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Recently, biomacromolecules have received considerable attention in hemostatic materials. Collagen, an ideal candidate for hemostatic sponges due to its involvement in the clotting process, has been facing challenges in extraction from raw materials, which is time-consuming, expensive, and limited by cultural and religious restrictions associated with traditional livestock and poultry sources. To address these issues, this study explored a new shortcut method that using wild Halocynthia roretzi (HR), a marine fouling organism, as a raw material for developing HR collagen fiber sponge (HRCFs), which employed urea to disrupt hydrogen bonds between collagen fiber aggregates. This method simplifies traditional complex manufacturing processes while utilized marine waste, thus achieving dual green in terms of raw materials and manufacturing processes. FTIR results confirmed that the natural triple-helical structure of collagen was preserved. HRCFs exhibit a blood absorption ratio of 2000–3500 %, attributed to their microporous structure, as demonstrated by kinetic studies following a capillary model. Remarkably, the cytotoxicity and hemolysis ratio of HRCFs are negligible. Furthermore, during in vivo hemostasis tests using rabbit ear and kidney models, HRCFs significantly reduce blood loss and shorten hemostasis time compared to commercial gelatin sponge and gauze, benefiting from the capillary effect and collagen's coagulation activity. This study provides new insights into the design of collagen-based hemostatic biomaterials, especially in terms of both raw material and green manufacturing processes.

Halocynthia roretzi, a member of Ascidiacea, is covered with its own tunic, which is composed of polysaccharides, such as cellulose Iβ and sulfated chitin. H. roretzi has an open-vessel system, whose blood vessels and hemocytes are found in the tunic, so that the mechanical environment of the tunic could be carefully controlled because of its influence on hemocyte behaviors. While active deformation of the tunic and related phenomena have been previously reported, the mechanical environment in the tunic, which directly influences its deformation, has been rarely investigated. Meanwhile, the developments of actuators based on cellulose and chitin have been frequently reported. However, a cellulose–sulfated chitin actuator has not been proposed. In this study, the mechanical environment of the tunic, which has been rarely investigated despite its importance in the active deformation of the tunic, was evaluated using finite element analysis. A finite element model of the tunic, based on its histological characteristics as well as deformation patterns, was developed. The results showed that the shape of the tunic, the pattern of fiber distribution, and control of the water content influenced the mechanical environment. [ABSTRACT FROM AUTHOR]

Knowledge of dietary composition is crucial for understanding the trophic ecology of a mariculture species and the available food in aquafarms, which is fundamental for assessing ecosystem health status and determining ecological response to ecosystem variation to develop a better management plan for marine farming. For suspension-feeding invertebrates, the growth and development rely heavily on the availability and quality of phytoplankton. However, their growth rate can be severely impacted by food deficiency or exposure to harmful algae blooms (HABs) and pathogenic parasites, which are major causes of mass mortality in mariculture. This study aimed to conduct a dietary analysis of three important mariculture species — Magallana gigas, Halocynthia roretzi, and Mizuhopecten yessoensis — on three farms in South Korea using DNA metabarcoding. Sequence processing and filtering generated 304 validated amplicon sequence variants from six fecal samples, taxonomically identifying 15 phyla, 18 classes, 32 orders, 39 families, 43 genera, and 46 species. The DNA recovery of Dinoflagellata was predominantly detected in all fecal samples, followed by Apicomplexa, Bacillariophyta, and Ciliophora. Of the three mariculture species, the fecal assemblage of M. gigas exhibited the highest richness and diversity, with a high read proportion of phytoplankton (specifically Dinoflagellata), while such proportions were relatively low in the M. yessoensis samples. Excessive read proportions of parasites (Apicomplexa) were detected in the fecal samples of two H. roretzi, indicating a possible link to as substantial decrease in H. roretzi production observed in South Korea. A total of 27 key species were detected in six fecal samples, including a high read proportion of Prorocentrum triestinum, which is responsible for HABs in Korean coastal waters. These results emphasize the potential of fecal DNA metabarcoding in the genetic monitoring of aquaculture farms/ecosystems and could be a useful reference for develop effective management plans to guarantee sustainable mariculture production.

