Omanicotyle heterospina n. gen. et n. comb. (Monogenea: Microcotylidae) from the gills of Argyrops spinifer (Forsskål) (Teleostei: Sparidae) from the Sea of Oman

Background The Sultanate of Oman’s aquaculture industry is expanding with an on-going assessment of potential new fish species for culture. The king soldier bream, Argyrops spinifer (Forsskål) (Sparidae), is one such species that is under consideration. During a routine health assessment of specimens caught in the Sea of Oman throughout the period November 2009 to March 2011, a number of gill polyopisthocotylean monogeneans were recovered. Methods A subsequent study of the monogeneans using a range of morphology-based approaches indicated that these were Bivagina heterospina Mamaev et Parukhin, 1974. In the absence of pre-existing molecular data, an expanded description of this species is provided, including a differential diagnosis with other species and genera belonging to the subfamily Microcotylinae Monticelli, 1892 with the subsequent movement of this species to a new genus to accommodate it. Results The polyopisthocotyleans collected from the gills of A. spinifer appear to be unique within the family Microcotylidae Taschenberg, 1879 in that, morphologically, they possess a pair of large, muscular vaginae each armed with a full crown of 16–18 robust spines and a unique dorsal region of folded tegument, which permits their discrimination from species of Bivagina Yamaguti, 1963. Sequencing of the SSU rDNA (complete 1968 bp) and LSU rDNA (partial 949 bp) places the specimens collected during this study within the subfamily Microcotylinae, but the LSU rDNA sequence differs from Bivagina and also from other microcotylid genera. Morphological features of B. heterospina sensu Mamaev et Parukhin, 1974 and the specimens collected from the current study are consistent with one another and represent a single species. The vaginal armature of these worms is unique and differs from all other genera within the Microcotylinae, including Bivagina, and its movement to Omanicotyle n. gen. to accommodate this species is proposed. Conclusions A new genus, Omanicotyle n. gen., is erected to accommodate Omanicotyle [Bivagina] heterospina n. comb. which represents the first monogenean to be described from Omani marine waters. Given the pathogenic potential of microcotylids on captive held fish stocks, a full assessment of Omanicotyle heterospina n. gen. et n. comb. is now required before large-scale production commences.


Background
In addition to the~150,000 tonnes of commercially important species of wild marine fish landed through Omani ports [1], the Omani aquaculture industry, which currently exceeds 500 tonnes p.a. and primarily concerns the production of Dicentrarchus labrax (L.) and Oreochromis niloticus niloticus (L.) [1], is exploring the potential of a number of native species suitable for aquaculture. Of the 990+ fish species known from Omani waters [2], approximately 50 species are under consideration for use in aquaculture, which will include an assessment of various species of seabream, grouper and snapper. Although global aquaculture production is expected to rise to meet the shortfall in wild catches, there is a parallel requirement to identify potential threats to the health and welfare of wild fisheries and aquaculture stocks so that contingencies to mitigate against their establishment or to minimise their potential impacts can be taken. During a recent assessment of these new aquaculture species by the Ministry of Fisheries Wealth, a sample of king soldier bream, Argyrops spinifer (Forsskål), was found to be infected with a microcotylid polyopisthocotylean monogenean, whose morphology is consistent with Bivagina heterospina Mamaev et Parukhin, 1974, which was previously described from the same host from Mokura Bay, Kuria Muria Islands in the Arabian Sea [3]. The morphological features of B. heterospina, however, appear unique and differ from other species of the genus Bivagina Yamaguti, 1963 and an expanded description of the microcotylid is provided in the current study. A new genus to accommodate this species is proposed.

Collection of host and parasite material
Thirty specimens of Argyrops spinifer, with a standard length of 26-52 cm, were collected by line angling in the Oman Sea off the coastal city of Muscat and landed at Muttrah (23°37′ 08.65″ N; 58°35′ 33.76 E) throughout the period November 2009 to June 2010. After landing, the fish were placed on ice and then directly transferred to the Ministry of Fisheries Wealth where the gills were excised. A total of 18 adult monogeneans (mean intensity of infection 0.6 ± 1.0 parasites fish -1 ; range 0-3) parasitising the gills, were removed using mounted surgical needles. Fifteen of the specimens were transferred directly into 90% ethanol for subsequent evaluation by staining, scanning electron and confocal laser scanning microscopy. The remaining three specimens were fixed in 95% ethanol for molecular studies.

