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The ascidian species Lissoclinum patella is found, amongst other places, around Heron Island on the Great Barrier Reef, Australia. L. patella has a cyanobacterial symbiont, Prochloron didemni , known to produce various cyclic peptides, including quantities of the cyclic pseudo-octapeptides, the patellamides. Patellamides are of pharmaceutical interest and have attracted the curiosity of coordination chemists because they can form quite stable mono- and di-nuclear transition metal complexes, particularly with copper(ii). For some patellamide derivatives, the binding of two CuII centres is cooperative and solution equilibria involving metal-free peptides, mono- and di-nuclear copper(ii) complexes, and various functions of these complexes have been described. These studies were also driven by the observation that the ascidians possess copper concentrations in excess of that in the seawater around Heron Island, and accumulation factors of approximately 104 have repeatedly been reported. New data presented here, based on inductively coupled plasma atomic emission spectroscopy (ICP-OES) and ICP-mass spectromety (MS) measurements, indicate that the 104 factor is overestimated and a factor >500 and up to approximately 3000 is more realistic. There are large quantities of cyclic peptides in Lissoclinum patella ascidians, produced by the obligate symbiont Prochloron. Ascidians accumulate copper but the generally assumed factor of 104 is much overestimated, and it is shown here that there is a significant accumulation factor >500 and up to approximately 3000. [ABSTRACT FROM AUTHOR]

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]

Globins have been studied as model proteins to elucidate the principles of protein evolution. This was achieved by understanding the relationship between amino acid sequence, three-dimensional structure, physicochemical properties, and physiological function. Previous molecular phylogenies of chordate globin genes revealed the monophyletic evolution of urochordate globins and suggested convergent evolution. However, to provide evidence of convergent evolution, it is necessary to determine the physicochemical and functional similarities between vertebrates and urochordate globins. In this study, we determined the expression patterns of Ciona globin genes using real-time RT-PCR. Two genes (Gb-1 and Gb-2) were predominantly expressed in the branchial sac, heart, and hemocytes and were induced under hypoxia. Combined with the sequence analysis, our findings suggest that Gb-1/-2 correspond to vertebrate hemoglobin-α/-β. However, we did not find a robust similarity between Gb-3, Gb-4, and vertebrate globins. These results suggested that, even though Ciona globins obtained their unique functions differently from vertebrate globins, the two of them shared some physicochemical features and physiological functions. Our findings offer a good example for understanding the molecular mechanisms underlying gene co-option and convergence, which could lead to evolutionary innovations.

Abstract Background Vertebrates develop their peripheral nervous system (PNS) from transient unique embryonic structures, the neural crest, and the ectodermal placodes that are located at the border of the forming central nervous system. By contrast, in the invertebrate chordates, amphioxus and ascidians, a large part of the PNS originates at the opposite of the embryo, in the ventral ectoderm. In both groups, a biphasic mechanism regulates ventral PNS formation: high BMP levels specify a neurogenic territory within which glutamatergic epidermal sensory neuron formation is controlled by the Notch pathway. Given these similarities and the phylogenetic relationships within chordates, it is likely that ventral PNS is an ancestral feature in chordates and that it has been lost in vertebrates. Results In order to get insights into the molecular control of ventral PNS formation and to test the hypothesis of their homology and potential contribution to the emergence of vertebrate PNS, we undertook a close comparison of ventral PNS formation in the ascidian Phallusia mammillata and the amphioxus Branchiostoma lanceolatum. Using timed RNA-seq series, we identified novel markers of the ventral PNS during different phases of its development in both species. By extensively determining the expression of paralogous and orthologous genes, we observed that only a minority of genes have a shared expression in the ventral PNS. However, a large fraction of ventral PNS orthologous genes are expressed in the dorsally forming PNS of vertebrates. Conclusions Our work has significantly increased the molecular characterization of ventral PNS formation in invertebrate chordates. The low observed conservation of gene expression in the ventral PNS suggests that the amphioxus and ascidian ventral PNS are either not homologous, or alternatively extensive drift has occurred in their regulatory mechanisms following a long period (600 My) of separate evolution and accelerated evolution in the ascidian lineage. The homology to genes expressed in the dorsally forming PNS of vertebrates suggests that ancestral sensory neurons gene networks have been redeployed in vertebrates.

Background: Injury response is key to successful regeneration. Yet, transcriptome analyses of injury response were performed only on a handful of regenerative organisms. Here, we studied the injury response of the solitary ascidian Polycarpa mytiligera, an emerging model system, capable of regenerating any body part. We used the siphon as a model for studying transcriptional changes following injury, and identified genes that were activated in the initial 24 hours post amputation (hpa). Results: Highly conserved genes, such as bone morphogenetic protein‐1 (BMP1), growth hormone secretagogue receptor (GHSR) and IL‐17, were upregulated by 12 hpa, yet their expression was sustained only in non‐regenerating tissue fragments. We optimized fluorescent in situ hybridization, and found that the majority of BMP1+ cells were localized to the rigid tunic that covers the animal. This highlights the importance of this tissue, particularly during injury response. BMP1 was overexpressed following injuries to other body regions, suggesting that it was a part of a common injury‐induced program. Conclusion: Our study suggests that, initially, specific injury‐induced genes were upregulated in P. mytiligera organs, yet, later, a unique transcriptional profile was observed only in regenerating tissues. These findings highlight the importance of studying diverse regenerating and non‐regenerating organisms for complete understanding of regeneration. Key Findings: Gene expression profiling of the injury response of the highly regenerative ascidian Polycarpa mytiligera.BMP1 and GHSR expression is induced following injury in regenerating versus non‐regenerating tissues of P. mytiligera.Following injury to the P. mytiligera siphon, the majority of BMP1+ cells were found in the tunic.Injury‐induced BMP1 expression was detectable following injuries to other body parts of P. mytiligera, suggesting a generic wound. [ABSTRACT FROM AUTHOR]

In animal development, most cell types stop dividing before terminal differentiation; thus, cell cycle control is tightly linked to cell differentiation programmes. In ascidian embryos, cell lineages do not vary among individuals, and rounds of the cell cycle are determined according to cell lineages. Notochord and muscle cells stop dividing after eight or nine rounds of cell division depending on their lineages. In the present study, we showed that a Cdk inhibitor, Cdkn1.b, is responsible for stopping cell cycle progression in these lineages. Cdkn1.b is also necessary for epidermal cells to stop dividing. In contrast, mesenchymal and endodermal cells continue to divide even after hatching, and Myc is responsible for maintaining cell cycle progression in these tissues. Expression of Cdkn1.b in notochord and muscle is controlled by transcription factors that specify the developmental fate of notochord and muscle. Likewise, expression of Myc in mesenchyme and endoderm is under control of transcription factors that specify the developmental fate of mesenchyme and endoderm. Thus, cell fate specification and cell cycle control are linked by these transcription factors. [ABSTRACT FROM AUTHOR]

