Alain de Chambrier Æ Sandrine C. Coquille Æ Jean Mariaux Æ Vasyl Tkach

Transcription

1 Syst Parasitol (2009) 73:49 64 DOI /s Redescription of Testudotaenia testudo (Magath, 1924) (Eucestoda: Proteocephalidea), a parasite of Apalone spinifera (Le Sueur) (Reptilia: Trionychidae) and Amia calva L. (Pisces: Amiidae) in North America and erection of the Testudotaeniinae n. subfam. Alain de Chambrier Æ Sandrine C. Coquille Æ Jean Mariaux Æ Vasyl Tkach Received: 29 July 2008 / Accepted: 20 October 2008 Ó Springer Science+Business Media B.V Abstract Testudotaenia testudo (Magath, 1924) is redescribed from the intestine of the softshell turtle Apalone spinifera (Le Sueur) (Trionychidae) and the bowfin Amia calva Linnaeus (Amiidae) from Reelfoot Lake, Tennessee, United States. A new subfamily, the Testudotaeniinae, is erected. The new taxon differs from all proteocephalidean subfamilies in the position of the genital organs in relation to the longitudinal internal musculature, i.e. the testes are cortical, rarely medullary; the ovary is partly medullary, with cortical lobes; the vitelline follicles are mainly medullary, with some follicles in the cortex; and the uterus is cortical. A key to the subfamilies of the order Proteocephalidea Mola, 1928 is provided. The most characteristic A. de Chambrier (&) S. C. Coquille J. Mariaux Department of Invertebrates, Natural History Museum Geneva, PO Box 6434, 1211 Geneva 6, Switzerland alain.dechambrier@ville-ge.ch S. C. Coquille Faculty of Sciences, Department of Zoology and Animal Biology, Geneva University, 30, quai Ernest-Ansermet, 1211 Geneva 4, Switzerland V. Tkach Department of Biology, University of North Dakota, 1, Campus Drive and Cornell Street, 101 Starcher Hall, Grand Forks, ND , USA V. Tkach Institute of Parasitology, Polish Academy of Sciences, 51/55 Twarda Street, Warszawa, Poland features of T. testudo are the precocious uterine aperture, the presence of internal uterine pores (as previously described for Proteocephalus paraguayensis (Rudin, 1917)), the eggs laid unripe, the very long strobila (up to 970 mm), and the presence of an anterior circular musculature in the suckers, which is considered as a good differential character. Three other species were found in Amia calva: Proteocephalus perplexus La Rue, 1911, P. ambloplitis (Leidy, 1887) and a new, undescribed form. Sequences of the partial nuclear 28S rrna gene of specimens of T. testudo from Apalone spinulifera and Amia calva confirm the conspecificity of samples from these two very distinct hosts, which may represent a capture phenomenon. As the subfamily Adenobrechmoinae Bursey, Goldberg & Kraus, 2006 and the genus Adenobrechmos Bursey, Goldberg & Kraus, 2006 are based on the presence of an apical organ, a character which reflects a rather common convergence, we consider the Adenobrechmoinae to be a junior synonym of the Proteocephalinae La Rue, 1911 and Adenobrechmos a junior synonym of Ophiotaenia La Rue, Adenobrechmos greeri Bursey, Goldberg & Kraus, 2006 thus becomes Ophiotaenia greeri (Bursey, Goldberg & Kraus, 2006) n. comb. Introduction During the course of an investigation into the parasite fauna of fishes and reptiles in the United States,

