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Sea squirts (Tunicata) are chordates and develop a swimming larva with a small and defined number of individually identifiable cells. This offers the prospect of connecting specific stimuli to behavioral output and characterizing the neural activity that links these together. Here, we describe the development of a microfluidic chip that allows live larvae of the sea squirt Ciona intestinalis to be immobilized and recorded. By generating transgenic larvae expressing GCaAMP6m in defined cells, we show that calcium ion levels can be recorded from immobilized larvae, while microfluidic control allows larvae to be exposed to specific waterborne stimuli. We trial this on sea water carrying increased levels of carbon dioxide, providing evidence that larvae can sense this gas. Research Highlights: Development of a microfluidic chip in which Ciona larvae can be immobilized, exposed to controlled stimuli, and recorded.A transgenic construct was designed to drive expression of the live cell calcium ion reporter GCaMP in Ciona palp cells and other sensory cells.Exposure of Ciona larvae to carbon dioxide‐enriched sea water leads to detectable changes in GCaMP activity. [ABSTRACT FROM AUTHOR]

Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermallyderived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem. [ABSTRACT FROM AUTHOR]

Physical and functional constraints on biological networks lead to complex topological patterns across multiple scales in their organization. A particular type of higher-order network feature that has received considerable interest is network motifs, defined as statistically regular subgraphs. These may implement fundamental logical and computational circuits and are referred to as "building blocks of complex networks". Their well-defined structures and small sizes also enable the testing of their functions in synthetic and natural biological experiments. Here, we develop a framework for motif mining based on lossless network compression using subgraph contractions. This provides an alternative definition of motif significance which allows us to compare different motifs and select the collectively most significant set of motifs as well as other prominent network features in terms of their combined compression of the network. Our approach inherently accounts for multiple testing and correlations between subgraphs and does not rely on a priori specification of an appropriate null model. It thus overcomes common problems in hypothesis testing-based motif analysis and guarantees robust statistical inference. We validate our methodology on numerical data and then apply it on synaptic-resolution biological neural networks, as a medium for comparative connectomics, by evaluating their respective compressibility and characterize their inferred circuit motifs. Author summary: Networks provide a useful abstraction to study complex systems by focusing on the interplay of the units composing a system rather than on their individual function. Network theory has proven particularly powerful for unraveling how the structure of connections in biological networks influence the way they may process and relay information in a variety of systems ranging from the microscopic scale of biochemical processes in cells to the macroscopic scales of social and ecological networks. Of particular interest are small stereotyped circuits in such networks, termed motifs, which may correspond to building blocks implementing fundamental operations, e.g., logic gates or filters. We here present a new tool that finds sets of motifs in networks based on an information-theoretic measure of how much they allow to compress the network. This approach allows us to evaluate the collective significance of sets of motifs, as opposed to only individual motifs. We apply our methodology to compare the neural wiring diagrams, termed "connectomes", of the tadpole larva Ciona intestinalis, the ragworm Platynereis dumerelii, and the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster at different developmental stages. [ABSTRACT FROM AUTHOR]

This study aimed to provide a comprehensive summary of the fundamental biological and certain population characteristics of Ciona intestinalis found along the Black Sea coasts of Türkiye. The research was conducted between September 2022 and April 15th, 2023, off the Sinop coast of the Black Sea, utilizing samples collected by commercial fishing vessels in the region. A total of 103 specimens of C. intestinalis were examined during the study. The average total length, width, and weight of C. intestinalis were 3.614 ± 0.089 cm, 1.967 ± 0.054 cm, and 2.004 ± 0.067 g, respectively. The length-weight relationship (LWR) and length-width relationship (LWdR) of C. intestinalis were established as W = 0.3996L1.2424 (R = 0.9389), Wd = 0.5362L + 0.0237 (R = 0.8897), and W = 0.9443Wd 1.0889 (R = 0.8786), respectively. This study presents, for the first time, the LWR, LWdR, and WdWR for C. intestinalis, a species of significant importance in the benthic ecosystem of the Black Sea. It is anticipated that this research will lay the groundwork for future investigations into the population dynamics and biological aspects of this species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Larvacean tunicates feature a spectacular innovation not seen in other animals - the trunk oikoplastic epithelium (OE). This epithelium produces a house, a large and complex extracellular structure used for filtering and concentrating food particles. Previously we identified several homeobox transcription factor genes expressed during early OE patterning. Among these are two Pax3/7 copies that we named pax37A and pax37B. The vertebrate homologs, PAX3 and PAX7 are involved in developmental processes related to neural crest and muscles. In the ascidian tunicate Ciona intestinalis , Pax3/7 plays a role in the development of cells deriving from the neural plate border, including trunk epidermal sensory neurons and tail nerve cord neurons, as well as in the neural tube closure. Here we have investigated the roles of Oikopleura dioica pax37A and pax37B in the development of the OE, by using CRISPR-Cas9 mutant lines and analyzing scRNA-seq data from wild-type animals. We found that pax37B but not pax37A is essential for the differentiation of cell fields that produce the food concentrating filter of the house: the anterior Fol, giant Fol and Nasse cells. Trajectory analysis supported a neuroepithelial-like or a preplacodal ectoderm transcriptional signature in these cells. We propose that the highly specialized secretory epithelial cells of the Fol region either maintained or evolved neuroepithelial features. This is supported by a fragmented gene regulatory network involved in their development that also operates in ascidian epidermal neurons. [Display omitted] • pax37B controls the development of the Fol region of the oikoplastic epithelium. • The pax37B TF network is similar to that of ascidian trunk epidermal neurons. • The pax37B + cells are secretory cells with a neuroepithelial-like origin. [ABSTRACT FROM AUTHOR]

