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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]

The extraction of collagen for packaging films typically requires a time-consuming process and the use of substantial chemicals. Herein, we present a full life cycle green preparation method for rapidly producing collagen-based food packaging films using Halocynthia roretzi (HR), a collagen-rich marine organism, as raw material. We first prepared the micro/nano-sized collagen fibers from HR tissue by utilizing urea and sonication as effective hydrogen-bond breakers. Subsequently, the collagen fiber was rapidly fabricated into a film through vacuum filtration. The resulting collagen fiber film (CFF) exhibited a uniform and dense surface, along with good tensile properties, water resistance, and biodegradability. In addition, the deposition of chitosan (CS) on the surface of CFF resulted in a remarkable preservation effect for both strawberries and pork. This full life cycle preparation method for collagen-based films provides a promising and innovative approach to the sustainable preparation of food packaging films. • CFFs obtained by disrupting hydrogen bonds in tissues with urea and sonication • CFFs are full life cycle green film materials • CFFs exhibited excellent food preservation properties as a packing material [ABSTRACT FROM AUTHOR]

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 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]

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]

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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Developmental fates are determined in relation to other fates in surrounding cells within the embryo, creating diversified tissue or cell types in the course of development. In ascidian embryos, maternally localized factors and inductive signals play essential and cooperative roles in fate determination at appropriate spatial and temporal positions during embryogenesis. Here, to clarify fate determination mechanisms of mesenchyme and muscle during mesenchyme induction by the fibroblast growth factor ( FGF) signal in Halocynthia roretzi, we examined the function of transcription factor Tbx6, which acts as a pivotal mediator of the maternally localized muscle differentiation determinant Macho-1. Our results suggest that the level of Tbx6 expression increases in muscle lineage cells through positive feedback regulation that promotes muscle differentiation as well as mesenchymal fate suppression. In addition, the FGF signal inactivated Tbx6 transcriptional activity and positive feedback, leading to induction of the mesenchymal lineage. Taken together, our finding suggests that Tbx6 is an important factor for determining mesenchyme and muscle fates. [ABSTRACT FROM AUTHOR]

Highlights: [•] We isolated two kinetoplastid organisms from sea squirts collected from Tongyeong, Korea. [•] Phylogenetic analysis identified these organisms as A. hoyamushi and P. sorokini. [•] Sea squirts injected with A. hoyamushi showed 100% STS, whereas P. sorokini did not. [•] We established optimum water temperatures and salinities for A. hoyamushi in Korean waters. [•] The optimum conditions for its growth should lead to methods for reducing the incidence of STS. [ABSTRACT FROM AUTHOR]

The process of establishing the anterior-posterior axis is an important event in the development of bilateral animals. Otx, which encodes a homeodomain transcription factor, is continuously expressed in the anterior part of the embryo in a wide range of animals. This pattern of expression is thought to be important for the formation of anterior neural structures, but the regulatory mechanism that sustains the expression is not known. Here, using embryos of the ascidian, Halocynthia roretzi, we investigated how the transcription of Otx is maintained in the cells of the anterior neural lineage during embryogenesis. We identified an enhancer region sufficient to mimic the Otx expression pattern from the gastrula to tailbud stages. Several putative transcription factor binding sites that are required for generating the Otx expression pattern were also identified. Distinct sets of sites were required at different developmental stages, suggesting that distinct transcriptional mechanisms regulate Otx transcription in each of the gastrula, neurula and tailbud stages. Along with previous studies on the transcriptional regulatory mechanism of Otx during the pre-gastrula stages, the present results provide the first overview of the mechanism that sustains Otx expression in the anterior neural lineage during ascidian embryogenesis and demonstrate the complexity of a developmental mechanism that maintains Otx transcription. [ABSTRACT FROM AUTHOR]

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. [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]

Abstract: The genetic structure and phylogeography of the solitary ascidian Halocynthia roretzi (sea pineapple) in Korea and Japan were studied based on mitochondrial DNA cytochrome oxidase subunit I (COI) gene nucleotide variations. The COI haplotypes found were divided into two clades in a statistical parsimony network. From the extensive haplotype diversity, limited nucleotide diversity, neutrality test, and mismatch distribution analysis, the species appears to have undergone rapid population expansion from an ancestral population with a small effective population size due to glacial bottlenecking in the late Pleistocene. Pairwise population F ST estimates inferred distinct genetic differentiation between the population from the northernmost east coast of Korea and the remaining pooled coastal water populations, while other populations generally exhibited low levels of differentiation. Analysis of molecular variance showed a weak but significant differentiation among three geographic population groups. Overall, the moderate population structure of H. roretzi may have been influenced by high levels of gene flow caused by hydrographic conditions such as sea currents. [Copyright &y& Elsevier]