2 50 Syst Parasitol (2009) 73:49 64 several species of proteocephalid tapeworms were found in spiny softshell turtles Apalone spinifera (Lesueur) and bowfins Amia calva Linnaeus collected from Reelfoot Lake, Tennessee. One of these tapeworm species, Testudotaenia testudo (Magath, 1924), was found in both hosts. Amia calva also hosted three additional proteocephalid species, namely Proteocephalus perplexus La Rue, 1911, P. ambloplitis (Leidy, 1887), both already known from this host (La Rue, 1911, 1914), and another, probably new, proteocephalidean cestode (hereafter new species 1 ) morphologically similar to members of the polyphyletic genus Proteocephalus (Weinland, 1858) (see de Chambrier et al., 2004). The morphological description of the latter taxon will be provided in a separate paper. T. testudo is redescribed here and a new subfamily is proposed to accommodate it. Materials and methods In June 2002, three eastern spiny softshells Apalone spinifera and four bowfins Amia calva were collected from Reelfoot Lake, Tennessee, N , W Turtles were collected in baited traps under a permit from the Tennessee Wildlife Resources Agency; the fish were collected by angling. Tapeworms were fixed in 4% hot (almost boiling) formaldehyde solution, with pieces of several worms placed in 99% pure ethanol for DNA analysis. Specimens used for morphological studies were then stained with Mayer s hydrochloric carmine solution, dehydrated in an ethanol series, cleared with eugenol (clove oil) and mounted in Canada balsam. Eggs were studied in whole-mounts. For histology, fragments of strobila and several scoleces were embedded in paraffin wax, cross-sectioned at lm, stained with Weigert s haematoxylin and counterstained with 1% eosin B (acidified with five drops of pure acetic acid per 100 ml of solution) following recently updated protocols (Scholz & Hanzelová, 1998; de Chambrier, 2001; de Chambrier et al., 2006). Specimens prepared for scanning electron microscopy (SEM) were dehydrated in a graded ethanol series, transferred to a graded amylacetate series, critical point dried in CO 2, sputtered with gold and examined with a Zeiss DSM 940A electron microscope at the Geneva Natural History Museum (MHNG). Microthrix terminology follows Thompson et al. (1980) and Hoberg et al. (1995). Acronyms of museum collections are as follows: MHNG INVE: Natural History Museum, Geneva, Switzerland, Invertebrate Collection; IPCAS: Institute of Parasitology, Academy of Sciences, České Budějovice, Czech Republic; USNPC: US National Parasite Collection, Beltsville, Maryland, USA. Measurements are given in micrometres unless otherwise stated. For two-dimensional measurements, length is given before width. Abbreviations: Ma, measurements according to Magath (1924); mean ± SD, mean ± standard deviation; N, number of measurements; CV, coefficient of variability (%); CI, consistency index; RC, rescaled consistency index. DNA was extracted from a few ethanol preserved proglottides with the DNeasy Tissue kit (Qiagen) according to the manufacturer instructions. About 1 kb long fragments of the 5 0 portion of the nuclear 28S rrna gene was amplified and sequenced as previously described by Zehnder & Mariaux (1999). New sequences were deposited with EMBL under accession numbers FM The sequences of T. testudo from fishes were aligned with known T. testudo sequences from turtles and all available sequences of proteocephalidean cestodes from Nearctic Amia and Ictalurus. Two species of the basal group of the Proteocephalidea (Gangesia and Acanthotaenia), as well as a member of the true Proteocephalus [Proteocephalus aggregate, see de Chambrier et al. (2004a)] were used as outgroups. The matrix comprised 20 taxa: Gangesia parasiluri Yamaguti, 1934 ex Silurus asotus L., Lake Suwa, Nagano Prefecture, Japan, 28.vi.1996, MHNG INVE 22436, AJ Acanthotaenia cf. shipleyi (Linstow, 1903) ex Varanus salvator Laurenti, Klang, Malaysia, 5.x. 2001, MHNG INVE 32837, AJ Proteocephalus percae (Müller, 1780) ex Perca fluviatilis L., Neuchâtel Lake, Switzerland, 12.vi.1996, MHNG INVE 36744, AJ Megathylacoides sp. ex Amia calva L., Reelfoot Lake, Tennessee, USA, 20.vi.2002, MHNG INVE 35373, FM Megathylacoides sp. ex A. calva L., Reelfoot Lake, Tennessee, USA, 20.vi.2002, MHNG INVE 35384, FM Proteocephalus ambloplitis (Leidy, 1887) ex Lepomis macrochirus Rafinesque, Pearl River, Mississippi, USA, AJ

