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Controlling global protein synthesis through the assembly of stress granules represents a strategy adopted by eukaryotic cells to face various stress conditions. TIA 1-related nucleolysin (TIAR), tristetraprolin (TTP), and Ras- GTPase-activating protein SH3-domain-binding protein (G3BP) are key components of stress granules, allowing the regulation of mRNA stability, and thus controlling not only stress responses but also cell proliferation and differentiation. In this study, we aimed at investigating the roles of tiar, ttp, and g3bp during embryogenesis of the solitary ascidian Ciona robusta under both physiological and stress conditions. We carried out CRISPR/Cas9 to evaluate the effects of gene knockout on normal embryonic development, and gene reporter assay to study the time and tissue specificity of gene transcription, together with whole-mount in situ hybridization and quantitative real time PCR. To induce acute stress conditions, we used iron and cadmium as "essential" and "non-essential" metals, respectively. Our results highlight, for the first time, the importance of tiar, ttp, and g3bp in controlling the development of mesendodermal tissue derivatives during embryogenesis of an invertebrate chordate. [ABSTRACT FROM AUTHOR]

Simple Summary: Ascidians (Tunicata) are a powerful model for studying the innate immune system. To better understand the dynamics of immune responses under bacterial challenge in Ciona robusta, we exposed ascidians to lipopolysaccharide (LPS) injection. Immunohistochemistry analysis on two components of the nuclear factor kappa B (NfκB) key signalling pathway, the Toll-like receptor 4 (TLR4) and NFκB, showed their over-expression on tissue of LPS-injected ascidians. Also, several enzymes related to immune responses were up-modulated following the LPS challenge. Our study suggests a broad and complex innate immune activation in the regulation of tunicate inflammatory responses. The close phylogenetic relationship between ascidians (Tunicata) and vertebrates makes them a powerful model for studying the innate immune system. To better understand the nature and dynamics of immune responses and the mechanisms through which bacterial infections are detected and translated into inflammation in Ciona robusta, we applied an approach combining in vivo lipopolysaccharide (LPS) stimulation, immune-labelling techniques and functional enzymatic analyses. The immunohistochemistry showed that Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were expressed during the inflammatory pharynx response 4 h post-LPS, with the formation of nodules in pharynx vessel lumen. Also, the endothelium vessels were involved in the inflammatory response. Observations of histological sections from naive and buffer-inoculated ascidians confirmed an immuno-positive response. Enzyme immune parameters—which included the activity of phenoloxidase, glutathione peroxidase, lysozyme, alkaline phosphatase and esterase—showed up-modulation 4 h after LPS injection, confirming their participation during ascidian inflammatory response. These findings provide new insights into the mechanisms underlying the LPS-induced C. robusta response and suggest that a broad innate immune mechanism, as in vertebrates, is involved in the regulation of inflammatory responses. Further findings in this direction are needed to cover knowledge gaps regarding the organized set of molecular and cellular networks involved in universal immune interactions with pathogens. [ABSTRACT FROM AUTHOR]

Subtropical coral reefs such as the lagoonal reefs of Norfolk Island in the remote South Pacific are vastly understudied, with many benthic species unrecorded in the scientific literature. Here we report on ascidian populations from 2021 to 2023 at Norfolk Islands inshore reefs. Quantitative assessments spanning that period were conducted to assess ascidian presence, cover, and benthic interactions. We show ascidian cover remained persistently low and stable across the lagoonal reefs during the study period. We find adjacent reef site, Cemetery Bay showed variation in ascidian cover over time, where we measure a 3.2-fold increase in cover over 2 years. Ascidians were associated primarily with sand and sediment substrates at all reef locations. Recorded interactions between hard corals and ascidians were low. This study provides valuable insights into the population dynamics of ascidians within subtropical reef ecosystems. Ongoing ascidian population monitoring can provide a comprehensive understanding of ascidian dynamics whilst also providing insight of theimpact of anthropogenic stressors on benthic communities. Together, this can aid in informing management and conservation strategies for subtropical reefs. [ABSTRACT FROM AUTHOR]

Abstract Background The tunicates form a group of filter-feeding marine animals closely related to vertebrates. They share with them a number of features such as a notochord and a dorsal neural tube in the tadpole larvae of ascidians, one of the three groups that make tunicates. However, a number of typical chordate characters have been lost in different branches of tunicates, a diverse and fast-evolving phylum. Consequently, the tunic, a sort of exoskeleton made of extracellular material including cellulose secreted by the epidermis, is the unifying character defining the tunicate phylum. In the larva of ascidians, the tunic differentiates in the tail into a median fin (with dorsal and ventral extended blades) and a caudal fin. Results Here we have performed experiments in the ascidian Phallusia mammillata to address the molecular control of tunic 3D morphogenesis. We have demonstrated that the tail epidermis medio-lateral patterning essential for peripheral nervous system specification also controls tunic elongation into fins. More specifically, when tail epidermis midline identity was abolished by BMP signaling inhibition, or CRISPR/Cas9 inactivation of the transcription factor coding genes Msx or Klf1/2/4/17, median fin did not form. We postulated that this genetic program should regulate effectors of tunic secretion. We thus analyzed the expression and regulation in different ascidian species of two genes acquired by horizontal gene transfer (HGT) from bacteria, CesA coding for a cellulose synthase and Gh6 coding for a cellulase. We have uncovered an unexpected dynamic history of these genes in tunicates and high levels of variability in gene expression and regulation among ascidians. Although, in Phallusia, Gh6 has a regionalized expression in the epidermis compatible with an involvement in fin elongation, our functional studies indicate a minor function during caudal fin formation only. Conclusions Our study constitutes an important step in the study of the integration of HGT-acquired genes into developmental networks and a cellulose-based morphogenesis of extracellular material in animals.

