Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species.

Genome structural variations within species are rare. How selective constraints preserve gene order and chromosome structure is a central question in evolutionary biology that remains unsolved. Our sequencing of several genomes of the appendicularian tunicate Oikopleura dioica around the globe reveals extreme genome scrambling caused by thousands of chromosomal rearrangements, although showing no obvious morphological differences between these animals. The breakpoint accumulation rate is an order of magnitude higher than in ascidian tunicates, nematodes, Drosophila, or mammals. Chromosome arms and sex-specific regions appear to be the primary unit of macrosynteny conservation. At the microsyntenic level, scrambling did not preserve operon structures, suggesting an absence of selective pressure to maintain them. The uncoupling of the genome scrambling with morphological conservation in O. dioica suggests the presence of previously unnoticed cryptic species and provides a new biological system that challenges our previous vision of speciation in which similar animals always share similar genome structures.

Specification of distinct cell types in a sensory-adhesive organ important for metamorphosis in tunicate larvae.

The papillae of tunicate larvae contribute sensory, adhesive, and metamorphosis-regulating functions that are crucial for the biphasic lifestyle of these marine, non-vertebrate chordates. We have identified additional molecular markers for at least 5 distinct cell types in the papillae of the model tunicate Ciona, allowing us to further study the development of these organs. Using tissue-specific CRISPR/Cas9-mediated mutagenesis and other molecular perturbations, we reveal the roles of key transcription factors and signaling pathways that are important for patterning the papilla territory into a highly organized array of different cell types and shapes. We further test the contributions of different transcription factors and cell types to the production of the adhesive glue that allows for larval attachment during settlement, and to the processes of tail retraction and body rotation during metamorphosis. With this study, we continue working towards connecting gene regulation to cellular functions that control the developmental transition between the motile larva and sessile adult of Ciona.

Genome-wide analysis of early vascular tunic repair and regeneration for Botrylloides digenesis reveals striking similarities to human wound healing.

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

Early transcriptional similarities between two distinct neural lineages during ascidian embryogenesis.

In chordates, the central nervous system arises from precursors that have distinct developmental and transcriptional trajectories. Anterior nervous systems are ontogenically associated with ectodermal lineages while posterior nervous systems are associated with mesoderm. Taking advantage of the well-documented cell lineage of ascidian embryos, we asked to what extent the transcriptional states of the different neural lineages become similar during the course of progressive lineage restriction. We performed single-cell RNA sequencing (scRNA-seq) analyses on hand-dissected neural precursor cells of the two distinct lineages, together with those of their sister cell lineages, with a high temporal resolution covering five successive cell cycles from the 16-cell to neural plate stages. A transcription factor binding site enrichment analysis of neural specific genes at the neural plate stage revealed limited evidence for shared transcriptional control between the two neural lineages, consistent with their different ontogenies. Nevertheless, PCA analysis and hierarchical clustering showed that, by neural plate stages, the two neural lineages cluster together. Consistent with this, we identified a set of genes enriched in both neural lineages at the neural plate stage, including miR-124, Celf3.a, Zic.r-b, and Ets1/2. Altogether, the current study has revealed genome-wide transcriptional dynamics of neural progenitor cells of two distinct developmental origins. Our scRNA-seq dataset is unique and provides a valuable resource for future analyses, enabling a precise temporal resolution of cell types not previously described from dissociated embryos.

Molecular control of cellulosic fin morphogenesis in ascidians.

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

Deep quantitative proteomics of North American Pacific coast star tunicate (Botryllus schlosseri).

Botryllus schlosseri, is a model marine invertebrate for studying immunity, regeneration, and stress-induced evolution. Conditions for validating its predicted proteome were optimized using nanoElute® 2 deep-coverage LCMS, revealing up to 4930 protein groups and 20,984 unique peptides per sample. Spectral libraries were generated and filtered to remove interferences, low-quality transitions, and only retain proteins with >3 unique peptides. The resulting DIA assay library enabled label-free quantitation of 3426 protein groups represented by 22,593 unique peptides. Quantitative comparisons of single systems from a laboratory-raised with two field-collected populations revealed (1) a more unique proteome in the laboratory-raised population, and (2) proteins with high/low individual variabilities in each population. DNA repair/replication, ion transport, and intracellular signaling processes were distinct in laboratory-cultured colonies. Spliceosome and Wnt signaling proteins were the least variable (highly functionally constrained) in all populations. In conclusion, we present the first colonial tunicate's deep quantitative proteome analysis, identifying functional protein clusters associated with laboratory conditions, different habitats, and strong versus relaxed abundance constraints. These results empower research on B. schlosseri with proteomics resources and enable quantitative molecular phenotyping of changes associated with transfer from in situ to ex situ and from in vivo to in vitro culture conditions.