3 Syst Parasitol (2009) 73: P. ambloplitis (Leidy, 1887) ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 35374, FM P. ambloplitis (Leidy, 1887) ex Ictalurus punctatus Rafinesque, Reelfoot Lake, Tennessee, USA, 20.vi.2002, MHNG INVE 36278, FM P. perplexus La Rue, 1911 ex I. punctatus Rafinesque, Reelfoot Lake, Tennessee, USA, 20.vi.2002, MHNG INVE 36277, sequence identical to MHNG INVE P. perplexus La Rue, 1911 ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 35366, FM P. perplexus La Rue, 1911 ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 36139, FM P. perplexus La Rue, 1911 ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 35321, sequence identical to MHNG INVE New species 1 ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 35548, FM Testudotaenia testudo, ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 36706, sequence identical to MHNG INVE T. testudo, ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 36707, sequence identical to MHNG INVE T. testudo, ex Apalone spinifera L., Reelfoot Lake, Tennessee, USA, 26.vi.2002, MHNG INVE 35316, sequence identical to MHNG INVE T. testudo, exa. spinifera L., Reelfoot Lake, Tennessee, USA, 26.vi.2002, MHNG INVE 35317, FM T. testudo, ex A. spinifera L., Reelfoot Lake, Tennessee, USA, 1.vii.2002, MHNG INVE 35319, sequence identical to MHNG INVE T. testudo, ex A. spinifera L., Reelfoot Lake, Tennessee, USA, 1.vii.2002, MHNG INVE 35320, FM The alignment was generated with Sequencher TM v4.1.2 (Gene Codes Corp.) and minor corrections were made by hand; the final alignment was 1,051 bp long. A parsimony analysis was performed using PAUP* v4.0b10 (Swofford, 2002) with the following settings: characters unordered and equally weighted, gaps treated as missing characters. Node support was assessed by bootstrapping (5,000 repeats). Uncorrected distances between sequences (gapped positions removed) were also calculated with PAUP*. Testudotaeniinae n. subfam. Diagnosis Proteocephalidea, Proteocephalidae, with features of family (Rego, 1995). Scolex with four deep suckers. Testes cortical, rarely medullary, arranged in two fields, in one or several layers. Ovary partially medullary, with cortical lobes. Vitelline follicles mainly medullary, with some follicles in cortex. Uterus cortical. Precocious uterine aperture present. Parasitic in turtles (Trionychidae) and freshwater fishes (Actinopterygii). Type- and only genus: Testudotaenia Freze, Testudotaenia Freze, 1965 Diagnosis Testudotaeniinae, with feature of subfamily. Tapeworms up to one metre long, with segmented strobila. Scolex small, bearing four distinctly separated suckers opening antero-laterally. Testes in two fields. Vagina always anterior to cirrus-sac. Band of vitelline follicles wider at level of ovary. Uterus with precocious longitudinal openings. Eggs laid unripe. Parasitic in testudine reptiles, occasionally in amiiform fishes. Type-species: T. testudo (Magath, 1924). Testudotaenia testudo (Magath, 1924) Freze, 1965 Type-host: Apalone spinifera(le Sueur) (eastern spiny softshell turtle) (Testunides: Trionychidae). Other host: Amia calva Linnaeus (bowfin) (Amiiformes: Amiidae). Prevalence: 100% (3/3) in Apalone spinifera, 50% (2/4) in Amia calva. Site: Intestine. Distribution: Lake City, Minnesota (type-locality), summer 1921; Louisiana (Acholonu, 1970); Reelfoot Lake, Tennessee (this study), United States. Material studied Ex Amia calva 2 specimens (coll. nos USNPC and , field number USA 06, 13 slides 3 whole-mounts and 10 serial sections; USA 12, 2 slides, serial sections, same specimen as MHNG INVE 36707); 2 specimens and fragments (coll. nos MHNG INVE 36707, 35369, , field number USA 12, 39 slides; USA 11, 1 slide, fragments; USA

