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BackgroundSelf-incompatibility, fusion/non-fusion reactions, and contact reactions (CRs) have all been identified as allorecognition phenomena in ascidians. CR is a reaction characteristic of the hemocytes ofHalocynthia roretzi, whereby they release phenol oxidase (PO) upon contact with non-self hemocytes. Thus, these cells may represent a primitive form of the vertebrate immune system. In the present study, we focused on the CR ofH. roretzihemocytes and sought to identify self-marker proteins that distinguish between self and non-self cells.ResultsWe initially generated a CR-inducing monoclonal antibody against the complete hemocyte membrane-protein complement (mAb11B16B10). This antibody was identified based on the differential induction of PO activity in individual organisms. The level of PO activity induced by this antibody in individual ascidians was consistent with the observed CR-induced PO activity. mAb11B16B10 recognized a series of 12 spots corresponding to a 100-kDa protein, with differing isoelectric points (pIs). A comparison of the 2D electrophoresis gels of samples from CR-reactive/non-reactive individuals revealed that some spots in this series in hemocytes were common to the CR-non-inducible individuals, but not to CR-inducible individuals. We cloned the corresponding gene and named itHalocynthia roretziself-marker-like protein-1 (HrSMLP1). This gene is similar to the glycoprotein DD3–3 found inDictyostelium,and is conserved in invertebrates.ConclusionWe generated a CR-inducing monoclonal antibody (mAb11B16B10) that recognized a series of novel membrane proteins (HrSMLP1) in the hemocytes ofH. roretzi. The combination of expressed spots of HrSMLP1 distinguishes non-self cells from self cells with respect to CR inducibility. Given that the HrSMLP1 gene is a single gene, it may represent a novel type of self-marker protein with a role in CR.

Ascidians are excellent, yet underused, marine sources of unique bioactive compounds of high nutritive content. However, reports regarding the lipid composition of ascidians are rare. In this study, using ultra-high-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry, we aimed to characterize the lipid profile of the tunics and inner body tissues of three species of ascidians, including Ciona intestinalis, Halocynthia roretzi, and Styela clava. We identified over 245 molecular species from 13 major lipid subclasses; glycerophospholipids (GP) and glycerolipids were the dominant lipid components in these three ascidian species (accounted for 66.30-90.60% of total lipids). Importantly, GP enriched in polyunsaturated fatty acids mainly existed in the inner body tissues of ascidians, which accounted for 18.17-32.47% of total lipids. Considering the high level of GP, we proposed that ascidians can be potentially used as health-promoting food for humans.

Azumiobodo hoyamushi, a kinetoplastid flagellate, is the causative agent of soft tunic syndrome, an infectious disease of the edible ascidian Halocynthia roretzi. The flagellate is thought to invade the tunic matrix via a damaged area of the tunic on the siphon wall. We hypothesized that the flagellate locates the tunic entry site by a chemotactic response to soluble substances diffused from the host ascidians. To investigate this hypothesis, we examined whether the flagellate shows a chemotactic response to tissue extracts (tunic and other tissues) from the host ascidian H. roretzi. We tested extracts from 5 tissues as well as hemolymph. Only the tunic extract showed significant positive chemotactic activity, and the activity decreased with increasing dilution. Furthermore, autoclaved tunic extract, extracts from diseased individuals, and extract from the styelid ascidian Styela clava also had chemotactic activity, although the activities were lower than that of tunic extract from healthy H. roretzi. Ultrafiltration of the tunic extract through a 3 kDa cutoff membrane completely abrogated the activity; the ultrafiltration retentate still showed activity. Thus, the soluble factors that attract the flagellate are present exclusively in the tunic extract, and the chemotactic factors are larger than 3 kDa. Our experiments also suggested that the tunic extract contains both heat-stable and heat-labile factors. We conclude that the flagellate locates the tunic entry site by chemotaxis toward soluble factors that diffuse from a damaged area of the tunic on the siphon wall.

