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Internal defense of ascidians relies, at least partially, on cells circulating in body fluids and infiltrating in tissues, referred to as hemocytes, although structure and composition of ascidian hemocytes still remain unclear. In the present study, we investigated hemocyte types and their functions of the solitary ascidian Halocynthia roretzi using flow cytometry. Based on morphology, cellular activities and intracellular parameters from the flow cytometry, we identified eight hemocyte types including, three granulocytes (Gr-1, Gr-2, and Gr-3), 4 hyalinocytes (Hy-1, Hy-1′, Hy-2, and Hy-3) and lymphocyte-like (Ly-like) cells. The granulocyte Gr-1 accounted for 30% of the total circulating hemocytes and exhibited highest density of lysosomes and oxidative activity. Gr-1 was deeply involved in phagocytosis and degradation of foreign material. Hyalinocytes consist of two main populations, Hy-1 and Hy-2, and each accounted for 30% of the circulating hemocyte. Hy-1 displayed lysosomal content, an inducible oxidative activity, and no proteases, while Hy-2 expressed highest density of intracellular proteases, no lysosomes and a low oxidative activity. It was believed that Hy-2 may represent an important link between cellular and humoral immune reactions. Hy-1 did not show phagocytosis activity. Hy-3 and the Ly-like cells presented a similar profile except for their size and complexity, and Hy-3 may represent an intermediate differentiation/maturation step between Ly-like cells and other hemocyte populations. This first characterization of the hemocyte populations of H. roretzi provides a solid basis to investigate further their respective roles and functions in physiological and pathological contexts.

In order to understand the co-culture impact of the sea squirt Halocynthia roretzi on the Pacific oyster Crassostrea gigas, we examined the filtration rate of the sea squirt in relation of water temperature () and body weight. The filtration rate increased in relation to body weight and water temperature, indicating a clear positive correlation with the two variables. Due to the clear positive correlation between filtration rates at each temperature groups for acceptable collinearity, each constants at regression equation was further analyzed for a unified model of filtration rate. Therefore the filtration rate of H. roretzi was estimated as: Fr (L/h/animal)

We examined the effect of starvation on the occurrence of tunic softness to determine the cause of mass mortality of cultured Halocynthia roretzi (Drasche) in the Tongyeong region, Korea. In terms of the survival rate of H. roretzi and the occurrence rate of tunic softness, H. roretzi starved for 35 days at water temperatures of 8, 12, and (room temperature of ) did not exhibit tunic softness at water temperatures of either or . for morphological changes, although the tunic of H. roretzi was shrunken and became visibly smaller with a darkening color in all experimental groups, as compared to the state prior to starvation, its tunics bulbs continuously. The ratio of RNA/DNA concentrations and protein contents for each of the tunic sections were lower in the starved group. Our results indicate that tunic softness is not related to feeding deficiency, as no histopathological symptoms were apparent in the digestive gland or tunic of H. roretzi due to starvation.

A Gram-negative, non-spore-forming, aerobic, motile and rod-shaped or ovoid bacterial strain, designated MA1-3(T), was isolated from a sea squirt (Halocynthia roretzi) collected from the South sea in South Korea. Strain MA1-3(T) was found to grow optimally at 25 °C, at pH 7.0-8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain MA1-3(T) fell within the clade comprising Colwellia species, clustering coherently with the type strains of Colwellia aestuarii, Colwellia polaris and Colwellia chukchiensis, showing sequence similarity values of 97.2, 96.4 and 95.6 %, respectively. It exhibited 16S rRNA gene sequence similarity values of 93.9-96.1 % to the type strains of the other Colwellia species. Strain MA1-3(T) was found to contain Q-8 as the predominant ubiquinone and C16:1 ω7c and/or C16:1 ω6c, C16:0 and C16:1 ω9c as the major fatty acids. The DNA G+C content of strain MA1-3(T) was determined to be 39.1 mol% and its mean DNA-DNA relatedness value with the type strain of C. aestuarii was 13 ± 5.4 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that the novel strain is separated from other Colwellia species. On the basis of the data presented, strain MA1-3(T) is considered to represent a novel species of the genus Colwellia, for which the name Colwellia meonggei sp. nov. is proposed. The type strain is MA1-3(T) (=KCTC 32380(T) = CECT 8302(T)).