Morphological methods
Whole mounts of specimens (n = 7) were stained with Mayer's paracarmine to highlight features of the internal anatomy. Specimens were measured using an eyepiece graticule and all measurements are given in micrometres as the range followed by the mean in parentheses, unless otherwise stated. The terminology of structures follows that of Williams [4]; lengths refer to measurements taken along the longitudinal axis of the worm.
Additional ethanol fixed specimens (n = 5) were prepared for scanning electron microscopy by rehydrating down through a graded ethanol series to water and then by transferring them into 0.2 M sodium cacodylate buffer pH 8 for 24 h, post-fixed in 1% osmium tetroxide for 2 hours at room temperature and then dehydrated through a graded ethanol series. Specimens were then transferred to 50:50 100% ethanol: hexamethyldisilazane followed by 2 changes of 100% hexamethyldisilazane (45 min each), air dried overnight, mounted on 12.5 mm aluminium stubs (Agar Scientific Ltd., Stansted, UK) and then sputter-coated with gold using an Edwards Sputter Coater S150B. Specimens were viewed under a Jeol JSM 6460 LV SEM, at an accelerating voltage of 10 kV.
A further three alcohol fixed specimens were processed for confocal laser scanning microscopy, by rinsing them in distilled water for 24 h and then transferring them to either 1) 40 mM chromotrope 2R (C2R) (Alfa Aesar, Heysham, UK) + 3 mM phosphotungstic acid (Sigma-Aldrich®, Poole, UK) + 0.5% acetic acid (Sigma-Aldrich®) for 4 h at room temperature [5] to stain the attachment clamps and copulatory spines; or 2) 5 μl (0.2U μl -1 methanol) Alexa Fluor® 594 phalloidin (Invitrogen Molecular Probes, Eugene, Oregon, USA) in 100 μl distilled water in the dark, at room temperature for 5 h to stain the muscular components of the worm. After staining, the specimens were rinsed and mounted in distilled water and then examined on a Leica TCS SP2 AOBS laser scanning confocal microscope.
Illustrations were prepared from images captured using a Zeiss AxioCam MRc digital camera mounted on top of an Olympus BX51 compound microscope using a ×0.75 interfacing lens and ×10 to ×100 oil immersion objectives and MRGrab 1.0.0.4 (Carl Zeiss Vision GmbH, 2001) software.

Molecular methods
Three individual monogeneans, previously fixed in 95% ethanol, were digested overnight at 56°C in DNA buffer containing 100 μg ml -1 proteinase K. Total DNA was extracted using a GeneMATRIX kit (EURx Poland) following the tissue protocol and used for PCR reactions. The small subunit ribosomal DNA (SSU rDNA) was amplified using the primers 18e, 390f, 870f/r and 18gM [6][7][8][9]. The D1-D2 domains of the large subunit ribosomal DNA (LSU rDNA) were amplified using the primers C1 and D2 [10]. PCR bands of the correct size were visualised and recovered from the PCR products using a GeneMATRIX PCR products extraction kit (EURx Poland). PCR reactions were performed in triplicate (three separate worms) according to the original descriptions and sequencing reactions were performed using BigDyeTM Terminator Cycle Sequencing chemistry utilising the same oligonucleotide primers used for the original PCRs. DNA sequencing was performed in both directions for all PCR products and contiguous sequences obtained manually using CLUSTAL_X [11] and BioEdit [12]. CLUSTAL_X was used for the initial sequence alignments and regions of ambiguous sequence alignments were manually edited using the BioEdit sequence alignment editor [12]. Alignment files of related microcotylids, consisting of 947 characters of LSU rDNA sequence data, were used in the phylogenetic analyses. Phylogenetic analyses were performed using the maximum likelihood methodology in PhyML [13] with the general time-reversible (GTR) substitution model selected and 1000 bootstrap repeats, and Bayesian inference (BI) analyses using MrBayes v. 3.0 [14]. Models of nucleotide substitution were evaluated for the data using MrModeltest v. 2.2 [15]. The most parameterrich evolutionary model based on the Akaike Information Criterion (AIC) was the GTR + I + G (GTR + proportion Invariant + Gamma) model of evolution. Posterior probability distributions were generated using the Markov Chain Monte Carlo (MCMC) method with four chains being run simultaneously for 1,000,000 generations. Burn in was set at 2500 and trees were sampled every 100 generations making a total of 7500 trees used to compile the majority rule consensus trees.

