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Globally, there is growing concern regarding the effects of the increasing anthropogenic pressures in marine communities. Artificial structures such as marinas and aquaculture facilities serve as invasion hotspots; hence, monitoring fouling communities on these structures can be valuable for detecting new invasions. In the current study, 24 settlement PVC plates were deployed for three months to compare the recruitment ability of these two artificial environments along the south coast of the offshore island of Madeira (NE Atlantic). The results showed higher variations in the species richness between regions (SW vs. SE) than between artificial habitats (sea-cages vs. marinas), although the community composition differed. Cnidaria and Bryozoa were the most representative groups in the aquaculture systems, while Bryozoa and Chordata were in the marinas. A sum of 18 NIS was recorded for the study, accounting for between 21.88% and 54.84% of the total number of species in the aquaculture facilities and marinas, respectively. The higher NIS percentage from the marinas was even more explicit in the SE coast, where Cradoscrupocellaria bertholletii, Parasmittina alba, and Botrylloides niger distinctly dominated fouling populations. The results suggest that at least some particular NIS previously reported in the studied marinas successfully colonized sea-cages. Future assessments need to address the potential role of aquaculture facilities as drivers for the secondary spread of NIS. Additionally, two new records are considered for Madeira: Eudendrium capillare and Ericthonius punctatus., SI

This article presents and describes data related to the monitoring of our in-land in-lab marine recirculating artificial seawater husbandry system for breeding colonial ascidians [1] over a timespan of three years. These datasets were collected both automatically as well as manually, and include abiotic parameters (salinity, pH, temperature, ORP), concentrations of noxious ions (NH4+, NO2−, NO3−, PO43−), the full lineaging of the colonies developing in the aquaculture setup, animal countings under four different feeding diets and animal survival in artificial seawater containing six different microbiota. Our aquaculture was used to breed two species of model colonial ascidians, Botrylloides diegensis [2] , [3] and Botryllus schlosseri [4] . All the datasets are provided as raw CSV files together with an analysis script to reproduce the figures of our accompanying research article [1] . These extensive datasets give detailed insights into the impact of culturing conditions on the breeding of colonial ascidians and could be used to investigate this intricate relationship.

Ascidians are marine filter feeder primitive chordates.Botrylloidesascidians possess diverse biological abilities like whole-body regeneration (WBR), hibernation/aestivation, blastogenesis, metamorphosis, and natural chimerism. However, the absence of distinctive morphological features often makes identification problematic.Botrylloides diegensisis an ascidian that has been misidentified in previous studies and is recorded in GenBank asBotrylloides leachiidue to the high morphological similarity between the sister species. More available sequences and strategies around identification would help resolve some of the confusion currently surrounding its ambiguous nature. We collected severalBotrylloidessamples from 7 locations around New Zealand - Dunedin, Christchurch, Picton, Nelson, Whangateau, New Plymouth and Invercargill - and barcoded the species based on Cytochrome Oxidase I, Histone 3, 18S, and 28S ribosomal RNA markers. Network and Bayesian trees confirmed the presence of threeBotrylloidesspecies:B. diegensis, B. jacksonianum, andB. aff. anceps. Additionally, morphotypes of three species were investigated regarding zooid size, area, tentacle numbers and colonial arrangement.

Botrylloides species are important members of the fouling community colonizing artificial substrates in harbors and marinas. During monitoring in 2017–2020 of non-indigenous species in Korea, one colonial ascidian species was distinctly different from other native colonial ascidians, such as B. violaceusand Botryllus schlosseri, in South Korea. This species was identified as B. diegensis. DNA barcodes with mitochondrial COI were used to identify one-toned and two-toned colonies of B. diegensis. Intraspecific variations between Korean and other regions of B. diegensis from the NCBI ranged from 0.0% to 1.3%. The Korean B. diegensis was clearly distinct from other species of Botrylloides at 15.8%–24.2%. In phylogenetic analysis results, Korean B. diegensis was established as a single clade with other regions of B. diegensis and was clearly distinct from Korean B. violaceus. After reviewing previous monitoring data, it was found that two-toned B. diegensis was already found in six harbors by July 2017. It has now spread into 14 harbors along the coastal line of South Korea. This means that B. diegensis might have been introduced to South Korea between 1999 and 2016.

Botryllids are colonial styelid ascidians for which morphological characters distinguishing genera and species are often difficult to identify and frequently ambiguous. Botrylloides perspicuus, Botrylloides giganteus, and Botrylloides pizoni have similar colony and zooid appearances and over recent years have been found in dispersed localities around the world. In order to determine how many valid Botrylloides species there are, and how they are geographically distributed, we performed both molecular and morphological analyses on fresh and archival museum specimens of these Botrylloides coming from six distant localities: southern Italy, southern California, Brazil, Galapagos Islands, New Zealand, and Australia. Moreover, we re-analysed the morphology of a number of formalin-preserved museum specimens. We concluded that both B. perspicuus and B. giganteus are indeed valid species and report morphological and molecular characters that distinguish these species; B. pizoni is concluded to be a junior synonym of B. giganteus. The geographic distribution, collection dates, and growth substrates of B. giganteus indicate that this species is rapidly spreading worldwide and has traits that may enhance invasiveness (i.e., presence in intertidal regions; high tolerance to air exposure, temperature and salinity variation). Therefore, B. giganteus should be a focus of both researchers and managers concerned with marine biosecurity. Interestingly, the morphological re-analyses of archival museum samples guided by molecular results, led to the taxonomic reassignment of several botryllid specimens as well as to the identification of a new species, Botrylloides conchyliatus sp. nov., which is here described. http://www.zoobank.org/urn:lsid:zoobank.org:pub:0D896E42-F460-45E2-B74C-576A011D9214.

