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A novel carotenoid with a unique 2,6-cyclo-ψ-end group, named roretziaxanthin (1), was isolated from the sea squirt Halocynthia roretzi as a minor carotenoid along with (3S,3′S)-astaxanthin, alloxanthin, halocynthiaxanthin, mytiloxanthin, mytiloxanthinone, etc. This structure was determined to be 3-hydroxy-1′,16′-didehydro-1′,2′-dihydro-2′,6′-cyclo-β,ψ-carotene-4,4′-dione by UV–VIS, MS, and NMR spectral data. The formation mechanism of roretziaxanthin in the sea squirt was discussed.

Marine ascidian is becoming one of the main sources of an antitumor drug that has shown high bioactivity and extensive application in cancer treatment. Halocynthia roretzi, an edible marine sea squirt, has been demonstrated to have various kinds of biological activities, such as anti-diabetic, anti-hypertension, and enhancing immunity. In this study, we reported that aqueous extracts from the edible parts of H. roretzi presented significantly inhibiting the efficiency on HepG-2 cell viability. The separate mixed compound exhibited strong effects of inhibitory proliferation and induced apoptosis via the generation of ROS along with the concurrent loss of mitochondrial membrane potential on tumor cells. Furthermore, we found that there existed a significantly synergistic effect of the ascidian-extracted compound mixture with the anti-cancer drug doxorubicin. In the presence of the extracts from H. roretzi, the dose of doxorubicin at the cellular level could be reduced by a half dose. The extracts were further divided by semipreparative-HPLC and the active ingredients were identified as a mixture of fatty amide, which was composed of hexadecanamide, stearamide, and erucamide by UHPLC-MS/MS. Our results suggest that the potential toxicity of ascidian H. roretzi in tumor cells, and the compounds extracted from H. roretzi could be potentially utilized on functional nutraceuticals or as an adjunct in combination with chemotherapy.

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.

Background:It has been previously reported that the tunic ofHalocynthia roretzi, mainly composed of cellulose, is actively deformed with mass transfer by the mechanical stimuli.Objective:In this study, how the tunic deforms in response to the mechanical environment was investigated.Method:The tunic specimen in the artificial seawater was still at 5˚C or underwent the mechanical stimuli at the temperature less than 10˚C. The mass and moisture content of the tunic, the concentrations of nitrate and dissolved organic matter in the artificial seawater used for the tunic, and the histological characteristics were evaluated.Results:The increase in mass of the tunic became lower as the region was closer to the bottom ofHalocynthia roretzi. However, the decrease in mass caused by the mechanical stimuli was not different between the adjacent regions. Also, the tunic of the siphon, the tubular tissue for influx and efflux of the seawater, increased the mass more slowly after the stimuli. The size of the layer covering the outside of the tunic was inversely related to the increment in mass. The change in mass was corresponding to that in water content. The concentrations of nitrate and dissolved organic matter in the artificial seawater were enhanced 5 days after the stimuli while the concentration ratio of dissolved organic matter to nitrate was kept constant.Conclusion:The water content in the tunic was used for controlling the mass response to the mechanical environment.

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.

The broad temperature range on the temperate coast of Korea has led fishermen to develop a unique and specialized procedure for the longline culture of ascidians. In Korea, warming of the coastal sea in winter has accelerated over the past few decades. After warmer winters, the rising temperatures of earlier springs have precluded the rearing of the ascidian spat in the culturing area and have imposed spring spat cultivation in colder-water nursery grounds. To examine the seasonal dynamics of energy reserves and the physiological strategies to optimize energy balance for the growth in the cultured sea squirt Halocynthia roretzi, its gross biochemical composition and physiological energetics were investigated monthly over two culturing periods from July 2005 to January 2006 and July 2013 to January 2014 in Geoje-Hansan Bay on the south coast of Korea. No indicators of the growth performance of sea squirts showed differences between the two culture practices (2005–2006 and 2013–2014) carried out using spat reared in different localities, i.e., the grow-out area and the colder nursery grounds. The seasonal patterns in accumulation and utilization of biochemical constituents in the sea squirt tissues were similar between the two periods. During the culturing period in the grow-out area, sea squirts retained physiological functions across the temperature range. Food energy acquisition and metabolic cost were positively related to water temperatures in the field conditions, probably due to the low and narrow range of suspended particulate matter (SPM) concentrations. A less-clear seasonal variability in food consumption rates yielded a seasonal discrepancy in scope for growth (SFG; i.e., negative during summer vs positive during autumn–winter). Consequently, the tissue weight and protein reserves of sea squirts varied concomitantly with the seasonal changes in SFG, supporting a fast growth during autumn–winter. Our results suggest that the spat reared in colder nursery grounds are suitable for cultivating sea squirts in the traditional grow-out area and support their sustainable culturing performance as an adaptation strategy to the winter–spring warming conditions that are unique to coastal seas.

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.

This study was designed to examine antioxidant and moisturizing effect of purified GAGS from ascidian tunics as functional cosmetic ingredient. A skin-tonner and essence which contain 1.0% of GAG were manufactured conventionally. These product then were tested for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, collagenase inhibitory activity, collagen-formation stimulator, and fibroblast-proliferation promoter abilities. The DPPH scavenging activity for skin-tonner and essence were 20.5% and 12.4% respectively. Collagenase inhibitory activity of skin-tonner was higher (94.6%) than the essence (80.1%). Skin-tonner and essence with GAGs showed collagen-formation stimulation ability, which were 129.9% and 120.2% respectively. These products also demonstrated significant (p

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.