Type material examined
The following museum type specimens were examined: 1 paratype (CNHE 203) and 8
Other localities: Sea of Oman landed at the port of Muttrah (23°37′ 08.65″ N; 58°35′ 33.76 E) (current study). General: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN) [16], details of this species have been submitted to ZooBank with the Life Science Identifier (LSID) zoobank.org:pub: CDEC0135-BA59-4754-B213-5C748A02C818. In addition, a species profile including taxonomic traits, host details and additional metadata are provided on http:// www.monodb.org [17].
Description  prepared for SEM (see Figure 4a). Anterior region containing the buccal organs, pharynx and genital atrium, delineated from the main body by a sharp narrowing of the worm (Figures 1a, 2a, and 3a). Paired muscular buccal organs lacking discernible spines, 137 (113-158) long, 79 (45-100) wide, with septum 36 (25-60) long ( Figure 3a); buccal cavity leading to a muscular circular pharynx 44 (40-48) long, 44 (35-53) wide. Oesophagus 124 (100-175) long. Intestinal caecae not equal in length, one terminates post-testicular, the other extends into the peduncle terminating just before the haptor. Short diverticula present in the anterior section posterior to the genital atrium, not evident throughout its entire length as obscured by vitellaria. Vitellaria irregular, not well defined, yellowish-brown in colouration, co-extensive with intestinal caecae, extending from the genital atrium to mid-way along the haptoral peduncle, each granule 40 (20-60) long, 21 (18)(19)(20)(21)(22)(23)(24)(25) wide. Pigment granules distributed throughout the body, a light scattering anterior to the vaginae, below this they concentrate into two dark, lateral bands that run the length of the body extending into and running the length of the haptor. Genital atrium unarmed, 77 (68-93) wide, 74 (63-88) long at its base; projects away from the body as a small cone. Two large, muscular vaginae 110 (90-130) wide, 120 (100-128) long, each armed with a crown of 16-18 robust spines, 24-28 long, which curve slightly towards the centre of the structure (Figures 1a,e and 2c,d). The muscles forming the wall of each vagina, 28 (25)(26)(27)(28)(29)(30)   length of a single primary filament, the anterior end of the worm pointing towards the distal tip, with their clamps attached to the second lamellae; the posterior part of the haptor curling round the filament. The function of the numerous C2R positive droplets along the lateral margins of the anterior haptor is unknown and can only be speculated upon. Given their high number and aggregation, it is unlikely that these represent the precursors for the synthesis of new clamps, i.e. they are not in close association with developing clamps. It could be suggested that these function as papillae increasing the surface area of this part of the haptor and make a contribution to the attachment of the worm. Unfortunately, this area was not visible on the specimens prepared for SEM; further specimens are therefore required to determine whether these structures are visible externally. According to Mamaev [18], parasites belonging to the subfamily Microcotylinae Monticelli, 1892 have either a single medio-lateral or rarely a dorso-laterally positioned vagina or occasionally two dorso-laterally situated vaginae. Given the large size of the vaginae, which occupy almost the entire width of the worm, it cannot be said that they are strictly dorso-lateral or medial.