4 52 Syst Parasitol (2009) 73: , 69 slides, fragments); fragments of strobila (coll. no. IPCAS C-482, field number USA 06, 7 slides). Ex Apalone spinifera 2 specimens (coll. nos. MHNG INVE and 35318, field numbers respectively USA 07, 3 whole-mounts, and USA 08, 4 whole-mounts). Redescription (Figs. 1 13) [Based on 5 specimens.] Cestodes flattened dorsoventrally. Strobila large, anapolytic, mm long (Ma = mm), up to 2.34 mm wide (Ma = 1.6 mm) (n = 2), consisting of proglottides: immature proglottides (up to appearance of spermatozoa in vas deferens), mature proglottides (up to appearance of eggs in uterus), pre-gravid proglottides (up to appearance of hooks in oncospheres), and gravid proglottides. Strobila covered uniformly with filiform microtriches more than 2 long (Fig. 13). Immature proglottides ( ,335; length/width ratio 1: ); mature proglottides (485 1, ; ratio 1: ); pregravid proglottides (860 1, ,980 2,200; ratio 1: ); gravid proglottides (1,170 1, ,960 2,345 (Ma = 2, ,600); ratio 1: ). Scolex aspinose, with slightly conical apical part, (Ma = 420) long, (Ma = 630) in diameter (n = 2), wider than neck which is (Ma = 240) wide. Scolex formed by 4 deep suckers, each (Ma = 120) wide (n = 8, mean = 250) (Figs. 1, 2). Anterior margin of suckers with internal circular musculature (Figs. 2, 3). Concentration of gland-cells at apex of scolex, c.50 in diameter (n = 2) (Fig. 1). Proliferation zone 3,380 4,765 long, wide. Internal longitudinal musculature well developed, consisting of isolated bundles of fibres (Figs. 6 8, 10). Osmoregulatory canals almost impossible to observe in mature and gravid proglottides. In immature proglottides, ventral canals much wider than dorsal, situated 17 22% of proglottis width from lateral margins, crossing cirrus-sac in its distal region, longitudinally overlapping marginal testes. Testes spherical to oval, (Ma = ), numbering (mean = 152 ± 18; n = 10; CV = 12%) (Ma = ), in 2 fields, more numerous laterally, not crossing anterior margin of ovary (Figs. 4, 5), forming 1 2 incomplete layers medially (Figs. 5, 6). Testes sometimes reaching laterally to vitelline follicles and to anterior margin of proglottides, producing spermatozoa even in gravid proglottides. Cirrus-sac pyriform (Figs. 4, 5), (mean = 685 ± 55; n = 41; CV = 8%) (Ma = 560) long, (mean = 240 ± 25; n = 41; CV = 11%) (Ma = 270) wide (length/width ratio 2.9 ± 0.31; n = 41; CV = 11%), representing 26 34% (mean = 31 ± 2%; n = 41; CV = 8%) of proglottis width. Internal sperm duct (vas deferens) thin-walled, forming numerous loops; ejaculatory duct coiled, thick-walled; cirrus occupies more than half of cirrus-sac length (Fig. 5). Genital ducts passing between osmoregulatory canals. Vas deferens strongly coiled, reaching to mid-line of proglottis. Genital atrium present. Genital pore irregularly alternate, markedly pre-equatorial, situated at 8 15% (mean = 11 ± 2; n = 41; CV = 16%) (Ma = \17%) of proglottis length. Vagina always anterior (n = 690) (Ma = always anterior) to cirrus-sac, thin-walled. Vaginal sphincter elongate, near genital atrium, its length representing c.1/4 to 1/5 of cirrus-sac length (Fig. 5). Mehlis glands in diameter (n = 30), representing 5 12% of proglottis width (Fig. 5). Ovary bilobed, with follicular lobes, occupying 50 63% (mean = 56 ± 3%; n = 41; CV = 5%) (Ma = c.50%) of proglottis width (Fig. 5). Ovary with isthmus in medulla and lobes in cortex (Fig. 7). Vitelline follicles arranged in 2 lateral bands, wider at level of ovary, with numerous follicles crossing longitudinal internal musculature, penetrating dorsal and ventral cortex, rarely present preporally (14%, n = 297), not overlapping vagina and cirrus-sac, representing porally 56 73% (mean = 66 ± 4%; n = 18; CV = 7%) and aporally 74 90% (mean = 82 ± 5%; n = 018; CV = 6%) of proglottis length, respectively (Figs. 4, 5). Formation of uterus. Uterus cortical, already present in immature proglottides, with development of type 2, according to de Chambrier et al. (2004a), i.e. in immature proglottides, uterine stem has concentration of numerous intensely-staining cells on both sides and occupies up to 22% of proglottis width. Lumen present in uterine stem in last immature proglottides, from which it develops dense lateral elongate digitations, which are terminally ramified or not, or develops into median field of undifferentiated

5 Syst Parasitol (2009) 73: Figs. 1 3 Testudotaenia testudo (Magath, 1924) from Amia calva Linnaeus. 1. Scolex, ventral view, USNPC Frontal section of scolex, INVE Detail of sucker, INVE Abbreviations: ao, concentration of gland-cells; cg, cells with finely granular cytoplasm; cm, circular musculature. Scale-bars: 1, 500 lm; 2, 3, 100 lm chromophilic cells (Fig. 4). In mature proglottides, lumen gradually appears into each digitiform diverticulum and extends from base to apex. Apex of diverticulum bordered by numerous chromophilic cells. Transverse uterine diverticula grow into cortical parenchyma. In pre-gravid proglottides, eggs completely fill uterine stem and thin-walled diverticula, occupying up to 36% of proglottis width (Fig. 5). Uterus in gravid proglottides with diverticula on each side comprising numerous chromophilic cells (Fig. 9), occupying most of gravid proglottis width (de Chambrier et al., 2004a). In gravid proglottides, diverticula occupy up to 47% of proglottis width, without eggs. Uterus with (n = 10) (Ma = 15 20) lateral branches on each side. Uterus opens via single, ventral, longitudinal slit situated along almost whole length of proglottis (Figs. 9, 11, 12). Narrow internal uterine pores present, similar to those described for Proteocephalus paraguayensis (Rudin, 1917) (see Figs. 6c, d in de Chambrier, 1990), emerging from diverticula into uterine stem (Fig. 12). Eggs laid through longitudinal uterine opening before they are ripe, irregular in shape, with spherical embryophore of (Ma = 29) in diameter; oncosphere spherical, (Ma = 19 21) in diameter,

