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BackgroundGonad differentiation is an essential function for all sexually reproducing species, and many aspects of these developmental processes are highly conserved among the metazoa. The colonial ascidian, Botryllus schlosseri is a chordate model organism which offers two unique traits that can be utilized to characterize the genes underlying germline development: a colonial life history and variable fertility. These properties allow individual genotypes to be isolated at different stages of fertility and gene expression can be characterized comprehensively.ResultsHere we characterized the transcriptome of both fertile and infertile colonies throughout blastogenesis (asexual development) using differential expression analysis. We identified genes (as few as 7 and as many as 647) regulating fertility in Botryllus at each stage of blastogenesis. Several of these genes appear to drive gonad maturation, as they are expressed by follicle cells surrounding both testis and oocyte precursors. Spatial and temporal expression of differentially expressed genes was analyzed by in situ hybridization, confirming expression in developing gonads.ConclusionWe have identified several genes expressed in developing and mature gonads in B. schlosseri. Analysis of genes upregulated in fertile animals suggests a high level of conservation of the mechanisms regulating fertility between basal chordates and vertebrates.

Objective: Eudistoma kaverium, an ascidian belonging to the family Polycitoridae is previously recorded from Indian waters. The work done on this species is limited only to taxonomy and ecology. As ascidians are potential source of anticancer compounds, we have purified fractions of E. kaverium using HPLC and evaluated its anticancer activity. Method: Chromatography based fractionation of the crude extract was carried out and mass spectrometry was used to identify the active molecule. Cell viability was checked using MTT assay. Flow cytometry, ROS generation, Hoechst staining and DNA fragmentation was performed to confirm the anticancer activity of the compound. Results: A column fraction EK-7, which was identified as a sterol Cholesta-4,6-dien-3-ol using GC–MS analysis, exhibited potent cytotoxicity with IC50 value of 6.5 μM in MCF-7 and 10.2 μM against HeLa cells. Moreover, EK-7 triggered the cell cycle arrest in the sub G0/G1 stage and induced apoptosis twenty-four hours post-treatment. Hoechst staining of EK-7-treated HeLa cells portrayed apoptotic events such as changes in cell morphology, chromatin condensation, membrane swelling, and development of apoptotic bodies. The changes in light scattering by the EK-7-treated HeLa cells indicate the general characteristics of cell death as a result of apoptosis. The EK-7-treated HeLa cells show cell cycle arrest in G0/G1, G2/M and S phases and the increased amount of sub G0/G1 population indicates the increase in the apoptotic induced cell population. The isolated compound Cholesta-4,6-dien-3-ol (EK-7), based on the GC–MS analysis and library search from E. kaverium displayed potent anticancer activity which is mediated through apoptosis targeting the G0/G1 phase. Conclusion: Based on the experimental data, it shows that EK-7(Cholesta-4,6-dien-3-ol) a sterol derivative has potent anticancer activity mediated through apoptosis. If this research is continued further with preclinical and clinical trials, EK-7(Cholesta-4,6-dien-3-ol) could be possibly used as a chemotherapeutic agent in treating cancer.

In vertebrate embryos, dorsal midline tissues, including the notochord, the prechordal plate, and the floor plate, play important roles in patterning of the central nervous system, somites, and endodermal tissues by producing extracellular signaling molecules, such as Sonic hedgehog (Shh). In Ciona, hedgehog.b, one of the two hedgehog genes, is expressed in the floor plate of the embryonic neural tube, while none of the hedgehog genes are expressed in the notochord. We have identified a cis-regulatory region of hedgehog.b that was sufficient to drive a reporter gene expression in the floor plate. The hedgehog.b cis-regulatory region also drove ectopic expression of the reporter gene in the endodermal strand, suggesting that the floor plate and the endodermal strand share a part of their gene regulatory programs. The endodermal strand occupies the same topographic position of the embryo as does the vertebrate hypochord, which consists of a row of single cells lined up immediately ventral to the notochord. The hypochord shares expression of several genes with the floor plate, including Shh and FoxA, and play a role in dorsal aorta development. Whole-embryo single-cell transcriptome analysis identified a number of genes specifically expressed in both the floor plate and the endodermal strand in Ciona tailbud embryos. A Ciona FoxA ortholog FoxA.a is shown to be a candidate transcriptional activator for the midline gene battery. The present findings suggest an ancient evolutionary origin of a common developmental program for the midline structures in Olfactores.

Oxytocin (OT) and vasopressin (VP) superfamily neuropeptides are distributed in not only vertebrates but also diverse invertebrates. However, no VPergic innervation of invertebrates has ever been documented. In the ascidian, Ciona intestinalis Type A (Ciona robusta), an OT/VP superfamily peptide was identified, and the Ciona vasopressin (CiVP) induces oocyte maturation and ovulation. In the present study, we characterize the innervation and phenotypes of genetically modified Ciona: CiVP promoter-Venus transgenic and CiVP mutants. CiVP promoter-Venus transgenic Ciona demonstrated that CiVP gene was highly expressed in the cerebral ganglion and several nerves. Fluorescence was also detected in the ovary of young CiVP promoter-Venus transgenic ascidians, suggesting that the CiVP gene is also expressed temporarily in the ovary of young ascidians. Furthermore, a marked decrease of post-vitellogenic (stage III) follicles was observed in the ovary of CiVP mutants, whereas pre-vitellogenic (stage I) and vitellogenic (stage II) follicles were increased in the mutant ovary, compared with that of wildtype Ciona. Gene expression profiles showed that the expression of various genes, including genes related to ovarian follicle growth, was altered in the ovary of CiVP mutants. Altogether, these results indicated that CiVP, mainly as a neuropeptide, plays pivotal roles in diverse biological functions, including growth of early-stage ovarian follicles via regulation of the expression of a wide variety of genes. This is the first report describing a VP gene promoter-transgenic and VP gene-edited invertebrate and also on its gene expression profiles and phenotypes.

