Syst Parasitol (2015) 92:181–195 DOI 10.1007/s11230-015-9597-5

Anisakid (Nematoda: ) from the marine fishes Plectropomus laevis Lace´pe`de (Serranidae) and Sphyraena qenie Klunzinger (Sphyraenidae) off New Caledonia, including two new species of Hysterothylacium Ward & Magath, 1917

Frantisˇek Moravec . Jean-Lou Justine

Received: 26 June 2015 / Accepted: 2 August 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Based on light and scanning electron presence of narrow lateral alae, a short caecum and a microscopical studies, two new species of Hysterothy- long ventricular appendix, the length (762–830 lm) lacium Ward & Magath, 1917 (Nematoda: Anisaki- and shape of the spicules, the number (37–38 pairs) dae) are described from the digestive tract of and arrangement of the genital papillae, and by the tail perciform fishes off New Caledonia: H. alatum n. sp. tip which lacks any distinct cuticular projections from Plectropomus laevis (Lace´pe`de) (Serranidae) visible under the light microscope. In addition, and and H. sphyraenae n. sp. from Sphyraena qenie unidentifiable at the species level, conspicuously large Klunzinger (Sphyraenidae). The former species (45.71–66.10 mm long) larvae of (H. alatum) is mainly characterised by its large body Railliet & Henry, 1912, were found in the body cavity (male 42.05 mm, gravid females 51.18–87.38 mm of P. laevis, which serves as a paratenic host for this long), the shape of the dorsal lip, conspicuously broad parasite. cervical alae, a short caecum and a long ventricular appendix, the length of the spicules (925 lm), the number (25 pairs) and distribution of the genital papillae and the tail tip bearing numerous minute Introduction cuticular protuberances. The other species (H. sphyraenae) is mainly characterised by the Our knowledge of the species diversity of anisakid nematodes (Anisakidae) which parasitise marine fishes and elasmobranchs in New Caledonian waters remains incomplete, since only four nominal species of these parasites have previously been recorded in this region: scoliodontis (Baylis, 1931) F. Moravec (&) from the shark Galeocerdo cuvier (Pe´ron & Lesueur) Institute of Parasitology, Biology Centre of the Czech (Carcharhinidae), Hysterothylacium cenaticum (Bruce Academy of Sciences, Branisˇovska´ 31, & Cannon, 1989) from Tetrapturus audax (Philippi) 370 05 Cˇ eske´ Budeˇjovice, Czech Republic e-mail: [email protected] (Istiophoridae), Raphidascaris (Ichthyascaris) ne- mipteri Moravec & Justine, 2005 from Nemipterus J.-L. Justine furcosus (Valenciennes) (Nemipteridae), and ´ Institut Syste´matique, Evolution, Biodiversite´, ISYEB, R.(Ichthyascaris) etelidis Moravec & Justine, 2012 UMR7205 CNRS, EPHE, MNHN, UPMC, Muse´um National d’Histoire Naturelle, Sorbonne Universite´s, from Etelis coruscans Valenciennes and Pristipo- CP51, 57 rue Cuvier, 75231 Paris cedex 05, France moides filamentosus (Valenciennes) (both Lutjanidae) 123 182 Syst Parasitol (2015) 92:181–195

(Moravec & Justine, 2005, 2006, 2012). Anisakid Family Anisakidae Railliet & Henry, 1912 nematodes unidentified to species or genera, mostly as larval stages, have also been reported from fishes belonging to different families by Justine et al. (2010a, Hysterothylacium alatum n. sp. b, 2012) and Shamsi et al. (2015). Recent examinations of anisakid nematodes col- Type-host: Blacksaddled colargrouper Plectropomus lected from two species of perciform fishes, the laevis (Lace´pe`de) (Perciformes: Serranidae); fork blacksaddled coralgrouper Plectropomus laevis length 550 mm. (Lace´pe`de) (Serranidae) and the blackfin barracuda Site in host: Digestive tract (pyloric caecum). Sphyraena qenie Klunzinger (Sphyraenidae), revealed Type-locality: Off New Caledonia, Passe de Teramba that they represent two new species of Hysterothy- off La Foa (collected 27.iii.2003). lacium Ward & Magath, 1917 plus one species of Prevalence and intensity: 1 of 6 fish infected; 6 larval Contracaecum Railliet & Henry, 1912, which specimens. are described herein. Type-material: Holotype and allotype (anterior and The blacksaddled coralgrouper P. laevis (maximum posterior body ends mounted on SEM stub and body length 125 cm, weight 24.2 kg) and the blackfin remaining parts of bodies preserved in 70 % ethanol) barracuda S. qenie (maximum body length 200 cm, plus 2 paratypes in the Helminthological Collection of weight up to 50 kg) are both tropical marine, reef- the Institute of Parasitology, Biology Centre of the associated fishes. The former has an Indo-Pacific Czech Academy of Sciences, Cˇ eske´ Budeˇjovice (Cat. distribution, whereas the latter is distributed in the No. N–1090); 2 paratypes in the Muse´um National Indo-Pacific from the Red Sea and East Africa to the d’Histoire Naturelle, Paris (MNHN JNC357A). central Indian Ocean, French Polynesia and the eastern Etymology: The specific name alatum (=winged) is a Pacific (Mexico, Panama). Both species are targeted Latin adjective relating to a characteristic feature of by commercial and recreational fishermen (Froese & this nematode, i.e. the presence of conspicuously Pauly, 2015). broad cervical alae.