A Gram-stain-negative, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated P-MA1-7T, was isolated from a sea squirt (Halocynthia roretzi) collected from the South Sea, Korea. Strain P-MA1-7T grew optimally at 25 °C, at pH 7.0–8.0 and in the presence of 2–3 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain P-MA1-7T fell within the cluster comprising the type strains of four species of the genus Litoreibacter , exhibiting 16S rRNA gene sequence similarity values of 97.0–98.5 % to these four type strains and less than 95.9 % sequence similarity to the strains of the other species examined. Strain P-MA1-7T contained Q-10 as the predominant ubiquinone and C18 : 1ω7c as the predominant fatty acid. The major polar lipids of strain P-MA1-7T were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G+C content of strain P-MA1-7T was 58.3 mol% and DNA–DNA relatedness values of strain P-MA1-7T with the type strains of the four species of the genus Litoreibacter were in the range of 8–21 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain P-MA1-7T was separate from other species of the genus Litoreibacter . On the basis of these data, strain P-MA1-7T is considered to represent a novel species of the genus Litoreibacter , for which the name Litoreibacter halocynthiae sp. nov. is proposed. The type strain is P-MA1-7T ( = KCTC 32213T = CCUG 63416T).

The edible ascidian, Halocynthia roretzi is a commercially important fisheries resource in Korea. However, there have been outbreaks of mass mortality due to soft tunic syndrome. It was discovered recently that the cause of death is infection by a protozoan parasite Azumiobodo hoyamushi. Alamar blue assay and microscopic counting were used to estimate anti-protozoal effects of 20 drugs having different action mechanisms. Through comparison of alamar blue assay and microscopic counting, 6 drugs were found to be potential in protozoan-killing effects: amphotericin B, formalin, hydrogen peroxide, bithionol, benzalkonium chloride, bronopol (24hr-EC 50≤20 ㎍/㎖). The preliminary data can be used as a basis to develop anti-protozoal agents against A. hoyamushi.

This study investigated the food biochemical characterization of commercial seasoned sea squirt Halocynthia roretzi (CSS). The proximate composition of CSS was 77.2-82.7% moisture, 7.1-9.1% crude protein, 0.3-2.6% crude lipid and 3.5-6.3% ash. Taste compound contents of CSS were 2.3-5.4% salinity (saltiness), 0.42-1.12 g/100 g total acidity (sourness) and 114.9-330.2 mg/100 g amino nitrogen (taste intensity). The Hunter color values of CSS were 23.79-32.50 for lightness, 9.97-20.45 for redness, 14.01-20.96 for yellowness and 64.50-76.63 for color difference. The odor intensity of CSS was 35.0-62.0. According to these results, there were large differences in proximate composition, taste compounds, Hunter color values and odor intensity of CSS. Viable cell counts ranged from 6.20 to 7.69 log (CFU/g), and most of the viable cells comprised of lactic acid-forming bacteria. CSS was not detected in the coliform group.

This study was conducted to develop and characterize sea squirt Halocynthia roretzi sikhae. According to the results for pH, total acidity, lactic acid bacteria, amino nitrogen and sensory evaluation of sea squirt sikhae during fermentation for 6 days at , the optimum fermentation periods were 4 days for sourness-disliking customers and 5 days for sour-disliking customers. No differences in the proximate compositions of sea squirt sikhaes fermented for 4 days (4D) and for 5 days (5D) were found. There was a difference in the eproximate compositions of commercial seasoned sea squirts, 4D and 5D, sea squirt sikhaes. The results of salinity, total acidity, amino nitrogen and sensory evaluation of two kinds of sikhae suggest that the taste was stronger for 5D than for 4D, both of which were superior to commercial seasoned sea squirts. There was, however, no difference in color of 4D, 5D and commercial seasoned sea squirts. The results of E. coli analyses suggest that sea squirt sikhae is a safe food in terms of sanitation.

The hemocytes of the tunicate Halocynthia roretzi are classified into six types based on their size, cellular form, and fine structure of the cytoplasmic granules: hyalinocytes, granulocytes, phagocytes, nephrocytes, morula cells, and multi-vacuole cells. Based on cell size, they are ordered multi-vacuole cells ()>nephrocytes ()>phagocytes ()>granulocytes ()>morula cells ()>hyalinocytes (). The proportion of hemocytes is ranked in the order multi-vacuole cells (54.8%)>nephrocytes (16.9%)>granulocytes (9.9%)>morula cells (8.8%)>phagocytes (6.1%)>hyalinocytes (3.5%).

