Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France

MEEGID 2376

No. of Pages 9, Model 5G

10 June 2015 Infection, Genetics and Evolution xxx (2015) xxx–xxx 1

Contents lists available at ScienceDirect

Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid 6 7

5

Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France

8

D. Jouet a,⇑, L. Kolárˇová b, C. Patrelle a, H. Ferté a, K. Skírnisson c

3 4

9 10 11 12 13 1 9 5 2 16 17 18 19 20 21 22 23 24 25 26 27 28

a b c

EA 4688 «VECPAR», UFR de Pharmacie, Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51096 Reims, France Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University in Prague and General Hospital in Prague, Czech Republic Institute for Experimental Pathology, Keldur, University of Iceland, Reykjavík, Iceland

a r t i c l e

i n f o

Article history: Received 9 March 2015 Received in revised form 8 June 2015 Accepted 9 June 2015 Available online xxxx Keywords: Trichobilharzia Radix balthica Morphology Molecular biology Anser anser Flyway

a b s t r a c t Parasitological investigations carried out on birds in Iceland and France highlight the presence of four species of avian schistosomes from greylag geese (Anser anser L.): the european nasal species Trichobilharzia regenti and three visceral species, among which an unknown species isolated from blood vessels of the large intestine and liver. Morphological and molecular analyzes of different parasite stages (eggs, adults) revealed new species of Trichobilharzia genus – Trichobilharzia anseri sp. nov. Studies on host–parasite relationship under natural conditions, showed that the life-cycle includes the snail Radix balthica (syn. R. peregra) as intermediate host. The cercariae, already isolated in Iceland from two ponds of the Reykjavik capital area – the Family park and Tjörnin Lake – are the same as those isolated in 1999 by Kolárˇová et al. during the first study on Icelandic parasitic agents of cercarial dermatitis. Ó 2015 Published by Elsevier B.V.

30 31 32 33 34 35 36 37 38 39 40

41 42 43

1. Introduction

44

The trematode family Schistosomatidae (Stiles & Hassall, 1898) comprises fifteen genera of which five are parasites of mammals: Schistosoma, Schistosomatium, Heterobilharzia, Bivitellobilharzia, Orientobilharzia and ten are parasites of birds: Allobilharzia, Anserobilharzia, Austrobilharzia, Bilharziella, Dendritobilharzia, Gigantobilharzia, Jilinobilharzia, Macrobilharzia, Ornitobilharzia and Trichobilharzia. Trichobilharzia spp. represents the largest genus with more than 40 species described (Brant et al., 2006; Brant and Loker, 2009; Brant et al., 2013; Horák et al., 2002; Kolárˇová et al., 2006; Lockyer et al., 2003; Olson et al., 2003; Snyder and Loker, 2000). Species of Trichobilharzia are recognized as the major causative agents of human cercarial dermatitis or swimmer’s itch, which develops as a consequence of transcutaneous penetration of the larvae (ocellate furcocercariae) (Horák et al., 2015; Kolárˇová et al., 2013a). Humans represent an impasse for the parasite development. After penetration into human skin, the development of avian schistosomes is incomplete and the parasites die in various host organs (Haas and Pietsch, 1991; Gay et al., 1999; Horák and Kolárˇová, 2000, 2001; Bayssade-Dufour et al., 2001, 2002;

45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

⇑ Corresponding author. E-mail address: [email protected] (D. Jouet).

Zbikowska, 2003). However, the infection by cercariae leads to direct (skin allergic response and maybe other yet unknown health complications) and indirect (economic losses in person work hours and in tourist industry) consequences (Kolárˇová et al., 1989, 1992, 1997, 1999; Soldánová et al., 2013; Zbikowska et al., 2001). Species identification for avian schistosomes, particularly for Trichobilharzia species, remains complex, in part this is due to incompletely described species (Blair and Islam, 1983). In addition, there are few discriminating morphological characters for adult worms and even fewer for discriminating the cercariae, especially among closely related species. Traditionally, schistosome species have been circumscribed not only on relative measurements and relative positions of internal organs, but also the organ specificity in the final host (Horák et al., 2002), egg morphology (Skírnisson and Kolárˇová, 2008), the distribution of sensory papille on cercariae (Podhorsky´ et al., 2009) and intermediate host specificity (Blair and Islam, 1983). However, any one of the above features is rarely sufficient for species identification. The introduction of molecular tools as species markers, such as 28S and internal transcribed spacers (ITS) rDNA or mitochondrial COI, have greatly improved our ability to discriminate among similar species. More importantly, such tools have allowed an accurate connection across different parasite life cycle stages (cercariae, eggs, adults) and across different times or geographic locations.

http://dx.doi.org/10.1016/j.meegid.2015.06.012 1567-1348/Ó 2015 Published by Elsevier B.V.

Please cite this article in press as: Jouet, D., et al. Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.06.012

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86

MEEGID 2376

No. of Pages 9, Model 5G

10 June 2015 2

D. Jouet et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx

124

With such fine scale detail, we have been able to detect haplotypic differentiation for morphologically very similar species (Aldhoun et al., 2009; Brant and Loker, 2009; Brant et al., 2006; Dvorˇák et al., 2002; Jouet et al., 2009, 2010; Littlewood and Johnston, 1995; Lockyer et al., 2003; Mollaret et al., 1997; Olson et al., 2003; Snyder and Loker, 2000; Webster et al., 2007). In part as an effort to understand the epidemiology of cercarial dermatitis, detailed investigations have been carried out in France and Iceland to study and identify the avian schistosome species circulating in the intermediate snail and definitive waterfowl hosts (Aldhoun et al., 2009; Jouet et al., 2010; Kolárˇová et al., 2006, 2013b; Skírnisson and Kolárˇová, 2008; Skírnisson et al., 2009). In ducks (Anas spp., Aythya spp., Mergus spp.), at least seven schistosomes species have been isolated: Bilharziella polonica, T. regenti, Trichobilharzia franki, Trichobilharzia szidati, Trichobilharzia mergi, and three species of Trichobilharzia spp. Geese and swans (Anatidae) were also found to be involved in the circulation of schistosomes such as Anserobilharzia brantae (Brant et al., 2013) and Allobilharzia visceralis (Kolárˇová et al., 2006), respectively. In the present study, we focus on schistosomes isolated from greylag geese (Anser anser) and ocellate furcocercariae from Radix balthica (syn. R. peregra) (Lymnaeidae) in Iceland and France. Greylag goose is a common bird in Iceland. Two populations are distinguished: (i) a sedentary population (600 birds), distributed in and around the capital area of Reykjavík in SW Iceland, descendents of wild migratory geese that started to breed in the city centre in the late 1950’s; (ii)a migrating population (100.000 birds) that is distributed throughout the country. In France, greylag geese belong predominantly to migrating populations from Northern and Eastern Europe, but with a few populations nesting sporadically over the territory. The aim of our study is to describe a new visceral schistosome species of the Trichobilharzia genus. Detailed morphological and molecular approaches were performed for the description. The fluke completes its life-cycle in Iceland using A. anser and R. balthica (syn. R. peregra) as definitive and intermediate hosts, respectively, and was isolated as well in France along a known flyway of the greylag goose.

