Jean-Lou Justine. Syst Parasitol (2011) 78:41 55 DOI /s

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1 Syst Parasitol (2011) 78:41 55 DOI /s Protocotyle euzetmaillardi n. sp. (Monogenea: Hexabothriidae) from the bigeye sixgill shark Hexanchus nakamurai Teng (Elasmobranchii: Hexanchidae) off New Caledonia Jean-Lou Justine Received: 25 May 2010 / Accepted: 20 July 2010 Ó Springer Science+Business Media B.V Abstract Protocotyle euzetmaillardi n. sp. is described from the gills of the sixgill shark Hexanchus nakamurai Teng caught in deep-sea off New Caledonia, South Pacific. The new species is compared with the two other species of the genus (both from the only other species in this shark genus, H. griseus (Bonn.)), namely P. grisea (Cerfontaine, 1899) Euzet & Maillard, 1974, redescribed from vouchers, and P. taschenbergi (Maillard & Oliver, 1966) Euzet & Maillard, 1974, redescribed from its type-specimens. The anatomy of the reproductive system is detailed; all three species have a characteristic oötype with longitudinal cells ( ootype côtelé of Euzet & Maillard). The following unique combination of characters differentiates the new species from its two congeners: posterior lobe of seminal vesicle absent, diverticulum of oviduct present and small body size. Furthermore, its tubular ovary does not include a region with sperm, which is present in both of the other species, and its eggs have only one filament, whereas eggs in the uterus have one or two filaments in P. grisea and one filament in J.-L. Justine (&) UMR 7138 Systématique, Adaptation, Évolution, Muséum National d Histoire Naturelle, 57, rue Cuvier, Paris Cedex 05, France justine@mnhn.fr J.-L. Justine Aquarium des Lagons, BP 8185, Nouméa, Nouvelle-Calédonie P. taschenbergi. The latter features differ from existing diagnoses of Protocotyle, in which eggs with two filaments and the presence of a tubular ovary dilated with sperm are key characteristics. Résumé Protocotyle euzetmaillardi n. sp., des branchies du requin Hexanchidae Hexanchus nakamurai Teng pêché en mer profonde au large de la Nouvelle-Calédonie, Pacifique Sud, est décrit. La nouvelle espèce est comparée aux deux autres espèces du genre (toutes deux de la seule autre espèce de ce genre de requin, H. griseus (Bonn.)), P. grisea (Cerfontaine, 1899) Euzet & Maillard, 1974, redécrit à partir de vouchers, and P. taschenbergi (Maillard & Oliver, 1966) Euzet & Maillard, 1974, redécrit à partir des spécimens types. L anatomie de l appareil reproducteur est détaillée; les trois espèces ont un ootype caractéristique avec des cellules longitudinales («ootype côtelé» de Euzet & Maillard). La combinaison suivante de caractères, unique, différencie la nouvelle espèce de ses congénères : lobe postérieur de la vésicule séminale absent, diverticule de l oviducte présent, petit taille corporelle. Son ovaire tubulaire ne comprend pas de partie avec sperme, qui est présente chez les deux autres espèces. Ses œufs ont un seul filament, alors que les œufs dans l utérus en ont un ou deux chez P. grisea, et un seul chez P. taschenbergi. Ceci diffère des précédents diagnostics génériques de Protocotyle, dans lesquels les œufs avec deux filaments et la

2 42 Syst Parasitol (2011) 78:41 55 présence d un ovaire tubulaire dilaté avec sperme étaient des caractéristiques clés. Introduction Hexanchus Rafinesque (sixgill sharks) includes only two species, H. griseus (Bonnaterre) and H. nakamurai Teng, both of which are circumglobal bathydemersal species, from and m depth, respectively (Froese & Pauly, 2010). Only two species of monogeneans are known from H. griseus, namely the hexabothriids Protocotyle grisea (Cerfontaine, 1899) Euzet & Maillard, 1974 and P. taschenbergi (Maillard & Oliver, 1966) Euzet & Maillard, 1974 (Euzet & Maillard, 1974). No monogenean has previously been recorded from H. nakamurai. I describe below a new species of Protocotyle Euzet & Maillard, 1974 from H. nakamurai and compare it with the two known species, redescribed below, with emphasis on the reproductive system. Materials and methods Three specimens of Hexanchus nakamurai were caught in the deep-sea, and are detailed here with their registration numbers, date, locality, depth, gender, length and weight: JN1 (uninfected), 26.i.2002, Baie du Santal, Lifou Island, CHONDRICAL cruise, station PAL1 ( S, E), depth m, female, length 1.66 m; JNC2615 (infected), 3.vii.2008, near Passe de Dumbéa, off Nouméa ( S, E), depth unknown, female, length 1.02 m, weight 3.6 kg; and JNC2632 (uninfected), 14.viii.2008, near Passe de Boulari, off Nouméa, depth 390 m, length 1 m, weight 3 kg. Monogeneans were collected alive, flattened and fixed in cold ethanol, stained with carmine and processed as described by Justine (2005). Drawings were made using an Olympus BH2 microscope equipped with a drawing tube and differential interference contrast (DIC) optics. Measurements were made from pencil drawings with the help of a custom-made transparent rule, previously calibrated with a stage micrometer. Measurements of sclerites are external perimeters including the points; in Figs. 3 and 5, sclerites are drawn with the same orientation as in the whole specimen in the same figure. Drawings were scanned and redrawn on a computer with Adobe Illustrator; colours were added to drawings with the following conventions: cyan for female organs, magenta for male organs, green for sperm from partner, and brown for the intestine. Silhouettes of specimens were drawn from scans of slides to show relative sizes (Fig. 1). All measurements are given in micrometres unless otherwise indicated. 10 mm MNHN 111H Tc47 Protocotyle taschenbergi (types) MNHN 111H Tc48 MNHN 110H Tc46 A1 A3 A2 A4 MNHN 110H Tc45 Protocotyle grisea (vouchers) MNHN JNC2615 Protocotyle euzetmaillardi (types) Fig. 1 Silhouettes of specimens of Protocotyle spp. on slides, drawn as seen directly (not inverted by the microscope)