It is a relatively common occurrence for forensic pathologists and anthropologists to be presented with what are believed to be human remains as part of their day-to-day practice. Despite this, the literature pertaining to such challenges is not extensive, and much knowledge of this is largely based on experience. As such, we present a case of what appeared to be a severed foot located on a beach, which examination revealed was a marine animal known as a sea squirt (ascidian). While marine scientists have been aware of such mimicry, to our knowledge, this has not been previously described in the forensic pathology literature. The external examination and post-mortem CT scan revealed the nonhuman nature of the remains, and an imminent police investigation was prevented, saving time and resources. Nonhuman remains may include animals and inorganic objects, and their discovery may invoke anxiety in the finder. A timely forensic pathology or anthropology examination will assist in alleviating such concern. Forensic pathologists and anthropologists should be prepared to face a variety of presented remains and objects. [ABSTRACT FROM AUTHOR]

Ascidians are marine filter-feeder chordates. Botrylloides ascidians possess diverse biological properties such as whole-body regeneration (WBR), hibernation/aestivation, blastogenesis, metamorphosis, and natural chimerism. However, the absence of distinctive morphological features often makes identification difficult. Botrylloides diegensis is an ascidian that has been misidentified in previous studies and is recorded in GenBank as Botrylloides leachii owing to the high morphological similarity between the sister species. More available sequences and strategies for identification would help resolve some of the confusion surrounding its ambiguous nature. We collected several Botrylloides samples from seven locations in Aotearoa New Zealand (Dunedin, Christchurch, Picton, Nelson, Whangateau, New Plymouth, and Invercargill) and barcoded the species based on Cytochrome Oxidase I, Histone 3, 18S, and 28S ribosomal RNA markers. Network and Bayesian trees confirmed the presence of three Botrylloides species: B. diegensis, Botrylloides jacksonianum, and Botrylloides affinis anceps. [ABSTRACT FROM AUTHOR]

Discovering macromolecules and understanding the associated mechanisms involved in underwater adhesion are essential for both studying the fundamental ecology of benthos in aquatic ecosystems and developing biomimetic adhesive materials in industries. Here, we employed quantitative proteomics to assess protein expression variations during the development of the distinct adhesive structure - stolon in the model fouling ascidian, Ciona robusta. We found 16 adhesive protein candidates with increased expression in the stolon, with ascidian adhesive protein 1 (AAP1) being particularly rich in adhesion-related signal peptides, amino acids, and functional domains. Western blot and immunolocalization analyses confirmed the prominent AAP1 signals in the mantle, tunic, stolon, and adhesive footprints, indicating the interfacial role of this protein. Surface coating and atomic force microscopy experiments verified AAP1′s adhesion to diverse materials, likely through the specific electrostatic and hydrophobic amino acid interactions with various substrates. In addition, molecular docking calculations indicated the AAP1′s potential for cross-linking via hydrogen bonds and salt bridges among Von Willebrand factor type A domains, enhancing its adhesion capability. Altogether, the newly discovered interfacial protein responsible for permanent underwater adhesion, along with the elucidated adhesion mechanisms, are expected to contribute to the development of biomimetic adhesive materials and anti-fouling strategies. Discovering macromolecules and studying their associated mechanisms involved in underwater adhesion are essential for understanding the fundamental ecology of benthos in aquatic ecosystems and developing innovative bionic adhesive materials in various industries. Using multidisciplinary analytical methods, we identified an interfacial protein - Ascidian Adhesive Protein 1 (AAP1) from the model marine fouling ascidian, Ciona robusta. The interfacial functions of AAP1 are achieved by electrostatic and hydrophobic interactions, and the Von Willebrand factor type A domain-based cross-linking likely enhances AAP1's interfacial adhesion. The identification and validation of the interfacial functions of AAP1, combined with the elucidation of adhesion mechanisms, present a promising target for the development of biomimetic adhesive materials and the formulation of effective anti-fouling strategies. [Display omitted] [ABSTRACT FROM AUTHOR]

The early stages of regeneration after injury are similar to those of wound healing. The ascidian Botrylloides diegensis can regenerate an entire adult from a small fragment of vascular tunic following the removal of all zooids in an injury-induced regeneration model. We investigated the molecular and cellular changes following injury to determine the differences between the healing process and the initiation of whole-body regeneration (WBR). We conducted transcriptome analysis at specific time points during regeneration and wound healing to identify differentially expressed genes (DEGs) and the unique biological processes associated with each state. Our findings revealed 296 DEGs at 10 h post-injury (hpi), with 71 highly expressed in healed tissue and 225 expressed during the WBR process. These DEGs were predicted to play roles in tissue reorganization, integrin signaling, extracellular matrix organization, and the innate immune system. Pathway analysis of the upregulated genes in the healed tunic indicated functional enrichment related to tissue repair, as has been observed in other species. Additionally, we examined the cell types in the tunic and ampullae in both tissue states using histology and in situ hybridization for six genes identified by transcriptome analysis. We observed strong mRNA expression in cells within the WBR tunic, and in small RNA-positive granules near the tunic edge. We hypothesized that many of these genes function in the compaction of the ampullae tunic, which is a pivotal process for WBR and dormancy in B. diegensis, and in an immune response. These findings establish surprising similarities between ascidian regeneration and human wound healing, emphasizing the potential for future investigations into human regenerative and repair mechanisms. This study provides valuable insights into the gene sets specifically activated during regeneration compared to wound healing, shedding light on the divergent activities of these processes. [Display omitted] • RNA-seq uncovers parallels between whole-body regeneration and wound healing in B. diegensis. • Similarities in gene expression and tunic cell behavior mirror epidermal wound healing. • Innate immune responses activated post-injury, in both healing and regeneration. • Cellular responses involve tunic contraction, vascular budding, and angiogenesis. • Extracellular vesicles and granules may contribute to communication and regeneration processes. [ABSTRACT FROM AUTHOR]

Serotonin (5 hydroxytryptamine, 5-HT) is a biogenic amine of ancient origin that is widespread among animals. It plays multiple roles during development and in adults as neurotransmitter at synaptic level and neuro hormone controlling complex behaviors in both vertebrates and invertebrates. Tunicates occupy a key phylogenetic position to understand the evolution of serotonin functions since they are the sister group of vertebrates. The presence of serotonin in tunicates was first reported in adults of the ascidian Ciona robusta (formerly Ciona intestinalis) in the 1946. Since then, serotonin systems have been in many tunicate species and its functions during embryogenesis and metamorphosis explored. We reviewed the current knowledge about serotonin in these animals first by comparing its presence and localization in larvae and adults of different species. Then, we focused on the model organism Ciona for which data regarding sequences and expression patterns of genes involved in serotonin synthesis and function have been reported. Overall, we provided a comprehensive overview of serotonergic machinery in tunicates and gave hints for future studies in this field. [ABSTRACT FROM AUTHOR]

