Syst Parasitol (2008) 71:145–158 DOI 10.1007/s11230-008-9156-4 Two new species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae) from the deep-sea grouper Epinephelus morrhua (Val.) (Perciformes: Serranidae) off New Caledonia Jean-Lou Justine Received: 6 March 2008 / Accepted: 25 March 2008 Ó Springer Science+Business Media B.V. 2008 Abstract Three species of monogeneans were collected from Epinephelus morrhua, a deep-sea grouper from the external slope of the coral reef, off New Caledonia, South Pacific, and are the first parasites recorded from this fish species. Pseudorhabdosynochus morrhua n. sp. is characterised by: a sclerotised vagina with small sclerotised chambers and a short secondary canal; squamodiscs with central rows of rodlets which form closed ovals; and a scaly tegument. P. variabilis n. sp. has: a male quadriloculate organ with a characteristic structure; a sclerotised vagina in which the primary canal, secondary canal and accessory structure are very long and the different parts have various arrangements in different specimens; its squamodiscs have central rows of rodlets which form closed circles; and a smooth tegument. A prominent vaginal structure, comparable to that of P. variabilis, has been found only in P. dolicocolpos Neifar & Euzet, 2007, but the species can be distinguished by details of the vagina and other structures. A species of Haliotrema Johnson & Tiegs, 1922 (Ancyrocephalidae) is reported but not described. Specimens of each of the three species were present in similar numbers. J.-L. Justine (&) Équipe Biogéographie Marine Tropicale, Unité Systématique, Adaptation, Évolution (CNRS, UPMC, MNHN, IRD), Institut de Recherche pour le Développement, BP A5, 98848 Noumea Cedex, New Caledonia e-mail: justine@ird.nc Résumé Trois espèces de monogènes ont été récoltées d’Epinephelus morrhua, un mérou de profondeur de la pente externe du récif corallien au large de la Nouvelle-Calédonie, Pacifique Sud, et sont les premiers parasites mentionnés de cette espèce de poisson. Pseudorhabdosynochus morrhua n. sp. est caractérisé par un vagin sclérifié avec des petits chambres sclérifiées et un canal secondaire court, des squamodisques avec des ovales fermés au centre, et un tégument écailleux. P. variabilis n. sp. a un organe tétraloculé mâle avec une structure caractéristique, un vagin sclérifié dans lequel le canal primaire, le canal secondaire et la structure accessoire sont très longs et dont les différentes parties sont disposées de manière variable selon les spécimens. Ses squamodisques ont des cercles fermés au centre et son tégument est lisse. Une structure vaginale originale comparable à celle de P. variabilis a été trouvée seulement chez P. dolicocolpos Neifar & Euzet, 2007, mais les espèces peuvent être distinguées par des détails du vagin et d’autres structures. Une espèce d’Haliotrema Johnson & Tiegs, 1922 (Ancyrocephalidae) est mentionnée mais non décrite. Les spécimens de chacune des trois espèces étaient en nombre similaire. Introduction Groupers (Serranidae: Epinephelinae) living at moderate depth (0–100 m) in the coral reefs and lagoon of New Caledonia harbour a rich fauna of monogeneans, mainly composed of species of the diplectanid genus 123 146 Pseudorhabdosynochus Yamaguti, 1958 (see Justine, 2007a, and references therein). Several other species of groupers are present on the outer slope of the coral reef at depths between 100–400 m (Laboute & Grandperrin, 2000). I describe here two diplectanid monogeneans from one of these deep-sea groupers, Epinephelus morrhua (Valenciennes); this species also harboured a new species of the ancyrocephalid genus Haliotrema Johnson & Tiegs, 1922, which is not described here. Apparently, no helminth parasite has been recorded from this fish (e.g. Gibson et al., 2005) and thus these monogeneans are the first described from this host. Materials and methods Three specimens of E. morrhua were caught by line on the outer slope off the reef barrier facing Nouméa, New Caledonia. The fish were kept in seawater in an icebox cooled with frozen water bottles and immediately brought back to the laboratory; monogeneans were collected within 3–4 hours of capture. A unique number (JNC) was assigned to each fish. The three fish were JNC2450 (Fork Length 445 mm, Weight 1,200 g, 15 January 2008, off Grand Récif Kué, 22°350 S, 166°300 E, depth 120–300 m), JNC 2453 (FL 460 mm, W 1,300 g, same locality as above, depth c.300 m), and JNC2462 (FL 