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 breedingBotrylloides 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 asBotryllus schlosseri, as well as of particular laboratory spaces. Overall, we show thatBotrylloides diegensiscan 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.HighlightsFirst artificial seawater based recirculating aquaculture for colonial ascidiansOver 3 years of continuous breedingSemi-automated setup with minimized maintenanceGood biomass production for strain propagation4 different culture conditions for optimized breeding for species of interest

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., Clonal ascidians are able to undergo whole body regeneration (WBR), where entire new bodies can be regenerated from blood vessel fragments. Here, the authors provide evidence in Botrylloides diegensis supporting pou3 and vasa expressing blood-borne cells isolated with anti-IA6 antibody as candidate stem cells responsible for WBR.

Botrylloides crystallinus n. sp., collected on sublittoral hard substrata in North Mediterranean Sea is here described. The species presents a seasonal life cycle with a spring-summer regression of the zooids and their buds as known in the congeneric species B. leachii (Savigny, 1816), from which however differs in many unusual morphological traits.

Based on mtCOI sequences comparisons, recent studies reassigned the ‘dwarf Botrylloides leachii’ from the Levant as Botrylloides nigrum. Here we conducted a survey of the literature and of deposited mtCOI sequences of botryllid ascidians, elucidating ambiguities in their taxonomy. We found that the species, dwarf morph of Botrylloides leachii, Botrylloides nigrum, Botryllus aster and Botryllus arenaceus are grouped together on a single molecular taxon. Then, results of three additional markers (18S, 28S, H3) contradicted literature suggestions, revealing minute distances between Botrylloides leachii and the ‘dwarf Botrylloides leachii’. Moreover, only Botrylloides leachii and the ‘dwarf Botrylloides leachii’ develop giant ampullae as an allorecognition response. Our results raise the possibility that inadequate identification, together with faults in molecular assignment, including queries regarding the efficacy of the mtCOI as the exclusive barcoding tool in botryllid ascidians, is the major culprits responsible for the emerged inconsistencies between the mtCOI sequences and traditional taxonomy. Thus, we assign the Levantine dwarf form as Botrylloides aff. leachii.

Past tests of settlement by marine invertebrate larvae on different colours were not designed to distinguish between responses to substrate colour and substrate brightness. Using colour vision testing methods, the design of this study provided a true test for responses to colour by including both coloured and grayscale settlement plates. The dominant fouling taxa at the test sites (the solitary ascidian, Ciona intestinalis; the colonial ascidians Botryllus schlosseri or Botrylloides violaceous; and a bryozoan Bugula sp.) showed no significant differences in settlement between blue, red or green plates. In contrast, the ascidians responded to substrate brightness with significantly lower settlement on lighter plates, while the bryozoans showed no significant preference relative to substrate brightness. The methods used here are a model for future tests of responses to colour during settlement. In addition, the contrasting responses to brightness are at odds with previous laboratory experiments that showed larvae of both ascidians and bryozoans are negatively phototactic at settlement. These results suggest that other cues must supersede any phototactic responses in the bryozoans, or alternatively that the range of light intensities near darker and lighter substrata were not part of the tests in the earlier reports.

Interactions between anthropogenic disturbances and introduced and native species can shift ecological communities, potentially leading to the successful establishment of additional invaders. Since its discovery in New Jersey in 1988, the Asian shore crab (Hemigrapsus sanguineus) has continued to expand its range, invading estuarine and coastal habitats in eastern North America. In estuarine environments, H. sanguineus occupies similar habitats to native, panopeid mud crabs. These crabs, and a variety of fouling organisms (both NIS and native), often inhabit man-made substrates (like piers and riprap) and anthropogenic debris. In a series of in situ experiments at a closed dock in southwestern Long Island (New York, USA), we documented the impacts of these native and introduced crabs on hard-substrate fouling communities. We found that while the presence of native mud crabs did not significantly influence the succession of fouling communities compared to caged and uncaged controls, the presence of introduced H. sanguineus reduced the biomass of native tunicates (particularly Molgula manhattensis), relative to caged controls. Moreover, the presence of H. sanguineus favored fouling communities dominated by introduced tunicates (especially Botrylloides violaceous and Diplosoma listerianum). Altogether, our results suggest that H. sanguineus could help facilitate introduced fouling tunicates in the region, particularly in locations where additional solid substrates have created novel habitats.

The mode of sexual reproduction and embryogenesis was compared in 3 species of Botrylloides: B. simodensis, B. lenis, and B. violaceus. In all species, a testis and an egg (occasionally 2 eggs), the former being anterior to the latter, mature in the mantle on either side of a zooid. The egg is surrounded by 2 follicular layers and is attached by a vesicular follicle stalk (oviduct) to the atrial brood pouch. The egg is ovulated into the brood pouch, where it is fertilized and undergoes embryogenesis. The egg of B. simodensis is heavily yolked and measures about 180 μm in diameter. The course of embryogenesis in this species is that typical of ascidians. A mature tadpole larva is produced and shed in about 5 days; then, the mother zooid degenerates. The larva is smallest of the three species and has 8 ampullae. The metamorphosed oozooid bears a single bud on the right side only. Extraembryonic nutrition seems to be very limited. Both Botrylloides lenis and B. violaceus are species which display extreme examples of viviparity. Their eggs are devoid of yolk granules, measuring about 90 μm in diameter in the former species and 60 μm in the latter. The course of embryogenesis is similar in these 2 species. The neurula stage is characterized by a spherical vesicular shape owing to precocious differentiation of the embryonic pharynx, whose ectoderm becomes vacuolated. At the posterior end of the neurula, the mesodermal cells are located in a mass, from which the tail is extended later. In B. lenis, embryogenesis takes about 20 days. At the neurula stage of the embryo, the mother zooid becomes a mantle sac as a result of visceral disintegration. During further embryogenesis, the growth of buds of successive generations in the colony is characteristically arrested. A swimming larva of this species is somewhat larger than that of B. simodensis. It has 14–24 ampullae, and the oozooid carries a single bud on its right side. In B. violaceus, the gestation period lasts for more than a month. At the early gastrula stage of the embryo, the body of the mother zooid fully disintegrates. Only the brood pouch bearing the embryo survives and remains connected with the colonial vascular system. In this species, sexual reproduction does not affect the growth of buds in the colony. The swimming larva is gigantic, being furnished with 24–34 ampullae, and the oozooid always bears 3 buds, 2 on the right side and one on the left side.