Pitx genes play important roles in a variety of developmental processes in vertebrates. In an ascidian species, Halocynthia roretzi, Hr- Pitx, the only Pitx gene of this species, has been reported to be expressed in the left epidermis at the tailbud stage. In the present study, first, we have shown that Hr- Pitx is also expressed in the papilla-forming region at the neurula to tailbud stages, and then we addressed transcription regulatory mechanisms for the expression of Hr- Pitx in the papilla-forming region. We have identified the genomic region ranging from 850 to 1211 bp upstream from the translation start site of the Hr- Pitx gene as an enhancer region that drives the transcription of Hr- Pitx in the papilla-forming region. Within the enhancer region, putative transcriptional factor binding sites for Otx as well as Fox were shown to be required for its activity. Finally, we carried out knocking down experiments of Hr- Otx function using an antisense morpholino oligonucleotide, in which the knocking down of Hr- Otx function resulted in reduction of the enhancer activity and loss of the expression of Hr- Pitx in the papilla-forming region. In Xenopus laevis, it has been reported that Pitx genes are expressed downstream of Otx function during development of the cement gland, an adhesive organ of its larva. Taken together, it is suggested that the expression regulatory mechanism of Pitx, involving Otx as the upstream gene, in the developing adhesive organ is conserved between ascidians and vertebrates. [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: The influence of sea squirt aquaculture on benthic–pelagic coupling was evaluated in semi-enclosed Korean coastal waters with an in situ benthic chamber and results show for the first time that suspended sea squirt cultures play an important role in benthic–pelagic coupling in the coastal zone. Measurements of primary production, vertical particulate fluxes, and benthic fluxes were made at two stations, a sea squirt (Halocynthia roretzi) farm (SSF) and an area of organic-matter-enriched sediment in Jinhae Bay. The vertical material fluxes of organic carbon, nitrogen, and biogenic silicate (BSi) were significantly higher at SSF than in Jinhae Bay, indicating massive biodeposits in the surface sediments at SSF. The organic carbon oxidation rates (Cox) were estimated after correction for CaCO3 dissolution. The average Cox at SSF (204 mmol C m−2 d−1) was significantly higher than that in the organic-enriched Jinhae Bay sediment (77 mmol C m−2 d−1). The organic carbon burial fluxes were determined using vertical profiles of organic carbon of up to 30 cm and the sedimentation rate calculated from the excess 210Pb distribution. At both stations, ∼95% of the settled organic carbon was oxidized and only ∼5% was buried in the deep sediment layer. The benthic fluxes of dissolved inorganic nitrogen and phosphate at SSF were 2–12 times higher than in Jinhae Bay, corresponding to 85%, and 270%, respectively, of the requirements for primary production. [Copyright &y& Elsevier]

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]

Abstract: 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. [ABSTRACT FROM AUTHOR]

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]

Recently, a serious disease spread extensively in aquaculture sites of the ascidian Halocynthia roretzi in Korea. To understand circumstances of ascidians in Korean aquaculture sites, residue levels of organotin compounds were analyzed, and detection of a marine birnavirus (MABV) in tissues of H. roretzi was attempted. Korean H. roretzi showed high concentrations of butyltins (mono, di, and tributyltins), especially in the gill, hepatopancreas, and digestive tract. However, there was no significant difference in the residues of butyltins in the hepatopancreas between diseased and non-diseased ascidians. The positive rate of MABV detection was high in the hepatopancreas, but also no significant difference was observed between diseased and non-diseased individuals. These observations suggest that an accumulation of tributyltin and a latency of MABV in H. roretzi tissues does not directly relate to the occurrence of the disease. [ABSTRACT FROM AUTHOR]

