Halorotetin A: A Novel Terpenoid Compound Isolated from Ascidian Halocynthia rotetzi Exhibits the Inhibition Activity on Tumor Cell Proliferation.

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.

Aqueous Extract of Sea Squirt (Halocynthia roretzi) with Potent Activity against Human Cancer Cells Acts Synergistically with Doxorubicin.

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.

A Novel Carotenoid with a Unique 2,6-Cyclo-ψ-End Group, Roretziaxanthin, from the Sea Squirt Halocynthia roretzi.

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-ß,ψ-carotene-4,4'-dione by UV-VIS, MS, and NMR spectral data. The formation mechanism of roretziaxanthin in the sea squirt was discussed.

Vitelline membrane proteins promote left-sided nodal expression after neurula rotation in the ascidian, Halocynthia roretzi.

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.

Wavy movements of epidermis monocilia drive the neurula rotation that determines left-right asymmetry in ascidian embryos.

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.

Eccentric position of the germinal vesicle and cortical flow during oocyte maturation specify the animal-vegetal axis of ascidian embryos.

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.

Microinjection of Exogenous DNA into Eggs of Halocynthia roretzi.

Exogenous gene expression assays during development, including reporters under the control of 5' upstream enhancer regions of genes, constitute a powerful technique for understanding the mechanisms of tissue-specific gene expression regulation and determining the characteristics, behaviors, and functions of cells that express these genes. The simple marine chordate Halocynthia roretzi has been used for these transgenic analyses for a long time and is an excellent model system for such studies, especially in comparative analyses with other ascidians. In this study, I describe simple methods for microinjecting H. roretzi eggs with exogenous DNA, such as a promoter construct consisting of a 5' upstream region and a reporter gene, which are prerequisites for transgenic analyses. I also describe basic knowledge regarding this ascidian species, providing reasons why it is an ideal subject for developmental biology studies.

Transgenic Techniques for Investigating Cell Biology During Development.

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

Tunic extract of the host ascidian attracts the causal agent of soft tunic syndrome, Azumiobodo hoyamushi (Kinetoplastea: Neobodonida).

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

Genome-wide survey of miRNAs and their evolutionary history in the ascidian, Halocynthia roretzi.

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.

Flow cytometric characterization of hemocytes of the solitary ascidian, Halocynthia roretzi.

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.

The kinetoplastid parasite Azumiobodo hoyamushi, the causative agent of soft tunic syndrome of the sea squirt Halocynthia roretzi, resides in the East Sea of Korea.

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

Dual green hemostatic sponges constructed by collagen fibers disintegrated from Halocynthia roretzi by a shortcut method.

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.

Full life cycle green preparation of collagen-based food packaging films using Halocynthia roretzi as raw material.

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.

Active Contraction in the Stable Mechanical Environment of the Tunic of the Ascidian, Halocynthia roretzi, a Polysaccharide-Based Tissue with Blood Circulatory System.

Halocynthia roretzi, a member of Ascidiacea, is covered with its own tunic, which is composed of polysaccharides, such as cellulose Iß 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.

Asterias pectinifera-Derived Collagen Peptides Mixed with Halocynthia roretzi Extracts Exhibit Anti-Photoaging Activities during Exposure to UV Irradiation, and Antibacterial Properties.

Asterias pectinifera, a species of starfish and cause of concern in the aquaculture industry, was recently identified as a source of non-toxic and highly water-soluble collagen peptides. In this study, we investigated the antioxidant and anti-photoaging functions of compounds formulated using collagen peptides from extracts of Asterias pectinifera and Halocynthia roretzi (AH). Our results showed that AH compounds have various skin protective functions, including antioxidant effects, determined by measuring the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl radicals, as well as anti-melanogenic effects, determined by measuring tyrosinase inhibition activity. To determine whether ethosome-encapsulated AH compounds (E(AH)) exert ultraviolet (UV)-protective effects, human dermal fibroblasts or keratinocytes were incubated with E(AH) before and after exposure to UVA or UVB. E(AH) treatment led to inhibition of photoaging-induced secretion of matrix metalloproteinase-1 and interleukin-6 and -8, which are associated with inflammatory responses during UV irradiation. Finally, the antibacterial effects of AH and E(AH) were confirmed against both gram-negative and gram-positive bacteria. Our results indicate that E(AH) has the potential for use in the development of cosmetics with a range of skin protective functions.

The Impact of Ascidian (Halocynthia roretzi)-derived Plasmalogen on Cognitive Function in Healthy Humans: A Randomized, Double-blind, Placebo-controlled Trial.

OBJECTIVES: Plasmalogen, phospholipids with previously shown associations with dementia, has attracted attention as a substance found in some studies to improve cognitive function. The effects of ascidian-derived plasmalogens on cognitive performance improvement were assessed in a randomized, double-blind, placebo-controlled study including Japanese adult volunteers with mild forgetfulness. METHODS: Participants consumed either the active food containing ascidian-derived plasmalogen (1 mg as plasmalogen) or the placebo food for 12 weeks, and their cognitive performance was assessed by Cognitrax. Participants were randomly allocated into the intervention (ascidian-derived plasmalogen; 8 males, and 17 females; 45.6 ± 11.1 years) or the placebo (9 males, and 15 females; mean age, 46.4 ± 10.8 years) group. RESULTS: Compared to the placebo group, the intervention group showed a significant increase score in composite memory (eight weeks: 3.0 ± 16.3 points, 12 weeks: 6.7 ± 17.5 points), which was defined as the sum of verbal and visual memory scores. CONCLUSIONS: These results indicate the consumption of ascidian-derived plasmalogen maintains and enhances memory function. This study was registered at the University Hospital Medical Information Network Clinical Trial Registry (UMIN-CTR, registry no. UMIN000026297). This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

A self-marker-like protein governs hemocyte allorecognition in Halocynthia roretzi.

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.

Hox gene cluster of the ascidian, Halocynthia roretzi, reveals multiple ancient steps of cluster disintegration during ascidian evolution.

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.

Genetic Distances in Three Ascidian Species determined by PCR Technique.

Seven oligonucleotides primers were shown to generate the shared loci, specific loci, unique shared loci to each species and shared loci by the three species which could be obviously scored. In the present study, 7 oligonucleotides primers produced 401 total loci in the Styela clava (SC) species, 390 in the Halocynthia roretzi (HR) and 434 in the Styela plicata (SP), respectively. Seven oligonucleotides primers generated 275 specific loci in the SC, 341 in the HR and 364 in the SP species, respectively. The oligonucleotides primer BION-23 generated 28 unique loci to each species in the SP species. Especially, the oligonucleotides primer BION-25 produced 7 unique loci to each species, which were identifying each species in the SP species. BION-17 distinguished 21 shared loci by the three ascidian species, major and/or minor fragments of sizes, which were identical in almost all of the samples. Based on the average bandsharing values of all samples, the similarity matrix ranged from 0.519 to 0.774 in the SC species, from 0.261 to 0.683 in the HR species and from 0.346 to 0.730 in the SP species. As regards average bandsharing value (BS) results, individuals from SC species (0.661±0.081) exhibited higher bandsharing values than did individuals from HR species (0.555±0.074) (P<0.05). The dendrogram obtained by the seven oligonucleotides primers indicates three genetic groups. In three ascidian species, the shortest genetic distance (0.071) exhibiting significant molecular difference was also between individual no. 20 and no. 21 within the SP species.