Halocynthia roretzi, the edible ascidian, has been demonstrated to be an important source of bioactive natural metabolites. Here, we reported a novel terpenoid compound named Halorotetin A that was isolated from tunic ethanol extract of H. roretzi by silica gel column chromatography, preparative layer chromatography (PLC), and semipreparative-HPLC. 1H and 13C NMRs, 1H-1H COSY, HSQC, HMBC, NOESY, and HRESIMS profiles revealed that Halorotetin A was a novel terpenoid compound with antitumor potentials. We therefore treated the culture cells with Halorotetin A and found that it significantly inhibited the proliferation of a series of tumor cells by exerting cytotoxicity, especially for the liver carcinoma cell line (HepG-2 cells). Further studies revealed that Halorotetin A affected the expression of several genes associated with the development of hepatocellular carcinoma (HCC), including oncogenes (c-myc and c-met) and HCC suppressor genes (TP53 and KEAP1). In addition, we compared the cytotoxicities of Halorotetin A and doxorubicin on HepG-2 cells. To our surprise, the cytotoxicities of Halorotetin A and doxorubicin on HepG-2 cells were similar at the same concentration and Halorotetin A did not significantly reduce the viability of the normal cells. Thus, our study identified a novel compound that significantly inhibited the proliferation of tumor cells, which provided the basis for the discovery of leading compounds for antitumor drugs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Halocynthia roretzi, a member of Ascidiacea, is covered with its own tunic, which is composed of polysaccharides, such as cellulose Iβ and sulfated chitin. H. roretzi has an open-vessel system, whose blood vessels and hemocytes are found in the tunic, so that the mechanical environment of the tunic could be carefully controlled because of its influence on hemocyte behaviors. While active deformation of the tunic and related phenomena have been previously reported, the mechanical environment in the tunic, which directly influences its deformation, has been rarely investigated. Meanwhile, the developments of actuators based on cellulose and chitin have been frequently reported. However, a cellulose–sulfated chitin actuator has not been proposed. In this study, the mechanical environment of the tunic, which has been rarely investigated despite its importance in the active deformation of the tunic, was evaluated using finite element analysis. A finite element model of the tunic, based on its histological characteristics as well as deformation patterns, was developed. The results showed that the shape of the tunic, the pattern of fiber distribution, and control of the water content influenced the mechanical environment.

Halocynthia roretzi has been cultured and utilized as a nutritious seafood in Southeastern Asia for many years because of its abundance active molecules. Here, we reported that feeding of H. roretzi tissues resulted in improving oral glucose tolerance in rats. To screen the active components, we identified 950 chemical compounds from H. roretzi metabolome. Furthermore, 11 molecules with the activity of regulating blood glucose and lipids were selected based on structural identity. Among them, two natural products, hesperetin and astaxanthin were selected for further quantitative detection. The results showed that concentrations of hesperetin and astaxanthin in the tissues of H. roretzi were calculated to be 13.7 ± 9.5 and 167.9 ± 48.4 µg/100 g, respectively. The high content of astaxanthin in H. roretzi indicates its potential roles and functions in anti-diabetes activity. Taken together, our results showed that H. roretzi could be used as a functional food supplement for regulating lipid/glucose metabolism.

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. [ABSTRACT FROM AUTHOR]

The annual consumption of fishery products, particularly sea squirt (Halocynthia roretzi), per person has steadily increased in South Korea. However, the quantitative risk of Vibrio parahaemolyticus following intake of sea squirt has not been analyzed. This study focuses on quantitative predictions of the probability of consuming sea squirt and getting of V. parahaemolyticus foodborne illness. The prevalence of V. parahaemolyticus in sea squirt was evaluated, and the time spent by sea squirt in transportation vehicles, market displays, and home refrigerators, in addition to the temperature of each of these, were recorded. The data were fitted to the @RISK program to obtain a probability distribution. Predictive models were developed to determine the fate of V. parahaemolyticus under distribution conditions. A simulation model was prepared based on experimental data, and a dose-response model for V. parahaemolyticus was prepared using data from literature to estimate infection risk. V. parahaemolyticus contamination was detected in 6 of 35 (17.1%) sea squirt samples. The daily consumption quantity of sea squirt was 62.14 g per person, and the consumption frequency was 0.28%. The average probability of V. parahaemolyticus foodborne illness following sea squirt consumption per person per day was 4.03 × 10−9. The objective of this study was to evaluate the risk of foodborne illness caused by Vibrio parahaemolyticus following sea squirt consumption in South Korea.

