A change in cis-regulatory logic underlying obligate versus facultative muscle multinucleation in chordates.

Vertebrates and tunicates are sister groups that share a common fusogenic factor, Myomaker (Mymk), that drives myoblast fusion and muscle multinucleation. Yet they are divergent in when and where they express Mymk. In vertebrates, all developing skeletal muscles express Mymk and are obligately multinucleated. In tunicates, Mymk is expressed only in post-metamorphic multinucleated muscles, but is absent from mononucleated larval muscles. In this study, we demonstrate that cis-regulatory sequence differences in the promoter region of Mymk underlie the different spatiotemporal patterns of its transcriptional activation in tunicates and vertebrates. Although in vertebrates myogenic regulatory factors (MRFs) such as MyoD1 alone are required and sufficient for Mymk transcription in all skeletal muscles, we show that transcription of Mymk in post-metamorphic muscles of the tunicate Ciona requires the combinatorial activity of MRF, MyoD and Early B-cell Factor (Ebf). This macroevolutionary difference appears to be encoded in cis, likely due to the presence of a putative Ebf-binding site adjacent to predicted MRF binding sites in the Ciona Mymk promoter. We further discuss how Mymk and myoblast fusion might have been regulated in the last common ancestor of tunicates and vertebrates, for which we propose two models.

Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis.

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.

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.

Identification of astrocyte-like cells in an adult ascidian during regeneration of the central nervous system.

The mechanisms underlying regeneration of the central nervous system (CNS) following lesions have been studied extensively in both vertebrate and invertebrate models. To shed light on regeneration, ascidians, a sister group of vertebrates and with remarkable ability to regenerate their brains, constitute an appropriate model system. Glial cells have been implicated in regeneration in vertebrates; however, their role in the adult ascidian CNS regeneration is unknown. A model of degeneration and regeneration using the neurotoxin 3-acetylpyridine (3AP) in the brain of the ascidian Styela plicata was used to identify astrocyte-like cells and investigate their role. We studied the CNS of control ascidians (injected with artificial sea water) and of ascidians whose CNS was regenerating (1 and 10 days after the injection with 3AP). Our results show that the mRNA of the ortholog of glutamine synthetase (GS), a glial-cell marker in vertebrates, is increased during the early stages of regeneration. Confirming the identity of GS, the protein was identified via immunostaining in a cell population during the same regeneration stage. Last, a single ortholog of GS (GSII) is present in ascidian and amphioxus genomes, while two types exist in fungi, some invertebrates, and vertebrates, suggesting that ascidians have lost the GSI type. Taken together, our findings revealed that a cell population expressing glial-cell markers may play a role in regeneration in adult ascidians. This is the first report of astrocyte-like cells in the adult ascidian CNS, and contributes to understanding of the evolution of glial cells among metazoans.

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.

Women in tunicate research: Pioneers of the past and their present legacy.

The search for female scientists who pioneered the research on tunicates is hindered by the tradition of reporting only the first initials of authors' names on scientific publications using only the initials of their first names. While this practice has the theoretical merit of broadening the readership by preventing the possible bias that could be caused by the gender of the author(s) in some of the readers, it rendered the identification of female researchers active in, or before, the first half of the 20th century quite challenging. Sifting through several dozen electronic records, and with the help of references and/or quotes found online, we have stitched together the information that we were able to retrieve on the life of female scientists who authored some of the earliest publications on tunicates, and we have organized them in (approximate) chronological order. We have also compiled brief synopses of the findings of scientists active in the field of tunicate biology in more recent times, and organized them by subdiscipline.

Development and circuitry of the tunicate larval Motor Ganglion, a putative hindbrain/spinal cord homolog.

The Motor Ganglion (MG) is a small collection of neurons that control the swimming movements of the tunicate tadpole larva. Situated at the base of the tail, molecular and functional comparisons suggest that may be a homolog of the spinal cord and/or hindbrain ("rhombospinal" region) of vertebrates. Here we review the most current knowledge of the development, connectivity, functions, and unique identities of the neurons that comprise the MG, drawn mostly from studies in Ciona spp. The simple cell lineages, minimal cellular composition, and comprehensively mapped "connectome" of the Ciona MG all make this an excellent model for studying the development and physiology of motor control in aquatic larvae.

Filter-feeding gelatinous macrozooplankton response to climate change and implications for benthic food supply and global carbon cycle.