In budding tunicates, aging accompanies a decrease in the gene expression of mitochondrial transcription factor A (Tfam), and the in vivo transfection of Tfam mRNA stimulates the mitochondrial respiratory activity of aged animals. The gene expression of both the transcriptional repressor Yin-Yang-1 (YY1) and corepressor Sirtuin6 (Sirt6) increased during aging, and the cotransfection of synthetic mRNA of YY1 and Sirt6 synergistically downregulated Tfam gene expression. Pulldown assays of proteins indicated that YY1-associated factor 2 (YAF2) was associated with both YY1 and SIRT6. Protein cross-linking confirmed that YAF2 bound YY1 and SIRT6 with a molar ratio of 1:1. YY1 was bound to CCAT- or ACAT-containing oligonucleotides in the 59 flanking region of the Tfam gene. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) showed that SIRT6 specifically induced the histone H3 lysine 9 (H3K9) deacetylation of the Tfam upstream region. YY1 and YAF2 accelerated SIRT6-induced H3K9 deacetylation. YY1 and Sirt6 mRNA transfection attenuated mitochondrial respiratory gene expression and blocked MitoTracker fluorescence. In contrast, the SIRT6 inhibitor and Tfam mRNA antagonized the inhibitory effects of YY1 and Sirt6, indicating that Tfam acts on mitochondria downstream of YY1 and Sirt6. We concluded that in the budding tunicate Polyandrocarpa misakiensis, YY1 recruits SIRT6 via YAF2 to the TFAM gene, resulting in aging-related mitochondrial downregulation. [ABSTRACT FROM AUTHOR]

Oxytocin (OT) and vasopressin (VP) superfamily neuropeptides are distributed in not only vertebrates but also diverse invertebrates. However, no VPergic innervation of invertebrates has ever been documented. In the ascidian, Ciona intestinalis Type A (Ciona robusta), an OT/VP superfamily peptide was identified, and the Ciona vasopressin (CiVP) induces oocyte maturation and ovulation. In the present study, we characterize the innervation and phenotypes of genetically modified Ciona : CiVP promoter-Venus transgenic and CiVP mutants. CiVP promoter-Venus transgenic Ciona demonstrated that CiVP gene was highly expressed in the cerebral ganglion and several nerves. Fluorescence was also detected in the ovary of young CiVP promoter-Venus transgenic ascidians, suggesting that the CiVP gene is also expressed temporarily in the ovary of young ascidians. Furthermore, a marked decrease of post-vitellogenic (stage III) follicles was observed in the ovary of CiVP mutants, whereas pre-vitellogenic (stage I) and vitellogenic (stage II) follicles were increased in the mutant ovary, compared with that of wildtype Ciona. Gene expression profiles showed that the expression of various genes, including genes related to ovarian follicle growth, was altered in the ovary of CiVP mutants. Altogether, these results indicated that CiVP, mainly as a neuropeptide, plays pivotal roles in diverse biological functions, including growth of early-stage ovarian follicles via regulation of the expression of a wide variety of genes. This is the first report describing a VP gene promoter-transgenic and VP gene-edited invertebrate and also on its gene expression profiles and phenotypes. [ABSTRACT FROM AUTHOR]

This research work aimed at investigating the chemistry and biosynthetic potential of ascidian-associated symbionts that originated from Manado Bay, North Sulawesi. We initially enriched the symbiotic cells associated with the Manadonese ascidian Lissoclinum patella. Subsequently we identified the presence of Prochloron didemni in both unenriched and salt-enriched samples by examining the 16S rRNA gene and the chlorophyll A oxygenase (CAO) gene. Investigation of the secondary metabolites by HPLC, LCMS/MS, and NMR showed the presence of ulithiacyclamide along with patellamide E in the unenriched symbiotic cells. Interestingly, ulithiacyclamide was detected in the enriched cells from the same Ascidian specimen. Molecular docking showed the high binding affinity of both compounds to estrogen receptor beta (ER-β) protein. This is a subtype of the nuclear receptor superfamily expressed abnormally in ovarian cancer cells. Furthermore, we isolated a patE gene variant encoding for a precursor with patellamide E (VTVCITFC) and ulithiacyclamide (CTLCCTLC) core peptides from the enriched cells. This represents a new core peptide combination. The outcome of this work provides a basis for producing useful cyclic peptides, in sustainable way, through symbiont cultivation. This could become a platform for bioengineering to generate diverse compound analogues. [ABSTRACT FROM AUTHOR]