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. Electronic supplementary material The online version of this article (10.1186/s40851-017-0078-3) contains supplementary material, which is available to authorized users.

Highly crystalline oriented Halocynthia roretzi cellulose Iβ films were investigated by IR-spectroscopy between −180 and +10 °C. Changes in the IR-spectra induced by temperature were compared to published changes induced by mechanical stretching. This made it possible to conclude that frequency shifts in the O–H stretching region of the IR-spectra due to temperature were not predominantly an indirect effect of thermal expansion leading to greater O–O distances, but were due directly to the effect of temperature on the O–H···O hydrogen bonds. Temperature induced frequency shifts of C–H stretching bands were consistent with the presence of weak inter-sheet C–H···O bonds. Furthermore, no phase transition in cellulose Iβ was found between −180 and +10 °C.

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 ...

The infectious kinetoplastid Azumiobodo hoyamushi causes 'soft tunic syndrome', a serious problem in aquaculture of the edible ascidian Halocynthia roretzi. Infection tests using dis- eased tunics demonstrated that juvenile (0.8 yr old) individuals never developed soft tunic syn- drome, but all individuals in the other age groups (1.8, 2.8, and 3.8 yr old) showed the disease symptoms. In the infection tests, tunic softening was first observed at the tunic around siphons. Based on ultrastructural observation of the inner wall of the branchial siphon, the tunic lining the inner wall in juveniles (0.5 yr old) was completely covered with cuticle, which had a dense struc- ture to prevent bacterial and protist invasion. In contrast, the tunic was often partly damaged and not covered with cuticle in healthy adults (≥2.5 yr old). The damaged tunic in the siphon wall could be an entrance for A. hoyamushi into the tunic of adult hosts.

Fresh sea squirt meat requires a modified processing and preservation process because it has a short shelf life due to its high mois ture content and strong proteolytic enzyme activity. In this study, bottled sea squirt meat prepared in vegetable oil (BSMO) to enhance the consumer acceptability was exposed to γ-ray (Co60, 10 kGy/h) irradiation to extend the shelf life without the use of a heating process. Response surface methodology was used to determine the optimal mixing ratio of BSMO containing 5% dehydrated fresh meat. Texture analysis and nutritional evaluation were also performed on a control and BSMO samples. The volatile basic nitrogen (VBN) content and total cell count were measured to determine the shelf life of irradiated BSMO products during chilled storage at 4°C for 60 days. According to a panel of 10 trained tasters (aged 20-29 years), the optimal mixing formulation was 80 g meat in 60 mL of mixed vegetable oil (30 mL of olive oil and 30 mL of sesame oil). The highest rated formulation, according to a panel of nine trained tasters (aged ≥30 years), was 80 g meat in 60 mL of mixed vegetable oil (42 mL of olive oil and 18 mL of sesame oil). Moisture, ash, and protein contents in BSMO did not change significantly ( P < 0.05) compared with the control. A higher lipid content (0.84 ± 0.23 to 2.13 ± 0.61; P < 0.05) was observed due to the presence of vegetable oil on the surface of BSMO. The vegetable oil raised the hardness, springiness, cohesiveness, gumminess, chewiness, and resilience of BSMO. BSMO products remained edible after 50 days of storage at 4°C based on the VBN content (BSMO 1: 27.92 ± 0.96 mg/100 g, BSMO 2: 24.84 ± 1.95 mg/100 g) and total cell count (BSMO 1: 4.60 ± 0.80 log CFU/mL, BSMO 2: 3.65 ± 0.20 log CFU/mL) when compared with standard levels of VBN (25.00 mg/100 g) and total cell count (5 log CFU/mL), respectively. The results showed that irradiated BSMO products could help to expand the processed seafood market and increase the popularity of seafood among the younger generations.

【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.】

Mass mortality of the edible sea squirt Halocynthia roretzi since the 1990s in the southern and eastern seas of Korea has caused large economic losses. The disease is characterized by symptoms of initially softened and thinned tunics that eventually rupture. Thus, the disease is called soft tunic syndrome (STS); however, the causative agent in these regions is unknown. In the present study, two kinetoplastid organisms were isolated from STS sea squirts collected from culture farms in Tongyeong located in the East Sea of Korea. Phylogenetic analysis of 18S rRNA sequences identified these organisms as Azumiobodo hoyamushi and Procryptobia sorokini. These kinetoplastids were injected into healthy sea squirts and cultured at 15 °C for 13 days. Sea squirts injected with A. hoyamushi showed 100% STS whereas, P. sorokini did not induce disease, thereby confirming A. hoyamushi as the causative agent of STS. A. hoyamushi flourishes in vitro at 10–15 °C, and dies at temperatures below 5 °C or above 20 °C. The optimum salinity level for growth is 30–35 psu, and death occurs below 25 psu. These optima coincide with marine temperature and salinity levels between March and June on the southern coasts of Korea, the period when the syndrome occurs at the highest frequency. The identification here of A. hoyamushi as the causative agent of STS and our findings regarding its optimum growth conditions should lead to methods for reducing the incidence of STS.

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)

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.

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.

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.

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.