During embryonic development, tissues and organs are gradually shaped into their functional morphologies through a series of spatiotemporally tightly orchestrated cell behaviors. A highly conserved organ shape across metazoans is the epithelial tube. Tube morphogenesis is a complex multistep process of carefully choreographed cell behaviors such as convergent extension, cell elongation, and lumen formation. The identity of the signaling molecules that coordinate these intricate morphogenetic steps remains elusive. The notochord is an essential tubular organ present in the embryonic midline region of all members of the chordate phylum. Here, using genome editing, pharmacology and quantitative imaging in the early chordate Ciona intestinalis we show that Ano10/Tmem16k, a member of the evolutionarily ancient family of transmembrane proteins called Anoctamin/TMEM16 is essential for convergent extension, lumen expansion, and connection during notochord morphogenesis. We find that Ano10/Tmem16k works in concert with the plasma membrane (PM) localized Na+/Ca2+ exchanger (NCX) and the endoplasmic reticulum (ER) residing SERCA, RyR, and IP3R proteins to establish developmental stage specific Ca2+ signaling molecular modules that regulate notochord morphogenesis and Ca2+ dynamics. In addition, we find that the highly conserved Ca2+ sensors calmodulin (CaM) and Ca2+/calmodulin-dependent protein kinase (CaMK) show an Ano10/Tmem16k-dependent subcellular localization. Their pharmacological inhibition leads to convergent extension, tubulogenesis defects, and deranged Ca2+ dynamics, suggesting that Ano10/Tmem16k is involved in both the "encoding" and "decoding" of developmental Ca2+ signals. Furthermore, Ano10/Tmem16k mediates cytoskeletal reorganization during notochord morphogenesis, likely by altering the localization of 2 important cytoskeletal regulators, the small GTPase Ras homolog family member A (RhoA) and the actin binding protein Cofilin. Finally, we use electrophysiological recordings and a scramblase assay in tissue culture to demonstrate that Ano10/Tmem16k likely acts as an ion channel but not as a phospholipid scramblase. Our results establish Ano10/Tmem16k as a novel player in the prevertebrate molecular toolkit that controls organ morphogenesis across scales. The notochord is an essential tubular organ that defines the chordate phylum. This study reveals that an ion channel called Anoctamin 10 orchestrates the robust morphogenesis of the notochord in the urochordate Ciona intestinalis. [ABSTRACT FROM AUTHOR]

Human transport of species across oceans disrupts natural dispersal barriers and facilitates hybridization between previously allopatric species. The recent introduction of the North Pacific sea squirt, Ciona robusta , into the native range of the North Atlantic sea squirt, Ciona intestinalis , is a good example of this outcome. Recent studies have revealed an adaptive introgression in a single chromosomal region from the introduced into the native species. Here, we monitored this adaptive introgression over time, examining both the frequency of adaptive alleles at the core and the hitchhiking footprint in the shoulders of the introgression island by studying a thousand Ciona spp. individuals collected in 22 ports of the contact zone, 14 of which were sampled 20 generations apart. For that purpose, we developed a KASP multiplex genotyping approach, which proved effective in identifying native, nonindigenous and hybrid individuals and in detecting introgressed haplotypes. We found no early generation hybrids in the entire sample, and field observations suggest a decline in the introduced species. At the core region of the introgression sweep, where the frequency of C. robusta alleles is the highest and local adaptation genes must be, we observed stable frequencies of adaptive alien alleles in both space and time. In contrast, we observed erosion of C. robusta ancestry tracts in flanking chromosomal shoulders on the edges of the core, consistent with the second phase of a local sweep and a purge of hitchhiked incompatible mutations. We hypothesize that adaptive introgression may have modified the competition relationships between the native and invasive species in human-altered environments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Voltage‐clamp fluorometry (VCF) enables the study of voltage‐sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage‐gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine‐conjugated flavonoid (4‐oxo‐2‐phenyl‐4H‐chromene‐7‐yl)‐phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis HV1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)‐carboxytetramethylrhodamine‐(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane‐bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 became a powerful tool for genetic engineering and in vivo knockout also in the invertebrate chordate Ciona intestinalis. Ciona (ascidians, tunicates) is an important model organism because it shares developmental features with the vertebrates, considered the sister group of tunicates, and offers outstanding experimental advantages: a compact genome and an invariant developmental cell lineage that, combined with electroporation mediated transgenesis allows for precise and cell type specific targeting in vivo. A high polymorphism and the mosaic expression of electroporated constructs, however, often hamper the efficient CRISPR knockout, and an optimization in Ciona is desirable. Furthermore, seasonality and artificial maintenance settings can profit from in vitro approaches that would save on animals. Here we present improvements for the CRISPR/Cas9 protocol in silico , in vitro and in vivo. Firstly, in designing sgRNAs, prior sequencing of target genomic regions from experimental animals and alignment with reference genomes of C. robusta and C. intestinalis render a correction possible of subspecies polymorphisms. Ideally, the screening for efficient and non-polymorphic sgRNAs will generate a database compatible for worldwide Ciona populations. Secondly, we challenged in vitro assays for sgRNA validation towards reduced in vivo experimentation and report their suitability but also overefficiency concerning mismatch tolerance. Thirdly, when comparing Cas9 with Cas9:Geminin, thought to synchronize editing and homology-direct repair, we could indeed increase the in vivo efficiency and notably the access to an early expressed gene. Finally, for in vivo CRISPR, genotyping by next generation sequencing (NGS) ex vivo streamlined the definition of efficient single guides. Double CRISPR then generates large deletions and reliable phenotypic excision effects. Overall, while these improvements render CRISPR more efficient in Ciona, they are useful when newly establishing the technique and very transferable to CRISPR in other organisms. [Display omitted] • We present improvements for the CRISPR/Cas9 protocol in silico , in vitro and in vivo. • Non-polymorphic sgRNAs can be designed by sequencing the experimental population and their efficiency validated in vitro. • Double CRISPR generates detectable large deletions in vitro and ex vivo. • In vivo, Cas9:Geminin improves the editing efficiency and NGS detects efficient single guides. [ABSTRACT FROM AUTHOR]