125

2. Material and methods

126

2.1. Examination of snails

127

144

R. balthica snails were collected from various localities between 1997 and 2014 in France and Iceland. In Iceland, our investigations included two ponds in Reykjavik: 4000 m2 pond in the Family park, established in 1993 in Laugardalur, and Lake Tjörnin, a 87,000 m2 pond in the center of the capital that is a reserve and feeding site for birds. In addition to these Icelandic ponds, we also sampled and examined snails from several water bodies recognized as areas of risk for cercarial dermatitis in different parts of the island, such as Landmannalaugar, Lake Botnsvatn, Lake My´vatn and Oslandstjörn. In France, most collections were conducted at Annecy and Der-Chantecoq Lakes as well as from smaller recreational ponds (Beauvais, Bourgogne, Ile de France). Since 1997, approx. thousand of snails were sampled and examined for cercarial emergence as described by Jouet et al. (2008) and Kolárˇová et al. (2010). Ocellate furcocercariae were preserved in 95% ethanol and frozen ( 20 °C) until the DNA analysis. Positive snail hosts were frozen directly at 20 °C in individual sterile bags for storage and specific identification.

145

2.2. Examination of birds

146

In both countries representatives of both sedentary and migrating greylag geese populations were collected. In Iceland 80 birds

87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123

128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143

147

were examined; 44 from the sedentary Reykjavík population, usually found dead or diseased and brought to the Keldur Institute by the local authorities; 36 from the migrating population, hunted in autumn during the open season; and 29 geese in France collected by members of network SAGIR and ONCFS (Office National de la Chasse et de la Faune Sauvage). Most of the birds were kept frozen until autopsy but a few birds in Iceland were examined as fresh specimens. Examination for the presence of visceral and nasal schistosomes was performed as described by Skírnisson and Kolárˇová (2008). Eggs and fragments of adults of schistosomes were analyzed by a morphological approach and then fixed in 95% ethanol and frozen for molecular investigations.

148

2.3. Identification of parasites

161

Morphological characterization of cercariae was made on fresh unfixed and on fixed and mounted material. Cercariae were fixed in hot 4% formaldehyde, stained with borax-carmine and mounted in Canada balsam. Because the isolation of the long intact adult schistosomes is difficult, only parasite fragments were obtained. Some of the specimens were conserved for staining in chlorhydric carmine and hematoxylin. The shape and size of the eggs and worms were examined morphologically under a microscope and their dimensions measured on native fresh mounts prior to fixation in 95% ethanol. Morphological details were photographed using a digital camera (Leica DC 300) attached to a microscope (Leica DMLB or Olympus BX50) equipped for differential interference contrast microscopy (DIC; Nomarski interference and phase contrast).

162

2.4. DNA and phylogenetic analysis

176

All stages of parasites (cercariae, adults and eggs) were preserved in 95% ethanol and frozen at 20 °C for DNA analysis. The sequenced samples are listed in Table 1. Also because precise identification of the host is necessary, part of the foot of some positive snail hosts was frozen directly at 20 °C for molecular analyses. After removing ethanol from the samples, DNA was extracted using the Qiamp DNA Mini Kit (Qiagen, Germany) following manufacturer’s instructions. During the first step (tissue lysis), fragments of worms and snails were crushed one by one using a piston pellet (Treff, Switzerland). Polymerase chain reactions (PCR) and sequencing of the D2 domain of the 28S subunit, internal transcribed spacer (ITS2) of ribosomal DNA and COI domain of the mitochondrial DNA were performed under conditions described by Jouet et al. (2010). The ITS2 region was also used for snail identification under conditions previously described (Jouet et al., 2008). PCR products were directly sequenced in both directions with the primers used for DNA amplification (Genoscreen, France). The sequences are deposited in GenBank under the accession numbers KP901348 to KP901395. Sequences were aligned using ClustalW that is included in MEGA version 5 software (Tamura et al., 2011), then checked by eye. The combined D2 domain, ITS2 of the rDNA and the COI domain of the mDNA (1595 bp) were used for tree construction using sequences obtained during this study and sequences of avian schistosomes available in GenBank. Phylogenetic trees were constructed using the Neighbor–Joining (NJ), the Maximum Likelihood (ML), and Minimum Evolution (ME) methods using the MEGA5 software. For each NJ, ML and ME analyses, the most appropriate nucleotide substitution model was determined (GTR+G), gaps were treated as missing data and internal node support was assessed by bootstrapping over 500 replicates.

177

Please cite this article in press as: Jouet, D., et al. Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.06.012

149 150 151 152 153 154 155 156 157 158 159 160

163 164 165 166 167 168 169 170 171 172 173 174 175

178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207

MEEGID 2376

No. of Pages 9, Model 5G

10 June 2015 3

D. Jouet et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx Table 1 Isolates of bird schistosomes used for molecular analysis. Taxa

Stage

Host

ICR9 ICR11 ICR13 ICR17 ICR19 FCI1 F15IS ANS12 ANS22 ANS24 ANS29 ANS32 ANS34 ANS36 ANS40 ANS41 SKI2 SKI4 SKI11 BAL1 ICE18 ICE41 ICE58 ICE80 ICE99 ICE128 ICE134

Cercariae Cercariae Cercariae Cercariae Cercariae Cercariae Cercariae Egg Egg Egg Egg Egg Egg Adult Adult Egg Egg Egg Adult Adult Egg Adult Egg Adult Adult Adult Egg

Radix Radix Radix Radix Radix Radix Radix Anser Anser Anser Anser Anser Anser Anser Anser Anser Anser Anser Anser Anser Anser Anser Anser Anser Anser Anser Anser

Origin

balthica balthica balthica balthica balthica balthica balthica anser anser anser anser anser anser anser anser anser anser anser anser anser anser anser anser anser anser anser anser

Iceland Iceland Iceland Iceland Iceland Iceland Iceland France France France France France France France France France Iceland Iceland Iceland Iceland Iceland Iceland Iceland Iceland Iceland Iceland Iceland

208

3. Results

209

3.1. Cercariae and intermediate host

210

Among the furcocercariae isolated from R. balthica (syn. R. peregra) in France and Iceland, we identified four different species of avian schistosomes: (i) T. regenti, only in France; (ii) T. mergi and Trichobilharzia sp. in France and Iceland; (iii) and Trichobilharzia anseri cercariae, only detected in two ponds in Reykjavik, Iceland, Family Park and Tjörnin Lake. The morphology of T. anseri cercariae (Fig. 1) and their measurements (Table 2) corresponded to the first description of avian schistosome cercariae from Iceland reported by Kolárová et al. (1999). Interestingly, throughout our study period, we found that these unusually large cercariae are the