6 54 Syst Parasitol (2009) 73:49 64 Figs. 4, 5 Testudotaenia testudo (Magath, 1924) from Amia calva. 4. Immature proglottis, ventral view, INVE Gravid proglottis, ventral view (eggs not present). Abbreviations: ci, cirrus; cs, cirrus-sac; vs, vaginal sphincter. Scale-bars: 500 lm with 3 pairs of hooks long (measured in mounted slides). Remarks We were unable to locate Magath s (1924) typematerial of Ophiotaenia testudo (now Testudotaenia testudo), either in the USNPC (Beltsville, MD) or in the Harold W. Manter Collection (Lincoln, NE). According to Achonolu (1970), the Magath s specimens are believed lost. Instead we used the measurements of the original description for comparative purposes. Our material (from both Apalone spinifera and Amia calva) corresponds with the original description, although Magath s material is slightly smaller. Magath (1924, Figs. 7 9) figured a cross-section of O. testudo without pointing out the particular position of the genital organs in relation to the longitudinal internal musculature. Brooks (1978) redescribed Proteocephalus testudo (=T. testudo) from Trachemys scripta elegans. His material differs from that described by Magath (1924) in the absence of an apical organ, in the ratio of

7 Syst Parasitol (2009) 73: Figs Testudotaenia testudo (Magath, 1924) from Amia calva. 6, 7. Cross-sections of mature proglottides, INVE At level of middle of proglottis; 7. At level of ovary. 8. Cross-sections of pre-gravid proglottis at level of uterus, INVE Ventral view of a gravid uterus, INVE (diagrammatic). Note the shape of the lateral ramified diverticula and the uterine aperture. 10. Dorsal view of internal longitudinal musculature, INVE (diagrammatic). Abbreviations: du, uterine diverticula; lm, longitudinal internal musculature; ov, ovary; te, testes; ut, uterus; vi, vitelline follicles. Scale-bars: 6 9, 500 lm; 10, 250 lm the cirrus-sac length to the proglottis width (42 59 vs 26 34%), in the anterior and posterior position of the vagina in relation to the cirrus-sac versus always anterior in the present study, and in the diameter of oncospheres (18 27 vs lm). It is likely that the material described by Brooks (1978) from Trachemys scripta elegans as Proteocephalus testudo may represent another species. Testudotaenia testudo has been reported from Minnesota and Louisiana by Achonolu (1970) and from Oklahoma by McKnight (1959). Tennessee is a new locality for this species.

8 56 Syst Parasitol (2009) 73:49 64 Figs Testudotaenia testudo (Magath, 1924) from Amia calva. INVE 36707, SEM photomicrographs of gravid proglottis. 11. Ventral view showing the uterine aperture (arrow). 12. Detail view of uterine aperture. 13. Microtriches at level of the ventral median surface (star in 11). Scale-bars: 11, 500 lm; 12, 100 lm; 13, 2 lm The most characteristic features of T. testudo are the presence of uterine apertures in pre-gravid proglottides, the presence of internal uterine pores, the eggs laid unripe, a large body size and the presence of an anterior circular musculature in the suckers. The precocious uterine aperture has previously been observed in Solenotaenia viperis Beddard, 1913 (syn. of Crepidobothrium viperis) by Beddard (1913). Other members of Crepidobothrium Monticelli, 1899 also possess a precocious uterine aperture (de Chambrier, 1989a, b). This structure seems to be functionally identical in Testudotaenia (see present paper) and Crepidobothrium (see de Chambrier, 1989a), although the two genera are not morphologically similar. In both genera, the eggs are laid unripe and precociously through a large longitudinal aperture, and the uterus is empty in pre-gravid proglottides. There is no clear explanation for this unusual egg dispersion mechanism and it may have evolved independently in Crepidobothrium and Testudotaenia. Although Proteocephalus paraguayensis (Rudin, 1917) exhibits a similar uterine aperture, as well as internal uterine pores (de Chambrier, 1990), its eggs remain in the uterus until the proglottides become gravid and the uterine aperture opens widely. An explanation for this difference could be in the diameter of the internal uterine pores; although the diameter of embryophores is similar in both groups (c lm), the diameter of the internal uterine pore reaches only lm in P. paraguayensis versus lm in T. testudo. A large body size is rare within the Proteocephalidea. Most proteocephalideans are small and attain lengths of up to mm (Freze, 1965). Some species, such as P. paraguayensis, Ophiotaenia calmettei (Barrois, 1898), O. phillipsi (Burt, 1937) and O. magna (Hannum, 1925), reach 600 mm (Barrois, 1898; Burt, 1937; de Chambrier, 1990; Hannum, 1925), and only Thaumasioscolex didelphidis Cañeda- Guzmán, de Chambrier & Scholz, 2001 (1,047 mm long) and Megathylacoides procerum Sneed, 1950 (1,113 mm long) are larger than T. testudo (see Cañeda-Guzmán et al., 2001; Freze, 1965). Although circular musculature is present in the suckers of several unrelated lineages of the Proteocephalidea, its appearance and the level of development vary in unrelated taxa. This musculature can be: only slightly developed [as in Brooksiella praeputialis (Rego, Santos & Silva, 1974) and Proteocephalus jarara (Fuhrmann, 1927) (Figs. 14, 15)]; hypertrophied, forming a double crescent [as in Mariauxiella pimelodi de Chambrier & Rego, 1995 (Fig. 16)]; horseshoe-shaped posteriorly [as in Mariauxiella piscatorum de Chambrier & Vaucher, 1999 and Manaosia bracodemoca (Woodland, 1935) de Chambrier, 2003 (Fig. 18)]; or horseshoe-shaped anteriorly [as in Megathylacoides lamothei García- Prieto, 1990) (Fig. 17) and Barsonella lafoni de Chambrier, Scholz, Beletew & Mariaux, 2009] (see de Chambrier et al., 1991, 2004b, 2009; de Chambrier & Rego, 1995; de Chambrier & Vaucher, 1999; de Chambrier, 2003; Scholz et al., 2003). This character is very constant within a species and may be used as a good discriminating feature.