It is often suggested that gelatinous zooplankton may benefit from anthropogenic pressures of all kinds and in particular from climate change. Large pelagic tunicates, for example, are likely to be favored over other types of macrozooplankton due to their filter-feeding mode, which gives them access to small preys thought to be less affected by climate change than larger preys. In this study, we provide model-based estimate of potential community changes in macrozooplankton composition and estimate for the first time their effects on benthic food supply and on the ocean carbon cycle under two 21st-century climate-change scenarios. Forced with output from an Earth System Model climate projections, our ocean biogeochemical model simulates a large reduction in macrozooplankton biomass in response to anthropogenic climate change, but shows that gelatinous macrozooplankton are less affected than nongelatinous macrozooplankton, with global biomass declines estimated at -2.8% and -3.5%, respectively, for every 1°C of warming. The inclusion of gelatinous macrozooplankon in our ocean biogeochemical model has a limited effect on anthropogenic carbon uptake in the 21st century, but impacts the projected decline in particulate organic matter fluxes in the deep ocean. In subtropical oligotrophic gyres, where gelatinous zooplankton dominate macrozooplankton, the decline in the amount of organic matter reaching the seafloor is reduced by a factor of 2 when gelatinous macrozooplankton are considered (-17.5% vs. -29.7% when gelatinous macrozooplankton are not considered, all for 2100 under RCP8.5). The shift to gelatinous macrozooplankton in the future ocean therefore buffers the decline in deep carbon fluxes and should be taken into account when assessing potential changes in deep carbon storage and the risks that deep ecosystems may face when confronted with a decline in their food source.

Botryllin, a Novel Antimicrobial Peptide from the Colonial Ascidian Botryllus schlosseri.

By mining the transcriptome of the colonial ascidian Botryllus schlosseri, we identified a transcript for a novel styelin-like antimicrobial peptide, which we named botryllin. The gene is constitutively transcribed by circulating cytotoxic morula cells (MCs) as a pre-propeptide that is then cleaved to mature peptide. The synthetic peptide, obtained from in silico translation of the transcript, shows robust killing activity of bacterial and unicellular yeast cells, causing breakages of both the plasma membrane and the cell wall. Specific monoclonal antibodies were raised against the epitopes of the putative amino acid sequence of the propeptide and the mature peptide; in both cases, they label the MC granular content. Upon MC degranulation induced by the presence of nonself, the antibodies recognise the extracellular nets with entrapped bacteria nearby MC remains. The obtained results suggest that the botryllin gene carries the information for the synthesis of an AMP involved in the protection of B. schlosseri from invading foreign cells.

Searching for Novel Sources of Hydrogen Sulfide Donors: Chemical Profiling of Polycarpa aurata Extract and Evaluation of the Anti-Inflammatory Effects.

Hydrogen sulfide (H2S) is a signaling molecule endogenously produced within mammals' cells that plays an important role in inflammation, exerting anti-inflammatory effects. In this view, the research has shown a growing interest in identifying natural H2S donors. Herein, for the first time, the potential of marine extract as a source of H2S-releasing agents has been explored. Different fractions obtained by the Indonesian ascidian Polycarpa aurata were evaluated for their ability to release H2S in solution. The main components of the most active fraction were then characterized by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and NMR spectroscopy. The ability of this fraction to release H2S was evaluated in a cell-free assay and J774 macrophages by a fluorimetric method, and its anti-inflammatory activity was evaluated in vitro and in vivo by using carrageenan-induced mouse paw edema. The anti-inflammatory effects were assessed by inhibiting the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), and interleukin-6 (IL-6), coupled with a reduction in nitric oxide (NO) and IL-6 levels. Thus, this study defines the first example of a marine source able to inhibit inflammatory responses in vivo through the release of H2S.

Artificial seawater based long-term culture of colonial ascidians.

Tunicates are highly diverse marine invertebrate filter-feeders that are vertebrates' closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually by budding, typically leading to the formation of colonies where animals, called zooids, are interconnected through an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions. To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates. Herein we present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 â€‹km away from their natural habitat. We show that colonies adapt well to in-lab culturing provided that a suitable marine microbiome is present, and that a specific strain can be isolated, propagated and efficiently used for research over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species, such as Botryllus schlosseri, as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.

Central nervous system regeneration in ascidians: cell migration and differentiation.

Adult ascidians have the capacity to regenerate the central nervous system (CNS) and are therefore excellent models for studies on neuroregeneration. The possibility that undifferentiated blood cells are involved in adult neuroregeneration merits investigation. We analyzed the migration, circulation, and role of hemocytes of the ascidian Styela plicata in neuroregeneration. Hemocytes were removed and incubated with superparamagnetic iron oxide nanoparticles (SPION), and these SPION-labeled hemocytes were injected back into the animals (autologous transplant), followed by neurodegeneration with the neurotoxin 3-acetylpyridine (3AP). Magnetic resonance imaging showed that 1, 5, and 10 days after injury, hemocytes migrated to the intestinal region, siphons, and CNS. Immunohistochemistry revealed that the hemocytes that migrated to the CNS were putative stem cells (P-element-induced wimpy testis + or PIWI + cells). In the cortex of the neural ganglion, migrated hemocytes started to lose their PIWI labeling 5 days after injury, and 10 days later started to show ß-III tubulin labeling. In the neural gland, however, the hemocytes remained undifferentiated during the entire experimental period. Transmission electron microscopy revealed regions in the neural gland with characteristics of neurogenic niches, not previously reported in ascidians. These results showed that migration of hemocytes to the hematopoietic tissue and to the 3AP-neurodegenerated region is central to the complex mechanism of neuroregeneration.