Halocynthia roretzi, the edible ascidian, has been demonstrated to be an important source of bioactive natural metabolites. Here, we reported a novel terpenoid compound named Halorotetin A that was isolated from tunic ethanol extract of H. roretzi by silica gel column chromatography, preparative layer chromatography (PLC), and semipreparative-HPLC. 1H and 13C NMRs, 1H-1H COSY, HSQC, HMBC, NOESY, and HRESIMS profiles revealed that Halorotetin A was a novel terpenoid compound with antitumor potentials. We therefore treated the culture cells with Halorotetin A and found that it significantly inhibited the proliferation of a series of tumor cells by exerting cytotoxicity, especially for the liver carcinoma cell line (HepG-2 cells). Further studies revealed that Halorotetin A affected the expression of several genes associated with the development of hepatocellular carcinoma (HCC), including oncogenes (c-myc and c-met) and HCC suppressor genes (TP53 and KEAP1). In addition, we compared the cytotoxicities of Halorotetin A and doxorubicin on HepG-2 cells. To our surprise, the cytotoxicities of Halorotetin A and doxorubicin on HepG-2 cells were similar at the same concentration and Halorotetin A did not significantly reduce the viability of the normal cells. Thus, our study identified a novel compound that significantly inhibited the proliferation of tumor cells, which provided the basis for the discovery of leading compounds for antitumor drugs.

Abstract Background Self-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 of Halocynthia 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 of H. roretzi hemocytes and sought to identify self-marker proteins that distinguish between self and non-self cells. Results We 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 it Halocynthia roretzi self-marker-like protein-1 (HrSMLP1). This gene is similar to the glycoprotein DD3–3 found in Dictyostelium, and is conserved in invertebrates. Conclusion We generated a CR-inducing monoclonal antibody (mAb11B16B10) that recognized a series of novel membrane proteins (HrSMLP1) in the hemocytes of H. 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.

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.

Background: Self-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 of Halocynthia 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 of H. roretzi hemocytes and sought to identify self-marker proteins that distinguish between self and non-self cells. Results: We 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 it Halocynthia roretzi self-marker-like protein-1 (HrSMLP1). This gene is similar to the glycoprotein DD3–3 found in Dictyostelium, and is conserved in invertebrates. Conclusion: We generated a CR-inducing monoclonal antibody (mAb11B16B10) that recognized a series of novel membrane proteins (HrSMLP1) in the hemocytes of H. 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. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract 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.

Recently, biomacromolecules have received considerable attention in hemostatic materials. Collagen, an ideal candidate for hemostatic sponges due to its involvement in the clotting process, has been facing challenges in extraction from raw materials, which is time-consuming, expensive, and limited by cultural and religious restrictions associated with traditional livestock and poultry sources. To address these issues, this study explored a new shortcut method that using wild Halocynthia roretzi (HR), a marine fouling organism, as a raw material for developing HR collagen fiber sponge (HRCFs), which employed urea to disrupt hydrogen bonds between collagen fiber aggregates. This method simplifies traditional complex manufacturing processes while utilized marine waste, thus achieving dual green in terms of raw materials and manufacturing processes. FTIR results confirmed that the natural triple-helical structure of collagen was preserved. HRCFs exhibit a blood absorption ratio of 2000-3500 %, attributed to their microporous structure, as demonstrated by kinetic studies following a capillary model. Remarkably, the cytotoxicity and hemolysis ratio of HRCFs are negligible. Furthermore, during in vivo hemostasis tests using rabbit ear and kidney models, HRCFs significantly reduce blood loss and shorten hemostasis time compared to commercial gelatin sponge and gauze, benefiting from the capillary effect and collagen's coagulation activity. This study provides new insights into the design of collagen-based hemostatic biomaterials, especially in terms of both raw material and green manufacturing processes., Competing Interests: None., (© 2024 The Authors. Published by Elsevier Ltd.)