558 mm, W 2,500 g, 4 March 2008, off Récif Mbere, 22°180 S, 166°120 E, depth c.170 m). The parasitological material was then assigned a corresponding JNC linked to the respective fish host. Gills were extracted and immediately processed: live monogeneans were individually picked off the gills with fine needles and prepared. Specimens were routinely processed for carmine staining, including initial flattening between a slide and a coverslip in ethanol (referred to as ‘carmine’ – see Justine 2005a), according to Justine (2005a), or with ammonium picrate-glycerine, according to Malmberg (1957), but slightly modified (referred to as ‘picrate’ – see Justine 2005a). A few specimens were observed in Berlese’s fluid. The rest of the gills were then fixed in hot (60°C) 4% formalin, and later monogeneans were removed with fine needles and processed for carmine staining without flattening; these are referred to as ‘unflattened carmine’. ‘Picrate’ slides were made with a single or several worms; ‘carmine’ and ‘unflattened carmine’ slides were made with a single worm per slide for the preparation of the different 123 Syst Parasitol (2008) 71:145–158 monogenean species. For JNC2462, certain specimens were fixed alive in glutaraldehyde or ethanol 100% for electron microscopy or molecular studies, respectively, and the specimens were later quickly examined with the microscope in a drop of glutaraldehyde or water; results of this triage were incorporated in Table 1. Monogeneans were drawn using a BH2 microscope equipped with a drawing tube and differential interference contrast optics. Measurements were made from the pencil drawings with the help of a custom-made transparent rule, previously calibrated with a stage micrometer. Drawings were scanned and redrawn on a computer with Adobe Illustrator. All drawings of bodies, and all drawings of other parts, are at the same scale for all species. The method of measurement of the sclerotised parts (see fig. 1 in previous papers (Justine, 2005a, 2007a) was described by Justine (2005a); measurements of the right-hand haptoral hard-parts and left-hand equivalents were pooled. All measurements are given in micrometres as: the holotype (h), and mean and standard deviation (when n [ 30) followed by the range and number of measurements in parentheses. Measurements of ammonium picrate preparations (p) and of specimens flattened in ethanol stained with carmine (c) may vary significantly (Justine, 2005a) and are separated when indicated; measurements of soft parts are generally given only for ‘carmine’ specimens. ‘Carmine’ specimens were preferred to ‘unflattened carmine’ specimens for the descriptions. The squamodiscs are described according to a new method (Justine, 2008; Sigura & Justine, 2008). Squamodiscs, observed with a differential interference microscope under oil immersion, were either drawn in detail or schematically (a short line for each rodlet); counts of rodlets in each row were tabulated, as a series of numbers. In the Tables, each line shows the total number of rodlets in each row, the first number representing the anteriormost (innermost) row and the last number the posteriormost (outermost) row. Rows forming circles are indicated by ‘c’, v-shaped rows are indicated by ‘‘v’’, and incomplete rows are indicated by ‘i’. The total number of rows (excluding incomplete rows) and the total number of rodlets are also shown for each squamodisc. This tabulation method provides, in a compact format, detailed information on the structure of squamodiscs, including intraspecific variations, and is useful for comparing species. Only squamodiscs in excellent condition are used, but specimens with a single good squamodisc can be used. The method is best Syst Parasitol (2008) 71:145–158 147 Table 1 Numbers of monogeneans collected in three specimens of Epinephelus morrhua Fish Pseudorhabdosynochus morrhua n. sp. Pseudorhabdosynochus variabilis n. sp. Haliotrema sp. JNC2450 22 18 9 JNC2453 JNC2462 Total d 55% d 45% m 45% m 37% m 18% 29 25 38 d 54% d 46% m 32% m 27% m 41% 17 17 16 d 50% d 50% m 34% m 34% m 32% 68 60 63 d 53% d 47% m 36% m 31% Total 49 92 50 191 m 33% JNC 2450, 2453: quasi-exhaustive search of all gills for both fish; JNC2462, limited, quick search d, percentage of the diplectanids; m, percentage of the total number of monogeneans explained by comparing a line in a Table and the actual drawing of the same squamodisc: compare Table 2 and Fig. 2A, B and Table 4 and