Search Results (70)

Allorecognition, or distinguishing between the self and nonself within the same species, is observed in both animals and plants, particularly in the context of immune reactions and self-incompatibility in sexual reproduction. Polymorphic recognition molecules are known to be responsible for such allorecognition during fertilization. Previous studies have reported that in ascidians and flowering plants, inbreeding avoidance relies on a pair of polymorphic recognition molecules with a receptor-ligand relationship that are encoded at a single locus, the S locus (Self-incompatibility locus), but the process by which such pairs of recognition molecules emerge and evolve to become polymorphic is not known. Here, a population genetics study was carried out as a novel approach for investigating allorecognition. To study the process by which self-recognition emerges, we simulated a situation in which an allorecognizing genotype is generated from a nonallorecognizing genotype through mutation and then analyzed whether the two genotypes could coexist. The conditions under which the numbers of allorecognition alleles could increase over evolutionary time were investigated, and the generational dynamics of nonallorecognizing genotypes were analyzed. Subsequent modeling was carried out to reproduce the allorecognition mechanism in Ciona, and consistency between the simulation results and experimental data was observed. Our approach provides new insight into the evolutionary process of allorecognition. Full article

K-mer analysis is a powerful tool for understanding genome structure and evolution. A “k-mer” refers to a short DNA sequence made up of k nucleotides (where k is a specific integer), while an “m-mer” is a similar concept but with a shorter sequence length. The functional mechanisms of CG-containing k-mers, as well as their potential role in evolutionary processes, remain unclear. To explore this issue, we analyzed 8-mers in several species with varying genomic complexities and evolutionary divergences: Homo sapiens, Saccharomyces cerevisiae, Bombyx mori, Ciona intestinalis, Danio rerio, and Caenorhabditis elegans, which were grouped by CG dinucleotide content (0CG, 1CG, and 2CG). We examined the relative frequencies of shorter m-mers (with m = 3 and 4) within each CG-defined group, using information-theoretic, distance-based, and angular metrics. Our results show that 0CG motifs follow random patterns, while 1CG and 2CG motifs display significant deviations, likely due to functional constraints such as nucleosome-binding and CpG island association. The observed unimodal distribution of 8-mers arises from the convergence of the three CG-defined groups. Among them, the 2CG group shows the highest divergence in m-mer composition, followed by 1CG, reflecting varying degrees of selective pressure. Furthermore, species-specific differences in CG-classified 8-mer patterns could provide valuable insights into phylogenetic relationships. Through extensive comparison, we explore how CG content and sequence composition influence genomic organization and contribute to evolutionary divergence across different taxa. These findings deepen our understanding of short motif functions, genome organization, and sequence evolution. Full article

The mitochondrial alternative oxidase (AOX) from the tunicate Ciona intestinalis has been explored as a potential therapeutic enzyme for human mitochondrial diseases, yet its systemic effects remain poorly understood. Here, we investigate the metabolic and physiological consequences of AOX expression during the development of Drosophila cultured under dietary stress. We show that the combination of strong, ubiquitous AOX expression and a low-nutrient condition leads to pupal lethality and severe defects in larval musculature, characterized by actin aggregation and muscle shortening. These structural abnormalities correlate with a decrease in larval biomass and motility. Interestingly, the muscle defects and the motility impairments vary in severity among individuals, predicting survival rates at the pupal stage. AOX expression in specific tissues (muscle, nervous system or fat body) does not individually recapitulate the lethal phenotype observed with ubiquitous expressions of the enzyme, indicating a complex metabolic imbalance. Metabolomic analysis revealed that the low-nutrient diet and AOX expression have opposite effects on most metabolites analyzed, especially in the levels of amino acids. Notably, supplementation of the low-nutrient diet with the essential amino acids methionine and/or tryptophan partially rescues pupal viability, body size, muscle morphology, and locomotion, whereas supplementation with proline and/or glutamate does not, highlighting a specific perturbation in amino acid metabolism rather than general bioenergetic depletion. These findings demonstrate that AOX expression disrupts metabolic homeostasis, with developmental and physiological consequences that must be considered when evaluating AOX for therapeutic applications. Full article

