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The spread of the invasive ascidian Didemnum vexillum represents a major ecological and economic threat in many parts of the world. Recorded for the first time in Norway in 2020, this species is now established in several localities, often characterized by busy port activities. The new detection and expansion of D. vexillum raise significant concerns in Norway, especially due to the potential extension of its distribution range into higher latitudes. This study presents the results of an in situ monitoring of the growth of eleven D. vexillum colonies in Stavanger between December 2020 and September 2022. Colony growth was calculated from underwater photographs taken approximately once a month. Overall, our findings indicate significant growth of most colonies after 624 days, increasing in size from 78 cm 2 to 1008 cm 2 . Seasonal variations in seawater temperature are a major factor affecting the size and growth of the colonies. Most of the growth occurred during the summer months, when seawater temperature was between 13 °C and 18 °C. A rapid decline in colony growth rates was observed in November, as temperatures dropped below 13 °C, followed by colony regression below ~8 °C. These results therefore confirm the general understanding of D. vexillum as rapidly spreading species, characterized by a high colony growth rate. Its ongoing spread toward northern Norway is likely to have negative impacts on key coastal ecosystems (e.g., kelp forests, rhodolith beds, seagrass meadows), as well as on several industries critical to Norway's economy (e.g., oil industry, aquaculture, fisheries). Based on the information provided here, it is essential to continue implementing measures to prevent further spread to the north and to develop localized mitigation strategies., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Maintenance and breeding of experimental organisms are fundamental to life sciences, but both initial and running costs, and hands-on zootechnical demands can be challenging for many laboratories. Here, we first aimed to further develop a simple protocol for reliable inland culture of tunicate model species of the Ciona genus. We cultured both Ciona robusta and Ciona intestinalis in controlled experimental conditions, with a focus on dietary variables, and quantified growth and maturation parameters. From statistical analysis of these standardized datasets, we gained insights into the post-embryonic developmental physiology of Ciona and inferred an improved diet and culturing conditions for sexual maturation. We showed that body length is a critical determinant of both somatic and sexual maturation, which suggests the existence of systemic control mechanisms of resource allocation towards somatic growth or maturation and supports applying size selection as a predictor of reproductive fitness in our inland culture to keep the healthiest animals at low density in the system. In the end, we successfully established a new protocol, including size selection, to promote both sperm and egg production. Our protocol using small tanks will empower researchers to initiate inland Ciona cultures with low costs and reduced space constraints.

AbstractColonial animals are composed of clonal individuals that remain physically connected and physiologically integrated. Salps are tunicates with a dual life cycle, including an asexual solitary stage that buds sexual colonies composed of jet-propelling zooids that efficiently swim together as a single unit by multijet propulsion. Colonies from different species develop distinct architectures characterized by their zooid arrangement patterns, but this diversity has received little attention. Thus, these architectures have never been formally defined using a framework of variables and axes that would allow comparative analyses. We set out to define an ontology of the salp colony architecture morphospace and describe the developmental pathways that build the different architectures. To inform these definitions, we collected and photographed live specimens of adult and developing colonies through offshore scuba diving. Since all salp colonies begin their development as a transversal double chain, we characterized each adult colonial architecture as a series of developmental transitions, such as rotations and translations of zooids, relative to their orientation at this early shared stage. We hypothesize that all adult architectures are either final or intermediate stages within three developmental pathways toward bipinnate, cluster, or helical forms. This framework will enable comparative studies on the biomechanical implications, ecological functions, evolutionary history, and engineering applications of the diversity of salp colony architectures.

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 ​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., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ascidians are invertebrate chordates and the closest living relative to vertebrates. In ascidian embryos a large part of the central nervous system arises from cells associated with mesoderm rather than ectoderm lineages. This seems at odds with the traditional view of vertebrate nervous system development which was thought to be induced from ectoderm cells, initially with anterior character and later transformed by posteriorizing signals, to generate the entire anterior-posterior axis of the central nervous system. Recent advances in vertebrate developmental biology, however, show that much of the posterior central nervous system, or spinal cord, in fact arises from cells that share a common origin with mesoderm. This indicates a conserved role for bi-potential neuromesoderm precursors in chordate CNS formation. However, the boundary between neural tissue arising from these distinct neural lineages does not appear to be fixed, which leads to the notion that anterior-posterior patterning and neural fate formation can evolve independently.

Tunicates encompass a large group of marine filter-feeding animals and more than half of them are able to reproduce asexually by a particular form of nonembryonic development (NED) generally called budding. The phylogeny of tunicates suggests that asexual reproduction is an evolutionarily plastic trait, a view that is further reinforced by the fact that budding mechanisms differ from one species to another, involving nonhomologous tissues and cells. In this review, we explore more than 150 years of literature to provide an overview of NED diversity and we present a comparative picture of budding tissues across tunicates. Based on the phylogenetic relationships between budding and nonbudding species, we hypothesize that NED diversity is the result of seven independent acquisitions and subsequent diversifications in the course of tunicate evolution. While this scenario represents the state-of-the-art of our current knowledge, we point out gray areas that need to be further explored to refine our understanding of tunicate phylogeny and NED. Tunicates, with their plastic evolution and diversity of budding, represent an ideal playground for evolutionary developmental biologists to unravel the genetic and molecular mechanisms regulating nonembryonic development, as well as to better understand how such a profound innovation in life-history has evolved in numerous metazoans., (© 2020 Wiley Periodicals, Inc.)

MicroRNAs are frequently clustered in the genome and polycistronically transcribed, regulating targeted genes in diverse signaling pathways. The miR-17-92 cluster is a typical miRNA cluster, playing crucial roles in the organogenesis and homeostasis of physiological processes in vertebrates. Here, we identified three miRNAs (csa-miR-92a, csa-miR-92b, and csa-miR-92c) that belonged to the miR-92 family and formed a miRNA cluster in the genome of a urochordate marine ascidian Ciona savignyi . Except for miR-92a and miR-92b, other homologs of the vertebrate miR-17-92 cluster members could not be identified in the Ciona genome. We further found that the mature sequences of urochordate miR-92 family members were highly conserved compared with the vertebrate species. The expression pattern revealed that three miR-92 family members had consistent expression levels in adult tissues and were predominantly expressed in heart and muscle tissue. We further showed that, at the embryonic and larval stages, csa-miR-92c was expressed in the notochord of embryos during 18-31 h post fertilization (hpf) by in situ hybridization. Knockout of csa-miR-92c resulted in the disorganization of notochord cells and the block of lumen coalescence in the notochord. Fibroblast growth factor (FGF), mitogen-activated protein kinase (MAPK), and wingless/integrated (Wnt)/planar cell polarity (PCP) signaling pathways might be involved in the regulatory processes, since a large number of core genes of these pathways were the predicted target genes of the miR-92 family. Taken together, we identified a miR-92 cluster in urochordate Ciona and revealed the expression patterns and the regulatory roles of its members in organogenesis. Our results provide expression and phylogenetic data on the understanding of the miR-92 miRNA cluster's function during evolution.

