Taxonomy and morphology of four “ ophrys-related ” scuticociliates ( Protista , Ciliophora , Scuticociliatia ) , with the description of a new genus , Paramesanophrys gen . nov

Generally, “ophrys-related” scuticociliates belong to a specialised group of ciliated protozoa that may act as commensals or pathogens of fishes and crustaceans. In the present study, four “ophrystaxa” scuticociliates, i.e., Paramesanophrys typica gen. et sp. nov., Mesanophrys carcini (Grolière & Léglise, 1977) Small & Lynn in Aescht, 2001, Metanophrys sinensis Song & Wilbert, 2000, and Metanophrys similis Song et al., 2002, were collected from Chinese coastal waters or mariculture ponds and investigated. Paramesanophrys gen. nov. is assigned to the family Orchitophryidae and differs from its other genera mainly by the position of the paroral membrane relative to membranelle 1–3, i.e., the membrane extends anteriorly to the posterior end of membranelle 3. The type species P. typica gen. et sp. nov., is defined by an elongated body with the posterior end depressed where the caudal cilium is located; 20 or 21 somatic kineties; double-rowed membranelle 1 with eight to ten basal bodies in each kinety; irregularly multi-rowed membranelle 2 and membranelle 3; scutica comprising c. seven or eight kinetosome pairs; a single macronuclear nodule; and marine habitat. The redescription of the three previously known species can be summarized as follows: 1) improved diagnosis is provided for Metanophrys sinensis Song & Wilbert, 2000 based on the original description and the present study; 2) some population-dependent characteristics of our new Mesanophrys carcini isolate are presented; 3) Metanophrys similis, collected from the South China Sea, resembles the original Qingdao population. European Journal of Taxonomy 191: 1–18 (2016) 2


Introduction
Ciliates in the subclass Scuticociliatia, commonly found in ecosystems worldwide, exhibit a great biological diversity and play important roles in marine ecosystems (Thompson & Kaneshiro 1968;Foissner & Wilbert 1981;Cawthorn et al. 1996;Lynn & Strüder-Kypke 2005;Fan et al. 2011aFan et al. , b, 2014;;Pan et al. 2013a, b;Castro et al. 2014;Foissner et al. 2014;Zhan et al. 2014).Many of them are common pathogens of fishes and invertebrates and can cause severe disease or even death in economically important aquaculture animals (Pérez-Uz & Song 1995;Song & Wilbert 2002;Fan et al. 2009Fan et al. , 2010;;Mallo et al. 2014;Ofelio et al. 2014).However, due to their small body size and a high degree of similarity in ciliature, the taxonomy of this group of organisms remains difficult and confusing (Thompson 1964;Agatha et al. 1993;Song 2000;Song & Wilbert 2000;Pan et al. 2010).Recent investigations in Chinese seas have shown a high diversity of scuticociliates, and the discovery of new taxa has highlighted the necessity to conduct further studies on this group (Wang et al. 2008a(Wang et al. , b, 2009;;Gao et al. 2010Gao et al. , 2012aGao et al. , b, 2013;;Pan et al. 2011Pan et al. , 2015a, b), b).
In the present study, a new genus, Paramesanophrys gen.nov., is established and detailed morphological information is provided for four scuticociliates, including one new species, Paramesanophrys typica gen.et sp.nov., and three nominal "ophrys" species.
Cells were observed in vivo using an oil immersion objective with brightfield and Nomarski differential interference contrast optics.Mixtures of a saturated mercury dichloride solution and Bouin's fluid were used to fix samples.The protargol silver staining method (Wilbert & Song 2008;Pan et al. 2013a) was applied to reveal the infraciliature.Measurements were performed at magnifications of 100-1250×.Drawings were produced with the help of a camera lucida.Systematics and terminology are mainly used in accordance with Lynn (2008) and Small & Lynn (1985).

Diagnosis
Orchitophryidae with cytostome above mid-body; buccal apparatus consisting of three Parauronemalike membranelles; PM with zigzag structure, extending anteriorly to posterior end of M3; M1 composed of two rows of kinetids; scutica comprising basal body pairs arranged in a line parallel to somatic kineties; single caudal cilium.

Etymology
The generic epithet, Paramesanophrys, refers to the similarity of the oral apparatus to that of the genus Mesanophrys.

Etymology
The epithet of this new species, typica (Greek, the type/typical, gender feminine), refers to the fact that it is the type of the new genus, Paramesanophrys gen.nov.

Type slides
A protargol slide with the holotype specimen encircled in black ink is deposited in the Laboratory of Protozoology, Ocean University of China (PXM-2011042101).A paratype slide is deposited in the Natural History Museum, London, UK (2016.3.10.1).
Movement by continuous swimming in water without pause or gliding slowly on substrate.
Ten or 11 somatic kineties, consisting of dikinetids in anterior two-thirds and monokinetid in posterior third of body (Fig. 5G, arrow).M1 slightly separated from apex, composed of two rows of kinetids with 7-9 basal bodies each (Fig. 5E-F).M2 composed of five or six longitudinal rows, each containing about 6-8 basal bodies (Fig. 5E-F).M3 located close to M2, much shorter than M2 and composed of three short, irregularly arranged rows of kinetosomes (Fig. 5E-F).PM extending anteriorly to posterior end of M2.Scutica Y-shaped, with c. four pairs of kinetosomes (Fig. 5E-F).

Ecological features
Salinity 32‰, pH 7.9 and water temperature about 11 °C.Song & Wilbert, 2000 Figs 4E, 6; Table 2 This species was described by Song & Wilbert (2000) in detail based on their Qingdao population.In the current work, it is reported for the first time from South China Sea.An improved diagnosis is provided herein based on all these data; the improved parts are highlighted in bold.