The edible ascidian, sea squirt Halocynthia roretzi (Drasche) is marine invertebrate that is a valuable source of foods and bioactive compounds. A severe disease of the sea squirt characterized by degeneration of tunic fibers formed of bundled cellulose microfibrils has occurred. We hypothesized that bacteria lyse the cellulose fibril structure, cellulase activity may be a causative agent of the disease. Among the bacteria isolated from diseased sea squirt, Pseudoalteromonas sp. NO3 had cellulase activity based on a Congo red overlay assay and starch-reducing activity. Sea squirts exhibited 40–100% cumulative mortality after injection with Pseudoalteromonas sp. NO3 using doses of 2×106−2×108 colony forming unit (CFU)/individual. Dead sea squirts possess thinner and ruptured tunics, which were similar to natural outbreaks. These results suggest that Pseudoalteromonas sp. NO3 possessing cellulase activity is one of the causes of tunic softness syndrome in sea squirt.

Azumiobodo hoyamushi, the causative agent of soft tunic syndrome, was likely introduced to farming sites of the edible ascidian Halocynthia roretzi via ascidian spat. The source of infection is thought to be cysts of A. hoyamushi that reside in the substrates on which the ascidian spat are attached, but not the spat themselves. Thus, there is a need to develop methods to prevent contamination of the substrates with A. hoyamushi during seed production of the ascidian. We evaluated the protozoacidal effects of sodium hypochlorite and povidone-iodine against the flagellate and temporary cyst forms of A. hoyamushi. Additionally, we evaluated the effects of these disinfectants on the development of fertilized ascidian eggs. The flagellate form of A. hoyamushi was completely inactivated by povidone-iodine (5 ppm, 1 min) and sodium hypochlorite (1 ppm, 1 min). The temporary cysts of A. hoyamushi were completely inactivated by both disinfectants (5 ppm, 1 min). Disinfection with 50 ppm povidone-iodine for 15 min or 5 ppm sodium hypochlorite for 15 min had no effect on ascidian embryogenesis. Thus, horizontal transmission of A. hoyamushi via the substrates can be efficiently prevented by disinfecting ascidian eggs or tools used for spawning with povidone-iodine baths ranging from 5 ppm for 1 min to 50 ppm for 15 min without any side effects.

Background Recent studies have reported that soft tunic syndrome (STS) in the edible ascidian Halocynthia roretzi is caused by the kinetoplastid parasite Azumiobodo hoyamushi. In this study, we attempted to detect and quantify the pathogen in benthic animals. Methods Four species of ascidians, three species of echinoderms, two species of bivalves, one species each of sponge and algae, as well as seawater, were collected in 2014 and 2015 from an ascidian farm on the southern coast of Korea by SCUBA diving. Samples were collected from ascidian hanging culture ropes or the sea bottom. Inhalent siphons were excised for the analysis of ascidians, and soft body tissues were excised from the other species. Membrane filters were used to filter collected seawater. Tissues and membrane filters were analysed using culture testing, PCR testing, and qPCR diagnoses. Results Only organisms belonging to Ascidiacea are susceptible to A. hoyamushi infection. The infection rate (% infected of the total number collected) and infection intensity (number of cells infected/g tissue wet weight) varied depending on the seasonal variation in seawater temperatures. Most ascidians examined were infected with A. hoyamushi and showed higher infection intensity in cold water seasons (April 2014 and February 2015), followed by a dramatic drop during warm water seasons (August and November, 2014). In addition, infection intensity of A. hoyamushi during the warm water period was higher in ascidians from the sea bottom than those from the hanging culture rope. Conclusions Among benthic organisms that inhabit the southern coast of Korea, most ascidians are susceptible to A. hoyamushi infection. Seasonal cycle of infection rates and intensities of the pathogen correspond well with the STS disappearance and onset cycle observed in ascidian farms. The high intensity of A. hoyamushi infection in the ascidians on the sea bottom of ascidian farms during summer suggest further studies on the role of the pathogen in resumption of STS occurrence in late fall or early winter in the southern coast of Korea.