Alternative splicing is widespread throughout eukaryotic genomes and greatly increases transcriptomic diversity. Many alternative isoforms have functional roles in developmental processes and are precisely temporally regulated. To facilitate the study of alternative splicing in a developmental context, we created MeDAS, a Metazoan Developmental Alternative Splicing database. MeDAS is an added-value resource that re-analyses publicly archived RNA-seq libraries to provide quantitative data on alternative splicing events as they vary across the time course of development. It has broad temporal and taxonomic scope and is intended to assist the user in identifying trends in alternative splicing throughout development. To create MeDAS, we re-analysed a curated set of 2232 Illumina polyA+ RNA-seq libraries that chart detailed time courses of embryonic and post-natal development across 18 species with a taxonomic range spanning the major metazoan lineages from Caenorhabditis elegans to human. MeDAS is freely available at https://das.chenlulab.com both as raw data tables and as an interactive browser allowing searches by species, tissue, or genomic feature (gene, transcript or exon ID and sequence). Results will provide details on alternative splicing events identified for the queried feature and can be visualised at the gene-, transcript- and exon-level as time courses of expression and inclusion levels, respectively., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human activities alter patterns of biodiversity, particularly through species extinctions and range shifts. Two of these activities are human mediated transfer of species and contemporary climate change, and both allow previously isolated genotypes to come into contact and hybridize, potentially altering speciation rates. Hybrids have been shown to survive environmental conditions not tolerated by either parent, suggesting that, under some circumstances, hybrids may be able to expand their ranges and perform well under rapidly changing conditions. However, studies assessing how hybridization influences contemporary range shifts are scarce. We performed crosses on Pyura herdmani and Pyura stolonifera (Chordata, Tunicata), two closely related marine invertebrate species that are ecologically dominant and can hybridize. These sister species live in sympatry along the coasts of southern Africa, but one has a disjunct distribution that includes northern hemisphere sites. We experimentally assessed the performance of hybrid and parental crosses using different temperature regimes, including temperatures predicted under future climate change scenarios. We found that hybrids showed lower performance than parental crosses at the experimental temperatures, suggesting that hybrids are unlikely to expand their ranges to new environments. In turn, we found that the more widespread species performed better at a wide array of temperatures, indicating that this parental species may cope better with future conditions. This study illustrates how offspring fitness may provide key insights to predict range expansions and how contemporary climate change may mediate both the ability of hybrids to expand their ranges and the occurrence of speciation as a result of hybridization., (© 2020 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2020 European Society For Evolutionary Biology.)

Fourteen aromatic metabolites ( 6 - 19 ) were isolated from an aqueous extract of the solitary tunicate Cnemidocarpa irene collected in Hokkaido, Japan. The structures of the metabolites were determined based on the spectroscopic interpretations, including one- and two-dimensional NMR, mass spectra, UV, and circular dichroism data. The biopterin analogue 10 modulated the behavior of mice after intracerebroventricular injection and showed a weak affinity to ionotropic glutamate receptor subtypes. Analyses of fluorescent coelomic fluid of the tunicate revealed that pterin 12 was responsible for the fluorescence of the blood cells, while β-carbolines 1 and 3 were fluorescent compounds in the serum. The metabolic profiles in adults, juveniles, larvae, and eggs of the animal differed substantially, suggesting that the metabolism of the animal, especially biosynthesis of aromatic secondary metabolites, changes over different life stages.

Ciona robusta (Ciona intestinalis type A), a model organism for biological studies, belongs to ascidians, the main class of tunicates, which are the closest relatives of vertebrates. In Ciona, a project on the ontology of both development and anatomy is ongoing for several years. Its goal is to standardize a resource relating each anatomical structure to developmental stages. Today, the ontology is codified until the hatching larva stage. Here, we present its extension throughout the swimming larva stages, the metamorphosis, until the juvenile stages. For standardizing the developmental ontology, we acquired different time-lapse movies, confocal microscope images and histological serial section images for each developmental event from the hatching larva stage (17.5 h post fertilization) to the juvenile stage (7 days post fertilization). Combining these data, we defined 12 new distinct developmental stages (from Stage 26 to Stage 37), in addition to the previously defined 26 stages, referred to embryonic development. The new stages were grouped into four Periods named: Adhesion, Tail Absorption, Body Axis Rotation, and Juvenile. To build the anatomical ontology, 203 anatomical entities were identified, defined according to the literature, and annotated, taking advantage from the high resolution and the complementary information obtained from confocal microscopy and histology. The ontology describes the anatomical entities in hierarchical levels, from the cell level (cell lineage) to the tissue/organ level. Comparing the number of entities during development, we found two rounds on entity increase: in addition to the one occurring after fertilization, there is a second one during the Body Axis Rotation Period, when juvenile structures appear. Vice versa, one-third of anatomical entities associated with the embryo/larval life were significantly reduced at the beginning of metamorphosis. Data was finally integrated within the web-based resource "TunicAnatO", which includes a number of anatomical images and a dictionary with synonyms. This ontology will allow the standardization of data underpinning an accurate annotation of gene expression and the comprehension of mechanisms of differentiation. It will help in understanding the emergence of elaborated structures during both embryogenesis and metamorphosis, shedding light on tissue degeneration and differentiation occurring at metamorphosis.

Colonial ascidians are the only chordates able to undergo whole body regeneration (WBR), during which entire new bodies can be regenerated from small fragments of blood vessels. Here, we show that during the early stages of WBR in Botrylloides diegensis, proliferation occurs only in small, blood-borne cells that express integrin-alpha-6 (IA6), pou3 and vasa. WBR cannot proceed when proliferating IA6+ cells are ablated with Mitomycin C, and injection of a single IA6+ Candidate stem cell can rescue WBR after ablation. Lineage tracing using EdU-labeling demonstrates that donor-derived IA6+ Candidate stem cells directly give rise to regenerating tissues. Inhibitors of either Notch or canonical Wnt signaling block WBR and reduce proliferation of IA6+ Candidate stem cells, indicating that these two pathways regulate their activation. In conclusion, we show that IA6+ Candidate stem cells are responsible for whole body regeneration and give rise to regenerating tissues.

