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The evolution of specialized cell‐types is a long‐standing interest of biologists, but given the deep time‐scales very difficult to reconstruct or observe. microRNAs have been linked to the evolution of cellular complexity and may inform on specialization. The endothelium is a vertebrate‐specific specialization of the circulatory system that enabled a critical new level of vasoregulation. The evolutionary origin of these endothelial cells is unclear. We hypothesized that Mir‐126, an endothelial cell‐specific microRNA may be informative. We here reconstruct the evolutionary history of Mir‐126. Mir‐126 likely appeared in the last common ancestor of vertebrates and tunicates, which was a species without an endothelium, within an intron of the evolutionary much older EGF Like Domain Multiple (Egfl) locus. Mir‐126 has a complex evolutionary history due to duplications and losses of both the host gene and the microRNA. Taking advantage of the strong evolutionary conservation of the microRNA among Olfactores, and using RNA in situ hybridization, we localized Mir‐126 in the tunicate Ciona robusta. We found exclusive expression of the mature Mir‐126 in granular amebocytes, supporting a long‐proposed scenario that endothelial cells arose from hemoblasts, a type of proto‐endothelial amoebocyte found throughout invertebrates. This observed change of expression of Mir‐126 from proto‐endothelial amoebocytes in the tunicate to endothelial cells in vertebrates is the first direct observation of the evolution of a cell‐type in relation to microRNA expression indicating that microRNAs can be a prerequisite of cell‐type evolution. Research Highlights: Direct observation of cell‐type evolution.High conservation of sequence enables for simple RISH experiment of expression.Mir‐126 follows the evolution of hematopoetic cells to endothelial cells. [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]

Background: Fish is a great nutritious food and provides quality protein and a variety of vitamins and minerals. This contributes significantly to the economy and food security in Iran. However, there are safety concerns related to the presence of zoonotic parasites. Objectives: The objective of this study is, therefore, to review fish‐borne zoonotic parasites in Iran. Methods: Keywords such as fish‐borne, parasites, zoonotic, Iran, and some names of fish‐borne zoonotic parasites were searched in databases including PubMed, Science Direct, Elsevier, SID, Magiran, Irandoc, Google Scholar and the World Health Organization. Results: The most common fish‐borne parasites with zoonotic potential identified in reports in the literature were the protozoa Balantidium spp., Myxobolus spp. and Sarcosystis sp.; the trematodes Heterophyes heterophyes and Clinostomum complanatum; the cestodes Ligula intestinalis and Diphyllobothrium latum; the nematodes Pseudoterranova sp., Anisakis spp., Contracaecum spp., Raphidascaris spp., Eustrongylides spp. and Capillaria sp.; and the acanthocephal Corynosoma spp. Conclusions: The potential risk factors for the transmission of fish‐borne zoonotic parasites to humans are consumption of raw or undercooked infected fish, contact with contaminated water and contact with infected fish. There is a need for epidemiological surveillance of fish for parasites with zoonotic potential and of occurrence of infections in humans to better understand the public health significance and design prevention programs. [ABSTRACT FROM AUTHOR]

Backgrounds: Elucidation of peptide-receptor pairs is a prerequisite for many studies in the neuroendocrine, endocrine, and neuroscience fields. Recent omics analyses have provided vast amounts of peptide and G protein-coupled receptor (GPCR) sequence data. GPCRs for homologous peptides are easily characterized based on homology searching, and the relevant peptide-GPCR interactions are also detected by typical signaling assays. In contrast, conventional evaluation or prediction methods, including high-throughput reverse-pharmacological assays and tertiary structure-based computational analyses, are not useful for identifying interactions between novel and omics-derived peptides and GPCRs. Summary: Recently, an approach combining machine learning-based prediction of novel peptide-GPCR pairs and experimental validation of the predicted pairs have been shown to breakthrough this bottleneck. A machine learning method, logistic regression for human class A GPCRs and the multiple subsequent signaling assays led to the deorphanization of human class A orphan GPCRs, namely, the identification of 18 peptide-GPCR pairs. Furthermore, using another machine learning algorithm, the support vector machine (SVM), the peptide descriptor-incorporated SVM was originally developed and employed to predict GPCRs for novel peptides characterized from the closest relative of vertebrates, Ciona intestinalis Type A (Ciona robusta). Experimental validation of the predicted pairs eventually led to the identification of 11 novel peptide-GPCR pairs. Of particular interest is that these newly identified GPCRs displayed neither significant sequence similarity nor molecular phylogenetic relatedness to known GPCRs for peptides. Key Messages: These recent studies highlight the usefulness and versatility of machine learning for enabling the efficient, reliable, and systematic identification of novel peptide-GPCR interactions. [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]