The edible ascidian Halocynthia roretzi is a commercially important fisheries resource in Korea. However, for the last several years, outbreaks of mass mortalities of the species have been occurring along the south and east coasts of Korea, where most ascidians are produced. Although it is known that tunic-softness syndrome is associated with these mortality events, the agent causing the syndrome has not yet been confirmed. To determine the agent causing tunic-softness syndrome, healthy and diseased ascidians were collected in March 2011 from Tongyeong, on the south coast of Korea, and were used for biological and pathological investigations. The results showed that diseased ascidians exhibited remarkably reduced body fluid, fatness index, and tunic index compared with healthy specimens. Interestingly, bi-flagellated protozoans were observed specifically in the tissue imprints and tunic cultures of diseased ascidians at an occurrence rate of 97.5%. Histological observation showed that the thickness of the tunics of diseased ascidians was reduced by half, and irregular structure and breakdown of the tunic fiber bundles were observed. In particular, flagellate-like cells were observed in the diseased ascidians. Our study clearly shows that bi-flagellated protists are present only in the softened ascidians, suggesting that the flagellates are partly or entirely associated with soft-tunic syndrome. Accordingly, further investigations to verify the effects of the flagellates found in the present study on soft-tunic syndrome should be conducted.

【The sea squirt Halocynthia roretzi is mainly cultured in Tongyeong, Southern coastal area of Korea. This study presents the physiological rates of respiration, excretion, feeding and assimilation efficiency of the sea squirt Halocynthia roretzi to analyze the SFG(scope for growth) and net growth efficiency, determined during 2007. Oxygen consumption and nitrogen excretion rates increased with a rise in temperature during the summer period whereas feeding rates decreased. The O:N ratio was high during winter(October to February). Assimilation efficiency showed an annual average of 75.4% during the experimental period, except during a period of elevated temperature in July to September(average $25^{\circ}C$ ). Net growth efficiency( $K_2$ ) was 8.7 to 64.2% except for May to September, when temperature increased at the aquaculture farm. SFG was negative from May to September, reflecting high temperatures and low feeding rates during this period; its highest positive values occurred during winter.】

An etiological study was conducted to clarify whether the flagellate-like cells found in histological preparations of the tunic of diseased Halocynthia roretzi (Drasche) were the causative agent of soft tunic syndrome in this ascidian. When pieces of softened diseased tunic were incubated overnight in sterile seawater, live flagellated cells, which were actively swimming in the seawater, were observed in 47 out of 61 diseased ascidians (77%), but not in moribund or abnormal individuals with normal tunics (n = 36) nor in healthy animals (n = 19). The flagellate was morphologically very similar to those observed in histological sections of the diseased tunic. By contrast, flagellates were not found in tunic pieces of healthy, moribund, and abnormal individuals that did not exhibit softening of the tunic. Light and electron microscopy revealed that the flagellate has polykinetoplastic mitochondria with discoidal cristae. The cytomorphologies of the flagellate were the same as those of the flagellate-like cells in the diseased tunic. We cultured the flagellate from the softened tunic in vitro and confirmed that the tunics of healthy ascidians, which were immersion-challenged with suspensions of the subcultured flagellates, became softened 17 d after exposure, including the final 12 d in aerated, running seawater. The occurrence of flagellates was also confirmed by incubating pieces of soft tunic from experimentally infected animals in seawater overnight. These results indicate that the flagellate is the causative agent of soft tunic syndrome.

To examine the functional properties of conjugated linoleic acid (CLA) and ascidian tunic extracts in fish, we compared mackerel fed ascidian tunic extract and CLA (CA25) and a control group. The daily growth index of CA25 was 1.92 compared to 1.86 in the control group. The viscerosomatic index of CA25 was 36.7% lower than that of the control group. After 8 weeks, the protein content decreased from 19.7 to 17.5% in the CA25 group. The ascidian tunic extract content in the viscera was much higher than in muscle (0.13 vs. 0.03 mg/100 g) after 8 weeks. At the start, the n-3 fatty acid content of the experimental fish was 25.2% in muscle and 23.7% in viscera. The CLA content in muscle in the CA25 group was 2.1% after 4 weeks and 2.3% after 8 weeks. By contrast, the CLA content in viscera did not change after 8 weeks.