9 Syst Parasitol (2009) 73: Figs Brooksiella praeputialis (Rego, Santos & Silva, 1974) de Chambrier et al., 2004, from Cetopsis coecutiens (Lichtenstein), slightly developed anterior circular musculature of suckers. 15. Proteocephalus jarara (Fuhrmann, 1927) de Chambrier et al., 1991, from Bothrops jararaca (Wied-Neuwied), slightly developed circular musculature. 16. Mariauxiella pimelodi de Chambrier & Rego, 1995, from Pimelodus ornatus Kner, hypertrophied circular musculature forming a double crescent. 17. Megathylacoides lamothei (Garcia- Prieto, 1990), from Ictalurus meridionalis (Günther), hypertrophied circular musculature horseshoe-shaped anteriorly. 18. Mariauxiella piscatorum de Chambrier & Vaucher, 1999, from Hemisorubim platyrhynchos (Valenciennes), hypertrophied circular musculature horseshoe-shaped posteriorly. Abbreviations: cm, circular musculature. Scale-bars: 14, 200 lm; 15 18, 250 lm Differential diagnosis The Testudotaeniinae differs from the 12 recognised proteocephalidean subfamilies (Ephedrocephalinae Mola, 1929; Gangesiinae Mola, 1929; Monticelliinae Mola, 1929; Proteocephalinae La Rue, 1911; Marsypocephalinae Woodland, 1933; Zygobothriinae Woodland, 1933; Endorchiinae Woodland, 1934; Peltidocotylinae, Woodland, 1934; Rudolphiellinae Woodland, 1935; Sandonellinae Khalil, 1960; Acanthotaeniinae Freze, 1963; and Corallobothriinae Freze, 1965) (Mola, 1929; Woodland, 1933a, 1934b, 1935b;