3 Syst Parasitol (2011) 78: Protocotyle grisea (Cerfontaine, 1899) Euzet & Maillard, 1974 Syns Squalonchocotyle grisea Cerfontaine, 1899; Neoerpocotyle grisea (Cerfontaine, 1899) Price, 1942; Erpocotyle grisea (Cerfontaine, 1899) Maillard & Oliver, 1966 Type-host: Hexanchus griseus (Bonnaterre). Site: Gills. Material examined: Two specimens deposited by Maillard & Oliver, each on one slide, MNHN 110H Tc45 and 110H Tc46 (Fig. 1). Brief redescription (Figs. 1, 2; Table 1) Haptoral hard parts comprise 3 pairs of similarly shaped sclerites (Fig. 2F H); median largest; posterior slightly smaller; anterior smallest. Appendix with 2 hamuli (Fig. 2I). Testes numerous, occupy intercaecal region of posterior part of body. Two thin sperm ducts (vasa efferentia), visible only anteriorly at level of ovary, reunite medially to form seminal vesicle, dorsal and just anterior to oötype. Seminal vesicle convoluted, thin-walled, contains spermatozoa, continues anteriorly, dorsal to uterus, becomes progressively thinner and connects to cirrus; no posterior lobe (Fig. 2A). Cirrus unarmed, comprising proximal enlarged part with sperm, thin mid-part and elongate, thin-walled distal bulb (Fig. 2C). Ovary located at mid-length of body proper, lobate proximally, tubular distally; distal tubular part of ovary (Fig. 2B) with few oöcytes, followed by empty zone, then elongate, dilated portion containing conspicuous amount of spermatozoa and finally curved part with many oöcytes (presumably fertilised). No difference seen between oöcytes anterior and posterior to zone containing spermatozoa. Oviduct (Fig. 2A) complex, comprising 3 parts: proximal oviduct thin, short, convoluted, curves dorsally around distal extremity of ovary, forms small diverticulum; median oviduct wider, straight, continues obliquely connecting with confluence of distal part of oviduct and genito-intestinal canal; distal oviduct receives median vitelloduct. Ovovitelloduct curves and connects with oötype. Two lateral vitelloducts unite to form posteriorly directed common vitelloduct. Oötype wall with longitudinal rows of large cells ( ootype côtelé of Euzet & Maillard, 1974). Mehlis gland-cells connect with proximal part of oötype. Oötype joins uterus. Uterus straight, enlarges distally to form sac. Few eggs visible in uterus. Two long vaginal ducts, linear, each connect to transverse vitelloduct, run anteriorly parallel to each other on either side of uterus, bend perpendicularly at extremity and open via 2 vaginal pores lateral to genital atrium. Uterine eggs exhibit variation: specimen Tc45, 16 eggs in uterus, including 9 with single filament at posterior pole (Fig. 2E) and 7 with 2 (anterior and posterior) polar filaments (Fig. 2D); specimen Tc45, with only single malformed egg in uterus; operculum of egg not detected. Remarks The two specimens are highly flattened, and are thus about three times greater in size than was reported by Maillard & Oliver (1966) for the same material (Table 1). These authors reported only measurements from unflattened specimens. However, because of the size of this species, the anatomy is visible only in highly flattened specimens. Figure 3 of Euzet & Maillard (1974), labelled Protocotyle n. g. obviously represents P. grisea; the arrow from vagina erroneously points to the uterus. Figure 19 of Boeger & Kritsky (1989), labelled with the character state, also represents P. grisea. The present redescription agrees with these two drawings. The strong sphincter of the uterus mentioned by Maillard & Oliver (1966; their Fig. 4) was not seen in either specimen, but the uterine wall is more folded at the level of the base of the cirrus. Maillard & Oliver (1966) did not mention a pars prostatica; in both specimens the base of the cirrus is surrounded by cells, possibly glandular, but similar cells are present around the distal part of the seminal vesicle. Protocotyle taschenbergi (Maillard & Oliver, 1966) Euzet & Maillard, 1974 Syns Onchocotyle appendiculata Kuhn, 1829 in Taschenberg (1879) (according to Maillard & Oliver, 1966); Erpocotyle taschenbergi Maillard & Oliver, 1966 Type-host: Hexanchus griseus (Bonnaterre). Site: Gills.