Fig. 4A, B. In addition, when the number of rows is constant in most specimens, which is the case for both species described here, results can be expressed as the mean number and range of rodlets in each row (Tables 3, 5). Note about the fish: Epinephelus morrhua has a wide distribution from the Red Sea and western Indian Ocean to the central Pacific Ocean. It is a deep-water species found at depths of 80–370 m. According to various authors, the maximum total length is from 70– 90 cm (Heemstra & Randall, 1993), so the specimens studied here were relatively small. The fish is occasionally found in the fish market at Nouméa (under the local name ‘‘loche à bandes noires’’) and has a good reputation for not being responsible for ciguatera seafood poisoning. This species was recently transferred (Craig & Hastings, 2007) to Mycteroperca Gill; however, a discussion on the possible implications of this new phylogeny of groupers for the understanding of relationships within Pseudorhabdosynochus is not within of the scope of this paper. Abbreviations: MNHN, Muséum national d’Histoire Naturelle, Paris; BMNH, Natural History Museum, London; USNPC, United States National Parasite Collection, Beltsville; SAMA AHC, South Australian Museum Adelaide, Australian Helminthological Collection; HCIP, Helminthological Collection, Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, České Budějovice; MPM, Meguro Parasitological Museum, Tokyo; h, holotype; c, carmine; p, picrate, uc, unflattened carmine. Pseudorhabdosynochus morrhua n. sp. Type-host: Epinephelus morrhua (Valenciennes). Type-locality: Outer slope of the barrier reef, off Nouméa, New Caledonia. Site: Between secondary gill lamellae. Type-material: Holotype, JNC2453A5, 23 January 2008, outer slope of the barrier reef off Nouméa, New Caledonia, 22°350 S, 166°300 E, depth c.300 m. Material examined: 63 specimens, including 30 ‘carmine’ (c), 18 ‘picrate’ (p), 9 ‘unflattened carmine’ (uc), 6 ‘berlese’ (b). Material deposited: Holotype (c) and 28 paratypes (17 c, 11 p), MNHN, JNC2450, JNC2453; vouchers (c, p, uc), MNHN, JNC2450, JNC2453, JNC2462; 1 paratype (c), BMNH 2008.3.17.1; 1 paratype (c), USNPC 100953; 1 paratype (c), SAMA AHC 29477; 1 paratype (c), HCIP M-464; 1 paratype (c), MPM 18869 (Slide Box A5121). Prevalence: 100% (3/3) Intensity: See Table 1; maximum intensity c.30. Etymology: From the host name, used as a name in apposition. 123 148 Description (Figs. 1–2) Body elongate; length h 340, c 352 (250–480, n = 20), width h 195, c 145 (80–195, n = 19). Tegument scaly; scales on ventral and dorsal faces from level of pharynx to squamodiscs. Anterior region with 3 pairs of head organs and 2 pairs of eye-spots; anterior eye-spots pair generally disrupted; posterior eye-spot often disrupted, distance between outer margins of posterior eye-spot pair h 22 c 20 (16–23, n = 6). Haptor differentiated from rest of body, as wide as body, width h 175, c 154 (130–180, n = 21), provided with 2 similar squamodiscs, 2 pairs of lateral hamuli, 3 bars and 14 marginal hooklets. Squamodiscs round in shape, made up of rows of rodlets; central rows forming closed ovals; rodlets adjacent in central rows, progressively more loosely connected in peripheral rows; last row with very thin, separated rodlets; ventral and dorsal squamodiscs similar; ventral squamodisc length h 62, c 53 (40–62, n = 16), width h 55, c 56 (42–63, n = 16), with h 11, always 11 (n = 15) rows of rodlets and 2 closed ovals; total number of rodlets h 87, mean 82 (72–94, n = 14, individual variations in Table 2); dorsal squamodisc, length h 56, c 53 (37–63, n = 15), width h 57, c 53 (35–58, n = 15), with h 12, generally 12 (10–12, n = 14) rows of rodlets and always 2 closed ovals; total number of rodlets h 95, mean 91 (80–103, n = 11, individual variations in Table 2). Number of rodlets in each row of squamodiscs for all specimens computed in Table 3. Ventral hamulus with handle and distinct guard, outer length h 36, c 34 (30–38, n = 27), p 38 (34–42, n = 22), inner length h -, c 27 (23–31, n = 24), p 28 (26–32, n = 22). Dorsal hamulus with indistinct guard, outer length h 34, c 32 ± 1.7 (26–35, n = 38), p 34 (31–36, n = 31), inner length h 19, c 19 (17–21, n = 27), p 19 (18–21, n = 22). Dorsal (lateral) bars slightly curved, with wide flattened medial extremity and cylindrical lateral extremity, length h 57, c 54 ± 2.7 (46–58, n = 48), p 59 (50-65, n = 22), maximum width h 18, c 16 ± 2.1 (10–19, n = 34), p 18 (14–22, n = 22). Ventral bar flat, with slightly constricted median portion and thin extremities, length h 87, c 82 (71–90, n = 24), p 87 (73–93, n = 11), maximum width h 12, c 10 (8–12, n = 19), p 11 (8–17, n = 11); ventral bar straight or bowshaped according to the specimens (Fig. 1E, F), with groove visible on its ventral side. 123 Syst Parasitol (2008) 71:145–158 Pharynx subspherical, length h 34, c 32 (23–42, n = 19), width h 29, c 26 (20–32, n = 19). Oesophagus apparently absent, such that intestinal bifurcation immediately follows pharynx. Caeca simple, terminate blindly at level of posterior margin of vitelline field. Testis subspherical, intercaecal, length h 37, c 43 (27–65, n = 9), width h 53, c 49 (30–65, n = 9). Vas deferens emerges from antero-sinistral part of testis, enlarges into seminal vesicle; seminal vesicle in middle region of body, transforms into duct; duct forms bends then transforms into small bulb, followed by duct; duct enlarges then connects with quadriloculate organ. Prostatic reservoir small, connects with quadriloculate organ. Quadriloculate organ with fourth (posterior) chamber slightly more sclerotised than 3 anterior chambers; first chamber with very thin anterior wall; fourth chamber ends in short sclerotised cone, prolonged by sclerotised tube; tube slightly wider at extremity than at base; end of tube prolonged by thin unsclerotised filament of variable length. Inner length of quadriloculate organ h 44, c 39 (35–44, n = 15), p 65 (58–74, n = 9); cone length h 7, c 7 (5–9, n = 21), p 6 (5–7, n = 7); tube length h 16, c 16 (12–18, n = 22), p 16 (13–19, n = 9); tube diameter h 3, c 3 (n = 9), p 3 (n = 21); filament length h 35, c 0–42 (n = 19), p 0–45 (n = 7). Ovary subequatorial, intercaecal, pre-testicular, encircles right caecum. Ovary width h 53, c 48 (40–60, n = 8). Oviduct passes medially to form oötype, surrounded by Mehlis’ gland; oötype short, opens into uterus. Uterus dextral. Unsclerotised vagina inconspicuous, elongate (Fig. 1D, 2O). Duct from sclerotised vagina to oötype inconspicuous. Vitelline fields extend posteriorly from posterior to pharyngeal level in 2 lateral bands, confluent in posttesticular region and terminate anterior to peduncle. Bilateral connections from vitelline fields to oötype inconspicuous. Egg unknown. Sclerotised vagina (nomenclature of parts according to Justine 2007a; see Fig. 1D) sinistral, a complex sclerotised structure; aspect changes slightly according to specimen and orientation (Fig. 2). Sclerotised vagina comprises anterior trumpet, followed by primary canal, primary chamber, secondary canal and secondary chamber; trumpet in continuity with unsclerotised vagina (Fig. 1D, 2O); primary canal coiled once anteriorly, with thin wall; canal continues into primary chamber; primary Syst Parasitol (2008) 71:145–158 149 A, 50 µm c t f B B-H, 50 µm USV Tr Ca1 AS C vl scew Ch2 pcew Ca2 D Ch1 A G E F H I Fig. 1 Pseudorhabdosynochus morrhua n. sp. from Epinephelus morrhua: (A) Ventral view, composite drawing, mainly from holotype; (B) Quadriloculate organ, paratype, ventral view (c, cone; t, tube; f, filament); (C) Sclerotised vagina, holotype, ventral view; (D) Sclerotised vagina, paratype, dorsal view (USV, unsclerotised vagina; Tr, trumpet; Ca1, primary canal; Ch1, primary chamber; Ca2, secondary canal; Ch2, secondary chamber; AS, accessory structure; measurements: vl, sclerotised vagina length; pcew, primary chamber external width; scew, secondary chamber external width); (E, F) Ventral bars in specimens with curved and straight bar; (G) Lateral (dorsal) bar; (H) Dorsal hamulus; (I) Ventral hamulus. (A–C) carmine; (D–I) picrate 123 150 Syst Parasitol (2008) 71:145–158 Table 2 Pseudorhabdosynochus morrhua n. sp., rodlets in squamodiscs Specimen Number of rodlets in each row (from centre to periphery) Closed/ Total Rodlet total JNC2450a6 vs i3-v8-9-9-7-8-7-9-6-5-1 1/11 72 JNC2450a5 vs v5-7-8-9-8-9-8-7-5-4-2 1/11 72 JNC2450a8 vs v7-v9-9-9-9-9-8-8-7-6-1 2/11 82 JNC2450a12 vs v8-v9-9-10-10-10-9-7-7-6-4 2/11 89 JNC2450a15 vs v6-v8-9-9-9-6-7-7-5-5-2 2/11 73 JNC2450a13 vs v7-9-9-11-10-10-8-7-6-6-2 1/11 85 JNC2453a13 vs v7-v10-11-11-9-9-9-8-9-6-4 2/11 83 JNC2453a11 vs v8-v10-10-11-11-11-8-7-6-6-1 2/11 89 JNC2453a9 vs v7-v10-10-10-10-10-10-8-8-6-5 2/11 94 JNC2453a8 vs v8-v10-10-11-11-9-10-9-8-6-2 2/11 94 JNC2453a7 vs v7-v10-10-10-9-9-9-7-6-6-3 2/11 86 JNC2453a6 vs v7-v8-9-9-9-8-8-8-7-5-2 2/11 80 JNC2453a5* vs v7-v10-10-10-9-10-8-7-6-6-4 