Dopamine-Derived Guanidine Alkaloids from a Didemnidae Tunicate: Isolation, Synthesis, and Biological Activities.

Mellpaladines A-C (1-3) and dopargimine (4) are dopamine-derived guanidine alkaloids isolated from a specimen of Palauan Didemnidae tunicate as possible modulators of neuronal receptors. In this study, we isolated the dopargimine derivative 1-carboxydopargimine (5), three additional mellpaladines D-F (6-8), and serotodopalgimine (9), along with a dimer of serotonin, 5,5'-dihydroxy-4,4'-bistryptamine (10). The structures of these compounds were determined based on spectrometric and spectroscopic analyses. Compound 4 and its congeners dopargine (11), nordopargimine (15), and 2-(6,7-dimethoxy-3,4-dihydroisoquinolin-1-yl)ethan-1-amine (16) were synthetically prepared for biological evaluations. The biological activities of all isolated compounds were evaluated in comparison with those of 1-4 using a mouse behavioral assay upon intracerebroventricular injection, revealing key functional groups in the dopargimines and mellpaladines for in vivo behavioral toxicity. Interestingly, these alkaloids also emerged during a screen of our marine natural product library aimed at identifying antiviral activities against dengue virus, SARS-CoV-2, and vesicular stomatitis Indiana virus (VSV) pseudotyped with Ebola virus glycoprotein (VSV-ZGP).

An ascidian Polycarpa aurata-derived pan-inhibitor against coronaviruses targeting Mpro.

Coronaviruses (CoVs) are responsible for a wide range of illnesses in both animals and human. The main protease (Mpro) of CoVs is an attractive drug target, owing its critical and highly conserved role in viral replication. Here, we developed and refined an enzymatic technique to identify putative Mpro inhibitors from 189 marine chemicals and 46 terrestrial natural products. The IC50 values of Polycarpine (1a), a marine natural substance we studied and synthesized, are 30.0 ± 2.5 nM for SARS-CoV-2 Mpro and 0.12 ± 0.05 µM for PEDV Mpro. Our research further demonstrated that pretreatment with Polycarpine (1a) inhibited the betacoronavirus SARS-CoV-2 and alphacoronavirus PEDV multiplication in Vero-E6 cells. As a result, Polycarpine (1a), a pan-inhibitor of Mpro, will function as an effective and promising antiviral option to combat CoVs infection and as a foundation for further therapeutic research.

Absolute Configuration of 12S-Deoxynortryptoquivaline from Ascidian-Derived Fungus Aspergillus clavatus Determined by Anisotropic NMR and Chiroptical Spectroscopy.

Tryptoquivalines are highly toxic metabolites initially isolated from the fungus Aspergillus clavatus. The relative and absolute configuration of tryptoquivaline derivates was primarily established by comparison of the chemical shifts, NOE data, and ECD calculations. A de novo determination of the complete relative configuration using NMR spectroscopy was challenging due to multiple spatially separated stereocenters, including one nonprotonated carbon. In this study, we isolated a new tryptoquivaline derivative, 12S-deoxynortryptoquivaline (1), from the marine ascidian-derived fungus Aspergillus clavatus AS-107. The correct assignment of the relative configuration of 1 was accomplished using anisotropic NMR spectroscopy, while the absolute configuration was determined by comparing calculated and experimental ECD spectra. This case study highlights the effectiveness of anisotropic NMR parameters over isotropic NMR parameters in determining the relative configuration of complex natural products without the need for crystallization.

A Survey of Recently Discovered Naturally Occurring Organohalogen Compounds.

The discovery of naturally occurring organohalogen compounds has increased astronomically in the 55 years since they were first discovered─from fewer than 50 in 1968 to a combined 7,958 described examples in three comprehensive reviews. The present survey, which covers the period 2021-2023, brings the number of known natural organohalogens to approximately 8,400. The organization is according to species origin, and coverage includes marine and terrestrial plants, fungi, bacteria, marine sponges, corals, cyanobacteria, tunicates, and other marine organisms.

Discovery of the Lipopeptides Albubactins A-H from Streptomyces albidoflavus RKJM0023 via Chemical Elicitation with Rhamnolipids and Synthesis of Albubactin A.