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 order to explore the influence mechanism of early biofilm and ecological factors on larvae adhesion and population establishment of ascidian in the aquaculture pond of Stichopus japonicus, laboratory simulation experiment was adopted to investigate the effects of temperature and salinity on the adhesion of four ascidians' larvae including Styela Clava, Ciona Savignyi, Ciona Intestinalis, and Molgula manhattensis on the basis of field investigation. The results showed that biofilm exhibited positive effect on the adhesion of four larvae, and the adhering rates of four larvae all increased with the increasing temperature in the range from 12°C to 27°C, while salinity showed little effect on the fixation from 29‰ to 32‰. Under the same environment, M. manhattensis showed higher adhesion competitiveness than the other three species, which might explain why M. manhattensis was the dominant species in the sessile ascidians community in the ponds farming S. japonicus. The study was helpful to understand the dialectical relationship between environmental factors and biological factors in the aquaculture environment and provided a theoretical basis for exploring the mechanism of marine biofilm formation. [ABSTRACT FROM AUTHOR]

Spermatogenesis is a complex and continuous process of germ-cell differentiation. This complex process is regulated by many factors, of which gene regulation in spermatogenic cells plays a decisive role. Spermatogenesis has been widely studied in vertebrates, but little is known about spermatogenesis in protochordates. Here, for the first time, we performed single-cell RNA sequencing (scRNA-seq) on 6832 germ cells from the testis of adult Ciona intestinalis. We identified six germ cell populations and revealed dynamic gene expression as well as transcriptional regulation during spermatogenesis. In particular, we identified four spermatocyte subtypes and key genes involved in meiosis in C. intestinalis. There were remarkable similarities and differences in gene expression during spermatogenesis between C. intestinalis and two other vertebrates (Chinese tongue sole and human). We identified many spermatogenic-cell-specific genes with functions that need to be verified. These findings will help to further improve research on spermatogenesis in chordates. [ABSTRACT FROM AUTHOR]

Although sensitivity to pollutants is well known to be species specific, closely related species are often assumed to respond similarly to them. We tested this assumption, comparing the sensitivity of Ciona intestinalis and Ciona robusta to two common marine pollutants: Bisphenol A (BPA) and tris(1-chloro-2-propyl)phosphate (TCPP). In particular, we focused on ascidian embryonic development and determined whether C. intestinalis and C. robusta displayed different responses. Our results demonstrate that closely related species can display either similar or very different sensitivity based on the tested contaminant. Ciona intestinalis and C. robusta had similar sensitivity to BPA, as their median effective concentration (EC50) and median lethal concentration (LC50) values were comparable. In contrast, TCPP showed very different teratogenic potential in the two analyzed species. Ciona robusta proved more vulnerable to this flame retardant as its teratogenic index was more than twice that calculated for C. intestinalis. Chemical modes of action as well as genetic differences could determine the diverse responses to environmental stressors. These results underline the presence of species-specific differences in embryonic sensitivity to contaminants and point out the importance of evaluating chemicals' teratogenic profile in several species. [ABSTRACT FROM AUTHOR]

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

Global shipping facilitates the introduction of fouling organisms to new geographic areas. The increase in maritime transport in recent decades has led to unprecedented development, where marine coastal waters have become one of the most invaded habitats around the globe. Among the most successful invaders are ascidians. Despite the geographic and oceanographic isolation of Iceland, it is far from being excluded as a recipient region. Ascidians have successfully been able to establish stable populations in Iceland that are slowly expanding around the SW region. Here we report the first record of Ascidiella scabra in Icelandic waters, and the spread of seven non-indigenous ascidians across the S-SW Icelandic harbours. We compare their relative abundances to our survey from 2018. The bulk of these ascidians is not only present in the temperate Atlantic Ocean, but also in the Northwest Pacific and temperate Australasia, revealing that global homogenization of the ascidiofauna is emerging in anthropized coastal areas. The rising of sea surface temperature, driven by climate change, is likely to continue to support the development of this scenario leading to biodiversity loss, which could endanger living resources with severe economic impacts. [ABSTRACT FROM AUTHOR]

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

Ascidians are sessile marine invertebrates and many are invasive. One notorious example is the solitary species Microcosmus squamiger, which has spread from a hypothesized native region in southeastern Australia to temperate waters worldwide and is considered a global marine invader. This study is the first report of M. squamiger from Jeju Island, South Korea. We collected specimens (N = 20) from Unjin and Wimi harbors on Jeju Island and dissected them to characterise their siphons, pharynx, and dorsal tubercle using stereomicroscopy and scanning electron microscopy. The shape of the dorsal tubercle (the peritubercular area filled with two spiral coils) and the length (15-20 µm) and shape (roof tile or fingernail) of the siphon spine were consistent with previous identifications of the species. Additionally, molecular verification was performed by extracting genomic DNA from four specimens, two each from Unjin and Wimi harbors. By sequencing a section of the mitochondrial cytochrome oxidase subunit I gene and analysing the generated sequences using a maximum likelihood-based phylogenetic tree, we compared to individuals previously identified as M. squamiger and confirmed the existence of two clades. Therefore, the presence of M. squamiger was verified by morphological and phylogenetic identification. Although M. squamiger is currently distributed globally (except in Antarctica), records from India (2006), Okinawa, and Japan (2007 and 2014) suggest that it was introduced into Asia in the last decade. Though no economic or biological damage caused by this species has been reported in South Korea, the high density of M. squamiger (50-60 individuals/rope-meter) is a potential hazard to other native species via fouling and competition for food and space. Therefore, further research is needed to determine the possible vectors and to continue to monitor its possible spread. [ABSTRACT FROM AUTHOR]