Background Plastic contamination is one of the concerns of our age. With more than 150 million tons of plastic floating in the oceans, and a further 8 million tons arriving to the water each year, in recent times the scientific community has been studying the effects these plastics have on sea life both in the field and with experimental approaches. Laboratory based studies have been using both natural sea water and artificial sea water for testing various aspects of plastic contamination, including the study of chemicals leached from the plastic particles to the water. We set out to test this equivalence, looking at the leaching of heavy metals form plastic particles. Methods We obtained leachates of polyvinyl chloride plastic pre-production nurdles both in natural and artificial sea water and determined the elements in excess from untreated water by Inductively coupled plasma – optical emission spectrometry. We then used these different leachates to assess developmental success in the tunicate Ciona intestinalis by treating fertilised eggs through their development to hatched larvae. Results Here we report that chemical analysis of polyvinyl chloride plastic pre-production pellet leachates shows a different composition in natural and artificial sea water. We find that the zinc leaching from the plastic particles is up to five times higher in natural seawater than in artificial seawater, and this can have an effect in the toxicological studies derived. Indeed, we observe different effects in the development of C. intestinalis when using leachates in natural or artificial sea water. We also observe that not all artificial sea waters are suitable for studying the development of the tunicate C. intestinalis. Conclusions Our results show that, at least in this case, both types of water are not equivalent to produce plastic leachaetes and suggest that precaution should be taken when conclusions are derived from results obtained in artificial sea water.

With the continuous development of experimental biology, the limitations of commonly utilized model organisms are becoming increasingly apparent. Discrepancies between research conducted on laboratory animals and humans significantly impede the translational application of findings derived from animal experiments. This review introduces ascidian Ciona intestinalis as a novel model organism, an invertebrate that is evolutionarily closest to vertebrates and is a sister group to vertebrates. The review summarizes recent research progress on Ciona intestinalis in various fields to illustrate the significant advantages and promising application prospects of it as a model organism. The research progress outlined in the review mainly encompasses: (1) The whole-genome sequencing of Ciona intestinalis has been determined and numerous related databases have been established. Various embryonic gene editing technologies have been successfully applied, making it an animal model easy to manipulate genetically and study the functions and interactions of target genes visually. (2) In the field of neurobiology, Ciona intestinalis boasts a central nervous system structure similar to that of vertebrates and possesses numerous homologous neuropeptides and hormone molecules. These features grant it an edge in exploring the mechanisms and functional evolution of endocrine and neuroendocrine-related molecules. Additionally, the sensitivity and habituation of its larvae to light stimulation provide an avenue for exploring mechanisms related to behavioral plasticity. (3) In the field of immunology, Ciona intestinalis possesses a mature innate immune system and has evolved precursor genes to the adaptive immune system, with a relatively simple coding of immune-related genes. These features make it an exemplary model organism for immunological studies. (4) In the field of developmental biology, many studies have focused on the notochord development process in Ciona intestinalis and the regulatory mechanisms of gene expression within it, indicating common evolutionary developmental strategies among chordates. Additionally, insights into its heart development also significantly enhance our comprehension on the genetic network of human heart development. (5) In medical research, the ability of Ciona intestinalis to regenerate its neural complex and siphon, as well as the resilience of its heart to recover contractile function from substantial damage, renders it a valuable animal model for the study of regeneration and heart injury. It also has unique advantages as a research model for Alzheimer's disease and new drug development. Furthermore, its brief five-month lifespan facilitates the observation and recording of the entire aging process and the exploration of the effects of various factors on aging. In summary, this review aims to demonstrate that Ciona intestinalis stands out as a model organism with unique attributes and is expected to play a significant role in a wider range of scientific research areas.

