Hamacreadium cribbi n. sp. (Digenea: Opecoelidae) from Lethrinusminiatus (Forster) (Perciformes: Lethrinidae) from New Caledonian waters

A new species of Hamacreadium Linton, 1910, H. cribbi n. sp. is described from Lethrinus miniatus (Forster) from the waters off New Caledonia. It is compared with the other species of Hamacreadium reported from lethrinids and is characterised by the size of its eggs which tend to be larger [72–93 (84) vs 54–81 (56) µm long] than those of other species. Other characteristics, such as body size and shape and internal ratios, differentiate H. cribbi from other species; these differences are discussed in detail.


Introduction
Hamacreadium Linton, 1910 is one of a group of plagioporine genera which are poorly defined morphologically and is 'characterized by a combination of rather generalized plagioporine characters. Pre-eminent among those are the excretory vesicle extending into the forebody, deeply lobed ovary, distinctly submedian genital pore, and the vitelline follicles entering the forebody and extending beyond the testes to the posterior extremity' (Cribb, 2005). The opecoelids are a group in need of a thorough molecular examination, and as a start we have sequenced a variety of worms, and have found that the Hamacreadium-like species found in Lethrinus miniatus (Forster) clusters with, but is distinct from worms identified as H. mutabile Linton, 1910(see Justine et al., 2012bAndres et al., 2014;Bray et al., 2016). To facilitate the discussion of this species in our phylogenetic studies we here describe and name this form.

Materials and methods
Fish were collected along the external slope of the reef, off Nouméa, New Caledonia. Digeneans were collected live, immediately fixed in nearly boiling saline (Cribb & Bray, 2010;Justine et al., 2012a) and then transferred to 80% ethanol. Whole-mounts were stained with Mayer's paracarmine, dehydrated in an ethanol series, cleared in beechwood creosote and mounted in Canada balsam. Measurements were made through a drawing tube on an Olympus BH-2 microscope, using a Digicad Plus digitising tablet and Carl Zeiss KS100 software adapted by Imaging Associates, and are quoted in micrometres. The following abbreviations are used: BMNH, British Museum (Natural History) Collection at the Natural History Museum, London, UK; MNHN JNC, Muséum National d'Histoire Naturelle, Paris, France.
Testes 2, oval to rounded, smoothly irregular, contiguous or slightly separated, in mid-hindbody, oblique. Cirrus-sac large, claviform, sigmoid, much wider proximally, reaches dorsally to ventral sucker, occasionally very slightly into hindbody, may be restricted to forebody in flattened specimens. Seminal vesicle long, coiled in posterior part of cirrus-sac. Pars prostatica and ejaculatory duct not clearly differentiated, long, coiled, complex coiling distally. Genital atrium distinct. Genital pore sinistral, ventral to left caeca or close, about halfway between bifurcation and ventral sucker.
Ovary usually lobate (3-5 lobes; see Fig. 2), oblique to and overlapping anterior testis. Seminal receptacle oval, dorsal to ovary. Mehlis' gland preovarian. Laurer's canal opens dorsally to Mehlis' gland. Uterus pre-ovarian, mainly intercaecal. Eggs tanned, operculate. Metraterm thick-walled, reaches level of ventral sucker. Vitellarium follicular, fields reach from bifurcal level of ventral sucker, to close to  posterior extremity, lateral to caeca and encroaching slightly over dorsal and ventral surface of caeca, almost confluent in bifurcal and post-testicular regions, usually continuous at level of ventral sucker, occasionally interrupted on one or both sides. Excretory pore terminal. Vesicle I-shaped, reaches to about level of genital pore.