In an effort to monitor ascidian recruitment in mussel aquaculture facilities, a series of settlement plates (20 × 20 cm) were placed in a mussel farm located in the Amvrakikos Gulf (Ionian Sea). The plates were vertically deployed on floating facilities in the water column at regular intervals (depths of 0.2 m, 1.5 m, and 3 m) to monitor the settlement and proliferation of ascidians. Furthermore, measurements of seawater physicochemical parameters such as temperature, salinity, dissolved oxygen, and chlorophyll-a concentration were conducted together with the record of ascidian species in each sampling from January 2021 to November 2021. The correlation of these parameters with ascidian species provides information on their effect on the periodicity of ascidians’ recruitment. The results demonstrated a significant correlation between ascidian presence and water temperature. The potential influence of other important environmental parameters such as chlorophyll-a was not revealed, likely due to the limited number of values and samples included in the analyses. While increased chlorophyll levels, reflecting increased primary productivity or nutrient availability, are associated with increased growth and reproduction of all ascidian species, the effect of temperature was more potent and species-specific. Ciona robusta, Styela plicata, Microcosmus squamiger, and Phallusia mammillata were mainly detected at temperatures below 25 °C, whereas Clavelina oblonga was prevalent at temperatures above 25 °C. The absence of most ascidians at temperatures above 25 °C was possibly attributed to decreased settlement success and to the increased competition from C. oblonga at higher temperatures. The deployment of settlement plates in correlation with seawater physiochemical parameters can provide valuable data on ascidian settlement dynamics and support the development of targeted management practices for biofouling control. Full article

Metamorphosis is a common developmental process in invertebrate development. It is essential for the degeneration of larval organs, formation of adult organs, and adaptation transformation of the living environment. However, the underlying molecular regulatory mechanism remains to be elucidated. In this study, we used tail regression of ascidian Styela clava as a model to understand the gene regulation pathway and molecular mechanism in organ metamorphosis. The TGFβ signaling pathway was screened and demonstrated to be involved in tail regression based on RNA sequencing on the different larval stages and verification with inhibitor treatment experiments. We further investigated the downstream gene network of the TGFβ signaling pathway through comparative transcriptome data analysis on the TGFβ pathway inhibition samples. Together with qRT-PCR verification, we identified four critical gene functional categories, including ion transporters/water channel, extracellular matrix structural constituent, extracellular matrix organization, and cell polarity establishment. Furthermore, a cross-species comparative analysis between Ciona robusta and S. clava was performed to understand the conservation and divergence of gene regulation in ascidians. Overall, our work identifies a crucial gene regulation pathway in ascidian tail regression and provides several potential downstream targets for understanding the molecular mechanism of larval metamorphosis. Full article

Anti-PD-1 and anti-PD-L1 antibodies have achieved great clinical success in cancer immunotherapy, and peptide and small molecule inhibitors of PD-1/PD-L1 binding also attract much attention. Ascidians are not only seafood, but are also an important source of bioactive substances, including anti-tumor components. In this study, ascidian enzymatic hydrolysates were found to contain PD-1/PD-L1 inhibitory components. Affinity ultrafiltration (AUF) coupled with the nanoLC-MS/MS method was first applied in screening for PD-L1 peptide inhibitors from ascidian enzymatic hydrolysates. Two anti-PD-L1 ascidian peptides, C5 (LDVVIHTVTYGDR) and S2 (VLRDNIQGITKPAIR), were filtered out from the ascidians Ciona intestinalis and Styela clava, respectively. C5 and S2 showed moderate anti-PD-1/PD-L1 effects with the IC₅₀ values of 33.9 µM (C5) and 112.8 μM (S2), respectively, by homogenous time-resolved fluorescence (HTRF) binding assay, and the K_D values of 22.9 µM (C5) and 29.1 µM (S2), respectively, by surface plasmon resonance (SPR) assay. The results of this study suggest that ascidian enzymatic hydrolysates may be a potential source of bioactive peptides with anti-PD-1/PD-L1 activity. Full article

The notochord is an axial structure required for the development of all chordate embryos, from sea squirts to humans. Over the course of more than half a billion years of chordate evolution, in addition to its structural function, the notochord has acquired increasingly relevant patterning roles for its surrounding tissues. This process has involved the co-option of signaling pathways and the acquisition of novel molecular mechanisms responsible for the precise timing and modalities of their deployment. To reconstruct this evolutionary route, we surveyed the expression of signaling molecules in the notochord of the tunicate Ciona, an experimentally amenable and informative chordate. We found that several genes encoding for candidate components of diverse signaling pathways are expressed during notochord development, and in some instances, display distinctive regionalized and/or lineage-specific patterns. We identified and deconstructed notochord enhancers associated with TGF-β and Ctgf, two evolutionarily conserved signaling genes that are expressed dishomogeneously in the Ciona notochord, and shed light on the cis-regulatory origins of their peculiar expression patterns. Full article