Abstract Tunicates are evolutionary model organisms bridging the gap between vertebrates and invertebrates. A genomic sequence in Ciona intestinalis (CiOX) shows high similarity to vertebrate orexin receptors and protostome allatotropin receptors (ATR). Here, molecular phylogeny suggested that CiOX is divergent from ATRs and human orexin receptors (hOX1/2). However, CiOX appears closer to hOX1/2 than to ATR both in terms of sequence percent identity and in its modelled binding cavity, as suggested by molecular modelling. CiOX was heterologously expressed in a recombinant HEK293 cell system. Human orexins weakly but concentration-dependently activated its Gq signalling (Ca2+ elevation), and the responses were inhibited by the non-selective orexin receptor antagonists TCS 1102 and almorexant, but only weakly by the OX1-selective antagonist SB-334867. Furthermore, the 5-/6-carboxytetramethylrhodamine (TAMRA)-labelled human orexin-A was able to bind to CiOX. Database mining was used to predict a potential endogenous C. intestinalis orexin peptide (Ci-orexin-A). Ci-orexin-A was able to displace TAMRA-orexin-A, but not to induce any calcium response at the CiOX. Consequently, we suggested that the orexin signalling system is conserved in Ciona intestinalis, although the relevant peptide-receptor interaction was not fully elucidated.

Pneumatosis cystoides intestinalis, or intestinal emphysema, is a condition characterized by the presence of multiple cystic structures within the gut wall and on the serosal surface of the intestine. Intestinal emphysema represents an accidental finding in swine, although it can be clinically relevant in humans. Its etiology is unknown, and many theories have been proposed. Among them, a bacterial etiology is considered the most likely. Therefore, in this study, the V3-V4 region of the 16S rRNA gene was sequenced from 19 swine ileal tracts, 12 with intestinal emphysema and 7 without lesions, to detect a possible bacterial agent. In parallel, prevalence was estimated. Escherichia–Shigella (13.15%), Clostridium_sensu_stricto_1; s__uncultured_bacterium (7.09%), and Fusobacterium; s_uncultured bacterium (6.60%) were the most abundant species identified. No statistically relevant differences were observed between the pathological and physiological groups. Prevalence ranged from 1.25 to 5.12% depending on the batch. Our results suggest that the gut wall bacterial microbiota greatly match the normal gut microbiota, and that the etiological agent of intestinal emphysema may be (1) undetectable due to the chronicity of the lesions, (2) not considered statistically relevant in comparing the two groups (p < 0.05) and likewise in causing lesions, and (3) undetectable due to contamination. Regarding prevalence, the condition is moderately frequent. [ABSTRACT FROM AUTHOR]

With the continuous development of experimental biology, the limitations of commonly utilized model organisms are becoming increasingly apparent. Discrepancies between research conducted on laboratory animals and humans significantly impede the translational application of findings derived from animal experiments. This review introduces ascidian Ciona intestinalis as a novel model organism, an invertebrate that is evolutionarily closest to vertebrates and is a sister group to vertebrates. The review summarizes recent research progress on Ciona intestinalis in various fields to illustrate the significant advantages and promising application prospects of it as a model organism. The research progress outlined in the review mainly encompasses: (1) The whole-genome sequencing of Ciona intestinalis has been determined and numerous related databases have been established. Various embryonic gene editing technologies have been successfully applied, making it an animal model easy to manipulate genetically and study the functions and interactions of target genes visually. (2) In the field of neurobiology, Ciona intestinalis boasts a central nervous system structure similar to that of vertebrates and possesses numerous homologous neuropeptides and hormone molecules. These features grant it an edge in exploring the mechanisms and functional evolution of endocrine and neuroendocrine-related molecules. Additionally, the sensitivity and habituation of its larvae to light stimulation provide an avenue for exploring mechanisms related to behavioral plasticity. (3) In the field of immunology, Ciona intestinalis possesses a mature innate immune system and has evolved precursor genes to the adaptive immune system, with a relatively simple coding of immune-related genes. These features make it an exemplary model organism for immunological studies. (4) In the field of developmental biology, many studies have focused on the notochord development process in Ciona intestinalis and the regulatory mechanisms of gene expression within it, indicating common evolutionary developmental strategies among chordates. Additionally, insights into its heart development also significantly enhance our comprehension on the genetic network of human heart development. (5) In medical research, the ability of Ciona intestinalis to regenerate its neural complex and siphon, as well as the resilience of its heart to recover contractile function from substantial damage, renders it a valuable animal model for the study of regeneration and heart injury. It also has unique advantages as a research model for Alzheimer's disease and new drug development. Furthermore, its brief five-month lifespan facilitates the observation and recording of the entire aging process and the exploration of the effects of various factors on aging. In summary, this review aims to demonstrate that Ciona intestinalis stands out as a model organism with unique attributes and is expected to play a significant role in a wider range of scientific research areas. [ABSTRACT FROM AUTHOR]

The pharyngeal endoderm, an innovation of deuterostome ancestors, contributes to pharyngeal development by influencing the patterning and differentiation of pharyngeal structures in vertebrates; however, the evolutionary origin of the pharyngeal organs in vertebrates is largely unknown. The endostyle, a distinct pharyngeal organ exclusively present in basal chordates, represents a good model for understanding pharyngeal organ origins. Using Stereo-seq and single-cell RNA sequencing, we constructed aspatially resolved single-cell atlas for the endostyle of the ascidian Styela clava. We determined the cell composition of the hemolymphoid region, which illuminates a mixed ancestral structure for the blood and lymphoid system. In addition, we discovered a cluster of hair cell-like cells in zone 3, which has transcriptomic similarity with the hair cells of the vertebrate acoustico-lateralis system. These findings reshape our understanding of the pharynx of the basal chordate and provide insights into the evolutionary origin of multiplexed pharyngeal organs. [ABSTRACT FROM AUTHOR]