Background: Extensive evidence suggests that gut microbiota may interact with the kidneys and play central roles in the pathogenesis of disease. However, the association of gut microbiota-kidneys in diarrhea remains unclear. Methods: A diarrhea mouse model was constructed by combining adenine with Folium sennae. We analyzed the characteristics of the gut content microbiota and short chain fatty acids (SCFAs); and explored the potential link between gut content microbiota, SCFAs, intestinal inflammatory response and kidney function. Results: Characteristic bacteria Lactobacillus intestinalis and Bacteroides acidifaciens were enriched in the gut contents of mice. The productions of SCFAs were remarkably inhibited. Model mice presented an increased trend of creatinine (Cr), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), a decreased trend of blood urea nitrogen (BUN) and secretory immunoglobulin A (SIgA). The pathological analysis proved obvious damage to the kidney structure. Lactobacillus intestinalis and Bacteroides acidifaciens exisited in the correlations with acetic acid, intestinal inflammatory response and kidney function. Conclusions: Adenine combined with Folium sennae-induced diarrhea, altered the structure and function of the gut content microbiota in mice, causing the enrichment of the characteristic bacteria Lactobacillus intestinalis and Bacteroides acidifaciens. The interactions between Lactobacillus intestinalis, Bacteroides acidifaciens and acetic acid, intestinal inflammation, and kidney function might be involved in the process of gut-kidney impairment in adenine, combined with Folium sennae-induced diarrhea. [ABSTRACT FROM AUTHOR]

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

Voltage-sensing phosphatase (Vsp) is a unique membrane protein that translates membrane electrical activities into the changes of phosphoinositide profiles. Vsp orthologs from various species have been intensively investigated toward their biophysical properties, primarily using a heterologous expression system. In contrast, the physiological role of Vsp in native tissues remains largely unknown. Here we report that zebrafish Vsp (Dr-Vsp), encoded by tpte gene, is functionally expressed on the endomembranes of lysosome-rich enterocytes (LREs) that mediate dietary protein absorption via endocytosis in the zebrafish mid-intestine. Dr-Vsp-deficient LREs were remarkably defective in forming endosomal vacuoles after initial uptake of dextran and mCherry. Dr-Vsp-deficient zebrafish exhibited growth restriction and higher mortality during the critical period when zebrafish larvae rely primarily on exogenous feeding via intestinal absorption. Furthermore, our comparative study on marine invertebrate Ciona intestinalis Vsp (Ci-Vsp) revealed co-expression with endocytosis-associated genes in absorptive epithelial cells of the Ciona digestive tract, corresponding to zebrafish LREs. These findings signify a crucial role of Vsp in regulating endocytosis-dependent nutrient absorption in specialized enterocytes across animal species. The physiological role of Vsp in zebrafish is assessed, revealing Vsp expression in the mid-intestine for dietary protein absorption. A comparative study on marine invertebrate Ciona intestinalis suggests conservation of Vsp function in the GI tract. [ABSTRACT FROM AUTHOR]

Metamorphosis is the dramatic and irreversible reconstruction of animal bodies transitioning from the larval stage. Because of the significant impact of metamorphosis on animal life, its timing is strictly regulated. Invertebrate chordate ascidians are the closest living relatives of vertebrates. Ascidians exhibit metamorphosis that converts their swimming larvae into sessile adults. Ascidian metamorphosis is triggered by a mechanical stimulus generated when adhesive papillae adhere to a substrate. However, it is not well understood how the mechanical stimulus is generated and how ascidian larvae sense the stimulus. In this study, we addressed these issues by a combination of embryological, molecular, and genetic experiments in the model ascidian Ciona intestinalis Type A, also called Ciona robusta. We here showed that the epidermal neuronal network starting from the sensory neurons at the adhesive papillae is responsible for the sensing of adhesion. We also found that the transient receptor potential (TRP) channel PKD2 is involved in sensing the stimulus of adhesion. Our results provide a better understanding of the mechanisms underlying the regulation of the timing of ascidian metamorphosis. [ABSTRACT FROM AUTHOR]

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ANISEED (www.aniseed.cnrs.fr) is the main model organism database for tunicates, the sister-group of vertebrates. This release gives access to annotated genomes, gene expression patterns, and anatomical descriptions for nine ascidian species. It provides increased integration with external molecular and taxonomy databases, better support for epigenomics datasets, in particular RNA-seq, ChIP-seq and SELEX-seq, and features novel interactive interfaces for existing and novel datatypes. In particular, the cross-species navigation and comparison is enhanced through a novel taxonomy section describing each represented species and through the implementation of interactive phylogenetic gene trees for 60% of tunicate genes. The gene expression section displays the results of RNA-seq experiments for the three major model species of solitary ascidians. Gene expression is controlled by the binding of transcription factors to cis-regulatory sequences. A high-resolution description of the DNA-binding specificity for 131 Ciona robusta (formerly C. intestinalis type A) transcription factors by SELEX-seq is provided and used to map candidate binding sites across the Ciona robusta and Phallusia mammillata genomes. Finally, use of a WashU Epigenome browser enhances genome navigation, while a Genomicus server was set up to explore microsynteny relationships within tunicates and with vertebrates, Amphioxus, echinoderms and hemichordates.