4 44 Syst Parasitol (2011) 78:41 55 Anterior Median F F-H, 1000 µm Fertilised (?) oöcytes Posterior G Diverticulum H D E Oviduct (proximal) D-E, 100 µm Oviduct (median) I Ovary, part with sperm I, 50 µm Vitellarium Uterus Seminal vesicle Vaginal ducts (2) Vasa efferentia (2) Ootype côtelé Transverse vitelloducts (2) Median vitelloduct Ovovitelloduct Mehlis gland Oviduct (distal) Genito-intestinal canal Oviduct (median) Fertilised (?) oocytes Diverticulum Ovary, empty part Mature oöcytes Ovary, lobate part C Ovary, part with sperm Ovary, empty part Mature oocytes A, 1000 µm B, C, 200 µm A B Fig. 2 Protocotyle grisea. A, central reproductive system; B, tubular part of ovary and oviduct; C, cirrus; D, eggs in uterus, with two filaments; E, egg in uterus, with single filament; F H, haptoral sclerites; I, hamuli. All drawings, MNHN 110H Tc45, except eggs (D,E), MNHN 110H Tc46

5 Syst Parasitol (2011) 78: Table 1 Measurements (lm) of Protocotyle grisea and P. taschenbergi Species P. grisea P. grisea P. grisea P. taschenbergi P. taschenbergi P. taschenbergi P. taschenbergi P. taschenbergi Source Maillard & Oliver, 1966 Voucher Tc45; this paper Voucher Tc46; this paper Maillard & Oliver, 1966 (original description) Type-specimen Tc47; this paper Type-specimen TC48-1; this paper Type-specimen Tc48-2; this paper Type-specimen Tc48-3; this paper Body and soft parts Total body length 8,000 9,000 24,250 18,750 2,900 3,700 3,040 7,640 7,060 4,200 Body proper length 18,000 15,000 2,640 6,640 6,060 3,480 Body width at level 2,000 2,500 3,750 3, ,220 1, of ovary Anterior sucker length 9 width Pharynx length 9 width Haptor Haptor length 6,250 3, ,000 1, Haptor width 900 1,500 1, Anterior sclerite length 1,450 1,720 1,680 1,720 c Median sclerite length 1,750 2,500 2,550 1,950 2,010 c Posterior sclerite length 1,600 2,320 2,330 1,820 1,860 c Appendix length 9 2, No appendix width Hamulus outer length Length Length Hamulus inner length Male organs Testicular field length 3,300 2,900 1,900 Testes number ? 140? Cirrus length ca. 1,000 1,400 1, Eggs Egg proper length 200 No egg Filaments number and length Two, 150 & 75 No egg One, or Two, & Two, 100 & 50 One,

6 46 Syst Parasitol (2011) 78:41 55 Anterior D Median E D-F, 200 µm Posterior F G, 50 µm G H C C, 400 µm I A, B, 1 mm A B H, I, 100 µm Fig. 3 Protocotyle taschenbergi. A, whole body (specimen without appendix); B, haptor, specimen with appendix; C, appendix; D F, sclerites; G, hamuli; H, cirrus; I, eggs in uterus, with single filament. All type-specimens; A,D F,I, MNHN 111H Tc48-1; B,C,G,H, MNHN 111H Tc48-2 Material examined: Type-specimens, deposited on 2 slides, MNHN 111H Tc47 (1 unflattened specimen) and 111H Tc48 (3 specimens, including 2 highly flattened (1 without appendix, here designated as Tc48-1, 1 with appendix, Tc48-2) and 1 unflattened (Tc48-3) (Fig. 1). Brief redescription (Figs. 1, 3, 4; Table 1) Haptoral hard parts comprise 3 pairs of similarly shaped sclerites (Fig. 3D F); in Tc48-2, median largest, posterior slightly smaller, anterior smallest; in