This study examined the antidiabetic and antihyperlipidemic activities of an ascidian (Halocynthia roretzi) in rats in vivo. Rats were fed on experimental diet including dried ascidian powder (200 mg/kg body weight) for 4 weeks, and then the triglyceride and total cholesterol levels in blood were analyzed. On the ascidian tunic powder diet, the triglyceride level decreased by more than 20.9% and the total cholesterol content decreased by more than 24.4%. In comparison, the triglyceride and total cholesterol level in the blood of rats fed ascidian meat powder decreased only slightly. Therefore, the ascidian tunic powder might be a healthy food with antidiabetic and antihyperlipidemic effects.

The mortality of the tunicate Halocynthia roretzi due to softness syndrome, which exhibits tunic thinning as well as loss of muscle elasticity, has caused serious problems for the aquaculture industry in Korea. The effects of softness syndrome can be better understood by studying the defense mechanisms of Halocynthia roretzi . This study used flow cytometry to assess hemocyte parameters in H. roretzi . Flow cytometry provided a means to rapidly quantify tunicate defense mechanisms by measuring temperature and zymosan-dependent effects on phagocytosis activity. Also, immune parameters, such as morphology of cell sub-populations (size and granularity), total hemocyte counts (THC), hemocyte viability, and phagocytosis activity using flow cytometry analysis, of normal tunicates and softness syndrome tunicates were compared. The results showed that, in normal tunicates, the phagocytic rate and hemocyte viability are higher than those of tunicates with softness syndrome. Total hemocyte numbers in softness syndrome tunicates were 4 times higher than that of normal tunicates. It is suggested that the tunicate immune system is associated with softness syndrome and that flow cytometry is a powerful tool for characterizing immune response.

In this study we investigated the antioxidant activities and quality characteristics of Halocynthia aurantium and Halocynthia roretzi. The pH of H. aurantium was higher than that of H. roretzi. The volatile basic nitrogens of H. roretzi and H. aurantium were 22.41 mg% and 16.80 mg%, respectively. Lightness and yellowness of H. roretzi were higher than those of H. aurantium, but redness of H. aurantium was higher. The results of sensory evaluation showed that the H. aurantium was better for color, odor, taste and acceptability. Total combined amino acid contents of H. roretzi and H. aurantium were and , respectively. Our results showed that H. roretzi had relatively higher contents of Asp, Glu, Gly, DPPH radical scavenging activity, ABTS radical scavenging activity and reducing power. Also total phenol content of H. roretzi was higher than that of H. aurantium. The organoleptic properties of the H. aurantium were superior but the antioxidant activities were relatively lower than those of H. aurantium. For commercial usage, additional study would be helpful in the two ascidians to recommend.

Since 2007, mass mortalities of cultured ascidians Halocynthia roretzi (Drasche) have occurred in Miyagi Prefecture, Japan. The mortalities occur from November through August, and the tunics of affected animals become abnormally weak and soft. The number of farming areas where mass mortalities have occurred has increased rapidly: 3 in 2007, 6 in 2008, and 14 in 2009. When an outbreak of the disease occurred, mortality reached 17 to 100%. Prominent histopathological changes in the diseased ascidians were found in the tunics; the tunics of affected animals were usually much thinner than those of healthy individuals, and the tunic matrix showed marked disintegration with irregular arrangements of fiber layers or the presence of hollow spaces. In addition, flagellate-like cells (10-14 microm x 2-3 microm) stained with hematoxylin were observed in the tunics of 31 out of 36 diseased animals (86%), but not in apparently healthy animals (n=38). Experimental infection with the disease was successfully conducted by immersing small pieces of tunic samples from diseased ascidians into aquaria with healthy ascidians. The flagellate-like cells were confirmed in the tunics of all the experimentally infected animals. These results indicate that the mass mortalities of ascidians accompanied by abnormally softened tunics were caused by an infectious agent, and suggest the involvement of the flagellate-like cells in the disease.

Lipids extracted from Halocynthia roretzi contain n-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid, as well as carotenoids. The aim of the present study was to evaluate the effect of H. roretzi lipids on white adipose tissue (WAT) weight and high blood glucose levels in diabetic/obese KK-Ay mice. H. roretzi lipids were fed to the diabetic/obese KK-Ay mice for 5 weeks. In the mice treated with the H. roretzi lipids compared to control mice, WAT weight was reduced, blood glucose levels and leptin mRNA expression in the epididymal WAT were significantly decreased, serum leptin levels also tended to decrease, and serum adiponectin levels tended to increase. These results demonstrate that H. roretzi lipids have beneficial health effects on diabetic/obese KK-Ay mice.