Here we present a parallel study of mRNA and microRNA expression during oral siphon (OS) regeneration in Ciona robusta, and the derived network of their interactions. In the process of identifying 248 mRNAs and 15 microRNAs as differentially expressed, we also identified 57 novel microRNAs, several of which are among the most highly differentially expressed. Analysis of functional categories identified enriched transcripts related to stress responses and apoptosis at the wound healing stage, signaling pathways including Wnt and TGFβ during early regrowth, and negative regulation of extracellular proteases in late stage regeneration. Consistent with the expression results, we found that inhibition of TGFβ signaling blocked OS regeneration. A correlation network was subsequently inferred for all predicted microRNA-mRNA target pairs expressed during regeneration. Network-based clustering associated transcripts into 22 non-overlapping groups, the functional analysis of which showed enrichment of stress response, signaling pathway and extracellular protease categories that could be related to specific microRNAs. Predicted targets of the miR-9 cluster suggest a role in regulating differentiation and the proliferative state of neural progenitors through regulation of the cytoskeleton and cell cycle.

The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos. We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona.

Vertebrate nervous systems can generate a remarkable diversity of behaviors. However, our understanding of how behaviors may have evolved in the chordate lineage is limited by the lack of neuroethological studies leveraging our closest invertebrate relatives. Here, we combine high-throughput video acquisition with pharmacological perturbations of bioamine signaling to systematically reveal the global structure of the motor behavioral repertoire in the Ciona intestinalis larvae. Most of Ciona's postural variance can be captured by 6 basic shapes, which we term "eigencionas." Motif analysis of postural time series revealed numerous stereotyped behavioral maneuvers including "startle-like" and "beat-and-glide." Employing computational modeling of swimming dynamics and spatiotemporal embedding of postural features revealed that behavioral differences are generated at the levels of motor modules and the transitions between, which may in part be modulated by bioamines. Finally, we show that flexible motor module usage gives rise to diverse behaviors in response to different light stimuli. Vertebrate nervous systems can generate a remarkable diversity of behaviors, but how did these evolve in the chordate lineage? A study of the protochordate Ciona intestinalis reveals novel insights into how a simple chordate brain uses neuromodulators to control its behavioral repertoire. [ABSTRACT FROM AUTHOR]

Marine symbiotic Palaemonidae, comprising over 600 species, live in association with marine invertebrates of different phyla, like Cnidaria, Echinodermata, Mollusca, Porifera, and Tunicata. A phylogenetic study is performed on a clade of bivalve- and ascidian-associated endosymbiotic shrimp species (Caridea: Palaemonidae), using morphological and molecular data. A Total Evidence approach is used in order to include all currently known ingroup species in an evolutionary framework. Ancestral state reconstruction analyses are performed to identify host-switching events and ancestral ranges. The clade, including Ascidonia, Conchodytes, Dactylonia, Odontonia, and Pontonia, and various smaller genera, is recovered as monophyletic, with an ascidian-associated ancestral host state. At least six interphylum host switches are tentatively identified, with members of Odontonia and Notopontonia switching back to an ascidian host affiliation after the ancestral host switch of the clade including Conchodytes, Odontonia and related genera, from an ascidian- to a bivalve host. The clade including Ascidonia and Pontonia was recovered to have an ancestor with an East Pacific/Atlantic distribution. The other studied genera remained in the original ancestral Indo-West Pacific range. We hypothesize that similar internal environments of shrimp hosts from different phyla will function as hot spots for interphylum host switching in various lineages of symbionts. [ABSTRACT FROM AUTHOR]

Microplastics are contaminants of global environmental concern. They can be ingested by a variety of organisms when they enter the food web. Several studies have reported trophic transfer of microplastics from low trophic levels to higher ones. Bioaccumulation has been suggested to occur but few studies have demonstrated it for marine environments. In this article, in controlled laboratory conditions, we exposed filter‐feeder ascidian juveniles to microplastics in the presence or in absence of mixotrophic cryptomonad flagellates. Cryptomonads can efficiently ingest microbeads, and their presence significantly increased the concentration of microplastics in the digestive tract of the ascidians. Our results demonstrate the occurrence of microplastic bioaccumulation in the lower levels of the marine trophic chain and suggest that unicellular organisms can be key actors in microplastic trophic transfer at the microscale level. Research Highlights: •Marine mixotrophic cryptomonad flagellates can ingest microbeads of polystyrene with a diameter of 10 µm. •The copresence of cryptomonads and microbeads significantly increased the microplastics in the digestive tract of filter‐feeding ascidians. [ABSTRACT FROM AUTHOR]