7 Syst Parasitol (2011) 78: Pharynx Fertilised (?) oöcytes Ovary, tubular part Vaginal pores (2) Cirrus Uterus Oviduct (proximal) Eggs Basal part of cirrus Vas deferens Vitellarium Ovary, part with sperm Seminal vesicle Egg C, 100 µm A, B, 500 µm Mature oöcytes C Vaginal ducts (2) Anterior lobe of seminal vesicle Uterus Vaginal ducts (2) Posterior blind lobe of seminal vesicle Vitellarium Ootype côtelé Transverse vitelloducts (2) Vasa efferentia (2) Ovovitelloduct Median vitelloduct Mehlis gland Oviduct (distal) Genito-intestinal canal Ovary, tubular part Ootype côtelé Vasa efferentia (2) Oviduct (proximal) Ovary, part with sperm Mature oöcytes Ovary, lobate part A Posterior blind lobe of seminal vesicle Testis B Fig. 4 Protocotyle taschenbergi. A, anterior reproductive system; B, central reproductive system; C, tubular part of ovary and oviduct. All, MNHN 111H Tc48-1, type-specimen

8 48 Syst Parasitol (2011) 78:41 55 Tc48-1, no marked differences between pairs. Appendix (Fig. 3B,C) with 2 hamuli (Fig. 3G). Testes occupy intercaecal area of posterior part of body. Two sperm ducts (vasa efferentia) thin, visible only anteriorly at level of ovary, reunite medially to form seminal vesicle dorsal and just anterior to oötype. Seminal vesicle thin-walled, contains abundant spermatozoa, extends anteriorly and posteriorly as 2 elongate lobes dorsal to uterus; anterior lobe highly convoluted, becomes progressively thinner and connects with cirrus; posterior lobe simple, blind, ends at level of tubular region of ovary (Fig. 4A,B). Cirrus unarmed; proximal part with transverse folds; distal part an elongate thin-walled bulb with longitudinal folds (Fig. 3H). Ovary located at mid-length of body proper, proximally lobate, distally tubular; distal tubular part of ovary (Fig. 4B,C) with few oöcytes, followed by dilate region containing conspicuous amount of spermatozoa, then curved region with many oöcytes (presumably fertilised). Unfertilised oöcytes apparently slightly larger than those presumed to be fertilised. Oviduct complex (Fig. 4B) comprises 2 parts: proximal oviduct thin, convoluted, curves dorsally around distal extremity of ovary, forms posterior branch, then continues obliquely and connects anterosinistrally with confluence of distal region and genito-intestinal canal; distal oviduct directed dextrally, receives common vitelloduct. Ovovitelloduct loops once on dextral side and connects with oötype; structures of ovovitelloduct and distal oviduct indiscernible. Two lateral vitelloducts open into posteriorly directed common vitelloduct. Oötype wall with longitudinal rows of large cells ( ootype côtelé of Euzet & Maillard, 1974). Mehlis gland-cells conspicuous, open into proximal part of oötype. Oötype joins uterus. Uterus straight, enlarges distally to form sac containing eggs. Two long, linear vaginal ducts, each connected to transverse vitelloduct, run anteriorly parallel to each other on either side of uterus, end at 2 vaginal pores lateral to genital atrium (Fig. 4A). Eggs seen only in uterus; operculum not seen; specimen Tc48-3 with 3 eggs, Tc48-1 with 3 eggs; always with single polar posterior filament (Fig. 3I). Remarks None of the deposited type-specimens correspond to the drawing of the original description (Fig. 5 in Maillard & Oliver, 1966); the measurements indicated in the original description correspond to the unflattened specimens (Tc47 and Tc43-3), but these are thick, overstained and no detail of anatomy is visible in them; moreover, specimen Tc47 has an unflattened haptor in which the sclerotised parts cannot be seen. No holotype was designated and thus all specimens are syntypes. The only well-flattened and complete specimen (with an appendix) is Tc48-2. The internal anatomy is readily visible and similar in specimens Tc48-1 and In the present redescription, the ovary is considered to have a distal tubular part; Maillard & Oliver (1966) thought that a seminal receptacle was a direct continuation of the ovary. These are two quite different interpretations of the same structure. The seminal vesicle is here considered to have an anterior branch and a posterior blind lobe; Maillard & Oliver (1966) thought that the canal déférent médian constituted a posterior cul-de-sac. Again, these are different interpretations of the same structure. Protocotyle euzetmaillardi n. sp. Type-host: Hexanchus nakamurai Teng, 1962; photographs of the infected specimen available in FishBase. Type-locality: Off Nouméa, New Caledonia, deepsea. Site: Gills. Type-material: Holotype MNHH JNC2651A1 (1 slide), 3 paratypes MNHN JNC2651A2 A4 (3 slides each with one specimen). Specimens A1 A3 highly flattened and complete, specimen A4 folded, less flattened, obscured by staining, haptor damaged. All collected 3.vii.2008, near Passe de Dumbéa ( S, E). Prevalence: 1/3 (33%) Etymology: This species is named for Louis Euzet and Claude Maillard, in recognition of their prominent contribution to the systematics of the Hexabothriidae. Description (Figs. 1, 5, 6; Table 2) [Based on 4 specimens; measurements in Table 2.] Body elongate-fusiform. Haptor symmetrical, armed with 6 suckers, each provided with hook-shaped sclerite, and appendix bearing single pair of terminal suckers and single pair of hamuli. Haptoral sclerites