Halocynthia roretzi, a solitary ascidian, can swell and deflate its solid, leather-like tunic largely, but it is not clear whether the tunic deforms actively. Cellulose Iβ, which is predominant in higher plants, is also present in the tunic as a highly crystalline form. The elastic modulus of its whisker crystalline is considerably high. Substantial amounts of chitin sulfate-like, water-soluble polysaccharide were also present. These polysaccharides alone would barely cause tunic deformation. If the tunic's flexible deformation is self-controlled, it would have another element in addition to the polysaccharide, and it would be an ideal model for designing a novel material. The hypothesis of this study was that the H. roretzi tunic has a nervous system, myocytes, and elastic fibers controlling its flexible deformation. Using acetylcholine and touch as stimuli, the tunic samples were found to deform at acetylcholine concentrations ≥20 µM, indicating the existence of a nervous system, and to have mechanosensitivity. The Bodian, Kluver-Barrera, acetylcholinesterase, immunohistochemical (α-smooth muscle actin), and Elastica-Masson staining methods indicated the presence of a nervous system, myocytes, a region rich in α-smooth muscle actin, and elastic fibers, validating the hypothesis. The self-controlled system in the tunic is useful to understand the properties of the tunic and to design a novel actively deforming material.

To examine the trophic relationship between the sea squirt Halocynthia roretzi and the Pacific oyster Crassostrea gigas cocultured in suspension, their δ 13 C and δ 15 N values were monitored monthly on the southern coast of Korea for a period of 2 yr. Suspended particulate organic matter was fractionated as coarse (>20 µm, CPOM) and fine particles (

Summary We studied hemocyte differentiation in the juveniles of the ascidian Halocynthia roretzi by light and electron microscopy. In the 2d juveniles post-metamorphosis, the hemocoel opens and hemocytes firstly differentiate in it. Those hemocytes were classified into vacuolated cells, granular cells, giant cells, and undifferentiated mesodermal cells. Around day 5 after metamorphosis the mesodermal cells gradually transform to hemoblasts. Therefore the juveniles have only three categories of differentiated hemocytes and the mesodermal cells or the hemoblasts. The scarcity of the hemocyte types and an abundance of immature hemocytes in the juveniles allowed us to follow their differentiation pathways distinctly. We revealed the process of cell differentiation characteristic to each hemocyte category. The circulating hemoblasts most likely function as hematopoietic stem cells in the early stages of ascidian hematopoiesis.

This paper was written in honor of Dr. Noriyuki Satoh who was awarded A.O. Kowalewsky medal, an international prize of the St. Petersburg Society of Naturalists. We congratulate and thank this eminent zoologist for his outstanding scientific achievements, for his life’s work in the discipline founded by A.O. Kowalewsky.

In the vicinity of Yashiro Island in the Inland Sea of Japan, the solitary ascidian (tunicate) Halocynthia roretzi with tunics of various colors were collected. Samples of these animals were sorted into three groups on the basis of visual observation of tunic color. The red group includes animals with dark-red, light-red, or orange tunics. The pink group includes animals with tunic colors ranging between red and white. The white group includes only animals with completely white tunics. Animals in the white group lacked color internally, with the exception of the hepatopancreas and the gonads in breeding season; the epidermis and gill basket were white. In contrast, animals of both the red group and the pink group were colored internally, with red-orange epidermis and yellow gill basket. Alloreactivity was tested by mixed-hemocyte incubation between different animals belonging to the same color group and between animals belonging to different color groups. Alloreactivity between animals of the white group was 56.3%, between animals of the pink group was 60.0%, and between animals of the red group was 69.3%. The relatively high frequency of compatible combinations among the white animals is discussed.

The molecular species of collagen in the muscle layer of the ascidian Halocynthia roretzi was examined by biochemical techniques. Two types of collagen which showed distinct patterns from each other on SDS-PAGE were isolated from the pepsin-solubilized collagen by differential salt precipitation and phosphocellulose column chromatography. They were demonstrated to be genetically distinct from each other by peptide mapping and by amino acid analysis. These results indicate that at least two molecular species of collagen are present in the muscle layer of the ascidian.