10 58 Syst Parasitol (2009) 73:49 64 Khalil, 1960; Freze, 1963, 1965) by a distinct arrangement of the genital organs in relation to the internal longitudinal musculature: cortical testes arranged in two fields, in one or several layers; an ovary with cortical lobes and a medullary isthmus; and vitelline follicles which are mainly medullary, with some follicles in the cortex; and a cortical uterus (Beddard, 1913; Schmidt, 1986; Rego, 1994, 1995;Regoetal.,1998;deChambrier & Vaucher, 1997, 1999). The relative position of these organs in various recognised subfamilies is summarised in Fig. 20. Molecular analysis A total of 128 characters were parsimony informative after the removal of gapped positions. A branch and bound parsimony analysis yielded a single shortest tree 223 steps long (CI = 0.68, RC = 0.54) (Fig. 19). All morphologically recognised species formed monophyletic assemblages with the exception of Proteocephalus perplexus La Rue, 1911 (intraspecific distance 0 1.9%), for which the sequence from a Canadian specimen branched separately from those of samples from the USA. The position of the Canadian sample was, however, poorly supported. All Testudotaenia (0 0.2%) from fishes and turtles formed an unambiguously monophyletic group. P. ambloplitis (0 1%) was found to be the sister taxon of P. perplexus and the new Proteocephalinae species (from Amia calva), the sister taxon of Testudotaenia. Megathylacoides Jones, Kerly & Sneed, 1956 (0.1%) was the most basal proteocephalidean from North American fishes included in the analyses. Discussion Although molecular tools have permitted significant progress in our understanding of the proteocephalidean systematics (Mariaux, 1998; Hoberg et al., 1999; Zehnder & Mariaux, 1999; Kodedová et al., 2000; Zehnder, 2000; Zehnder et al., 2000; Zehnder & de Chambrier, 2000; de Chambrier et al., 2004a; Hypša et al., 2005; Scholz et al., 2007), the current classification remains mainly based on the seminal series of Woodland s papers published almost a century ago (Woodland, 1925a, b, c, 1933a, b, 1934a, b, 1935a, b). His subfamily classification is essentially based on the position of the different genital organs in relation to the internal longitudinal musculature (Fig. 20). Based on this criterion, the new subfamily, Testudotaeniinae, is well defined because of a unique combination of key characters, i.e. medullary vitelline follicles associated with cortical testes, uterus and ovary. It must be emphasised that the current systematic structure of the Proteocephalidea remains provisional. We are aware that some of the subfamilies (and genera) will likely prove to be artificial grouping of unrelated taxa, especially when molecular tools are used. However, every attempt to reorganise the order (i.e. Zehnder & Mariaux, 1999; de Chambrier et al., 2004; Hypša et al., 2005) on a sound phylogenetic bases has failed for all but a few limited groups of species. On the other hand, as many as seven subfamilies defined by Woodland s characters (i.e. the Acanthotaeniinae, Corallobothriinae, Ephedrocephalinae, Gangesiinae, Peltidocotylinae, Rudolphiellinae and Sandonellinae) have not been invalidated by recent investigations (Zehnder & Mariaux, 1999; Scholz et al., 2003; Hypša et al., 2005; de Chambrier et al., 2008), and we believe that the cortical/medullary organisation of the reproductive systems will certainly remain an important systematic character. Nevertheless, Proteocephalus currently remains a polyphyletic assemblage (Zehnder & Mariaux, 1999; Scholz et al., 2003; Hypša et al., 2005). We anticipate that several species currently placed in this genus will need to be assigned to different genera and even subfamilies. Our molecular tree shows that new species 1 from Amia calva, which is clearly not a member of the new subfamily Testudotaeniinae, is the sister-group of Testudotaenia. It might, logically, belong to a new subfamily. The exact status of this taxon should be clarified together with its description, but, given the above considerations of the diagnoses of the subfamilies and the relatively weak sister-group relationship between Testudotaenia and the new species, this does not affect the erection of the Testudotaeniinae. The Adenobrechmoinae Bursey, Goldberg & Kraus, 2006 was not included in our comparison. Bursey et al. (2006) described a new genus and species, Adenobrechmos greeri Bursey, Goldberg & Kraus, 2006, a parasite of the scincid lizard Sphenomorphus aignanus in Papua New Guinea. They justified the erection of their new genus (and subfamily Adenobrechmoinae) by the presence of a glandular apical organ. Contrary to the opinion of

11 Syst Parasitol (2009) 73: Fig. 19 Phylogenetic tree showing the monophyly of Testudotaenia from fish and turtles and its relationship to the other proteocephalideans found in Amia calva. Parsimony analysis, single shortest tree, see text for details. Number on branches are bootstrap values [50% (5000 repeats)

12 60 Syst Parasitol (2009) 73:49 64 Fig. 20 Survey of subfamilies of the Proteocephalidea and position of their genital organs in relation to the internal longitudinal musculature (modified from Euzet, 1982). Abbreviations: lm, longitudinal internal musculature; ov, ovary; te, testes; ut, uterus; vi, vitelline follicles Bursey et al. (2006), the presence of such organs has already been described in numerous proteocephalidean species belonging to various subfamilies from a large variety of hosts and geographical regions (e.g. Proteocephalus glanduligerus (Janicki, 1928), P. sophiae de Chambrier & Rego, 1994, P. joanae de Chambrier & Paulino, 1997, P. vitellaris (Verma, 1928), P. tigrinus Woodland, 1925 and Ophiotaenia hylae Johnston, 1912) (Proteocephalinae), Nomimoscolex piraeeba Woodland, 1934, Vaucheriella bicheti de Chambrier, 1987, N. suspectus Zehnder, de Chambrier, Vaucher & Mariaux, 2000 (Zygobothriinae) (see Johnston, 1912; Woodland, 1925c; Janicki, 1928; Verma, 1928; Woodland, 1934b; de Chambrier, 1987, 2004; de Chambrier & Rego, 1994; de Chambrier & Paulino, 1997; Scholz et al., 1998; Zehnder et al., 2000). This character reflects a rather common convergence but certainly does not justify the erection of a new supraspecific taxon. Considering the medullary position of testes, ovary, uterus and vitelline follicles, coupled with a scolex with uniloculate suckers and testes in one field, this taxon could be placed in Proteocephalus Weinland, However, almost all proteocephalidean cestodes found in reptiles and amphibians are members of Ophiotaenia La Rue, 1911 (see Freze, 1965; de Chambrier et al., 2006; Ammann & de Chambrier, 2008; Coquille & de Chambrier, 2008), which differs from Proteocephalus essentially in the presence of two testicular fields and a preformed uterus (Freze, 1965). Both Proteocephalus and Ophiotaenia have been demonstrated to be polyphyletic in previous phylogenetic analyses (Zehnder & Mariaux, 1999; de Chambrier et al., 2004a; Hypša et al., 2005). Pending a complete revision of the group, we choose to provisionally place the concerned taxon in Ophiotaenia. Therefore, we consider the Adenobrechmoinae Bursey, Goldberg & Kraus, 2006 to be a junior synonym of the Proteocephalinae and Adenobrechmos Bursey, Goldberg & Kraus, 2006 a junior synonym of Ophiotaenia. Consequently, Adenobrechmos greeri Bursey, Goldberg & Kraus,