211 212 213 214 215 216 217 218 219

Accession numbers D2

ITS2

ITS1

COI

KP901348 KP901349 KP901350 KP901351 KP901352 KP901353 KP901354 FJ793861 KP901355 KP901356 FJ793862 FJ793863 – FJ793865 FJ793866 FJ793864 KP901357 KP901358 KP901359 KP901360 KP901361 KP901362 KP901363 – KP901364 KP901365 KP901366

KP901367 – – – – KP901368 – FJ793910 – – FJ793911 FJ793912 KP901369 FJ793914 FJ793915 FJ793913 KP901370 KP901371 KP901372 – – KP901373 KP901374 KP901375 – – –

– – – – – – – KP901376 – – KP901377 KP901378 – – – KP901379 – – – – – – – – – – –

KP901380 – – – – KP901381 – – – – – KP901382 – KP901383 KP901384 KP901385 – – – – – – – – – – –

dominant morphotype of cercariae shed by R. balthica in the Family Park pond. Body surface and tail stem are covered with fine spines. In fixed cercariae, the acetabulum is near the body center and the two eyespots are located in the anterior half of the body. The intestines have two short caeca that terminates behind eyespots. There are two pairs of circumacetabular penetration glands where the second pair surrounds the acetabulum dorsally, then ends behind it. The distal body part behind acetabulum is filled with three pairs of postacetabular penetration glands. Both types of gland ducts pass the cercarial body through head organ, terminating at the anterior end of body. Excretory system is composed of seven pairs of flame cells, two pairs of cilia clusters and excretory ducts. Flame cell formula without clusters of cilia is: 2(3 + 3 + (1)) = 14, and with

Fig. 1. Cercariae of Trichobilharzia anseri. Scale bar: 200 lm.

Please cite this article in press as: Jouet, D., et al. Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.06.012

220 221 222 223 224 225 226 227 228 229 230 231 232 233

MEEGID 2376

No. of Pages 9, Model 5G

10 June 2015 4

D. Jouet et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx

Table 2 Measurements of Trichobilharzia anseri cercariae in comparison with various species of Trichobilharzia spp. isolated from Radix balthica (syn. R. peregra) (in lm). Reference

Total length Body length Body width Acetabulum diameter Tail stem length Tail stem width Furca length

234 235 236 237

238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260

T. anseri

T. sp. ‘‘peregra’’

T. mergi

T. salmanticensis

T. regenti

Simon Martin & Simon Vicente (1999)

Horák et al. (1998)

1013–1176 300–386 119–122 35–48 410–460 55–61 303–330

759 225 55 27 331 49 206

This paper (Fresh)

This paper (Fixed)

Jouet et al. (2010)

Kolárˇová et al. (2013)

1361.4 ± 232 457.5 ± 57 126.1 ± 11 40.5 ± 11 580.6 ± 89 86.8 ± 7 323.3 ± 14

1092.9 ± 141 285.2 ± 25 88 ± 28 27.5 ± 8 523.5 ± 78 48.7 ± 11 284.2 ± 39

864 257 73 28 379 50 227

750 256 81 27 310 30 185

clusters: 2[3 + 3 + (2) + (1)] = 18. Striated furcae are equipped with finfolds, which are well developed almost along their entire length. The cercariae are photosensitive and are able to attach to the wall of glass container, as described by Kolárˇová et al. (2010). 3.2. Adult schistosomes and definitive host Examination of greylag geese for the presences of schistosomes revealed both nasal and visceral flukes. The nasal schistosomes were identified as the European T. regenti and were detected in 3 of 46 (6.5%) geese in Iceland and 3 of 29 (10.3%) in France. In our view, the low prevalence could be related to an accidental infestation. The visceral schistosomes were identified to three different species, each distinguishable by the size and the shape of the eggs that were isolated: (i) T. anseri, previously referred to as Trichobilharzia sp. II by Skírnisson and Kolárˇová (2008) and A. anser Aa3 by Jouet et al. (2009) of which eggs (Fig. 2) and adults were isolated; (ii) an unknown species, previously referred to as Dendritobilharzia sp. and A. anser Aa2 by the same authors, however only the eggs were recovered, which are much smaller in comparison to previous species. Both of these species were isolated in France and Iceland alone or co-parasited; and (iii) A. brantae that was isolated only in geese examined from France (Brant et al., 2013). T. anseri was found in 8 of 29 (27.6%) geese in France, mainly from the Champagne-Ardenne region, and 36 of 80 (45%) geese in Iceland with 29 of 44 (66%) from the sedentary population in the Reykjavík area and 7 of 36 (20%) from the migrating population, collected in Hrútafjörður and Reykhólar, NW Iceland and

My´rdalur in South Iceland. Because no intact worms of T. anseri were recovered, the total length could not be measured. The longest male fragment (anterior part) was 2.949 mm, the longest female fragment measured 2.519 mm. Male (Fig. 3c,d and Table 3) has a filiform body of almost uniform width. Tegument is longitudinally and transversally striated; the oral sucker, acetabulum and inner part of gynaecophoric canal bear well developed spines. The oral sucker encompasses a subterminally–situated oral opening that leads to a long esophagus ending in cecal bifurcation located two-thirds of the distance between the anterior end and the acetabulum. Cecal reunion is posterior to vesicula seminalis (v.s.) interna, in front of canalis gynaecophorus (ventral grove), at the level of prostatic channel. The reunited intestine continues along the gynaecophoric canal to the posterior area in a sinuous course between the testes and ends before a slightly rounded tail. In fresh worms, the intestine is sometimes filled by dark brown pigment, probably hematin, from hemoglobin digestion. Contrary to the body length, the gynaecophoric canal is short and slightly broadened (0.294  0.074 mm), and includes spiny ventral groove and genital papilla. The reproductive system consists of numerous testes (>120), collecting ducts and vesicula seminalis. Testes are spherical or elliptical and commence in a relatively long distance posterior to the end of gynaecophoric canal (0.326 mm); they continue in one row to the body end. They are located one after another and a common cecum interlocks between them, such that each curve has one to four testicles at each side. The elongated seminal vesicle occupies most of the space between the acetabulum and ventral groove and is well divided into external and internal parts. The vesicula seminalis (v.s.) interna is two

Fig. 2. Eggs of Trichobilharzia anseri. (A) egg with miracidium (260  64 lm); (B) egg without miracidium (245  55 lm).

Please cite this article in press as: Jouet, D., et al. Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.06.012

261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289

MEEGID 2376

No. of Pages 9, Model 5G

10 June 2015 D. Jouet et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx

5

Fig. 3. Anterior and posterior end of a female (a,b) and a male (c,d) of Trichobilharzia anseri. A, acetabulum; CB, caeca bifurcation; CG, canalis gynaecophorus; CR, cecal reunion; GO, genital opening; GP, genital papilla; I, intestine; O, ovary; OE, esophagus; OS, oral sucker; RS, receptaculum seminis; T, testes; U, uterus; VI, vitellaria; VSE, vesicula seminalis externa; VSI, vesicula seminalis interna. Egg with miracidium (e).

