to the Radix genus will have and shape) is very complicated because of the continuous to be confirmed by molecular analysis, due to the difficulties variability and plasticity of these characteristics depending encountered in morphological identification. This molecular on environmental conditions (Pfenninger et al., 2006). According identification of snails and cercariae allowed us to show the to Bargues et al. (2001, 2003) and Pfenninger et al. (2006) presence on a same site, the Annecy Lake, of the two species T. the molecular approach might be effective for identifying franki and T. franki ‘‘peregra’’ (haplotypes EAN77 and EAN79, species of this genus. In the future, identification of snails EAN80). belonging to this genus needs to be confirmed by molecular Concerning the recently described American species, molec- analyses. ular analyses show significant interspecific variations between In our study, ITS-2 was chosen for phylogenetic analysis. In the T. querquedulae, T. physellae and T. franki ‘‘peregra’’, thus genus Radix Montfort, 1810, six valid species are recognized: R. excluding this haplotype from membership of these two species. labiata (Rossmaessler, 1835), R. ampla (Hartmann, 1821), R. lagotis This separation between the European and American species is (Schrank, 1803), Radix sp., R. auricularia (Linnaeus, 1758), R. confirmed by the differences concerning the life cycle of the balthica (Linnaeus, 1758) (=R. peregra (Mu¨ ller, 1774), =R. ovata parasites: European species use lymnaeid snails as intermediate (Draparnaud, 1805)). In our study, the molluscs collected in France hosts, whereas T. querquedulae and T. physellae use physid and Iceland belong to two species: R. balthica (= peregra, ovata) and snails. R. auricularia. Consequently, it seems important for us to revise the In the past, only three Trichobilharzia species were recognized membership of the species T. franki for the haplotypes isolated and molecularly identified in Europe (Hora´ k et al., 2002). On the from R. peregra. Our results showed that, molecularly, they basis of these species, furcocercariae isolated from L. stagnalis, R. differed from T. franki,butalsofromT. querquedulae and T. auricularia and R. peregra were identified as pertaining to the physellae, for different domains such as the D2 and ITS of the species T. szidati (=ocellata), T. franki and T. regenti, respectively, by ribosomal DNA, but also for the COX1 domain of the mitochon- the nature of their intermediate host. This specific relationship drial DNA. This seems to argue in favour of their membership of a between intermediate host and larval stages of bird schistosomes, new species. However, it is impossible to define them as such on formerly used as a criterion for differentiation of the cercariae at the simple basis of the morphology of the cercariae or their the specific level, was subsequently questioned by the discovery molecular analysis, or on the description of a specific host. Only a of several parasitic species in the same intermediate host (Jouet search for adults flukes corresponding to these haplotypes can et al., 2008). This phenomenon could be explained by mechanisms finally clarify our assumption and thus lead to the description of of pressure of selection and adaptation of the parasite to a new a new species of Trichobilharzia,and,therefore,explainthe host. Following this view, cercariae with similar molecular complete life cycle of this schistosome, to avoid any confusion in sequences and isolated from snails of different species (R. the future. [(Fig._1)TD$IG] D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) 1218–1227 1223

Fig. 1. Phylogenetic tree based on the D2 domain of rDNA of ocellate furcocercariae and sequences of bird schistosomes (genera Bilharziella, Gigantobilharzia, Dendritobilharzia and Trichobilharzia) constructed using the maximum likelihood method in Phyml online (HKY+G model of substitution). The scale shows the number of nucleotide substitutions per site between DNA sequences. Sequences of B. polonica were set as outgroup. The node support is given in neighbour-joining, maximum likelihood and maximum parsimony bootstraps. 1224[(Fig._2)TD$IG] D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) 1218–1227

Fig. 2. Phylogenetic tree based on the ITS of rDNA of ocellate furcocercariae constructed using the maximum likelihood method in Phyml online (HKY+G model of substitution). The scale shows the number of nucleotide substitutions per site between DNA sequences. Sequences of Trichobilharzia szidati were set as outgroup. The node support is given in neighbour-joining and maximum likelihood bootstraps. [(Fig._3)TD$IG] D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) 1218–1227 1225

Fig. 3. Phylogenetic tree based on the COX1 domain of mtDNA of ocellate furcocercariae constructed using the maximum likelihood method in Phyml online (HKY+G model of substitution). The scale shows the number of nucleotide substitutions per site between DNA sequences. Sequences of Trichobilharzia szidati were set as outgroup. The node support is given in neighbour-joining and maximum likelihood bootstraps.

Table 5 Estimation of pairwise distance between species T. franki haplotypes ‘‘auricularia and peregra’’, T. physellae, T. querquedulae, T. regenti and T. szidati for the D2 and ITS region of the rDNA and the COX1 domain of the mtDNA.

D2 ITS COX1

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1 T. franki ‘‘auricularia’’ 2 T. franki ‘‘peregra’’ 0.005 0.024 0.110 3 T. physellae 0.005 0.004 0.016 0.023 0.100 0.106 4 T. querquedulae 0.002 0.004 0.004 0.019 0.022 0.011 0.088 0.100 0.095 5 T. regenti 0.012 0.010 0.010 0.010 0.060 0.053 0.058 0.047 0.118 0.100 0.119 0.129 6 T. szidati 0.013 0.011 0.011 0.011 0.010 0.100 0.097 0.093 0.089 0.104 0.133 0.103 0.131 0.119 0.127 1226[(Fig._4)TD$IG] D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) 1218–1227

Fig. 4. Phylogenetic tree based on the ITS-2 domain of rDNA of snails constructed using the maximum likelihood method in Phyml online (HKY+G model of substitution). The scale shows the number of nucleotide substitutions per site between DNA sequences. Lymnaea stagnalis was set as outgroup according to Bargues et al. (2001). The node support is given in neighbour-joining, maximum likelihood and maximum parsimony bootstraps.

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