13 Syst Parasitol (2009) 73: becomes Ophiotaenia greeri (Bursey, Goldberg & Kraus, 2006) n. comb. The finding of T. testudo in an evolutionary ancient fish, Amia calva, is rather surprising. There is no other example of a proteocephalidean species known to parasitise such different groups of hosts. Proteocephalidean species usually exhibit an oioxenic specificity, at least in the Neotropical region (de Chambrier & Vaucher, 1999), and rarely a stenoxenic specificity. As stenoxeny is uncommon in the New World proteocephalideans, it is interesting to note the other proteocephalideans of fishes from North America are often stenoxenic: both Proteocephalus ambloplitis and P. perplexus are found in Amia, Lepomis and Ictalurus. The situation is different for Palaearctic species of Proteocephalus, such as P. longicollis (Zeder, 1800), P. percae (Müller, 1780) and P. torulosus (Batsch, 1786), which may parasitise fish hosts belonging to different fish families or even orders (Scholz & Hanzelová, 1998). We assume that the presence of T. testudo in Amia calva represents an isolated local capture phenomenon, as both hosts were collected in the same place and habitat. They both are opportunist feeders and have a similar diet consisting of fishes, frogs, crayfishes, insects and shrimp. Key to subfamilies of the Proteocephalidea (modified after Khalil, 1960) 1 Genital organs entirely cortical... Monticelliinae Genital organs at least partly medullary Genital organs entirely medullary... 3 Genital organs partly medullary and partly cortical Vitelline follicles in two lateral compact bands... 4 Vitelline follicles in two compact bands posterior to ovary... Sandonellinae 4 Rostellum present... 5 Rostellum absent Rostellum without hooks; parasites of reptiles... Acanthotaeniinae Rostellum usually with hooks; parasites of siluriform fishes... Gangesiinae 6 Metascolex present... Corallobothriinae Metascolex absent... Proteocephalinae 7 Testes medullary... 8 Testes cortical Uterus medullary... Zygobothriinae Uterus cortical, with branches in medulla... Endorchiinae 9 Vitelline follicles medullary Vitelline follicles cortical Uterus cortical... Peltidocotylinae Uterus medullary Vitelline follicles in one entire dorsal field... Ephedrocephalinae Vitelline follicles in two lateral dorsal rows... Rudolphiellinae 12 Uterus cortical... Testudotaeniinae Uterus medullary... Marsypocephalinae Acknowledgements We are grateful to André Puiz for providing SEM photomicrographs, Janik Pralong and Florence Marteau for technical assistance, and to Gilles Roth for his help with drawings (all from Geneva). We thank Tomáš Scholz (České Budějovice) for fruitful suggestions and helpful comments. We also thank Susan Lim (University of Malaya, Kuala Lumpur) and Takeshi Shimazu (Nagano Prefectural College, Japan) for providing samples. Scott Snyder (University of Nebraska at Omaha) and Stephen Curran (Gulf Coast Research Laboratory, University of Southern Mississippi) kindly helped with the field collecting of turtles and fish. This project was supported in part by the National Science Foundation PBI award Nos and References Achonolu, A. D. (1970). On Proteocephalus testudo (Magath, 1924) (Cestoda: Proteocephalidae) from Trionyx spinifer (Chelonia) in Louisiana. Journal of Wildlife Diseases, 6, Ammann, M., & de Chambrier, A. (2008). Ophiotaenia gilberti sp. n. (Cestoda: Proteocephalidea), a parasite of Thamnodynastes pallidus (Serpentes: Colubridae) from Paraguay. Revue Suisse de Zoologie, 115, Barrois, T. (1898). Sur quelques Ichthyotaenia parasites de serpents. Bulletin de la Société des Sciences de l Agriculture et des Arts de Lille, 2, 6 9. Beddard, F. E. (1913). Contributions to the anatomy and systematic arrangement of the Cestoidea. IX. On a new genus of ichthyotaeniids. Proceedings of the Zoological Society of London, (1913), Brooks, D. R. (1978). Systematic status of proteocephalid cestodes from reptiles and amphibians in North America with descriptions of three new species. Proceedings of the Helminthological Society of Washington, 45, Bursey, C. R., Goldberg, S. R., & Kraus, F. (2006). New genus, new species of Cestoda (Proteocephalidae) from the lizard Sphenomorphus aignanus (Squamata: Scincidae) from

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