Description (Figs. 1–3) Materials and methods General. Body large, elongate, yellowish in colour. Fish were caught off New Caledonia by various Anterior part of body slender. Broad asymmetrical means. The nematodes intended for morphological cervical alae, widest in region of c. 1/3 of oesophageal studies were fixed in hot 70 % ethanol. For light length, extend posteriorly to about level of middle of microscopical examination (LM), the specimens were oesophagus (Figs. 1D, 2A); from there they continue cleared with glycerine. Drawings were made with the posteriorly to tail as narrow cuticular ridges (Figs. 2E, aid of a Zeiss drawing attachment. Specimens used for F, 3A). Deirids not observed. Anterior end with 3 large scanning electron microscopical examination (SEM) lips provided with broad lateral flanges; each lip has were postfixed in 1 % osmium tetroxide (in phosphate narrow base. Posterior part of dorsal lip roughly buffer), dehydrated through a graded acetone series, rectangular, broad, with margins of lateral flanges critical-point-dried and sputter-coated with gold; they almost parallel, bears 2 lateral double papillae; were examined using a JEOL JSM-7401F scanning anterior part of dorsal lip much narrower, anteriorly electron microscope at an accelerating voltage of 4 kV rounded, separated from posterior part of lip by 2 (GB low mode). All measurements are in micrometres fairly deep lateral depressions (Figs. 1B, 2B–D). unless otherwise indicated. The classification system Subventral lips with 1 double, 1 single papilla and of the Ascaridoidea adopted follows Keys to the amphid. Interlabia well developed, conical, reaching Nematode Parasites of Vertebrates (Anderson et al., anteriorly to about level of labial papillae (Figs. 1C, 2009; Gibbons, 2010). The fish nomenclature follows 2B–D). Oesophagus cylindrical, long, ending in small FishBase (Froese & Pauly, 2015). spherical ventriculus; ventricular appendix narrow,

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Fig. 1 Hysterothylacium alatum n. sp. ex Plectropomus laevis. A, Anterior end of young female, lateral view; B, Dorsal lip; C, Cephalic end of gravid female, sublateral view; D, Anterior body end of largest female, ventral view; E, Tail of largest female, lateral view; F, Tail tip of largest female, ventral view; G, Tail of male, ventral view; H, Egg; I, Vulva, lateral view; J, Posterior end of male, ventral view; K, Tail of male, lateral view; L, Posterior end of male, lateral view

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Fig. 2 Hysterothylacium alatum n. sp., SEM micrographs. A, Anterior end of female, ventral view; B–D, Cephalic end, apical, dorsal and subdorsal views, respectively; E, F, Posterior end of male, lateral and ventral views, respectively. Abbreviations: a, double cephalic papilla; b, single cephalic papilla; c, amphid; d, dorsal lip; i, interlabium very long. Anterior intestinal caecum short, slightly maximum width 190. Nerve-ring and excretory pore longer than length of ventriculus (Fig. 1A, D). Cae- 653 and 680, respectively, from anterior extremity. cum to ventricular appendix length ratio 1:8–18. Ventriculus 109 9 136; ventricular appendix Nerve-ring encircles oesophagus at about 1/6 of its 5.30 mm long, maximum width 204. Intestinal cae- length. Excretory pore at level of nerve-ring or just cum 299 long, with maximum width 82. Caecum to posterior to it (Fig. 1A, D). Tail conical; its tip with ventricular appendix length ratio 1:18. Posterior end of numerous minute but distinct protuberances. body curved ventrally. Spicules equal, alate, 925 mm long, representing 2.2 % of body length (Fig. 1L). Male [Based on 1 specimen, holotype.] Length of Total of 25 pairs of genital papillae present, 17 being body 42.05 mm, maximum width 558. Lips 105 long; pre-anals, 1 adanal, and 7 postanals; papillae of several length of interlabia 45. Length of oesophagus posteriormost pre-anal pairs, adanal pair and of 1.90 mm, representing 4.5 % of body length; postanal pairs very small; no double papillae present 123 Syst Parasitol (2015) 92:181–195 185

appendix 4.64–5.85 (4.64) mm long, maximum width 136 (136). Intestinal caecum 320–571 (571) long, maximum width 82–95 (95). Caecum to ventricular appendix length ratio 1:8–18 (1:8). Vulva pre-equato- rial, 26.38–46.44 (46.44) mm from anterior end of body, at 52–53 % (53 %) of body length; posterior vulval lip protrudes distinctly (Fig. 1I). Vagina long (in allotype 3.40 mm long, 163 wide), directed posteriorly from vulva. Uterus forms numerous coils, contains numerous almost spherical eggs 48–51 in diameter in allotype (Fig. 1H); only immature eggs present in paratype. Tail conical, 231–680 long (Fig. 1E); tip rounded, bears numerous small cuticular protuberances (Fig. 1E, F).