Palynomorph assemblages and mineral microfossils from four southern Red Sea coastal environments, 1. mangrove swamp 2. middle Holocene paleochannel 3. algal mat, and 4. Sabia Island coral reef environments, were studied to establish a baseline data set. The palynomorphs recovered were grouped into pollen and spores, dinoflagellate cysts, and algal remains, fungal spores, hyphae and fruit bodies, protists, and invertebrates. A variety of cuticles, tracheids and other woody fragments commonly occur in almost all the samples. All groups of palynomorphs occur in all these environments; however, their relative abundances may vary. Mangrove swamp environment has a higher diversity and numerical abundance of palynomorphs where protists and invertebrates are common. Crustacean and annelid remains are common to abundant. The palynomorph assemblages in the paleochannel section and algal mat are impoverished. Algal mat samples are characterized by the presence of abundant green filamentous, branched alga. Sabia Island coral reef environment mostly has a rare presence of pollen, spores, fungal remains and dinoflagellate cysts and algal remains. Protists and invertebrates dominate the assemblage. Ascidian and sponge spicules are mineral microfossils occur only in this environment and few specimens of phytoliths occur in this assemblage as well. [ABSTRACT FROM AUTHOR]

Tunicates encompass a large group of marine filter‐feeding animals and more than half of them are able to reproduce asexually by a particular form of nonembryonic development (NED) generally called budding. The phylogeny of tunicates suggests that asexual reproduction is an evolutionarily plastic trait, a view that is further reinforced by the fact that budding mechanisms differ from one species to another, involving nonhomologous tissues and cells. In this review, we explore more than 150 years of literature to provide an overview of NED diversity and we present a comparative picture of budding tissues across tunicates. Based on the phylogenetic relationships between budding and nonbudding species, we hypothesize that NED diversity is the result of seven independent acquisitions and subsequent diversifications in the course of tunicate evolution. While this scenario represents the state‐of‐the‐art of our current knowledge, we point out gray areas that need to be further explored to refine our understanding of tunicate phylogeny and NED. Tunicates, with their plastic evolution and diversity of budding, represent an ideal playground for evolutionary developmental biologists to unravel the genetic and molecular mechanisms regulating nonembryonic development, as well as to better understand how such a profound innovation in life‐history has evolved in numerous metazoans. Research Highlights: Tunicate phylogeny shows convergent gains of asexual reproduction.We provide a comparative overview of budding diversity in tunicates.We hypothesize that NED diversity results from 7 acquisitions and we raise questions for evo‐devo studies. [ABSTRACT FROM AUTHOR]

Deuterostome invertebrates, including echinoderms, hemichordates, cephalochordates, and urochordates, exhibit common and species-specific morphological, developmental, physiological, and behavioral characteristics that are regulated by neuroendocrine and nervous systems. Over the past 15 years, omics, genetic, and/or physiological studies on deuterostome invertebrates have identified low-molecular-weight transmitters, neuropeptides and their cognate receptors, and have clarified their various biological functions. In particular, there has been increasing interest on the neuroendocrine and nervous systems of Ciona intestinalis Type A, which belongs to the subphylum Urochordata and occupies the critical phylogenetic position as the closest relative of vertebrates. During the developmental stage, gamma-aminobutylic acid, D-serine, and gonadotropin-releasing hormones regulate metamorphosis of Ciona. In adults, the neuropeptidergic mechanisms underlying ovarian follicle growth, oocyte maturation, and ovulation have been elucidated. This review article provides the most recent and fundamental knowledge of the neuroendocrine and nervous systems of Ciona , and their evolutionary aspects. • Deuterostomes possess a variety of endocrine, neuroendocrine, and nervous systems. • Ciona , a deuterostome invertebrate, is the closest relative of vertebrates. • Metamorphosis is regulated by neurotransmitters and neuropeptides in Ciona larva. • Ovarian follicle development in Ciona adults is regulated by neuropeptides. • Recent studies highlight common and unique neuroendocrine and nervous systems of Ciona in deuterostomes. [ABSTRACT FROM AUTHOR]

Calcium ion (Ca2+) is involved in the protein-mediated larval adhesion of fouling ascidians, yet the effects of environmental Ca2+ on larval adhesion remain largely unexplored. Here, the larvae of fouling ascidian C. robusta were exposed to different concentrations of Ca2+. Exposures to low-concentration (0 mM and 5 mM) and high-concentration (20 mM and 40 mM) Ca2+ significantly decreased the adhesion rate of larvae, which was primarily attributed to the decreases in adhesive structure length and curvature. Changes in the expressions of genes encoding adhesion-, microvilli-, muscle contraction-, and collagen-related proteins provided a molecular-level explanation for adhesion rate reduction. Additionally, larvae likely prioritized their energy towards immunomodulation in response to Ca2+ stresses, ultimately leading to adhesion reduction. These findings advance our understanding of the influencing mechanisms of environmental Ca2+ on larval adhesion, which are expected to provide references for the development of precise antifouling strategies against ascidians and other fouling species. [Display omitted] • Exposures to high- and low-concentrations of Ca2+ reduced larval adhesion rate. • Adhesive structure changes attributed to the reduction in adhesion rate. • Expression changes of adhesion-related genes explained adhesion reduction. • Collagen, microvilli, and muscle-related genes were related to adhesion reduction. • Larvae prioritized energy towards immunomodulation, leading to adhesion reduction. [ABSTRACT FROM AUTHOR]

