Study of Myxosporea (Myxozoa), infecting worldwide mullets with description of a new species

Existing data on Myxozoa parasites infecting mullets were reviewed. The validity of nine species names was updated. Sixteen species were registered during analysis of original material collected in the Mediterranean, Black, Azov, and Japan Seas in 2004–2005. A new bivalvulid myxozoan parasite, Myxobolus adeli n. sp., was described from the inner organs of the golden grey mullet Liza aurata (Risso, 1810) collected in the Mediterranean (Ebro Delta, Spain), Black Sea (Kerch Strait, Ukraine), and Azov Sea (Genichesk, Ukraine) coastal waters. It is characterized by the presence of elongated, spindle-like cysts 0.5–1.3 mm in size, filled with wide transverse-oval spores about 6.2 × 7.2 × 4.6 μm in size, with two equal polar capsules measuring about 3.0 × 1.8 μm and short polar filament, turned into four coils. The obtained data show that this species differs from all previously described Myxobolus spp. with equal polar capsules. Comparative study of Myxobolus spp. recorded in worldwide mullets indicates a close relationship with M. adeli n. sp. and Myxobolus improvisus Isjumova, 1964 registered in mullets. Probably, the last species includes representatives of some different species, infecting freshwater and marine hosts.


Introduction
The mullets (Mugiliformes: Mugilidae) have a worldwide distribution and inhabit tropical and temperate waters (Nelson 1984). According to current data (FishBase) the Mugilidae family includes 24 genera and 72 species, inhabiting tropical, subtropical, and the southern part of the Atlantic, Indian, and Pacific oceans. Many mullet species have comparatively trivial areas, but one of them-grey mullet Mugil cephalus (Linnaeus, 1758)-can be cosmopolitan, spreading including the coastal waters of Europe, Asia, Africa, Australia, America, and Oceania. Mullets have been used as a considerable source of food in different parts of the world. The importance of mullet for aquaculture and the pathologic potential of some parasites, in particular Myxosporea, motivate their detailed study. Myxosporea represents one of the important groups of parasites infecting worldwide mullets (Lom and Dyková 1992;Kent et al. 2001). So far, a few revisionary studies of parasites infecting worldwide mullets have been conducted by Paperna (1975). Twelve species of Myxozoa were reviewed by Paperna and Overstreet (1981). The genera Sphaerospora, Henneguya, Myxidium, Myxosoma, Myxobolus, Kudoa, infecting mullets, were revisionary studied by Sitjà-Bobadilla and Alvarez-Pellitero (1994), Jajasri and Hoffman (1982), Landsberg and Lom (1991), Eiras 2002, Eiras et al. (2005, and Moran et al. (1999).
In the last decades, geography of the mullet parasites studies and knowledge about myxosporeans infecting worldwide mullets were considerably widened. The aim of this paper is to investigate the biodiversity of myxozoans based on existing data and original material obtained during parasitological investigations of mullets in the Mediterranean, Black, Azov, and Japan Seas. Studies were supported by INTAS project .

Material and methods
The original study was carried out on data obtained during parasitological investigations of 3,362 fish specimens. Mullets were caught in May-June and October-November 2004 In the Mediterranean coastal region of Spain (Ebro River Estuary and Santa Pola Bay) 1,550 specimens of mullets belonging to five genera were dissected. In the Ponto-Azov region, Ukraine (coastal waters near Kerch, Genichesk, Berdiansk, and Mariupol), 1,498 mullets representing four genera were dissected. Material from the Japan Sea was presented by results of parasitological dissections of 314 mullets from two genera caught in the Russian coastal regions of Japan Sea (Razdolnaya River, Kievka Bay, Posiete Bay, Artemovka). Parasitological analysis was performed based on partial parasitological dissection (Bykhovskaya-Pavlovskaya 1985). Fresh spores were fixed on slides in a glycerine jelly medium according to Donets and Schulman (1973). Spores were photographed and measured on digital images. Descriptions of the spores were based on the references of Schulman et al. (1997) and Lom and Arthur (2006). Live and Giemsa-stained spores were observed and measured under MBI-3 and Olympus BX50F4 microscope equipped with Analysis Pro 2.11 software.
For ultrastructural analyses, infected tissues were fixed in a 2.5 % (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for several days at 4°C. After washing twice with 0.1 M sodium cacodylate buffer and post-fixation in 2.0 % (v/v) osmium tetroxide in cacodylate buffer for 1 h at 4°C, the pieces were dehydrated and embedded in Epon-Araldite solution using a standard procedure (Vávra and Maddox 1976). Blocks of embedded tissues were sectioned with an LKB III ultra-microtome. Semi-thin sections were stained with methylene blue. Ultrathin sections were mounted on copper grids, double-stained with uranyl acetate and lead citrate, and examined in a JEM 100B electron microscope operated at 80 kV.