Discussion
This species was reported from this host species by (Justine et al., 2010b) under the name Neolebouria sp.
A. These authors also reported the following species as hosts: the Spotcheek Emperor Lethrinus rubrioperculatus Sato; the Pacific Yellowtail Emperor Lethrinus atkinsoni Seale, the Drab Emperor Lethrinus ravus Carpenter & Randall, and the Slender Emperor, Lethrinus variegatus Valenciennes. This description is based solely on the worms from L. miniatus, and it is to be noticed that worms from the other hosts may differ, possibly at the specific level. The eggs are consistently larger in the specimens from L. miniatus than in any of the other hosts, or indeed any of the other similar species discussed below (Fig. 3). The worms from Lethrinus atkinsoni are consistently broader (Fig. 4), and the relative distance between the ventral sucker and the ovary is greater (Fig. 5). Several species of Hamacreadium have been reported from Lethrinus spp., including two reports from L. miniatus. Unfortunately, neither is accompanied by descriptions. Durio & Manter (1968) reported the type-species of Hamacreadium, H. mutabile, from L. miniatus from New Caledonia, but molecular evidence and other evidence (see below) suggests that this species occurs only in lutjanid fishes. Shen (1985) reported H. lethrini Yamaguti, 1934 from L. miniatus from off the Xisha Islands (or Paracel Islands) in the South China Sea. These species will be discussed below, along with the other Hamacreadium spp. from Lethrinus spp.
1. Hamacreadium balistesi Nagaty & Abdel Aal, 1962 was originally reported from the Spangled Emperor Lethrinus nebulosus (Forsskål) and the White-banded Triggerfish Rhinecanthus aculeatus (Linnaeus) (Balistidae) from the Red Sea (Nagaty & Abdel Aal, 1962b) and was considered as synonym of H. mutabile by Bray & Cribb (1989). As is discussed below, H. mutabile is probably a specific parasite of lutjanids. The cirrus-sac in H. balistesi is said to be 'preacetabular'. Nagaty & Abdel Aal (1962b) mention that the vitelline fields 'may be interrupted at acetabular level' and differentiate it from H. interruptus by, inter alia, 'vitelline follicles constantly arranged or may be interrupted at acetabular level instead of their constant interrupted arrangement', but see the Fischthal & Kuntz (1965) description of H. interruptus Nagaty, 1941 (discussed below). Ramadan (1983) considered H. balistesi a synonym of H. interruptus. The ventral sucker to ovary distance is relatively distinctly greater than in H. cribbi n. sp. (Fig. 5) and the eggs are smaller (Fig. 3). The excretory system is neither described nor illustrated.
3. Hamacreadium egyptia el Abdou, Heckmann, Beltagy & Ashour, 2001 was described from Lethrinus nebulosus and the Sky Emperor Lethrinus mahsena (Forsskål) in the Red Sea (el Abdou et al., 2001). Although reported from two hosts, measurements of only one specimen are given and these measurements vary between the description and the table. The illustrations and the tiny gonads clearly show that the worm is immature and we consider it unrecognisable.
5. Hamacreadium interruptus Nagaty, 1941 was originally reported from the Pink Ear Emperor Lethrinus lentjan (Lacépède) (as Lethrinus mahsenoides Valenciennes) from the Red Sea (Nagaty, 1941). Fischthal & Kuntz (1965)   They considered Plagioporus longivesicula Yamaguti, 1952 and Hamacreadium lethrini Nagaty & Abdel Aal, 1962 as synonyms. Hafeezullah & Dutta (1980) described H. interruptus from an unidentified marine fish off Chiria Tapu, Andaman Islands. The lack of an identified host reduces the value of the description and it is not used in comparison here. Tadros et al. (1978) reported this species from the type-host also in the Red Sea. Nagaty (1941) described and illustrated a large lateral gap in the vitelline fields at the level of the ventral sucker. Fischthal & Kuntz (1965) stated that the vitelline fields are 'interrupted at acetabular level on both sides in 12, on left side only in 4, on right side only in 1, and uninterrupted on both sides in 3'. The eggs are smaller than those in H. cribbi n. sp. (Fig. 3). The excretory system is neither described nor illustrated. 6. Hamacreadium khalili Ramadan, 1983 was originally reported from L. mahsena and L. nebulosus from the Red Sea (Ramadan, 1983). El-Labadi et al. (2006) reported the species from the Lavender Jobfish Pristipomoides sieboldii (Bleeker) (Lutjanidae) and the Yellow-edged Lyretail Variola louti (Forsskål) (Serranidae), from the Gulf of Aqaba in the Red Sea, without descriptive matter. The species is differentiated from H. interruptus in a key by 'Testes globular and lobulated, cirrus pouch triangular'. The vitelline follicles are 'aggregated in two sets, a posterior and an anterior, with a gap between them'. The eggs are smaller than those in H. cribbi n. sp. (Fig. 3). The anterior extent of the excretory vesicle was not traced.