Non-gravid female [Based on 2 specimens.] Length of body 11.48–14.21 mm, maximum width 258–299. Maximum width of cervical alae 27–41. Lips 75–78 long; length of interlabia 33–36. Length of oesophagus 1.71–1.90 mm, representing 13.4–14.9 % of body length; maximum width 82–109. Nerve-ring and excretory pore 354–367 and 405–408, respectively, from anterior extremity. Ventriculus 68–109 9 82–95; ventricular appendix 1.67–1.85 mm long, maximum width 41. Intestinal caecum 190–204 long, maximum width 54–68. Caecum to ventricular appendix length ratio 1:9. Vulva in larger specimen pre-equatorial, 5.98 mm from anterior end of body, at 42 % of body Fig. 3 Hysterothylacium alatum n. sp., SEM micrographs. A, length; vulval lips not protruding. Uterus empty. Tail Tail of male, ventrolateral view; B, Tail tip of male, lateral view 204–218 long; tip rounded, bears numerous small cuticular protuberances. (Figs. 1G, J–L, 2E, F, 3A). One papilla-like ventro- median organ located on anterior cloacal lip present Remarks (Figs. 1G,J, L, 2E, F, 3A). Tail conical, 136 long; tail tip bearing numerous minute protuberances (Figs. 1G, According to Moravec et al. (2012), a total of 67 valid J–L, 2E, F, 3A, B). species of Hysterothylacium have been reported from marine, estuarine and freshwater fishes. However, an Gravid female [Based on 2 complete and 1 incomplete additional three species have since been described: ovigerous specimens; measurements of allotype in H. gibsoni Xu, Li & Zhang, 2014, H. simile Li, Zhang parentheses.] Length of body 51.18–87.38 (87.38) mm, & Liu, 2013 and H. zhoushanense Li, Liu & Zhang, maximum width 598–789 (789). Length of asymmet- 2012 (emend.) (see Li et al., 2012, 2013; Xu et al., rical cervical alae in allotype (3.67/2.72) mm, maxi- 2014). Consequently, at present this genus contains 70 mum width (299). Lips 136–190 (190) long; length of recognised species. interlabia 68–99 (99). Length of oesophagus 3.33–5.30 Of these, as in the new species, the following ten (5.30) mm, representing 6.0–6.5 (6.0) % of body species possess a markedly short caecum and a long length; maximum width 204 (204). Nerve-ring and ventricular appendix, spicules of about 1 mm in length excretory pore 612–843 (843) mm and 680–925, and the tail tip bearing numerous small cuticular respectively, from anterior extremity. Ventriculus protuberances or spines: H. amoyense (Hsu¨, 1933), 109–136 9 122–136 (136 9 122); ventricular H. anguillae Moravec, Taraschewski, Appelhoff & 123 186 Syst Parasitol (2015) 92:181–195