Background: The tunicates form a group of filter-feeding marine animals closely related to vertebrates. They share with them a number of features such as a notochord and a dorsal neural tube in the tadpole larvae of ascidians, one of the three groups that make tunicates. However, a number of typical chordate characters have been lost in different branches of tunicates, a diverse and fast-evolving phylum. Consequently, the tunic, a sort of exoskeleton made of extracellular material including cellulose secreted by the epidermis, is the unifying character defining the tunicate phylum. In the larva of ascidians, the tunic differentiates in the tail into a median fin (with dorsal and ventral extended blades) and a caudal fin. Results: Here we have performed experiments in the ascidian Phallusia mammillata to address the molecular control of tunic 3D morphogenesis. We have demonstrated that the tail epidermis medio-lateral patterning essential for peripheral nervous system specification also controls tunic elongation into fins. More specifically, when tail epidermis midline identity was abolished by BMP signaling inhibition, or CRISPR/Cas9 inactivation of the transcription factor coding genes Msx or Klf1/2/4/17, median fin did not form. We postulated that this genetic program should regulate effectors of tunic secretion. We thus analyzed the expression and regulation in different ascidian species of two genes acquired by horizontal gene transfer (HGT) from bacteria, CesA coding for a cellulose synthase and Gh6 coding for a cellulase. We have uncovered an unexpected dynamic history of these genes in tunicates and high levels of variability in gene expression and regulation among ascidians. Although, in Phallusia, Gh6 has a regionalized expression in the epidermis compatible with an involvement in fin elongation, our functional studies indicate a minor function during caudal fin formation only. Conclusions: Our study constitutes an important step in the study of the integration of HGT-acquired genes into developmental networks and a cellulose-based morphogenesis of extracellular material in animals. [ABSTRACT FROM AUTHOR]

The sea squirt Ciona robusta (formerly Ciona intestinalis type A) has been the subject of many interdisciplinary studies. Known as a vanadium-rich ascidian, C. robusta is an ideal model for exploring microbes associated with the ascidian and the roles of these microbes in vanadium accumulation and reduction. In this study, we discovered two bacterial strains that accumulate large amounts of vanadium, CD2-88 and CD2-102, which belong to the genera Pseudoalteromonas and Vibrio, respectively. The growth medium composition impacted vanadium uptake. Furthermore, pH was also an important factor in the accumulation and localization of vanadium. Most of the vanadium(V) accumulated by these bacteria was converted to less toxic vanadium(IV). Our results provide insights into vanadium accumulation and reduction by bacteria isolated from the ascidian C. robusta to further study the relations between ascidians and microbes and their possible applications for bioremediation or biomineralization. [ABSTRACT FROM AUTHOR]

Controlling global protein synthesis through the assembly of stress granules represents a strategy adopted by eukaryotic cells to face various stress conditions. TIA 1-related nucleolysin (TIAR), tristetraprolin (TTP), and Ras-GTPase-activating protein SH3-domain-binding protein (G3BP) are key components of stress granules, allowing the regulation of mRNA stability, and thus controlling not only stress responses but also cell proliferation and differentiation. In this study, we aimed at investigating the roles of tiar, ttp, and g3bp during embryogenesis of the solitary ascidian Ciona robusta under both physiological and stress conditions. We carried out CRISPR/Cas9 to evaluate the effects of gene knockout on normal embryonic development, and gene reporter assay to study the time and tissue specificity of gene transcription, together with whole-mount in situ hybridization and quantitative real time PCR. To induce acute stress conditions, we used iron and cadmium as “essential” and “non-essential” metals, respectively. Our results highlight, for the first time, the importance of tiar, ttp, and g3bp in controlling the development of mesendodermal tissue derivatives during embryogenesis of an invertebrate chordate.

Abstract The cholecystokinin (CCK)/gastrin family peptides are involved in regulation of feeding and digestion in vertebrates. In the ascidian Ciona intestinalis type A (Ciona robusta), cionin, a CCK/gastrin family peptide, has been identified. Cionin is expressed exclusively in the central nervous system (CNS). In contrast, cionin receptor expression has been detected in the CNS, digestive tract, and ovary. Although cionin has been reported to be involved in ovulation, its physiological function in the CNS remains to be investigated. To elucidate its neural function, in the present study, we analyzed the expression of cionin and cionin receptors in the CNS. Cionin was expressed mainly in neurons residing in the anterior region of the cerebral ganglion. In contrast, the gene expressin of the cionin receptor gene CioR1, was detected in the middle part of the cerebral ganglion and showed a similar expression pattern to that of VACHT, a cholinergic neuron marker gene. Moreover, CioR1 was found to be expressed in cholinergic neurons. Consequently, these results suggest that cionin interacts with cholinergic neurons as a neurotransmitter or neuromodulator via CioR1. This study provides insights into a biological role of a CCK/gastrin family peptide in the CNS of ascidians.

The cholecystokinin (CCK)/gastrin family peptides are involved in regulation of feeding and digestion in vertebrates. In the ascidian Ciona intestinalis type A (Ciona robusta), cionin, a CCK/gastrin family peptide, has been identified. Cionin is expressed exclusively in the central nervous system (CNS). In contrast, cionin receptor expression has been detected in the CNS, digestive tract, and ovary. Although cionin has been reported to be involved in ovulation, its physiological function in the CNS remains to be investigated. To elucidate its neural function, in the present study, we analyzed the expression of cionin and cionin receptors in the CNS. Cionin was expressed mainly in neurons residing in the anterior region of the cerebral ganglion. In contrast, the gene expressin of the cionin receptor gene CioR1, was detected in the middle part of the cerebral ganglion and showed a similar expression pattern to that of VACHT, a cholinergic neuron marker gene. Moreover, CioR1 was found to be expressed in cholinergic neurons. Consequently, these results suggest that cionin interacts with cholinergic neurons as a neurotransmitter or neuromodulator via CioR1. This study provides insights into a biological role of a CCK/gastrin family peptide in the CNS of ascidians. [ABSTRACT FROM AUTHOR]

Ascidian larvae undergo tail elongation and notochord lumenogenesis, making them an ideal model for investigating tissue morphogenesis in embryogenesis. The cellular and mechanical mechanisms of these processes have been studied; however, the underlying molecular regulatory mechanism remains to be elucidated. In this study, assays for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) were applied to investigate potential regulators of the development of ascidian Ciona savignyi larvae. Our results revealed 351 and 138 differentially accessible region genes through comparisons of ATAC-seq data between stages 21 and 24 and between stages 24 and 25, respectively. A joint analysis of RNA-seq and ATAC-seq data revealed a correlation between chromatin accessibility and gene transcription. We further verified the tissue expression patterns of 12 different genes. Among them, Cs-matrix metalloproteinase 24 (MMP24) and Cs-krüppel-like factor 5 (KLF5) were highly expressed in notochord cells. Functional assay results demonstrated that both genes are necessary for notochord lumen formation and expansion. Finally, we performed motif enrichment analysis of the differentially accessible regions in different tailbud stages and summarized the potential roles of these motif-bearing transcription factors in larval development. Overall, our study found a correlation between gene expression and chromatin accessibility and provided a vital resource for understanding the mechanisms of the development of ascidian embryos. [ABSTRACT FROM AUTHOR]