A cis-regulatory change underlying the motor neuron-specific loss of Ebf expression in immotile tunicate larvae.

Many species in the tunicate family Molgulidae have independently lost their swimming larval form and instead develop as tailless, immotile larvae. These larvae do not develop structures that are essential for swimming such as the notochord, otolith, and tail muscles. However, little is known about neural development in these nonswimming larvae. Here, we studied the patterning of the Motor Ganglion (MG) of Molgula occulta, a nonswimming species. We found that spatial patterns of MG neuron regulators in this species are conserved, compared with species with swimming larvae, suggesting that the gene networks regulating their expression are intact despite the loss of swimming. However, expression of the key motor neuron regulatory gene Ebf (Collier/Olf/EBF) was reduced in the developing MG of M. occulta when compared with molgulid species with swimming larvae. This was corroborated by measuring allele-specific expression of Ebf in hybrid embryos from crosses of M. occulta with the swimming species M. oculata. Heterologous reporter construct assays in the model tunicate species Ciona robusta revealed a specific cis-regulatory sequence change that reduces expression of Ebf in the MG, but not in other cells. Taken together, these data suggest that MG neurons are still specified in M. occulta larvae, but their differentiation might be impaired due to reduction of Ebf expression levels.

From the individual to the colony: Marine invertebrates as models to understand levels of biological organization.

The developmental and evolutionary principles of coloniality in marine animals remain largely unexplored. Although many common traits have evolved independently in different groups of colonial animals, questions about their significance for colonial life histories remain unanswered. In 2018 (Nov. 25 - Dec. 8), the inaugural course on the Evolution of Coloniality and Modularity took place at the Center for Marine Biology of the University of São Paulo (CEBIMAR-USP), Brazil. During the intensive two-week graduate-level course, we addressed some of the historical ideas about animal coloniality by focal studies in bryozoans, tunicates, cnidarians, and sponges. We discussed many historical hypotheses and ways to test these using both extant and paleontological data, and we carried direct observations of animal colonies in the different phyla to address questions about coloniality. We covered topics related to multi-level selection theory and studied colonial traits, including modular miniaturization, polymorphism, brooding, and allorecognition. Course participants carried out short research projects using local species of animals to address questions on allorecognition and regeneration in ascidians and sponges, fusion and chimerism in anthoathecate hydrozoans, and evolution of polymorphism in bryozoans. Although many questions remain unanswered, this course served as a foundation to continue to develop a developmental and evolutionary synthesis of clonal and modular development in colonial marine organisms.

An introgression breakthrough left by an anthropogenic contact between two ascidians.

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

Marine Natural Products from Tunicates and Their Associated Microbes.

Marine tunicates are identified as a potential source of marine natural products (MNPs), demonstrating a wide range of biological properties, like antimicrobial and anticancer activities. The symbiotic relationship between tunicates and specific microbial groups has revealed the acquisition of microbial compounds by tunicates for defensive purpose. For instance, yellow pigmented compounds, "tambjamines", produced by the tunicate, Sigillina signifera (Sluiter, 1909), primarily originated from their bacterial symbionts, which are involved in their chemical defense function, indicating the ecological role of symbiotic microbial association with tunicates. This review has garnered comprehensive literature on MNPs produced by tunicates and their symbiotic microbionts. Various sections covered in this review include tunicates' ecological functions, biological activities, such as antimicrobial, antitumor, and anticancer activities, metabolic origins, utilization of invasive tunicates, and research gaps. Apart from the literature content, 20 different chemical databases were explored to identify tunicates-derived MNPs. In addition, the management and exploitation of tunicate resources in the global oceans are detailed for their ecological and biotechnological implications.

Botryllus schlosseri as a Unique Colonial Chordate Model for the Study and Modulation of Innate Immune Activity.