Tunicates are marine invertebrates that compose the closest phylogenetic group to the vertebrates. This chordate subphylum contains a particularly diverse range of reproductive methods, regenerative abilities and life-history strategies. Consequently, tunicates provide an extraordinary perspective into the emergence and diversity of chordate traits. To gain further insights into the evolution of the tunicate phylum, we have sequenced the genome of the colonial StolidobranchianBotrylloides leachii.We have produced a high-quality (90 % BUSCO genes) 159 Mb assembly, containing 82 % of the predicted total 194 Mb genomic content. TheB. leachiigenome is much smaller than that ofBotryllus schlosseri(725 Mb), but comparable to those ofCiona robustaandMolgula oculata(both 160 Mb). We performed an orthologous clustering between five tunicate genomes that highlights sets of genes specific to some species, including a large group unique to colonial ascidians with gene ontology terms including cell communication and immune response.By analysing the structure and composition of the conserved gene clusters, we identified many examples of multiple cluster breaks and gene dispersion, suggesting that several lineage-specific genome rearrangements occurred during tunicate evolution. In addition, we investigate lineage-specific gene gain and loss within the Wnt, Notch and retinoic acid pathways. Such examples of genetic change within these highly evolutionary conserved pathways commonly associated with regeneration and development may underlie some of the diverse regenerative abilities observed in the tunicate subphylum. These results supports the widely held view that tunicate genomes are evolving particularly rapidly.

For organisms with complex life cycles, longer time spent in the plankton by dispersing propagules can cause reduced survival, growth and fecundity, which could alter interactions between neighbours in the post-dispersal environment. We compared post-settlement performance of bryozoan Watersipora subtorquata colonies that developed from larvae of different natural and experimental planktonic durations over ca. 15 wk of colony growth. Settlers were situated either near established adults of the ascidian Botrylloides leachii or without competition. In creased larval planktonic durations reduced colony growth in the absence of competition; colonies that developed from longer or delayed larval durations were 2 to 3 times smaller than those that developed from shorter durations. Colonies that developed from longer larval periods (natural or experimental) also experienced higher mortality (75 to 100%) than those that settled quickly (20 to 42%), but these effects varied between experiments and seasons. In winter, W. subtorquata colonies of longer larval planktonic durations experienced greater mortality when adjacent to established B. leachii, whereas differences in colony growth due to planktonic duration were reduced by adjacent B. leachii. The influence of B. leachii varied between experiments in different seasons, however, and did not alter colony performance in summer. Our findings demonstrate that while increased larval planktonic duration can be costly for post-dispersal growth and survival, some differences can be mediated by species inter-actions and environmental variability. This suggests that while connectivity among populations that take longer to disperse may be limited, it may also be influenced in complex ways by the post-recruitment environment and not simply dispersal duration.

Between 2005 and 2012, 61 marinas and harbours around the English coast were surveyed to record the occurrence of non-native species (NNS) of sessile invertebrates. From these surveys, geographic distributions are described for eight species of ascidians, six bryozoans and five other species. A mean of 6.7 sessile invertebrate NNS per site (range 0–13 species) was recorded. At the 43 sites on the English Channel coast, the mean was 7.8 NNS per site, and all of the ten English sites that had ≥ 10 NNS were in the western or central region of the Channel coast. Ten sites on the Channel coast surveyed in 2004 were re-visited at least once in 2009 or 2010, and the mean number of sessile invertebrate NNS had increased from 6.0 to 7.6 species per site. Combining data from all visits in 2005–2012 for the sites surveyed in 2004, the mean number of NNS recorded per site rose to 9.2. Very rapid rates of spread along the English coast and beyond are inferred for the erect bryozoan Tricellaria inopinata d’Hondt & Occhipinti Ambrogi, 1985 and the ascidian Corella eumyota Traustedt, 1882. At least five recently arrived species that were limited to a small number of sites in 2012 are expected to increase markedly in geographic range and prevalence: the ascidians Asterocarpa humilis (Heller, 1878), Botrylloides diegensis Ritter and Forsyth, 1917, and Didemnum vexillum Kott, 2002; and the bryozoans Schizoporella japonica Ortmann, 1890 and Watersipora subatra (Ortmann, 1890). Rapid assessment surveys of marinas and similar sites proved to be an effective means of documenting the arrival and spread of NNS over a relatively wide geographic range and of distinguishing different rates of progression.