A wide variety of bioactive peptides have been identified in the central nervous system and several peripheral tissues in the ascidian Ciona intestinalis type A (Ciona robusta). However, hemocyte endocrine peptides have yet to be explored. Here, we report a novel 14-amino-acid peptide, CiEMa, that is predominant in the granular hemocytes and unilocular refractile granulocytes of Ciona. RNA-seq and qRT-PCR revealed the high CiEma expression in the adult pharynx and stomach. Immunohistochemistry further revealed the highly concentrated CiEMa in the hemolymph of the pharynx and epithelial cells of the stomach, suggesting biological roles in the immune response. Notably, bacterial lipopolysaccharide stimulation of isolated hemocytes for 1–4 h resulted in 1.9- to 2.4-fold increased CiEMa secretion. Furthermore, CiEMa-stimulated pharynx exhibited mRNA upregulation of the growth factor (Fgf3/7/10/22), vanadium binding proteins (CiVanabin1 and CiVanabin3), and forkhead and homeobox transcription factors (Foxl2, Hox3, and Dbx) but not antimicrobial peptides (CrPap-a and CrMam-a) or immune-related genes (Tgfbtun3, Tnfa, and Il17-2). Collectively, these results suggest that CiEMa plays roles in signal transduction involving tissue development or repair in the immune response, rather than in the direct regulation of immune response genes. The present study identified a novel Ciona hemocyte peptide, CiEMa, which paves the way for research on the biological roles of hemocyte peptides in chordates. [ABSTRACT FROM AUTHOR]

Brain-derived neurotrophic factor (BDNF) plays an important role in neurogenesis, synaptic plasticity, and cognition. BDNF is a neurotrophin that binds to tropomyosin receptor kinase B (TrkB), a specific receptor on target cell surfaces; it acts on neuronal formation, development, growth, and repair via transcription factors, such as cAMP response element-binding protein (CREB), and it is involved in learning and memory. BDNF expression is decreased in patients with Alzheimer's disease (AD). Exercise and the intake of several different foods or ingredients can increase BDNF expression, as confirmed with lutein, xanthophylls (polar carotenoids), and ethanolamine plasmalogen (PlsEtn), which are present at high levels in the brain. This study examined the effects of combining lutein and PlsEtn using lutein-rich Chlorella and ascidian extracts containing high levels of PlsEtn bearing docosahexaenoic acid, which is abundant in the human brain, on the activation of the BDNF–TrkB–CREB signaling pathway in the hippocampus of Sprague-Dawley rats. Although activation of the BDNF–TrkB–CREB signaling pathway in the hippocampus was not observed in Chlorella or ascidian PlsEtn monotherapy, activation was observed with combination therapy at an equal dose. The results of this study suggest that the combination of Chlorella and ascidian PlsEtn may have a preventive effect against dementia, including AD. [ABSTRACT FROM AUTHOR]

Serotonin (5 hydroxytryptamine, 5-HT) is a biogenic amine of ancient origin that is widespread among animals. It plays multiple roles during development and in adults as neurotransmitter at synaptic level and neuro hormone controlling complex behaviors in both vertebrates and invertebrates. Tunicates occupy a key phylogenetic position to understand the evolution of serotonin functions since they are the sister group of vertebrates. The presence of serotonin in tunicates was first reported in adults of the ascidian Ciona robusta (formerly Ciona intestinalis) in the 1946. Since then, serotonin systems have been in many tunicate species and its functions during embryogenesis and metamorphosis explored. We reviewed the current knowledge about serotonin in these animals first by comparing its presence and localization in larvae and adults of different species. Then, we focused on the model organism Ciona for which data regarding sequences and expression patterns of genes involved in serotonin synthesis and function have been reported. Overall, we provided a comprehensive overview of serotonergic machinery in tunicates and gave hints for future studies in this field.

The close phylogenetic relationship between ascidians (Tunicata) and vertebrates makes them a powerful model for studying the innate immune system. To better understand the nature and dynamics of immune responses and the mechanisms through which bacterial infections are detected and translated into inflammation in Ciona robusta, we applied an approach combining in vivo lipopolysaccharide (LPS) stimulation, immune-labelling techniques and functional enzymatic analyses. The immunohistochemistry showed that Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were expressed during the inflammatory pharynx response 4 h post-LPS, with the formation of nodules in pharynx vessel lumen. Also, the endothelium vessels were involved in the inflammatory response. Observations of histological sections from naive and buffer-inoculated ascidians confirmed an immuno-positive response. Enzyme immune parameters—which included the activity of phenoloxidase, glutathione peroxidase, lysozyme, alkaline phosphatase and esterase—showed up-modulation 4 h after LPS injection, confirming their participation during ascidian inflammatory response. These findings provide new insights into the mechanisms underlying the LPS-induced C. robusta response and suggest that a broad innate immune mechanism, as in vertebrates, is involved in the regulation of inflammatory responses. Further findings in this direction are needed to cover knowledge gaps regarding the organized set of molecular and cellular networks involved in universal immune interactions with pathogens.

Allorecognition, the ability of an organism to distinguish kin from non-kin, or self from non-self, has been studied extensively in a group of invertebrate chordates, the colonial ascidians called botryllids (Subphylum Tunicata, Class Ascidiacea, Family Styelidae). When two conspecific botryllid colonies come in contact, there are two potential outcomes to an allorecognition reaction: fusion or rejection. The rejection outcome of allorecognition varies by species, and has been classified by type (referred to as R-Type). R-Type is defined according to how far the fusion process progresses before the rejection begins, since the rejection reaction appears as an interference of the fusion process. Here, we map the evolution of R-Types onto an extended and robust phylogeny of the botryllids. In this study, we have reconstructed the largest phylogenomic tree of botryllids, including 97 samples and more than 40 different species, and mapped on it nine of the 13 species for which the R-Type is known. Based on the R-Type known in a single outgroup species (Symplegma reptans), we infer that at least R-Type B and E-like could be ancestral to the Botrylloides/Botryllus group. However, the application of ancestral character state reconstructions does not provide conclusive results since several clades show more than one equiparsimonious R-Type state. Notably, all R-Type A species are clustered together and certainly evolved later than other R-Types. Our phylogenomic tree has been built on 177 nuclear loci and nearly all clades are well supported. Moreover, our phylogenetic analyses also take into account the results of species delimitation analyses based on the mitochondrial COI gene and of careful morphological analyses of the samples. The implementation of this integrated taxonomic approach, combining morphological as well as nuclear and mitochondrial data, has allowed the description of six new species, and the identification of a number of putative unnamed taxa. Thus, our results also demonstrate the existence of an unexplored hidden diversity within botryllids. [ABSTRACT FROM AUTHOR]

