Pseudorhabdosynochus sulamericanus (Monogenea, Diplectanidae), a parasite of deep-sea groupers (Serranidae) occurs transatlantically on three congeneric hosts (Hyporthodus spp.), one from the Mediterranean Sea and two from the western Atlantic

Fish

Hyporthodus haifensis is a relatively rarely collected fish, and morphological differentiation from seemingly similar groupers is difficult (Craig, Sadovy de Mitcheson & Heemstra, 2012; Heemstra & Randall, 1993). Specimens of Hyporthodus haifensis were obtained from the fish markets of Sfax, Tunisia and Tripoli, Libya (Table 1). Field identifications of these specimens were made by the authors (AC & LN) with usual keys (Craig, Sadovy de Mitcheson & Heemstra, 2012; Heemstra & Randall, 1993; Louisy, 2015). One of our fish (Hh4; Table 1) was transported frozen from Tunisia to Paris where its identification was confirmed by thorough morphological analysis (Dr. B Séret, MNHN-IRD, pers. comm., 2015), and it was deposited as a voucher specimen in the ichthyological collection of the MNHN (registration number MNHN 2015-0242). For nine specimens of H. nigritus and eight specimens of H. niveatus (Table 2), tissue samples (fin clips) were collected from Gulf of Mexico commercial or recreational fisheries catches during routine fish population monitoring surveys conducted by the Florida Fish and Wildlife Conservation Commission (FWC); Ids in Table 2 correspond to collection numbers of FWC. Morphological identifications of Florida specimens were made by trained FWC fisheries biologists. Fish nomenclature follows FishBase (Froese & Pauly, 2016).

Molecular barcoding of fish

We used the QIAamp DNA Mini Kit (Qiagen), per the manufacturer’s instructions, to perform DNA extraction. The 5′ region of the cytochrome oxidase I (COI) mitochondrial gene was amplified with the primers FishF1 (5′-TCAACCAACCACAAAGACATTGGCAC-3′) and FishR1 (5′-TAGACTTCTGGGTGGCCAAAGAATCA-3′) (Ward et al., 2005). PCR reactions were performed in 20 µl, containing 1 ng of DNA, 1x CoralLoad PCR buffer, 3 mM MgCl₂, 66 µM of each dNTP, 0.15 µM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94 °C, followed by 40 cycles at 94 °C for 30 s, 48 °C for 40 s, and 72 °C for 50 s, with a final extension at 72 °C for 7 min. PCR products were purified (Ampure XP Kit; Beckman Coulter) and sequenced in both directions on a 3730xl DNA Analyzer 96-capillary sequencer (Applied Biosystems). We used CodonCode Aligner version 3.7.1 software (CodonCode Corporation, Dedham, MA, USA) to edit sequences, which were 670 bp in length, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers KT023566, KT023567, KT023568 and KU739501 –KU739517. Species identification was confirmed with the BOLD identification engine (Ratnasingham & Hebert, 2007).

Monogeneans

The host specimens of H. haifensis were not in a perfect state of freshness and the monogeneans were not alive when they were collected. We used seawater to rinse parasites from host gills into Petri dishes, and we further isolated them under a stereomicroscope with incident lighting to prepare them for additional microscopic evaluation. The majority of specimens were mounted in Berlese fluid (hereafter designated ‘b’), a technique which flattens the specimens. A few unflattened monogeneans were dehydrated in an ethanol series, stained with carmine, cleared with clove oil and mounted in Canada balsam (hereafter ‘uc’).

Most monogeneans collected from H. haifensis belonged to a single, abundant, species of Pseudorhabdosynochus (Table 1); the very few specimens from other species are noted but not otherwise considered here.