The colonial marine invertebrate Botrylloides leachii belongs to the Tunicata subphylum, the closest invertebrate relatives to the vertebrate group and the only known class of chordates that can undergo whole-body regeneration (WBR). This dramatic developmental process allows a minute isolated fragment of B. leachii’s vascular system, or a colony excised of all adults, to restore a functional animal in as little as 10 days. In addition to this exceptional regenerative capacity, B. leachii can reproduce both sexually, through a tadpole larval stage, and asexually, through palleal budding. Thus, three alternative developmental strategies lead to the establishment of filter-feeding adults. Consequently, B. leachii is particularly well suited for comparative studies on regeneration and should provide novel insights into regenerative processes in chordates.

Whole-body regeneration (WBR)—the formation of an entire adult from only a small fragment of its own tissue—is extremely rare among chordates. Exceptionally, in the colonial ascidian Botrylloides leachii (Savigny, 1816) a fully functional adult is formed from their common vascular system after ablation of all adults from the colony in just 10 d, thanks to their high blastogenetic potential. While previous studies have identified key genetic markers and morphological changes, no study has yet focused on the hematological aspects of regeneration despite the major involvement of the remaining vascular system and the contained hemocytes in this process. To dissect this process, we analyzed colony blood flow patterns using time-lapse microscopy to obtain a quantitative description of the velocity, reversal pattern, and average distance traveled by hemocytes. We also observed that flows present during regeneration are powered by temporally and spatially synchronized contractions of the terminal ampullae. In addition, we revised previous studies of B. leachii hematology as well as asexual development using histological sectioning and compared the role played by hemocytes during WBR. We found that regeneration starts with a rapid healing response characterized by hemocyte aggregation and infiltration of immunocytes, followed by increased activity of hemoblasts, recruitment of macrophage-like cells for clearing the tissues of debris, and their subsequent disappearance from the circulation concomitant with the maturation of a single regenerated adult. Overall, we provide a detailed account of the hematological properties of regenerating B. leachii colonies, providing novel lines of inquiry toward the decipherment of regeneration in chordates.

(250 words)Whole-body regeneration, the formation of an entire adult from only a small fragment of its own tissue, is extremely rare among chordates. Exceptionally, in the colonial ascidianBotrylloides leachii, a fully functional adult is formed from their common vascular system, upon ablation of all adults from the colony, in just 10 days thanks to their high blastogenetic potential. While previous studies have identified key genetic markers and morphological changes, no study has yet focused on the haematological aspects of regeneration despite the major involvement of the remaining vascular system and the contained haemocytes in this process. To dissect this process, we analysed colony blood flow patterns using time-lapse microscopy to obtain a quantitative description of the velocity, reversal pattern, and average distance travelled by haemocytes. We also observed that flows present during regeneration are powered by temporally and spatially synchronized contractions of the terminal ampullae. In addition, we revised previous studies onB.leachiihaematology as well as asexual development using histological sectioning, and compared the role of haemocytes during whole-body regeneration. We found that regeneration starts with a rapid healing response characterized by blood clotting and infiltration of immunocytes, followed by increased activity of haemoblasts, recruitment of macrophage-like cells for clearing the tissues of debris, and their subsequent disappearance from the circulation concomitant with the maturation of a single regenerated adult. Overall, we provide a uniquely detailed account of the haematological properties of regeneratingB.leachiicolonies, providing novel lines of inquiry towards the decipherment of regeneration in chordates.

Individuals of colonial animals (e.g. zooids) are in continuous turnover. In ascidians colonial or solitary species have evolved by convergence multiple times. Colonial Botryllus and Botrylloides are well-studied genera that exhibit colony-wide developmental mechanisms that regulate synchronous and orchestrated cycles of budding and turnover of zooids. The origins of modular developmental mechanisms that facilitated the evolution of coloniality in this group remain unclear. To reconstruct ancestral states of coloniality we studied Symplegma brakenhielmi, a sister taxon of the botryllids. S. brakenhielmi zooids are embedded in a common tunic and present a similar vascular system as the botrylloides, however development and turnover of zooids occurs asynchronously and in a more independent manner. We generated a table of common stages of budding in Symplegma and Botryllus for comparative studies of asexual development. We tested dependent processes of budding among individuals of the colony by systemic bud or zooid removals. Although our results showed a higher degree of independence in bud development in S. brakenhielmi, we found a subtle colony-wide regulatory mechanism of modular development, i.e. new buds expedited development after the removal of all buds in the colony. Next, we characterized external morphology, ultrastructure, and abundance of circulatory blood cells in the vascular system of S. brakenhielmi. Macrophage-like cells (MLCs) are involved in zooid resorption and turnover. Proportions of MLCs in the blood of S. brakenhielmi corresponded to the peak of occurrence of this cell type during the budding cycle of B. schlosseri. We found several new blood cell types in S. brakenhielmi, including two cell types that resemble circulatory progenitor stem cells of other botryllid colonial ascidians. These cells showed features of undifferentiated cells and expressed mitotic marker Phospho-histone H3. Comparative studies of S. brakenhielmi and B. schlosseri allow us to discuss possible changes in the regulation of modular development (i.e. regulation of life and death in the colony), and a possible contribution of circulatory blood cells in budding processes. We propose that the higher degree of developmental independence in S. brakenhielmi budding is a result of its ancestral solitary mode of development.