Osmoregulation is essential for organisms to adapt to the exterior environment and plays an important role in embryonic organogenesis. Tubular organ formation usually involves a hyperosmotic lumen environment. The mechanisms of how the cells respond and regulate lumen formation remain largely unknown. Here, we reported that the nuclear factor of activated T cells-5 (NFAT5), the only transcription factor in the NFAT family involved in the cellular responses to hypertonic stress, regulated notochord lumen formation in chordate Ciona. Ciona NFAT5 (Ci-NFAT5) was expressed in notochord, and its expression level increased during notochord lumen formation and expansion. Knockout and expression of the dominant negative of NFAT5 in Ciona embryos resulted in the failure of notochord lumen expansion. We further demonstrated that the Ci-NFAT5 transferred from the cytoplasm into nuclei in HeLa cells under the hyperosmotic medium, indicating Ci-NFAT5 can respond the hypertonicity. To reveal the underly mechanisms, we predicted potential downstream genes of Ci-NFAT5 and further validated Ci-NFAT5-interacted genes by the luciferase assay. The results showed that Ci-NFAT5 promoted SLC26A6 expression. Furthermore, expression of a transport inactivity mutant of SLC26A6 (L421P) in notochord led to the failure of lumen expansion, phenocopying that of Ci-NFAT5 knockout. These results suggest that Ci-NFAT5 regulates notochord lumen expansion via the SLC26A6 axis. Taken together, our results reveal that the chordate NFAT5 responds to hypertonic stress and regulates lumen osmotic pressure via an ion channel pathway on luminal organ formation. [ABSTRACT FROM AUTHOR]

Due to similarities in iodine concentrations and peroxidase activities, the thyroid in vertebrates is considered to originate from the endostyle of invertebrate chordates even though it is a glandular (mucus-producing) organ for aquatic suspension feeding. Among chordates with an endostyle, urochordates are useful evolutionary research models for the study of vertebrate traits. The ascidian Ciona intestinalis forms an endostyle with specific components of glandular- and thyroid-related elements, and molecular markers have been identified for these components. Since we previously examined a simple endostyle in the larvacean Oikopleura dioica, the expression of the thyroid-related transcription factor genes, Ciona Nkx2-1 and FoxE, was perturbed by TALEN-mediated gene knockout in the present study to elucidate the shared and/or divergent features of a complex ascidian endostyle. The knockout of Ciona Nkx2-1 and FoxE exerted different effects on the morphology of the developing endostyle. The knockout of Nkx2-1 eliminated the expression of both glandular and thyroidal differentiation marker genes, e.g., vWFL1, vWFL2, CiEnds1, TPO, and Duox, while that of FoxE eliminated the expression of the differentiation marker genes, TPO and CiEnds1. The supporting element-related expression of Pax2/5/8a, Pax2/5/8b, FoxQ1, and β-tubulin persisted in the hypoplastic endostyles of Nkx2-1- and FoxE-knockout juveniles. Although the gene regulation of ascidian-specific CiEnds1 remains unclear, these results provide insights into the evolution of the vertebrate thyroid as well as the urochordate endostyle. [ABSTRACT FROM AUTHOR]

Acute and chronic dermatological injuries need rapid tissue repair due to the susceptibility to infections. To effectively promote cutaneous wound recovery, it is essential to develop safe, low-cost, and affordable regenerative tools. Therefore, we aimed to identify the biological mechanisms involved in the wound healing properties of the glycosaminoglycan dermatan sulfate (DS), obtained from ascidian Styela plicata, a marine invertebrate, which in preliminary work from our group showed no toxicity and promoted a remarkable fibroblast proliferation and migration. In this study, 2,4-DS (50 µg/mL)-treated and control groups had the relative gene expression of 84 genes participating in the healing pathway evaluated. The results showed that 57% of the genes were overexpressed during treatment, 16% were underexpressed, and 9.52% were not detected. In silico analysis of metabolic interactions exhibited overexpression of genes related to: extracellular matrix organization, hemostasis, secretion of inflammatory mediators, and regulation of insulin-like growth factor transport and uptake. Furthermore, in C57BL/6 mice subjected to experimental wounds treated with 0.25% 2,4-DS, the histological parameters demonstrated a great capacity for vascular recovery. Additionally, this study confirmed that DS is a potent inducer of wound-healing cellular pathways and a promoter of neovascularization, being a natural ally in the tissue regeneration strategy. [ABSTRACT FROM AUTHOR]

Sperm chemotaxis, in which sperms are attracted to conspecific eggs via species‐specific attractants, plays an important role in fertilization. This phenomenon has been observed in various animals and species‐specific sperm attractants have been reported in some species. However, the mechanisms involved in the reception and recognition of the species‐specific attractant by the sperms is poorly studied. Previously, we found that the plasma membrane‐type Ca2+/ATPase (PMCA) is the receptor for the sperm‐activating and ‐attracting factor (SAAF) in the ascidian Ciona intestinalis. To determine the role of PMCA in species‐specific sperm chemotaxis, we identified the amino acid sequences of PMCAs derived from six Phlebobranchia species. The testis‐specific splice variant of PMCA was found to be present in all the species investigated and the ascidian‐specific sequence was detected near the 3′‐terminus. Moreover, dN/dS analysis revealed that the extracellular loops 1, 2, and 4 in ascidian PMCA underwent a positive selection. These findings suggest that PMCA recognizes the species‐specific structure of SAAF at the extracellular loops 1, 2, and 4, and its testis‐specific C‐terminal region is involved in the activation and chemotaxis of ascidian sperms. Reasearch Highlights: •The three‐dimensional structure of plasma membrane‐type Ca2+/ATPase (PMCA) suggested that the extracellular loops 1, 2, and 4 in PMCA are involved in the recognition and reception of sperm‐activating and ‐attracting factor in ascidians. [ABSTRACT FROM AUTHOR]

Background: Vertebrates develop their peripheral nervous system (PNS) from transient unique embryonic structures, the neural crest, and the ectodermal placodes that are located at the border of the forming central nervous system. By contrast, in the invertebrate chordates, amphioxus and ascidians, a large part of the PNS originates at the opposite of the embryo, in the ventral ectoderm. In both groups, a biphasic mechanism regulates ventral PNS formation: high BMP levels specify a neurogenic territory within which glutamatergic epidermal sensory neuron formation is controlled by the Notch pathway. Given these similarities and the phylogenetic relationships within chordates, it is likely that ventral PNS is an ancestral feature in chordates and that it has been lost in vertebrates. Results: In order to get insights into the molecular control of ventral PNS formation and to test the hypothesis of their homology and potential contribution to the emergence of vertebrate PNS, we undertook a close comparison of ventral PNS formation in the ascidian Phallusia mammillata and the amphioxus Branchiostoma lanceolatum. Using timed RNA-seq series, we identified novel markers of the ventral PNS during different phases of its development in both species. By extensively determining the expression of paralogous and orthologous genes, we observed that only a minority of genes have a shared expression in the ventral PNS. However, a large fraction of ventral PNS orthologous genes are expressed in the dorsally forming PNS of vertebrates. Conclusions: Our work has significantly increased the molecular characterization of ventral PNS formation in invertebrate chordates. The low observed conservation of gene expression in the ventral PNS suggests that the amphioxus and ascidian ventral PNS are either not homologous, or alternatively extensive drift has occurred in their regulatory mechanisms following a long period (600 My) of separate evolution and accelerated evolution in the ascidian lineage. The homology to genes expressed in the dorsally forming PNS of vertebrates suggests that ancestral sensory neurons gene networks have been redeployed in vertebrates. [ABSTRACT FROM AUTHOR]