Abstract Stereotyped left–right asymmetry both in external and internal organization is found in various animals. Left-right symmetry is broken by the neurula rotation in the ascidian, Halocynthia roretzi. Neurula embryos rotate along the anterior–posterior axis in a counterclockwise direction, and the rotation stops when the left side of the embryo is oriented downwards, resulting in contact of the left-side epidermis with the vitelline membrane at the bottom of perivitelline space. Then, such contact induces the expression of nodal and its downstream Pitx2 gene in the left-side epidermis. Vitelline membrane is required for the promotion of nodal expression. Here, we showed that a chemical signal from the vitelline membrane promotes nodal gene expression, but mechanical stimulus at the point of contact is unnecessary since the treatment of devitellinated neurulae with an extract of the vitelline membrane promoted nodal expression on both sides. The signal molecules are already present in the vitelline membranes of unfertilized eggs. These signal molecules are proteins but not sugars. Specific fractions in gel filtration chromatography had the nodal promoting activity. By mass spectrometry, we selected 48 candidate proteins. Proteins that contain both a zona pellucida (ZP) domain and epidermal growth factor (EGF) repeats were enriched in the candidates of the nodal inducing molecules. Six of the ZP proteins had multiple EGF repeats that are only found in ascidian ZP proteins. These were considered to be the most viable candidates of the nodal -inducing molecules. Signal molecules are anchored to the entire vitelline membrane, and contact sites of signal-receiving cells are spatially and mechanically controlled by the neurula rotation. In this context, ascidians are unusual with respect to mechanisms for specification of the left-right axis. By suppressing formation of epidermis monocilia, we also showed that epidermal cilia drive the neurula rotation but are dispensable for sensing the signal from the vitelline membrane. Highlights • Neurula rotation determines L-R asymmetry in ascidian embryos. • Contact of left-side epidermis with vitelline membrane promotes nodal expression. • The signal molecule is a protein and not a sugar and is present before fertilization. • 48 proteins including six ZP domain proteins were listed as candidates of the signal. • Epidermal cilia are dispensable for sensing the signal from the vitelline membrane. [ABSTRACT FROM AUTHOR]

Tadpole larvae of the ascidian, Halocynthia roretzi , show morphological left–right asymmetry in the brain structures and the orientation of tail bending within the vitelline membrane. Neurula embryos rotate along the anterior–posterior axis in a counterclockwise direction, and then this rotation stops when the left side of the embryo is oriented downwards. Contact of the left-side epidermis with the vitelline membrane promotes nodal gene expression in the left-side epidermis. This is a novel mechanism in which rotation of whole embryos provides the initial cue for breaking left–right symmetry. Here we show that epidermal monocilia, which appear at the neurula rotation stage, generate the driving force for rotation. A ciliary protein, Arl13b, fused with Venus YFP was used for live imaging of ciliary movements. Although overexpression of wild-type Arl13b fusion protein resulted in aberrant movements of the cilia and abrogation of neurula rotation, mutant Arl13b fusion protein, in which the GTPase and coiled-coil domains were removed, did not affect the normal ciliary movements and neurula rotation. Epidermis cilia moved in a wavy and serpentine way like sperm flagella but not in a rotational way or beating way with effective stroke and recovery stroke. They moved very slowly, at 1/7 Hz, consistent with the low angular velocity of neurula rotation (ca. 43°/min). The tips of most cilia pointed in the opposite direction of embryonic rotation. Similar motility was also observed in Ciona robusta embryos. When embryos were treated with a dynein inhibitor, Ciliobrevin D, both ciliary movements and neurula rotation were abrogated, showing that ciliary movements drive neurula rotation in Halocynthia. The drug also inhibited Ciona neurula rotation. Our observations suggest that the driving force of rotation is generated using the vitelline membrane as a substrate but not by making a water current around the embryo. It is of evolutionary interest that ascidians use ciliary movements to break embryonic left–right symmetry, like in many vertebrates. Meanwhile, ascidian embryos rotate as a whole, similar to embryos of non-vertebrate deuterostomes, such as echinoderm, hemichordate, and amphioxus, while swimming. • Neurula rotation determines L-R asymmetry in ascidian embryos. • Epidermis monocilia generate the driving force of neurula rotation. • Epidermis monocilia move slowly in a wavy and serpentine way. • Cilia generate counter-force by crawling on the inner surface of vitelline membrane. • Involvement of cilia in specification of L–R asymmetry is of evolutionary interest. [ABSTRACT FROM AUTHOR]