Voltage-sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), which is similar to phosphatase and tensin homolog (PTEN). How the VSD regulates the innate enzyme component of VSP remains unclear. Here, we took a combined approach that entailed the use of electrophysiology, fluorometry, and structural modeling to study the electrochemical coupling in Ciona intestinalis VSP. We found that two hydrophobic residues at the lowest part of S4 play an essential role in the later transition of VSD-CCR coupling. Voltage clamp fluorometry and disulfide bond locking indicated that S4 and its neighboring linker move as one helix (S4-linker helix) and approach the hydrophobic spine in the CCR, a structure located near the cell membrane and also conserved in PTEN. We propose that the hydrophobic spine operates as a hub for translating an electrical signal into a chemical one in VSP. [ABSTRACT FROM AUTHOR]

To evolve fish farming in an eco-efficient way, feed production must become less dependent on forage fish-based ingredients and make more use of low trophic level organisms, including microalgae, higher plants, as filter feeding organisms and other ingredients with low competition to established food value chains. Diets nearly free of fish meal and fish oil are not a novelty but are often composed of complex mixtures, containing supplements to meet the farmed animal's nutritional requirements. Sustaining a growing aquaculture production, maintaining at the same time fish health, welfare, and profitability, and meeting strict environmental and food safety demands, is challenging and requires new technologies, great investments, and more knowledge. A benchmarking feeding trial was performed to demonstrate the main effects of four low trophic raw materials on Atlantic salmon (Salmo salar) growth, metabolism, skin health and fillet quality. To this end, a diet was produced to contain commercially relevant levels of fresh high quality organic FM and FO and was used as a control in the trial (FMFO). Heterotrophically produced Schizochytrium limacinum biomass was used to replace organic FO (HM diet). Spray dried cell wall disrupted biomass of the phototrophically cultured diatom Phaeodactylum tricornutum replaced partly FM and FO (PM diet). Black soldier fly (Hermetia illucens) larvae meal and tunicate (Ciona intestinalis) meal, were used to produce the diets BSFL and TM, respectively, replacing large parts of FM as compared to the FMFO. A fifth test diet was produced combining all test raw materials and removing all FM and FO (0FM0FO diet). All test ingredients were well accepted sustaining high growth rates (TGC values near 4) and feed efficiency (FCR values below 0.9) in salmon showing good gut health and normal metabolic responses. However, none of the treatments reached the growth performance of FMFO. Additional differences between test and control treatments were identified in dietary nutrient apparent digestibility, fish biometrics, blood metabolites and fillet and skin composition. Extensive raw material and dietary chemical characterisation was performed to provide insight on potential shortcomings in the novel low trophic level ingredients which can possibly be overcome combining complementary raw materials. [ABSTRACT FROM AUTHOR]

The tunicate (Ciona intestinalis) βγ-crystallin represents an intermediate case between the calcium-binding proteins ancestral to the vertebrate βγ-crystallin fold and the vertebrate structural crystallins. Unlike the structural βγ-crystallins in the vertebrate eye lens, this βγ-crystallin strongly binds Ca2+. Furthermore, Ca2+ binding greatly stabilizes the protein, an effect that has previously been observed in microbial βγ-crystallins but not in those of vertebrates. This relationship between binding and protein stabilization makes the tunicate βγ-crystallin an interesting model for studying the evolution of the human βγ-crystallin. We also compare and contrast the binding sites of tunicate βγ-crystallin with those of other βγ-crystallins to develop hypotheses about the functional origin of the lack of Ca2+-binding sites in human crystallins.

Abstract Background One of the sustainable development goals focuses on the biomass-based production as a replacement for fossil-based commodities. A novel feedstock with vast potentials is tunicate biomass, which can be pretreated and fermented in a similar way to lignocellulose. Ciona intestinalis is a marine filter feeder that is cultivated to produce fish feed. While the inner tissue body is used for feed production, the surrounding tunic remains as a cellulose-rich by-product, which can be further separated into outer and inner tunic. Ethanol production from organosolv-pretreated whole-tunic biomass was recently validated. The aim of the present study was to evaluate the potential of organosolv pretreated outer-tunic biomass for the production of biofuels and cellobiose that is a disaccharide with prebiotic potential. Results As a result, 41.4 g/L of ethanol by Saccharomyces cerevisiae, corresponding to a 90.2% theoretical yield, was achieved under the optimal conditions when the tunicate biomass was pretreated at 195 °C for 60 min at a liquid-to-solid ratio of 50. In addition, cellobiose production by enzymatic hydrolysis of the pretreated tunicate biomass was demonstrated with a maximum conversion yield of 49.7 wt. %. Conclusions The utilisation of tunicate biomass offers an eco-friendly and sustainable alternative for value-added biofuels and chemicals. The cultivation of tunicate biomass in shallow coastal sea improves the quality of the water and ensures sustainable production of fish feed. Moreover, there is no competition for arable land, which leaves the latter available for food and feed production.