For illustration of parasites we used an Olympus BH2 microscope equipped with drawing apparatus and differential interference contrast (DIC) optics. The measurements of sclerotised parts, all in micrometres, were taken with the help of a custom-made transparent ruler and are expressed as the range followed in parentheses by the mean, the standard deviation when n ≥ 29, and (n) the number of observations; measurements were taken as in Fig. 1 in Chaabane, Neifar & Justine (2015). The measurements of the right-hand haptoral hard-parts and left-hand equivalents were pooled. Because measured lengths may vary as a function of how specimens are prepared and the degree to which they are flattened (Justine, 2005b), here they are given separately for specimens prepared, respectively, in Berlese (b) and carmine (uc). The terminology for different parts of the male quadriloculate organ and the vagina is that of Justine (2007a). We scanned drawings and used Adobe Illustrator software (version CS5) to refine lines and in some cases to add colour fill to better graphically differentiate structural elements. Museum abbreviations used are as follows: MNHN, Muséum National d’Histoire Naturelle, Paris; BMNH, Natural History Museum, London.

COI sequences of monogeneans

We used a QIAmp DNA Micro Kit (Qiagen) to extract DNA from a whole monogenean specimen (from fish Hh4; Table 1). The specific primers JB3 (=COI-ASmit1) (forward 5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4.5 (=COI-ASmit2) (reverse 5′-TAAAGAAAGAACATAATGAAAATG-3′) were used to amplify a fragment of 424 bp of the COI gene (Bowles, Blair & McManus, 1995; Littlewood, Rohde & Clough, 1997). PCR reaction was performed in 20 µl, containing 1 ng of DNA, 1x CoralLoad PCR buffer, 3 mM MgCl₂, 0.25 mM dNTP, 0.15 µM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). Thermocycles consisted of an initial denaturation step at 94 °C for 2 min, followed by 37 cycles of denaturation at 94 °C for 30 s, annealing at 48 °C for 40 s, and extension at 72 °C for 50 s. The final extension was conducted at 72 °C for 5 min. Sequences were edited with CodonCode Aligner software version 3.7.1 (CodonCode Corporation, Dedham, MA, USA), compared to the GenBank database content with BLAST, and deposited in GenBank under accession number KT023569.

Trees and distances

A tree was constructed from all available COI sequences of species of the genus Hyporthodus, including sequences already available in GenBank and our new sequences. The tree was inferred using Maximum Likelihood method. The best evolutionary model for the data set was estimated in MEGA7 (Kumar, Stecher & Tamura, in press) under the Bayesian Information Criterion (BIC) to be Hasegawa–Kishino–Yano model (Hasegawa, Kishino & Yano, 1985) with a discrete Gamma distribution (HKY + G). The tree was computed in MEGA7, with 100 bootstrap replications. A tree inferred from the same data, using the Neighbour-Joining method (Saitou & Nei, 1987) and evolutionary distances computed using the Kimura-2 parameter (Kimura, 1980) with 1,000 bootstrap replicates, was also constructed with MEGA7. Genetic distances (Kimura-2 parameter distance) were estimated with MEGA7. All codon positions were used.

Identification of fish hosts, Haifa grouper

When we began our study, no sequence of H. haifensis was available in GenBank, and identification of our first COI sequences via BOLD yielded confusing results, probably because of sequences in the database derived from misidentified specimens. We obtained COI sequences for one specimen from Tunisia (now deposited in the MNHN collections) and two additional specimens from Libya, and the three sequences were identical or very similar (1 bp difference); sequences were also identical or very similar (1 bp difference) to three sequences of H. haifensis (KJ709537, KJ709538 and KJ709539) recently added to GenBank (Landi et al., 2014), from off Sicily, i.e., geographically close to Tunisia and Western Libya. We conclude with certainty, from identical COI sequences and convergent morphological identification, that our fish specimens belong to the species H. haifensis.

Comparison of barcode sequences from Hyporthodus species

We obtained 8 and 9 COI new sequences from H. niveatus and H. nigritus, respectively. In both cases, these sequences were similar to or identical with sequences deposited under the same names in GenBank. A ML analysis (Fig. 1) produced distinct branches for the species H. octofasciatus, H. haifensis, H. acanthistius, H. niveatus, H. nigritus and H. flavolimbatus; H. ergastularius and H. septemfasciatus were not well resolved, but this might be due to misidentification of some sequences, as previously suggested (Schoelinck et al., 2014); a NJ bootstrap analysis produced the same tree topology (Fig. 1). With the exception of the two latter species, all species, and especially H. haifensis, H. nigritus and H. niveatus, were each in separate clades with high support (100%). Specimens of H. nigritus and of H. niveatus were grouped with specimens previously identified under the same names (a single sequence in the case of H. nigritus, 6 sequences in the case of H. niveatus). Hyporthodus haifensis was not closely related neither to H. niveatus (5.6–6% distance) nor to H. nigritus (6.8–7% distance), and the three species were not sister-species (Fig. 1); however, precise phylogenetic relationships between the three species could not be determined because of very low support of several nodes in the phylogenetic analysis.