Bilateria share sequential steps in their digestive systems, and digestion occurs in a pre-absorption step within a chamber-like structure. Previous studies on the ascidian Ciona intestinalis type A, an evolutionary research model of vertebrate organs, revealed that Ciona homologs of pancreas-related exocrine digestive enzymes (XDEs) are exclusively expressed in the chamber-like bulging stomach. In the development of the gastrointestinal tract, genes for the pancreas-related transcription factors, namely Ptf1a, Nr5a2, and Pdx, are expressed near the stomach. Recent organ/tissue RNA-seq studies on two Ciona species reported that transcripts of the XDE homologs exist in the intestinal regions, as well as in the stomach. In the present study, we investigated the spatial gene expression of XDE homologs in the gastrointestinal region of the C. intestinalis type A. Whole-mount in situ hybridization using adult and juvenile specimens revealed apparent expression signals of XDE homologs in a small number of gastrointestinal epithelial cells. Furthermore, two pancreas-related transcription factor genes, Nr5a2 and Pdx, exhibited multi-regional expression along the Ciona juvenile intestines. These results imply that ascidians may form multiple digestive regions corresponding to the vertebrate pancreas. [ABSTRACT FROM AUTHOR]

Intestinal absorption is essential for heterotrophic bilaterians with a tubular gut. Although the fundamental features of the digestive system were shared among chordates with evolution, the gut morphologies of vertebrates diverged and adapted to different food habitats. The ascidian Ciona intestinalis type A, a genome-wide research model of basal chordates, is used to examine the functional morphology of the intestines because of its transparent juvenile body. In the present study, the characteristic gene expression patterns (GEP) of Ciona absorptive proteins, e.g., brush border membrane enzymes for terminal digestion (lactase, maltase, APA, and APN) and transporters (SGLT1, GLUT5, PEPT1, and B0AT1), were investigated in juveniles and young adults, with a special reference to the absorption of other nutrients by pinocytosis- and phagocytosis-related proteins (megalin, cubilin, amnionless, Dab2, Rab7, LAMP, cathepsins, and MRC1). Whole-mount in situ hybridization revealed that these GEP showed multi-regional and repetitive features along the Ciona gastrointestinal tract, mainly in the stomach and several regions of the intestines. In young adults, many absorption-related genes, including pinocytosis-/phagocytosis-related genes, were also expressed between the stomach and mid-intestine. In the gastrointestinal epithelium, absorption-related genes showed zonal GEP along the epithelial structure. Comparisons of GEP, including other intestinal functions, such as nutrient digestion and intestinal protection, indicated the repetitive assignment of a well-coordinated set of intestinal GEP in the Ciona gastrointestinal tract. [ABSTRACT FROM AUTHOR]

The vase tunicate, Ciona intestinalis (Linnaeus, 1767), is a social but non-colonial ascidian that is implicated in biofouling of aquatic structures and destruction of the shellfish industry through competition for planktonic nutrients and substrate settling habitats. The sequencing of the C. intestinalis genome has provided insight into the phylogenetic origins of this species, indicating that this lineage and its allies represent a sister taxon to the chordates. Although the practical use of this genomic information at controlling this invasive species is equivocal, a number of new studies on the neurological and neuroendocrine aspects of C. intestinalis have suggested new molecular targets that may be exploited for practical applications on the control of C. intestinalis to protect and enhance the shellfish industry from this invasive species. As a result, we have developed a novel behavioural assay for C. intestinalis, which can be employed to investigate novel agents that inhibit growth and development in this species. [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 FROM AUTHOR]

Background: Ascidians significantly change their body structure through metamorphosis, but the spatio‐temporal cell dynamics in the early metamorphosis stage has not been clarified. A natural Ciona embryo is surrounded by maternally derived non‐self‐test cells before metamorphosis. However, after metamorphosis, the juvenile is surrounded by self‐tunic cells derived from mesenchymal cell lineages. Both test cells and tunic cells are thought to be changed their distributions during metamorphosis, but the precise timing is unknown. Results: Using a metamorphosis induction by mechanical stimulation, we investigated the dynamics of mesenchymal cells during metamorphosis in a precise time course. After the stimulation, two‐round Ca2+ transients were observed. Migrating mesenchymal cells came out through the epidermis within 10 min after the second phase. We named this event "cell extravasation." The cell extravasation occurred at the same time as the backward movement of posterior trunk epidermal cells. Timelapse imaging of transgenic‐line larva revealed that non‐self‐test cells and self‐tunic cells temporarily coexist outside the body until the test cells are eliminated. At the juvenile stage, only extravasated self‐tunic cells remained outside the body. Conclusions: We found that mesenchymal cells extravasated following two‐round Ca2+ transients, and distributions of test cells and tunic cells changed in the outer body after tail regression. Key Findings: We found mesenchymal cells were released from multiple points in the epidermis of the trunk (extravasation) after mechanical stimulation.Extravasation occurs simultaneously with backward movement of the epidermis.Before metamorphosis, mesenchymal cells already migrated beneath the epidermis.Three‐dimensional timelapse imaging revealed that extravasated cells were divided into two groups: faster migrating cell groups than before metamorphosis and slower cell groups.After extravasation, non‐self‐external cells are eliminated and self‐extravasated cells surrounded the outer body. Both cells are thought to be responsible for defense. [ABSTRACT FROM AUTHOR]