Nanoplastics are known to represent a threat to marine ecosystems. Their combination with other contaminants of emerging concerns (CECs) may amplify ecotoxic effects, with unknown impacts on marine biodiversity. This study investigates the effects, single and combined, of bisphenol A (BPA)—one of the most hazardous CECs—and polystyrene nanoparticles (PS NPs)—as a proxy for nanoplastics, being among the most commonly found asmarine debris—on cholinesterase (ChE) activities of the ascidian Ciona robusta. ChE activity was first measured in the siphons, tunic, and viscera of wild-caught adult specimens and exposed in vitro to BPA (0.01, 0.21, 0.69 mM) and PS NPs (0.0096–0.096 mM; 8.096 × 10⁹–10¹⁰ particles, respectively) alone and combined for 15 min of incubation. PS NPs’ behavior in milliQ water and in the ChE assay reaction buffer was characterized alone, combined with BPA, and analyzed through ζ-potential measurements via Dynamic Light Scattering. The results revealed that ChE activity was predominant in the viscera and siphons of C. robusta; PS NPs did not affect the ChE activity alone or combined, while BPA caused a concentration-dependent inhibition of ChE activity in the viscera. No changes in ζ-potential were observed for PS NPs alone or combined with BPA in the ChE buffer, suggesting no interaction. Further investigations are needed to understand the potential neurotoxic consequences for C. robusta and ecological risk scenarios due to exposure to BPA and nanoplastics in marine coastal waters. Full article

We previously reported that five astacin-like metalloproteases with thrombospondin type-1 repeats (Tasts) located on the sperm surface are a promising candidate as the protease involved in sperm penetration of the vitelline coat (VC) during fertilization of the ascidian Ciona intestinalis type A (Phlebobranchia). However, whether such a protease is involved in the fertilization of other ascidians is unknown. Here, we investigated the effects of four metalloprotease inhibitors on the fertilization of the ascidian Halocynthia roretzi (Stolidobranchia). Three metalloprotease inhibitors, GM6001, TAPI-0, and TAPI-1, strongly inhibited fertilization at 33 and 11 μM, whereas TAPI-2 weakly inhibited fertilization at 33 μM. In contrast, GM6001NC (negative control) had no effect on fertilization at 100 μM. Furthermore, GM6001 had no inhibitory effect on the fertilization of VC-deprived eggs. The metalloprotease appears to function at the middle or late stage of fertilization. Ten Tast genes were identified in the H. roretzi genome database, among which four genes (HrTast1, HrTast2b, HrTast2c, and HrTast3c) possessed a single transmembrane domain in the N-terminal region. These four genes are transcribed in the testis and ovary, as revealed by RT-PCR. Anti-HrTast2c IgG raised against a peptide corresponding to the Zn-binding consensus sequence weakly inhibited fertilization at 0.5 mg/mL. These results led us to propose that sperm astacin-like metalloproteases may be involved in sperm penetration of the VC during H. roretzi fertilization. Full article

Animals show diverse processes of gametogenesis in the evolutionary pathway. Here, we characterized the spermatogenic cells in the testis of the marine invertebrate Ciona robusta. Ciona sperm differentiate in a non-cystic type of testis, comprising many follicles with various sizes and stages of spermatogenic cells. In the space among follicles, we observed free cells that were recognized by antibody against Müllerian inhibiting substance, a marker for vertebrate Sertoli cells. We further categorized the spermatogenic cells into four round stages (RI to RIV) and three elongated stages (EI to EIII) by morphological and immunohistochemical criteria. An antibody against a vertebrate Vasa homolog recognized a few large spermatogonium-like cells (RI) near the basal wall of a follicle. Consistent with the period of meiosis, a synaptonemal complex protein SYCP3 was recognized from early spermatocytes (RII) to early spermatids (E1). Acetylated tubulins were detected in spermatids before flagellar elongation at the RIV stage and became distributed along the flagella. Electron microscopy showed that the free cells outside the testicular follicle possessed a characteristic of vertebrate Sertoli cells. These results would provide a basis for basic and comparative studies on the mechanism of spermatogenesis. Full article