Abstract Background In tunicates, the capacity to build an adult body via non-embryonic development (NED), i.e., asexual budding and whole body regeneration, has been gained or lost several times across the whole subphylum. A recent phylogeny of the family Styelidae revealed an independent acquisition of NED in the colonial species Polyandrocarpa zorritensis and highlighted a novel budding mode. In this paper, we provide the first detailed characterization of the asexual life cycle of P. zorritensis. Results Bud formation occurs along a tubular protrusion of the adult epidermis, the stolon, in a vascularized area defined as budding nest. The bud arises through a folding of the epithelia of the stolon with the contribution of undifferentiated mesenchymal cells. This previously unreported mode of bud onset leads to the formation of a double vesicle, which starts to develop into a zooid through morphogenetic mechanisms common to other Styelidae. The budding nest can also continue to accumulate nutrients and develop into a round-shaped structure, designated as spherule, which represents a dormant form able to survive low temperatures. Conclusions To understand the mechanisms of NED and their evolution, it is fundamental to start from a robust phylogenetic framework in order to select relevant species to compare. The anatomical description of P. zorritensis NED provides the foundation for future comparative studies on plasticity of budding and regeneration in tunicates.

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.

Photoreceptor cells (PRC) are neurons highly specialized for sensing light stimuli and have considerably diversified during evolution. The genetic mechanisms that underlie photoreceptor differentiation and accompanied the progressive increase in complexity and diversification of this sensory cell type are a matter of great interest in the field. A role of the homeodomain transcription factor Onecut (Oc) in photoreceptor cell formation is proposed throughout multicellular organisms. However, knowledge of the identity of the Oc downstream-acting factors that mediate specific tasks in the differentiation of the PRC remains limited. Here, we used transgenic perturbation of the Ciona robusta Oc protein to show its requirement for ciliary PRC differentiation. Then, transcriptome profiling between the trans-activation and trans-repression Oc phenotypes identified differentially expressed genes that are enriched in exocytosis, calcium homeostasis, and neurotransmission. Finally, comparison of RNA-Seq datasets in Ciona and mouse identifies a set of Oc downstream genes conserved between tunicates and vertebrates. The transcription factor Oc emerges as a key regulator of neurotransmission in retinal cell types.

Ascidians host abundant and diverse bacterial symbionts, and ascidian-associated bacteria have been shown to benefit ascidian hosts. However, these host-symbiont interactions are often overlooked in coral reef ecosystems, where ascidians frequently occur as key components. In this study, we explored the bacterial communities associated with Atriolum robustum, a common ascidian from Xisha coral reef, China, using Illumina HiSeq high-throughput sequencing of the 16S rRNA gene amplicon. A total of 1411 OTUs were identified from A. robustum, revealing a high bacterial diversity with a Shannon index of 6.26 ± 1.04. Proteobacteria (in particular Alphaproteobacteria and Gammaproteobacteria) were dominant in A. robustum, followed by Bacteroidetes and Chloroflexi. Ten OTUs comprised the core microbiome of A. robustum, matching taxa with reported capabilities for heavy metal resistance, nutrient cycling, and defense against pathogens. Predictive metabolic profiles of the bacterial community associated with A. robustum, using Tax4Fun, showed a high abundance of genes involved in carbohydrate metabolism and amino acid metabolism. A comparison of the bacterial composition and predictive functions associated with A. robustum and other reported ascidians, suggested that horizontal transmission played a key role in the formation of bacterial communities in A. robustum. It was found that A. robustum from Xisha coral reef harbored a great diversity of bacteria, and bacterial symbionts had a potential functional role in metabolism and in the immune response of their ascidian host, and so played a beneficial role in maintaining the health of the coral reef ecosystem. [ABSTRACT FROM AUTHOR]

Ascidians are prolific colonizers of new environments and possess a range of well-studied features that contribute to their successful spread, but the role of their symbiotic microbial communities in their long-term establishment is mostly unknown. In this study, we utilized next-generation amplicon sequencing to provide a comprehensive description of the microbiome in the colonial ascidian Clavelina oblonga and examined differences in the composition, diversity, and structure of symbiont communities in the host's native and invasive ranges. To identify host haplotypes, we sequenced a fragment of the mitochondrial gene cytochrome c oxidase subunit I (COI). C. oblonga harbored a diverse microbiome spanning 42 bacterial and three archaeal phyla. Colonies in the invasive range hosted significantly less diverse symbiont communities and exhibited lower COI haplotype diversity than colonies in the native range. Differences in microbiome structure were also detected across colonies in the native and invasive range, driven largely by novel bacteria representing symbiont lineages with putative roles in nitrogen cycling. Variability in symbiont composition was also observed among sites within each range. Together, these data suggest that C. oblonga hosts a dynamic microbiome resulting from (i) reductions in symbiont diversity due to founder effects in host populations and (ii) environmental selection of symbiont taxa in response to new habitats within a range. Further investigation is required to document the mechanisms behind these changes and to determine how changes in microbiome structure relate to holobiont function and the successful establishment of C. oblonga worldwide. [ABSTRACT FROM AUTHOR]

The solitary European sea squirt Ascidiella aspersa was introduced to northern Japan, causing dense coverage of the aquacultured Japanese scallop Mizuhopecten yessoensis. There are concerns that allelopathy by the sea squirt will suppress the growth of the scallop. Herein, we studied the allelopathic and defensive potential of A. aspersa using hemolytic assay. An alkyl sulfate, viz. 3,7,11,15-tetramethyl-hexadecan-1,19-disulfate (1), was isolated as a hemolytic compound, and 1 showed mild toxicity against the Japanese scallop and brine shrimp. [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.