Ciona intestinalis is an invertebrate animal model system that is well characterized and has many advantages for the study of cardiovascular biology. The regulatory mechanisms of cardiac myocyte proliferation in Ciona are intriguing since regeneration of functional tissue has been demonstrated in other organs of Ciona in response to injury. To identify genes that are differentially expressed in response to Ciona cardiac injury, microarray analysis was conducted on RNA from adult Ciona hearts with normal or damaged myocardium. After a 24- or 48- h recovery period, total RNA was isolated from damaged and control hearts. Initial results indicate significant changes in gene expression in hearts damaged by ligation in comparison to control hearts. Ligation injury shows differential expression of 223 genes as compared to control with limited false discovery (5.8%). Among these 223 genes, 117 have known human orthologs of which 68 were upregulated and 49 were downregulated. Notably, Fgf9/16/20, insulinlike growth factor binding protein and Ras-related protein Rab11b were significantly upregulated in injured hearts, whereas expression of a junctophilin ortholog was decreased. Histological analyses of injured myocardium were conducted in parallel to the microarray study which revealed thickened myocardium in injured hearts. Taken together, these studies will connect differences in gene expression to cellular changes in the myocardium of Ciona, which will help to promote further investigations into the regulatory mechanisms of cardiac myocyte proliferation across chordates. [ABSTRACT FROM AUTHOR]

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

Background Plastic contamination is one of the concerns of our age. With more than 150 million tons of plastic floating in the oceans, and a further 8 million tons arriving to the water each year, in recent times the scientific community has been studying the effects these plastics have on sea life both in the field and with experimental approaches. Laboratory based studies have been using both natural sea water and artificial sea water for testing various aspects of plastic contamination, including the study of chemicals leached from the plastic particles to the water. We set out to test this equivalence, looking at the leaching of heavy metals form plastic particles. Methods We obtained leachates of PVC plastic pre-production nurdles both in natural and artificial sea water and determined the elements in excess from untreated water by Inductively coupled plasma – optical emission spectrometry. We then used these different leachates to assess developmental success in the tunicate Ciona intestinalis by treating fertilised eggs through their development to hatched larvae. Results Here we report that chemical analysis of PVC plastic pre-production pellet leachates shows a different composition in natural and artificial sea water. We find that the Zn leaching from the plastic particles is reduced up to five times in artificial sea water, and this can have an effect in the toxicological studies derived. Indeed, we observe different effects in the development of C. intestinalis when using leachates in natural or artificial sea water. We also observe that not all artificial sea waters are suitable for studying the development of the tunicarte C. intestinalis. Conclusions Our results show that, at least in this case, both types of water are not equivalent to produce plastic leachaetes and suggest that precaution should be taken when conclusions are derived from results obtained in artificial sea water.

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

The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) is a membrane protein containing a voltage-sensing domain (VSD) that is homologous to VSDs from voltage-gated ion channels responsible for cellular excitability. Previously published crystal structures of Ci-VSD in putative resting and active conformations suggested a helical-screw voltage sensing mechanism in which the S4 helix translocates and rotates to enable exchange of salt-bridge partners, but the microscopic details of the transition between the resting and active conformations remained unknown. Here, by combining extensive molecular dynamics simulations with a recently developed computational framework based on dynamical operators, we elucidate the microscopic mechanism of the resting-active transition at physiological membrane potential. Sparse regression reveals a small set of coordinates that distinguish intermediates that are hidden from electrophysiological measurements. The intermediates arise from a noncanonical helical-screw mechanism in which translocation, rotation, and side-chain movement of the S4 helix are only loosely coupled. These results provide insights into existing experimental and computational findings on voltage sensing and suggest ways of further probing its mechanism. Understanding the molecular basis of voltage-sensing is of great importance in biology. Here, the authors use computational analysis and simulations to reveal atomic level insights into the mechanism of an isolated voltage-sensing domain. [ABSTRACT FROM AUTHOR]

The majority of voltage-gated ion channels contain a defined voltage-sensing domain and a pore domain composed of highly conserved amino acid residues that confer electrical excitability via electromechanical coupling. In this sense, the voltage-gated proton channel (Hv1) is a unique protein in that voltage-sensing, proton permeation and pH-dependent modulation involve the same structural region. In fact, these processes synergistically work in concert, and it is difficult to separate them. To investigate the process of Hv1 voltage sensor trapping, we follow voltage-sensor movements directly by leveraging mutations that enable the measurement of Hv1 channel gating currents. We uncover that the process of voltage sensor displacement is due to two driving forces. The first reveals that mutations in the selectivity filter (D160) located in the S1 transmembrane interact with the voltage sensor. More hydrophobic amino acids increase the energy barrier for voltage sensor activation. On the other hand, the effect of positive charges near position 264 promotes the formation of salt bridges between the arginines of the voltage sensor domain, achieving a stable conformation over time. Our results suggest that the activation of the Hv1 voltage sensor is governed by electrostatic–hydrophobic interactions, and S4 arginines, N264 and selectivity filter (D160) are essential in the Ciona-Hv1 to understand the trapping of the voltage sensor. [ABSTRACT FROM AUTHOR]