Immunohistochemical studies on the neural complex (neural gland, dorsal strand, and cerebral ganglion) of an ascidian, Halocynthia roretzi, were performed by using an antiserum against porcine ACTH. The antiserum recognized a considerable number of the cells scattered along the tubular structure of the dorsal strand and a few cells in the cerebral ganglion. Immunoelectron microscopic studies revealed that the ACTH-like substance resided within secretory granules with diameter of 300-500 nm. Furthermore, those ACTH-immunoreactive cells were demonstrated to be different from PRL-immunoreactive cells, the presence of which had previously been reported.

This study identifies a complement component C3-like protein in the solitary tunicate, Styela plicata. Three different polyclonal antibodies raised against C3 molecules from two species (humans and the tunicate, Halocynthia roretzi) were used to identify the C3-like protein in S. plicata hemolymph. The C3 cross-reactive protein is a 170 kDa heterodimer composed of polypeptides (116 and 80 kDa) that have molecular weights comparable to those of C3α and C3β from other species. Amino acid sequencing and amino acid composition analysis confirmed that the C3-like protein from S. plicata is closely related to C3 from H. roretzi.

To understand the effect of heated effluent from a nuclear power plant on marine organism, experimental culture of Halocynthia roretzi was carried out at heated effluent of Wolsung nuclear power plant from January to December 1996. Temperature was and salinity was during the culture period, The Growth of H. roretzi on lower area of Bonggil-ri (St. 1) was not normal in height, breadth and weight due to heated effluent. Daily growth rate (DGR) of H. roretzi about Kampo area (St, 4) was significant other station, and St. 1 was significant from other station except St. 4, Mytilus edulis was major fouling organism (over ) that were M. edulis, Dideninum moseleyi, Styela clava in experiment culture farm. St. 1 was higher (mean 143 individual) and St. 4 was lower (mean 56 individual) appearance attached of M. edulis. Growth of H. roretzi reduced when attached number of M. eduiis was increased, because correlation between DGR and number of M. edulis was negative.

Background: The kinetoplastid parasite, Azumiobodo hoyamushi, is the causative agent of soft tunic syndrome (STS) in ascidians and leads to their mass mortality in Korean waters. This study was conducted to quantify A. hoyamushi density during the development of STS in the tunics of ascidians (Halocynthia roretzi) using real-time polymerase chain reaction (qPCR). Findings: The infection intensity of A. hoyamushi, as measured by qPCR, varied depending on the part of the tunic analyzed, as well as the stage of STS development. The highest infection intensity was recorded in the tunics of the siphons. The infection intensity of A. hoyamushi in the siphons was only 2.9 cell/tunic (area, 0.25 cm(2)) or 106.0 cell/gram tunic (GT) in the early phase of STS, but this value increased dramatically to 16,066 cells/tunic (0.25 cm(2)) or 617,004 cell/GT at the time of death. The number of A. hoyamushi parasites increased gradually and their distribution spread from the siphons to the other parts of the tunics. Conclusions: qPCR enabled the quantitation of A. hoyamushi and the results revealed that parasite density increased as STS progressed. In addition, our results suggested that the siphons might function as the portal of entry for A. hoyamushi during infection.

金沢大学理学部, The blood cells of a solitary ascidian, Halocynthia roretzi exhibit the allogeneic cellular reaction in vitro denoted as the "contact reaction"(Fuke, 1980). Nine cell types have been recognized in the blood and body fluid of H. roretzi (Fuke and Fukumoto, 1993). In the present study, the precise role of each cell type in allogeneic reactions is investigated in vitro. The vacuolated cells show devacuolation after contact with almost all other cell types from different reactive animals. These cells include hyaline amoebocytes, granular amoebocytes, macrogranular cells, small amoebocytes and giant cells (large basophilic cells), as well as vacuolated cells. The hyaline amoebocytes and small amoebocytes, which belong to the phagocyte series exhibit contact reactions with the cells of phagocyte series from other specimens of H. roretzi. They also show a contact reaction when in contact with granular amoebocytes. Therefore, almost all cell types have the materials responsible for individuality on their cell surface and can directly show the allogeneic cellular reactivity when they contact each other. To determine whether the contact reaction is involved in cell death, the loss of plasma membrane integrity was examined using fluorescent dyes. The number of cells showing uptake of ethidium bromide increased immediately after mixing of allogeneic cells. Almost the same cell types as those showing allogeneic behavior by light microscopy, as described above, exhibited loss of cell membrane integrity. These results are discussed in the context of immune systems in invertebrates and vertebrates.