Epithelial outpocketing, tunic softening, mesenchymal cell death, dedifferentiation/transdifferentiation, and resistance to environmental stress are major events that occur during asexual reproduction by budding in the tunicate, Polyandrocarpa misakiensis. To identify the molecules underlying these events and compare them with those operating in regeneration, differential gene expression profiles were developed in buds and zooids. Among approximately 40,000 contigs, 21 genes were identified as potentially being involved in asexual reproduction. Genes related to tunic softening, phagocytosis-stimulating opsonin, and stress resistance were activated in the very early stage of budding. At the later stage of budding when buds separated from the parent and entered the developmental stage, genes for cell adhesion, cell death, and differentiation were activated. The transcription factor AP2 was spatio-temporally expressed in a similar pattern to the tunic-softening gene endoglucanase (EndoG). AP2 mRNA activated EndoG when introduced into zooids by electroporation. Eight out of 21 budding-related genes were significantly activated by AP2 mRNA. Polyandrocarpa zooids possess regenerative potential other than budding. Zooidal regeneration accompanied cell death/phagocytosis, cell-cell adhesion/communication, and dedifferentiation/redifferentiation. Consistent with morphological features, eight related genes including SP8 transcription factor were activated during zooidal regeneration. Most of these genes were identical to those induced by AP2 mRNA, indicating that asexual reproduction in P. misakiensis shares AP2-regulated downstream genes with zooidal regeneration. The present results suggest that SP8 may be indispensable for both budding and regeneration and that the potential dedifferentiation-related gene SOXB1 plays a minor role in zooidal regeneration., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ovarian follicle development is an essential process for continuation of sexually reproductive animals, and is controlled by a wide variety of regulatory factors such as neuropeptides and peptide hormones in the endocrine, neuroendocrine, and nervous systems. Moreover, while some molecular mechanisms underlying follicle development are conserved, others vary among species. Consequently, follicle development processes are closely related to the evolution and diversity of species. Ciona intestinalis type A ( Ciona rubusta ) is a cosmopolitan species of ascidians, which are the closest relative of vertebrates. However, unlike vertebrates, ascidians are not endowed with the hypothalamus-pituitary-gonadal axis involving pituitary gonadotropins and sexual steroids. Combined with the phylogenetic position of ascidians as the closest relative of vertebrates, such morphological and endocrine features suggest that ascidians possess both common and species-specific regulatory mechanisms in follicle development. To date, several neuropeptides have been shown to participate in the growth of vitellogenic follicles, oocyte maturation of postvitellogenic follicles, and ovulation of fully mature follicles in a developmental stage-specific fashion. Furthermore, recent studies have shed light on the evolutionary processes of follicle development throughout chordates. In this review, we provide an overview of the neuropeptidergic molecular mechanism in the premature follicle growth, oocyte maturation, and ovulation in Ciona , and comparative views of the follicle development processes of mammals and teleosts.

Botryllus schlosseri is a model colonial ascidian and a marine invader. It is currently recognized as a species complex comprising five genetically divergent clades, with clade A globally distributed and clade E found only in Europe. This taxon has also been recently redescribed by designation of a clade A specimen as the neotype. To clarify the taxonomic status of clade E and its relationship to clade A, we examine the entire mitochondrial genome and study the morphology of clade E. The mitogenome of clade E has an identical gene order to clade A, but substantially differs in the size of several non-coding regions. Remarkably, the nucleotide divergence of clade A-clade E is incompatible with the intraspecies ascidian divergence, but similar to the congeneric one and almost identical to the divergence between species once considered morphologically indistinguishable (e.g. the pair Ciona intestinalis (Linnaeus, 1767)- Ciona robusta Hoshino & Tokioka, 1967, and the pair Botrylloides niger Herdman, 1886- Botrylloides leachii (Savigny, 1816)). Clade E differs morphologically from the Botryllus schlosseri neotype mainly in the number and appearance of the stomach folds, and the shape of the anal opening, the first intestinal loop and the typhlosole. Our integrative taxonomical approach clearly distinguishes clade E as a species separate from Botryllus schlosseri , with unique morphological and molecular characters. Therefore, we here describe clade E as the new species Botryllus gaiae sp. nov. [ABSTRACT FROM AUTHOR]