Oceanographic studies revealed the abundance of minute plastic particles in coastal regions. Such particles, called microplastics, are abundant in sizes smaller than 100 µm ESD (Equivalent Spherical Diameter) and can be collected and ingested by planktonic copepods. Those animals are the most abundant metazoans on our planet. Abundantly co-occurring with planktonic copepods in subtropical and temperate neritic waters are doliolids (Tunicata, Thaliacea), which can dominate subtropical shelves because of their high asexual reproductive performance. Our studies were designed to examine the effects of polystyrene beads at low abundance, compared with phytoplankton, on abundantly occurring gonozooids of Dolioletta gegenbauri. Our findings reveal that such abundance of microplastic particles, in the presence of environmental concentrations of phytoplankton, reduces rates of feeding, growth, and oxygen consumption of this tunicate. Feeding rates on phytoplankton in the presence of beads were reduced by up to 58%, growth rates by up to 85%, and oxygen consumption rates by up to 33%. We conclude that such microplastic particles could limit the often in situ encountered pronounced proliferation of this tunicate species (Deibel in: Bone (ed) The biology of pelagic tunicates, Oxford University Press, Oxford, 1998).

Endocrine Disrupting Chemicals (EDCs) are molecules able to interfere with the vertebrate hormonal system in different ways, a major one being the modification of the activity of nuclear receptors (NRs). Several NRs are expressed in the vertebrate brain during embryonic development and these NRs are suspected to be responsible for the neurodevelopmental defects induced by exposure to EDCs in fishes or amphibians and to participate in several neurodevelopmental disorders observed in humans. Known EDCs exert toxicity not only on vertebrate forms of marine life but also on marine invertebrates. However, because hormonal systems of invertebrates are poorly understood, it is not clear whether the teratogenic effects of known EDCs are because of endocrine disruption. The most conserved actors of endocrine systems are the NRs which are present in all metazoan genomes but their functions in invertebrate organisms are still insufficiently characterized. EDCs like bisphenol A have recently been shown to affect neurodevelopment in marine invertebrate chordates called ascidians. Because such phenotypes can be mediated by NRs expressed in the ascidian embryo, we review all the information available about NRs expression during ascidian embryogenesis and discuss their possible involvement in the neurodevelopmental phenotypes induced by EDCs., (© 2019 Wiley Periodicals, Inc.)

Human occupation of coastal areas promotes the establishment of non-native species but information on bioinvasions is usually biased toward the Northern Hemisphere. We assessed non-native species' importance in sessile communities at six marinas along the most urbanized area of the Southwestern Atlantic coastline. We found 67 species, of which 19 are exotic. The most frequent species was the exotic polychaete Branchiomma luctuosum, while the most abundant was the exotic bryozoan Schizoporella errata that monopolized the substrata in three marinas. Along with S. errata, the exotic polychaete Hydroides elegans and ascidian Styela plicata dominated space in the three remaining marinas, while native species were in general rare. We show that communities associated with artificial substrata along this Brazilian urbanized area are dominated by exotic species and that using abundance data along with species identity can improve our understanding of the importance of exotic species for the dynamics of biological communities., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, and trophic interactions primarily due to methodological challenges, including the ability to culture them. This is particularly true for the doliolid, Dolioletta gegenbauri. D. gegenbauri commonly occurs in productive subtropical continental shelf systems worldwide, often at bloom concentrations capable of consuming a large fraction of daily primary production. In this study, we describe cultivation approaches for collecting, rearing, and maintaining D. gegenbauri for the purpose of conducting laboratory-based studies. D. gegenbauri and other doliolid species can be captured live using obliquely towed conical 202 µm mesh plankton nets from a drifting ship. Cultures are most reliably established when water temperatures are below 21 °C and are started from immature gonozooids, maturing phorozooids, and large nurses. Cultures can be maintained in rounded culture vessels on a slowly rotating plankton wheel and sustained on a diet of cultured algae in natural seawater for many generations. In addition to the ability to establish laboratory cultures of D. gegenbauri, we demonstrate that the collection condition, algae concentration, temperature, and exposure to naturally conditioned seawater are all critical to the culture establishment, growth, survival, and reproduction of D. gegenbauri.

Biofouling is one of the challenges that can strongly affect the finfish farm economy. Although several studies on biofouling in aquaculture have been conducted in the Mediterranean Sea, they focused on specific taxa or were limited to a particular period of sampling. The present study investigated for the first time the development, composition and variation in a biofouling community in a finfish farm with immersion time, season and depth. The results indicate that all these factors influence biofouling succession and recruitment. Moreover, the species that had a crucial role in structuring the community and in the farm cleaning activities were the ascidian Styela plicata and the bivalve Mytilus galloprovincialis . Compared with the literature data, the results highlight the heterogeneity in the composition of the biofouling present in the Mediterranean Sea. Moreover, such knowledge of the biofouling community could provide important information about management efforts and the costs that farmers will face when siting new fish farms.

Wnt/β-catenin signaling is an ancient pathway in metazoans and controls various developmental processes, in particular the establishment and patterning of the embryonic primary axis. In vertebrates, a graded Wnt activity from posterior to anterior endows cells with positional information in the central nervous system. Recent studies in hemichordates support a conserved role for Wnt/β-catenin in ectoderm antero-posterior patterning at the base of the deuterostomes. Ascidians are marine invertebrates and the closest relatives of vertebrates. By combining gain- and loss-of-function approaches, we have determined the role of Wnt/β-catenin in patterning the three ectoderm derivatives of the ascidian Ciona intestinalis, central nervous system, peripheral nervous system and epidermis. Activating Wnt/β-catenin signaling from gastrulation led to a dramatic transformation of the ectoderm with a loss of anterior identities and a reciprocal anterior extension of posterior identities, consistent with studies in other metazoans. Surprisingly, inhibiting Wnt signaling did not produce a reciprocal anteriorization of the embryo with a loss of more posterior identities like in vertebrates and hemichordate. Epidermis patterning was overall unchanged. Only the identity of two discrete regions of the central nervous system, the anteriormost and the posteriormost regions, were under the control of Wnt. Finally, the caudal peripheral nervous system, while being initially Wnt dependent, formed normally. Our results show that the Ciona embryonic ectoderm responds to Wnt activation in a manner that is compatible with the proposed function for this pathway at the base of the deuterostomes. However, possibly because of its fast and divergent mode of development that includes extensive use of maternal determinants, the overall antero-posterior patterning of the Ciona ectoderm is Wnt independent, and Wnt/β-catenin signaling controls the formation of some sub-domains. Our results thus indicate that there has likely been a drift in the developmental systems controlling ectoderm patterning in the lineage leading to ascidians., Competing Interests: The authors have declared that no competing interests exist.