2/11 87 JNC2453a4 vs v6-v6-9-9-10-9-7-7-6-5-2 2/11 76 JNC2450a8 JNC2450a15 ds ds v8-v10-11-11-9-8-9-9-7-6-6-3 v6-v8-8-9-9-8-7-7-7-5-4-2 2/12 2/12 97 80 JNC2453a12 ds v7-v9-9-10-9-9-8-8-7-6-4-3 2/12 89 JNC2453a11 ds v6-v9-10-9-9-8-8-10-10-8-6-4 2/12 97 JNC2453a10 ds v7-v10-10-9-10-9-9-9-i5-7-6-4 2/12 90 JNC2453a9 ds v8-v10-11-11-10-10-9-9-8-7-6-4 2/12 103 JNC2453a8 ds v6-v10-10-10-10-10-10-8-7-6-4-2 2/12 93 JNC2453a7 ds v6-v8-10-10-10-9-10-9-7-6-4 2/11 89 JNC2453a6 ds v7-v9-11-10-8-8-8-7-6-5-4-3 2/12 86 JNC2453a5* ds v7-v9-10-10-9-9-8-9-8-7-5-4 2/12 95 JNC2453a4 ds v6-v9-9-8-9-9-9-8-8-6-4-2 2/12 87 vs ventral squamodisc, mean 82 (72–94), n = 14) ds dorsal squamodisc, mean 91 (80–103, n = 11) * Holotype, drawn in Fig. 2A, B; i, incomplete rows; v, closed oval rows chamber sclerotised, small, ovoid, aligned and in continuity with primary canal; wall thick; interior crest sometimes visible within primary chamber; secondary canal inserted into anterior part of primary chamber; secondary canal short; secondary chamber sclerotised, spherical, ventral to and larger than primary chamber; accessory structure, curved, weakly sclerotised, inserted into secondary chamber. External and internal surfaces of primary and secondary chamber smooth. Duct from sclerotised vagina to oötype connects to secondary chamber (Fig. 2H). Total length of sclerotised vagina (measured from extremity of trumpet to posterior extremity, i.e. not 123 taking in account curved length along bend or coil of primary canal) h 32, c 28 (21–31, n = 23), p 31 (21–35, n = 10); external width of primary chamber h 4, c 4 (3.5–5, n = 22), p 5 4–6, n = 10); external width of secondary chamber h 6, c 6 (5–6, n = 21), p 7 (5.5–7.5, n = 10). Orientation of sclerotised vagina: trumpet always anterior. Differential diagnosis Several species of Pseudorhabdosynochus share with P. morrhua n. sp. the following characteristics of the sclerotised vagina: a trumpet followed by a thinwalled primary canal, a small primary chamber, a Syst Parasitol (2008) 71:145–158 151 A B C D 50 µm E K F G L H M I N J O Fig. 2 Pseudorhabdosynochus morrhua n. sp. from Epinephelus morrhua: (A, B) Ventral (A) and dorsal (B) squamodiscs, holotype, ventral view (C, D) Ventral (C) and dorsal (D) tegumental scales, same specimen, ventral view (E–O) Sclerotised vagina, variation according to specimen and orientation (E–I), carmine, ventral view; (J, K), carmine, dorsal view; (L–O), picrate, dorsal view. O, unsclerotised vagina drawn. All paratypes except A, B short secondary canal and a secondary chamber of size similar to the primary chamber. P. hirundineus Justine, 2005 from Variola louti (Forsskål) and P. epinepheli (Yamaguti, 1938) Yamaguti, 1958 from several species differ in the shape and orientation of the primary chamber (not in continuity with the primary canal) and the elongate shape of the secondary chamber (Beverley-Burton & Suriano, 1981; Justine, 2005b; Kritsky & BeverleyBurton, 1986; Yamaguti, 1938, 1958). P. euitoe Justine, 2007 from E. maculatus (Bloch) and P. chauveti Sigura & Justine, 2008 from 123 152 Syst Parasitol (2008) 71:145–158 Table 3 Pseudorhabdosynochus morrhua n. sp., number of rodlets in each row of the squamodisc Ventral squamodisc (n = 14) Row 1 2 3 4 5 6 7 8 9 10 11 Mean 7 9 9 10 9 9 8 8 7 6 2 Range 3–8 6–10 8–11 9–11 7–11 6–11 7–10 7–9 5–9 4–6 1–5 Dorsal squamodisc (n = 11) Row 1 2 3 4 5 6 7 8 9 10 11 12 Mean 7 9 10 10 9 9 9 8 7 6 5 3 Range 6–8 8–10 8–11 8–11 8–10 8–10 7–10 7–10 5–10 5–8 4–6 2–4 E. cyanopodus (Richardson) differ in the presence of a sclerotised ring on the trumpet and the elongate shape of the secondary chamber (Justine, 2007a; Sigura & Justine, 2008). P. malabaricus Justine & Sigura, 2007 and P. maternus Justine & Sigura, 2007, both from E. malabaricus Bloch & Schneider, differ in that their secondary chamber is smaller than the primary chamber (Justine & Sigura, 2007). P. minutus Justine, 2007 from Cephalopholis sonnerati (Valenciennes) differs in its smaller secondary chamber and smaller body size (Justine, 2007b). Finally, P. morrhua has a general vaginal structure which is found in several other species, but can be distinguished from all these species by differences in the proportions of the vaginal parts. Pseudorhabdosynochus variabilis n. sp. Type-host: Epinephelus morrhua (Valenciennes). Type-locality: Outer slope of the barrier reef, off Nouméa, New Caledonia. Site: Between secondary gill lamellae. Type-material: Holotype, JNC2453B2, 