Description of Zhanjiang population
Body 25-30 × 10-15 µm in vivo, usually elongate-oval in outline, with anterior end distinctly pointed and posterior rounded (Fig. 6A-B).Body asymmetrical in outline when viewed ventrally, with anterior end slightly curved sideways (Fig. 6A-B).Ventral side almost straight, while dorsal side convex.Buccal field 2/ 5 to ½ of body length, with cytostome located anterior to equatorial plane of body (Fig. 6C).Cilia densely packed, about 7-8 µm long.Caudal cilium about 15 µm in length (Fig. 6B).Pellicle thin and slightly notched, with extrusomes about 2-3 µm long and dense beneath cortex (Fig. 6D).Endoplasm colourless to greyish, containing several food vacuoles and bar-or dumbbell-like crystals, which are usually 3 µm long and located in anterior and posterior regions of body (Fig. 6A-B, G).One large round to oval macronucleus approximately centrally located, with many small, irregularly shaped nucleoli on surface.Contractile vacuole about 5 µm in diameter and caudally positioned near ventral side (Fig. 6B).Movement with no special features, including swimming moderately fast, sometimes continuously swimming in water without pause.
Ten somatic kineties arranged longitudinally, and dikinetids about ¾ of length of each in anterior part (Fig. 6J).Buccal apparatus consists of three Parauronema-like membranelles (Fig. 6E-F).M1 slightly below apex and composed of two rows of kinetids with 7-10 basal bodies each, and longer than M2.M2 two-rowed, containing about five basal bodies in each row.M3 located close to M2, normally with three short, obliquely arranged rows of basal bodies.Scutica Y-shaped, with several pairs of kinetosomes (Fig. 6I).Silverline system in quadrangular mesh-pattern (Fig. 6H).Contractile vacuole pore located near posterior end of kinety 2.

Ecological features
Salinity 21‰, pH 7.3 and water temperature 26 °C.Song et al., 2002 Fig. 7; Table 2 This species is reported for the first time from the South China Sea.The morphological and behavioural characteristics of the Zhanjiang population closely resemble those of the Qingdao population; therefore, only the morphometric characterisation and important features are supplied.
Twelve somatic kineties with dikinetids arranged in approximately anterior half of each row and monokinetids positioned posteriorly (Fig. 7H).M1 positioned near apex and comprised of three longitudinal rows of kinetids with six basal bodies each (Fig. 7G, I).M2 three-rowed, as long as M1 and also composed of about six basal bodies in each longitudinal row (Fig. 7G, I).M3 located close to M2 and normally comprised of three short, parallel arranged rows of basal bodies (Fig. 7G, I).PM extends to about anterior third of body (Fig. 7G).Scutica, with about 5-7 basal bodies, arranged in long line.

Discussion
About Paramesanophrys gen.nov.and P. typica gen.et sp.nov.
The family Orchitophryidae is characterised as follows: small-to medium-sized body; ovoid-shaped; caudal cilium often present; oral region in anterior ⅓ to ½ of body; scutica aligned along midventral postoral region; bacterivorous and histophagous; marine habitats, always as facultative parasites of crustaceans, asteroids, fish and free-swimming (Lynn 2008).Paramesanophrys gen.nov.should be assigned to Orchitophyridae based on its morphological characters and habitat.
Besides having a unique Paramesanophrys-type PM, Paramesanophrys typica gen et sp.nov.also has the scutica comprising c. seven or eight kinetosome pairs aligned in a line parallel to the somatic kineties and a conspicuous pellicle depression in the middle of caudal margin.This combination of features clearly separates it from all known scuticociliates at the species level.

Mesanophrys carcini (Grolière & Léglise, 1977) Small & Lynn in Aescht, 2001
The main characteristics that aid in identifying this species are the slender body, short buccal field, oral apparatus and somatic infraciliature (Song & Wilbert 2000).The characteristics of the Qingdao population are different from those of the population reported by Song & Wilbert (2000) in having a larger body size (on average 55 × 20 μm vs. 40 × 12 μm) and a variable number of somatic kineties (10 or 11 vs. constantly 11; Table 3).These variations are considered population-dependent (Song & Wilbert 2000).

Metanophrys sinensis Song & Wilbert, 2000
Our population is virtually identical to the two Qingdao populations (Song & Wilbert 2000;Ma & Song 2003), that is, they agree in body size and shape, habitat, infraciliature, silverline system and marine habitat, except the proportion of buccal field length to body length (40-50% in the present study vs. ca.30-40% in the previous studies) and the presence of extrusomes (vs.not observed in previous descriptions) (Song & Wilbert 2000;Ma & Song 2003; Table 3).Nevertheless, it is believed that they are conspecific because of their close similarity in other living characteristics and infraciliature.

Metanophrys similis Song et al., 2002
The Zhanjiang population is identical to the original description (Song et al. 2002) according to the body size, ciliature and habitat; hence, the identity of this species is not in doubt.Compared with the original description, the population described in this paper has a different body shape (plump pyriform vs. slender body shape in Song et al. 2002; Table 3), which may be due to different nutritional conditions (Song et al. 2002).(2000) present study Song et al. (2002) present study Song & Wilbert (2000) Ma & Song (2003) present study

Fig. 1 .
Fig. 1.Sampling sites.A. Coastal waters of the Yellow Sea at Qingdao, Shandong province.B. A coastal mariculture-region in Zhanjiang, Guangdong province.C. Coastal waters of Daya Bay, Guangdong province.

Table 1 .
Paramesanophrys typica gen.et sp.nov.Comparison of collection data for four species.We failed to extract DNA from Paramesanophrys typica gen.et sp.nov.due to the low number of specimens of this species.If possible, we will try to acquire sequence data from it in the future.