Differential diagnosis
Omanicotyle n. gen. can be differentiated from the other 24 genera in the subfamily Microcotylinae on the basis of vaginal number (single or paired) and its relative armature, and also by armature of the genital atrium.  [18] as the type species, following the reallocation of the other species in the genus to other genera]; Pseudobivagina Mamaev, 1986;and Pseudoneobivagina Mamaev, 1986. The remaining genera, which also include Omanicotyle n. gen., all have armed vaginae, i.e. Bivagina Yamaguti, 1963 (certain species within the genus); Kahawaia Lebedev, 1969; and Neobivaginopsis Villalba, 1987. Omanicotyle n. gen. can be readily discriminated from the other genera. The vaginae in Bivagina are typically small but heavily muscularised structures armed with a crescent of short spines; the vaginal ducts connecting the two vaginae are typically clearly visible. Kahawaia possesses two cuticularised, pyriform pads armed with spines, interpreted as vaginae. The vaginae of Neobivaginopsis are large, muscular, contractile structures the openings of which have lightly sclerotised borders. The vaginae of Omanicotyle n. gen. by comparison, are large, muscular structures with a full corona of spines, which near abut against each other and occupy almost the entire width of the worm and as such obscure the vaginal ducts (which could not be seen in the specimens examined for the current study).
Omanicotyle n. gen. can also be discriminated from almost all the other genera on the relative armature of the genital atrium and/or that of the cirrus/penis when present. The following genera have both an armed genital atrium and an armed cirrus: Caballeraxine; Diplostamenides; Lutianicola; Neobivagina; Neobivaginopsis; Pseudobivagina; Pseudoneobivagina; Sciaenacotyle; and Sebasticotyle. Atriostella possesses an armed genital atrium but an unarmed cirrus, whilst species belonging to the genera Diplasiocotyle, Kahawaia, Jaliscia, Microcotyle, Paranaella, Polymicrocotyle and Solostamanides possess an armed genital atrium but no differentiated cirrus. The genera Gamacallum, Magniexcipula and Monomacracanthus have an unarmed genital atrium but an armed penis/cirrus, whilst species belonging to Paramicrocotyloides, Pauciconfibula and Pseudoaspinatrium have both an unarmed genital atrium and an unarmed cirrus. Only the genera Bivagina and Omanicotyle n. gen. have an unarmed genital atrium and no differentiated cirrus. The latter two genera, however, can be readily separated on the size and armature of their vaginae, as discussed above.

Molecular results
Both the SSU and LSU regions of the rDNA were successfully sequenced. A nucleotide BLAST search showed that the SSU rDNA of O. heterospina n. gen. et n. comb. was most similar to Microcotyle sebastis with a 99% identity, and the LSU rDNA was most similar to B. pagrosomi, both polyopisthocotylean monogeneans belong to the family Microcotylidae. The phylogenetic analyses produced similar tree topologies ( Figure 5), O. heterospina n. gen. et n. comb. consistently grouped with B. pagrosomi as a strongly supported sister group to the Microcotyle clade.