The S4 segment of voltage-sensing domains (VSDs) directly responds to voltage changes by reorienting within the electric field as a permion. A narrow hydrophobic "gasket" or charge transfer center at the core of most VSDs focuses the electric field into a narrow region and catalyzes the sequential and reversible translocation of S4 positive gating charge residues across the electric field while preventing the permeation of physiological ions. Mutating specific S4 gating charges can cause ionic leak currents through the VSDs. These gating pores or omega currents play important pathophysiological roles in many diseases of excitability. Here, we show that mutating D129, a key countercharge residue in the Ciona intestinalis voltagesensing phosphatase (Ci-VSP), leads to the generation of unique anionic omega currents. Neutralizing D129 causes a dramatic positive shift of activation, facilitates the formation of a continuous water path through the VSD, and creates a positive electrostatic potential landscape inside the VSD that contributes to its unique anionic selectivity. Increasing the population or dwell time of the conducting state by a high external pH or an engineered Cd2+ bridge markedly increases the current magnitude. Our findings uncover a new role of countercharge residues in the impermeable VSD of Ci-VSP and offer insights into mechanisms of the conduction of anionic omega currents linked to countercharge residue mutations. [ABSTRACT FROM AUTHOR]

Background: We aimed to determine the prevalence of Giardia intestinalis and Cryptosporidium spp. in patients who admitted hospital with diarrhea and to gain information about the transmission of these parasites in Ağrı, Türkiye. Methods: This study included 184 patients who applied to Ağrı-Diyadin State Hospital, Türkiye in 2022. The immunochromatographic card test was used for detection of the G. intestinalis and Cryptosporidium spp. Nested PCR-RFLP analysis of the COWP gene and sequence analysis of the gp60 gene were used to genotype and subtype Cryptosporidium spp., whereas Nested PCR and sequence analyses of ß-giardin gene were used genotype G. intestinalis. Results: Of the 184 stool specimens examined, 12 (14.29%) and 7 (3.80%) were positive for G. intestinalis and Cryptosporidium spp., respectively. The Cryptosporidium species were identified as C. parvum belonging to the IId subtype family. The G. intestinalis were identified assemblages A. Conclusion: Assemblage A, which is associated with diarrhea, is responsible for giardiasis and C. parvum IId subtype, often found in sheep, goats and cattle, is responsible for cryptosporidiosis in Ağrı, Türkiye. [ABSTRACT FROM AUTHOR]

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In 1830, Cuvier and Geoffroy Saint‐Hilaire confronted each other in a famous debate on the unity of the animal kingdom, which permeated the zoology of the 19th century. From that time, a growing number of naturalists attempted to understand the large‐scale relationships among animals. And among all the questions, that of the origin of vertebrates was one of the most controversial. Analytical methods based on comparative anatomy, embryology and paleontology were developed to identify convincing homologies that would reveal a logical sequence of events for the evolution of an invertebrate into the first vertebrate. Within this context, several theories have clashed on the question of the identity of the ancestor of vertebrates. Among the proposals, a group of rather discrete organisms, the ascidians, played a central role. Because he had discovered an ascidian with a particularly atypical larval development, the Molgula, Henri de Lacaze‐Duthiers, a rigorous and meticulous naturalist, became involved in the ascidian hypothesis. While the visionary mind of Lacaze‐Duthiers led him to establish a particularly innovative methodology and the first marine biology station in Europe, at Roscoff, the tailless tadpole of the Molgula prevented him from recognizing the ancestor of vertebrates. This old 19th century story echoes the ever‐present questions driving the field of Eco‐Evo‐Devo. Research Highlights: In the 19th century, ascidians were important in the debate over vertebrates' origins.Henri de Lacaze‐Duthiers discovered Molgula and its tailless tadpole and this prevented him from recognizing the relationships between ascidians and vertebrates.This old story echoes the ever‐present questions driving Eco‐Evo‐Devo. [ABSTRACT FROM AUTHOR]

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

Ciona intestinalis is an invertebrate animal model system that is well characterized and has many advantages for the study of cardiovascular biology. The regulatory mechanisms of cardiac myocyte proliferation in Ciona are intriguing since regeneration of functional tissue has been demonstrated in other organs of Ciona in response to injury. To identify genes that are differentially expressed in response to Ciona cardiac injury, microarray analysis was conducted on RNA from adult Ciona hearts with normal or damaged myocardium. After a 24- or 48-h recovery period, total RNA was isolated from damaged and control hearts. Initial results indicate significant changes in gene expression in hearts damaged by ligation in comparison to control hearts. Ligation injury shows differential expression of 223 genes as compared to control with limited false discovery (5.8%). Among these 223 genes, 117 have known human orthologs of which 68 were upregulated and 49 were downregulated. Notably, Fgf9/16/20, insulin-like growth factor binding protein and Ras-related protein Rab11b were significantly upregulated in injured hearts, whereas expression of a junctophilin ortholog was decreased. Histological analyses of injured myocardium were conducted in parallel to the microarray study which revealed thickened myocardium in injured hearts. Taken together, these studies will connect differences in gene expression to cellular changes in the myocardium of Ciona, which will help to promote further investigations into the regulatory mechanisms of cardiac myocyte proliferation across chordates.