Morphology of monogeneans: Pseudorhabdosynochus sulamericanus Santos, Buchmann & Gibson, 2000

• Taxonomic summary

Synonym: Pseudorhabdosynochus sp. of Chaabane, Neifar & Justine, 2015.

Type-host: Hyporthodus niveatus (Valenciennes, 1828).

Type-locality: Off Brazil (Santos, Buchmann & Gibson, 2000).

Other hosts: Hyporthodus nigritus (Holbrook, 1855) (Kritsky, Bakenhaster & Adams, 2015); Hyporthodus haifensis (Ben-Tuvia, 1953) (this paper).

Other localities: Off Florida (Kritsky, Bakenhaster & Adams, 2015); off Sfax, Tunisia, and Tripoli, Libya (this paper).

Infection site: Gill lamellae.

Prevalence: In our specimens from Tunisia and Libya, 4/4 (100 %), see Table 1.

Material examined: 343 voucher specimens from H. haifensis from off Tunisia and Libya (Table 1), MNHN HEL555; 2 paratypes from H. niveatus off Brazil (BMNH 1999.1.6.1-3); 2 voucher specimens from H. niveatus off Florida (MNHN HEL459, HEL460).

• Redescription (Figs. 2– 6)

Redescription (based on 36 specimens in Berlese and 18 unflattened specimens in carmine from H. haifensis from off Tunisia and Libya; for measurements of other specimens, see Table 3). Adult length uc 634 (500–800, n = 14), b 727 (350–980, n = 16) long, including haptor; maximum width uc 182 (100–270, n = 14), b 230 (115–310, n = 16) at level of ovary (Fig. 2A). Tegument scaly in posterior region (Fig. 6F). Anterior region with 3 pairs of head organs and 2 pairs of dorsal eye-spots, distance between outer margins of anterior eye-spots uc 26 (20–29, n = 7), b 30 (25–38, n = 5), of posterior eye-spots uc 31 (18–39, n = 12), b 34 (28–42, n = 7). Pharynx medial, subspherical. Oesophagus very short or absent. Two simple lateral intestinal caeca not united posteriorly. Haptoral peduncle present. Haptor trapezoidal, width uc 181 (150–200, n = 8), b 208 (180–240, n = 6). Dorsal squamodisc, length uc 84 (75–100, n = 10), b 99 (85–120, n = 13), width uc 75 (59–90, n = 10), b 104 (70–122, n = 13) (Fig. 6D). Ventral squamodisc, length uc 89 (73–150, n = 11), b 100 (78–120, n = 15), width uc 89 (73–150, n = 11), b 100 (78–120, n = 15) (Figs. 5F and 6C). Squamodiscs with 15–16 concentric rows of rodlets; 1 innermost row u-shaped. Rodlets with visible spurs (‘éperons’) (Figs. 6C and 6D). Ventral anchors with handle and distinct guard, outer length uc 44 (40–48, n = 6), b 49 ± 3.1 (40–54, n = 58), inner length uc 41 (30–47, n = 4), b 44 ± 3.1 (32–50, n = 54) (Figs. 5B and 6E). Dorsal anchors with indistinct guard, outer length uc 41 (35–45, n = 7), b 44 ± 2.6 (36–48, n = 51), inner length uc 29 (25–31, n = 3), b 29 ± 2.8 (24–36, n = 33) (Figs. 5C and 6E). Lateral dorsal bars, with flattened medial end, length uc 60 ± 2.3 (55–65, n = 29), b 82 ± 9.3 (60–115, n = 67), maximum width uc 22 ± 3.2 (15–28, n = 29), b 30 ± 4.4 (18–38, n = 67) (Figs. 5E and 6E). Ventral bar long, sometimes V-shaped, with constricted median portion, length uc 93 (82–120, n = 12), b 118 ± 11 (88–135, n = 29), maximum width, uc 17 (13–28, n = 12), b 20 ± 4. 2 (13–26, n = 30) (Figs. 5A, 5D and 6E); for V-shaped ventral bars, measurements were taken as in Fig. 5D.