Weyl, 2012, H. arnoglossi Petter & Maillard, 1987, shorter (560–680 lm vs 925 lm), the medioventral H. fabri (Rudolphi, 1819), H. pelagicum Deardorff & precloacal organ is absent (vs present), the ventricular Overstreet, 1982, H. reliquens (Norris & Overstreet, appendix is much shorter (506–835 lm long in males, 1975), H. rhacodes Deardorff & Overstreet, 1978, 556–661 lm in females vs 5,300 lm in male, H. scomberoidei Bruce & Cannon, 1989, H. scombero- 4,638–5,848 lm in females), the length ratio of the mori Bruce, 1990 and H. zhoushanense (see Norris & caeca and ventricular appendix is different (1:5.0–6.5 Overstreet, 1975; Deardorff & Overstreet, 1978, 1981, in males, 1:3.4–5.4 in females vs 1:18 in male, 1:8–18 1982; Petter & Maillard, 1987; Bruce & Cannon, in females), the vulval lips are not protruding (vs 1989; Bruce, 1990; Li et al., 2008, 2012, 2013; protruding) and the female tail tip bears rather large Moravec et al., 2012; Xu et al., 2014). cuticular spines (vs numerous small protuberances) Of these ten species, only H. scomberoidei,a (see Deardorff & Overstreet, 1981). Both species also parasite of Scomberoides commersonianus Lace´pe`de differ in the type-host family (Scombridae vs Ser- (Carangidae) off Australia, possesses lateral alae ranidae) and geographical regions (Atlantic vs conspicuously expanded in the cervical region, as in Pacific). the new species. In all of the other nine species, the Fairly broad lateral alae are typical also of lateral alae are absent (H. amoyense), strongly reduced H. aduncum (Rudolphi, 1802), H. auctum (Rudolphi, (H. anguillae, H. reliquens, H. fabri) or very narrow 1802), H. sinense Li, An & Zhang, 2007 and H. spirale (H. arnoglossi, H. pelagicum, H. rhacodes, Rossin, Datri, Incorvaia & Timi, 2011, but all of these H. scomberomori, H. zhoushanense). Hysterothy- species have a relatively long caecum (Hartwich, lacium scomberoidei differs from the new species 1975; Moravec et al., 1985; Moravec & Nagasawa, mainly in the shape of the dorsal lip (lateral flanges of 2000; Rossin et al., 2011; Li et al., 2007, 2013). the posterior part of lip with straight rims vs rims Hysterothylacium alatum n. sp. has previously been rounded), a conspicuously shorter ventricular appen- reported from P. laevis off New Caledonia as dix (1.79–2.16 mm in males, 1.74–2.71 mm in Hysterothylacium sp. by Justine et al. (2010a). Figus females vs 5.30 mm in male, 4.64–5.85 mm in et al. (2005) recorded adult Hysterothylacium sp. from females), a non-elevated vulva (vs vulval lips the serranid fish Serranus cabrilla (Linnaeus) in the markedly elevated), a shorter body in gravid females southwestern Mediterranean, which, as visible from (23.5–52.5 mm long vs 51.2–87.4 mm) and by larger photos provided, seems to be morphologically similar eggs (53–60 lm vs 48–51 lm in diameter) (see Bruce to H. alatum n. sp. However, judging from the & Cannon, 1989). different host genera and remote geographical regions, Conspicuously broad lateral alae in the cervical both these forms probably belong to different species. region are also present in H. fortalezae (Klein, 1973), From off New Caledonia, two other nematode H. nipponense Moravec & Nagasawa, 1998 (syn. species were recently described from a congeneric H. japonicum Moravec & Nagasawa, 1998) and host, Plectropomus leopardus (Lace´pe`de), namely the H. tetrapteri (Bruce & Cannon, 1989) (Klein, 1973; trichosomoidid Huffmanela plectropomi Justine, 2011 Deardorff & Overstreet, 1981; Bruce & Cannon, 1989; and the capillariid Capillaria plectropomi Moravec & Moravec & Nagasawa, 1998a, b; Xu et al., 2014). Justine, 2014 (Justine, 2011; Moravec & Justine, However, H. nipponense and H. tetrapteri differ 2014). It is likely that these two species can also markedly from the new species in having a very long parasitise both Plectropomus leopardus and P. laevis caecum, whereas the caecum of H. fortalezae is short, in the shared environment, as suggested by the finding resembling thus that of H. alatum n. sp. Hysterothy- of unidentified capillariids in both species by Justine lacium fortalezae is a parasite of Scomberomorus spp. et al. (2010a). (Scombridae) and allegedly also a carangid and a serranid in the West Atlantic region (off Brazil and the USA) (Klein, 1973; Deardorff & Overstreet, 1981). Hysterothylacium sphyraenae n. sp. However, in contrast to the new species, the body size of H. fortalezae is much smaller (males Type-host: Blackfin barracuda Sphyraena qenie Klun- 12.0–23.4 mm, females 10.0–15.4 mm long vs male zinger (Perciformes: Sphyraenidae); fork length 42.1 mm, females 51.2–87.4 mm), the spicules are 630 mm, weight 1.5 kg 123 Syst Parasitol (2015) 92:181–195 187