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

Interferon regulatory factors (IRFs) are key transcription factors that function in the immune system via the interferon (IFN) pathway. In the current study, we identified and characterized three IRFs (CsIRFL1, CsIRFL2, and CsIRFL3) from ascidian Ciona savignyi. Phylogenetic analysis showed that CsIRFL1 was clustered with two IRFs from Ciona robusta and shrimp IRF apart from the vertebrate IRFs, whereas CsIRFL2 and CsIRFL3 were grouped with an unnamed protein from Oikopleura dioica into a sub-branch highly identifying with the vertebrate IRF4, IRF8, and IRF9. Gene expression analysis revealed that CsIRFL1 and CsIRFL2 expressed in all the examined adult tissues (stomach, intestines, eggs, hemocytes, gonad, heart, and pharynx) and predominantly in hemocytes. However, the expression of CsIRFL3 was undetectable in the tested adult tissues. Furthermore, in situ hybridization showed that CsIRFL1 and CsIRFL2 mainly expressed in immunocytes within hemolymph, including phagocytes, macrophage-like cells, morula cells, and amoebocytes, suggesting CsIRFL1 and CsIRFL2 were involved in ascidian immune responses. We then performed LPS and poly(I:C) challenge assay and found that CsIRFL1 highly expressed in the cultured hemocytes following LPS infection for 24 h. After viral analogue poly(I:C) stimulation, the expression of CsIRFL2 was dramatically upregulated from 12 to 24 h. Meanwhile, two critical components of the IFN signaling pathways, STAT and TBK1, showed the increased expression as well after poly(I:C) induction, indicating that CsIRFL2 and IFN pathways genes were activated under the infection of viral analogue. Thus, our findings suggested that CsIRFL2 was a potential transcriptional regulatory factor that participated in regulating the ascidian anti-virus immune response. • Three IRF genes (CsIRFL1, CsIRFL2, and CsIRFL3) was cloned and characterized from ascidian Ciona savignyi. • CsIRFL1 and CsIRFL2 predominantly expressed in hemocytes, especially in immunocytes within hemolymph. • CsIRFL1 was distributed in the cytoplasm and the nucleus of HEK293T cells; CsIRFL2 was localized in the nucleus of HEK293T cells. • In cultured hemocytes, CsIRFL2 were significantly upregulated following stimulation with poly(I:C) for 24 h. [ABSTRACT FROM AUTHOR]

Fluctuating environmental changes impose tremendous stresses on sessile organisms in marine ecosystems, in turn, organisms develop complex response mechanisms to keep adaptive homeostasis for survival. Physiological plasticity is one of the primary lines of defense against environmental challenges, and such defense often relies on the antioxidant defense system (ADS). Hence, it is imperative to understand response mechanisms of ADS to fluctuating environments. Invasive species provide excellent models to study how species cope with environmental stresses, as invasive species encounter sudden, and often recurrent, extensive environmental challenges during the whole invasion process. Here, we studied the roles of ADS on rapid response to recurrent cold challenges in a highly invasive tunicate (Ciona robusta) by simulating cold stresses during its invasion process. We assessed antioxidative indicators, including malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), as well as transcriptional changes of ADS-related genes to reveal the physiological plasticity under recurring cold stresses. Our results demonstrated that physiological homeostasis relied on the resilience of ADS, which further accordingly tuned antioxidant activity and gene expression to changing environments. The initial cold stress remodeled baselines of ADS to promote the development of stress memory, and subsequent stress memory largely decreased the physiological response to recurrent environmental challenges. All results here suggest that C. robusta could develop stress memory to maintain physiological homeostasis in changing or harsh environments. The results obtained in this study provide new insights into the mechanism of rapid physiological adaption during biological invasions.

Ocean acidification (OA) refers to the decrease in ocean water pH resulting from the increasing absorption of atmospheric CO2. This will cause changes in the ocean’s carbonate chemistry system with a resulting impact on reproduction of marine organisms. Reproduction is the fundamental process that allows the conservation of the species; in free-spawning marine invertebrates, this process is highly sensitive to changes in seawater quality and chemistry. To date, the majority of the studies concerned OA effects on reproduction has been focused on embryo and larval development. Despite several evidence for the impairment of reproductive success by environmental perturbations through altering gamete quality, sperm physiological responses to OA are poorly investigated. In this study, we evaluated the effects of exposure to acidified seawater (AcSW) (pH 7.8), which approximate the predicted global averages for oceanic surface waters at the end of this century, on sperm quality of the mussel Mytilus galloprovincialis and the ascidian Ciona robusta by evaluating several endpoints, such as motility, vitality, mitochondrial activity, oxidative state, and intracellular pH (pHi). Following sperm exposure to AcSW, the percentage of motile spermatozoa, mitochondrial activity and pHi decreased in comparison to the current seawater pH of 8.1, whereas vitality and oxidative state were unaffected by the low external pH in both the species. In broadcast spawners, a relationship between sperm intracellular pH and the initiation of motility are well known. Spermatozoa are immotile in the testes and motility is induced after the spermatozoa are released into seawater; the alkaline pH of seawater, in fact, increases the pHi activating motility and mitochondrial respiration. The results of this study suggest that the lowering of seawater pH as predicted to occur for 2100, through the inhibition of pHi increase, prevent sperm motility activation. Sperm motility is a key determinant of fertilization success; consequently, a corresponding drop in fertilization success would be expected with important implications for the fitness and the survival of marine invertebrates.