290 291 292 293 294 295 296 297 298 299 300 301 302 303

times longer than v.s. externa. The ejaculatory duct ends by a well developed genital papilla that arises posterior to the end of the v.s. interna, and transverses the prostatic region where cecal reunion occurs. The genital opening is on the right side of the anterior part of the gynaecophoric canal. Since only dead worms were examined, we could not observe the excretory system. Female (Fig. 3a,b and Table 4) is thinner than the male. Similarly as for males, the body is filiform with spatulated posterior tail. The oral sucker and acetabulum are the same as in the males; the esophagus ends by cecal bifurcation which begins not far anterior to the acetabulum. The cecal reunion is located at the level of receptaculum seminis. The intestine is filled with dark pigment and continues a sinuous path between vitelline follicles until close to the posterior part of body. The reproductive system consists of

an elongate ovary, slightly curved, followed by a seminal receptacle. Both organs are distinctly visible in the anterior part of the body. Between them and arising from seminal receptacle opens the Laurer’s canal. Behind the seminal receptacle are numerous vitelline follicles (more than 100) aligned until to the posterior end of the body, interlocked by vitelloduct. The uterus is located between the acetabulum and ovary, sometimes containing a single immature egg, which ends at the genital opening situated just posterior to the acetabulum. Eggs (Figs. 2 and 3e and Table 4) are spindle-shaped with a long straight axis with the greatest width near the middle. The poles are long and slender. One of approx. 30% of the total egg length is conical and tapers gradually to a sharp point that ends in a usually curved, but sometimes straight, process on the top. This end was

Please cite this article in press as: Jouet, D., et al. Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.06.012

304 305 306 307 308 309 310 311 312 313 314 315 316 317

MEEGID 2376

No. of Pages 9, Model 5G

10 June 2015 6

D. Jouet et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx

Table 3 Measurements of Trichobilharzia anseri male.

Maximum fragment length Width at the anterior end Width at the level of acetabulum Width before spatulate end Width at posterior end Width behind canalis gynec. Length of spatulate end Oral sucker Acetabulum to anterior end Acetabulum Gyn.can. to ant. end Gyn.can. to acetab. Gyn. canal width Gyn. canal length Esophagus (= caeca bifurcation) VSE to ant.end Ves. sem. externa Ves. sem. interna Genital papilla Number of testes Size of testes 1st testis to gyn. can. Last testis to post. end

318 319 320 321 322

N° measured

Mean ± SD (mm)

1 7 7

2.949 0.044 ± 0.006 0.052 ± 0.005

3 3 7 3 9 8 8 7 7 9 9 8

0.044 ± 0.011 0.082 ± 0.030 0.031 ± 0.004 0.283 ± 0.058 0.036 ± 0.007 0.540 ± 0.079 0.035 ± 0.007 1.043 ± 0.061 0.535 ± 0.027 0.074 ± 0.022 0.295 ± 0.080 0.395 ± 0.036

6 7 7 7 2 6 8 1

0.089 ± 0.018 0.137 ± 0.026  0.023 ± 0.003 0.278 ± 0.028  0.019 ± 0.003 0.017 ± 0.003 >110 0.034 ± 0.014  0.025 ± 0.008 0.326 ± 0.168 0.038

situated at the front of female genital tract. The other pole is even longer, reaching approx. 35% of the total egg length. This pole does not markedly taper until close to the rounded, but, sharp end. The poles are often slightly flexed sideward (Skírnisson and Kolárˇová, 2008).

323

3.3. Sequencing and phylogenetic analysis

324

Based on the ITS2 sequences, the snails shedding T. anseri ocellate furcocercariae in Iceland is R. balthica (Linnaeus, 1758) syn. R. peregra (Müller, 1774), R. ovata (Draparnaud, 1805) (Bargues et al., 2001; Glöer, 2002). The molecular analysis of the combined D2–ITS2–COI domains confirmed that cercariae from the snails, and adults and eggs from A. anser belong to Trichobilharzia (Fig. 4). Moreover, these analyses showed clearly that DNA sequences of all these stages belonged to the same clade, thus providing evidence that we have a single species that is distinct from all other species with known genetic data

325 326 327 328 329 330 331 332 333

Table 4 Measurements of Trichobilharzia anseri female.

Maximum fragment Length Maximum width Width around the egg Width at ovarian region Width before spatulate end Length of spatulate end Oral sucker Acetab.to anter.end Acetabulum Esophagus Ovary Ovary to acetabulum Ovary length Receptaculum Seminis Vittelaria to sem. recept. Vitellaria Immature egg in the uterus Mature egg in the intestine

N° measured

Mean ± SD (mm)

1 4 5 11 4 1 12 10 11 10 6 12 5 9 5 5 4 61

2.519 0.053 ± 0.009 0.042 ± 0.007 0.052 ± 0.012 0.052 ± 0.009 0.140 0.041 ± 0.006  0.036 ± 0.004 0.534 ± 0.084 0.038 ± 0.008  0.034 ± 0.005 0.386 ± 0.070 0.802 ± 0.133 0.436 ± 0.075 0.399 ± 0.064 x 0.016 ± 0.003 0.163 ± 0.068 0.112 ± 0.037 0.020 ± 0.005  0.019 ± 0.003 0.191 ± 0.035  0.043 ± 0.005 0.255 ± 0.019  0.059 ± 0.007

(e.g. T. regenti, T. franki, Trichobilharzia querquedulae, Trichobilharzia physellae, T. szidati, T. mergi and Trichobilharzia stagnicolae). For the ITS2 domain (345 positions), our sequences correspond with the haplotype of cercariae (GenBank Accn: FJ469784– FJ469791) previously isolated from R. balthica in the Family Park in Iceland (Aldhoun et al., 2009). With respect to the partial mitochondrial COI (692 bp) fragment, intraspecies variation was about 5 changes (0.7%) compared to the interspecific variation of 85–93 changes (12.3–13.4%) observed with other species of the genus. In view of the above distinct molecular data, and morphological characters these specimens belong to a new species within the genus Trichobilharzia. The flukes originating from A. anser were named T. anseri.

334

3.4. Systematic summary

348

335 336 337 338 339 340 341 342 343 344 345 346 347

Species: T. anseri sp. n. Type host: A. anser L. (greylag goose)

349 350

Site of infection: Mesenteric vessels of the intestine and liver. Intermediate host: R. balthica L. (syn. R. peregra) Type locality: Family Park and Tjörnin, Reykjavik, Iceland.