The effect of seed coating salt-stressed tomato with the bacterium Achromobacter xylosoxidans BOA4 and/or irrigation with an extract of the marine algae Enteromorpha intestinalis (EI) is herein evaluated. The plant shoots and roots were harvested separately on day 50, following extensive saline stress. The addition of BOA4 and/or EI extract resulted in an average increase of 33% in plant shoot DW, but an averaged decrease of 44% in the root to shoot biomass ratio. Anthocyanin content increased by over 34% and 44% with EI and BOA4 plus EI treatments, respectively. Since enhanced protein tyrosine nitration (PTN) is a known plant response to salt stress, the PTN level was inspected through 3-nitrotyrosine content determination. This was drastically increased by salt stress; however, BOA4, EI or both caused an averaged PTN decrease of 30% in stressed roots or shoots. This PTN response could be associated with tomato phenotypic characteristics and is postulated to be inversely correlated to cytokinin contents in stressed plants, namely cis-zeatin-type-cis-zeatin (cZ) plus cis-zeatin riboside (cZR), and isopentenyladenine (iP). The latter showed a drastic average increase by 3.6-fold following BOA4 and/or EI treatments of salinized tomato. This increment could be related to cytokinin biosynthesis induced by the applied bio-stimulants; IP and derivatives are the main cytokinins in seaweeds, and Achromobacter xylosoxidans BOA4 was shown to produce up to 17.5 pmol mL−1 of isopentenyladenine. This work is the first report on the influence of bio-stimulants, used to improve salt stress tolerance, on plant PTN levels; BOA4 and/or EI treatments decreased PTN, while increasing cis-zeatin-type and iP cytokinins in tomato, the latter showed an enhanced tolerance to salt stress. [ABSTRACT FROM AUTHOR]

The set of four closely related solitary ascidians Ciona spp. were once considered a single cosmopolitan species, Ciona intestinalis , but are now recognized as genetically and morphologically distinct species. The possibility of ecological differences between the species was not widely considered in studies preceding the schism of Ciona spp. Consequently, there may be an over-generalization of the ecology of Ciona spp., with potential implications for the broad range of studies targeting these species, encompassing the evolution, development, genomics, and invasion biology of Ciona spp. We completed a comprehensive review of the ecology of Ciona spp. to establish the similarities and differences between the widely distributed Ciona robusta and C. intestinalis (and what little is known of the two other species, Ciona sp. C and Ciona sp. D). When necessary, we used study locations and the species' geographic ranges to infer the species in each study in the review. As expected, ecological similarities are the norm between the two species, spanning both abiotic and biotic interactions. However, there are also important differences that have potential implications for other aspects of the biology of Ciona spp. For example, differences in temperature and salinity tolerances likely correspond with the disparities in the geographic distribution of the species. Asymmetries in topics studied in each species diminish our ability to fully compare several aspects of the ecology of Ciona spp. and are priority areas for future research. We anticipate that our clarification of common and unique aspects of each species' ecology will help to provide context for future research in many aspects of the biology of Ciona spp. [ABSTRACT FROM AUTHOR]

A compilation of marine halacarid mites (Halacaridae) found with ascidians (sea squirts) has been carried out based on published records. In this paper, we listed 19 species found with ascidians, altogether. These 19 species belong to 7 genera, viz. Agaue (3 species), Arhodeoporus (1 species), Copidognathus (8 species), Halacarus (1 species), Lohmannella (2 species), Rhombognathides (1 species), and Thalassarachna (3 species). Most of these reports are from mixed habitats and all these species which were found among ascidians were reported also from other habitats. Most of the cases of halacarids on acidians may be occasional invaders. [ABSTRACT FROM AUTHOR]

Protozoan parasites interact with a wide variety of organisms ranging from bacteria to humans, representing one of the most common causes of parasitic diseases and an important public health problem affecting hundreds of millions of people worldwide. The current treatment for these parasitic diseases remains unsatisfactory and, in some cases, very limited. Treatment limitations together with the increased resistance of the pathogens represent a challenge for the improvement of the patient's quality of life. The continuous search for alternative preclinical drugs is mandatory, but the mechanisms of action of several of these compounds have not been described. Electron microscopy is a powerful tool for the identification of drug targets in almost all cellular models. Interestingly, ultrastructural analysis showed that several classes of antiparasitic compounds induced similar autophagic phenotypes in trypanosomatids, trichomonadids, and apicomplexan parasites as well as in Giardia intestinalis and Entamoeba spp. with the presence of an increased number of autophagosomes as well as remarkable endoplasmic reticulum profiles surrounding different organelles. Autophagy is a physiological process of eukaryotes that maintains homeostasis by the self-digestion of nonfunctional organelles and/or macromolecules, limiting redundant and damaged cellular components. Here, we focus on protozoan autophagy to subvert drug effects, discussing its importance for successful chemotherapy. [ABSTRACT FROM AUTHOR]