Environmental Risk Assessment (ERA) often relies on a restricted set of species as bio-indicators, introducing uncertainty when modeling complex environmental variables. This may lead to oversimplified or erroneous risk assessments. Ascidians, marine filter-feeding sessile chordates, are valuable models for scientific research in various biological fields such as stem cell biology, embryogenesis, regeneration, innate immunity, and developmental biology. Their global distribution, sensitivity to pollutants, high abundance, mass sexual reproduction, and habitation in coastal areas impacted by anthropogenic pollution make them excellent indicators for monitoring marine pollution and global environmental changes, including biological invasions and species diversity diminution cases. Despite their potential as environmental bioindicators, ascidians remain underutilized in ERAs (≤0.13% of ERA studies), particularly in the field of chemical pollution impact assessment, primarily due to a lack of standardization. This underrepresentation poses a challenge for accurate modeling, especially in models relying on a broad range of species (e.g., Species Sensitivity Distributions). Given these constraints, expanding the use of ascidians in ERAs could improve the comprehension and precision of environmental changes and their assessments. This underscores the necessity for future research to establish standardized testing protocols and choose the most suitable ascidian species for inclusion in ERAs. Full article

Functional melanocortin receptor (MCR) genes have been identified in the genomes of early chordates, e.g., the cyclostomata. Whether they appear in the most ancient chordates such as cephalochordate and urochordata, however, remains unclear due to missing genetic data. Herein, we studied five putative (from NCBI database), sequence-based predicted MCR-like receptors from urochordata and cephalochordate, including Styela clava, Ciona intestinalis, Branchiostoma floridae, and Branchiostoma belcheri. The BLAST and phylogenetic analyses suggested a relationship between these specific receptors and vertebrate MCRs. However, several essential residues for MCR functions in vertebrates were missing in these putative chordata MCRs. To test receptor functionality, several experimental studies were conducted. Binding assays and functional analyses showed no specific binding and no ligand-induced cAMP or ERK1/2 signaling (with either endogenous α-MSH or synthetic ligands for MC4R), despite successfully expressing four receptors in HEK 293T cells. These four receptors showed high basal cAMP signaling, likely mediated by ligand-independent Gs coupling. In summary, our results suggest that the five predicted MCR-like receptors are, indeed, class A G protein-coupled receptors (GPCRs), which in four cases show high constitutive activity in the Gs-cAMP signaling pathway but are not MCR-like receptors in terms of ligand recognition of known MCR ligands. These receptors might be ancient G protein-coupled receptors with so far unidentified ligands. Full article

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. Full article

The urochordate Ciona robusta exhibits numerous functional and morphogenetic traits that are shared with vertebrate models. While prior investigations have identified several analogies between the gastrointestinal tract (i.e., gut) of Ciona and mice, the molecular mechanisms responsible for these similarities remain poorly understood. This study seeks to address this knowledge gap by investigating the transcriptional landscape of the adult stage gut. Through comparative genomics analyses, we identified several evolutionarily conserved components of signaling pathways of pivotal importance for gut development (such as WNT, Notch, and TGFβ-BMP) and further evaluated their expression in three distinct sections of the gastrointestinal tract by RNA-seq. Despite the presence of lineage-specific gene gains, losses, and often unclear orthology relationships, the investigated pathways were characterized by well-conserved molecular machinery, with most components being expressed at significant levels throughout the entire intestinal tract of C. robusta. We also showed significant differences in the transcriptional landscape of the stomach and intestinal tract, which were much less pronounced between the proximal and distal portions of the intestine. This study confirms that C. robusta is a reliable model system for comparative studies, supporting the use of ascidians as a model to study gut physiology. Full article

Throughout embryonic development, the shaping of the functional and morphological characteristics of embryos is orchestrated by an intricate interaction between transcription factors and cis-regulatory elements. In this study, we conducted a comprehensive analysis of deuterostome cis-regulatory landscapes during gastrulation, focusing on four paradigmatic species: the echinoderm Strongylocentrotus purpuratus, the cephalochordate Branchiostoma lanceolatum, the urochordate Ciona intestinalis, and the vertebrate Danio rerio. Our approach involved comparative computational analysis of ATAC-seq datasets to explore the genome-wide blueprint of conserved transcription factor binding motifs underlying gastrulation. We identified a core set of conserved DNA binding motifs associated with 62 known transcription factors, indicating the remarkable conservation of the gastrulation regulatory landscape across deuterostomes. Our findings offer valuable insights into the evolutionary molecular dynamics of embryonic development, shedding light on conserved regulatory subprograms and providing a comprehensive perspective on the conservation and divergence of gene regulation underlying the gastrulation process. Full article