Summary: Ascidians (Urochordate) are hermaphroditic marine invertebrates that release sperm and eggs to the surrounding seawater. However, several ascidians, including Ciona intestinalis and Halocynthia roretzi, show strict self-sterility due to a self/nonself-recognition mechanism in the interaction between sperm and the vitelline coat (VC) of the eggs. We have previously reported that sperm intracellular Ca2+ level drastically increased immediately after sperm binding to the VC of self eggs but not nonself eggs in C. intestinalis type A, which was potently inhibited by lowering the external Ca2+ concentration, suggesting that sperm Ca2+ influx occurs after sperm self-recognition on the VC. Here, we investigated whether self-sterility was abolished by lowering the external Ca2+ concentration in C. intestinalis. The results showed that the block to self-fertilization was removed by low-Ca2+ (∼1 mM) seawater without decreasing the fertilization rate. Such an effect was not observed with Mg2+ or K+. These results led us to conclude that a low-Ca2+ environment is sufficient to block the self-recognition signal upon fertilization. As low-Ca2+ seawater showed no effect on H. roretzi self-sterility, we propose that the mechanism of self-sterility in Ciona must be distinctive from that in Halocynthia. [ABSTRACT FROM AUTHOR]

• DOM exposure results in higher lipophilic contaminant uptake trend in mucous feeder. • Despite favorable emulsification properties terrDOM does not lead to more uptake. • The pattern in Ciona matches observations made during a previous study on Mytilus. • Less contaminant accumulated in mucous feeder Ciona than in Mytilus. The widely reported increase of terrestrial dissolved organic matter (terrDOM) in northern latitude coastal areas ("coastal darkening") can impact contaminant dynamics in affected systems. One potential impact is based on differences in chemical adsorption processes of the molecularly larger terrDOM compared to marine DOM (marDOM) that leads to increased emulsification of lipophilic contaminants with terrDOM. Filter feeders filter large amounts of water and DOM daily and thus are directly exposed to associated contaminants through both respiration and feeding activity. Thus, increased exposure to terrDOM could potentially lead to an increase in bioaccumulation of lipid soluble contaminants in filter feeders. To assess the effect of DOM on bioaccumulation in filter feeders, we exposed the mucous based filter feeding ascidian Ciona intestinalis (formerly known as Ciona intestinalis Type B), to the lipophilic veterinary drug teflubenzuron (log K OW : 5.39) in combination with four DOM treatments: TerrDOM, marDOM, a mix of the two called mixDOM, and seawater without DOM addition. The exposure lasted for 15 days, after which the individuals in all DOM treatments showed a trend towards higher bioaccumulation of Teflubenzuron than those in the seawater control. However, there was considerable overlap in posterior distributions. Against our expectations, marDOM resulted in the highest bioaccumulation factor (BAF), followed by mixDOM, with terrDOM resulting in the lowest BAF except for seawater (kinetic BAF L/kg median, 2.5 %–97.5 % percentile marDOM 94, 74–118; mixDOM 82, 63–104; terrDOM 79; 61-99; seawater 61, 44–79). All BAFs were below the level of concern according to the EU REACH regulation (BAF < 2000 L / kg) and, therefore, likely not environmentally problematic in the examined context. However, the results show that DOM can act as a dietary vector; thus, different combinations of contaminants, DOM, and filter feeding organisms should be tested further. [ABSTRACT FROM AUTHOR]

Aging increases oxidative and inflammatory stress caused by a reduction in metabolism and clearance, thus leading to the development of age-associated diseases. The quality of our daily diet and exercise is important for the prevention of these diseases. Marine resources contain various valuable nutrients, and unique glycerophospholipid plasmalogens are found abundantly in some marine invertebrates, including ascidians. One of the major classes, the ethanolamine class (PlsEtn), exists in a high ratio to phospholipids in the brain and blood, while decreased levels have been reported in patients with age-associated diseases, including Alzheimer’s disease. Animal studies have shown that the administration of marine PlsEtn prepared from marine invertebrates improved PlsEtn levels in the body and alleviated inflammation. Animal and human studies have reported that marine PlsEtn ameliorates cognitive impairment. In this review, we highlight the biological significance, relationships with age-associated diseases, food functions, and healthcare materials of plasmalogens based on recent knowledge and discuss the contribution of marine plasmalogens to health maintenance in aging.

Association of plasmalogens (Pls), a subclass of phospholipids, with Alzheimer disease (AD) has been reported. However, the efficacy of Pls against AD as well as the mechanisms for actions of Pls remain to be clarified. In this study, we examined the neurotrophic activity of Pls extracted from ascidian in PC12 cells. In addition, the memory-improving effects of Pls were examined in mice. Pls induced neurite outgrowth, as well as cAMP-response element (CRE)-, serum-response element (SRE)-, and activator protein 1(APl)-mediated transcription in PC12 cells. Additionally, during the passive avoidance tests performed in mice, Pls improved the JV-methyl-D-aspartate (NMDA) receptor antagonist MK-801-induced impairment of emotional memory. These results suggest that Pls extracted from ascidian are potentially useful for the treatment and prevention of AD. [ABSTRACT FROM AUTHOR]

Fertilization is an essential process in terrestrial organisms for creating a new organism with genetic diversity. Before gamete fusion, several steps are required to achieve successful fertilization. Animal spermatozoa are first activated and attracted to the eggs by egg-derived chemoattractants. During the sperm passage of the egg's extracellular matrix or upon the sperm binding to the proteinaceous egg coat, the sperm undergoes an acrosome reaction, an exocytosis of acrosome. In hermaphrodites such as ascidians, the self/nonself recognition process occurs when the sperm binds to the egg coat. The activated or acrosome-reacted spermatozoa penetrate through the proteinaceous egg coat. The extracellular ubiquitin–proteasome system, the astacin-like metalloproteases, and the trypsin-like proteases play key roles in this process in ascidians. In the present review, we summarize our current understanding and perspectives on gamete recognition and egg coat lysins in ascidians and consider the general mechanisms of fertilization in animals and plants. [ABSTRACT FROM AUTHOR]

Abstract Background Inductive signaling interactions between different cell types are a major mechanism for the further diversification of embryonic cell fates. Most blastomeres in the model chordate Ciona robusta become restricted to a single predominant fate between the 64-cell and mid-gastrula stages. The deeply stereotyped and well-characterized Ciona embryonic cell lineages allow the transcriptomic analysis of newly established cell types very early in their divergence from sibling cell states without the pseudotime inference needed in the analysis of less synchronized cell populations. This is the first ascidian study to use droplet scRNAseq with large numbers of analyzed cells as early as the 64-cell stage when major lineages such as primary notochord first become fate restricted. Results and conclusions We identify 59 distinct cell states, including new subregions of the b-line neural lineage and the early induction of the tail tip epidermis. We find that 34 of these cell states are directly or indirectly dependent on MAPK-mediated signaling critical to early Ciona patterning. Most of the MAPK-dependent bifurcations are canalized with the signal-induced cell fate lost upon MAPK inhibition, but the posterior endoderm is unique in being transformed into a novel state expressing some but not all markers of both endoderm and muscle. Divergent gene expression between newly bifurcated sibling cell types is dominated by upregulation in the induced cell type. The Ets family transcription factor Elk1/3/4 is uniquely upregulated in nearly all the putatively direct inductions. Elk1/3/4 upregulation together with Ets transcription factor binding site enrichment analysis enables inferences about which bifurcations are directly versus indirectly controlled by MAPK signaling. We examine notochord induction in detail and find that the transition between a Zic/Ets-mediated regulatory state and a Brachyury/FoxA-mediated regulatory state is unexpectedly late. This supports a “broad-hourglass” model of cell fate specification in which many early tissue-specific genes are induced in parallel to key tissue-specific transcriptional regulators via the same set of transcriptional inputs.