Invertebrates lack hypothalamic-pituitary-gonadal axis, and have acquired species-specific regulatory systems for ovarian follicle development. Ascidians are marine invertebrates that are the phylogenetically closest living relatives to vertebrates, and we have thus far substantiated the molecular mechanisms underlying neuropeptidergic follicle development of the cosmopolitan species, Ciona intestinalis Type A. However, no ovarian factor has so far been identified in Ciona. In the present study, we identified a novel Ciona-specific peptide, termed PEP51, in the ovary. Immunohistochemical analysis demonstrated the specific expression of PEP51 in oocyte-associated accessory cells, test cells, of post-vitellogenic (stage III) follicles. Immunoelectron microscopy revealed that PEP51 was localized in the cytosol of test cells in early stage III follicles, which lack secretory granules. These results indicate that PEP51 acts as an intracellular factor within test cells rather than as a secretory peptide. Confocal laser microscopy verified that activation of caspase-3/7, the canonical apoptosis marker, was detected in most PEP51-positive test cells of early stage III. This colocalization of PEP51 and the apoptosis marker was consistent with immunoelectron microscopy observations demonstrating that a few normal (PEP51-negative) test cells reside in the aggregates of PEP51-positive apoptotic test cells of early stage III follicles. Furthermore, transfection of the PEP51 gene into COS-7 cells and HEK293MSR cells resulted in activation of caspase-3/7, providing evidence that PEP51 induces apoptotic signaling. Collectively, these results showed the existence of species-specific ovarian peptide-driven cell metabolism in Ciona follicle development. Consistent with the phylogenetic position of Ciona as the closest sister group of vertebrates, the present study sheds new light on the molecular and functional diversity of the regulatory systems of follicle development in the Chordata. [ABSTRACT FROM AUTHOR]

We present a data set reporting the checklist of the species belonging to the class Ascidiacea for the 9 Italian marine biogeographical units, updating the one previously published by Mastrototaro & Tursi in 2010. In detail, the updated checklist includes 138 taxa at species level (66 Aplousobranchia, 30 Phlebobranchia and 42 Stolidobranchia). Records refer to various marine habitats, including lagoons, harbours and semi-enclosed environments as well as deep habitats. The previous checklist reported 129 taxa (66 Aplousobranchia, 27 Phlebobranchia and 36 Stolidobranchia): in detail, 12 species were added, of which four newly described species, seven non-indigenous species and a new record for the Italian waters. On the other hand, three species reported in the previous checklist were removed: two because currently considered dubious, and one because its occurrence in the Mediterranean Sea is not actually confirmed. Finally, two species were renamed because they are considered synonyms of older ones and five species are currently accepted with new combinations. In addition, five species expanded their biogeographic distribution. The checklist data set will be dynamically updated with new records, and it will be freely available from Lifewatch Italy at https://www.lifewatchitaly.eu/en/initiatives/checklist-fauna-italia-en/checklist. This note describes the state of the art of the Ascidian checklist data set until February 2022.

Subtropical coral reefs such as the lagoonal reefs of Norfolk Island in the remote South Pacific are vastly understudied, with many benthic species unrecorded in the scientific literature. Here we report on ascidian populations from 2021 to 2023 at Norfolk Islands inshore reefs. Quantitative assessments spanning that period were conducted to assess ascidian presence, cover, and benthic interactions. We show ascidian cover remained persistently low and stable across the lagoonal reefs during the study period. We find adjacent reef site, Cemetery Bay showed variation in ascidian cover over time, where we measure a 3.2-fold increase in cover over 2 years. Ascidians were associated primarily with sand and sediment substrates at all reef locations. Recorded interactions between hard corals and ascidians were low. This study provides valuable insights into the population dynamics of ascidians within subtropical reef ecosystems. Ongoing ascidian population monitoring can provide a comprehensive understanding of ascidian dynamics whilst also providing insight of theimpact of anthropogenic stressors on benthic communities. Together, this can aid in informing management and conservation strategies for subtropical reefs.

Allorecognition, the ability of an organism to distinguish kin from non-kin, or self from non-self, has been studied extensively in a group of invertebrate chordates, the colonial ascidians called botryllids (Subphylum Tunicata, Class Ascidiacea, Family Styelidae). When two conspecific botryllid colonies come in contact, there are two potential outcomes to an allorecognition reaction: fusion or rejection. The rejection outcome of allorecognition varies by species, and has been classified by type (referred to as R-Type). R-Type is defined according to how far the fusion process progresses before the rejection begins, since the rejection reaction appears as an interference of the fusion process. Here, we map the evolution of R-Types onto an extended and robust phylogeny of the botryllids. In this study, we have reconstructed the largest phylogenomic tree of botryllids, including 97 samples and more than 40 different species, and mapped on it nine of the 13 species for which the R-Type is known. Based on the R-Type known in a single outgroup species (Symplegma reptans), we infer that at least R-Type B and E-like could be ancestral to the Botrylloides/Botryllus group. However, the application of ancestral character state reconstructions does not provide conclusive results since several clades show more than one equiparsimonious R-Type state. Notably, all R-Type A species are clustered together and certainly evolved later than other R-Types. Our phylogenomic tree has been built on 177 nuclear loci and nearly all clades are well supported. Moreover, our phylogenetic analyses also take into account the results of species delimitation analyses based on the mitochondrial COI gene and of careful morphological analyses of the samples. The implementation of this integrated taxonomic approach, combining morphological as well as nuclear and mitochondrial data, has allowed the description of six new species, and the identification of a number of putative unnamed taxa. Thus, our results also demonstrate the existence of an unexplored hidden diversity within botryllids.

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. [ABSTRACT FROM AUTHOR]

Progress of molecular biology resulted in the accumulation of information on biomolecular interactions, which are complex enough to be termed as networks. Dynamical behavior generated by complex network systems is considered to be the origin of the biological functions. One of the largest missions in modern life science is to obtain logical understanding for the dynamics of complex systems based on experimentally identified networks. However, a network does not provide sufficient information to specify dynamics explicitly, i.e. it lacks information of mathematical formulae of functions or parameter values. One has to develop mathematical models under assumptions of functions and parameter values to know the detail of dynamics of network systems. In this review, on the other hand, we introduce our own mathematical theory to understand the behavior of biological systems from the information of regulatory networks alone. Using the theory, important aspects of dynamical properties can be extracted from networks. Namely, key factors for observing/controlling the whole dynamical system are determined from network structure alone. We also show an application of the theory to a real biological system, a gene regulatory network for cell-fate specification in ascidian. We demonstrate that the system was completely controllable by experimental manipulations of the key factors identified by the theory from the information of network alone. This review article is an extended version of the Japanese article, Controlling Cell-Fate Specification System Based on a Mathematical Theory of Network Dynamics, published in SEIBUTSU BUTSURI Vol. 60, p. 349-351 (2020). [ABSTRACT FROM AUTHOR]

The extracellular ubiquitin–proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the "sperm reaction". However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi. [ABSTRACT FROM AUTHOR]

Ascidian larvae undergo tail elongation and notochord lumenogenesis, making them an ideal model for investigating tissue morphogenesis in embryogenesis. The cellular and mechanical mechanisms of these processes have been studied; however, the underlying molecular regulatory mechanism remains to be elucidated. In this study, assays for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) were applied to investigate potential regulators of the development of ascidian Ciona savignyi larvae. Our results revealed 351 and 138 differentially accessible region genes through comparisons of ATAC-seq data between stages 21 and 24 and between stages 24 and 25, respectively. A joint analysis of RNA-seq and ATAC-seq data revealed a correlation between chromatin accessibility and gene transcription. We further verified the tissue expression patterns of 12 different genes. Among them, Cs-matrix metalloproteinase 24 (MMP24) and Cs-krüppel-like factor 5 (KLF5) were highly expressed in notochord cells. Functional assay results demonstrated that both genes are necessary for notochord lumen formation and expansion. Finally, we performed motif enrichment analysis of the differentially accessible regions in different tailbud stages and summarized the potential roles of these motif-bearing transcription factors in larval development. Overall, our study found a correlation between gene expression and chromatin accessibility and provided a vital resource for understanding the mechanisms of the development of ascidian embryos.