Substitution effect of sea tangle (ST) with tunic of sea squirt (SS) in diet on growth and carcass composition of juvenile abalone was determined. One thousand four hundred and seventy abalones were distributed into 21 containers. Six formulated diets in triplicate were prepared. A 200 g/kg ST was included into the ST0 diet. The 200, 400, 600, 800 and 1000 g/kg of ST were substituted with the same amount of tunic of SS, referred to as the ST200, ST400, ST600, ST800 and ST1000 diets, respectively. Finally, Undaria was prepared to compare effect of the formulated diets on performance of abalone. The experimental diets were fed to abalone for 16 weeks. Weight gain of abalone fed the ST400 diet was higher than that of abalone fed the ST0, ST600, ST800 and ST1000 diets and Undaria. Weight gain of abalone fed the formulated diets was higher than that of abalone fed the Undaria. The chemical composition of the carcass of abalone was affected by dietary substitution of ST with tunic of SS. In conclusion, ST could be completely substituted with tunic of SS without retardation in performance of abalone. Abalone fed the ST400 diet substituting 400 g/kg ST with tunic of SS achieved the best growth.

Seven oligonucleotides primers were shown to generate the shared loci, specific loci, unique shared loci to each species and shared loci by the three species which could be obviously scored. In the present study, 7 oligonucleotides primers produced 401 total loci in the Styela clava (SC) species, 390 in the Halocynthia roretzi (HR) and 434 in the Styela plicata (SP), respectively. Seven oligonucleotides primers generated 275 specific loci in the SC, 341 in the HR and 364 in the SP species, respectively. The oligonucleotides primer BION-23 generated 28 unique loci to each species in the SP species. Especially, the oligonucleotides primer BION-25 produced 7 unique loci to each species, which were identifying each species in the SP species. BION-17 distinguished 21 shared loci by the three ascidian species, major and/or minor fragments of sizes, which were identical in almost all of the samples. Based on the average bandsharing values of all samples, the similarity matrix ranged from 0.519 to 0.774 in the SC species, from 0.261 to 0.683 in the HR species and from 0.346 to 0.730 in the SP species. As regards average bandsharing value (BS) results, individuals from SC species (0.661±0.081) exhibited higher bandsharing values than did individuals from HR species (0.555±0.074) (P

Ascidians are increasingly being used as a system for investigating cell biology during development. The extreme genetic and cellular simplicity of ascidian embryos in combination with superior experimental tractability make this an ideal system for in vivo analysis of dynamic cellular processes. Transgenic approaches to cellular and sub-cellular analysis of ascidian development have begun to yield new insights into the mechanisms regulating developmental signaling and morpho-genesis. This chapter focuses on the targeted expression of fusion proteins in ascidian embryos and how this technique is being deployed to garner new insights into the cell biology of development.

Soft tunic syndrome is a fatal disease in the edible ascidian Halocynthia roretzi, causing serious damage to ascidian aquaculture in Korea and Japan. In diseased individuals, the tunic, an integumentary extracellular matrix of ascidians, softens and eventually tears. This is an infectious disease caused by the kinetoplastid flagellate Azumiobodo hoyamushi. However, the mechanism of tunic softening remains unknown. Because cellulose fibrils are the main component of the tunic, we compared the contents and structures of cellulose in healthy and diseased tunics by means of biochemical quantification and X-ray diffractometry. Unexpectedly, the cellulose contents and structures of cellulose microfibrils were almost the same regardless of the presence or absence of the disease. Therefore, it is unlikely that thinning of the microfibrils occurred in the softened tunic, because digestion should have resulted in decreases in crystallinity index and crystallite size. Moreover, cellulase was not detected in pure cultures of A. hoyamushi in biochemical and expressed sequence tag analyses. These results indicate that cellulose degradation does not occur in the softened tunic.

Background Hox gene clusters with at least 13 paralog group (PG) members are common in vertebrate genomes and in that of amphioxus. Ascidians, which belong to the subphylum Tunicata (Urochordata), are phylogenetically positioned between vertebrates and amphioxus, and traditionally divided into two groups: the Pleurogona and the Enterogona. An enterogonan ascidian, Ciona intestinalis (Ci), possesses nine Hox genes localized on two chromosomes; thus, the Hox gene cluster is disintegrated. We investigated the Hox gene cluster of a pleurogonan ascidian, Halocynthia roretzi (Hr) to investigate whether Hox gene cluster disintegration is common among ascidians, and if so, how such disintegration occurred during ascidian or tunicate evolution. Results Our phylogenetic analysis reveals that the Hr Hox gene complement comprises nine members, including one with a relatively divergent Hox homeodomain sequence. Eight of nine Hr Hox genes were orthologous to Ci-Hox1, 2, 3, 4, 5, 10, 12 and 13. Following the phylogenetic classification into 13 PGs, we designated Hr Hox genes as Hox1, 2, 3, 4, 5, 10, 11/12/13.a, 11/12/13.b and HoxX. To address the chromosomal arrangement of the nine Hox genes, we performed two-color chromosomal fluorescent in situ hybridization, which revealed that the nine Hox genes are localized on a single chromosome in Hr, distinct from their arrangement in Ci. We further examined the order of the nine Hox genes on the chromosome by chromosome/scaffold walking. This analysis suggested a gene order of Hox1, 11/12/13.b, 11/12/13.a, 10, 5, X, followed by either Hox4, 3, 2 or Hox2, 3, 4 on the chromosome. Based on the present results and those previously reported in Ci, we discuss the establishment of the Hox gene complement and disintegration of Hox gene clusters during the course of ascidian or tunicate evolution. Conclusions The Hox gene cluster and the genome must have experienced extensive reorganization during the course of evolution from the ancestral tunicate to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene arrangement on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to tunicates as well as later ascidian lineage-specific events.