Serotonin (5-hydroxytryptamine (5-HT)) is a biogenic monoamine with pleiotropic functions. It exerts its roles by binding to specific 5-HT receptors (5HTRs) classified into different families and subtypes. Homologs of 5HTRs are widely present in invertebrates, but their expression and pharmacological characterization have been scarcely investigated. In particular, 5-HT has been localized in many tunicate species but only a few studies have investigated its physiological functions. Tunicates, including ascidians, are the sister group of vertebrates, and data about the role of 5-HTRs in these organisms are thus important for understanding 5-HT evolution among animals. In the present study, we identified and described 5HTRs in the ascidian Ciona intestinalis. During development, they showed broad expression patterns that appeared consistent with those reported in other species. Then, we investigated 5-HT roles in ascidian embryogenesis exposing C. intestinalis embryos to WAY-100635, an antagonist of the 5HT1A receptor, and explored the affected pathways in neural development and melanogenesis. Our results contribute to unraveling the multifaceted functions of 5-HT, revealing its involvement in sensory cell differentiation in ascidians.

Ascidians are a highly evolved invertebrate group enjoying a wide global distribution and are well known for their symbiotic association with microbial communities. Here, we analysed the genomic features of an ascidian associated bacterium, Bacillus aryabhattai MCCB 387 and compared its genomic features with the other three free-living B. aryabhattai reported from different ecological niches to underpin the gene repertoires involved in successful colonisation and symbiosis. In general, many 'symbiosis factors' genes have been observed in all four isolates demonstrating their potential to live with eukaryotic hosts. However, ascidian-associated B. aryabhattai MCCB 387 showed certain additional and unique features and metabolic specificity, which might have favoured and enhanced the ecological fitness of a host association. On detailed analysis, the gene for fibronectin proteins which helps to bind with integrins and facilitate cell–cell contact and possible colonisation is seen in ascidian-associated B. aryabhattai MCCB 387. The presence of one complete set of CRISPR-Cas region, Type I restriction-modification system and Toxin-antitoxin systems in MCCB 387 shows the genomic features favouring the symbiotic mode of life. MCCB 387 genome revealed a comparatively high number of Biosynthetic Gene Clusters (smBGCs) supporting the strain's secondary metabolite production potential, which might enhance fitness toward host-symbiont interaction. The genome of B. aryabhattai MCCB 387 is enriched with Mobile Genetic Elements (MGEs), suggestive of increased horizontal gene transfer between the host-microbial communities. Enriched MGEs might contribute to acquiring new genetic features and genome plasticity for the symbiont. Taken together, these genomic features might confer B. aryabhattai MCCB 387 an edge over other potential competitors, which likely enhances its ecological fitness and host colonisation. [ABSTRACT FROM AUTHOR]

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]

Botrylloides niger (class Ascidiacea) is an invasive marine filter-feeding invertebrate that is believed to originate from the West Atlantic region. This species of colonial tunicate has been observed in several locations along the coasts of Israel and around the Suez Canal, but it has not yet been reported on the coasts of the Northeastern Mediterranean Sea (NEMS), suggesting an ongoing Lessepsian migration. However, the extent of this invasion might be concealed by reports of other potentially misidentified species of Botrylloides, given that the strong morphological similarities within this genus renders taxonomical identification particularly challenging. In this study, we performed a phylogeographic and morphological analysis of B. niger in the NEMS. We collected 238 samples from 8 sampling stations covering 824 km of the coastlines of NEMS. We reported 14 different morphotypes, of which the orange-brown, orange, and brown-striped morphs were the most abundant. Using the mitochondrial cytochrome C oxidase I (COI) as a DNA barcode marker, we identified 4 haplotypes. The COI haplotypes clustered with the reference B. niger sequences from GenBank and differed significantly from the sister Botrylloides species. We confirmed our identification using three additional barcoding markers (Histone 3, 18S rRNA, and 28S rRNA), which all matched with over 99% similarity to reference sequences. In addition, we monitored a station for a year and conducted a temporal analysis of the collected colonies. The colonies were absent during the winter and spring, while new colonies were established in the summer and expanded during autumn. We performed demographic population analysis on our spatial data that identified a possible population subdivision at a sampling site, which might have been caused by local freshwater input. Herein, we present the first report on the presence of Botrylloides niger in the NEMS. This study represents a key step toward understanding the diversity and the propagation of this highly invasive species of colonial ascidians, both within the Mediterranean basin as well as globally.

The endostyle is a pharyngeal organ with an opening groove and cilia in invertebrate chordates (amphioxus and ascidian) and cyclostomate (lamprey), serving as a filter-feeding tract and thyroid-secreting location. Emerging evidence implies its complex cellular composition and potentially versatile functions. Multiple cell types in the endostyle have been thought to be progenitors of complex organs in advanced vertebrates. To describe the expression profile and the potential functions, bulk RNA sequencing on the endostyle in ascidian Styela clava was conducted and distinct markers were selected by multileveled comparative analysis. Transcriptional data assay and qRT-PCR-verified results showed the regional expression patterns of Hox genes in the longitudinal axis. Organ-specific markers of the endostyle was proposed by comparing expression with the main organs of the ascidian. A cross-species transcriptional profile projection between the endostyle and organs from Danio rerio and Homo sapiens indicates a robust homogenous relationship to the thyroid and digestive system of the endostyle. The high similarity between the endostyle and the head kidney in zebrafish/the bone marrow in human implies uniquely profound functions of the pharyngeal organ in proto-vertebrates. Our result revealed that the transcriptional profile of the human parathyroid gland was similar to the ascidian endostyle, indicating the evolutionary origin of vertebrate hormone secretion organs.

In Phlebobranchiata ascidians, oocytes and spermatozoa are stored in the oviduct and spermiduct, respectively, until spawning occurs. Gametes in the gonoducts are mature and fertilizable; however, it was found that the gametes of the ascidians Phallusia philippinensis and Ciona intestinalis could not undergo fertilization in the gonoductal fluids. The body fluids of the ascidians, especially in the gonoducts, were much more acidic (pH 5.5–6.8) than seawater (pH 8.2), and the fertilization rate was low under such acidic conditions. Hence, we examined the effect of pH on gametes. Pre-incubation of gonoductal eggs at pH 8.2 prior to insemination increased fertilization rates, even when insemination was performed under low pH conditions. Furthermore, an increase in ambient pH induced an increase in the intracellular pH of the eggs. It was also found that an increase in ambient pH triggered the release of sperm attractants from the egg and is therefore necessary for sperm chemotaxis. Hence, acidic conditions in the gonoductal fluids keep the gametes, especially eggs, infertile, and the release of eggs into seawater upon spawning induces an increase in ambient pH, which enables egg fertilization.

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.