9 Syst Parasitol (2011) 78: H, 50 µm B, 200 µm I, 100 µm E-G, 200 µm A, 1000 µm H B D C, D, 50 µm C Anterior E Median F Posterior G J, 30 mm A I J Fig. 5 Protocotyle euzetmaillardi n. sp. A, whole body; B, extremity of appendix; C,D, hamuli (C, holotype; D, well-oriented); E G, haptoral sclerites; H, anterior part of cirrus; I, eggs in uterus, with single filament; J, specimen with anastomosis of intestinal caeca. All holotype, except D, J, paratype JNC2615A3

10 50 Syst Parasitol (2011) 78:41 55 Pharynx Ovary, tubular part Oesophagus Vaginal pores (2) Fertilised (?) oöcytes Oviduct (median) Cirrus Uterus sac Basal part of cirrus (obscured by eggs) Eggs Oviduct (proximal) Diverticulum Vas deferens C Uterus C, 50 µm Vitellarium A, B, 500 µm Intestine Vaginal ducts (2) Intestine Uterus Vaginal ducts (2) Seminal vesicle Vasa efferentia (2) Ootype côtelé Vitelloduct A B Seminal vesicle Vitellarium Ootype côtelé Transverse vitelloducts (2) Vasa efferentia (2) Mehlis gland Median vitelloduct Oviduct junction Ovovitelloduct Oviduct (distal) Genito-intestinal canal Oviduct (median) Oviduct (proximal) Diverticulum Ovary, tubular part Ovary, lobate part Fig. 6 Protocotyle euzetmaillardi n. sp. A, anterior reproductive system; B, central reproductive system; C, tubular part of ovary and oviduct. All holotype

11 Syst Parasitol (2011) 78: Table 2 Measurements (lm) of Protocotyle euzetmaillardi n. sp Measurements Holotype JNC2615A1 Paratype JNC2615A2 Paratype JNC2615A3 Paratype JNC2615A4 Body and soft parts Total body length 6,040 6,120 5,360 4,560 Body proper length 4,960 5,080 4,400 3,660 Body width at level of ovary 1,080 1,260 1,080 9,60 Anterior sucker length 9 width Pharynx length 9 width Haptor Haptor length 1,080 1, Haptor width 1,760 1,800 1,460 Damaged Anterior sclerite length Median sclerite length Posterior sclerite length Appendix length 9 width Appendix sucker length 9 width Hamulus outer length Length Length Hamulus inner length 59 Male organs Testicular field length 1,950 1,800 1,500 Testes number [63 [49 [43 Cirrus length [225 [270 Eggs Egg length Egg width Filaments number and length One, One, One, One (Fig. 5E G) in 3 pairs arranged symmetrically, each with same shape and with point almost at right-angles to distal end of sclerite shaft; median pair slightly longer than anterior and posterior pairs. Appendix elongate. Pair of hamuli with V-shaped root situated near distal end of appendix; hamuli generally badly oriented for drawing accurate profile (Fig. 5C); morphology readily visible only in single hamulus from paratype JNC2615A3 (Fig. 5D). Pair of terminal suckers oblong. Anterior sucker terminal. Pharynx subspherical. Oesophagus very short, such that intestine bifurcates at level of pharynx. Caeca externally diverticulate, confluent posterior to testes, end as single, simple, medial caecum which extends into haptor and appendix (Fig. 5A). In single specimen, union of caeca at occurs at 2/3 of body length (Fig. 5J). Testes numerous, occupy intercaecal area of posterior part of body (Fig. 5A). Two sperm ducts (vasa efferentia) thin, visible only anteriorly at level of ovary, reunite medially to form seminal vesicle just antero-dorsal to oötype. Seminal vesicle (Fig. 6A,B) convoluted, thin-walled, contains spermatozoa, continues anteriorly, dorsal to uterus, becomes progressively narrower and connects with cirrus; no posterior lobe. Cirrus (Fig. 5H) unarmed; median region with transverse folds; distal region a thin-walled bulb with longitudinal folds. Connection between basal part of cirrus and seminal vesicle obscured by eggs in uterus in all specimens, thus making measurement of cirrus length and description of prostatic part impossible. Ovary located at mid-length of body proper, proximally lobate, distally tubular. Oviduct (Fig. 6B) complex, comprising three parts: proximal oviduct narrow, convoluted, curves dorsally around distal extremity of ovary, forms very small posteriorly directed diverticulum; median oviduct wider, straight, passes obliquely and connects antero-sinistrally,