A wide variety of bioactive peptides have been identified in the central nervous system and several peripheral tissues in the ascidian Ciona intestinalis type A (Ciona robusta). However, hemocyte endocrine peptides have yet to be explored. Here, we report a novel 14-amino-acid peptide, CiEMa, that is predominant in the granular hemocytes and unilocular refractile granulocytes of Ciona. RNA-seq and qRT-PCR revealed the high CiEma expression in the adult pharynx and stomach. Immunohistochemistry further revealed the highly concentrated CiEMa in the hemolymph of the pharynx and epithelial cells of the stomach, suggesting biological roles in the immune response. Notably, bacterial lipopolysaccharide stimulation of isolated hemocytes for 1–4 h resulted in 1.9- to 2.4-fold increased CiEMa secretion. Furthermore, CiEMa-stimulated pharynx exhibited mRNA upregulation of the growth factor (Fgf3/7/10/22), vanadium binding proteins (CiVanabin1 and CiVanabin3), and forkhead and homeobox transcription factors (Foxl2, Hox3, and Dbx) but not antimicrobial peptides (CrPap-a and CrMam-a) or immune-related genes (Tgfbtun3, Tnfa, and Il17-2). Collectively, these results suggest that CiEMa plays roles in signal transduction involving tissue development or repair in the immune response, rather than in the direct regulation of immune response genes. The present study identified a novel Ciona hemocyte peptide, CiEMa, which paves the way for research on the biological roles of hemocyte peptides in chordates.

Brain-derived neurotrophic factor (BDNF) plays an important role in neurogenesis, synaptic plasticity, and cognition. BDNF is a neurotrophin that binds to tropomyosin receptor kinase B (TrkB), a specific receptor on target cell surfaces; it acts on neuronal formation, development, growth, and repair via transcription factors, such as cAMP response element-binding protein (CREB), and it is involved in learning and memory. BDNF expression is decreased in patients with Alzheimer’s disease (AD). Exercise and the intake of several different foods or ingredients can increase BDNF expression, as confirmed with lutein, xanthophylls (polar carotenoids), and ethanolamine plasmalogen (PlsEtn), which are present at high levels in the brain. This study examined the effects of combining lutein and PlsEtn using lutein-rich Chlorella and ascidian extracts containing high levels of PlsEtn bearing docosahexaenoic acid, which is abundant in the human brain, on the activation of the BDNF–TrkB–CREB signaling pathway in the hippocampus of Sprague-Dawley rats. Although activation of the BDNF–TrkB–CREB signaling pathway in the hippocampus was not observed in Chlorella or ascidian PlsEtn monotherapy, activation was observed with combination therapy at an equal dose. The results of this study suggest that the combination of Chlorella and ascidian PlsEtn may have a preventive effect against dementia, including AD.

Cranial neurogenic placodes have been considered vertebrate innovations. However, anterior neural plate border (ANB) cells of ascidian embryos share many properties with vertebrate neurogenic placodes; therefore, it is now believed that the last common ancestor of vertebrates and ascidians had embryonic structures similar to neurogenic placodes of vertebrate embryos. Because BMP signaling is important for specifying the placode region in vertebrate embryos, we examined whether BMP signaling is also involved in gene expression in the ANB region of ascidian embryos. Our data indicated that Admp, a divergent BMP family member, is mainly responsible for BMP signaling in the ANB region, and that two BMP-antagonists, Noggin and Chordin, restrict the domain, in which BMP signaling is activated, to the ANB region, and prevent it from expanding to the neural plate. BMP signaling is required for expression of Foxg and Six1/2 at the late gastrula stage, and also for expression of Zf220, which encodes a zinc finger transcription factor in late neurula embryos. Because Zf220 negatively regulates Foxg, when we downregulated Zf220 by inhibiting BMP signaling, Foxg was upregulated, resulting in one large palp instead of three palps (adhesive organs derived from ANB cells). Functions of BMP signaling in specification of the ANB region give further support to the hypothesis that ascidian ANB cells share an evolutionary origin with vertebrate cranial placodes. [ABSTRACT FROM AUTHOR]

The most anterior structure of the ascidian larva is made of three palps with sensory and adhesive functions essential for metamorphosis. They derive from the anterior neural border and their formation is regulated by FGF andWnt. Given that they also share gene expression profiles with vertebrate anterior neural tissue and cranial placodes, their study should shed light on the emergence of the unique vertebrate telencephalon. We show that BMP signaling regulates two phases of palp formation in Ciona intestinalis. During gastrulation, the anterior neural border is specified in a domain of inactive BMP signaling, and activating BMP prevented its formation. During neurulation, BMP defines ventral palp identity and indirectly specifies the inter-papilla territory separating the ventral and dorsal palps. Finally, we show that BMP has similar functions in the ascidian Phallusia mammillata, for whichwe identified novel palpmarkers. Collectively, we provide a better molecular description of palp formation in ascidians that will be instrumental for comparative studies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A colonial ascidian of the genus Distaplia caused a mass mortality of the pen shell Atrina maura (Sowerby, 1835) during June 2016 in the southwest of the Gulf of California (Mexico), with a significant socioeconomic cost. Tentatively identified in previous works as Distaplia cf. stylifera, a precise taxonomic determination was still lacking. In the present work, based on a detailed morphological study, it is confirmed that this aggressive species is Distaplia stylifera (Kowalevsky, 1874). Originally described from the Red Sea, the species currently has a wide circumtropical distribution (with the exception of the Eastern Pacific to date) and is reported as introduced in parts of its range. The present account thus represents an important range extension of this species. However, when revising the original description and later observations, the reported variability of several characters makes it likely that the binomen is in fact a complex of species, as is common in other ascidians with wide distributions. A complete morphological and genetic study including populations from the entire range of distribution would be necessary to settle the status of D. stylifera. Taxonomic uncertainties hinder a correct interpretation of biogeographical patterns and inference on the origin of the studied population. Nevertheless, the known introduction potential of the species, coupled with an explosive growth in an anthropized environment, and the lack of any previous reports in the Eastern Pacific, strongly suggest that the investigated population represents yet another instance of ascidian introduction. From the point of view of management, its invasive behavior is cause for great concern and warrants mitigation measures. [ABSTRACT FROM AUTHOR]