Styela clava is an edible sea squirt farmed in Korea that has gradually invaded other seas, negatively impacting the ecology and economy of coastal areas. Extracts from S. clava have shown wide bioactivities, and ascidians have the unique capability among animals of biosynthesizing cellulose. Thus, S. clava is a relevant candidate for valorization. Herein, we aimed at surveying and characterizing polysaccharides in both tunic and flesh of this ascidian. To this end, we enzymatically hydrolyzed both tissues, recovering crystalline cellulose from the tunic with high aspect ratios, based on results from microscopy, X-ray diffraction, and infrared spectroscopy analyses. Alkaline hydroalcoholic precipitation was applied to isolate the polysaccharide fraction that was characterized by gel permeation chromatography (with light scattering detection) and NMR. These techniques allowed the identification of glycogen in the flesh with an estimated Mw of 7 MDa. Tunic polysaccharides consisted of two fractions of different Mw. Application of Diffusion-Ordered NMR allowed spectroscopically separating the low-molecular-weight fraction to analyze the major component of an estimated Mw of 40–66 kDa. We identified six different sugar residues, although its complexity prevented the determination of the complete structure and connectivities of the residues. The two more abundant residues were N-acetylated and possibly components of the glycosaminoglycan-like (GAG-like) family, showing the remaining similarities to sulfated galactans. Therefore, Styela clava appears as a source of nanocrystalline cellulose and GAG-like polysaccharides.

Metamorphosis is the dramatic conversion of an animal body from larva to adult. In ascidians, tadpole-shaped, swimming larvae become sessile juveniles by losing their tail during metamorphosis. This study investigated the cellular and molecular mechanisms underlying this metamorphic event called tail regression, in the model ascidian Ciona. The ascidian tail consists of internal organs such as muscle, notochord, nerve cord, and the outer epidermal layer surrounding them. We found that the epidermis and internal organs show different regression strategies. Epidermal cells are shortened along the anterior-posterior axis and gather at the posterior region. The epidermal mass is then invaginated into the trunk by apical constriction. The internal tissues, by contrast, enter into the trunk by forming coils. During coiling, notches are introduced into the muscle cells, which likely reduces their rigidness to promote coiling. Actin filament is the major component necessary for the regression events in both the epidermis and internal tissues. The shortening and invagination of the epidermis depend on the phosphorylation of the myosin regulatory light chain (mrlc) regulated by rho-kinase (ROCK). The coiling of internal tissues does not require ROCK-dependent phosphorylation of mrlc, and they can complete coiling without epidermis, although epidermis can facilitate the coiling of internal tissues. We conclude that tail regression in ascidians consists of active morphogenetic movements in which each tissue's independent mechanism is orchestrated with the others to complete this event within the available time window. • Ascidian tadpole larvae lose their tail by tail regression during metamorphosis. • Tail epidermis regresses by becoming narrow along the anterior-posterior length and subsequent invagination into the trunk. • Tail mesodermal tissues regress by coiling. • Epidermal regression is dependent on ROCK-mediated phosphorylation of myosin. • The epidermis is dispensable for coiling of mesoderm; however, it facilitates coiling's progression. [ABSTRACT FROM AUTHOR]

Tunicates occupy the evolutionary position at the boundary of invertebrates and vertebrates. It exhibits adaptation to broad environmental conditions and is distributed globally. Despite hundreds of years of embryogenesis studies, the genetic basis of the invasive habits of ascidians remains largely unknown. The leathery sea squirt, Styela clava, is an important invasive species. We used the chromosomal‐level genome and transcriptome of S. clava to explore its genomic‐ and molecular‐network‐based mechanisms of adaptation to environments. Compared with Ciona intestinalis type A (C. robusta), the size of the S. clava genome was expanded by 2‐fold, although the gene number was comparable. An increase in transposon number and variation in dominant types were identified as potential expansion mechanisms. In the S. clava genome, the number of genes encoding the heat‐shock protein 70 family and members of the complement system was expanded significantly, and cold‐shock protein genes were transferred horizontally into the S. clava genome from bacteria. The expanded gene families potentially play roles in the adaptation of S. clava to its environments. The loss of key genes in the galactan synthesis pathway might explain the distinct tunic structure and hardness compared with the ascidian Ciona species. We demonstrated further that the integrated thyroid hormone pathway participated in the regulation of larval metamorphosis that provides S. clava with two opportunities for adapting to their environment. Thus, our report of the chromosomal‐level leathery sea squirt genome provides a comprehensive genomic basis for the understanding of environmental adaptation in tunicates. [ABSTRACT FROM AUTHOR]

After the accidental release of crude oil in marine environment, dispersants are applied on sea surface transferring oil into the water column where it can be broken down by biodegradation, thereby reducing potential pollution to coastal areas. Before they can be used in the wild, the ecotoxicity of such dispersants is usually evaluated with toxicity assays using algae, crustacean and fishes. Nowadays, there is a need to find alternative species to reduce the use of vertebrates both for ethical considerations and for reducing the cost of bioassays. Ciona robusta is a solitary ascidian that inhabits shallow waters and marine coastal areas. This species has been recently adopted as valuable biological model for ecotoxicity studies, thanks to its rapid embryonic and larval development, resemblance to vertebrates, and low risk of ethical issues. On this ground, the lethal and sublethal toxicity of two dispersants has been evaluated on Ciona juveniles. At this stage, the organisms become filter-feeders and the morphological alterations of the organs can be easily observed. The median lethal concentrations at 96 h (96hLC50) for Dispersant 1 (non-ionic surfactant) and for Dispersant 2 (mixture of non-ionic surfactants and anionic surfactants) are 41.6 mg/L (38.6–44.9) and 92.5 mg/L (87.7–97.5), respectively. The Ciona juvenile model was compared to Dicentrarchus labrax fish juveniles test, and it showed increased sensitivity for Ciona to these compounds. These results suggest that 96 h mortality test bioassay could be a good alternative method to 96 h mortality assay with D. labrax, limiting the use of vertebrates for dispersant toxicity. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