Site in host: Stomach. (1.52) mm long, maximum width 136 (109). Intestinal Type-locality: Fish caught by trolling, off Noumea´, caecum 204 (150) long, with maximum width 109 New Caledonia (collected 14.iv.2003). (109). Caecum to ventricular appendix length ratio 1:7 Prevalence and intensity: Single fish examined; 3 (1:10). Posterior end of body curved ventrally. nematode specimens. Spicules equal, alate, 762 (830) long, representing Type-material: Holotype and allotype in the Muse´um 4.1 (4.4) % of body length; fairly broad ventral National d’Histoire Naturelle, Paris (MNHN spicular ala forms distinct spike somewhat anterior JNC394E). to mid-length of spicule (Fig. 4G). Total of 38 (37) Etymology: The specific name of this nematode relates pairs of small subventral papillae present, 28 (27) to the genitive form of the generic name of the host. being pre-anals and 10 (10) postanals; papillae of several posteriormost pre-anal and postanal pairs very Description (Figs. 4–6) small; papillae of 4th subventral postanal pair doubled. One papilla-like ventromedian organ located on General. Medium-sized nematodes with transversely anterior cloacal lip present (Figs. 4D, E, G, 5C, E– striated cuticle (Figs. 5A–G, 6B), green when alive, G, 6B). Posterior cloacal lip with granular surface whitish in ethanol. Maximum width near middle of (Figs. 4E, 5F, G). Tail conical, 150 (150) long, body. Lips almost equal in size (dorsal lip slightly markedly narrowed at its distal third; tip obtusely smaller than ventrolateral lips), slightly shorter than pointed, without caudal projections visible under LM, wide, with narrow bases; their lateral flanges widest but with few poorly developed protuberances visible just posterior to middle of lips; pulp with 2 anteriorly under SEM (Figs. 4D, E, G, 5C, E–G, 6A). protruding lobes. Dorsal lip with 2 subdorsal double papillae; each subventral lip with 1 double subventral Female [Based on 1 specimen containing immature papilla, 1 small single papilla and amphid situated eggs, allotype.] Length of body 19.11 mm, maximum laterally (Figs. 4B, C, 5A, B, D). Interlabia well width 571. Lips 93 long. Length of interlabia 39. developed, low, about 1/3 length of lips (Figs. 4C, 5A, Length of oesophagus 2.56 mm, representing 13.4 % B, D). Cervical alae very narrow, starting about level of body length; maximum width 272. Nerve-ring and of lip bases, extending posteriorly in both sexes as excretory pore 490 and 558, respectively, from cordons almost to end of tail (Figs. 4E, 5C, E–G). anterior extremity. Ventriculus 109 9 109; ventricu- Deirids just posterior to level of nerve-ring (Fig. 4B). lar appendix 1.32 mm long, maximum width 95. Oesophagus long, slightly broader posteriorly than Intestinal caecum 245 long, maximum width 68. anteriorly. Nerve-ring encircles oesophagus at about Caecum to ventricular appendix length ratio 1:5. one sixth of its length. Ventriculus small, almost Vulva pre-equatorial, 7.11 mm from anterior end of spherical; ventricular appendix long, narrow. Intesti- body, at 37 % of body length. Vagina about 1.63 mm nal caecum markedly short. Excretory pore just long, directed posteriorly from vulva. Uterus forms posterior to nerve-ring (Fig. 4A). Tail of both sexes numerous coils and contains only immature eggs. Tail conical; tip lacks distinct cuticular outgrowths visible conical, 340 long; tip obtusely pointed, without any under LM. caudal projections visible under LM (Fig. 4F).

Male [Based on 2 specimens; holotype; measurements Remarks of paratype in parentheses.] Length of body 18.39 (18.71), maximum width 558 (544). Deirids slightly Of the 70 previously described, recognised species of asymmetrical, 544/589 (544/571) from anterior Hysterothylacium (see above), 19 are characterised by extremity. Width of cervical alae 27(27). Lips 96 the presence of a markedly short intestinal caecum and (93) long; length of interlabia 39 (45). Length of a long ventricular appendix; these features also typical oesophagus 2.51 (2.46) mm, representing 13.7 of H. alatum n. sp. and H. sphyraenae n. sp. However, (13.2) % of body length; maximum width 122 (109). in contrast to H. sphyraenae n. sp., some of these Nerve-ring 517 (503) from anterior extremity; excre- species possess conspicuously broad cervical alae tory pore just posterior to nerve-ring. Ventriculus (H. alatum, H. fortalezae and H. scomberoidei) or their 95 9 105 (136 9 150); ventricular appendix 1.41 spicules are much shorter, i.e. not exceeding 0.5 mm 123 188 Syst Parasitol (2015) 92:181–195

Fig. 4 Hysterothylacium sphyraenae n. sp. ex Sphyraena genie. A, Anterior body end of female, lateral view; B, Cephalic end of male, dorsal view; C, Same, enlarged; D, Posterior end of male, ventral view; E, Tail of male, ventral view; F, Tail of female, lateral view; G, Posterior end of male, lateral view

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Fig. 5 Hysterothylacium sphyraenae n. sp., SEM micrographs. A, Dorsal lip, dorsal view; B, Ventrolateral lip, ventrolateral view; C; Posterior end of male, lateral view; D, Cephalic end, lateral view; E, Posterior end of male, ventral view; F, G, Tail of male, ventrolateral and lateral views, respectively (arrow indicates postanal double papilla). Abbreviations: a, double cephalic papilla; b, single cephalic papilla; c, amphid; e, precloacal median organ; g, cloacal aperture; i, interlabium