Invasive marine species pose a potential risk to biodiversity. One strategy used to minimize this risk includes early detection combined with quantitative estimation. Quantitative estimation can be used to distinguish the environmental factors that need to be prioritized in the management and control of invasive species. In this study, quantification of environmental DNA (eDNA) and a visual survey were compared to determine whether eDNA-based quantitative PCR (qPCR; real-time PCR) can replace the visual survey of the well-known invasive ascidians Ciona robusta and Didemnum vexillum. Visual surveys and seawater eDNA collection were performed five times over a 16-month period at two sites in South Korea (Gunsan and Yeosu). The visual survey included species coverage on artificial substrates and eDNA-based qPCR analysis was performed using specific markers (primers and probes). The designed markers were validated in silico/in vitro, and the limit of detection (LOD)/limit of quantification (LOQ) was confirmed by qPCR to avoid false-positive results. The results showed that when coverage was high, the number of eDNA copies/rxn was also tended to be high; there was a positive correlation for C. robusta, but not for D. vexillum. Therefore, eDNA-based quantitative studies have the potential to complement or replace the traditional surveys, of morphological analysis. [ABSTRACT FROM AUTHOR]

Tunicates belongs to Urochordate that inhibit coral reefs ecosystem. The uniqueness of the tunic can be seen from the body shape like a tube with 2 siphons and the body is covered by a protective layer known as the tunic. The present study aims to observe marine tunicate at waters of Badi Island, Pangkajene Regency, South Sulawesi. This research using the Belt Transect method with a length of 20 m and a width of 4 m at a depth of 5 m with 4 station points according to the wind directions. All tunicate inside transect was noted and identified. Identification was based on morphological characteristics. The result shows that there were 18 species of tunicates present in the coral reefs ecosystem of Badi Island.

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. [ABSTRACT FROM AUTHOR]

The glycoprotein hormone (GPH) system is fundamentally significant in regulating the physiology of chordates, such as thyroid activity and gonadal function. However, the knowledge of the GPH system in the primitive chordate ascidian species is largely lacking. Here, we reported an ancestral GPH system in the ascidian (Styela clava), which consists of GPH α subunit (Sc-GPA2), GPH β subunit (Sc-GPB5), and the cognate leucine-rich repeat-containing G protein-coupled receptor (Sc-GPHR). Comparative structure analysis revealed that distinct from vertebrate GPH β subunits, Sc-GPB5 was less conserved, showing an atypical N-terminal sequence with a type II transmembrane domain instead of a typical signal peptide. By investigating the presence of recombinant Sc-GPA2 and Sc-GPB5 in cell lysates and culture media of HEK293T cells, we confirmed that these two subunits could be secreted out of the cells via distinct secretory pathways. The deglycosylation experiments demonstrated that N-linked glycosylation only occurred on the conserved cysteine residue (N78) of Sc-GPA2, whereas Sc-GPB5 was non-glycosylated. Although Sc-GPB5 exhibited distinct topology and biochemical properties in contrast to its chordate counterparts, it could still interact with Sc-GPA2 to form a heterodimer. The Sc-GPHR was then confirmed to be activated by tethered Sc-GPA2/GPB5 heterodimer on the Gs-cAMP pathway, suggesting that Sc-GPA2/GPB5 heterodimer-initiated Gs-cAMP signaling pathway is evolutionarily conserved in chordates. Furthermore, in situ hybridization and RT-PCR results revealed the co-expression patterns of Sc-GPA2 and Sc-GPB5 with Sc-GPHR transcripts, respectively in ascidian larvae and adults, highlighting the potential functions of Sc-GPA2/GPB5 heterodimer as an autocrine/paracrine neurohormone in regulating metamorphosis of larvae and physiological functions of adults. Our study systematically investigated the GPA2/GPB5-GPHR system in ascidian for the first time, which offers insights into understanding the function and evolution of the GPH system within the chordate lineage. [ABSTRACT FROM AUTHOR]

Simple Summary: Formation of the complex organ has long been an arresting topic in evolution. The prototype of the complex organ is more likely to exist in creatures of ancient evolutionary hierarchy. In sister groups to vertebrates, including cephalochordate and urochordate, and the basal vertebrate, cyclostomate, a specialized pharyngeal organ is uniquely possessed by animals in these groups, named the endostyle. The endostyle serves as a filter-feeding and thyroid hormone-synthesizing function and is recognized as a prototype of the thyroid gland. Here, we comprehensively uncovered the expression profile of the endostyle in Styela clava, a wildly distributed ascidian with strong vitality. Cross-species comparison assures the function of the endostyle as a thyroid and parathyroid gland and the key composition of the digestive tract. Meanwhile, the high correlation with blood-producing organs, such as the head kidney and bone marrow in advanced vertebrates, implies a potential function of the endostyle. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The spindle assembly checkpoint (SAC) is a surveillance system that preserves genome integrity by delaying anaphase onset until all chromosomes are correctly attached to spindle microtubules. Recruitment of SAC proteins to unattached kinetochores generates an inhibitory signal that prolongs mitotic duration. Chordate embryos are atypical in that spindle defects do not delay mitotic progression during early development, implying that either the SAC is inactive or the cell-cycle target machinery is unresponsive. Here, we show that in embryos of the chordate Phallusia mammillata, the SAC delays mitotic progression from the 8th cleavage divisions. Unattached kinetochores are not recognized by the SAC machinery until the 7th cell cycle, when the SAC is acquired. After acquisition, SAC strength, which manifests as the degree of mitotic lengthening induced by spindle perturbations, is specific to different cell types and is modulated by cell size, showing similarity to SAC control in early Caenorhabditis elegans embryos. We conclude that SAC acquisition is a process that is likely specific to chordate embryos, while modulation of SAC efficiency in SAC proficient stages depends on cell fate and cell size, which is similar to non-chordate embryos. [ABSTRACT FROM AUTHOR]