In marine systems subject to vessel traffic, the likelihood of an invasion by an exotic species typically increases with the arrival of each infested boat. In this paper, recreational boating activity patterns and boat fouling by tunicates were compared between eastern Canada and central New Zealand, and the relative importance of boat characteristics on fouling was evaluated using Boosted Regression Tree analysis. For Canada, we also determined the relative importance of boat characteristics and propagule exposure (i.e., the interaction between tunicate density in source region and time in water) on patterns of boat fouling. Approximately half of boats examined during the fall in eastern Canada and during the summer in central New Zealand were fouled by tunicates. Although there was a greater richness of tunicate species on New Zealand boats, the two countries had several species or genera in common, including Botryllus schlosseri, Ciona spp. and Botrylloides spp. The time since last boat maintenance was longer in New Zealand than in Canada. However, boat fouling and boat-mediated spread may be facilitated in Canada by boating activity patterns there, as the movement of boats among multiple marinas is considerably greater than in New Zealand. Among the boat characteristics, voyage type, the time that boats spent in water (Canada) and time since last application of antifouling paint (New Zealand) were among the best predictors of boat fouling. However, our results from Canada showed that propagule exposure was more important than boat characteristics in predicting the presence of the invasive colonial tunicate, B. schlosseri. This study shows the importance of small boats as potential vectors for tunicates and demonstrates that predictive models for the spread of biofouling species should be based on regional boating patterns, boating characteristics, and local propagule exposure.

Approximately 200 grey PVC settlement plates were hung along at sites along the Dutch coast at a depth of 1 meter. The cover of each species that could be identified on the plates was recorded every three months from March 2009 to March 2010 to study the role of native versus non-native ascidians in the succession of marine fouling communities. Three native ascidian species, Ciona intestinalis, Ascidiella aspersa and Botryllus schlosseri, and five species of cryptogenic and/or non-native origin, Molgula socialis, Styela clava, Diplosoma listerianum, Botrylloides violaceus and Didemnum vexillum, were recorded. After the Cnidaria, Ascidiacea were found to be the group that explained most of the differences found in species communities on settlement plates at different stages in the succession. Regardless of the fact that non-native ascidian species were very abundant at times, their role in the succession of the studied marine fouling communities appears to be limited. After the polyp stage of the moon jelly Aurelia aurita and hydroid species of the genus Obelia, the two native ascidian species Ciona intestinalis and Ascidiella aspersa explained most of the differences that were found between the successive stages in the fouling communities. In general, the three native ascidian species appeared to determine the succession of the fouling communities more than the five cryptogenic and/or nonnative ascidian species. Whether this is linked to species being either native or non-native is questionable however. The various ascidian species differed strongly in the period of settlement, the ability to settle on prior settlers and the ability to survive the winter. The non-native ascidians settled mostly in June to December, while the native ones settled from March to December. Although the native ascidians M. socialis and B. schlosseri were found to be restricted in their settlement by the presence of prior settlers, the non-native species B. violaceus and D. vexillum were not. Native and non-native ascidian species survived the winter equally well. Native C. intestinalis and A. aspersa and non-native B. botrylloides and D. vexillum were most successful in surviving winter water temperatures of just below 0°C. Thus the results of this study, based on one year of data, suggest that the roles of ascidian species in the succession of fouling communities vary, and that being native or non-native is not necessarily linked to the degree to which species influence the development of marine fouling communities. Their individual seasonality, abilities to overgrow prior settlers and survival of winter temperatures appear to be more important.

Botrylloides simodensis, belonging to the family Botryllidae, is a common species on the rocky shore around Shimoda. Since this species was first reported in 1981, it has been noticed that some B. simodensis colonies have distinctly different ground colors and color patterns from colonies including type specimens (holotype and all of paratypes) of B. simodensis. However, as there is no remarkable difference among them in morphology other than the colonial colors and color patterns, they have been considered to belong to the same species. In the present work, colonies of B. simodensis sensu lato were divided into three groups, two major and one minor, based on their color-morphs and periods of sexual reproduction. In one of the major groups, B. simodensis sensu strict, the period of sexual reproduction was limited from July to September, as shown in the original description. The other major group engaged in sexual reproduction for much longer than the former group, and they frequently made clusters of vascular vessels and underwent vascular budding. Therefore, the group with the long period of sexual reproduction was described as a new species of the genus Botrylloides here. In addition, phylogenetic analysis based on mtDNA sequences of these groups indicated that each group had a peculiar sequence, and that they were genetically separated.

Whether the non-native seaweed Sargassum muticum can displace or impact native eel- grass Zostera marina has been debated in the literature, based on differing substrate requirements of the 2 species. Field observations in Bamfield, British Columbia, Canada, revealed that the non-native S. muticum successfully inhabits an eelgrass bed through colonizing siphons of the native clam Tresus capax. Numerical or physical facilitation of S. muticum into the eelgrass bed by T. capax may be tem- pered by seaweed quality or condition. We used field sampling to investigate whether seaweed condi- tion differs between 2 attachment substrates (clam siphon and rock) as a proxy for habitat quality. At- tachment substrate promoted different morphologies of S. muticum; individuals attached to clam siphons expressed morphologies consistent with sheltered areas compared to individuals attached to rock, which expressed wave-exposed morphologies. Habitat association with the different morpholo- gies supported differences in the epibiont communities colonizing S. muticum. Further, S. muticum subsequently facilitated incursion of the non-native tunicates Styela clava and Botrylloides violaceous into the eelgrass bed through habitat provisioning. By facilitating 2 additional invaders, S. muticum enhances the level of invasion in the eelgrass bed. This non-native seaweed has the potential to disrupt the persistence of eelgrass in this system.

Two botryllid species, Botryllus schlosseri and Botrylloides leachii are listed in a recent literature review on the ascidian species inventory from the Red Sea (Shenkar, Mar Biodiv 42:459–469, 2012). However, a detailed literature search does not reveal any scientific documentation for the existence of these species in the Red Sea.