Omics studies contribute to the elucidation of genomes and profiles of gene expression. In the ascidian Ciona intestinalis Type A (Ciona robusta), mass spectrometry (MS)-based peptidomic studies have detected numerous Ciona -specific (nonhomologous) neuropeptides as well as Ciona homologs of typical vertebrate neuropeptides and hypothalamic peptide hormones. Candidates for cognate G protein-coupled receptors (GPCRs) for these peptides have been found in the Ciona transcriptome by two ways. First, Ciona homologous GPCRs of vertebrate counterparts have been detected by sequence homology searches of cognate transcriptomes. Second, the transcriptome-derived GPCR candidates have been used for machine learning-based systematic prediction of interactions not only between Ciona homologous peptides and GPCRs but also between novel Ciona peptides and GPCRs. These data have ultimately led to experimental evidence for various Ciona peptide-GPCR interactions. Comparative transcriptomics between the wildtype and Ciona vasopressin (CiVP) gene-edited Ciona provide clues to the biological functions of CiVP in ovarian follicular development and whole body growth. Furthermore, the transcriptomes of follicles treated with peptides, such as Ciona tachykinin and cionin (a Ciona cholecystokinin homolog), have revealed key regulatory genes for Ciona follicle growth, maturation, and ovulation, eventually leading to the verification of essential and novel molecular mechanisms underlying these biological events. These findings indicate that omics studies, combined with artificial intelligence and single-cell technologies, pave the way for investigating in greater details the nervous, neuroendocrine, and endocrine systems of ascidians and the molecular and functional evolution and diversity of peptidergic regulatory networks throughout chordates. [ABSTRACT FROM AUTHOR]

The ascidian larval tail contains muscle cells for swimming. Most of these muscle cells differentiate autonomously. The genetic program behind this autonomy has been studied extensively and the genetic cascade from maternal factors to initiation of expression of a muscle structural gene, Myl.c , has been uncovered; Myl.c expression is directed initially by transcription factor Tbx6-r.b at the 64-cell stage and then by the combined actions of Tbx6-r.b and Mrf from the gastrula to early tailbud stages. In the present study, we showed that transcription of Myl.c continued in late tailbud embryos and larvae, although a fusion protein of Tbx6-r.b and GFP was hardly detectable in late tailbud embryos. A knockdown experiment, reporter assay, and in vitro binding assay indicated that an essential cis -regulatory element of Myl.c that bound Tbx6-r.b in early embryos bound Tbx15/18/22 in late embryos to maintain expression of Myl.c. We also found that Tbx15/18/22 was controlled by Mrf , which constitutes a regulatory loop with Tbx6-r.b. Therefore, our data indicated that Tbx15/18/22 was activated initially under control of this regulatory loop as in the case of Myl.c , and then Tbx15/18/22 maintained the expression of Myl.c after Tbx6-r.b had disappeared. RNA-sequencing of Tbx15/18/22 morphant embryos revealed that many muscle structural genes were regulated similarly by Tbx15/18/22. Thus, the present study revealed the mechanisms of maintenance of transcription of muscle structural genes in late embryos in which Tbx15/18/22 takes the place of Tbx6-r.b. [Display omitted] • Transcription of a muscle specific gene, Myl.c , continues in late ascidian embryos. • A critical transcription factor, Tbx6-r.b, is undetectable in late embryos. • Tbx15/18/22 takes the place of Tbx6-r.b to maintain Myl.c expression. • Many muscle structural genes are under the control of Tbx15/18/22 in late embryos. • Tbx15/18/22 maintains gene expression in differentiated muscle cells. [ABSTRACT FROM AUTHOR]

Despite a rich taxonomic literature on the symbionts of ascidians, the nature of these symbioses remains poorly understood. In the Egyptian Red Sea, the solitary ascidian Phallusia nigra hosted a symbiotic amphipod and four copepod species, with densities as high as 68 mixed symbionts per host. Correlation analyses suggested no competition or antagonism between symbionts. Ascidian mass, ash-free dry mass per wet mass (AFDM/WM), and both symbiont density and diversity per host, differed significantly among three reefs from El Gouna, Egypt. However, there was no correlation between amphipod, total copepod, or total symbiont densities and host mass or AFDM/WM. A host condition index based on body to tunic mass ratio was significantly related to symbiont density overall, but this positive pattern was only strong at a single site studied. Despite assumptions based on the habit of some of the symbiont groups, our analyses detected little effect of symbionts on host health, suggesting a commensal relationship. [ABSTRACT FROM AUTHOR]

Omics studies contribute to the elucidation of genomes and profiles of gene expression. In the ascidian Ciona intestinalis Type A (Ciona robusta), mass spectrometry (MS)-based peptidomic studies have detected numerous Ciona-specific (nonhomologous) neuropeptides as well as Ciona homologs of typical vertebrate neuropeptides and hypothalamic peptide hormones. Candidates for cognate G protein-coupled receptors (GPCRs) for these peptides have been found in the Ciona transcriptome by two ways. First, Ciona homologous GPCRs of vertebrate counterparts have been detected by sequence homology searches of cognate transcriptomes. Second, the transcriptome-derived GPCR candidates have been used for machine learning-based systematic prediction of interactions not only between Ciona homologous peptides and GPCRs but also between novel Ciona peptides and GPCRs. These data have ultimately led to experimental evidence for various Ciona peptide-GPCR interactions. Comparative transcriptomics between the wildtype and Ciona vasopressin (CiVP) gene-edited Ciona provide clues to the biological functions of CiVP in ovarian follicular development and whole body growth. Furthermore, the transcriptomes of follicles treated with peptides, such as Ciona tachykinin and cionin (a Ciona cholecystokinin homolog), have revealed key regulatory genes for Ciona follicle growth, maturation, and ovulation, eventually leading to the verification of essential and novel molecular mechanisms underlying these biological events. These findings indicate that omics studies, combined with artificial intelligence and single-cell technologies, pave the way for investigating in greater details the nervous, neuroendocrine, and endocrine systems of ascidians and the molecular and functional evolution and diversity of peptidergic regulatory networks throughout chordates.

Colonial tunicates are the only chordates that regularly regenerate a fully functional whole body as part of their asexual life cycle, starting from specific epithelia and/or mesenchymal cells. In addition, in some species, whole-body regeneration (WBR) can also be triggered by extensive injuries, which deplete most of their tissues and organs and leave behind only small fragments of their body. In this manuscript, we characterized the onset of WBR in Botryllus schlosseri, one colonial tunicate long used as a laboratory model. We first analyzed the transcriptomic response to a WBR-triggering injury. Then, through morphological characterization, in vivo observations via time-lapse, vital dyes, and cell transplant assays, we started to reconstruct the dynamics of the cells triggering regeneration, highlighting an interplay between mesenchymal and epithelial cells. The dynamics described here suggest that WBR in B. schlosseri is initiated by extravascular tissue fragments derived from the injured individuals rather than particular populations of blood-borne cells, as has been described in closely related species. The morphological and molecular datasets here reported provide the background for future mechanistic studies of the WBR ontogenesis in B. schlosseri and allow to compare it with other regenerative processes occurring in other tunicate species and possibly independently evolved.

