During a routine survey of the Pacific oyster Crassostrea gigas in Tongyoung (previously Chungmu) on the southern coast of Korea, basophilic inclusions were observed in the gonadal tissues. They were detected from March to May at a prevalence rate of 3.3 to 7.1%. The inclusion bodies were Feulgen-positive and stained orange-red with phloxine tartrazine. Electron microscopic observation revealed non-enveloped, icosahedral particles 40 to 45 nm in diameter. These morphological characteristics resemble those of papova virus-like inclusions previously described from Pacific and eastern (American) oysters C. virginica in North America. Although many mitochondrial bodies and intact sperm cells were observed around the inclusion body, no host reaction, such as hemocytic infiltration, was detected. KEY WORDS: Pacific oyster · Crassostrea gigas · Viral gametocytic hypertrophy · Papova-like virus · Seminal glandResale or republication not permitted without written consent of the publisher embedding and cut into 4 µm thick sections. Some sections were stained with Harris's hematoxylin and eosin (H&E), some with Feulgen stain and some with phloxine tartrazine, for bright field light microscopy (Olympus Vanox AHBS3) examination.Ultrastructural examination. Samples showing inclusion bodies within the gonadal epithelia were selected for processing for transmission electron microscopy. Tissue specimens from the same oysters, preserved in Carson's fixative solution were rinsed in 0.2 M cacodylate buffer at pH 7.2 for 48 h at 4°C before post-fixing in 2.5% glutaraldehyde solution, and 1% osmium tetroxide. After dehydration through graded alcohols, the tissues were embedded in Epon resin compound (Ouken, Japan). Hardening was carried out at 35°C for 12 h, 45°C for 12 h, and 60°C for 48 h. Semi-thin sections (200 nm) were stained with toluidine blue, and ultra-thin sections (60 nm) were stained with uranyl acetate and lead citrate. Ultra-thin sections were examined with a JEOL 1200 EX-2 transmission electron microscope (TEM) at 80 kV. RESULTSBasophilic inclusions in gonoducts were observed in specimens from all sites except Stn 4. Infections were observed from March to May at prevalences of 3.3 to 7.1%. Prevalence at Stn 3 was higher than at the other sites ( Table 1). The heaviest infection included 62 inclusion bodies in a single tissue section; however, no host reaction was observed. The longest axis of the inclusion bodies varied from 15 to 60 µm, and the shape was oval to spherical. Some inclusion bodies had dense staining margins (Figs. 2 & 3). Feulgen staining confirmed the presence of dense nucleic acid (Fig. 4), and orange-red staining with phloxine tartrazine (Fig. 5) demonstrated a deoxyribonucleic acid (DNA) composition. Advanced infections led to disruption of the nuclear membrane and putative release of the viral particles (Fig. 4, arrowhead).Non-enveloped, icosahedral viral particles, 40 to 45 nm in diameter, filled the inclusion bodies (Figs. 6 & 7). These morphological characteristics resemble those of Pa...

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In 2004, epizootiological studies were conducted on mass mortalities of tunicates Halocynthia roretzi in Goje, Korea. The clinical characteristics of infected H. roretzi were weakness of the tunic, loss of elasticity, and finally death involving a rupture of the tunic. Histological studies revealed severe hemocyte infiltration in the connective tissue surrounding the intestine and mantle of infected H. roretzi. Hypertrophied eosinophilic hemocytes containing several cytoplasmic vacuoles were observed in the connective tissue surrounding the intestine, gill and mantle. Ultrastructural examination revealed the presence of a parasite in the cytoplasm of hemocytes. Secondary cells were observed in the primary cell of the parasite. Spore formation within primary cells suggests that the parasite may be an intrahemocytic paramyxean parasite (IPP) and may cause mass mortality of H. roretzi. KEY WORDS: Intrahemocytic paramyxean parasite · Tunicate · Halocynthia roretzi · Histology · UltrastructureResale or republication not permitted without written consent of the publisher Dis Aquat Org 72: [65][66][67][68][69] 2006 Halocynthia roretzi is an important aquaculture species in Korea and Japan. It is an edible tunicate as well as a source of collagen. Mass mortalities of H. roretzi have occurred over the last 2 decades along the southern and eastern coasts of Korea causing serious economic damage.Mortalities of more than 50 000 t of cultured Halocynthia roretzi were reported from Korea in 2003, and this occurence was the incentive for the present epidemiological study. Diseases causing mortalities in urochordates have rarely been reported (Monniot 1990). In the present study, we describe histological and ultrastructural characteristics of an intrahemocytic paramyxean parasite (IPP) that is associated with the mass mortality of H. roretzi. This paper provides the first description of an IPP infecting the hemocytes of edible tunicates H. roretzi cultured on the southern coast of Korea. MATERIALS AND METHODSTunicates (n = 30) Halocynthia roretzi 1 to 2 yr old were collected from private aquaculture farms located in Goje, Korea, in June 2004.Histological examination. Halocynthia roretzi tissues were fixed in Davidson's solution (Howard & Smith 1983), processed for paraffinembedding and cut into 4 µm thick sections. Sections were stained with Harris's hematoxylin and eosin (H&E) for light microscopy examination.Ultrastructural examination. Halocynthia roretzi tissues were fixed in 2.5% glutaraldehyde in 0.2 M cacodylate buffer at pH 7.2 for 1 h at 4°C. After 2 washes in cacodylate buffer, the tissues were post-fixed in osmium tetroxide in the same buffer at 4°C. Samples were dehydrated through graded alcohols, rinsed twice for 15 min in propylene oxide, and embedded in Epon resin compound (Ouken). Semi-thin sections (200 µm) were stained with toluidine blue, and ultra-thin sections (60 µm) were stained with uranyl acetate and lead citrate. Ultra-thin sections were carefully examined with a Hitachi 7100 transmission ...

Tintinnida is a diverse taxon that accommodates over 1,500 morphospecies, which is an important component of marine planktonic food webs. However, evolutionary relationships of tintinnids are poorly known because molecular data of most groups within this order are lacking. In our study, the small subunit (SSU) rRNA genes representing 10 genera, 5 families of Tintinnida were sequenced, including the first SSU rRNA gene sequences for Coxliella, Dadayiella, Epiplocyloides, and Protorhabdonella, and phylogenetic trees were constructed to assess their intergeneric relationships. Phylogenies inferred from different methods showed that (1) Three newly sequenced Eutintinnus species fell into Eutintinnus clade forming a sister group to the clade containing Amphorides, Steenstrupiella, Amphorellopsis, and Salpingella; (2) Surprisingly, the genetic distances between Amphorides amphora and Amphorellopsis acuta population 1 was even smaller than that between the two populations of Amphorellopsis acuta, casting doubt on the validity of Amphorides and Amphorellopsis as presently defined; (3) The SSU rRNA sequences of Dadayiella ganymedes and Parundella aculeata were almost identical. Therefore, Parundella ganymedes novel combination is proposed; (4) Coxliella, which is currently assigned within Metacylididae, branched instead with some Tintinnopsis species. Furthermore, the validation of Coxliella, which was considered to be a "questionable" genus, was confirmed based on evidences from morphology, ecology, and molecular data; (5) Protorhabdonella and Rhabdonella showed rather low intergeneric distance and grouped together with strong support suggesting that Rhabdonellidae is a well-defined taxon; and (6) Epiplocyloides branched with species in Cyttarocylididae indicating their close relationship.

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