Abstract Background Unidirectional regeneration in the basal chordate Ciona intestinalis involves the proliferation of adult stem cells residing in the branchial sac vasculature and the migration of progenitor cells to the site of distal injury. However, after the Ciona body is bisected, regeneration occurs in the proximal but not in the distal fragments, even if the latter include a part of the branchial sac with stem cells. A transcriptome was sequenced and assembled from the isolated branchial sacs of regenerating animals, and the information was used to provide insights into the absence of regeneration in distal body fragments. Results We identified 1149 differentially expressed genes, which were separated into two major modules by weighted gene correlation network analysis, one consisting of mostly upregulated genes correlated with regeneration and the other consisting of only downregulated genes associated with metabolism and homeostatic processes. The hsp70, dnaJb4, and bag3 genes were among the highest upregulated genes and were predicted to interact in an HSP70 chaperone system. The upregulation of HSP70 chaperone genes was verified and their expression confirmed in BS vasculature cells previously identified as stem and progenitor cells. siRNA-mediated gene knockdown showed that hsp70 and dnaJb4, but not bag3, are required for progenitor cell targeting and distal regeneration. However, neither hsp70 nor dnaJb4 were strongly expressed in the branchial sac vasculature of distal fragments, implying the absence of a stress response. Heat shock treatment of distal body fragments activated hsp70 and dnaJb4 expression indicative of a stress response, induced cell proliferation in branchial sac vasculature cells, and promoted distal regeneration. Conclusions The chaperone system genes hsp70, dnaJb4, and bag3 are significantly upregulated in the branchial sac vasculature following distal injury, defining a stress response that is essential for regeneration. The stress response is absent from distal fragments, but can be induced by a heat shock, which activates cell division in the branchial sac vasculature and promotes distal regeneration. This study demonstrates the importance of a stress response for stem cell activation and regeneration in a basal chordate, which may have implications for understanding the limited regenerative activities in other animals, including vertebrates.

Flagellar motility in sperm is activated and regulated by factors related to the eggs at fertilization. In the ascidian Ciona intestinalis, a sulfated steroid called the SAAF (sperm activating and attracting factor) induces both sperm motility activation and chemotaxis. Cyclic AMP (cAMP) is one of the most important intracellular factors in the sperm signaling pathway. Adenylyl cyclase (AC) is the key enzyme that synthesizes cAMP at the onset of the signaling pathway in all cellular functions. We previously reported that both transmembrane AC (tmAC) and soluble AC (sAC) play important roles in sperm motility in Ciona. The tmAC plays a major role in the SAAF-induced activation of sperm motility. On the other hand, sAC is involved in the regulation of flagellar beat frequency and the Ca2+-dependent chemotactic movement of sperm. In this study, we focused on the role of sAC in the regulation of flagellar motility in Ciona sperm chemotaxis. The immunochemical analysis revealed that several isoforms of sAC protein were expressed in Ciona sperm, as reported in mammals and sea urchins. We demonstrated that sAC inhibition caused strong and transient asymmetrization during the chemotactic turn, and then sperm failed to turn toward the SAAF. In addition, real-time Ca2+ imaging in sperm flagella revealed that sAC inhibition induced an excessive and prolonged Ca2+ influx to flagella. These results indicate that sAC plays a key role in sperm chemotaxis by regulating the clearance of [Ca2+]i and by modulating Ca2+-dependent flagellar waveform conversion. [ABSTRACT FROM AUTHOR]

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

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

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

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

Background and Aim: Giardia intestinalis is one of the most prevalent intestinal parasites in humans and animals, and children in close contact with livestock are particularly at risk of infection. This study aimed to detect assemblages of G. intestinalis and determine the origin of zoonotic transmission of Giardia in children and calves in different parts of Babylon province, Iraq. Materials and Methods: One hundred stool samples from children (68 boys and 32 girls) and 100 fecal samples from calves (46 males and 54 females) of different ages were randomly collected. Molecular techniques were used to estimate the prevalence of G. intestinalis in children and calves. A nested polymerase chain reaction (PCR) was performed by targeting the triose phosphate isomerase gene in the samples to detect G. intestinalis assemblages. Results: The overall rates of infection with G. intestinalis in children and calves were 21% and 34%, respectively, using the conventional microscopic method. The results illustrated that 61.90% (13/21) and 38.09% (8/21) of positive samples from children were allocated to assemblages A and B, respectively (p > 0.05). In calves, assemblages A and B were detecte in 82.35% (28/34) and 17.64% (6/34) of positive samples from calves, respectively (p = 0.001). Ten PCR products were sequenced and submitted to the GenBank database. Phylogenetic analysis detected five human sequences each belonging to G. intestinalis assemblages A (OM850335-OM850339) and B (OM850340-OM850344). Similarly, five calf sequences each belonged to G. intestinalis assemblages A (ON75756-ON757660) and B (ON757661-ON757665). Conclusion: The detection of large numbers of G. intestinalis assemblage A in both humans and cattle indicated that cattle could be a main source of zoonotic G. intestinalis infection in children in Babylon province, Iraq. [ABSTRACT FROM AUTHOR]

While thousands of environmental metagenomes have been mined for the presence of novel biosynthetic gene clusters, such computational predictions do not provide evidence of their in vivo biosynthetic functionality. Using fluorescent in situ enzyme assay targeting carrier proteins common to polyketide (PKS) and nonribosomal peptide synthetases (NRPS), we applied fluorescence-activated cell sorting to tunicate microbiome to enrich for microbes with active secondary metabolic capabilities. Single-cell genomics uncovered the genetic basis for a wide biosynthetic diversity in the enzyme-active cells and revealed a member of marine Oceanospirillales harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. This demonstrates activity-guided single-cell genomics as a tool to guide novel biosynthetic discovery.