23 January 2008, outer slope of the barrier reef off Nouméa, New Caledonia, 22°350 S, 166°300 E, depth c.300 m. Material examined: 48 specimens, including 31 ‘carmine’ (c), 8 ‘picrate’ (p), 7 ‘unflattened carmine’ (uc), 2 ‘berlese’ (b). Material deposited: Holotype (c) and 27 paratypes (22 c, 5 p), MNHN, JNC2450, JNC2453; vouchers (c, p, uc), MNHN, JNC2450, JNC2453, JNC2462; 1 paratype (c), BMNH 2008.3.17.2; 1 paratype (c), USNPC 100954; 1 paratype (c), SAMA AHC 29478; 1 paratype (c), HCIP M-463; 1 paratype (c), MPM 18870 (Slide Box A5122). Prevalence: 100% (3/3) 123 Intensity: See Table 1; maximum intensity c.30. Etymology: Latin for variable, with reference to the sclerotised vagina. Description (Figs. 3–5) Body length h 450, c 388 (300–500, n = 22), width h 175, c 206 (160–240, n = 19). Tegument smooth. Anterior region with 3 pairs of head organs and 2 pairs of eye-spots; distance between outer margins of anterior eye-spot pair h 30, c 31 (23–40, n = 20), of posterior eye-spot pair h 32, c 29 (20–37, n = 22). Haptor differentiated from rest of body, narrower than body, width h 125, c 127 (110–145, n = 20), provided with 2 similar squamodiscs, 2 pairs of lateral hamuli, 3 bars and 14 marginal hooklets. Squamodiscs round in shape, made up of rows of rodlets; central rows forming closed circles; rodlets progressively thinner from centre to periphery; rodlets adjacent in 5–6 central rows, separated in external (posterior) rows; last row with very thin rodlets; ventral and dorsal squamodiscs similar; ventral squamodisc length h 46, c 48 (44–52, n = 16), width h 49, c 48 (44–51, n = 17), with h 12, almost always 12 (n = 12) rows of rodlets and 3 closed ovals; total number of rodlets h 88, mean 88 (85–90, n = 7, individual variations in Table 4); dorsal squamodisc, length h 47, c 48 (40–54, n = 17), width h 48, c 45 (40–48, n = 17), with h 12, almost always 12 (n = 12) rows of rodlets and 2 closed ovals; total number of rodlets h 89, mean 89 (83–96, n = 10, individual variations in Table 4). Number of rodlets in each row of squamodiscs for all specimens computed in Table 5. Ventral hamulus with handle and distinct guard (handle often bent hampering measurement of outer length), outer length h -, c 33 (30–37, n = 12), p 36 153 A, 50 µm Syst Parasitol (2008) 71:145–158 B c t B-D, 50 µm f C vl Ch1 Tr Ca1 Ca2 pc sc ew sc el A ew Ch2 AS D Fig. 3 Pseudorhabdosynochus variabilis n. sp. from Epinephelus morrhua: (A) Ventral view, composite drawing, mainly from holotype; (B) Quadriloculate organ, holotype, ventral view; (c, cone; t, tube; f, filament; arrow, characteristic thickening of wall inside fourth chamber at base of cone); (C) Sclerotised vagina, holotype, ventral view; (D) Sclerotised vagina, paratype, dorsal view (Tr, trumpet; Ca1, primary canal; Ch1, primary chamber; Ca2, secondary canal; Ch2, secondary chamber; AS, accessory structure; measurements: vl, sclerotised vagina length; pcew, primary chamber external width; scew, secondary chamber external width; scel, secondary chamber external length). All carmine (33–37, n = 10), inner length h 28, c 31 (28–35, n = 16), p 34 (32–37, n = 10). Dorsal hamulus with indistinct guard, outer length h 33, c 33 ± 1.3 (29–35, n = 46), p 33 (33–34, n = 2), inner length h 20, c 21 ± 1.5 (18–24, n = 35), p 21 (19–23, n = 9). Dorsal (lateral) bars straight, with wide flattened 123 154 Syst Parasitol (2008) 71:145–158 Table 4 Pseudorhabdosynochus variabilis n. sp., rodlets in squamodiscs Specimen Number of rodlets in each row (from centre to periphery) Closed/ Total Rodlet total JNC2450a20 vs c4-c5-c8-10-10-10-9-8-8-7-5-4 3/12 88 JNC2453b1** vs c4-c7-c7-10-11-9-10-7-8-8-5-4 3/12 90 JNC2453b10 vs c6-c8-10-10-9-8-9-7-6-5-4-3 2/12 85 JNC2453b16 vs c5-c7-c7-10-9-9-9-9-8-7-5-3 3/12 88 JNC2453b2* vs c5-c7-c8-10-9-11-8-8-7-7-5-3 3/12 88 JNC2453b3 vs c4-c6-c7-9-11-10-10-8-7-7-6-4 3/12 89 JNC2453b5 vs c4-c5-c8-10-10-10-9-7-7-6-6-3 3/12 85 JNC2450a20 ds c5-c8-10-10-9-9-7-7-6-5-4-3 2/12 83 JNC2453b1** ds c5-c8-10-11-11-9-8-9-6-5-4-3 2/12 89 JNC2453b10 ds c4-c6-c8-10-9-8-10-10-10-7-6-4 3/12 92 JNC2453b13 ds c5-c8-10-10-10-9-9-8-7-6-5-4 2/12 91 JNC2453b14 ds c6-c8-12-11-9-10-10-8-7-6-5-4 2/12 96 JNC2453b16 ds c5-c8-11-11-12-11-9-7-6-5-5-3 2/12 93 JNC2453b17 ds c5-c7-10-9-10-9-9-8-7-5-5-3 2/12 87 JNC2453b2* JNC2453b3 ds ds c6-c8-10-11-11-9-7-6-6-7-5-3 c5-c7-10-10-9-9-8-8-7-5-4-1 2/12 2/12 89 83 JNC2453b5 ds c4-c5-8-10-10-10-9-8-7-6-6-2 2/12 85 vs ventral squamodisc, mean 