Mass coral bleaching is challenging today's coral reefs. However, our understanding of dynamics in benthic space holders, following such disturbances, is limited. To address this, we quantified successional dynamics of the ascidian, Didemnum cf. molle using a series of temporally and spatially matched photoquadrats across both the 2016 and 2017 mass coral bleaching events on the Great Barrier Reef. Unlike corals, D. cf. molle appeared to flourish in the warm temperatures and rapidly expanded. Indeed, colony density increased nearly 6-fold over two years with one quadrat experiencing an increase of over 1000 ind. m -2 . However, this increase did not simply track the increase in space due to coral mortality, but may have benefitted from reduced predation or increased nutrient availability following mass coral mortality. This study highlights the potential for D. cf molle to expand under bleaching conditions and to become a more prominent component of future reef configurations., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

To understand the mechanisms involved in organisms' responses to toxicity from oil pollution, we studied the effect of acute exposure (24 h) to the marine water-soluble fraction of diesel oil (WFDO) on the ascidian Styela plicata. We evaluated the mortality and behavior by means of the siphon reflex, and the response of blood cells (hemocytes) contained in the pharynx, by means of the production of nitric oxide (NO) and reactive oxygen species (ROS), in addition to the activity of the antioxidant enzyme catalase (CAT). We also correlated oxidative stress with the activation of apoptotic pathways. No mortality occurred 24 h after the ascidians were exposed to 5% and 10% marine WFDO; however, the siphon reflex, a behavioral test based on the time that the animals took to close their siphons, increased. We also observed an inflammatory response, as estimated by the increase in the number of hemocytes in the pharynx. NO and ROS production and CAT activity were reduced, whereas caspase-3, a signaling molecule involved in apoptosis, was activated. This suggests that in ascidians acutely exposed to oil, another mechanism can occur in addition to oxidative stress. Another possibility is that WFDO may directly interact with cellular macromolecules and activate caspase-3, independently of generating oxidative stress. The results showed that components of diesel oil affected a marine organism, which showed reduced ROS production in the pharynx cells, including hemocytes, and activation of apoptotic pathways., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The distribution of intertidal organisms can depend on processes operating early in their life history. The ascidian Pyura praeputialis, a mid- to low-intertidal habitat-forming ecosystem engineer, was strongly associated with specific types of habitat (biogenic vs. bare rock). We examined field patterns and performed laboratory and field experiments to assess the nature of this association. Recruits were frequently found on the tunics of conspecifics and clumps of turfing coralline algae. Larvae preferred these same habitats in a series of laboratory settlement assays. Laboratory-reared juveniles (20- & 50-days-old) survived poorly on bare rock in the laboratory, while those on rugose surfaces - the tunic of adults and turfing corallines - showed high survivorship. Field-collected juveniles (<2 cm) affixed to these rugose habitats also exhibited high survivorship in the field. We conclude that both pre and post-settlement processes determine spatial pattern in this important habitat-forming taxon. The acute sensitivity of juveniles to desiccating conditions was unexpected in an intertidal organism., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Classical non-homologous end joining (c-NHEJ), a fundamental pathway that repairs double-strand breaks in DNA, is almost universal in eukaryotes and involves multiple proteins highly conserved from yeast to human [1]. The genes encoding these proteins were not detected in the genome of Oikopleura dioica, a new model system of tunicate larvaceans known for its very compact and highly rearranged genome [2-4]. After showing their absence in the genomes of six other larvacean species, the present study examined how O. dioica oocytes and embryos repair double-strand DNA breaks (DSBs), using two approaches: the injection of linearized plasmids, which resulted in their rapid end joining, and a newly established CRISPR Cas9 technique. In both cases, end joining merged short microhomologous sequences surrounding the break (mainly 4 bp long), thus inducing deletions larger than for the tunicate ascidian Ciona intestinalis and human cells. A relatively high frequency of nucleotide insertions was also observed. Finally, a survey of genomic indels supports the involvement of microhomology-mediated repair in natural conditions. Overall, O. dioica repairs DSBs as other organisms do when their c-NHEJ pathway is experimentally rendered deficient, using another mode of end joining with the same effect as alternative NHEJ (a-NHEJ) or microhomology-mediated end joining (MMEJ) [5-7]. We discuss how the exceptional loss of c-NHEJ and its replacement by a more mutation-prone mechanism may have contributed to reshaping this genome and even been advantageous under pressure for genome compaction., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The colonial ascidian Didemnum vexillum is a high-profile marine invader, with a geographically widespread distribution after introductions to several temperate regions. D. vexillum has been the focus of several eradication and control programmes globally and the need for specific biological knowledge that relates to establishment processes, persistence, impacts and potential for spread remains. The present study describes temporal patterns of D. vexillum percent cover on experimental substrates over 1.5-years in relation to seasonality of substratum availability, in conjunction with key physical (i.e. temperature and sedimentation) and biological factors (i.e. interspecific competition) at two sites in New Zealand. Colonies showed large fluctuations in percent cover between the two study sites and with reference to timing of bare substratum availability. Colonies generally exhibited an initial lag phase, with peak levels of D. vexillum cover reached during the second summer or autumn post-deployment. The long-term competitive dominance of colonies founded from the reattachment of fragments, as opposed to ambient larval recruitment alone, was also investigated. Increases in colony size as a result of ambient recruitment alone were initially much slower. However, after 12-months colony cover exceeded that of plates inoculated with fragments, suggesting the benefit of the apparent competitive dominance conferred by fragment inoculation is restricted to the early establishment phase. This information will provide increased understanding of the population dynamics of this species, as well as assist in the implementation of effective management strategies through knowledge of environmental drivers of prolific infestations., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A fundamental challenge in developmental biology is to understand how forces produced by individual cells are patterned in space and time and then integrated to produce stereotyped changes in tissue-level or embryo-level morphology. Ascidians offer a unique opportunity to address this challenge by studying how small groups of cells collectively execute complex, but highly stereotyped morphogenetic movements. Here we highlight recent progress and open questions in the study of ascidian morphogenesis, emphasizing the dynamic interplay of cell fate determination, cellular force generation and tissue-level mechanics., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Asexual propagation and whole body regeneration are forms of nonembryonic development (NED) widespread across animal phyla and central in life history and evolutionary diversification of metazoans. Whereas it is challenging to reconstruct the gains or losses of NED at large phylogenetic scale, comparative studies could benefit from being conducted at more restricted taxonomic scale, in groups for which phylogenetic relationships are well established. The ascidian family of Styelidae encompasses strictly sexually reproducing solitary forms as well as colonial species that combine sexual reproduction with different forms of NED. To date, the phylogenetic relationships between colonial and solitary styelids remain controversial and so is the pattern of NED evolution. In this study, we built an original pipeline to combine eight genomes with 18 de novo assembled transcriptomes and constructed data sets of unambiguously orthologous genes. Using a phylogenomic super-matrix of 4,908 genes from these 26 tunicates we provided a robust phylogeny of this family of chordates, which supports two convergent acquisitions of NED. This result prompted us to further describe the budding process in the species Polyandrocarpa zorritensis, leading to the discovery of a novel mechanism of asexual development. Whereas the pipeline and the data sets produced can be used for further phylogenetic reconstructions in tunicates, the phylogeny provided here sets an evolutionary framework for future experimental studies on the emergence and disappearance of complex characters such as asexual propagation and whole body regeneration.