352

Other localities: Hrútafjörður, Reykhólar, My´rdalur, Iceland. Der-Chantecoq Lake, Marne, France. Type material:

357

Cercariae: Natural History Museum of Vienna, Austria: holotype: Cercaria spp. Laugardalur, Reykjavík, Iceland NHMW EV 3690; paratype: Cercaria spp. Lake Tjornin NHMW EV 3689. Snail: Natural History Museum of Vienna, Austria: NHMW Moll.90.677 Adults: Parasite Division, Museum of Southwestern Biology, University of New Mexico (http://arctos.database.museum/ guid/MSB:Para): Allotype (anterior end of a female): MSB: Para 20889; Holotype (anterior end of a male): MSB: Para 20888; Paratype – (posterior part of a male); paratypes (anterior parts of males): MSB: Para 20890. Deposition of the nucleotide sequences: Sequences deposited in GenBank under accession numbers: KP901348 to KP901395 Etymology: The nomen ‘‘anseri’’ is derived from the scientific name of the definitive host.

361

351

353 354 355 356

358 359 360

362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378

4. Discussion

379

The utilization of molecular tools to complement morphological characterization in taxonomy has allowed species definitions to be more holistic – including morphology of larval stages, eggs and adult flukes (especially the position of the cecal reunion and internal organs), genetic data, life–cycle characteristics (in natural or experimental conditions), and site where adult flukes reside within the definitive host, including pathologies which arise from this infection. Genetic approach connects data from larval stages and eggs (often impossible to distinguish to species) to adult worms across time and space. These methods have been particularly successful elucidating species diversity and host use for parasites with complex life cycles, avoiding mistakes of the past, with description of species now considered to be incertae sedis (Blair and Islam, 1983). Experimental life cycle also allowed description of new species, but it is time consuming and often logistically difficult, relying on readily available hosts. Even if we are able to describe the life cycle stages, genetic data is still important because it is not possible to

380

Please cite this article in press as: Jouet, D., et al. Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.06.012

381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397

MEEGID 2376

No. of Pages 9, Model 5G

10 June 2015 D. Jouet et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx

*

7

* Trichobilharzia franki * Trichobilharzia querquedulae * Trichobilharzia physellae * Trichobilharzia regenti

*

*

Bilharziella polonica

ANS32 ANS40 ANS36 Trichobilharzia anseri * ANS41 FCI1 ICR9 Trichobilharzia szidati * Trichobilharzia mergi * Trichobilharzia stagnicolae * Anserobilharzia brantae * Allobilharzia visceralis * Gigantobilharzia huronensis Dendritobilharzia pulverulenta

*

0,05

Fig. 4. Phylogenetic tree based on the combined D2-ITS2-COI, constructed using the Maximum Likelihood algorithm. The scale shows the number of nucleotide substitutions per site between DNA sequences. Bilharziella polonica was set as outgroup. The node support is given in Neighbor-Joining, Maximum Likelihood and Minimum Evolution bootstraps. The ‘‘⁄’’ indicates node support of >95% bootstrap for NJ, ML and ME.

398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440

capture the range of intraspecific variation, even with good morphological descriptions (Kolárˇová et al., 2013a). We used this integrative approach for the description of a new species. Our results were compared with other schistosome genera, and with species within the genus Trichobilharzia. During the first prospective and descriptive study of dermatitis causing agents conducted in Iceland, cercariae of T. anseri were isolated and described initially by Kolárˇová et al. (1999), which recognized very large furcocercariae emitted by snails in Family Park, Reykjavík. However, when the authors conducted their life cycle studies, they failed to obtain infections because they used mallards instead of greylag geese. These same cercariae were repeatedly rediscovered at the same site between 2004 and 2013 (Aldhoun et al., 2009; Jouet et al., unpublished) and were characterized with their snail morphologically and molecularly. Taking into account the arrangement of the circumacetabular penetration glands, number of flame cells, eyespots and finfolds on the furcae, T. anseri cercariae belong to the Trichobilharzia spp., except for Trichobilharzia arcuata (Islam, 1986), australis australis (Blair and Islam, 1983), and Trichobilharzia corvi (Ito, 1960, cited by Blair and Islam (1983)) for which the excretory system is different. The intermediate hosts are freshwater snails in the family Lymnaeidae, R. balthica (syn. R. peregra). This species has been documented as the most widely used snail in the life cycle of avian schistosomes in Iceland and in France (Jouet et al., 2008, 2010; Skírnisson and Kolárˇová, 2008). Given the specificity of our avian schistosomes with their intermediate host, we can differentiate T. anseri cercariae from Trichobilharzia cameroni Wu, 1953, Trichobilharzia oregonensis (Macfarlane and Macy, 1946) Macy and More, 1955, Trichobilharzia jequitibaensis Leite, Costa et Costa, 1978, Trichobilharzia adamsi Edwards et Jansch, 1955, T. physellae (Talbot, 1936) McMullen and Beaver, 1945 and T. querquedulae (McLeod, 1937) McMullen and Beaver, 1945, which use snails in the family Physidae for their development. In addition, adults of T. physellae and T. querquedulae also differ by the anterior position of the cecal reunion, and the arrangement of testes, respectively. In addition, the morphological comparison of our cercariae to other Trichobilharzia spp. larvae, also emitted by R. balthica in Europe (Table 2), confirms their membership to a single species, especially by their macroscopically visible very large size (greater than 1 mm). Location of adult flukes in vessels of the intestine and liver classifies T. anseri as member of the visceral schistosome group.