Several botryllid colonies of the genus Botrylloides from the Mediterranean coast of Israel are analysed. Three species are described: B. leachii, which differs because of its smaller size, from specimens collected from other European seas and is considered a case of dwarfism due to the particular hydrological conditions of the study area; B. anceps, a species originally described from the Indo-Pacific region, which probably migrated from the Red Sea; and Botrylloides israeliense, a new species. The distinction between the genera Botryllus and Botrylloides is discussed, and the original Milne-Edwards diagnosis based on differences in the structure of the system is recognised as still valid. Lastly, the diagnostic features usually employed in specific taxonomic determinations of Botryllinae are analysed.

Botryllid tunicates have become increasingly important as model experimental organisms in a variety of biological and ecological fields, including invasion ecology. This paper summarizes existing botryllid culture methods and offers new ones for culturing Botryllus schlosseri (golden star tunicate) and Botrylloides violaceus (violet tunicate) under laboratory conditions. Techniques for maintaining adult colonies, obtaining larvae, and establishing juvenile cultures are presented. Considerations for designing laboratory experiments using botryllid tunicates are discussed. Key words : Botrylloides , Botryllus , culture techniques, golden star tunicate, violet tunicate Introduction Botryllid tunicates have become increasingly important as model experimental organisms in a variety of biological fields such as immunology, physiology, ecology, genetics, developmental biology, evolutionary biology, and aging (e.g., Boyd et al. 1986; Rinkevich and Shapira 1998; Manni et al. 2007 and references therein). Golden star tunicate

Most ascidian larvae settle and begin adhesion by means of three mucus secreting and sensory organs, the adhesive papillae or palps. However, the adhesive papillae of Botrylloides genus larvae, despite their name, have only a sensory function. By immunohistochemical localization of serotonin and β‐tubulin, we demonstrated that the adhesive papillae of Botrylloides leachi contain two distinct types of neurons with different localization and possibly a different function. The central neurons emerge from the tunic at the apex of papillae and probably have a mechanosensory function. The lateral neurons contain serotonin and may play a role in the mechanism of metamorphosis triggering. Moreover, by histological analysis we found numerous secreting cells, clustered in the centre of the area among the three papillae, forming a glandular organ. This organ could perform the attachment of the larva to the substrate, which is traditionally considered to be operated by the anterior epidermis of the larva, with a suck...

The phenomenon of whole body regeneration (WBR) from minute soma fragments is a rare event in chordates, confined to the subfamily of botryllid ascidians and is poorly understood on the cellular and molecular levels. We assembled a list of 1326 ESTs from subtracted mRNA, at early stages of Botrylloides leachi WBR, and classified them into functional categories. Sixty-seven (15%) ESTs with roles in innate immunity signaling were classified into a broad functional group, a result supported by domain search and RT–PCR reactions. Gene ontology analysis for human homologous to the immune gene category, identified 22 significant entries, of which “peptidase activity” and “protease inhibitor activity”, stood out as functioning during WBR. Analyzing expressions of serine protease Bl-TrSP, a representative candidate gene from the “peptidase activity” subgroup, revealed low transcript levels in naïve vasculature with upregulated expression during WBR. This was confirmed by in situ hybridization that further elucidated staining restricted to a circulating population of macrophage cells. Furthermore, Bl-TrSP was localized in regeneration niches within vasculature, in regenerating buds, and in buds, during blastogenesis. Functional inhibition of serine protease activity disrupts early remodeling processes of the vasculature microenvironment and hinders WBR. Comparison of genome-wide transcription of WBR with five other developmental processes in ascidians (including metamorphosis, budding and blastogenesis), revealed a broad conservation of immune signaling expressions, suggesting a ubiquitous route of harnessing immune-related genes within a broader range of tunicate developmental context. This, in turn, may have enabled the high diversity of life history traits represented by urochordate ascidians.

Subtidal benthic communities show distinct patterns of community structure related to substrate angle. Suspension-feeding invertebrate communities often dominate vertical and undercut rock surfaces, while macroalgae dominate horizontal surfaces. One factor that may shape this pattern is sea urchin grazing, which is often more intense on horizontal surfaces. The native Gulf of Maine ascidian Aplidium glabrum, like other native ascidians, is generally restricted to vertical and undercut rock walls, whereas the introduced ascidian Botrylloides violaceous is often abundant on horizontal surfaces. We tested the hypothesis that this pattern could be due to differing predation intensity on these two ascidians by Strongylocentrotus droebachiensis, a dominant omnivore in the Gulf of Maine. Feeding preference of S. droebachiensis on the native A. glabrum vs. B. violaceous was estimated in the laboratory and in field experiments. Laboratory results show that S. droebachiensis prefers to feed on the native ascidian A. glabrum over B. violaceous. In the field, potential differences in the impact of S. droebachiensis on the two species were masked by the much greater growth rate of B. violaceus compared to A. glabrum. These results may help explain observed patterns in ascidian distribution in the Gulf of Maine, and ultimately the overall success of B. violaceus as a major invader throughout New England.