Background: Metamorphosis takes place within the life cycle of most marine invertebrates. The marine ascidian is a classical model to study complex cellular processes and underlying molecular mechanisms involved in its larval metamorphosis. The detailed molecular signaling pathways remain elusive, though extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinase (JNK) have been revealed to regulate cell migration, differentiation, and apoptosis in ascidian larval organ regression and juvenile organ development. MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression at the post-transcriptional level. Large numbers of miRNAs have been demonstrated to be involved in many developmental and metamorphic processes. However, the identification of miRNAs in ascidian larval metamorphosis has not yet been investigated., Results: Totally, 106 known and 59 novel miRNAs were screened out through RNA-sequencing of three small RNA libraries from 18 to 21-h post-fertilization (hpf) tailbud embryos as well as from 42 hpf larvae (after tail regression) in Ciona savignyi. Expression profiling of miRNAs was confirmed by quantitative real-time PCR, showing that the expression levels of csa-miR-4040, csa-miR-4086, csa-miR-4055, csa-miR-4060, csa-miR-216a, csa-miR-216b, csa-miR-217, csa-miR-183, and csa-miR-92c were significantly higher in 42 hpf larvae, whereas those of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were higher in 18 and 21 hpf embryos; then, their expression in 42 hpf larvae became significantly low. For these 12 miRNAs, whose expression levels significantly changed, we predicted their target genes through the combination of miRanda and TargetScan. This prediction analysis revealed 332 miRNA-target gene pairs that were associated with the ERK, JNK, and transforming growth factor beta signaling pathways, suggesting that the identified miRNAs are involved in the regulation of C. savignyi larval metamorphosis via controlling the expression of their target genes. Furthermore, we validated the expression of five selected miRNAs by northern blotting. Among the selected miRNAs, the expression patterns of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were further examined by whole-mount in situ hybridization. The results showed that all three miRNAs were specifically expressed in a cell population resembling mesenchymal cells at the head and trunk part in swimming larvae but not in metamorphic larvae. Utilizing the luciferase assay, we also confirmed that miR-4000f targeted Mapk1, suggesting that the csa-miR-4018a/csa-miR-4018b/csa-miR-4000f cluster regulates larval metamorphosis through the Mapk1-mediated signaling pathway., Conclusions: Totally, 165 miRNAs, including 59 novel ones, were identified from the embryos and larvae of C. savignyi. Twelve of them showed significant changes in expression before and during metamorphosis. In situ hybridization and northern blotting results revealed that three miRNAs are potentially involved in the signaling regulatory network for the migration and differentiation of mesenchymal cells in larval metamorphosis. Furthermore, the luciferase reporter assay revealed that Mapk1 is a target of csa-miR-4000f. Our results not only present a list and profile of miRNAs involved in Ciona metamorphosis but also provide informative cues to further understand their function in ascidian larval metamorphosis.

In the present study, we describe the identified and characterized the gene and the transcript of a novel glutathione peroxidase-7 (GPx7) from the solitary ascidian Ciona robusta, an invertebrate chordate widely distributed in temperate shallow seawater. The putative nucleotide and amino acid sequences were compared with those of GPx7 from other metazoans and phylogenetic analysis suggests the presence of a high evolutionary pressure in the contest of neutral evolution. The mRNA of CrGPx7 is located in hemocytes and ovarian follicular cells, as revealed by in situ hybridization. The time course of CrGPx7 mRNA levels in the presence of Cd, Cu and Zn, showed upregulation in the final stages of the experiments, suggesting a role of GPx7 in late protection from oxidative stress. Our in silico analyses of the crgpx7 promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and xenobiotic-responsive elements (XRE), suggesting that the transcription of these genes directly depends on metals. Cell-free extract from C. robusta tissues show the presence of selenium-independent GPx activity that is inhibited by the presence of metals., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Exogenous gene expression assays during development, including reporters under the control of 5' upstream enhancer regions of genes, constitute a powerful technique for understanding the mechanisms of tissue-specific gene expression regulation and determining the characteristics, behaviors, and functions of cells that express these genes. The simple marine chordate Halocynthia roretzi has been used for these transgenic analyses for a long time and is an excellent model system for such studies, especially in comparative analyses with other ascidians. In this study, I describe simple methods for microinjecting H. roretzi eggs with exogenous DNA, such as a promoter construct consisting of a 5' upstream region and a reporter gene, which are prerequisites for transgenic analyses. I also describe basic knowledge regarding this ascidian species, providing reasons why it is an ideal subject for developmental biology studies.

Ascidians possess relatively small and compact genomes. This feature enables us to easily isolate cis-regulatory DNAs of genes of interest. Particularly, cis-regulatory DNAs of genes showing tissue- or cell-type-specific expression are routinely used for the artificial induction of gene expression. This strategy helps us to label cells, tissues, and organs of interest, and to investigate gene functions through overexpression, ectopic expression, and the disruption of functions by dominant-negative forms. Thus, cis-regulatory DNAs provide a powerful tool for tissue-specific genetic manipulation in studies of ascidian development and physiology. This chapter summarizes the types of cis-regulatory DNAs as a genetic manipulation tool, describes the methods used for isolating cis-regulatory DNAs, and provide reported examples of the use of cis-regulatory DNAs as molecular tools for investigating gene functions.