88 (85–90, n = 7) ds dorsal squamodisc, mean 89 (83–96, n = 10) * Holotype; ** paratype drawn in Fig. 4A, B. c, closed circle rows medial extremity and cylindrical lateral extremity, length h 43, c 45 ± 1.6 (42–49, n = 49), p 51 (47–55, n = 10), maximum width h 15, c 15 ± 1.8 (12–20, n = 49), p 17 (13–19, n = 2). Ventral bar with characteristic shape including constricted central portion between 2 wider parts, continued by elongate extremities; length h 53, c 53 (48–60, n = 24), p 56 (55–59, n = 5), maximum width h 13, c 12 (8–15, n = 23), p 17 (13–19, n = 10); groove visible on its ventral side. Pharynx subspherical, length h 29, c 29 (20–38, n = 23), width h 30, c 28 (19–33, n = 23). Oesophagus apparently absent, such that intestinal bifurcation immediately follows pharynx. Caeca simple, terminate blindly at level of posterior margin of vitelline field. Testis subspherical, intercaecal, length h 31, c 33 (25–40, n = 14), width h 38, c 51 (33–75, n = 14). Vas deferens emerges from antero-sinistral part of testis, enlarges into small seminal vesicle; seminal vesicle in middle region of body, transforms into duct; duct forms bends then transforms into small bulb, 123 followed by duct; duct enlarges to form vesicle, then connects with quadriloculate organ. Prostatic reservoir conspicuous, elongate, with obliquely striated muscles, connects with quadriloculate organ; length h 45, c 46 (40–50, n = 9), width h 11, c 12 (10–15, n = 9). Quadriloculate organ with fourth (posterior) chamber more sclerotised than 3 anterior chambers; first chamber with very thin anterior wall; fourth chamber ends in very sclerotised cone; characteristic thickening of wall present inside fourth chamber at base of cone (Fig. 3B, arrow); cone prolonged by sclerotised tube; end of tube prolonged by thin unsclerotised filament of variable length. Inner length of quadriloculate organ h 49, c 51 (47–58, n = 21); cone length h 20, c 18 (17–22, n = 10); tube length h 20, c 21 (19–23, n = 23), p 20 (17–24, n = 4); tube diameter h 3, c 3 (2–3.5, n = 22), p 3 (n = 4); filament length h 40, c 26–50 (n = 22). Ovary subequatorial, intercaecal, pre-testicular, encircles right caecum. Ovary width h 45, c 54 (38–75, n = 14). Oviduct passes medially to form oötype, surrounded by Mehlis’ gland; oötype short, opens into uterus. Uterus dextral. Female genital pore 155 50 µm Syst Parasitol (2008) 71:145–158 A B C D E F Fig. 4 Pseudorhabdosynochus variabilis n. sp. from Epinephelus morrhua: (A, B) Ventral (A) and dorsal (B) squamodiscs, same paratype, ventral view; (C) Lateral (dorsal) bar; (D) Ventral bar; (E) Ventral hamulus; (F) Dorsal hamulus. A, B, carmine; C–F, picrate Table 5 Pseudorhabdosynochus variabilis n. sp., number of rodlets in each row of the squamodisc Ventral squamodisc (n = 7) Row 1 2 3 7 5 6 7 8 9 10 11 12 Mean 5 6 8 10 10 10 9 8 7 7 5 3 Range 4–6 5–8 7–10 9–10 9–11 8–11 8–10 7–9 6–8 5–7 4–6 3–4 4 5 6 7 8 9 10 11 12 Dorsal squamodisc (n = 10) Row 1 2 3 Mean 5 7 10 10 10 9 9 8 7 6 5 3 Range 4–6 5–8 8–12 9–11 9–12 8–11 7–10 6–10 6–10 5–7 4–6 1–4 ventral (Fig. 5H). Unsclerotised vagina often inconspicuous, elongate (Fig. 5F, S). Duct from sclerotised vagina to oötype inconspicuous. Vitelline fields extend posteriorly from posterior to pharyngeal level in 2 lateral bands, confluent in post-testicular region and terminate anterior to peduncle. Bilateral connections from vitelline fields to oötype inconspicuous. Egg unknown. Sclerotised vagina (nomenclature of parts according to Justine 2007a; see Fig. 3D) sinistral, a complex sclerotised structure; aspect changes dramatically according to specimen and orientation (Fig. 5). 123 156 Syst Parasitol (2008) 71:145–158 B A C D E F G H I 50 µm L J K N M Q O P R S Fig. 5 Pseudorhabdosynochus variabilis n. sp. from Epinephelus morrhua: Sclerotised vagina, variation according to specimen and orientation. (A–I) carmine, ventral view; (J–P) carmine, dorsal view; (Q) picrate, dorsal view; (R, S) picrate, ventral view. All paratypes. H, female pore drawn; F, S, unsclerotised vagina drawn. All vaginae drawn with anterior of specimen facing up 123 Syst Parasitol (2008) 71:145–158 Sclerotised vagina comprises anterior trumpet, followed by primary canal, primary chamber, secondary canal, secondary chamber and accessory structure; anterior trumpet often ornamented with inconspicuous transversal crests; wall of primary canal very thin thoughout most of its length, thicker in posterior region; primary