Abstract: The sea squirt Halocynthia roretzi metabolizes fucoxanthin, and subsequently accumulates its derived carotenoids with characteristic structures. In the present study, we isolated halocynthiaxanthin and fucoxanthinol as carotenoids having antiproliferative activity from H. roretzi. Halocynthiaxanthin and fucoxanthinol inhibited the growth of HL-60 human leukemia cells in a dose- and time-dependent manner. Viability of HL-60 treated with 12.5 μM halocynthiaxanthin and fucoxanthinol was decreased by 12.1% and 5.7% of control after 48 h incubation, respectively. Furthermore, halocynthiaxanthin and fucoxanthinol induced apoptosis in HL-60 cells, MCF-7 human breast cancer cells, and Caco-2 human colon cancer cells. When HL-60 cells were incubated with 12.5 μM halocynthiaxanthin and fucoxanthinol for 48 h, relative DNA fragmentations were enhanced to 5- and 7-fold compared to that in control cells, respectively. The activities of apoptosis induction by halocynthiaxanthin and fucoxanthinol were higher than that of fucoxanthin which we have previously reported as a carotenoid possessing the ability to induce apoptosis. Fucoxanthinol exhibited the highest apoptosis-inducing activity among the three carotenoids. Furthermore, the expression levels of apoptosis-suppressing protein Bcl-2 were decreased in HL-60 cells treated with halocynthiaxanthin and fucoxanthinol. These results suggest that halocynthiaxanthin and fucoxanthinol exhibited potential antiproliferative effects via apoptosis induction in several cancer cell lines. [Copyright &y& Elsevier]

The immunoreceptor tyrosine-based activation motif (ITAM) plays an important role in signal transduction through antigen receptors in mammalian lymphocytes. We previously reported that an ITAM-containing receptor, ascidian hemocyte ITAM-containing receptor 1 (AhITAMR1), exists on the hemocyte surfaces of the ascidian Halocynthia roretzi, and is involved in both phagocytosis and hemocyte aggregation. In this study, we carried out differential display screening of upregulated genes during H. roretzi hemocyte aggregation and found that at least three genes are upregulated. One encodes glutathione S-transferase ω (GSTω), while the other two encode novel proteins. The expression of all three genes was induced by treatment with a specific monoclonal antibody against AhITAMR1, while their expression was inhibited by wortmannin, BAPTA-AM, and cyclosporin A. We also found that the expression of GSTω was induced by treatment with anti-T cell receptor antibody in mouse peripheral T cells. We propose that signal transduction pathways mediated by ITAM-containing receptors are conserved from ascidian hemocytes to mammalian T cells. [ABSTRACT FROM AUTHOR]

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]

Reactive oxygen intermediates (ROIs), including superoxide anions and hydrogen peroxide, are generated by phagocytes in invertebrates, as well as in vertebrates. To understand the molecular mechanisms underlying the generation of ROIs by hemocytes of the solitary ascidian Halocynthia roretzi, we established a method of measuring ROIs using luminol-dependent chemiluminescence (LDCL). LDCL analyses revealed that both zymosan and phorbol myristate acetate (PMA), but not lipopolysaccharide, β1,3-glucan, or formylpeptide, induced the generation of ROIs by H. roretzi hemocytes. The zymosan-induced LDCL was markedly inhibited by the addition of superoxide dismutase (SOD) or H. roretzi plasma. A calcium-chelating reagent, BAPTA-AM, completely inhibited the zymosan-induced LDCL. On the other hand, the PMA-induced LDCL was only slightly inhibited by the addition of SOD or BAPTA-AM. Spectroscopic analysis at a low temperature revealed that H. roretzi hemocytes had absorption spectra specific for type b cytochrome, a component of the NADPH oxidase complex in mammalian phagocytes. These results strongly suggest that H. roretzi hemocytes generate superoxide anions upon phagocytosis and that intracellular calcium ions and possibly an NADPH oxidase complex are involved in their generation by H. roretzi hemocytes. [Copyright &y& Elsevier]