Testis subspherical, posterior, intercaecal. Male copulatory organ quadriloculate, first (anterior) chamber as sclerotised as the three others; fourth chamber forming short cone, prolonged by thin sclerotised tube and filament (Figs. 2B, 2C, 6A and 6B). Inner length uc 49 (45–59, n = 17), b 71 ± 6.5 (56–82, n = 32). Cone length uc 5 (5–7, n = 17), b 5 ± 1.1 (4–10, n = 31). Tube length uc 13 (10–17, n = 16), b 14 ± 1.1 (12–17, n = 30); tube diameter uc 4 (3–4, n = 16), b 4 ± 0.6 (3–5, n = 31). Filament with extremity often bifid, length uc 3 (2.5–5, n = 15), b 4 ± 1.7 (0–7, n = 29).

Vitelline follicles lateral, coextensive with intestinal caeca and contiguous posteriorly to testis. Ovary on right side, looping dorsoventrally around right intestinal caecum. Eggs observed within genital ducts reniform, with thickest shell at proximal pole, polar filament absent, length b 120–128 (n = 2), width b 40–43 (n = 2).

Sclerotized vagina consists of slightly sclerotised funnel-shaped trumpet, followed by short primary canal with thick wall (Figs. 2D, 3 and 4). Primary canal surrounded by additional sclerotised material in its proximal part, which obscures internal relationships. Posterior end of primary canal directed to primary chamber, junction between two structures visible (in specimens from H. niveatus) or not (in specimens from H. haifensis). Primary chamber small, pear-shaped. Secondary canal (junction between primary chamber and secondary chamber) not seen. Secondary chamber spherical, heavily sclerotised. Accessory structure with internal canal, looping twice, inserted on secondary chamber. Total length of sclerotised vagina uc 28 (23–31, n = 4), b 35 ± 2.9 (30–42, n = 35). Diameter of secondary chamber uc 6 (6–7, n = 4), b 5 ± 0.5 (4–6, n = 36). In specimens from Hyporthodus niveatus, the structure is identical but the continuity from the primary canal to the primary chamber could be followed, in contrast with specimens from H. haifensis.

• Remarks on morphology

Most authors have emphasized the importance of the morphological structure of the sclerotised vagina for Pseudorhabdosynochus species identification (Chaabane, Neifar & Justine, 2015; Justine, 2005a; Justine, 2005b; Justine, 2007a; Justine, 2007b; Justine, 2008c; Justine, 2010; Justine, Dupoux & Cribb, 2009; Justine et al., 2010; Justine & Sigura, 2007; Knoff et al., 2015; Mendoza-Franco, Violante-González & Herrera, 2011; Neifar & Euzet, 2007), although the quadriloculate organ and the hard parts of the host attachment apparatus (haptor) including the squamodisc are additional characters for species diagnosis.

Several Pseudorhabdosynochus species have in common with P. sulamericanus the following vaginal characters: a wide and visible trumpet; diameter of secondary chamber clearly larger than that of primary chamber. These species are: P. dolicocolpos Neifar & Euzet, 2007, P. enitsuji Neifar & Euzet, 2007, P. morrhua Justine, 2008, and P. firmicoleatus Kritsky, Bakenhaster & Adams, 2015.

• P. dolicocolpos (from Mycteroperca costae off Tunisia and Senegal) has a long, coiled thin-walled primary canal (vs short, straight and sclerotised in P. sulamericanus); although the structure is similar, the general shape of the sclerotised vagina is very different. In addition, its male copulatory organ has a long tube (35–45 vs 10–17) (Neifar & Euzet, 2007).