12 52 Syst Parasitol (2011) 78:41 55 via distinct junction, with confluence of distal part and genito-intestinal canal; distal oviduct directed dextrally, receives common vitelloduct. Ovovitelloduct loops once on dextral side and connects with oötype; structure of ovovitelloduct and distal oviduct indiscernible. Two lateral vitelloducts unite to form posteriorly directed common vitelloduct. Oötype wall with longitudinal rows of large cells ( ootype côtelé of Euzet & Maillard, 1974). Mehlis gland-cells open into proximal region of oötype. Oötype joins uterus. Uterus straight, enlarges distally to form sac containing numerous eggs. Two long, linear vaginal ducts, each connected to transverse vitelloduct, run anteriorly parallel on either side of uterus, end as two vaginal pores lateral to genital atrium (Fig. 6A). Eggs numerous in all specimens, seen only in uterus; operculum not seen; single posterior polar filament always present (Fig. 5I). In JNC2615A3, egg in process of being formed within oötype clearly seen to have single posterior filament. Remarks The small diverticulum of the oviduct observed in this species (Fig. 6B) is similar to that of P. grisea and occurs in the same position as the seminal receptacle reported in various hexabothriids: I agree with Boeger & Kritsky (1989) that it is a reduced seminal receptacle. Spermatozoa are clearly stored in the dilated region of the distal ovary in P. grisea and P. taschenbergi; this region is absent in P. euzetmaillardi n. sp. and no spermatozoa were visible in the diverticulum. The posterior region of the seminal vesicle in this species has the same structure and shape as in P. grisea and therefore contrasts with that of P. taschenbergi, in which a conspicuous posterior lobe or cul-de-sac is present. Discussion The Hexabothriidae Price, 1942 is a family of polyopisthocotylean monogeneans exclusively parasitic on the gills of chondrichthyan fishes (sharks, rays and chimeras) and includes about 60 species. Euzet & Maillard (1974) gave an historical account of the Hexabothriidae, revised all species and discussed co-evolution of these monogeneans and their hosts; they recognised 11 genera. Two genera, Paraheteronchocotyle Mayes, Brooks & Thorson, 1981 and Callorhynchocotyle Suriano & Incorvaia, 1982, were added shortly after. Boeger & Kritsky (1989) performed a cladistic analysis, discussed the generic placement of many species and accepted the 11 genera listed in Euzet & Maillard and the two new genera; a fourteenth genus, Branchotenthes Bullard & Dippenaar, 2003, has subsequently been added (Bullard & Dippenaar, 2003; Glennon, Chisholm, & Whittington, 2005). From abundant material (respectively 490 and 435 specimens) collected from two large (2.55 and 4.15 m long) sixgill sharks Hexanchus griseus caught off the French Mediterranean coast (Laubier, Maillard, & Oliver, 1966), two species of hexabothriid monogeneans were collected (Maillard & Oliver, 1966), one rare (20/490), large species designated at that time as Erpocotyle grisea (Cerfontaine, 1900) and an abundant (470/490), smaller new species described as E. taschenbergi Maillard & Oliver, Euzet & Maillard (1974) transferred the two species from H. griseus to Protocotyle Euzet & Maillard, The haptor muscles and the mechanism of attachment of P. grisea were discussed and compared with those of other hexabothriids (Euzet & Maillard, 1976). Protocotyle was considered valid by Boeger & Kritsky (1989) in their revision. The literature on hexabothriids from hexanchid sharks is scarce, only two species being recorded, i.e. P. grisea and P. taschenbergi. The present paper adds a third species, P. euzetmaillardi n. sp. As certain characters seen in these three species do not fit previous definitions of the genus, I shall first discuss the generic diagnosis and then the specific diagnosis. Generic diagnosis Protocotyle was characterised by Euzet & Maillard (1974) as having an unarmed cirrus, parallel vaginae, a seminal receptacle formed as an enlargement of the oviduct, an oötype côtelé (i.e. with longitudinal rows of cells in its wall), and eggs with two polar filaments. The new species described here corresponds well with all of these characters, except for the number of egg filaments (see below). Boeger & Kritsky (1989) considered Protocotyle valid and provided a longer list of characters. The following characters from their list were found in