10. Hamacreadium lethrini Yamaguti, 1934, was originally reported from Lethrinus haematopterus, from the Pacific coast of Wakayama Prefecture, Japan (Yamaguti, 1934). Fischthal & Kuntz (1964) redescribed, but did not illustrate, the species from the Longfin Emperor Lethrinus erythropterus Valenciennes (as Lethrinus hypselopterus Bleeker) and the Humpback Red Snapper Lutjanus gibbus (Forsskål) (Lutjanidae) from Puerto Princesa, Palawan Island. Philippines. The species has been reported two further times, but with no descriptive matter. Dyer et al. (1988) reported as host the Thumbprint Emperor Lethrinus harak (Forsskål) from Okinawa, Japan and Shen (1985) reported Lethrinus miniatus and the Grey Large-eye Bream Gymnocranius griseus (Temminck & Schlegel) (Lethrinidae) as hosts off Xisha Islands (or Paracel Islands) in the South China Sea. The original description is very similar to H. cribbi n. sp., but the testes are described and illustrated as irregularly indented, not the usual condition in H. cribbi. Fischthal & Kuntz (1964) described the post-testicular distance as 0-585 lm, indicating that at least one of their specimens was damaged. The eggs are generally smaller than in H. cribbi (Fig. 3). The excretory system reaches to about the same level as in H. cribbi. 11. Hamacreadium mehsena Nagaty, 1941, reported from Lethrinus 'mehsena' (presumably Lethrinus mahsena) from the Red Sea (Nagaty, 1941) differs from Hamacreadium in that the vitellarium does not reach into the forebody. Pritchard (1966) placed the species in her new genus Apopodocotyle Pritchard, 1966, but Cribb (2005 replaced in its original genus. It appears closest to the genus Podocotyle Dujardin, 1845, but while the testes are described as oblique, they appear almost symmetrical in the illustration. This form cannot be confused with H. cribbi. The excretory system is neither described nor illustrated. 12. Hamacreadium mutabile Linton, 1910, was originally described from the Grey Snapper Lutjanus griseus (Linnaeus) (considered the type-host), the Schoolmaster Snapper Lutjanus apodus (Walbaum), the Yellowtail Snapper Ocyurus chrysurus (Bloch) (all Lutjanidae), the Porkfish Anisotremus virginicus (Linnaeus) (Haemulidae) and the Gray Angelfish Pomacanthus arcuatus (Linnaeus) (Pomacanthidae) from the Dry Tortugas, Florida, USA, in the Gulf of Mexico (Linton, 1910). Since then it has been reported from an unfeasible number of host-species and localities, including several lethrinids. Bray & Cribb (1989) presented a long list of putative new synonyms, including Hamacreadium balistesi, Hamacreadium interruptum, Hamacreadium lenthrium, Hamacreadium lethrini Yamaguti, 1934nec Nagaty & Abdel Aal, 1962, Hamacreadium lethrini Nagaty & Abdel Aal, 1962nec Yamaguti, 1934, Hamacreadium nagatyi, Hamacreadium nebulosae and Plagioporus longivesicula. The advent of molecular techniques has cast doubt on the likelihood of one species having such a wide host and locality range. Miller et al. (2011) having examined the host-specificity of fish digeneans on the Great Barrier Reef, concluded 'that no euryxenous host distribution should be accepted on the basis of morphology only'. It is worth noting that McCoy (1929McCoy ( , 1930 was able to experimentally infect only lutjanids with H. mutabile despite the 'numerous other species tested'. Recently, Andres et al. (2014) have registered rDNA sequence data on H. mutabile from L. griseus, from the northern Gulf of Mexico, which differs distinctly from that of H. cribbi (Fig. 6). Hamacreadium mutabile has been reported in lutjanids in New Caledonian waters (Justine et al., 2012b).
13. Hamacreadium nebulosae Nagaty & Abdel Aal, 1962 is known only from three specimens found in L. nebulosus in the Red Sea (Nagaty & Abdel Aal, 1962b). According to the measurements given the sucker-width ratio is about 1:1, whereas in the illustration this ratio is about 1:1.8. The excretory vesicle is said to reach to the mid-level of the ventral sucker. The eggs are distinctly smaller than those of H. cribbi (Fig. 3).

Molecular phylogeny
The description of this species was considered desirable as it is included in a wider study of opecoelid phylogeny and systematics based on LSU and SSU rDNA sequences. A small section of the resultant tree is included here as Figure 6 and details of the techniques are as in Bray et al. (2016). Hamacreadium mutabile is a widely reported parasite mainly of lutjanid fishes, and our worms from a lutjanid from New Caledonia, labelled as Hamacreadium 'mutabile', were identified as this species by Justine et al. (2012b). Morphologically this New Caledonian form appears practically identical to the Gulf of Mexico form, but the molecular evidence indicates that it is one of a group as closely related, but distinct, worms (Fig. 6). The Gulf of Mexico H. mutabile material (based only on a LSU rDNA sequence) is from the type-host in the eastern Gulf of Mexico, close to the type-locality of Dry Tortugas, Florida (Linton, 1910;Andres et al., 2014). Hamacreadium cribbi clusters weakly with H. mutabile, it is probably more realistic to consider the Hamacreadium species to be related by a polytomy.