The habitat and morphology of T. anseri distinguishes it from species of Trichobilharzia that inhabit the nasal areas of birds: Trichobilharzia aureliani Fain, 1956, T. arcuata Islam, 1986, T. australis Blair and Islam, 1983, T. duboisi Fain, 1959, T. nasicola Fain, 1955, T. regenti Horák, Kolárˇová et Dvorˇák, 1998, Trichobilharzia rodhaini Fain, 1955 and Trichobilharzia spinulata Fain, 1955. Adults of T. anseri have distinct morphological features from those of other visceral species that have known developmental stages and life cycles. Contrary to T. anseri, cecal reunion is just posterior to acetabulum in males of Trichobilharzia elvae (Miller, 1923) McMullen and Beaver, 1945, Trichobilharzia limnaeae Yamaguti, 1971, Trichobilharzia ocellata described by Chikami (1961), T. physellae (Talbot, 1936) McMullen and Beaver, 1945, T. stagnicolae (Talbot, 1936) McMullen and Beaver, 1945, T. szidati Neuhaus, 1952, and T. corvi (Yamaguti, 1941) McMullen and Beaver, 1945. T. anseri specimens also differ in the position of the cecal reunion from Trichobilharzia species which are regarded as species inquirendae (Blair and Islam, 1983): Trichobilharzia anatina Fain, 1955; Trichobilharzia cerylei, Fain 1955; Trichobilharzia lonchurae (Fischtal and Kuntz, 1973) Tsai et al., 1979; T. ocellata (La Valette, 1855) Brumpt, 1931; Trichobilharzia guandongensis Tsai et al., 1979. Otherwise, T. corvi, T. cerylei, T. lonchurae and T. guandongensis also differ by the ovoid shape of their eggs, as for Trichobilharzia filiformis (Szidat, 1938), McMullen and Beaver, 1945. Number and distribution of the testes also differentiate T. anseri from Trichobilharzia jianensis Liu, Chen, Jin, Tan et Yang, 1977, Trichobilharzia paoi (Kung, Wang et Chen, 1960) Tang et Tang, 1962, Trichobilharzia brevis Basch, 1966, T. franki Müller et Kimmig, 1994, Trichobilharzia salmanticensis Simon–Martin et Simon–Vicente, 1999, Trichobilharzia parocellata (Johnston and Simpson, 1939) Islam and Copeman, 1980 and Trichobilharzia berghei Fain, 1955. Lastly, other individual criteria such as general morphology of eggs and size and position of internal organs for adults, distinguish T. anseri from Trichobilharzia maegraithi Kruatrachue, Bhaibulaya, Chedapan et Harinasuta, 1967, T. mergi Kolárˇová, Skírnisson, Ferté et Jouet, 2013, Trichobilharzia longicauda Davis, 2006, T. ocellata (La Valette, 1855) Brumpt, 1931, including representatives of species inquirendae, i.e. Trichobilharzia alaskensis Harkema, McKeever et Becker, 1957, Trichobilharzia kowalewskii (Ejsmont, 1929) McMullen and Beaver, 1945, Trichobilharzia indica Baugh, 1963, Trichobilharzia zongshani Tsai et al., 1979, Trichobilharzia shoutedeni

Please cite this article in press as: Jouet, D., et al. Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.06.012

441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483

MEEGID 2376

No. of Pages 9, Model 5G

10 June 2015 8 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549

D. Jouet et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx

Fain, 1955, Trichobilharzia kossarewi Skrjabin et Zakharov, 1920, Trichobilharzia burnetti (Brackett, 1942) McMullen and Beaver, 1945, Trichobilharzia waubesensis (Brackett, 1942) McMullen and Beaver, 1945, Trichobilharzia horiconensis (Brackett, 1942) McMullen and Beaver, 1945, Trichobilharzia kegonsensis (Brackett, 1942) McMullen and Beaver, 1945 and Trichobilharzia littlebi (Byrd, 1956) Farley, 1971. T. anseri eggs are particularly specific. Although they have a spindle-shaped form common to Trichobilharzia eggs, the very elongated shape and size of the poles markedly differ from all other species of the genus. Based on molecular analysis, sequences obtained in the present study belong to the BTGD clade (Brant et al., 2006) and thus to the Trichobilharzia spp. Our results confirm that the cercariae isolated from R. balthica, and the eggs and adults isolated from A. anser belong to a new unique species, T. anseri, that is different from all the other species of the genus. Pairwise uncorrected p genetic distances for 28S D2 (0.4–3.2%), ITS2 (2.2–6.5%) and COI (13–14.7%) among species of Trichobilharzia spp. are consistent with this new species status (Brant and Loker, 2009; Vilas et al., 2005). In the tree (Fig. 4), compared with the other species of Trichobilharzia, the position of T. anseri is close to those of T. szidati, T. mergi and T. stagnicolae. Nevertheless, despite this close phylogenetic relationship, T. anseri differs from these species mainly in spectrum of the intermediate and definitive hosts, shape of the eggs, and morphology of the adults. Furthermore, it is the first species of Trichobilharzia (based on sequence data) to have come from a non-duck host. Similarly as for other schistosomes, the geographic distribution of T. anseri is delineated by its hosts, which seems entirely realized in Iceland, i.e., specifically (geolocally) in two areas of Reykjavik – the Family Park and Tjörnin pond, as we have shown, but probably also in other areas due to the presence of the parasite in the migrating geese populations. At present, no studies have highlighted the parasite larvae in another country. The very limited distribution of T. anseri larval stages to the only Reykjavik area could be explained by the realization of the parasitic cycle through the sedentary goose population residing in the city. However, considering occurrence of the flukes in migrating populations from other areas in Iceland and France, it can be suggested that T. anseri can also occur along the migratory routes of greylag geese. Thus the opportunity to distribute eggs exists. Recently, four main flyways were recognized in Europe for A. anser: (i) breeding range in Iceland and a wintering range in UK or Ireland; (ii) a sedentary population in Scotland; (iii) a breeding range in North-Western Europe and wintering in South-Western Europe; and (iv) a breeding range in Central Europe and wintering in North Africa (Comolet-Tirman, 2009). The discovery of T. anseri from greylag geese in France in the Champagne–Ardenne region could be explained either by the presence of migratory geese from Iceland or by geese infected in the north of the UK, which would imply the presence of the parasite larval stages in this geographical area. The fact that the cercariae were not found in snails in France could be due to the lack of adequate intermediate hosts in goose resting sites, not allowing establishment of the parasite life–cycle. It is also interesting to note that in France, greylag geese were infected by schistosomes, which originated from Iceland as well as from North America (Brant et al., 2013). So far, none of our studies have revealed shared parasites in birds between North America and Iceland. This finding could be related to the nature of intermediate hosts present in each of these sites; R. balthica in Iceland; R. auricularia in North America; and both species in France. Besides the fact that France seems to be a migratory crossroads for many species of birds, we can point out the importance of parasitic studies in the monitoring of avian populations migration. Migratory birds are recognized as vectors for potentially dangerous microorganisms for humans (Avian Influenza, West Nile Virus, Babesia spp., Haemoparasites) over long distances (Dieter et al., 2001; Gicik

and Arslan, 2003; Tsiodras et al., 2008). By using parasites as biological tags, it could be possible in the future to obtain a more detailed assessment of the movement of bird populations and associated risks.

550

5. Conclusions

554

Iceland and France are known to have highly favorable natural conditions for avian schistosomes and further investigations in these countries will probably revealed more species. Our study confirms that combination of molecular methods with traditional morphological observations of flukes facilitates taxonomical determination, life-cycle description and their geographic distribution. Using holistic approach, we describe a new species of Trichobilharzia spp., the life cycle of which takes place in greylag geese (A. anser) and R. balthica as definitive and intermediate host, respectively. The complete life-cycle of T. anseri occurred in Iceland, at least in the city of Reykjavík, but also in different ponds all along flyway of migrating populations. Kolárˇová et al. (1999) were able to show the pathogenic role of this species for abnormal hosts by experimental infection in mice. In the future, therefore, it would be interesting to determine the role of T. anseri cercariae as causative agent of cercarial dermatitis in humans. Given the increase of new species described, especially within Trichobilharzia, a deep taxonomical revision of the genus is required in order to establish the validity of previously described species.

555

6. Uncited reference

575

Olsen (2009).