Phallusia mammillata has recently emerged as a new ascidian model. Its unique characteristics, including the optical transparency of eggs and embryos and efficient translation of exogenously introduced mRNA in eggs, make the Phallusia system suitable for fluorescent protein (FP)-based imaging approaches. In addition, genomic and transcriptomic resources are readily available for this ascidian species, facilitating functional gene studies. Microinjection is probably the most versatile technique for introducing exogenous molecules such as plasmids, mRNAs, and proteins into ascidian eggs/embryos. However, it is not practiced widely within the community; presumably, because the system is rather laborious to set up and it requires practice. Here, we describe in as much detail as possible two microinjection methods that we use daily in the laboratory: one based on an inverted microscope and the other on a stereomicroscope. Along the stepwise description of system setup and injection procedure, we provide practical tips in the hope that this chapter might be a useful guide for introducing or improving a microinjection setup.

Ascidians are increasingly being used as a system for investigating cell biology during development. The extreme genetic and cellular simplicity of ascidian embryos in combination with superior experimental tractability make this an ideal system for in vivo analysis of dynamic cellular processes. Transgenic approaches to cellular and sub-cellular analysis of ascidian development have begun to yield new insights into the mechanisms regulating developmental signaling and morphogenesis. This chapter focuses on the targeted expression of fusion proteins in ascidian embryos and how this technique is being deployed to garner new insights into the cell biology of development.

Neurobiology in ascidians has made many advances. Ascidians have offered natural advantages to researchers, including fecundity, structural simplicity, invariant morphology, and fast and stereotyped developmental processes. The researchers have also accumulated on this animal a great deal of knowledge, genomic resources, and modern genetic techniques. A recent connectomic analysis has shown an ultimately resolved image of the larval nervous system, whereas recent applications of live imaging and optogenetics have clarified the functional organization of the juvenile nervous system. Progress in resources and techniques have provided convincing ways to deepen what we have wanted to know about the nervous systems of ascidians. Here, the research history and the current views regarding ascidian nervous systems are summarized.

With increasing coastal infrastructure and use of novel materials there is a need to investigate the colonisation of assemblages associated with new structures, how these differ to natural and other artificial habitats and their potential impact on regional biodiversity. The colonisation of Europe's first artificial surf reef (ASR) was investigated at Boscombe on the south coast of England (2009-2014) and compared with assemblages on existing natural and artificial habitats. The ASR consists of geotextile bags filled with sand located 220m offshore on a sandy sea bed at a depth of 0-5m. Successional changes in epibiota were recorded annually on differently orientated surfaces and depths using SCUBA diving and photography. Mobile faunal assemblages were sampled using Baited Remote Underwater Video (BRUV). Distinct stages in colonisation were observed, commencing with bryozoans and green algae which were replaced by red algae, hydroids and ascidians, however there were significant differences in assemblage structure with depth and orientation. The reef is being utilised by migratory, spawning and juvenile life-history stages of fish and invertebrates. The number of non-native species was larger than on natural reefs and other artificial habitats and some occupied a significant proportion of the structure. The accumulation of 180 benthic and mobile taxa, recorded to date, appears to have arisen from a locally rich and mixed pool of native and non-native species. Provided no negative invasive impacts are detected on nearby protected reefs the creation of novel yet diverse habitats may be considered a beneficial outcome.

The appendicularian Oikopleura dioica is a planktonic chordate that retains a tadpole shape throughout its life. Its simple and transparent body, invariant cell lineages, fast development and available genome and transcriptome resources make it a promising model organism for research in developmental biology. However, large-scale analysis of gene expression in O. dioica is limited owing to the laborious and time-consuming process of manual removal of the vitelline membrane, because devitellinisation of pre-hatching embryos causes failure of normal development. Therefore, in this study, modified procedures were developed for whole-mount in situ hybridisation (WISH) and immunohistochemistry (WIHC). This protocol enables rapid mRNA or protein detection without a manual devitellination step for each specimen. The critical procedure is brief treatment of the vitelline membrane of living embryos with 0.05% actinase E before fixation. Two minutes of treatment was optimal for the penetration of antisense RNA probes and antibodies through the vitelline membrane. This WISH protocol was applicable for chromogenic and fluorescent tyramide signal amplification reactions. Using the new protocol, we found eight genes with tissue-specific expression in the tail muscle, trunk epidermis, heart, pharynx, oesophagus, stomach or gill openings of developing larvae. This procedure also allowed for the detection of exogenous FLAG-tagged histone-enhanced green fluorescent protein by WIHC using anti-FLAG antibody. This study provides a useful and convenient tool for studying spatial and temporal gene expression patterns in this simple chordate model and should facilitate handling large amounts of genetic data from transcriptome-based approaches and other techniques such as treatments with chemical inhibitors.

Facilitation cascades occur when multiple foundation species in a community are involved in a hierarchy of positive interactions, and consist of a primary facilitator which positively affects secondary facilitators, each supporting a suit of dependent species. There is no theoretical limit to the number of levels in a facilitation cascade, yet the existence of more than two has rarely been examined. We manipulated biogenic substrate produced by a primary facilitator (cockle shells) and a secondary facilitator (barnacles and their empty tests) in a space-limited subtidal community to test the hypothesis that solitary ascidians would be the third-level facilitator. In the field, most ascidians were found on barnacles, and most barnacles occupied cockle shells. To produce this pattern, barnacles could nurse ascidians (a longer 'facilitation chain') or outcompete them from cockle shells (a shorter chain). Experimental results clearly supported the nursing hypothesis providing evidence for a facilitation cascade with three hierarchical levels of foundation species. Our findings confirm that like predation and competition, positive interspecific interactions nest into multi-tier hierarchies with numerous levels. While the number of foundation species should increase community stability and resilience as it increases diversity and reduces environmental stress, facilitation chain length may have the opposite effect.