The high crystallinity and the high microfibrils aspect ratio of tunicate cellulose (TC) indicate TC's excellent chemical and material applications. However, its quantity and quality from different ...

【This study was carried out to investigate nutritional and physiologically active characterizations of sikhae and the seasoned products from the sea squirt Halocynthia roretzi. The total taste values of sikhae fermented for 4 and 5 days were 10.9 and 15.4, respectively, which was lower than for commercially seasoned sea squirts. The sikhaes contained mostly glutamic and aspartic acids. The total amino acid contents of sikhaes fermented for 4 and 5 days were 5.5 and 6.0 g/100 g, respectively, which were lower than those of commercial seasoned-sea squirts or similar. An amount of 100 g of sikhae and its seasoned products contained P, K, Mg and Fe, and these minerals, which are deemed good for our health, were at 10% above the recommended daily requirements. The functional properties of sikhae fermented for 4 and 5 days were as follows: for ACE inhibiting activity, 69 and 69.5%, respectively; for antioxidative activity, 28.9 and 29.3%, respectively; for xanthine oxidase inhibitory activity, 52.8 and 53.1%, respectively; and for ${\alpha}$ -glucosidase inhibitory activity, 2.4 and 1.4%, respectively. Antimicrobial activity of the 5 day fermented sikhae against Vibrio parahaemolyticus and Staphyloccus aureus was detected in 8 mm and in 7 mm against Escherichia coli.】

Background miRNAs play essential roles in the modulation of cellular functions via degradation and/or translation attenuation of target mRNAs. They have been surveyed in a single ascidian genus, Ciona. Recently, an annotated draft genome sequence for a distantly related ascidian, Halocynthia roretzi, has become available, but miRNAs in H. roretzi have not been previously studied. Results We report the prediction of 319 candidate H. roretzi miRNAs, obtained through three complementary methods. Experimental validation suggests that more than half of these candidate miRNAs are expressed during embryogenesis. The majority of predicted H. roretzi miRNAs appear specific to ascidians or tunicates, and only 32 candidates, belonging to 25 families, are widely conserved across metazoans. Conclusion Our study presents a comprehensive identification of candidate H. roretzi miRNAs. This resource will facilitate the study of the mechanisms for miRNA-controlled gene regulatory networks during ascidian development. Further, our analysis suggests that the majority of Halocynthia miRNAs are specific to ascidian or tunicates, with only a small number of widely conserved miRNAs. This result is consistent with the general notion that animal miRNAs are less conserved between taxa than plant ones. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3707-5) contains supplementary material, which is available to authorized users.

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)

The vascular endothelial growth factor (VEGF)-VEGF Receptor (VEGFR) system is an important pathway for regulation of angiogenesis. However, its evolutionary development, particularly the step from invertebrates to vertebrates, is still largely unknown. Here, we molecularly cloned the VEGFR-like gene from Halocynthia roretzi, a species belonging to the Tunicata, the chordate subphylum recently considered the sister group of vertebrates. The cDNA encoded a homolog of human VEGFR, including the transmembrane domain, and the tyrosine kinase domain with a kinase-insert region, which was designated S. sq VEGFR (GenBank AB374180). Similar to Tunicates including ascidians in the phylogenetic tree, the Amphioxus, another chordate, is located close to vertebrates. However, S. sq VEGFR has a higher homology than the Amphioxus VEGFR-like molecule (GenBank AB025557) to human VEGFR in the kinase domain-2 region. The S. sq VEGFR mRNA was expressed at highest levels in circulatory system-containing tissues, suggesting that S. sq VEGFR plays an important role in the formation or maintenance of circulatory system in Tunicates, Halocynthia roretzi.