Fertilization is one of the most important events in living organisms to generate a new life with a mixed genetic background. To achieve successful fertilization, sperm and eggs must undergo complex processes in a sequential order. Fertilization of marine invertebrate Ciona intestinalis type A (Ciona robusta) has been studied for more than a hundred years. Ascidian sperm are attracted by chemoattractants from eggs and bind to the vitelline coat. Subsequently, sperm penetrate through the vitelline coat proteolytically and finally fuse with the egg plasma membrane. Here, we summarize the fertilization mechanisms of ascidians, particularly from sperm-egg interactions to sperm penetration of the egg coat. Since ascidians are hermaphrodites, inbreeding depression is a serious problem. To avoid self-fertilization, ascidians possess a self-incompatibility system. In this review, we also describe the molecular mechanisms of the self-incompatibility system in C. intestinalis type A governed by three allelic gene pairs of s-Themis and v-Themis.

The neural IgCAM family of cell adhesion molecules, which includes NCAM and related molecules, has evolved via gene duplication and alternative splicing to allow for a wide range of isoforms with distinct functions and homophilic binding properties. A search for neural IgCAMs in ascidians (Ciona intestinalis, Ciona savignyi, and Phallusia mammillata) has identified a novel set of truncated family members that, unlike the known members, lack fibronectin III domains and consist of only repeated Ig domains. Within the tunicates this form appears to be unique to the ascidians, and it was designated ACAM, for Ascidian Cell Adhesion Molecule. In C. intestinalis ACAM is expressed in the developing neural plate and neural tube, with strongest expression in the anterior sensory vesicle precursor. Unlike the two other conventional neural IgCAMs in C. intestinalis, which are expressed maternally and throughout the morula and blastula stages, ACAM expression initiates at the gastrula stage. Moreover, C. intestinalis ACAM is a target of the homeodomain transcription factor OTX, which plays an essential role in the development of the anterior central nervous system. Morpholino (MO) knockdown shows that ACAM is required for neural tube closure. In MO-injected embryos neural tube closure was normal caudally, but the anterior neuropore remained open. A similar phenotype was seen with overexpression of a secreted version of ACAM. The presence of ACAM in ascidians highlights the diversity of this gene family in morphogenesis and neurodevelopment.

Reproductive isolation is central to speciation, but interspecific crosses between two closely related species can produce viable and fertile hybrids. Two different species of tunicates in the same ascidian genus, Ciona robusta and Ciona intestinalis, can produce hybrids. However, wild sympatric populations display limited gene flow, suggesting the existence of obstacles to interspecific reproduction that remain unknown. Here, we took advantage of a closed culture system to cross C. robusta with C. intestinalis and established F1 and F2 hybrids. We monitored post-embryonic development, survival, and sexual maturation to characterize the genetic basis of simple traits, and further probe the physiological mechanisms underlying reproductive isolation. Partial viability of first and second generation hybrids suggested that both pre- and postzygotic mechanisms contributed to genomic incompatibilities in hybrids. We observed asymmetric fitness, whereby the C. intestinalis maternal lines fared more poorly in our system, pointing to maternal origins of species-specific sensitivity. We discuss the possibility that asymmetrical second generation inviability and infertility emerge from interspecific incompatibilities between the nuclear and mitochondrial genomes, or other maternal effect genes. This work paves the way to quantitative genetic approaches to study the mechanisms underlying genomic incompatibilities and other complex traits in the genome-enabled Ciona model.

Abstract Background Gut microbiota plays important roles in host animal metabolism, homeostasis and environmental adaptation. However, the interplay between the gut microbiome and urochordate ascidian, the most closet relative of vertebrate, remains less explored. In this study, we characterized the gut microbial communities of urochordate ascidian (Halocynthia roretzi) across the changes of season and starvation stress using a comprehensive set of omic approaches including 16S rRNA gene amplicon sequencing, shotgun metagenomics, metabolomic profiling, and transcriptome sequencing. Results The 16S rRNA gene amplicon profiling revealed that ascidians harbor indigenous gut microbiota distinctly different to the marine microbial community and significant variations in composition and abundance of gut bacteria, with predominant bacterial orders representing each season. Depressed alpha-diversities of gut microbiota were observed across starvation stress when compared to the communities in aquafarm condition. Synechococcales involving photosynthesis and its related biosynthesis was reduced in abundance while the enrichments of Xanthomonadales and Legionellales may facilitate bile acid biosynthesis during starvation. Metabolomics analysis found that long chain fatty acids, linolenic acid, cyanoamino acid, and pigments derived from gut bacteria were upregulated, suggesting a beneficial contribution of the gut microbiome to the ascidian under starvation stress. Conclusions Our findings revealed seasonal variation of ascidian gut microbiota. Defense and energy-associated metabolites derived from gut microbiome may provide an adaptive interplay between gut microbiome and ascidian host that maintains a beneficial metabolic system across season and starvation stress. The diversity-generating metabolisms from both microbiota and host might lead to the co-evolution and environmental adaptation. Graphical Abstract

Abstract Ascidians are sessile marine chordate invertebrates found along seashores worldwide and are typically regarded as invasive organisms. Knowledge concerning their global genetic structure and subsequent invasive potential is limited. Here, we identified three ascidians—Ciona robusta, Ciona savignyi, and Styela clava from the northeast region of China using morphological characteristics and mitochondrial cytochrome c oxidase subunit I (cox1) as genetic marker. We additionally used phylogenetics to aid in the identification of these three species. The results of a population genetic analysis showed that among the three species, the level of haplotype diversity was particularly high within C. savignyi, and nucleotide diversity varied moderately. We divided the three species separately into native and invasive populations using 170 cox1 sequences from global resources to explore population genetic structure and invasive potential. Although in the network analysis Ciona spp. formed haplogroups of native and invasive populations, some haplotypes were still shared. We found that the haplotypes did not cluster within the network of S. clava. Our AMOVA results also showed that Ciona spp. had a weak genetic structure, and less genetic differentiation was present in S. clava. These data suggest that there are extensive incursions of these three ascidians into different geographical regions. Global comparisons of ascidian populations will help in the understanding of their population genetic structure and invasive potential, hence providing important insights regarding conservation as well as management.

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.

Cell division orientation is thought to result from a competition between cell geometry and polarity domains controlling the position of the mitotic spindle during mitosis. Depending on the level of cell shape anisotropy or the strength of the polarity domain, one dominates the other and determines the orientation of the spindle. Whether and how such competition is also at work to determine unequal cell division (UCD), producing daughter cells of different size, remains unclear. Here, we show that cell geometry and polarity domains cooperate, rather than compete, in positioning the cleavage plane during UCDs in early ascidian embryos. We found that the UCDs and their orientation at the ascidian third cleavage rely on the spindle tilting in an anisotropic cell shape, and cortical polarity domains exerting different effects on spindle astral microtubules. By systematically varying mitotic cell shape, we could modulate the effect of attractive and repulsive polarity domains and consequently generate predicted daughter cell size asymmetries and position. We therefore propose that the spindle position during UCD is set by the combined activities of cell geometry and polarity domains, where cell geometry modulates the effect of cortical polarity domain(s).