The marine tunicate-derived Streptomyces albidoflavus RKJM0023 was cultured in the presence of a rhamnolipid mixture in an effort to elicit the production of silent natural products. MS/MS-based molecular networking analysis enhanced with nonparametric statistics highlighted the upregulation of a molecular cluster (Kruskal-Wallis p = 1.6 e-6 for 1) in which no MS/MS features had library matches. Targeted isolation of these features resulted in the discovery of nine new N-acylated lipopeptides, albubactins A-H (1-8) each containing a unique glutamine tripeptide and a C-terminal ethyl ester moiety. Three related albubactin acids A-C (9-11) lacking the ethyl ester were also identified. NMR spectroscopy and UPLC-HR-ESI-MS/MS demonstrated that the albubactins were obtained as mixtures that shared a common m/z and differed only in their acylated terminal groups. Due to the complex spectroscopic elucidation with many overlapping shifts, a total synthesis of albubactin A (1) was completed and used to determine the absolute configuration of the new albubactins.

Adrenoreceptor phylogeny and novel functions of nitric oxide in ascidian immune cells.

Nitric oxide (NO) is a simple molecule involved in many biological processes and functions in the cardiovascular, neural, and immune systems. In recent years, NO has also been recognized as a crucial messenger in communication between the nervous and immune systems. Together with NO, catecholamines are the main group of neurotransmitters involved in cross-talk between the nervous and immune systems. Catecholamines such as noradrenaline, can act on immune cells through adrenoreceptors (ARs) present on the cell surface, and NO can cross the cell membrane and interact with secondary messengers, modulating catecholamine production. Here, we analyzed the mutual modulation by noradrenaline and NO in Phallusia nigra immune cells for specific subtypes of ARs. We also investigated the involvement of protein kinases A and C as secondary messengers to these specific subtypes of ARs in the adrenergic signaling pathway that culminates in NO modulation, and the phylogenetic distribution of ARs in deuterostome genomes. This analysis provided evidence for single-copy orthologs of α1, α2 and ß-AR in ascidian genomes, suggesting that NO and NA act on a less diverse set of ARs in urochordates. Pharmacological assays showed that high levels of NO can induce ascidian immune cells to produce catecholamines. We also observed that protein kinases A and C are the secondary messengers involved in downstream modulation of NO production through an ancestral ß-AR. Taken together, these results provide new information on NO as a modulator of immune cells, and reveal the molecules involved in the signaling pathway of ARs. The results also indicate that ARs may participate in NO modulation. Finally, our results suggest that the common ancestor of urochordates possessed a less complex system of ARs required for immune action and diverse pharmacological responses, since the α-ARs are phylogenetically more related to D1-receptors than are the ß-ARs.

Regeneration of tunic cuticle is suppressed in edible ascidian Halocynthia roretzi contracting soft tunic syndrome.

Soft tunic syndrome is an infectious disease caused by the flagellate Azumiobodo hoyamushi, which severely damages the aquaculture of the edible ascidian Halocynthia roretzi. Tunic is a cellulosic extracellular matrix entirely covering the body in ascidians and other tunicates, and its dense cuticle layer covers the tunic surface as a physical barrier against microorganisms. When the tunic of intact H. roretzi individuals was cut into strips, electron-dense fibers (DFs) appeared on the cut surface of the tunic matrix and aggregated to regenerate a new cuticular layer in seawater within a few days. DF formation was partially or completely inhibited in individuals with soft tunic syndrome, and DF formation was also inhibited by the presence of some proteases, indicating the involvement of proteolysis in the process of tunic softening as well as cuticle regeneration. Using pure cultures of the causative flagellate A. hoyamushi, the expression of protease genes and secretion of some proteases were confirmed by RNA-seq analysis and a 4-methylcoumaryl-7-amide substrate assay. Some of these proteases may degrade proteins in the tunic matrix. These findings suggest that the proteases of A. hoyamushi is the key to understanding the mechanisms of cuticular regeneration inhibition and tunic softening.

Chemical and Pharmacological Prospection of the Ascidian Cystodytes dellechiajei.

Marine invertebrates are a traditional source of natural products with relevant biological properties. Tunicates are soft-bodied, solitary or colonial, sessile organisms that provide compounds unique in their structure and activity. The aim of this work was to investigate the chemical composition of the ascidian Cystodytes dellechiajei, selected on the basis of a positive result in biological screening for ligands of relevant receptors of the innate immune system, including TLR2, TLR4, dectin-1b, and TREM2. Bioassay-guided screening of this tunicate extract yielded two known pyridoacridine alkaloids, shermilamine B (1) and N-deacetylshermilamine B (2), and a family of methyl-branched cerebrosides (3). Compounds 2 and 3 showed selective binding to TREM2 in a dose-dependent manner. N-deacetylshermilamine B (2), together with its acetylated analogue, shermilamine B (1), was also strongly cytotoxic against multiple myeloma cell lines. TREM2 is involved in immunomodulatory processes and neurodegenerative diseases. N-deacetylshermilamine B (2) is the first example of a polycyclic alkaloid to show an affinity for this receptor.