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.

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.

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.

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.

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 (

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.

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.

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.

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 pteridine-containing bromophysostigmine alkaloids, urochordamine A and B, which were isolated from ascidians as larval metamorphosis promoters, were converted to more polar compounds, urochordamine A′ and B′, respectively, when left standing in protic solvents. These four compounds promoted larval metamorphosis of the ascidianHalocynthia roretzi in the order A′>A>B>B′, and induced metamorphosis of the pediveliger larvae of the musselMytilus edulis galloprovincialis.

Four new 3,4-dihydroxystyrene dimers (3, 4, 5, and 9) have been isolated from a marine sponge Jaspis sp. along with the known narains (1 and 2), of which 3–5 induced metamorphosis of ascidian Halocynthia roretzi larvae. Structures of these metabolites were elucidated on the basis of spectral data. Biogenesis of these compounds is also discussed.

Cytogenetic features of three ascidian species were studied with specimens obtained from Korean coastal areas. The gonadal tissue was processed in order to obtain mitotic and meiotic chromosomes. The chromosome numbers of Amaroucium pliciferum turned out to be 2n=26 and n=13 with 2sm, 3st, 8t and Halocynthia roretzi to be 2n=16 and n=8 with 6m, 2t. Dipioid number of chromosomes in Dendrodoa aggregata was 64 with 32t. The karyotypes of these Korean ascidians were reported for the first time.

'Soft tunic syndrome' causes mass mortality in the edible ascidian Halocynthia roretzi in Korean and Japanese aquaculture. In histopathological comparison, there were no specific differences between diseased specimens from Korea and Japan, indicating that soft tunic syndrome occurring in Korea and Japan is the same disease. No bacterial or protozoan cells were microscopically detected in either healthy or diseased tunics suggesting they are not the direct causes of soft tunic syndrome. Attempts were made to isolate virus from affected ascidians taking into account temperature conditions in which soft tunic syndrome is most prevalent in the field. However, no viruses were isolated from diseased or non-diseased specimens using chinook salmon embryo (CHSE-214), flounder fin (FFN) or epithelioma papillosum cyprini (EPC) cell lines.

α-hydroperoxy diethyl peroxide was commonly extracted from the tunic of the titled four different ascidian species. The structure was determined by spectroscopic data, polarographic data and oxidation reactions.

1. 1. Two new lysophosphatidylinositols have been isolated from the siphons of the ascidian Halocynthia roretzi. Their structures were studied by spectroscopic methods. 2. The major lipid contains eicosapentaenoic acid, while the minor lipid contains arachidonic acid. 3. The major compound was anti-fungal against Mortierella ramaniana at 0.2 mg/disk, and hemolytic against sheep erythrocytes at ed 50, 0.18 μM.

Ascidian tadpole larvae possess a primitive nervous system, which is a prospective prototype of the chordate nervous system. It is composed of relatively few cells but sufficient for complex larval behavior. Here we report on HrETR-1, a gene zygotically expressed in a large proportion of the developing neural cells of the ascidian, Halocynthia roretzi. HrETR-1 is an early neural marker which can be used for analyzing neural differentiation. HrETR-1 expression intensified in most neural cells of genes isolated to date, in both central and peripheral nervous systems including palps as early as the 110-cell stage. Using this gene as a probe, we characterized neural cells in the nervous system as well as confirming their origins. Also, we recognized three types of peripheral epidermal neurons which presumably correlate to the larval neurons previously reported for another ascidian. Among these, five bilateral neurons located in the anterior region of the trunk appeared to be derived from a8.26 blastomeres.

There has been a long history of speculation and debate concerning the origin of vertebrates (Garstang, 1928; Berrill, 1955; Jefferies, 1986; Gee, 1996). A widely accepted view is that the tunicates and cephalochordates, invertebrates known as protochordates because of their simple chordate body plans, are the closest relatives of the vertebrates. The features that distinguish vertebrates from protochordates are a head containing a cartilaginous or bony skeleton, a tripartite brain, and elaborate sensory organs. The intricate head of vertebrates is derived in part from the neural crest, whose appearance in chordate evolution coincides with that of the earliest vertebrates (Gans and Northcutt, 1983). Another characteristic feature of vertebrates is their large genomes, which are estimated to contain 60,000–80,000 protein-coding genes (Antequera and Bird, 1993; Brenner et al., 1993; Fields et al., 1994). Protochordates have smaller genomes with fewer genes. Molecular phylogenetic studies support the early separation of the tunicates from the chordate lineage leading to cephalochodates and vertebrates (Turbeville et al., 1994; Wada and Satoh, 1994). As the oldest living chordates, tunicates can provide useful information about vertebrate ancestry.