Ascidians, particularly those highly invasive ones, are typical fouling organisms to cause significantly negative ecological and economic influence in coastal ecosystems. Stolon, which is the unique structure of some solitary ascidians to complete the essential process of adhesion, possesses extremely high tolerance to environmental stresses during biofouling and invasions. However, the mechanisms underlying environmental tolerance remain largely unknown. Here, we used the quantitative proteomics technology, isobaric tags for relative and absolute quantitation (iTRAQ), to investigate the molecular response to environmental challenges (temperature and salinity) in the stolon of a highly invasive fouling ascidian, Ciona robusta. When compared with the control, a total of 75, 86, 123, and 83 differential abundance proteins were identified under low salinity, high salinity, low temperature, and high temperature stress, respectively. Bioinformatic analyses uncovered the key pathways under both temperature and salinity stresses, including “cytoskeleton,” “signal transduction,” and “posttranslational modification,” which were involved in stolon structure stability, protein synthesis, and stress response activation. Under the low salinity stress, the “extracellular matrix” pathway was identified to play a crucial role by regulating cell signal transduction and protein synthesis. To deal with the high salinity stress, stolon could store more energy by activating “carbohydrate/lipid transport” and “catabolism” pathways. The energy generated by “lipid metabolism” pathway might be beneficial to resist the low temperature stress. The upregulation of “cell cycle” pathway could inhibit cell growth, thus helping stolon conserve more energy against the high temperature stress. Our results here provide valuable references of candidate pathways and associated genes for studying mechanisms of harsh environmental adaptation and developing antifouling strategies in marine and coastal ecosystems.

Osmoregulation is essential for organisms to adapt to the exterior environment and plays an important role in embryonic organogenesis. Tubular organ formation usually involves a hyperosmotic lumen environment. The mechanisms of how the cells respond and regulate lumen formation remain largely unknown. Here, we reported that the nuclear factor of activated T cells-5 (NFAT5), the only transcription factor in the NFAT family involved in the cellular responses to hypertonic stress, regulated notochord lumen formation in chordate Ciona. Ciona NFAT5 (Ci-NFAT5) was expressed in notochord, and its expression level increased during notochord lumen formation and expansion. Knockout and expression of the dominant negative of NFAT5 in Ciona embryos resulted in the failure of notochord lumen expansion. We further demonstrated that the Ci-NFAT5 transferred from the cytoplasm into nuclei in HeLa cells under the hyperosmotic medium, indicating Ci-NFAT5 can respond the hypertonicity. To reveal the underly mechanisms, we predicted potential downstream genes of Ci-NFAT5 and further validated Ci-NFAT5-interacted genes by the luciferase assay. The results showed that Ci-NFAT5 promoted SLC26A6 expression. Furthermore, expression of a transport inactivity mutant of SLC26A6 (L421P) in notochord led to the failure of lumen expansion, phenocopying that of Ci-NFAT5 knockout. These results suggest that Ci-NFAT5 regulates notochord lumen expansion via the SLC26A6 axis. Taken together, our results reveal that the chordate NFAT5 responds to hypertonic stress and regulates lumen osmotic pressure via an ion channel pathway on luminal organ formation.

Plastic pollution is recognized as a global environmental threat and concern is increasing regarding the potential interactions of the smallest fragments, nanoplastics (1 µm), with either physical and chemical entities encountered in the natural environment, including toxic pollutants. The smallest size of nanoplastics (Ciona robusta embryos (22 h post fertilization, hpf) by looking at embryotoxicity through phenotypic alterations. We confirmed the ability of BPA to impact ascidian C. robusta embryo development, by affecting sensory organs pigmentation, either alone and in combination with PS NPs. Our findings suggest that no interactions are taking place between PS NPs and BPA in filtered sea water (FSW) probably due to the high ionic strength of seawater able to trigger the sorption surface properties of PS NPs. Further studies are needed to elucidate such peculiarities and define the risk posed by combined exposure to BPA and PS NPs in marine coastal waters.

The heart of ascidians (marine invertebrate chordates) has a tubular structure, and heartbeats propagate from one end to the other. The direction of pulsation waves intermittently reverses in the heart of ascidians and their relatives; however, the underlying mechanisms remain unclear. We herein performed a series of experiments to characterize the pacemaker systems in isolated hearts and their fragments, and applied a mathematical model to examine the conditions leading to heart reversals. The isolated heart of Ciona robusta autonomously generated pulsation waves at ∼20 to 25 beats min-1 with reversals at ∼1 to 10 min intervals. Experimental bisections of isolated hearts revealed that independent pacemakers resided on each side and also that their beating frequencies periodically changed as they expressed bimodal rhythms, which comprised an ∼1.25 to 5.5 min acceleration/deceleration cycle of a beating rate of between 0 and 25 beats min-1. Only fragments including 5% or shorter terminal regions of the heart tube maintained autonomous pulsation rhythms, whereas other regions did not. Our mathematical model, based on FitzHugh-Nagumo equations applied to a one-dimensional alignment of cells, demonstrated that the difference between frequencies expressed by the two independent terminal pacemakers determined the direction of propagated waves. Changes in the statuses of terminal pacemakers between the excitatory and oscillatory modes as well as in their endogenous oscillation frequencies were sufficient to lead to heart reversals. These results suggest that the directions of pulsation waves in the Ciona heart reverse according to the changing rhythms independently expressed by remotely coupled terminal pacemakers., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Acute and chronic dermatological injuries need rapid tissue repair due to the susceptibility to infections. To effectively promote cutaneous wound recovery, it is essential to develop safe, low-cost, and affordable regenerative tools. Therefore, we aimed to identify the biological mechanisms involved in the wound healing properties of the glycosaminoglycan dermatan sulfate (DS), obtained from ascidian Styela plicata, a marine invertebrate, which in preliminary work from our group showed no toxicity and promoted a remarkable fibroblast proliferation and migration. In this study, 2,4-DS (50 µg/mL)-treated and control groups had the relative gene expression of 84 genes participating in the healing pathway evaluated. The results showed that 57% of the genes were overexpressed during treatment, 16% were underexpressed, and 9.52% were not detected. In silico analysis of metabolic interactions exhibited overexpression of genes related to: extracellular matrix organization, hemostasis, secretion of inflammatory mediators, and regulation of insulin-like growth factor transport and uptake. Furthermore, in C57BL/6 mice subjected to experimental wounds treated with 0.25% 2,4-DS, the histological parameters demonstrated a great capacity for vascular recovery. Additionally, this study confirmed that DS is a potent inducer of wound-healing cellular pathways and a promoter of neovascularization, being a natural ally in the tissue regeneration strategy.