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and H. ogcocephali (Olsen, 1952), were described as lacking such ornamentation on the tip of the tail (e.g. Schmidt et al., 1974; Deardorff & Overstreet, 1981; Bruce & Cannon, 1989; Moravec & Nagasawa, 2000). However, it is worth noting that two of them, H. dollfusi and H. ogcocephali, were studied only under the LM and the tail tip of H. cornutum was observed by SEM only under low magnification (Bruce & Cannon, 1989). The tail tip of all specimens of H. sphyraenae studied under the LM appeared to lack any cuticular ornamentation, resembling that in H. cornutum, H. dollfusi and H. ogcocephali; only at a very high magnification did the tail tip reveal the presence of a few minute protuberances (Fig. 6A). Hysterothylacium sphyraenae differs from H. cor- nutum, a parasite of Thunnus spp., mainly in the absence of ventral, precloacal, cuticular ornamentation, the presence of a pair of double postanal papillae, the shape of the female tail (markedly elongate in H. cornutum)and its somewhat shorter spicules (Deardorff & Overstreet, 1982; Bruce & Cannon, 1989; Moravec & Nagasawa, 2000). It differs mainly from H. dollfusi, a specific parasite of the freshwater paddlefish Polyodon spathula (Walbaum) in North America, by the presence of lateral alae, more numerous pairs of pre-anal papillae (27–28 vs 19–20) and somewhat shorter spicules (0.76–0.83 mm vs 1.07–1.45 mm) (Schmidt et al., 1974). From H. ogco- cephali, a specific parasite of Ogcocephalus radiatus Fig. 6 Hysterothylacium sphyraenae n. sp., SEM micrographs. (Mitchill) in the North Atlantic region, by the shape of the A, Tail tip of male, lateral view; B; Postanal double and single papillae lips (conspicuously elongate in H. ogcocephali), more numerous pairs of postanal papillae (10 vs 3–4) and somewhat longer spicules (762–830 vs 330–660 lm) (H. gibsoni, H. physiculi Moravec & Nagasawa, 2000 (Deardorff & Overstreet, 1981). A characteristic feature and H. winteri Torres & Soto, 2004) or much longer, of the new species, not reported in other Hysterothy- i.e. not less than 1.3 mm [H. bifidalatum Petter & lacium spp., is the presence of a distinct ventral spike Maillard, 1987, H. chaunaxi (Olsen, 1952), H. pelag- formed by each spicular ala (Fig. 4G). icum and H. reliquens] (see e.g. Deardorff & Over- Hysterothylacium sphyraenae is the first valid street, 1981; Petter & Maillard, 1987; Bruce & nominal species of this genus described from fishes Cannon, 1989; Moravec & Nagasawa, 2000; Torres of the family Sphyraenidae. Williams & Williams & Soto, 2004; Xu et al., 2014). (1996) listed ‘‘H. marinum’’ from Sphyraena barracu- Although most of the remaining 11 species have da (Walbaum) in the Pacific Ocean, but this informa- narrow lateral alae, resembling H. sphyraenae, almost tion is based on evident species misidentification. The all of these species are characterised by the presence of taxonomic status of the anisakid originally established numerous, distinct cuticular spines or protuberances as Gordius marinus Linnaeus, 1767 remains uncertain on the tail tip, which are readily visible under the LM and the records of Contracaecum or Hysterothylacium (H. amoyense, H. anguillae, H. arnoglossi, H. fabri, marinum reported by Yamaguti (1935), Deardorff & H. rhacodes, H. scomberomori and H. zhoushanensis). Overstreet (1981) and Moravec & Nagasawa (2000) In contrast, three species, H. cornutum (Stossich, relate, in fact, to H. rigidum (Rudolphi, 1809). In 1904), H. dollfusi (Schmidt, Leiby & Kritsky, 1974) contrast to H. sphyraenae, H. rigidum is a specific 123 Syst Parasitol (2015) 92:181–195 191 parasite of the Lophiidae in Europe, reported also off Japan, and it is characterised by a very long caecum extending anteriorly nearly to the level of the nerve- ring, spicules usually longer than 1 mm and by the presence of distinct numerous cuticular spines at the tail tip in both sexes (Yamaguti, 1935; Hartwich, 1975; Deardorff & Overstreet, 1981; Moravec & Nagasawa, 2000). Unidentifiable ascaridoid larvae were inadequately described as Ascaris sphyranurae Linstow, 1907 from the body cavity of Sphyraena barracuda (reported as Sphyranura barracuda) off Tasmania, Australia by Linstow (1907a); apparently, S. barracuda only served as a paratenic host. Adults or larvae of Hysterothy- lacium, not identified to species, have frequently been reported from other species of congeneric hosts throughout the world (e.g. Petter & Sey, 1997; Williams & Williams, 1996; Al-Zubaidy et al., 2012).