Abstract 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.

Localized maternal mRNAs play important roles in embryogenesis, e.g. the establishment of embryonic axes and the developmental cell fate specification, in various animal species. In ascidians, a group of maternal mRNAs, called postplasmic/PEM RNAs, is localized to a subcellular structure, called the Centrosome-Attracting Body (CAB), which contains the ascidian germ plasm, and is inherited by the germline cells during embryogenesis. Posterior end mark (Pem), a postplasmic/PEM RNAs member, represses somatic gene expression in the germline during cleavage stages by inhibition of RNA polymerase II activity. However, the functions of other postplasmic/ PEM RNAs members in germline formation are largely unknown. In this study, we analyzed the functions of two postplasmic/PEM RNAs, Popk-1 and Zf-1, in transcriptional regulation in the germline cells. We show that Popk-1 contributes to transcriptional quiescence by controlling the size of the CAB and amount of Pem protein translated at the CAB. Our studies also indicated that zygotic expression of a germline gene starts around the onset of gastrulation and that the decrease of Pem protein is necessary and sufficient for the zygotic germline gene expression. Finally, further studies showed that the decrease of the Pem protein level is facilitated by Zf-1. Taken together, we propose that postplasmic/PEM RNAs such as Popk-1 and Zf-1 control the protein level of the transcriptional repressor Pem and regulate its transcriptional state in the ascidian germline. [ABSTRACT FROM AUTHOR]

Abstract 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 cm2) 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 cm2) 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.

The animal-vegetal (A-V) axis is already set in unfertilized eggs. It plays crucial roles in coordinating germ-layer formation. However, how the A-V axis is set has not been well studied. In ascidians, unfertilized eggs are already polarized along the axis in terms of cellular components. This polarization occurs during oocyte maturation. Oocytes within the gonad have the germinal vesicle (GV) close to the future animal pole. When the GVs of full-grown oocytes were experimentally translocated to the opposite pole by centrifugal force, every aspect that designates A-V polarity was reversed in the eggs and embryos. This was confirmed by examining the cortical allocation of the meiotic spindle, the position of the polar body emission, the polarized distribution of mitochondria and postplasmic/PEM mRNA, the direction of the cortical flow during oocyte maturation, the cleavage pattern and germ-layer formation during embryogenesis. Therefore, the eccentric position of the GV triggers subsequent polarizing events and establishes the A-V axis in eggs and embryos. We emphasize important roles of the cortical flow. This is the first report in which the A-V axis was experimentally and completely reversed in animal oocytes before fertilization. [ABSTRACT FROM AUTHOR]

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.