Results and discussion
Myxosporeans of the worldwide mullets By the present time, 64 myxosporean species from 13 genera and nine families infecting 16 mullet species belonging to six genera have been registered (Table 1). Five species were identified to the genus range. The majority of myxosporeans parasitizing mullets are attributed to the family Myxobolidae. Among them, 32 and two species belong to the genera Myxobolus and Henneguya, correspondingly. Eleven species belong to the family Myxidiidae, eight representatives of Zschokkella genus, and three species belong to the genus Myxidium. Ten species were found as representatives of the family Kudoidae belonging to a single genus Kudoa. The family Sphaerosporidae contains four species belonging to the genus Sphaerospora. One species from Alataspora and one from Pseudalataspora genera were registered as representatives of the Alatasporidae family. Sphaeromyxidae, Ortholineidae, Chloromyxidae, Polysporoplasmidae as well as the Sinuolineidae family are represented by single species of each genus (Sphaeromyxum, Ortholinea, Chloromyxum, Polysporoplasma).
The maximum of species richness of Myxosporea was registered in flathead mullet M. cephalus. Thirty six species of myxosporeans from eight genera were mentioned in named host. The area includes the Mediterranean basin, Red Sea, Atlantic Coast of Africa, Mexican Gulf, and Indian and Pacific Ocean coastal waters.

V. cunnesius
Intestinal musculature Indian Ocean: Visakhaptnam harbor (India) Kalavati and Anuradha (1993) species were found in the mesenterium and intestines; two in the heart, on fins, and scales. The urinary bladder, spleen, and liver were infected with a separate species of myxozoans. Eighteen species were detected in various organs (Table 1).

Original data of the author's investigations
We conducted taxonomical studies of mullet myxosporeans collected in the Mediterranean, Black, Azov, and Japan Seas in the summer and autumn 2004-2005. M. cephalus w a s pa r a s i t ol og i ca l l y s t u d i ed i n a l l re gi on s ; L. haematocheila-in the Japan, Black, and Azov Seas; L. aurata and L. saliens-in the Mediterranean, Black, and Azov Seas; and L. ramada and C. labrosus-exclusively in the Mediterranean Sea.
Totally, 16 species of myxosporeans have been registered. New information about myxosporean fauna for each region of investigations has been received.
Zschokkella admiranda from M. cephalus has been registered for the first time in the Mediterranean fauna. Sphaeromyxa sabrazesi, Kudoa unicapsula, Alataspora sp., Z. admiranda, Myxobolus adeli sp. n., M. parvus, M. muelleri, M. ichkeulensis, M. spinacurvatura, Myxobolus rohdei, M. exiguus, Myxobolus nile, Myxobolus episquamalus have been found in the coastal waters of Spain. M. cephalus appeared to be a new host for S. sabrazesi. L. aurata was registered as a new host for Sphaerospora dicentrarchi. L. ramada and C. labrosus were found as hosts for Polysporoplasma mugilis in the Mediterranean Sea. P. mugilis infecting L. aurata has been found for the first time in the Black Sea. S. dicentrarchi, M. ichkeulensis, and M. spinacurvatura infecting M. cephalus was firstly registered in the Black and Azov Seas. L. aurata was firstly registered as a new host for Z. admiranda. M. ichkeulensis, M. spinacurvatura, and M. episquamalus parasitizing M. cephalus has been found for the first time in the Japan Sea.
In the present paper, we describe the following new species: M. adeli sp. n. from L. aurata in the Mediterranean, Black, and Azov Seas.
Alataspora sp. (Table 3 Description. Vegetative stages presented by rounded or oval-shaped bisporous plasmodia with transparent ectoplasm and small-grained endoplasm. Spores are strongly elongated in the plane perpendicular to the sutural line. They have clearly expressed triangular part, cavity of which contains polar capsules and amoeboid germ. Elongated top parts of the valves form single winglike appendages slightly unequal in sizes. Suture line is straight and clear. Spherical polar capsules are located close to the anterior pole and open near the suture line to one side of spore. Amoeboid germ is located under polar capsules. Spore measurements presented in Table 3. Taxonomic summary. Based on the spore construction, Alataspora sp. occupies intermediate position between Fig. 1 (continued) representatives of Alataspora and Pseudalataspora genera. It resembles Alataspora solomoni Yurakhno, 1988, differing from it by unequal length of valves and larger spores and polar capsules. We consider Alataspora sp. a species inquirenda that needs a precise species description after obtaining of additional data.