A strongly divergent lineage, putatively a new cryptic species, of colonial ascidian was first detected as an anomalous sample in a population genomics study of the well-known worldwide invasive species Didemnum vexillum Kott, 2002. This putative new taxon, found in a marina in Roscoff, France, is indistinguishable from Didemnum vexillum in the external aspect and coexists with it in syntopy. However, morphological characters such as spicules and larvae allow a clear-cut distinction. In accordance with the preliminary results based on genome-wide analyses, morphological traits and mitochondrial sequences of the cytochrome oxidase I gene both support the establishment of a new species Didemnum pseudovexillum sp. nov. Previous unidentified sequences in public databases showed that the new species is also present in NW Mediterranean marinas. Didemnum pseudovexillum sp. nov. is assigned for the time being a cryptogenic species status, although its presently known disjoint distribution across two biogeographic regions and its presence in ports are suggestive of an introduced species. Further studies should be performed to ascertain its current distribution and putative natural range and settle its native vs. non-native status. This finding casts doubts on previous reports of Didemnum vexillum and also calls for caution when performing fast field surveys of non-indigenous species such as rapid assessment surveys (RAS) or BioBlitz surveys, based solely on external characters. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Protection of the germline from somatic differentiation programs is crucial for germ cell development. In many animals, whose germline development relies on the maternally inherited germ plasm, such protection in particular at early stages of embryogenesis is achieved by maternally localized global transcriptional repressors, such as PIE-1 of Caenorhabditis elegans , Pgc of Drosophila melanogaster and Pem of ascidians. However, zygotic gene expression starts in later germline cells eventually and mechanisms by which somatic gene expression is selectively kept under repression in the transcriptionally active cells are poorly understood. By using the ascidian species Halocynthia roretzi , we found that H3K27me3, a repressive transcription-related chromatin mark, became enriched in germline cells starting at the 64-cell stage when Pem protein level and its contribution to transcriptional repression decrease. Interestingly, inhibition of H3K27me3 together with Pem knockdown resulted in ectopic expression in germline cells of muscle developmental genes Muscle actin (MA4) and Snail , and of Clone 22 (which is expressed in all somatic but not germline cells), but not of other tissue-specific genes such as the notochord gene Brachyury , the nerve cord marker ETR-1 and a heart precursor gene Mesp , at the 110-cell stage. Importantly, these ectopically expressed genes are normally expressed in the germline sister cells (B7.5), the last somatic lineage separated from the germline. Also, the ectopic expression of MA4 was dependent on a maternally localized muscle determinant Macho-1. Taken together, we propose that H3K27me3 may be responsible for selective transcriptional repression for somatic genes in later germline cells in Halocynthia embryos and that the preferential repression of germline sister-lineage genes may be related to the mechanism of germline segregation in ascidian embryos, where the germline is segregated progressively by successive asymmetric cell divisions during cell cleavage stages. Together with findings from C. elegans and D. melanogaster , our data for this urochordate animal support the proposal for a mechanism, conserved widely throughout the animal kingdom, where germline transcriptional repression is mediated initially by maternally localized factors and subsequently by a chromatin-based mechanism. • H3K27me3 becomes enriched in ascidian germline cells from the 64-cell stage onwards. • Maternal germline silencer Pem suppresses RNAPII activity through the 110-cell stage. • H3K27me3- and Pem-deficient germline mis-expresses its sister muscle-lineage genes. • The mis-expression of muscle genes depends on a maternal determinant, Macho-1. [ABSTRACT FROM AUTHOR]

In ascidian embryos, the earliest transcription from the zygotic genome begins between the 8-cell and 16-cell stages. Gata.a, a maternally expressed Gata transcription factor, activates target genes specifically in the animal hemisphere, whereas the complex of β-catenin and Tcf7 antagonizes the activity of Gata.a and activates target genes specifically in the vegetal hemisphere. Here, we show that genes zygotically expressed at the 16-cell stage have significantly more Gata motifs in their upstream regions. These genes included not only genes with animal hemisphere-specific expression but also genes with vegetal hemisphere-specific expression. On the basis of this finding, we performed knockdown experiments for Gata.a and reporter assays, and found that Gata.a is required for the expression of not only genes with animal hemisphere-specific expression, but also genes with vegetal hemisphere-specific expression. Our data indicated that weak Gata.a activity that cannot induce animal hemisphere-specific expression can allow β-catenin/Tcf7 targets to be expressed in the vegetal cells. Because genes zygotically expressed at the 32-cell stage also had significantly more Gata motifs in their upstream regions, Gata.a function may not be limited to the genes expressed specifically in the animal or vegetal hemispheres at the 16-cell stage, and Gata.a may play an important role in the earliest transcription of the zygotic genome. • Upstream regions of genes activated in early ascidian embryos have many Gata motifs (83). • Maternal Gata.a regulates genes not only in animal cells but also in vegetal cells (82). • Weak Gata.a activity allows β-catenin/Tcf7 targets to be expressed in vegetal cells (83). [ABSTRACT FROM AUTHOR]