canal always forms bend in its posterior region; longitudinal axis of posterior region of primary canal in continuity with longitudinal axis of primary chamber; primary chamber sclerotised, small, pear-shaped; secondary canal inserted laterally into anterior part of primary chamber, just posteriorly to chamber–primary canal junction; secondary canal thin, very long, often bent; longitudinal axis of secondary canal in continuity with longitudinal axis of secondary chamber; secondary chamber sclerotised, fusiform, ventral to primary chamber; accessory structure inserted into secondary chamber, very long, often bent. External and internal surface of both primary and secondary chambers smooth. [Because the anterior regions of the primary canal and the secondary canal are both flexible, the spatial arrangement of the individual parts of the sclerotised vagina and the total length vary greatly in different specimens.] Duct from sclerotised vagina to oötype connects laterally to secondary chamber (Fig. 5Q, S). Total length of sclerotised vagina h 41, c 45 (26–57, n = 23), p 46 (39–52, n = 5); external width of primary chamber h 5, c 5 (3.5–6, n = 23), p 5 (5–6, n = 5); external length of secondary chamber h 12, c 12 (7–17, n = 23), p 13 (11–15, n = 5); external width of secondary chamber h 5, c 5 (3.5–6, n = 23), c 6 (5–6, n = 5). Sclerotised trumpet oriented in various directions between anteriorly and antero-sinistrally; rest of sclerotised vagina with various orientations. Differential diagnosis With its succession of trumpet, primary canal, primary chamber, secondary canal, secondary chamber and accessory structure, the sclerotised vagina of P. variabilis n. sp. corresponds perfectly to the general pattern of vaginal organisation in Pseudorhabdosynochus (see Justine, 2007a); the uniqueness of this species is that the primary canal, secondary canal and accessory structure are very long and that the apparent flexibility of the connecting parts allows a variety of spatial arrangements of the chambers. 157 The secondary canal of the sclerotised vagina of Pseudorhabdosynochus spp. is often very short, or even non-existent, when the two chambers are united within a single sclerotised structure. P. fuitoe Justine, 2007 has a long secondary canal but the general structure (Justine, 2007a) is very different from P. variabilis. Only P. dolicocolpos Neifar & Euzet, 2007 has a sclerotised vagina with a similar structure to P. variabilis (see Neifar & Euzet, 2007). However, P. variabilis can be distinguished from P. dolicocolpos by a relatively longer secondary canal and a shorter sclerotised vagina (c.50 vs 75–120 lm). In addition, P. variabilis has squamodiscs with 2–3 circular rows (vs 1–2 V-shaped rows) and its tegument is smooth (vs scaly). P. dolicocolpos has an asymmetrical haptor, a character not seen in P. variabilis. Discussion With only three species of monogeneans, the parasitic fauna of E. morrhua is relatively poor when compared to that of other groupers from the inner lagoon of comparable size, such as E. maculatus which has 12 monogenean species (Journo & Justine, 2006; Justine, 2007a). Studies on other species of deep-sea groupers are needed to confirm this difference. In other groupers examined in New Caledonia, the monogenean fauna generally includes a dominant species, often accounting for more than 50% of the specimens, and a range of other less abundant species (Poulin & Justine, 2008). In contrast, the three species found in E. morrhua each account for a third of the number of monogeneans. This usual observation has yet to be explained. Other parasites (copepods and larval isopods on the gills; acanthocephalans, tetraphyllidean cestodes, nematodes and several digenean species in the digestive tract; and trypanorhynch cestode larvae in the general body cavity) have also been collected from the same specimens of E. morrhua. The undescribed specimens of Haliotrema sp. have been deposited in the MNHN collections (MNHN JNC2450, JNC2453, JNC2462). Acknowledgements Sophie Olivier, student, participated in the fishing operations and parasitological survey and in the preparation of slides. Andreas Wallberg (Uppsala University, Sweden) participated in one fishing cruise. Sam Tereua (IRD) provided safe navigation and excellent fishing skills on board 123 158 the R/V ‘Coris’. Identification of the hosts was kindly confirmed (from photographs) by John E. 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