Understanding the mechanisms that sustain immunological nonreactivity is essential for maintaining tissue in syngeneic and allogeneic settings, such as transplantation and pregnancy tolerance. While most transplantation rejections occur due to the adaptive immune response, the proinflammatory response of innate immunity is necessary for the activation of adaptive immunity. Botryllus schlosseri, a colonial tunicate, which is the nearest invertebrate group to the vertebrates, is devoid of T- and B-cell-based adaptive immunity. It has unique characteristics that make it a valuable model system for studying innate immunity mechanisms: (i) a natural allogeneic transplantation phenomenon that results in either fusion or rejection; (ii) whole animal regeneration and noninflammatory resorption on a weekly basis; (iii) allogeneic resorption which is comparable to human chronic rejection. Recent studies in B. schlosseri have led to the recognition of a molecular and cellular framework underlying the innate immunity loss of tolerance to allogeneic tissues. Additionally, B. schlosseri was developed as a model for studying hematopoietic stem cell (HSC) transplantation, and it provides further insights into the similarities between the HSC niches of human and B. schlosseri. In this review, we discuss why studying the molecular and cellular pathways that direct successful innate immune tolerance in B. schlosseri can provide novel insights into and potential modulations of these immune processes in humans.

Chemical Diversity and Biological Activity of Secondary Metabolites Isolated from Indonesian Marine Invertebrates.

Marine invertebrates have been reported to be an excellent resource of many novel bioactive compounds. Studies reported that Indonesia has remarkable yet underexplored marine natural products, with a high chemical diversity and a broad spectrum of biological activities. This review discusses recent updates on the exploration of marine natural products from Indonesian marine invertebrates (i.e., sponges, tunicates, and soft corals) throughout 2007-2020. This paper summarizes the structural diversity and biological function of the bioactive compounds isolated from Indonesian marine invertebrates as antimicrobial, antifungal, anticancer, and antiviral, while also presenting the opportunity for further investigation of novel compounds derived from Indonesian marine invertebrates.

Transcriptional regulation of Rab32/38, a specific marker of pigment cell formation in Ciona robusta.

Through a myriad of pigments stored in different cells, animal pigmentation represents a crucial process to face disparate environmental and ecological challenges. In vertebrates, the small GTPase Rab32 and Rab38 have a conserved role in the transport of key melanogenic enzymes, as tyrosinase (tyr) and tyrosinase-related protein (tyrp), to the melanosomes in formation. We provide a survey on Rab32/38 evolution and its regulatory logics during pigment cell formation in Ciona robusta. Our phylogeny supports the existence of a single Rab32/38 gene in tunicates, which is probably the unique transporter for tyrosinase family members in this clade. Different deletions allow us to identify the minimal cis-regulatory element able to recapitulate the endogenous gene expression during pigment cell development in C. robusta. In this conserved region, we identified two putative binding sites for the transcription factor Mitf, which is known for its role as regulator of pigmentation in vertebrates. Mutational analysis revealed that both Mitf binding sites are essential for the activity of this regulatory region and we demonstrated that Mitf misexpression is able to induce ectopic activation of the Rab32/38 regulatory region in vivo. Our results strongly indicate that Mitf is involved in the regulation of Rab32/38 activity during Ciona pigment cell development.

Spawning induction, development and culturing of the solitary ascidian Polycarpa mytiligera, an emerging model for regeneration studies.

BACKGROUND: Ascidians (phylum Chordata, class Ascidiacea) represent the closest living invertebrate relatives of the vertebrates and constitute an important model for studying the evolution of chordate development. The solitary ascidian Polycarpa mytiligera exhibits a robust regeneration ability, unique among solitary chordates, thus offering a promising new model for regeneration studies. Understanding its reproductive development and establishing land-based culturing methods is pivotal for utilizing this species for experimental studies. Its reproduction cycle, spawning behavior, and developmental processes were therefore studied in both the field and the lab, and methods were developed for its culture in both open and closed water systems. RESULTS: Field surveys revealed that P. mytiligera's natural recruitment period starts in summer (June) and ends in winter (December) when seawater temperature decreases. Laboratory experiments revealed that low temperature (21 °C) has a negative effect on its fertilization and development. Although spontaneous spawning events occur only between June and December, we were able to induce spawning under controlled conditions year-round by means of gradual changes in the environmental conditions. Spawning events, followed by larval development and metamorphosis, took place in ascidians maintained in either artificial or natural seawater facilities. P. mytiligera's fast developmental process indicated its resemblance to other oviparous species, with the larvae initiating settlement and metamorphosis at about 12 h post-hatching, and reaching the juvenile stage 3 days later. CONCLUSIONS: Polycarpa mytiligera can be induced to spawn in captivity year-round, independent of the natural reproduction season. The significant advantages of P. mytiligera as a model system for regenerative studies, combined with the detailed developmental data and culturing methods presented here, will contribute to future research addressing developmental and evolutionary questions, and promote the use of this species as an applicable model system for experimental studies.