551 552 553

556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574

576

Acknowledgements

577

In Iceland, the authors would like to thank Gudmundur Björnsson, Thorvaldur Th. Björnsson, Omar Dabney and the staff of the Family Park in Reykjavík for valuable help with collecting greylag geese, and Jitka Aldhoun and Libor Mikes for help with dissections of geese in 2003. Financial support was provided by the Research Fund of the University of Iceland and the Jules Verne Program for French–Icelandic scientific cooperation. In France, financial support for this study was provided by ONCFS (Office National de la Chasse et de la Faune Sauvage). We thank the ONCFS staff of Der-Chantecoq, especially Yves Maupois and Daniel Delorme, and LVD10 (Olivier Gibout) for providing material. In the Czech Republic, the studies were financially supported by the Charles University in Prague (Research Programs PRVOUK No. P25/LF1/2 and UNCE No. 204017) and the Grant Agency of the Ministry of Health (IGA MZ CR NT 13108–4/2012). We are grateful to S. for having proofread the English.

578

References

594

Aldhoun, J., Kolárˇová, L., Horák, P., Skírnisson, K., 2009. Bird schistosome diversity in Iceland: molecular evidence. J. Helminthol. 83, 173–180. Bargues, M.D., Vigo, M., Horák, P., Dvorˇák, J., Patzner, R.A., Pointier, J.P., Jackiewicz, M., Meier-Brook, C., Mas Coma, S., 2001. European Lymnaeidae (Mollusca: Gastropoda), intermediate hosts of trematodiases, based on nuclear ribosomal DNA ITS-2 sequences. Infect. Genet. Evol. 1 (2), 85–107. Bayssade-Dufour, C., Martins, C., Vuong, P.N., 2001. Histopathologie pulmonaire d’un modèle mammifère et dermatite cercarienne humaine. Med. Mal. Infect. 31, 713–722. Bayssade-Dufour, C., Vuong, P.N., René, M., Martin-Loehr, C., Martins, C., 2002. Lésions viscérales de mammifères et oiseaux, exposés aux agents de dermatite cercarienne humaine. Bull. Soc. Pathol. Exot. 95 (4), 229–237. Blair, D., Islam, K.S., 1983. The life–cycle and morphology of Trichobilharzia australis n. sp. (Digenea: Schistosomatidae) from the nasal blood vessels of the black duck (Anas superciliosa) in Australia, with a review of the genus Trichobilharzia. Syst. Parasitol. 5, 89–117.

595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610

Please cite this article in press as: Jouet, D., et al. Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.06.012

579 580 581 582 583 584 585 586 587 588 589 590 591 592 593

MEEGID 2376

No. of Pages 9, Model 5G

10 June 2015 D. Jouet et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679

Brant, S.V., Jouet, D., Ferte, H., Loker, E.S., 2013. Anserobilharzia gen. n. (Digenea, Schistosomatidae) and redescription of A. brantae (Farr & Blankemeyer, 1956) comb. n. (syn. Trichobilharzia brantae), a parasite of geese (Anseriformes). Zootaxa 3670 (2), 193–206. Brant, S.V., Loker, E.S., 2009. Molecular systematics of the avian schistosome genus Trichobilharzia (Trematoda: Schistosomatidae) in North America. J. Parasitol. 95, 941–963. Brant, S.V., Morgan, J.A., Mkoji, G.M., Snyder, S.D., Rajapakse, R.P., Loker, E.S., 2006. An approach to revealing blood fluke life cycles, taxonomy, and diversity: provision of key reference data including DNA sequence from single life cycle stages. J. Parasitol. 92 (1), 77–88. Chikami, A., 1961. Studies on Trichobilharzia ocellata (La Valette, 1855). Jpn. J. Parasitol. 10, 106–118. Comolet-Tirman, J., 2009. L’oie cendrée Anser anser (L.1758) en France et en Europe. Dynamique de population, statuts de conservation, voies de migration et dates de migration prénuptiale. Service du Patrimoine Naturel, Dpt Ecologie et Gestion de la Biodiversité. Rapport SPN/DEGB/MNHN N°4. Dieter Jr, R.A., Dieter, R.S., Dieter 3rd, R.A., Gulliver, G., 2001. Zoonotic diseases: health aspects of Canadian geese. Int. J. Circumpolar Health. 60 (4), 676–684. Dvorˇák, J., Vancova, S., Hampl, V., Flegr, J., Horák, P., 2002. Comparison of European Trichobilharzia species based on ITS1 and ITS2 sequences. Parasitology 124, 307–313. Fain, A., 1956. Les schistosomes d’oiseaux du genre Trichobilharzia Skrjabin et Zakharov, 1920 au Ruanda-Urundi. Rev. Zool. Bot. Afr. 54, 147–178. Gay, P., Bayssade-Dufour, C., Grenouillet, F., Bourezane, Y., Dubois, J.P., 1999. Etude expérimentale de dermatites cercariennes provoquées par Trichobilharzia en France. Med. Mal. Infect. 29, 629–637. Gicik, Y., Arslan, M.O., 2003. The prevalence of helminths in the alimentary tract of geese (Anser anser domesticus) in Kars District, Turkey. Vet. Res. Commun. 27 (5), 391–395. Glöer, P., 2002. Die Süßwassergastropoden Nord- und Mitteleuropas. Bestimmungsschlüssel, Lebensweise, Verbreitung. Die Tierwelt Deutschlands, 73. Teil. 327 S. ConchBooks, Hackenheim. Haas, W., Pietsch, U., 1991. Migration of Trichobilharzia ocellata schistosomula in the duck and in the abnormal murine host. Parasitol. Res. 77 (7), 642–644. Horák, P., Kolárˇová, L., 2000. Survival of bird Schistosomes in mammalian lungs. Int. J. Parasitol. 30 (1), 65–68. Horák, P., Kolárˇová, L., 2001. Bird Schistosomes: do they die in mammalian skin? Trends Parasitol. 17 (2), 66–69. Horák, P., Kolárˇová, L., Adema, C.A., 2002. Biology of the schistosome genus Trichobilharzia. Adv. Parasitol. 52, 155–233. Horák, P., Mikeš, L., Lichtenbergová, L., Skála, V., Soldánová, M., Brant, S.V., 2015. Avian schistosomes and outbreaks of cercarial dermatitis. Clin. Microbiol. Rev. 28 (1), 165–190. Islam, K.S., 1986. The morphology and life–cycle of Trichobilharzia arcuata n. sp. (Schistosomatidae: Bilharziellinae) a nasal schistosome of water whistle ducks (Dendrocygna arcuata) in Australia. Syst. Parasitol. 8, 117–128. Jouet, D., Ferté, H., Depaquit, J., Rudolfová, J., Latour, P., Zaniella, D., Kaltenbach, M.L., Léger, N., 2008. Trichobilharzia spp. in natural conditions in Annecy Lake, France. Parasitol. Res. 103, 51–58. Jouet, D., Ferté, H., Hologne, C., Kaltenbach, M.L., Depaquit, J., 2009. Avian schistosomes in French aquatic birds: a molecular approach. J. Helminthol. 83 (2), 181–189. Jouet, D., Skírnisson, K., Kolárˇová, L., Ferté, H., 2010. Molecular diversity of Trichobilharzia franki in two intermediate hosts (Radix auricularia and Radix peregra): a complex of species. Infect. Genet. Evol. 10, 1218–1227. Kolárˇová, L., Gottwaldova, V., Cechova, D., Sevcova, M., 1989. The occurrence of cercarial dermatitis in Central Bohemia. Int. J. Hyg. Environ. Med. 189 (1), 1–13. Kolárˇová, L., Horák, P., Fajfrlik, K., 1992. Cercariae of Trichobilharzia szidati Neuhaus, 1952 (Trematoda: Schistosomatidae): the causative agent of cercarial dermatitis in Bohemia and Moravia. Folia Parasitol. 39 (4), 399–400. Kolárˇová, L., Horák, P., Sitko, J., 1997. Cercarial dermatitis in focus: schistosomes in the Czech Republic. Helminthologia 34 (3), 127–139. Kolárˇová, L., Horák, P., Skírnissson, K., 2010. Methodical approaches in the identification of areas with a potential risk of infection by bird schistosomes causing cercarial dermatitis. J. Helminthol. 84, 327–335. Kolárˇová, L., Horák, P., Skírnisson, K., Marecˇková, H., Doenhoff, M., 2013a. Cercarial dermatitis, a neglected allergic disease. Clin. Rev. Allergy Immunol. 45 (1), 63– 74.