Discussion
There are 24 genera in the subfamily Microcotylinae [17], which, according to Mamaev [18] includes microcotylids that possess a symmetric or sub-symmetric, well-delineated haptor, adults that lack haptoral anchors, intestinal limbs with lateral branches/appendages without anastomoses, an armed or unarmed genital atrium, and, usually a single, medio-lateral, vagina, rarely laterodorsal, but occasionally two, dorso-laterally or ventrolaterally positioned, vaginae. The subfamily Microcotylinae was revised by Unnithan [20] [21] and Sciaenacotyle [22] were subsequently added. The new genus Omanicotyle gen. n. bears microcotylid-like clamps each with a supplementary lanceolate process (see [18]; Figures 1d, 3c,d, and 5a-f ), lack haptoral anchors in the adult, and have intestinal crura of unequal lengths, with short, anterior lateral branches (Figure 1a). The extent of intestinal branching, however, is unknown as much of its length is obscured by vitellaria. No branching was seen in the haptoral peduncle region. Omanicotyle gen n. is erected principally on the basis of the structure and armament of its vaginae, which are evident as two large muscular organs (av. 110 μm wide) occupying almost the entire width of each specimen (Figures 1a,e and 2c,d). Each distinct vagina is armed with a complete ring of 16-18 robust spines, confirmed by their positive reaction to the C2Rbased sclerite stain [5] (Figure 3d). While two vaginae are a feature for several genera across the subfamily, the complexity and the degree of variation observed in the armament of these requires supporting molecular studies to unambiguously place species within a genus. Unfortunately, the Microcotylidae are not well represented in the databases with SSU rDNA sequences, however, more data are available for the D1-D2 regions of the LSU rDNA. In addition, the LSU rDNA is known to allow better phylogenetic resolution among monogenean family groups than the SSU counterpart [10]. From Figure 6, it can be seen that Omanicotyle heterospina n. gen. et n. comb. is well supported as a member of the subfamily Microcotylinae and as a sister taxon to the Microcotyle, grouping with B. pagrosomi (percentage similarity 95.2%; identities = 876/920 (95%); gaps = 10/920 (1%)). Other parts of the phylogeny, however, are not well resolved; Diplostamenides sciaenae (Microcotylinae), for example, groups with Cynoscionicola "branquialis" (Anchoromicrocotylinae Bravo-Hollis, 1981), which is weakly associated with Atrispinum acarne (Atriasterinae Maillard et Noisy, 1979). Additional gene sequence data for more taxa are, therefore, required to fully resolve the phylogenetic relationships amongst the Microcotylidae. Unfortunately, molecular data are also lacking for the purportedly closely related sister taxa of Bivagina, i.e. Pseudobivagina and Neobivagina. All three genera, however, can be separated on the degree of the genital atrium armature. Dillon & Hargis [23] separated Bivagina, with an unarmed genital atrium, from Neobivagina, with an armed genital atrium. Later, Mamaev [18] separated out Pseudobivagina, possessing a muscular copulatory organ armed dorsally with a semi-crown of rib-like spines, covered with longer ribs arranged in a dome configuration on the walls of the genital atrium. centrodonti and B. pagrosomi type material, photographs of B. tai provided by Professor K. Ogawa, and drawings provided in the literature [23][24][25] suggest that the proportionately small vaginae are predominantly armed with unequal sized spines in their lateral corners whilst the vaginae of Omanicotyle heterospina n. gen. et n. comb. are large, occupying the entire width of the worm and are armed with a full crown of robust, equal-sized spines. One additional feature that may be unique to Omanicotyle n. gen., which may have taxonomic significance, is the consistent, circular region of folded tegument on the dorsal surface ( Figure 4a). Likewise, it is not known whether numerous C2R-positive (i.e. suggesting they are of the same proteins forming the hook material) regions distributed along the margins of the anterior portion of the haptor are unique and a definitive statement on these must wait until a detailed confocal microscopy-based study on representative species from each genus in the Microcotylinae can be conducted. The function of both these latter structures also requires confirmation but must await the collection of further specimens.

Conclusions
The polyopisthocotylean Omanicotyle heterospina n. gen. et n. comb. collected from the gills of the sparid A. spinifer is the first monogenean to be described from the Sea of Oman and is assigned to a new genus within the subfamily Microcotylinae (Microcotylidae), based on consistent morphological and molecular differences discriminating it from other genera in the subfamily.
Morphologically, Omanicotyle n. gen., which possesses two large, fully-armed vaginae, can be discriminated from the genus Bivagina (e.g. B. pagrosomi) on differences in the armament of the vaginae and a characteristic circular, dorsal region of folded tegument. Sequencing of the LSU rDNA (949 bp) revealed only a 95.2% percentage similarity with B. pagrosomi (identities = 876/920 (95%); gaps = 10/920 (1%)), lending support to the proposal that these specimens are placed as a new taxon within the Microcotylinae. A full assessment of the disease potential of Omanicotyle heterospina n. gen. et n. comb. and how it may impact on the production of A. spinifer in Omani waters is advised before production begins.