The occurrence of soft tunic syndrome in wild populations of the ascidian Halocynthia roretzi was monitored by diving at 5 to 6 sites in Miyagi Prefecture in Japan in summer 2010 and 2011. These sites were located at varying distances from farming sites at which the disease had previously been detected. All dead ascidians were collected, and their tunics were examined for Azumiobodo hoyamushi, the causative agent of soft tunic syndrome, using 18S rRNA PCR. In both years

The sea squirt Halocynthia roretzi is an important marine food resource species that is found in the waters around Korea. We describe the isolation and characterization of 13 new polymorphic microsatellite loci in 96 sea squirt samples that were collected from the marine environment of Samcheok on the east coast of Korea. The number of alleles that were observed for each locus ranged from six to 32, and the value of expected and observed heterozygosities was 0.504-0.922 and 0.396-0.813, respectively. These markers will be useful tools for future population studies.

Hclocynthia roretzi (Drasche) is a mid- and infra-littoral edible ascidian, occurring in the Japan Sea and vicinity. It has not yet been recorded by modern ascidian taxonomists from the shores of Mie and Aichi Prefectures on the Pacific coast of central Japan, while it is known to occur nearby in Osaka and Sagami Bays. Results of a questionnaire addressed in 1996 to an the marine fishermen's associations of these two prefectures and some additional inquiries revealed that some shores have been inhabited by the ascidian since 1990 or earlier (i. e., as long as 20 or 30 years). The ascidian was obviously referable to H. roretzi "Type C" sensu Numakunai & Hoshino (1973, 1974). Surveys of historical documents and old literature showed that the ascidian may have occurred in this area early in the 10th century (in Mikawa Bay), in the middle of the Edo Period (in Ago May of the Kii Peninsula), and around 1897 (off Toba in Ise Bay). The possibility that H. roretzi is an index species of marine environmental changes in central Japan is briefly discussed.

Ascidians are known to accumulate high levels of vanadium in their blood cells. Recently, we found a vanadium-associated protein (VAP) in blood cells of a vanadium-rich ascidian, Ascidia sydneiensis samea. In this paper, we raised a monoclonal antibody against VAP, designated F8DH. Immunoblot analysis showed that F8DH recognized 2 related peptides of 15 kDa and 16 kDa of VAP. Using F8DH, VAP was shown to be in the cytoplasm of vanadocytes and compartment cells, both of which were reported to contain vanadium. F8DH also stained the vanadocytes distributed in the connective tissues around the alimentary canal, suggesting that vanadocytes in the connective tissue contained VAP. Furthermore, blood cells of 3 different species of ascidian having high levels of vanadium, A. sydneiensis samea, A. ahodori, and Ciona intestinalis, showed reactivity of F8DH but little reactivity was observed in 2 species having less vanadium, Halocynthia roretzi and Pyura michaelseni, suggesting that VAP recognized by F8DH...

Two distinct cDNAs encoding troponin C (TnC) isoforms were isolated from the ascidian, Halocynthia roretzi. One is expressed in adult body wall smooth muscle and heart muscle, and the other in larval striated muscle. The H. rorezti gene is composed of 7 exons separated by 6 introns, and Southern blot analysis showed that TnC is a single copy gene product. The two isoforms of TnC were derived through the alternative splicing of the third exon.

The plasma of a solitaly ascidian, Halocynthia roretzi, stimulates the proliferation of mouse thymocytes in the presence of a mitogen, phytohemagglutinin P (PHA-P). Eight factors capable of stimulating the proliferation of mouse thymocytes were isolated from H. roretzi plasma by procedures including hydrophobic chromatography, gel filtration, and anion-exchange chromatography. Four active fractions with molecular weights of 100–110 K, 75–90 K, 10–30 K, and 5–6 K were first separated by gel filtration. Each of these fractions was then separated into two fractions with different ionic properties by anion-exchange chromatography. The eight factors thus obtained have different heat stabilities, heparin-binding abilities, and PHA-P sensitivities.