13 Syst Parasitol (2011) 78: specimens of the three species examined: body elongate; distal cirrus elongate, unarmed; proximal cirrus expanded basally (not seen in P. euzetmaillardi); prostatic region present (see remark below); vaginal ducts parallel, undifferentiated; ovary proximally lobate; ovary with sinuous descending branch; ovary with sinuous ascending branch; oötype with longitudinal rows of large cells in walls; seminal receptacle reduced or absent; haptor symmetrical; sucker sclerites of similar shape and size; haptoral appendix marginal, with anchors; parasite of hexanchoid sharks. The prostatic region or pars prostatica is inconspicuous in the three species, and similar, possibly glandular, cells were seen around the basal part of the cirrus and the terminal part of the seminal vesicle. This character, mentioned by Boeger & Kritsky (1989; their Fig. 12) was not clearly seen. Two characters are different to those previously listed by Boeger & Kritsky (1989) and deserve mention. These are eggs with two filaments and the dilated terminal region of the ovary. The eggs seen in the uterus of the specimens examined had one or two filaments in P. grisea, but only one filament in P. taschenbergi and P. euzetmaillardi. Maillard & Oliver (1966) claimed that eggs had two filaments in P. taschenbergi, but did not provide figures. It may be that they were only able to observe mature eggs laid by live monogeneans (although this is not clearly stated in their text, it is a common procedure) and that laid eggs have a more complete structure (with two filaments) than unlaid eggs in the uterus. The character terminal portion of the ovary dilated was not found in P. euzetmaillardi, but was clearly visible in P. grisea and P. taschenbergi. This character is the single synapomorphy of Protocotyle in Boeger & Kritsky (1989) s cladistic analysis. However, close similarities of the structure of the genital system of P. euzetmaillardi suggests that this species belongs to the same genus as P. grisea and P. taschenbergi, and therefore this character should be considered as variable in this genus. It may be that the specimens of P. euzetmaillardi had not recently mated, thus having no sperm available to dilate the ovary; but, even if this hypothesis is true, it still remains that the character is variable. The very characteristic oötype with a longitudinal rows of cells ( ootype côtelé of Euzet & Maillard, 1974; ootype cannelé of Maillard & Oliver, 1966)is found only in three hexabothriid genera, namely Protocotyle, Squalonchocotyle Cerfontaine, 1899 and Rajonchocotyle Cerfontaine, 1899, and was considered a synapomorphy uniting these three genera by Boeger & Kritsky (1989). All three genera have a symmetrical haptor, similar sclerites and a haptoral appendix with hamuli. Differentiating characters are given in Table 3 and include the new information on the presence of eggs with one or two filaments in Protocotyle. Members of Squalonchocotyle have parallel vaginae, a branched ovary, eggs with two filaments and are found in squaloid sharks (Kitamura, Ogawa, Taniuchi & Hirose, 2006); members of Rajonchocotyle have Y-shaped vaginae, a lobate ovary, eggs without filaments and are found in rajoid rays; and members of Protocotyle have parallel vaginae, a lobate ovary, eggs with one or two filaments and are found in hexanchoid sharks. Specific diagnosis There are only three species in Protocotyle. Table 4 summarises the key characters. P. grisea is clearly differentiated from the two other species by its larger size and larger sclerites. The two smaller species are Table 3 Differentiating characters of Squalonchocotyle, Rajonchocotyle and Protocotyle, the three hexabothriid genera presenting an oötype with a longitudinal rows of cells ( ootype côtelé of Euzet & Maillard, 1974) Squalonchocotyle Rajonchocotyle Protocotyle Distal cirrus Unarmed Unarmed absent Ovary Branched Lobate Lobate Egg filaments Two None One or two Vaginal ducts Parallel Y-shaped Parallel Seminal receptacle Present Present Absent or reduced Hosts Squaloids Rajoids Hexanchoids