We investigated and compared, by laboratory experiments, the filter-feeding activity on bacteria by the solitary ascidian Styela plicata and the colonial ascidian Polyandrocarpa zorritensis. Clearance rates and retention efficiencies were estimated by using, as only food source, the bacterial species Vibrio alginolyticus selected on account of its importance in aquaculture pathogenicity. The Cmax was 1.4±0.17Lh -1 g -1 DW for S. plicata and 1.745Lh -1 g -1 DW for P. zorritensis. The highest retention efficiency was 41% corresponding to a removed bacterial biomass of 16.34+1.71 μgCL -1 g -1 DW for P. zorritensis and 81% corresponding to a bacterial biomass of 32.28+2.15 μgCL -1 g -1 DW for S. plicata. Styela plicata resulted higher efficient than P. zorritensis in removing V. alginolyticus from seawater in experimental tanks, thus representing a more suitable biofilter to restore the quality of microbiologically contaminated waters including those where aquaculture is practiced. Present laboratory experiments represent the first contribution to the comparison of the filtration activity of the two ascidians, as well as to characterize the filtration process on bacterioplankton and pone the basis for future field works aimed to restore bacteriological polluted seawater., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Substrate cover, water quality parameters and assemblages of corals, fishes, sponges, echinoderms, ascidians, molluscs, benthic foraminifera and macroalgae were sampled across a pronounced environmental gradient in the Jakarta Bay-Thousand Islands reef complex. Inshore sites mainly consisted of sand, rubble and turf algae with elevated temperature, dissolved oxygen, pH and chlorophyll concentrations and depauperate assemblages of all taxa. Live coral cover was very low inshore and mainly consisted of sparse massive coral heads and a few encrusting species. Faunal assemblages were more speciose and compositionally distinct mid- and offshore compared to inshore. There were, however, small-scale differences among taxa. Certain midshore sites, for example, housed assemblages resembling those typical of the inshore environment but this differed depending on the taxon. Substrate, water quality and spatial variables together explained from 31% (molluscs) to 72% (foraminifera) of the variation in composition. In general, satellite-derived parameters outperformed locally measured parameters., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Diverse animal taxa metamorphose between larval and juvenile phases in response to bacteria. Although bacteria-induced metamorphosis is widespread among metazoans, little is known about the molecular changes that occur in the animal upon stimulation by bacteria. Larvae of the tubeworm Hydroides elegans metamorphose in response to surface-bound Pseudoalteromonas luteoviolacea bacteria, producing ordered arrays of phage tail-like metamorphosis-associated contractile structures (MACs). Sequencing the Hydroides genome and transcripts during five developmental stages revealed that MACs induce the regulation of groups of genes important for tissue remodeling, innate immunity, and mitogen-activated protein kinase (MAPK) signaling. Using two MAC mutations that block P. luteoviolacea from inducing settlement or metamorphosis and three MAPK inhibitors, we established a sequence of bacteria-induced metamorphic events: MACs induce larval settlement; then, particular properties of MACs encoded by a specific locus in P. luteoviolacea initiate cilia loss and activate metamorphosis-associated transcription; finally, signaling through p38 and c-Jun N-terminal kinase (JNK) MAPK pathways alters gene expression and leads to morphological changes upon initiation of metamorphosis. Our results reveal that the intricate interaction between Hydroides and P. luteoviolacea can be dissected using genomic, genetic, and pharmacological tools. Hydroides' dependency on bacteria for metamorphosis highlights the importance of external stimuli to orchestrate animal development. The conservation of Hydroides genome content with distantly related deuterostomes (urchins, sea squirts, and humans) suggests that mechanisms of bacteria-induced metamorphosis in Hydroides may have conserved features in diverse animals. As a major biofouling agent, insight into the triggers of Hydroides metamorphosis might lead to practical strategies for fouling control., Competing Interests: The authors declare no conflict of interest.

Colonial ascidians are marine invertebrate chordates that are the closest invertebrate relative to the vertebrates. Colonies of Botryllus schlosseri undergo a continuous asexual reproduction process that involves the regeneration of entire new bodies, which include all somatic and germline tissues. This adult regenerative process depends on signaling pathways known to regulate the formation of tissues and organs during embryonic development. The formation of gonads within regenerated bodies depends on migration and homing of germ cell precursors to niches within the developing bodies, and Botryllus colonies can cycle between fertile and infertile states. The vasculature that connects all individuals within the colony is highly regenerative, and is a valuable tool for the study of angiogenesis in adult blood vessels. The tremendous regenerative capacity of the vasculature even results in regeneration of entire new bodies solely from fragments of blood vessels upon surgical removal of all bodies. The mechanism underlying this regeneration of whole bodies is not well understood, but appears to depend on proliferation of circulating, blood borne cells. Because of all of these features, colonial ascidians are ideal model organisms for the study of germ cell migration, fertility, vascular biology and regeneration., (Copyright © 2016. Published by Elsevier Ltd.)

Ascidians were collected along the shore of Iles du Salut and deeper on the slope in a program of evaluation of the biodiversity in Guiana. Most of the samples belong to already known species from the Caribbean area and Brazilian coast. The colonial forms dominate. The 6 new species have been dredged deeper than 50m but not found by SCUBA divers. In spite of an intensive sampling, the ascidian diversity in Guiana is low with 36 species recorded. This is the result of the abundance of sediment suspended in the water and uniformly deposited on all substrates which affects filtration rate and limits the settling of the ascidian larvae.

Didemnid ascidians are notorious marine invaders, fouling infrastructure in many ecosystems globally. However, there have been few reports of direct interactions with native species in their natural environment. The invasive colonial ascidian Didemnum perlucidum was discovered in the Swan River estuary (Western Australia) growing on the native seagrass Halophila ovalis. Given the known effects of other related Didemnum species it was expected that D. perlucidum could adversely affect the seagrass, with possible flow on effects to the rest of the ecosystem. This study aimed to document the distribution and abundance of D. perlucidum in the estuary, and to determine whether this species had a negative impact on H. ovalis or associated flora and fauna. D. perlucidum was largely present near areas of infrastructure, particularly mooring buoys, suggesting these were the source of D. perlucidum recruits on the seagrasses. It showed a clear seasonal pattern in abundance, with highly variable cover and colony size. D. perlucidum had a measurable effect on H. ovalis, with colonies enveloping all plant tissue, likely restricting the photosynthetic ability of individual leaves and total plant biomass. There were also significantly less seagrass-associated mud snails (Batillaria australis) where D. perlucidum cover was high. These results demonstrate the ability of invasive ascidians to colonise and affect native seagrasses and associated biota. Seagrasses are pivotal to the ecological function of many urban estuaries world-wide. Biodiversity in these systems is already vulnerable to multiple stressors from human activities but the potential stress of fouling ascidians may pose an additional and increasing threat in the future.

Background: We performed an analysis of the transcriptome during the blastogenesis of the chordate Botryllus schlosseri, focusing in particular on genes involved in cell death by apoptosis. The tunicate B. schlosseri is an ascidian forming colonies characterized by the coexistence of three blastogenetic generations: filter-feeding adults, buds on adults, and budlets on buds. Cyclically, adult tissues undergo apoptosis and are progressively resorbed and replaced by their buds originated by asexual reproduction. This is a feature of colonial tunicates, the only known chordates that can reproduce asexually., Results: Thanks to a newly developed web-based platform ( http://botryllus.cribi.unipd.it ), we compared the transcriptomes of the mid-cycle, the pre-take-over, and the take-over phases of the colonial blastogenetic cycle. The platform is equipped with programs for comparative analysis and allows to select the statistical stringency. We enriched the genome annotation with 11,337 new genes; 581 transcripts were resolved as complete open reading frames, translated in silico into amino acid sequences and then aligned onto the non-redundant sequence database. Significant differentially expressed genes were classified within the gene ontology categories. Among them, we recognized genes involved in apoptosis activation, de-activation, and regulation., Conclusions: With the current work, we contributed to the improvement of the first released B. schlosseri genome assembly and offer an overview of the transcriptome changes during the blastogenetic cycle, showing up- and down-regulated genes. These results are important for the comprehension of the events underlying colony growth and regression, cell proliferation, colony homeostasis, and competition among different generations.