Contracaecum sp. third-stage larvae

Host: Blacksaddled colargrouper Plectropomus laevis (Lace´pe`de) (Perciformes: Serranidae); fork length 860 mm, weight 13.2 kg. Site in host: Ensheated around intestine; bodies of some specimens ensheathed by thin-walled capsule. Locality: Off Noume´a, New Caledonia, Re´cif Abore´ (collected 2 July 2006). Prevalence and intensity: 1 of 6 fish examined; 5 nematode specimens. Fig. 7 Contracaecum sp. third-stage larva ex Plectropomus Voucher specimens: Muse´um National d’Histoire laevis. A, Anterior end of body, lateral view; B, Cephalic end, Naturelle, Paris (MNHN JNC1887). apical view; C, Cephalic end, lateral view; D, Tail, lateral view; E, Cephalic end, lateral view Description (Figs. 7, 8)

[Based on 5 specimens.] Body robust, whitish, tooth (Figs. 7B, E, 8A–C). Oesophagus narrow, 45.71–66.10 mm long and 1.22–1.59 mm wide. Cuticle 1.80–2.19 mm long, 163–204 wide. Ventriculus small, finely transversely striated (Fig. 8A–D), lateral alae rounded, size 136–204 9 122–190; posterior ventricular absent. Cephalic end rounded, with anteriormost part appendix conspicuously large, 5.17–5.83 mm long, somewhat elevated, separated from rest of body by 326–490 wide. Nerve-ring 313–488 from anterior groove (Figs. 7B, C, 8A–C). Oral aperture demarcated extremity. Intestinal caecum short, extends only short by 2 broad, lobular elevations (anlagen of lips) protruding distance anterior to ventriculus; length 408–558, maxi- out of mouth, 12–41 high, 1 dorsal and 1 U-shaped mum width 204–218 (Fig. 7A). Length ratio of caecum ventral; latter bearing small ventral cuticular tooth and ventricular appendix 1:10–14. Genital primordium (Figs. 7B, C, 8A–C). Four submedian cephalic papillae indistinct. Tail conical, 190–218 long, with few minute surround oral aperture (Figs. 7B, C, 8A–C). Excretory terminal cuticular spines not visible in all specimens pore situated immediately posterior to ventral cephalic (Figs. 7D, 8D).

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Fig. 8 Contracaecum sp. third-stage larva ex Plectropomus laevis, SEM micrographs. A, Cephalic end, lateral and apical views; B, Cephalic end, apical view (arrow indicates ventral tooth); C, Cephalic end, apical view (another specimen) (arrow indicates ventral tooth); D, Tail, ventral view. Abbreviations: a, cephalic papilla; c, cloacal aperture; e, excretory pore; o, oral aperture

Remarks Paraheterotyphlum fourth-stage larvae; Sprent, 1978) and in the large body measurements. However, adults The general morphology of these larvae, especially the of Paraheterotyphlum spp. are 61–158 mm long structure of the digestive tract and the location of the (Sprent, 1978), whereas those of Contracaecum spp. excretory pore, indicates that they belong to Contra- are at most 90 mm long in C. osculatum (Rudolphi, caecum. Species of this genus are parasites of fish- 1802) (see e.g. Baylis, 1937; Mozgovoy, 1953; eating birds and marine (rarely freshwater) mammals, Hartwich, 1975). However, according to Sprent except sirenians (Anderson et al., 2009). However, as (1978), the excretory pore of P. australe Johnston & compared with the known third-stage larvae of other Mawson, 1948 (the type-species of Paraheteroty- congeneric species, these larvae are considerably phlum) is located slightly posterior to the nerve-ring, larger (up to c.60 mm vs at most about 26 mm in whereas that in the present larvae is found just other congeners) (e.g. Anderson, 2000; Moravec et al., posterior to the cephalic larval tooth (Fig. 8A–C). 1993; Moravec & Van As, 2015) and their tail tip bears Therefore, these larvae appear to belong to Contra- a few minute cuticular spines, not reported for larvae caecum. In having minute cuticular spines at the tail of other Contracaecum spp. tip, they also resemble fourth-stage larvae of some On the other hand, they are rather similar to adults Hysterothylacium spp., but differ distinctly from them of the genus Paraheterotyphlum Johnston & Mawson, by the location of the excretory pore, the much less 1948, parasites of sea snakes in the Pacific region developed anlagen of lips and the markedly larger (Johnston & Mawson, 1948; Schmidt & Kuntz, 1973; body measurements. Sprent, 1978), particularly in having a very long Third-stage larvae of Contracaecum have fre- ventricular appendix and a short caecum, the presence quently been reported from the internal organs of of minute cuticular spines on the tail tip (found in many species of freshwater, brackish-water and marine