Background: Unidirectional regeneration in the basal chordate Ciona intestinalis involves the proliferation of adult stem cells residing in the branchial sac vasculature and the migration of progenitor cells to the site of distal injury. However, after the Ciona body is bisected, regeneration occurs in the proximal but not in the distal fragments, even if the latter include a part of the branchial sac with stem cells. A transcriptome was sequenced and assembled from the isolated branchial sacs of regenerating animals, and the information was used to provide insights into the absence of regeneration in distal body fragments. Results: We identified 1149 differentially expressed genes, which were separated into two major modules by weighted gene correlation network analysis, one consisting of mostly upregulated genes correlated with regeneration and the other consisting of only downregulated genes associated with metabolism and homeostatic processes. The hsp70, dnaJb4, and bag3 genes were among the highest upregulated genes and were predicted to interact in an HSP70 chaperone system. The upregulation of HSP70 chaperone genes was verified and their expression confirmed in BS vasculature cells previously identified as stem and progenitor cells. siRNA-mediated gene knockdown showed that hsp70 and dnaJb4, but not bag3, are required for progenitor cell targeting and distal regeneration. However, neither hsp70 nor dnaJb4 were strongly expressed in the branchial sac vasculature of distal fragments, implying the absence of a stress response. Heat shock treatment of distal body fragments activated hsp70 and dnaJb4 expression indicative of a stress response, induced cell proliferation in branchial sac vasculature cells, and promoted distal regeneration. Conclusions: The chaperone system genes hsp70, dnaJb4, and bag3 are significantly upregulated in the branchial sac vasculature following distal injury, defining a stress response that is essential for regeneration. The stress response is absent from distal fragments, but can be induced by a heat shock, which activates cell division in the branchial sac vasculature and promotes distal regeneration. This study demonstrates the importance of a stress response for stem cell activation and regeneration in a basal chordate, which may have implications for understanding the limited regenerative activities in other animals, including vertebrates. [ABSTRACT FROM AUTHOR]

Ultrasonic antifouling devices are installed globally on a variety of vessel types and are marketed as an environmentally friendly method for biofouling control. The aim of this study was to examine the effects of ultrasound on adults of three species of common solitary ascidians (Ciona intestinalis, Ascidiella aspersa and Styela plicata). After a brief (10 s) exposure to two ultrasound frequencies (30 and 35 kHz), alterations in the frequency of siphon closing events and the length of time the siphons remained closed/open were observed. The results revealed that ascidians are able to perceive ultrasound, showing frequency-dependent behavioural responses that vary depending on the species and size of individuals involving both tactile receptors and an acoustic system homologous to the vertebrate inner ear. Continuous (5 h) 30 kHz exposure caused other types of responses, the most interesting of which was the long-term opening of the oral siphon, indicating a lack of reactivity to mechanical stimuli. This effect suggests a stress condition that could lead to increased vulnerability to predators and filter-feeding impairment. Therefore, knowledge of the acoustic sensitivity of sessile marine species appears to be essential for better understanding the potential effects of noise pollution on marine ecosystems. [ABSTRACT FROM AUTHOR]

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

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]

Continual application of chemical insecticides causes a lot of environmental and health problems. Mosquitoes as vectors of diseases develop resistance toward them. Finding out safe alternatives is an urgent need. Algal extracts are a promising alternative approach as they are biodegradable and eco-friendly. The present study assessed the larvicidal efficacy of three ethanolic algal extracts (Chaetomorpha linum, Ulva intestinalis and Sargassum dentifolium) belonging to two different algal divisions against the 3rd larval instar of Culex pipiens L. (Diptera: Culicidae) under laboratory conditions. Toxicological studies showed that three species of brown and green algal extracts exhibited good larvicidal activity. Percentages of mortality increased with increasing concentrations of all algal extracts. Based on the LC50 values, C. linum (Chlorophyta) exhibited the highest larvicidal potency among the used extracts, followed by U. intestinalis (Chlorophyta) and S. dentifolium (Phaeophyta) with LC50, s of 224.45, 231.06 and 241.79 ppm, respectively, upon 48 hours exposure. GC-Mass analysis of the tested extracts revealed that the most abundant constituents in the ethanolic extracts of C. linum, U. intestinalis and S. dentifolium were palmitic acid (17.42 %), linolenic acid (23.93%) and Di-noctyl phthalate (19.03 %), respectively. The application of LC50 values of tested ethanolic algal extracts induced many biochemical and morphological aberrations in the treated C. pipiens larvae, compared to the untreated larvae. The biochemical changes were tracked through the analysis of the insect's main metabolites (carbohydrates, lipids and proteins), in addition to measuring the changes in acetylcholine esterase, GST, a- and ß-esterases after algal extracts treatment. Generally, variations were recorded in the lipid and carbohydrate after treatment with C. linum. Moreover, C. linum inhibited the activity of a and ß esterases enzymes to a great extent, compared to the untreated. Many histological abnormalities were noticed in the treated larvae. Overall, these results demonstrated that ethanolic algal extracts of C. linum, U. intestinalis and S. dentifolium might be used to control C. pipiens mosquitoes without harming humans or the environment. Hence, they could be incorporated into integrated vector management programs. [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]

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]