9

Kolárˇová, L., Rudolfová, J., Hampl, V., Skírnisson, K., 2006. Allobilharzia visceralis gen. nov., sp. nov. (Schistosomatidae-Trematoda) from Cygnus cygnus (L.) (Anatidae). Parasitol. Int. 55 (3), 179–186. Kolárˇová, L., Skírnisson, K., Ferté, H., Jouet, D., 2013b. Trichobilharzia mergi sp.nov. (Trematoda: Digenea: Schistsomatidae), a visceral schistosome of Mergus serrator (L.) (Aves: Anatidae). Parasitol. Int. 62 (3), 300–308. Kolárˇová, L., Skírnisson, K., Horák, P., 1999. Schistosome cercariae as the causative agent of swimmer’s itch in Iceland. J. Helminthol. 73 (3), 215–220. Littlewood, D.T., Johnston, D.A., 1995. Molecular phylogenetics of the four Schistosoma species groups determined with partial 28S ribosomal RNA gene sequences. Parasitology 111 (2), 167–175. Lockyer, A.E., Olson, P.D., Ostergaard, P., Rollinson, D., Johnston, D.A., Attwood, S.W., Southgate, V.R., Horák, P., Snyder, S.D., Le, T.H., Agatsuma, T., McManus, D.P., Carmichael, A.C., Naem, S., Littlewood, D.T., 2003. The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships of Schistosoma Weinland, 1858. Parasitology 126 (3), 203– 224. McMullen, D.B., Beaver, P.C., 1945. Studies on schistosome dermatitis. IX. The life cycles of three dermatitis-producing schistosomes from birds and a discussion of the subfamily Bilharziellinae (Trematoda: Schistosomatidae). Am. J. Hyg. 42, 128–154. Mollaret, I., Jamieson, B.G., Adlard, R.D., Hugall, A., Lecointre, G., Chombard, C., Justine, J.L., 1997. Phylogenetic analysis of the Monogenea and their relationships with Digenea and Eucestoda inferred from 28S rDNA sequences. Mol. Biochem. Parasitol. 90 (2), 433–438. Neuhaus, W., 1952. Biologie und Entwicklung von Trichobilharzia szidati n. sp. (Trematoda, Schistosomatidae), einem Erreger von Dermatitis beim Menschen. Z. Parasitenk. 15, 203–266. Olsen, G.H., 2009. Bacterial and parasitic diseases of Anseriformes. Vet. Clin. North Am. Exot. Anim. Pract. 12 (3), 475–490. Olson, P.D., Cribb, T.H., Tkach, V.V., Bray, R.A., Lttlewood, D.T., 2003. Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda). Int. J. Parasitol. 33 (7), 733–755. Podhorsky´, M., Hu˚zová, Z., Mikeš, L., Horák, P., 2009. Cercarial dimensions and surface structures as a tool for species determination of Trichobilharzia spp. Acta Parasitol. 54, 28–36. Skírnisson, K., Aldhoun, J.A., Kolárová, L., 2009. A review on swimmer’s itch and the occurrence of bird schistosomes in Iceland. J. Helminthol. 83 (2), 165–171. Skírnisson, K., Kolárˇová, L., 2008. Diversity of bird schistosomes in anseriform birds in Iceland based on egg measurements and egg morphology. Parasitol. Res. 103, 43–50. Snyder, S.D., Loker, E.S., 2000. Evolutionary relationships among the Schistosomatidae (Platyhelminthes: Digenea) and an Asian origin for Schistosoma. J. Parasitol. 86 (2), 283–288. Soldánová, M., Selbach, C., Kalbe, M., Kostadinova, A., Sures, B., 2013. Swimmer’s itch: etiology, impact, and risk factors in Europe. Trends Parasitol. 29 (2), 65–74. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Tsiodras, S., Kelesidis, T., Kelesidis, I., Bauchinger, U., Falagas, M.E., 2008. Human infections associated with wild birds. J. Infect. 56 (2), 83–98. Vilas, R., Criscione, C., Blouin, M., 2005. A comparison between mitochondrial DNA and the ribosomal internal transcribed regions in prospecting for cryptic species of Platyhelminth parasites. Parasitology 131, 839–846. Webster, B.L., Rudolfová, J., Horák, P., Littlewood, D.T., 2007. The complete mitochondrial genome of the bird schistosome Trichobilharzia regenti (Platyhelminthes: Digenea), causative agent of cercarial dermatitis. J. Parasitol. 93 (3), 553–561. Yamaguti, S., 1941. Studies on the helminth fauna of Japan. Part 32. Trematodes of birds. Jpn. J. Zool. 9, 321–341. Yamaguti, S., 1971. Synopsis of Digenetic Trematodes of Vertebrates. Keigaku Publishing Co, Tokyo, p. 1074. Zbikowska, E., 2003. Is there a potential danger of ‘‘swimmer’s itch’’ in Poland? Parasitol. Res. 89, 59–62. Zbikowska, E., Franckiewicz Grygon, B., Wojcik, A.R., 2001. The case of dermatitis in the human as a result of the influence of the cercaria of the bird schistosome. Wiad. Parazytol. 47 (3), 427–431.

Please cite this article in press as: Jouet, D., et al. Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.06.012

680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748