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

Taxonomic evaluations are needed to accurately determine the host selection of fish parasites. The present study is a multidisciplinary research in the field of basic and fish diseases sciences. The description of the hybrid species of Squalius orientalis and Alburnus derjugini and infection of Ligula intestinalis in these hybrid fish were reported for the first time from the Kürtün Dam Lake in northeast Turkey. A total of 450 fish were sampled in March, August, and October in 2020 using gillnets. Detailed morphological characteristics (n = 24) were compared to determine the difference among ancestors and hybrid species. The prevalence of L. intestinalis between the sampling periods and the size groups of fish (0 – 10, 11 – 15, and ≥16 cm in length) were examined. Moreover, the highest prevalence of the parasite was observed in October (78.94 %), with a size range of 0 – 10 cm in length (77.8 %). In addition, the total prevalence of the parasite was 48.44 %. The results revealed that most of the diagnostic metric and meristic features of hybrid fish were ranging between the data of S. orientalis and A. derjugini. According to previous reports, when hybrid individuals were compared with their ancestors in terms of prevalence, hybrid individuals were more susceptible to L. intestinalis infections. This study was unique as it provided the first record of L. intestinalis in a hybrid fish population. [ABSTRACT FROM AUTHOR]

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

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr.

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

Metronidazole (MTZ) is a clinically important antimicrobial agent that is active against both bacterial and protozoan organisms. MTZ has been used extensively for more than 60 years and until now resistance has been rare. However, a recent and dramatic increase in the number of MTZ resistant bacteria and protozoa is of great concern since there are few alternative drugs with a similarly broad activity spectrum. To identify key factors and mechanisms underlying MTZ resistance, we utilized the protozoan parasite Giardia intestinalis , which is commonly treated with MTZ. We characterized two in vitro selected, metronidazole resistant parasite lines, as well as one revertant, by analyzing fitness aspects associated with increased drug resistance and transcriptomes and proteomes. We also conducted a meta-analysis using already existing data from additional resistant G. intestinalis isolates. The combined data suggest that in vitro generated MTZ resistance has a substantial fitness cost to the parasite, which may partly explain why resistance is not widespread despite decades of heavy use. Mechanistically, MTZ resistance in Giardia is multifactorial and associated with complex changes, yet a core set of pathways involving oxidoreductases, oxidative stress responses and DNA repair proteins, is central to MTZ resistance in both bacteria and protozoa. [ABSTRACT FROM AUTHOR]

The protovertebrate Ciona intestinalis type A (sometimes called Ciona robusta) contains a series of sensory cell types distributed across the head-tail axis of swimming tadpoles. They arise from lateral regions of the neural plate that exhibit properties of vertebrate placodes and neural crest. The sensory determinant POU IV/Brn3 is known to work in concert with regional determinants, such as Foxg and Neurogenin, to produce palp sensory cells (PSCs) and bipolar tail neurons (BTNs), in head and tail regions, respectively. A combination of single-cell RNA-sequencing (scRNA-seq) assays, computational analysis, and experimental manipulations suggests that misexpression of POU IV results in variable transformations of epidermal cells into hybrid sensory cell types, including those exhibiting properties of both PSCs and BTNs. Hybrid properties are due to coexpression of Foxg and Neurogenin that is triggered by an unexpected POU IV feedback loop. Hybrid cells were also found to express a synthetic gene battery that is not coexpressed in any known cell type. We discuss these results with respect to the opportunities and challenges of reprogramming cell types through the targeted misexpression of cellular determinants. [ABSTRACT FROM AUTHOR]

Background: Giardia is a diarrheagenic eukaryotic parasite that consists of at least eight morphologically identical but genetically distinct genotypes. Human giardiasis is caused mainly by A and B assemblages. Aim and objectives: The study aimed to compare the performance of gdh polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and tpi assemblage-specific primers in genotyping of G. intestinalis. Materials and Methods: Stool samples of 315 children were microscopically screened for G. intestinalis. Positive samples were genotyped using tpi assemblage-specific primers and gdh semi-nested PCR-RFLP techniques. Results: The prevalence of Giardia was 18.1%. The detected genotypes using tpi and gdh approaches were assemblage A (15.8% vs. 12.7%) and assemblage B (36.8% vs. 74.5%) as single infections and mixed assemblages A and B (47.4% vs. 12.7%). The two approaches showed a moderate agreement (kappa index = 0.413, P < 0.001). PCR-RFLP of gdh gene revealed that sub-assemblages BIII and BIV were equally detected (30.9% each). The remaining samples were equally divided between sub-assemblage AII, mixed BIII and BIV, and mixed AII and BIII (12.7% each). A significant association was detected between the retrieved sub-assemblages and the presence of symptoms. Conclusions: Although both approaches confirmed the predominance of assemblage B, the use of assemblage-specific primers is more effective in elucidating the true picture of mixed assemblage infection. [ABSTRACT FROM AUTHOR]