14 54 Syst Parasitol (2011) 78:41 55 Table 4 Differentiating characters of the three species of Protocotyle from Hexanchus spp. Character P. grisea P. taschenbergi P. euzetmaillardi n. sp. Posterior branch of seminal vesicle Absent Present Absent Terminal portion of ovary Dilate Dilate Not dilate Diverticulum of oviduct Present Absent Present Extremity of vaginal duct Curved Rectilinear Rectilinear Body size (flattened) (mm) Large (15 25) Small (4 7) Small (4 6) Sclerite length (lm) 1,700 2, Cirrus length (lm) ca. 1, [270 Host H. grisea H. grisea H. nakamurai differentiated by the structure of the seminal vesicle (posterior lobe present in P. taschenbergi vs absent in P. euzetmaillardi), the dilated part of tubular ovary containing sperm (present in P. taschenbergi vs absent in P. euzetmaillardi), the size of sclerites, smaller ( lm) in P. taschenbergi than in P. euzetmaillardi ( lm) and their different hosts (H. grisea for P. taschenbergi vs H. nakamurai for P. euzetmaillardi). The sphincter in uterus allegedly present (Maillard & Oliver, 1966) in P. grisea was not seen in the highly flattened specimens and thus cannot be considered a reliable character. Monogeneans previously recorded from deep-sea sharks off New Caledonia included only the monocotylid Triloculotrema chisholmae Justine, 2009 from Squalus melanurus Fourmanoir & Rivaton (Justine, 2009), whereas parasites previously recorded from H. nakamurai off New Caledonia include a trypanorhynch cestode (Beveridge & Justine, 2007) and an isopod (Trilles & Justine, 2004). This is the first hexabothriid described from New Caledonian waters (Justine, 2010). Acknowledgements Cyndie Dupoux and Sophie Olivier participated in the fishing expedition and parasitological survey. The two uninfected sharks were collected during the 2002 CHONDRICAL cruise (head: Bernard Séret) and by the Aquarium des Lagons, Nouméa. Bernard Séret (MNHN, IRD, Paris) identified the sharks from photographs. References Beveridge, I., & Justine, J.-L. (2007). Paragrillotia apecteta n. sp. and redescription of P. spratti (Campbell & Beveridge, 1993) n. comb. (Cestoda, Trypanorhyncha) from hexanchid and carcharhinid sharks off New Caledonia. Zoosystema, 29, Boeger, W. A., & Kritsky, D. C. (1989). Phylogeny, coevolution, and revision of the Hexabothriidae Price, 1942 (Monogenea). International Journal for Parasitology, 19, Bullard, S. A., & Dippenaar, S. M. (2003). Branchotenthes robinoverstreeti n. gen. and n. sp. (Monogenea: Hexabothriidae) from gill filaments of the bowmouth guitarfish, Rhina ancylostoma (Rhynchobatidae), in the Indian Ocean. Journal of Parasitology, 89, Euzet, L., & Maillard, C. (1974). Les Monogènes Hexabothriidae Price, Historique, systématique, phylogenèse. Bulletin du Muséum National d Histoire Naturelle, 3 série, 206, Zoologie, 136, Euzet, L., & Maillard, C. (1976). The mechanism of attachment of some Hexabothriidae (Monogenea) to a host. Trudy Biologo-Pochvennogo Instituta, Novaya Seriya, 34, [Published in Russian with English Abstract; original French text communicated by the authors]. Froese, R., & Pauly, D. (Eds.). (2010). FishBase. World Wide Web electronic publication. Glennon, V., Chisholm, L. A., & Whittington, I. D. (2005). Branchotenthes octohamatus sp. n. (Monogenea: Hexabothriidae) from the gills of the southern fiddler ray, Trygonorrhina fasciata (Rhinobatidae) in South Australia: description of adult and larva. Folia Parasitologica, 52, Justine, J.-L. (2005). Species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae) from Epinephelus fasciatus and E. merra (Perciformes: Serranidae) off New Caledonia and other parts of the Indo-Pacific Ocean, with a comparison of measurements of specimens prepared using different methods, and a description of P. caledonicus n. sp. Systematic Parasitology, 62, Justine, J.-L. (2009). A new species of Triloculotrema Kearn, 1993 (Monogenea: Monocotylidae) from a deepsea shark, the blacktailed spurdog Squalus melanurus (Squaliformes: Squalidae), off New Caledonia. Systematic Parasitology, 74, Justine, J.-L. (2010). Parasites of coral reef fish: how much do we know? With a bibliography of fish parasites in New Caledonia. Belgian Journal of Zoology, 140(Suppl.), Kitamura, A., Ogawa, K., Taniuchi, T., & Hirose, H. (2006). Two new species of hexabothriid monogeneans from the ginzame Chimaera phantasma and shortspine spurdog Squalus mitsukurii. Systematic Parasitology, 65,

15 Syst Parasitol (2011) 78: Laubier, L., Maillard, C., & Oliver, G. (1966). Contribution à l étude des parasites du griset : Hexanchus griseus (Bonnaterre, 1788). Vie et Milieu, Série A: Biologie Marine, 17, Maillard, C., & Oliver, G. (1966). Monogenea, Hexabothriidae. Vie et Milieu, Série A: Biologie Marine, 17, Taschenberg, O. (1879). Weitere Beiträzur Kenntniss ectoparasitischer mariner Trematoden. Festschrift zur Feier des hundertjärigen Bestehens der Naturforschenden Gesellschaft in Halle A/S. Halle: Niemeyer, pp Trilles, J. P., & Justine, J.-L. (2004). Une nouvelle espèce de Cymothoidae et trois Aegidae (Crustacea, Isopoda) récoltés sur des poissons de mer profonde au large de la Nouvelle-Calédonie. Zoosystema, 26,