Invasive ascidians are a growing concern for ecologists and natural resource managers, yet few studies have documented their short- and long-term temporal patterns of abundance. This study focuses on the invasion of the Gulf of Maine by the colonial ascidians Botryllus schlosseri, Botrylloides violaceus, Diplosoma listerianum and Didemnum sp. A. We examined the time of arrival and potential vectors for these four invasive ascidians using survey data (collected from 1969 onwards) and literature documentation. We also compared the dominance and seasonal patterns of abundance of these species using data from two identical panel studies; one conducted from 1979 to 1980, the other from 2003 to 2005. Didemnum and Botrylloides were most likely first introduced into the Damariscotta River, Maine in the early 1970's through oyster aquaculture while Botryllus and Diplosoma were probably transported by commercial and recreational vessels. The overall abundance of colonial ascidians has increased since 1979 and 1980. Botryllus was the only invasive colonial ascidian present during the 1979 to 1980 study and accounted for an average of 6.16% cover on panels. From 2003 to 2005, the more recently arrived colonial ascidians Botrylloides and Didemnum accounted for 7.38% and 2.08% cover respectively, while Botryllus covered only 1.16%. Our results reveal a shift in seasonal abundance between 1979 to 1980 and 2003 to 2004. In 1979 and 1980, colonial ascidians had the highest percent cover in fall and winter while in 2003 and 2005 they had highest percent cover in summer and fall. Seasonal patterns of space occupation by colonial ascidians were correlated with seasonal changes in seawater temperature.

Over the last 50 yrs seven non-native ascidians have settled in The Netherlands, concentrated in the two periods 1974–1977 and 1991–2004 (i.e., Styela clava , Aplidium glabrum , Diplosoma listerianum , Didemnum sp., Botrylloides violaceus, Molgula complanata and Perophora japonica ). The year of the introduction of B. violaceus remains a matter of dispute because many of the Botrylloides specimens that are recorded in western Europe, have been identified as the closely resembling species B. leachi . Only Didemnum sp. has become a true invasive species and has become a threat to native ecosystems, especially in the province of Zeeland, by its ability to overgrow virtually all hard substrata present. This includes rocks, stones, sand, algae and almost all sessile marine animals. The sudden population expansion of the didemnid from 1996 onward, coincided with the cold winter of 1995–1996, which caused decreased population sizes of many marine animals. The resulting increase in the availability of suitable substrates for settlement and the strong decrease of grazing sea urchins, may have triggered the population expansion. Studying its population dynamics, the optimal growing temperature for Didemnum sp. appears to be 14–18 °C. Virtually all colonies die when the water temperature gets colder than 5 °C. Colonies growing on live marine animals seem to be more resistant to the cold, than those growing on rocks, stones and plants. Two potential predators of the didemnid have also been recorded in Dutch waters: the gastropods Trivia arctica and Lamellaria sp.

The external peripheral termini of vasculature in botryllid ascidians (ampullae) are important in the expression of effector mechanisms during histocompatibility reactions. We study here morphological changes to the microstructure during allorecognition in Botrylloides subpopulation I (SP1) from the Mediterranean coast of Israel, where special giant ampullae, called megaloampullae are developed hours after first allogeneic challenge. The basal part of each naive ampulla consists of a sheath of continuous squamous epithelial cells (1 μm×20 μm). At the tip of the ampullae, about 130 epithelial cells become columnar with wide apical pads (15 μm×5–10 μm), that contain electron-opaque “adhesive vesicles”. Foci of crystallizations were observed within the vesicles of some columnar cells. Ampullar epithelial cells develop internal folds that support tight attachments between circulating blood cells (most of them are pigment cells) and ampullar epithelium. During the histoincompatibility process, the tunic peripheral edge at the interaction area becomes highly convoluted. Filaments (up to 1.16 μm) accumulate in the interaction tunic matrix. Some of these filaments encircle destructed cells. Fragmented cell membranes are also found next to the reacting ampullae. The most characteristic feature of the Botrylloides SP1 rejection reaction is dilation of the ampullae. The ampullar epithelium becomes “curly” when megaloampullae are formed. Within the curly region, about 30% of the squamous epithelial cells are further stretched, up to 33 μm in length. Some additional mechanisms are suggested to explain the fast increase (up to seven times) in ampullar width that is recorded within a few hours from the first allogeneic contact.

Monoclonal antibodies are an important tool in the study of botryllid ascidians' immunology and developmental biology. Here we describe the development of a panel of 38 monoclonal antibodies that are specific to Botryllus schlosseri (Ascidiacea; subfamily Botryllinae) cell surface antigens. Many of these hybridomas recognize (by enzyme-linked immunosorbent assay and immunohistochemistry) epitopes of Botrylloides subpopulations (SP) II and III from the Mediterranean coast of Israel and show, on blood cell smear assays, reactions with subsets of Botryllus circulating blood cells. Fluorescence-activated cell sorting analyses using antibodies positive for botryllid tissues revealed up to 3.6% positive cells. ELISA screenings were performed with 64 new monoclonal antibodies on 5 different individual botryllid ascidian colonies (B. schlosseri, Botrylloides). The positive antibodies in this panel identified a large number of different antigenic determinants, some of which distinguish Botryllus versus Botrylloides colonies, and other, different colonies within these two species, or different cell types within tissues, embryos, and buds of individual colonies. Only 21 monoclonal antibodies tested positive with all colonies. Cross-reactivity with at least one Botrylloides colony was recorded in 49 hybridomas that identified Botryllus cells. This wide panel of monoclonal antibodies is the first such detailed set of monoclonals available for studies on botryllid ascidians.

The mode of allo-recognition among colonies was investigated in a viviparous ascidian, Botrylloides lentus. Each embryo is enveloped by a pouch of epithelial cells (brood pouch), and is brooded in the vascular lumen of the parental colony. That is, the parental colony tolerates the presence of semi-allogeneic conspecifics (embryos) in the vascular system. A rejection reaction occurs when incompatible colonies are brought into contact at their growing edges. The inflammatory rejection reaction is limited to a small area where the tunic of two colonies has partially fused. On the contrary, incompatible colonies fuse and their blood vessels become interconnected with one another, when they are brought into contact at artificially cut surfaces. This mode is the same as those of other viviparous species of Botrylloides, B. fuscus and B. violaceus. A relationship between viviparity and the loss of allo-recognition in the vascular system is suggested.