[Display omitted] • Consumption of ascidian of H. roretzi tissues significantly improves the glucose tolerance in diabetic rats. • Consumption of ascidian H. roretzi tissue significantly reduces plasma cholesterol and triglyceridesin levels in diabetic rats. • Ascidian H. roretzi tissue is a rich source of amino acid, unsaturated fatty acids, vitamins, and bioactive compounds. • Ascidian H. roretzi tissue contain 11 anti-diabetes molecules and high levels of natural astaxanthin. Halocynthia roretzi has been cultured and utilized as a nutritious seafood in Southeastern Asia for many years because of its abundance active molecules. Here, we reported that feeding of H. roretzi tissues resulted in improving oral glucose tolerance in rats. To screen the active components, we identified 950 chemical compounds from H. roretzi metabolome. Furthermore, 11 molecules with the activity of regulating blood glucose and lipids were selected based on structural identity. Among them, two natural products, hesperetin and astaxanthin were selected for further quantitative detection. The results showed that concentrations of hesperetin and astaxanthin in the tissues of H. roretzi were calculated to be 13.7 ± 9.5 and 167.9 ± 48.4 µg/100 g, respectively. The high content of astaxanthin in H. roretzi indicates its potential roles and functions in anti-diabetes activity. Taken together, our results showed that H. roretzi could be used as a functional food supplement for regulating lipid/glucose metabolism. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Tadpole larvae of the ascidian Halocynthia roretzi show morphological left-right asymmetry. The tail invariably bends towards the left side within the vitelline membrane. The structure of the larval brain is remarkably asymmetric nodal, a conserved gene that shows left-sided expression, is also expressed on the left side in H. roretzi but in the epidermis unlike in vertebrates. We show that nodal signaling at the late neurula stage is required for stereotypic morphological left-right asymmetry at later stages. We uncover a novel mechanism to break embryonic symmetry, in which rotation of whole embryos provides the initial cue for leftsided expression of nodal. Two hours prior to the onset of nodal expression, the neurula embryo rotates along the anteriorposterior axis in a counterclockwise direction when seen in posterior view, and then this rotation stops when the left side of the embryo is oriented downwards. It is likely that epidermis monocilia, which appear at the neurula rotation stage, generate the driving force for the rotation. When the embryo lies on the left side, protrusion of the neural fold physically prevents it from rotating further. Experiments in which neurula rotation is perturbed by various means, including centrifugation and sandwiching between glass, indicate that contact of the left epidermis with the vitelline membrane as a consequence of neurula rotation promotes nodal expression in the left epidermis. We suggest that chemical, and not mechanical, signals from the vitelline membrane promote nodal expression. Neurula rotation is also conserved in other ascidian species. [ABSTRACT FROM AUTHOR]

Abstract: Soft tunic syndrome of Halocynthia roretzi manifests as soft, weak, and rupturable tunics, causing mass mortality. Utilizing liquid chromatography–tandem mass spectrometry (LC–MS/MS), innate immune response was established by comparing hemolymph protein profiles of ascidians with healthy or softened tunics. Of 100 proteins in each individual ascidian, 59 proteins from healthy and 56 proteins from diseased ascidians were functionally classified. Proteins found only in diseased individuals included trypsin inhibitor and Hr-29, and with high exponentially modified protein abundance index (emPAI) values. From 41 proteins identified to be common to both healthy and diseased ascidians, 15 were associated with innate immune response. Ficolin 3, a component of the lectin-complement system, was significantly decreased in diseased ascidians, but a cell surface protein, type II transmembrane serine protease-1 (TTSP), was considerably elevated. These results suggest that trypsin inhibitor, ficolin 3, and TTSP are probably involved in the innate immune response related to this tunic disease. Beside, Hr-29 could be suggested as a biomarker for soft tunic syndrome. [Copyright &y& Elsevier]

The article discusses a study conducted to clarify whether flagellate-like cells found in histological preparations of the tunic of diseased Halocynthia roretzi (Drasche) were the causative agent of soft tunic syndrome in the ascidian. The flagellate was cultured from the softened tunic in vitro and confirmed that the tunics of healthy ascidians softened 17 days after exposure including the final 12 days in aerated, running sea water. The study concluded that the flagellate is the causative agent of soft tunic syndrome.

The article discusses a study concerning the link between the histopathological changes observed in the tunics of affected cultured ascidians Halocynthia roretzi and their mass mortality in Korea. The tunics of affected ascidians appear to be physically very soft and weak, when they are supposed to be normally very hard and stiff. Details of the histopathological examinations conducted on both diseased and healthy ascidians are explained and the results of the disease monitoring performed are summarized.