Important contributions to evolutionary developmental biology have been made using the comparative organismal approach. As examples, I describe insights obtained from studies of Molgula ascidians and Astyanax cavefish., (© 2016 Elsevier Inc. All rights reserved.)

Ascidians belong to tunicates, the sister group of vertebrates. Peripheral nervous systems (PNSs) including epidermal sensory neurons (ESNs) in the trunk and dorsal tail regions of ascidian larvae are derived from cells adjacent to the neural plate, as in vertebrates. On the other hand, peripheral ESNs in the ventral tail region are derived from the ventral ectoderm under the control of BMP signalling, reminiscent of sensory neurons of amphioxus and protostomes. In this study, we show that two distinct mechanisms activate a common gene circuit consisting of Msx, Ascl.b, Tox, Delta.b and Pou4 in the dorsal and ventral regions to differentiate ESNs. Our results suggest that ventral ESNs of the ascidian larva are not directly homologous to vertebrate PNSs. The dorsal ESNs might have arisen via co-option of the original PNS gene circuit to the neural plate border in an ancestral chordate.

In the last decade, new surveys were made in French Polynesia as part of the Moorea Biocode project to improve the knowledge about the regional fauna. Here we describe Ascidia paulayi sp. nov., a new species of Ascidiidae. Due its similarity with A. sydneiensis, a discussion about the species of this group complements the study.

How ocean acidification affects marine life is a major concern for science and society. However, its impacts on encrusting biofouling communities, that are both the initial colonizers of hard substrata and of great economic importance, are almost unknown. We showed that community composition changed significantly, from 92% spirorbids, 3% ascidians and 4% sponges initially to 47% spirorbids, 23% ascidians and 29% sponges after 100 days in acidified conditions (pH 7.7). In low pH, numbers of the spirorbid Neodexiospira pseudocorrugata were reduced ×5 compared to controls. The two ascidians present behaved differently with Aplidium sp. decreasing ×10 in pH 7.7, whereas Molgula sp. numbers were ×4 higher in low pH than controls. Calcareous sponge (Leucosolenia sp.) numbers increased ×2.5 in pH 7.7 over controls. The diatom and filamentous algal community was also more poorly developed in the low pH treatments compared to controls. Colonization of new surfaces likewise showed large decreases in spirorbid numbers, but numbers of sponges and Molgula sp. increased. Spirorbid losses appeared due to both recruitment failure and loss of existing tubes. Spirorbid tubes are comprised of a loose prismatic fabric of calcite crystals. Loss of tube materials appeared due to changes in the binding matrix and not crystal dissolution, as SEM analyses showed crystal surfaces were not pitted or dissolved in low pH conditions. Biofouling communities face dramatic future changes with reductions in groups with hard exposed exoskeletons and domination by soft-bodied ascidians and sponges., (© 2015 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

In the course of our continuous interest in identifying bioactive compounds from marine microbes, we have investigated a tunicate-derived fungus, Penicillium sp. CYE-87. A new compound with the 1,4-diazepane skeleton, terretrione D (2), together with the known compounds, methyl-2-([2-(1H-indol-3-yl)ethyl]carbamoyl)acetate (1), tryptamine (3), indole-3-carbaldehyde (4), 3,6-diisobutylpyrazin-2(1H)-one (5) and terretrione C (6), were isolated from Penicillium sp. CYE-87. The structures of the isolated compounds were established by spectral analysis, including 1D (1H, 13C) and 2D (COSY, multiplicity edited-HSQC and HMBC) NMR and HRESIMS, as well as comparison of their NMR data with those in the literature. The compounds were evaluated for their antimigratory activity against the human breast cancer cell line (MDA-MB-231) and their antiproliferation activity against HeLa cells. Compounds 2 and 6 showed significant antimigratory activity against MDA-MB-231, as well as antifungal activity against C. albicans.

Using RNA interference, we have selectively perturbed neurotransmitter-related features of the larval swimming behavior of Oikopleura dioica, a tunicate with a central nervous system comprising about 130 neurons. We injected dsRNA into fertilized eggs to knockdown the expression of the genes, respectively, encoding ChAT (choline acetyltransferase) and GAD (glutamic acid decarboxylase), enzymes critical for the biosynthesis of acetylcholine and GABA. These two neurotransmitters have conserved roles during evolution, particularly within chordate motor systems, where they mediate respectively neuromuscular and central inhibitory signals. In Oikopleura, interference with ChAT expression prevented the normal bidirectional, propagating tail movement characteristic of swimming, permitting only repeated unilateral tail bends. Proper swimming was never observed, and the resting period between episodes of activity was lengthened. This phenotype is most likely caused by the reduction of transcription observed for both the targeted ChAT gene and the VAChT gene (Vesicular Acetylcholine Transporter), both genes being transcribed from the same operon. Interference with GAD expression led to an uncoordinated version of swimming with a spiral movement trajectory, but with episodes similar in duration and cycle frequency to those of normal swimming. Our results suggest locomotor functions for ChAT and GABA that are more subtle than previously proposed for tunicates and opens the way for a genetic dissection of Oikopleura neuronal circuits, which are likely to be among the most simplified in the chordate phylum., (© 2015 Wiley Periodicals, Inc.)

Following the Challenger Expedition in the southern Hemisphere, several international surveys have studied Antarctic ascidians. Several pedunculate Molgula were successively described under various names. From the French part of the Antarctic continent and the Kerguelen area, numerous Molgula were recently collected. They are described here in different species, but closely allied. Their taxonomy is revised with an historical review of the most detailed publications and a link to the ancient names.

Among the Pyuridae family the genus Herdmania Lahille, 1888 differs from the diagnose of Pyura Molina, 1782 by the presence of needle-shape echinated spicules in all tissues. These striking elements formerly led to identify many specimens collected in tropical or temperate waters as Herdmania momus (Savigny, 1916). Later the genus was split into several species with a special attention to the different structure of the gonoducts. Newly collected specimens and a revision of previously described animals in Polynesia lead to the redescription of Herdmania papietensis Herdman 1882 from Polynesia and to compare it to the other Herdmania species known in the Pacific Ocean.