Dynamics of Chromatin Opening across Larval Development in the Urochordate Ascidian Ciona savignyi.

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

Evidence of extensive multispecies macrofouling in Eckström's topknot Zeugopterus regius (Bonnaterre, 1788), Pleuronectiformes, Scophthalmidae.

Diverse and extensive macrofouling of the left-hand (eyed) side has been observed in multiple films and photographs of different specimens of Eckström's topknot Zeugopterus regius. Identified foulers include macroalgae and tunicates. Photographs of unfouled specimens and preserved juveniles have also been inspected. Macrofouling is not universal in this species; unfouled fish were observed around the strongly tidal British Isles, whereas the worst-fouled topknots were seen in the eutrophic, microtidal northern Adriatic.

Bioactive Molecules from the Innate Immunity of Ascidians and Innovative Methods of Drug Discovery: A Computational Approach Based on Artificial Intelligence.

The study of bioactive molecules of marine origin has created an important bridge between biological knowledge and its applications in biotechnology and biomedicine. Current studies in different research fields, such as biomedicine, aim to discover marine molecules characterized by biological activities that can be used to produce potential drugs for human use. In recent decades, increasing attention has been paid to a particular group of marine invertebrates, the Ascidians, as they are a source of bioactive products. We describe omics data and computational methods relevant to identifying the mechanisms and processes of innate immunity underlying the biosynthesis of bioactive molecules, focusing on innovative computational approaches based on Artificial Intelligence. Since there is increasing attention on finding new solutions for a sustainable supply of bioactive compounds, we propose that a possible improvement in the biodiscovery pipeline might also come from the study and utilization of marine invertebrates' innate immunity.

Spatially resolved single-cell atlas of ascidian endostyle provides insight into the origin of vertebrate pharyngeal organs.

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.

Genomic Resources and Annotations for a Colonial Ascidian, the Light-Bulb Sea Squirt Clavelina lepadiformis.

Ascidian embryos have been studied since the birth of experimental embryology at the end of the 19th century. They represent textbook examples of mosaic development characterized by a fast development with very few cells and invariant cleavage patterns and lineages. Ascidians belong to tunicates, the vertebrate sister group, and their study is essential to shed light on the emergence of vertebrates. Importantly, deciphering developmental gene regulatory networks has been carried out mostly in two of the three ascidian orders, Phlebobranchia and Stolidobranchia. To infer ancestral developmental programs in ascidians, it is thus essential to carry out molecular embryology in the third ascidian order, the Aplousobranchia. Here, we present genomic resources for the colonial aplousobranch Clavelina lepadiformis: a transcriptome produced from various embryonic stages, and an annotated genome. The assembly consists of 184 contigs making a total of 233.6 Mb with a N50 of 8.5 Mb and a L50 of 11. The 32,318 predicted genes capture 96.3% of BUSCO orthologs. We further show that these resources are suitable to study developmental gene expression and regulation in a comparative framework within ascidians. Additionally, they will prove valuable for evolutionary and ecological studies.

The unknown unknown: A framework for assessing environmental DNA assay specificity against unsampled taxa.

Taxon-specific quantitative PCR (qPCR) assays are commonly used for environmental DNA sampling-based inference of animal presence. These assays require thorough validation to ensure that amplification truly indicates detection of the target taxon, but a thorough validation is difficult when there are potentially many non-target taxa, some of which may have incomplete taxonomies. Here, we use a previously published, quantitative model of cross-amplification risk to describe a framework for assessing qPCR assay specificity when there is missing information and it is not possible to assess assay specificity for each individual non-target confamilial. In this framework, we predict assay specificity against unsampled taxa (non-target taxa without sequence data available) using the sequence information that is available for other confamilials. We demonstrate this framework using four case study assays for: (1) An endemic, freshwater arthropod (meltwater stonefly; Lednia tumana), (2) a globally distributed, marine ascidian (Didemnum perlucidum), (3) a continentally distributed freshwater crustacean (virile crayfish; Faxonius virilis, deanae and nais species complex) and (4) a globally distributed freshwater teleost (common carp; Cyprinus carpio and its close relative C. rubrofuscus). We tested the robustness of our approach to missing information by simulating application of our framework for all possible subsamples of 20-all non-target taxa. Our results suggest that the modelling framework results in estimates which are largely concordant with observed levels of cross-amplification risk using all available sequence data, even when there are high levels of data missingness. We explore potential limitations and extensions of this approach for assessing assay specificity and provide users with an R Markdown template for generating reproducible reports to support their own assay validation efforts.