In vertebrates, melanins produced in specialized pigment cells are required for visual acuity, camouflage, sexual display and protection from ultra violet (UV) radiation. There are three pigment cell types that are classified based on their distinct embryonic origins. Retinal pigment epithelium (RPE) cells originate from the outer layer of the optic cup. Pigment cells of the pineal organ are formed from the developing diencephalon. Melanocytes are derived from the neural crest unique to vertebrate embryos. Some of these pigment cells also play roles that are independent of the activity of tyrosinase, the key melanogenesis enzyme, or melanin: production of substrate(s) for catecholamine synthesis, maintenance of endolymph composition in the cochlea, maintenance of photoreceptor cells in the retina and retinoid metabolism essential for the visual cycle. To deduce the evolutionary origins of vertebrate pigment cells and a possible archetypal genetic circuitry, which may have been modified and utilized to generate multiple pigment cell types, comparison of developmental mechanisms of pigment cells between vertebrates and closely related invertebrate ascidians are proposed to provide useful information. The tadpole-type larva of ascidians possesses two melanin-containing pigment cells, termed the otolith and ocellus pigment cells, in the brain that are believed to be required for photo- and geotactic responses during swimming. In this review, current knowledge on the development of the two ascidian pigment cells is summarized, i.e. complete cell lineage, structure and expression of genes encoding two melanogenesis enzymes, and molecular developmental mechanisms involving BMP-CHORDIN antagonism, and possible evolutionary relationships between ascidian and vertebrate pigment cells are discussed. [ABSTRACT FROM AUTHOR]

The Rel/NF-κB family is known to be involved in a wide variety of biological processes, including morphogenesis. In the present study, two protochordate cDNA clones encoding Rel/NF-κB proteins, named As-rel1 and As-rel2, were isolated from a fertilized egg cDNA library of the ascidian Halocynthia roretzi. The As-rel1 protein is a typical Rel/NF-κB family member, containing a Rel homology domain, a nuclear localization sequence and a C-terminal putative transcription activation domain, while the As-rel2 protein is a novel Rel/NF-κB family member that lacks a nuclear localization sequence and the C-terminal domain. Northern blot analyses showed that both transcripts were maternally expressed and that their expression changed during development of H. roretzi embryos. Although injection of the As-rel2 mRNA into H. roretzi fertilized eggs had little effect on embryonic development, injection of the As-rel1 mRNA interfered greatly with notochord formation, resulting in a shortened tail with a reduced number of notochord cells. In contrast, embryos co-injected with As-rel1 and As-rel2 mRNA developed normally, indicating that the As-rel2 protein rescued the defect in notochord formation induced by the injection of As-rel1 mRNA alone. These results strongly suggest that the As-rel1 protein functions as a suppressor in ascidian notochord formation, while the As-rel2 protein has an antagonistic effect on the action of the As-rel1 protein. [ABSTRACT FROM AUTHOR]

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. • Seasonal dynamics of energy reserves and physiological performance in the longline-cultured sea squirt were examined.. • The two culture practices carried out using spat reared in different localities displayed identical growth performance. • Sea squirts retained physiological functions across the temperature range during the culturing period in the grow-out area. • The sea squirt spat reared in colder nursery grounds support sustainable culture in the traditional grow-out area. [ABSTRACT FROM AUTHOR]

Background: In the previous paper published in 2017, we described the structure of Hox gene cluster of the ascidian, Halocynthia roretzi, and discussed the scenario for the disintegration of Hox gene clusters during evolution of ascidians. The description about the Hox gene cluster structure still represents the latest information, hence it has been left unchanged. In contrast, some points in Discussion, the description on the phylogenetic relationships among tunicates and the theoretical scenario for the disintegration of Hox gene cluster during evolution of ascidians, should be changed because the phylogenetic relationships among tunicates have recently been updated. The above mentioned points were made in accordance with the phylogenetic tree for tunicates based on the mitochondrial DNA sequences, which was the latest at the time of publication. In 2018, however, Kocot et al. and Delsuc et al. proposed new phylogenetic trees for tunicates based on a large number of nuclear gene sequences. The trees obtained by the two groups are essentially the same and different from the previous one in the phylogenetic positions of Appendicularia and Thaliacea, which leads to a change in the order of the emergence of ascidians and the Hox gene cluster disintegration during evolution of ascidians or tunicates. Results: We add here a note to update the previous description on the phylogenetic relationships among tunicates and the theoretical scenario, including one Figure, so as to coincide with the new phylogenetic relationships among tunicates based on the nuclear gene sequences. Conclusion: The previous summarized conclusion remains unchanged: we suggest that the Hox gene cluster of the ancestral ascidian experienced extensive genome shuffling during the course of evolution to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene organization on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to all ascidians and later lineage-specific events. [ABSTRACT FROM AUTHOR]