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fishes, which serve as paratenic hosts (Anderson, 2000; Anderson, R. C. (2000). Nematode parasites of vertebrates. Moravec, 2009, 2013). Unfortunately, the species Their development and transmission. Second edition. Wallingford and New York: CABI Publishing, 650 pp. identification of Contracaecum larvae from fishes Anderson, R. C., Chabaud, A. G., & Willmott, S. (Eds) (2009). based on morphometrical features is practically Keys to the nematode parasites of vertebrates. Archival impossible. Judging by the structure of the digestive volume. Wallingford: CAB International, 463 pp. tract, and especially the markedly short caecum and Baylis, H. A. (1937). On the ascarids parasitic in seals, with special reference to the genus Contracaecum. Parasitol- long ventricular appendix, the present larvae tend to ogy, 29, 121–128. resemble the adults of C. rectangulum (Linstow, 1907), Bruce, N. L. (1990). Redescription of the ascaridoid nematode as illustrated by Linstow (1907b) (see also Mozgovoy, Hysterothylacium scomberomori (Yamaguti) from Aus- 1953). This parasite of Antarctic seals was later tralian Spanish mackerel Scomberomorus commerson (Lace´pe`de). Memoirs of the Queensland Museum, 28, considered a junior synonym of C. osculatum (Rudol- 427–434. phi, 1802) by Baylis (1937), although Mozgovoy Bruce, N. L., & Cannon, L. R. G. (1989). Hysterothylacium, (1953) did not agree with this synonymy (the caecum is Iheringascaris and Maricostula new genus, nematodes much longer than the ventricular appendix in C. oscu- (Ascaridoidea) from Australian pelagic marine fishes. Journal of Natural History, 23, 1397–1441. latum). However, according to Linstow (1907b), the Deardorff, T. L., & Overstreet, R. M. (1978). Thynnascaris body lengths of C. rectangulum is only 40 mm and rhacodes sp. n. (Nematoda: Ascaridoidea) in fishes from 55–60 mm, respectively, for the male and females, the Israeli Mediterranean coast. Annales de Parasitologie which is not comparable with the present third-stage Humaine et Compare´e, 53, 519–525. Deardorff, T. L., & Overstreet, R. M. (1981). Review of Hys- larvae. Apparently, these larvae belong to another, terothylacium and Iheringascaris (both previ- perhaps undescribed species of Contracaecum. ously = Thynnascaris) (Nematoda: Anisakidae) from the northern Gulf of Mexico. Proceedings of the Biological Acknowledgements We thank Claude Chauvet (UNC, Society of Washington, 93(1980), 1035–1079. Noume´a) for providing fish, and Ronald Fricke (Staatliches Deardorff, T. L., & Overstreet, R. M. (1982). Hysterothylacium Museum fu¨r Naturkunde, Stuttgart, Germany) for identifying the pelagicum sp. n. and H. cornutum (Stossich, 1904) (Ne- barracuda (from photographs). Thanks are also due to the staff of matoda: Anisakidae) from marine fishes. Proceedings of the Laboratory of Electron Microscopy, Institute of Parasitology, the Helminthological Society of Washington, 49, 246–251. BC CAS, in Cˇ eske´ Budeˇjovice for their technical assistance, and to Figus, V., D’Amico, V., Loddo, S. L., Siddu, N. L., & Canestri Blanka Sˇkorı´kova´ of the same Institute for help with the Trotti, G. (2005). Elminti parassiti di Serranus cabrilla (L.) illustrations. The authors also thank two anonymous reviewers (Osteichthyes, Serranidae) del Golfo di Cagliari, Mediter- for their helpful suggestions, and to David Gibson, Natural History raneo sud-occidentale. Ittiopatologia, 2, 207–215. Museum, London, for the linguistic revision of the MS. Froese, R., & Pauly, D. (Eds) (2015). FishBase. World Wide Web electronic publication. http://www.fishbase.org, ver- Funding This study was partly supported by the Institute of sion 04/2015. Parasitology (with institutional support RVO 60077344) and the Gibbons, L. M. (2010). Keys to the nematode parasites of ver- Czech Science Foundation (Project No. P505/12/G112). tebrates. Supplementary volume. Wallingford: CABI, 416 pp. Hartwich, G. (1975). Schlauchwu¨rmer, Nemathelminthes Rund- Compliance with ethical standards oder Fadenwu¨rmer, Nematoda Parasitische Rundwu¨rmer von Wirbeltieren I. und . Die Tier- Conflict of interest The authors declare that they have no welt Deutschlands 62. Jena: VEB Gustav Fischer Verlag, conflict of interest. 256 pp. Johnston, T. H., & Mawson, P. M. (1948). Some new records of nematodes from Australian snakes. Records of the South Ethical approval All applicable institutional, national and Australian Museum, 9, 101–106. international guidelines for the care and use of were Justine, J.-L. (2011). Huffmanela plectropomi (Nematoda: Tri- followed. chosomoididae: Huffmanelinae) from the coralgrouper Plectropomus leopardus (Lace´pe`de) off New Caledonia. Systematic Parasitology, 79, 139–143. Justine, J.-L., Beveridge, I., Boxshall, G. 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