The developmental roles of factors associated with the nuclear complex of Halocynthia roretzi and Boltenia villosa oocytes were investigated by cutting mature oocytes into animal and vegetal merogons before and during GVBD. Animal and vegetal merogons were cultured in sea water until the GV cytoplasm had dispersed within the cytoplasm of control oocytes and then they were cross-fertilized and scored for their ability to undergo normal development. Halocynthia oocyte fragments produced from the animal region of oocytes containing intact GVs exhibited a low frequency of polyspermy, a high frequency of fertilization and cleavage, and a high frequency of expressing an epidermal antigen, Epi-2. In contrast, merogons produced from the vegetal region of Halocynthia oocytes in which GVs were intact exhibited a high frequency of polyspermy, did not undergo cell division, and expressed a high frequency of Epi-2 expression. When vegetal fragments were produced after the dispersal of approximately 50-70% of the GV nucleoplasm, these merogons exhibited a low frequency of polyspermy, high frequencies of cell division (including the formation of epidermal layer), and in most cases expressed Epi-2. Vegetal Boltenia fragments produced during GVBD in some cases developed into larvae. These results suggest that the ascidian GV nucleoplasm may contain factors required for fertilization and cell division and that epidermal determinants reside in the oocyte cytoplasm.

Four new antibacterial and antifungal sulfates, 2,6-dimethylheptyl sulfate [1], (4Z,7Z)-4,7-decadienyl sulfate [2], (4Z,7E)-4,7-decadienyl sulfate [3], and (3Z,6Z)-3,6,9-decatrienyl sulfate [4], have been isolated from the hepatopancreas of the ascidian Halocynthia roretzi. The structures were determined by spectral analysis.

A new bisguanidinium alkaloid, stellettadine A (1), containing a norsesquiterpene unit has been isolated from a marine sponge Stelletta sp. as a metamorphosis-inducing compound. Its structure including the absolute stereochemistry was elucidated on the basis of spectral data and chemical degradation. Compound 1 induced metamorphosis of the ascidian Halocynthia roretzi larvae with an ED100 value of 50 μM.

Two N, N -dimethylguamidinium styryl sulfates, ( E )- and ( Z )-narains, which induce metamorphosis of the ascidian Halocynthia roretzi larvae have been isolated from a marine sponge Jaspis sp. The structures were determined by spectral analysis.

Inactivation of the K inward current through the anomalous rectifier channel of the egg cell membrane of a tunicate,Halocynthia roretzi Drashe, was studied under voltage-clamp. The noise spectrum of the steady-state current recorded at hyperpolarized potentials was measured in solutions in which Na, Cs, Hydrazine, or Sr caused inactivation of the current. The unitary conductance estimated was independent of which cation caused inactivation. From the relation between the concentration of cations which caused inactivation and the extent of inactivation at fixed potentials, the binding of one inactivator to a channel was found to cause inactivation, and the potency of inactivation was Cs+>Hydrazine+>Na+>Li+, and Ba2+>Sr2+. The inactivation caused by Na+ was increased by K+ when [K] o was lower than 20mm, but was decreased by K+ in higher K-ASW (artificial sea water). One K+ was found to inactivate the channel cooperatively with one Na+. Increase of inactivation by K+ was a dominant effect in Cs-ASW. The inactivation was explained quantitatively by a model assuming cooperative plugging by a monovalent inactivator and a K+.

Considerable amounts of water-soluble polysaccharides were found in the test of the tunicate Halocynthia roretzi . After fractionation with ethanol, a chitin sulfate-like polysaccharide was isolated. From the results of chemical analysis, optical rotation, infrared spectrum and alkaline treatment, it is suggested that the predominant structure for this polysaccharide is (1→4)-2-acetamido-2-deoxy-6- O -sulfo- β -D-glucopyranan.

The nucleotide sequences of 5S rRNAs from sponge Halichondria japonica and tunicate Halocynthia roretzi were determined by chemical and enzymatic gel methods. Their phylogenetic positions among metazoans were derived from the 5S rRNA sequences by a computer analysis based on the maximum parsimony principle. It was suggested that the sponge is closely related to several invertebrates and the tunicate has affinity to vertebrates rather than invertebrates.