Human‐driven translocations of species have diverse evolutionary consequences such as promoting hybridization between previously geographically isolated taxa. This is well illustrated by the solitary tunicate, Ciona robusta, native to the North East Pacific and introduced in the North East Atlantic. It is now co‐occurring with its congener Ciona intestinalis in the English Channel, and C. roulei in the Mediterranean Sea. Despite their long allopatric divergence, first and second generation crosses showed a high hybridization success between the introduced and native taxa in the laboratory. However, previous genetic studies failed to provide evidence of recent hybridization between C. robusta and C. intestinalis in the wild. Using SNPs obtained from ddRAD‐sequencing of 397 individuals from 26 populations, we further explored the genome‐wide population structure of the native Ciona taxa. We first confirmed results documented in previous studies, notably (i) a chaotic genetic structure at regional scale, and (ii) a high genetic similarity between C. roulei and C. intestinalis, which is calling for further taxonomic investigation. More importantly, and unexpectedly, we also observed a genomic hotspot of long introgressed C. robusta tracts into C. intestinalis genomes at several locations of their contact zone. Both the genomic architecture of introgression, restricted to a 1.5 Mb region of chromosome 5, and its absence in allopatric populations suggest introgression is recent and occurred after the introduction of the non‐native species. Overall, our study shows that anthropogenic hybridization can be effective in promoting introgression breakthroughs between species at a late stage of the speciation continuum. [ABSTRACT FROM AUTHOR]

Positively charged amino acids respond to membrane potential changes to drive voltage sensor movement in voltage-gated ion channels, but determining the displacements of voltage sensor gating charges has proven difficult. We optically tracked the movement of the two most extracellular charged residues (R1 and R2) in the Shaker potassium channel voltage sensor using a fluorescent positively charged bimane derivative (qBBr) that is strongly quenched by tryptophan. By individually mutating residues to tryptophan within the putative pathway of gating charges, we observed that the charge motion during activation is a rotation and a tilted translation that differs between R1 and R2. Tryptophan-induced quenching of qBBr also indicates that a crucial residue of the hydrophobic plug is linked to the Cole-Moore shift through its interaction with R1. Finally, we show that this approach extends to additional voltage-sensing membrane proteins using the Ciona intestinalis voltage-sensitive phosphatase (CiVSP). [ABSTRACT FROM AUTHOR]

The Hv1 proton channel is unique among voltage-gated channels for containing the pore and gate within its voltage-sensing domain. Pore opening has been proposed to include assembly of the selectivity filter between an arginine (R3) of segment S4 and an aspartate (D1) of segment S1. We determined whether gating involves motion of S1, using Ciona intestinalis Hv1. We found that channel opening is concomitant with solution access to the pore-lining face of S1, from the cytoplasm to deep inside the pore. Voltage- and patch-clamp fluorometry showed that this involves a motion of S1 relative to its surroundings. S1 motion and the S4 motion that precedes it are each influenced by residues on the other helix, thus suggesting a dynamic interaction between S1 and S4. Our findings suggest that the S1 of Hv1 has specialized to function as part of the channel's gate.

The ascidian Ciona intestinalis is a marine invertebrate belonging to the sister group of the vertebrates, the tunicates. Its compact genome and simple, experimentally tractable embryos make Ciona well-suited for the study of cell-fate specification in chordates. Tunicate larvae possess a characteristic chordate body plan, and many developmental pathways are conserved between tunicates and vertebrates. Previous studies have shown that FGF signals are essential for neural induction and patterning at sequential steps of Ciona embryogenesis. Here we show that two different ETS family transcription factors, Ets1/2 and Elk1/3/4, have partially redundant activities in the anterior neural plate of gastrulating embryos. Whereas Ets1/2 promotes pigment cell formation in lateral lineages, both Ets1/2 and Elk1/3/4 are involved in the activation of Myt1L in medial lineages and the restriction of Six3/6 expression to the anterior-most regions of the neural tube. We also provide evidence that photoreceptor cells arise from posterior regions of the presumptive sensory vesicle, and do not depend on FGF signaling. Cells previously identified as photoreceptor progenitors instead form ependymal cells and neurons of the larval brain. Our results extend recent findings on FGF-dependent patterning of anterior-posterior compartments in the Ciona central nervous system.

Calcium imaging is an increasingly valuable technique for understanding neural circuits, neuroethology, and cellular mechanisms. The analysis of calcium imaging data presents challenges in image processing, data organization, analysis, and accessibility. Tools have been created to address these problems independently, however a comprehensive user-friendly package does not exist. Here we present Mesmerize, an efficient, expandable and user-friendly analysis platform, which uses a Findable, Accessible, Interoperable and Reproducible (FAIR) system to encapsulate the entire analysis process, from raw data to interactive visualizations for publication. Mesmerize provides a user-friendly graphical interface to state-of-the-art analysis methods for signal extraction & downstream analysis. We demonstrate the broad scientific scope of Mesmerize's applications by analyzing neuronal datasets from mouse and a volumetric zebrafish dataset. We also applied contemporary time-series analysis techniques to analyze a novel dataset comprising neuronal, epidermal, and migratory mesenchymal cells of the protochordate Ciona intestinalis. Calcium imaging is valuable for understanding neuro and cell biology, but is challenging to analyze, organize, and access. Here, the authors present an efficient, expandable and user-friendly platform, which encapsulates the entire analysis process all to way to interactive visualizations. [ABSTRACT FROM AUTHOR]