• P. enitsuji (from M. costae off Tunisia and Senegal) has a less conspicuous trumpet and a well-visible primary canal. In addition, its male copulatory organ has a long tube (55–70 vs 10–17) (Neifar & Euzet, 2007).

• P. morrhua (from M. morrhua off New Caledonia) has a less conspicuous trumpet and a thin-walled primary canal (vs sclerotised). In addition, the anterior chamber of its male copulatory organ has a very thin wall (vs as sclerotised as other chambers in P. sulamericanus) (Justine, 2008c).

• P. firmicoleatus (from H. flavolimbatus, type-host, and H. niveatus, both off Florida) was considered as closely resembling P. sulamericanus (Kritsky, Bakenhaster & Adams, 2015). However, Kritsky, Bakenhaster & Adams (2015) enumerated several morphological differences between the two species: absence of additional structure around the sclerotised vagina in P. firmicoleatus (vs present in P. sulamericanus), anchor morphology, tegumental scales (lacking in P. firmicoleatus) and number of rows of rodlets in the squamodisc (12 (11–13) in P. firmicoleatus vs 15 (14–17) in P. sulamericanus).

In none of these three species is there additional sclerotised material around the primary canal of the sclerotised vagina, as in P. sulamericanus. In P. sulamericanus, the male quadriloculate organ has the usual structure found in species of Pseudorhabdosynochus, but a minor difference can be detected at its distal extremity, i.e., a thin and short filament with bifid extremity. However, this detail itself could not be considered alone as a differential character for the species because it is variable; it was not mentioned in the original description or redescription (Kritsky, Bakenhaster & Adams, 2015; Santos, Buchmann & Gibson, 2000).

Pseudorhabdosynochus sulamericanus has an exceptional vaginal structure. In most Pseudorhabdosynochus species, there is a general pattern in which the continuity of the lumen can be followed from trumpet to secondary chamber through primary canal, primary chamber and secondary chamber (Justine, 2007a). This continuity is likely to correspond to the complex journey of inseminated sperm through the female organ, from the entrance (trumpet) to the secondary chamber which exits into a soft tube connected to the oötype (Justine, 2009). In specimens of P. sulamericanus from H. haifensis, we could discern neither the continuity between the primary canal and the primary chamber, nor the continuity from the primary chamber to the secondary chamber through the secondary canal. This is probably due to the presence of the additional sclerotised material which obscures vision. However, in specimens from H. niveatus, the primary canal—primary chamber continuity could be seen, but we evaluated far fewer of these specimens and so cannot be sure about the range of morphological variability in this structure. The additional sclerotised material is visible in the drawings of the original description and probably mentioned as “enclosed in muscular, funnel-shaped organ” (Santos, Buchmann & Gibson, 2000); it is mentioned in its redescription as “surrounded by variable small sclerites” (Kritsky, Bakenhaster & Adams, 2015); none of these authors used a DIC microscope which provides a better resolution of the hollow sclerotised organs.

The only other species of Pseudorhabdosynochus found on species of Hyporthodus are P. querni (Yamaguti, 1968) Kritsky & Beverley-Burton, 1986 from H. quernus off Hawaii, and P. firmicoleatus from H. flavolimbatus and H. niveatus, both off Florida. Pseudorhabdosynochus querni has a vaginal structure very different from that of P. sulamericanus (Yamaguti, 1968; Yang, Gibson & Zeng, 2005); P. firmicoleatus has a somewhat similar vaginal structure (Kritsky, Bakenhaster & Adams, 2015) but can also be distinguished by other characteristics (see above).

COI sequences of monogeneans

We obtained COI sequences of P. sulamericanus from H. haifensis from off Tunisia and Libya. The closest sequence in GenBank according to BLAST was from P. cyanopodus Sigura & Justine, 2008 (Schoelinck, Cruaud & Justine, 2012), a parasite from Epinephelus spp. in the South Pacific. The sequences differed by 17.6% (Kimura-2 parameter distance). Since no sequence of P. sulamericanus from the Americas was available, no further comparison was possible.