The genus Absidia is widely used in the biotransformation of different classes of natural products. This study evaluates the ability of the Absidia coerulea 3A9 marine derived strain isolated from the ascidian Distaplia stilyfera to perform biotransformations by conducting assays with (–)-cubebin, as substrate. The experiment was optimized using the experimental design proposed by Plackett–Burman for seven factors and eight experiments, to establish the biotransformation conditions that would allow maximum production of biotransformed dibenzylbutyrolactone (–)-hinokinin. An analytical method based on Reverse-Phase-High Performance Liquid Chromatography (RP-HPLC) was developed to quantify the fungal biotransformation product. The factor that influenced the (–)-hinokinin peak area the most positively was the percentage of seawater (%seawater) given that its %relative standard deviation (%RSD) showed a 32.92% deviation from the real value. [ABSTRACT FROM AUTHOR]

Taxonomic uncertainties and the lack of ecological knowledge can hinder the correct identification and the assignment of biogeographic status of marine species. The ascidian Asterocarpa humilis (Heller, 1878), originally described from New Zealand, has a broad distribution in shallow temperate areas of the Southern Hemisphere, having recently colonised areas of the Northern Hemisphere. A closely related species, Cnemidocarpa robinsoni Hartmeyer, 1916, has been reported in the South-Eastern Pacific and the South-Western Atlantic, and several authors considered it a junior synonym of A. humilis. We gathered for the first time morphological and genetic data from specimens from distant areas. We studied the morphology of specimens collected at seven locations of South America. We also re-examined specimens from museum collections and revised the available literature on these species. Genetic data were obtained from specimens from Argentina and compared with available sequences of A. humilis from Chile, New Zealand, England and France. Morphological and genetic analyses showed that all compared specimens were conspecific. Furthermore, specimens from different continents shared haplotypes and exhibited low genetic distance among them. These results, the biological characteristics of this ascidian, and its longstanding presence in different habitats from disjoint areas, allow us to question its native range. We support the idea that A. humilis is a cryptogenic and neocosmopolitan species that has been transported by maritime traffic through the Southern Hemisphere, revealing frequent processes of exchange through this wide area for more than a century, with presumably associated alterations in the marine biota. Morphological and genetic analyses in specimens of two similar ascidians from different worldwide areas, particularly from SW Atlantic, revealed that they belong to a single species: Asterocarpa humilis. Longstanding presence in natural and artificial habitats along disjoint areas of the Southern Hemisphere suggests its human transport and its neocosmopolitanism. We propose a cryptogenic status for this species in the Southern Hemisphere. [ABSTRACT FROM AUTHOR]

Marine invertebrates, particularly ascidians, constitute an important source of potential active and biofunctional natural products. The microbial diversity associated with ascidians is little recognized, although these microorganisms play a vital role in marine ecosystems. The objective of this study was to investigate bacterial population diversity in four ascidian samples: Phallusia nigra, Phallusia fumigata, Eudistoma viride, and Rhopalaea macrothorax, collected from the North Bay, Andaman and Nicobar Islands. Microbial strains identified up to the species level revealed 236 distinct species/ribotypes out of 298 bacterial strains. Of 298 ascidian-associated bacteria, 72 isolates belong to the class Gammaproteobacteria and the genus Endozoicomonas. The results from this investigation will contribute a broaden knowledge of microbial diversity associated to marine ascidians, and as a promising source for the discovery of new natural products. [ABSTRACT FROM AUTHOR]

Background: Inductive signaling interactions between different cell types are a major mechanism for the further diversification of embryonic cell fates. Most blastomeres in the model chordate Ciona robusta become restricted to a single predominant fate between the 64-cell and mid-gastrula stages. The deeply stereotyped and well-characterized Ciona embryonic cell lineages allow the transcriptomic analysis of newly established cell types very early in their divergence from sibling cell states without the pseudotime inference needed in the analysis of less synchronized cell populations. This is the first ascidian study to use droplet scRNAseq with large numbers of analyzed cells as early as the 64-cell stage when major lineages such as primary notochord first become fate restricted. Results and conclusions: We identify 59 distinct cell states, including new subregions of the b-line neural lineage and the early induction of the tail tip epidermis. We find that 34 of these cell states are directly or indirectly dependent on MAPK-mediated signaling critical to early Ciona patterning. Most of the MAPK-dependent bifurcations are canalized with the signal-induced cell fate lost upon MAPK inhibition, but the posterior endoderm is unique in being transformed into a novel state expressing some but not all markers of both endoderm and muscle. Divergent gene expression between newly bifurcated sibling cell types is dominated by upregulation in the induced cell type. The Ets family transcription factor Elk1/3/4 is uniquely upregulated in nearly all the putatively direct inductions. Elk1/3/4 upregulation together with Ets transcription factor binding site enrichment analysis enables inferences about which bifurcations are directly versus indirectly controlled by MAPK signaling. We examine notochord induction in detail and find that the transition between a Zic/Ets-mediated regulatory state and a Brachyury/FoxA-mediated regulatory state is unexpectedly late. This supports a "broad-hourglass" model of cell fate specification in which many early tissue-specific genes are induced in parallel to key tissue-specific transcriptional regulators via the same set of transcriptional inputs. [ABSTRACT FROM AUTHOR]

Colour variation in marine invertebrates is typically attributed to intraspecific variability; however, the use of molecular tools to investigate its genetic basis has uncovered many instances of cryptic speciation. The colonial ascidian Distaplia bermudensis occurs in a wide array of colour morphs, ranging from all white to a dark blue tunic with yellow siphon apertures; yet careful observations of zooid and larval morphologies have not revealed any consistent difference among these colour morphs. In this study, we sampled the 5 main colour morph groupings of D. bermudensis from Wilmington, North Carolina (USA) and sequenced fragments of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear 18S rRNA genes. Analyses of both genes revealed the presence of two distinct genetic lineages that corresponded to unique colour morphology groupings. We further characterized symbiotic microbial communities for four samples from each lineage by high‐throughput sequencing of partial 16S rRNA genes. Each genetic lineage also harboured unique symbiont communities that differed significantly in diversity and structure. Thus, the two genetic lineages detected herein corresponded to unique colour morphs and microbial communities, indicating that two distinct species coexist under the same scientific name. Further, our study reports the utility of symbiont profiling in resolving the taxonomic status of phenotypically variable ascidian species. [ABSTRACT FROM AUTHOR]

The diversity of actinobacteria associated with marine ascidian Phallusia nigra from Andaman Islands was investigated. A total of 10 actinobacteria were isolated and based on the biochemical and molecular characterization, the isolates were assigned to 7 different actinobacterial genera. Eight putatively novel species belonging to genera Rhodococcus, Kineococcus, Kocuria, Janibacter, Salinispora and Arthrobacter were identified based on 16S rDNA sequence similarity with the NCBI database. The organic extracts of ten isolates displayed considerable bioactivity against test pathogens, which were Gram-positive and Gram-negative in nature. PCR-based screening for type I and type II polyketide synthases (PKS-I, PKS-II) and nonribosomal peptide synthetases (NRPS) revealed that, 10 actinobacterial isolates encoded at least one type of polyketide synthases biosynthesis gene. Majority of the isolates found to produce industrially important enzymes; amylase, protease, gelatinase, lipase, DNase, cellulase, urease, phosphatase and l-asparaginase. The present study emphasized that, ascidians are a prolific resource for novel bioactive actinobacteria with potential for novel drug discovery. This result expands the scope to functionally characterize the novel ascidian associated marine actinobacteria and their metabolites could be a source for the novel molecules of commercial interest. [ABSTRACT FROM AUTHOR]

Botryllus schlosseri is a colonial ascidian with characteristics that make it an attractive model for studying immunology, stem cell biology, evolutionary biology, and regeneration. Transcriptome sequencing and the recent completion of a draft genome sequence for B. schlosseri have revealed a large number of genes, both with and without vertebrate homologs, but analyzing the spatial and temporal expression of these genes in situ has remained a challenge. Here, we report a robust protocol for in situ hybridization that enables the simultaneous detection of multiple transcripts in whole adult B. schlosseri using Tyramide Signal Amplification in conjunction with digoxigenin- and dinitrophenol-labeled RNA probes. Using this protocol, we have identified a number of genes that can serve as markers for developing and mature structures in B. schlosseri, permitting analysis of phenotypes induced in loss-of-function experiments.