Abstract: Lhx3 genes are members of one of the six subfamilies of the LIM class homeobox genes. In ascidians, Lhx3 is known to play a critical role in endoderm differentiation, while in vertebrates Lhx3 is involved in the development of pituitary and subsets of motor neurons. It has been shown recently, using RT-PCR analysis, that two transcriptional isoforms a and b are differentially expressed during the larval development of Ciona intestinalis (). The present study provides an in-depth description of Lhx3 gene expression during the development of the two remote ascidian species, C. intestinalis and Halocynthia roretzi; for this, 5′RACE and whole-mount in situ hybridization (WISH) were employed. In both species, maternal expression of Lhx3a, but not Lhx3b, is evident. In H. roretzi, the maternal Lhx3a transcripts have been detected by WISH in the animal half of early cleavage stage embryos. In both species, transcriptional isoform a is also zygotically expressed in the sensory vesicle and the visceral ganglion lineages from the neurula stage onward. By contrast, Lhx3b transcripts are expressed only zygotically and localized in the endoderm, notochord and mesenchyme lineages during cleavage stage. Lhx3a, but not Lhx3b, transcripts are subjected to trans-splicing. Additionally, in C. intestinalis, other variations in the 5′ region have been identified among Lhx3a transcripts. Although some differences are present, over-all developmental expression of Lhx3 is rather well conserved between the two ascidian species, which is quite different from that of vertebrate counterparts. [Copyright &y& Elsevier]

Abstract: Estrogen receptor-related receptors (ERRs) were the first orphan nuclear receptors identified on the basis of their sequence similarity to the estrogen receptors. Although unique ERRs were found in some marine invertebrates, the molecular functions of these receptors are not well understood. In the present study, we identified three transcript variants of the tunicate Halocynthia roretzi ERR (Hr-ERR), varying in their 3′ untranslated regions, and putatively encoding a unique receptor deriving from an ancestor protein common to vertebrate ERRα/β/γ. Maternal mRNA of Hr-ERR was detected throughout the entire egg cytoplasm and early embryos. Zygotic Hr-ERR was predominantly expressed in the heart, and at lower levels in muscle, stomach, gonad and digestive glands. Electrophoretic mobility shift assay demonstrated that Hr-ERR directly binds to the estrogen-response element (ERE) and ERR-response element (ERRE). Gene reporter assays also showed that Hr-ERR activates transcription through ERE and ERRE. Hr-ERR-mediated transactivation was modulated by various cofactors for mammalian ERRs, such as peroxisome proliferator-activated receptor γ coactivator 1-α and small heterodimer partner. In addition, the ERR antagonists 4-hydroxytamoxifen and diethylstilbestrol inhibited the Hr-ERR-mediated transactivation, whereas Hr-ERR activity on ERE was further induced by genistein, an ERRα agonist. Taken together, our results show that Hr-ERR is an unduplicated ERR that however, possesses functional properties common to ERRα and not to ERRβ/γ. [Copyright &y& Elsevier]

The posterior-vegetal cytoplasm (PVC) of fertilized ascidian eggs plays important roles in embryo development. It has been reported that some maternal RNAs are localized to the PVC. We identified four novel type I postplasmic mRNAs that are localized to the PVC through the use of data from a cDNA project of maternal mRNAs in the eggs of Halocynthia roretzi (MAGEST database). The mRNAs are HrGLUT, HrPEN-1, and HrPEM-3, which show similarity to a glucose transporter, a g1-related protein, and Ciona pem-3, respectively; and HrPEN-2, with no similarity. Maternal mRNAs of all four genes were identically localized to the PVC after ooplasmic segregation. During cleavage, they were concentrated in the centrosome-attracting body (CAB) and were then segregated into the small blastomeres located at the posterior pole. This localization pattern is common to all known type I postplasmic mRNAs found so far. HrGLUT, HrPEN-1, and HrPEM-3 were expressed zygotically in various tissues later in embryogenesis: HrGLUT and HrPEM-3 in the mesenchyme and nervous system, and HrPEN-1 in the ectodermal cells. [Copyright &y& Elsevier]