Simple and colonial ascidians were collected at different depths at Arraial do Cabo, Rio de Janeiro, between 2000 and 2003. The collection here presented includes representatives of the families Clavelinidae (Clavelina oblonga), Polycitoridae (Cystodytes dellechiajei), Polyclinidae (Polyclinum constellatum and Polyclinum molle sp. nov.), Holozoidae (Distaplia bermudensis), Ascidiidae (Ascidia sydneiensis and Phallusia nigra), Styelidae (Botrylloides giganteum, Botrylloides nigrum, Symplegma brakenhielmi, Symplegma rubra, Polyandrocarpa anguinea, Eusynstyela floridana, Eusynstyela tincta and Styela plicata), Pyuridae (Herdmania pallida and Microcosmus exasperatus). Didemnids were also collected in the area but were not considered here. Of the 17 species found, one (Polyclinum molle sp. nov.) is a new species in the family Polyclinidae and the others are all species with tropical distribution.

Fifty-one species are discussed of which five are new. Of the remaining 46 species, 20 previously known from one to three records and 11 more frequently recorded have their known geographic range and/or range of morphological variation extended and the synonymy of a further 15 species is revised. Taxonomic problems, including phylogeny, are addressed in Rhopalaea crassa, Pycnoclavella detorta, Ecteinascidia spp. and Plurella (see Phallusia polytrema). New species are Sigillina pulvinus, Polyclinum corbis, Perophora longicaulis, Stolonica brevigastra and Botrylloides saccus.

SUMMARYUnderstanding how the shape and motion of an aquatic animal affects the performance of swimming requires knowledge of the fluid forces that generate thrust and drag. These forces are poorly understood for the large diversity of animals that swim at Reynolds numbers (Re) between 100 and 102. We experimentally tested quasi-steady and unsteady blade-element models of the hydrodynamics of undulatory swimming in the larvae of the ascidian Botrylloides sp. by comparing the forces predicted by these models with measured forces generated by tethered larvae and by comparing the swimming speeds predicted with measurements of the speed of freely swimming larvae. Although both models predicted mean forces that were statistically indistinguishable from measurements, the quasi-steady model predicted the timing of force production and mean swimming speed more accurately than the unsteady model. This suggests that unsteady force (i.e. the acceleration reaction) does not play a role in the dynamics of steady undulatory swimming at Re≈102. We explored the relative contribution of viscous and inertial force to the generation of thrust and drag at 100102) and low (

Xenogeneic rejection reactions were histologically examined among four compound ascidians of the genus Botrylloides; B. simodensis, B. lentus, B. fuscus and B. violaceus, to compare with the allogeneic rejections of these species. When the incompatible conspecifics were brought into contact, hemolytic rejections occurred at the point where the tunic of the two colonies was partially fused. Xenogeneic contact at their growing edges induced hemolytic rejection in some combinations (B. simodensis-B. lentus, B. lentus-B. fuscus, and B. fuscus-B. violaceus), while conspicuous reaction was not found in the other combinations. Since the hemolytic rejection requires the partial fusion of tunic, the occurrence of hemolytic rejection suggests that the tunic cuticle of the colonies does not discriminate the facing colony from conspecifics. On the other hand, whereas cut surface contact between incompatible conspecifics induced intense rejection in B. simodensis, it resulted in fusion (formation of vascular connection) even in the combination in which the growing edge contact resulted in rejection. In xenogeneic combination, the cut surface contact of colonies always resulted in an intense rejection reaction except for B. fuscus-B. violaceus in which hemolytic reactions did not occur. The absence of hemolytic rejection suggests that the effector system for rejection reaction is not activated in this combination. Activity of phenoloxidase, a key enzyme of the rejection reaction, indicates lower reactivity in B. lentus, B. fuscus and B. violaceus than that in B. simodensis.

Morula cells (MCs), ubiquitous ascidian cells which participate in alloimmune reactions, were studied in allogeneic and xenogeneic assays performed with Botryllus schlosseri (Pallas, 1766) colonies from the Mediterranean and the Pacific coast, USA. Experiments were performed on the morphological level, on blood cells sampled or vitally labeled in situ and on histological sections. In non-interacting B. schlosseri during blastogenic cycles A to C, MCs congregate in tentacles of the zooids' siphons. During the takeover phase of blastogenesis they disappear, appearing again in the siphons of the newly developed generation of zooids. In both compatible and incompatible allogeneic reactions their numbers in interacting ampullae increased fourfold within 2 h after first contacts, reaching up to 50% of the total blood-borne cells in ampulla lumens. In rejection processes most of these cells died, while in autologous or allogeneic fusions they disappeared from interacting ampullae within days of the reaction. During allogeneic rejections, we followed cases (up to 1 week) in which MCs crossed all morphological/physiological barriers between noncompatible colonies and entered the tunic matrices and blood systems of the interacting partners, forming successful microchimerism. In xenogeneic assays with Botrylloides sp., MCs concentrated at the xenogeneic boundary, but they never crossed into the other species. One week after fusion or rejection interactions, large numbers of macrophages clearing dead cells were found in contact areas. This is the first evidence for compartmentalization of specific cellular defense reactions in the tunicate circulatory system.

Residents within any community can affect the larval settlement of both their own and other species. In marine sessile communities resident adults can affect larval settlement by preying on settling larvae, removing or adding space for the larvae to colonize, or stimulating or prohibiting larval settlement on available substratum nearby. To examine those processes by which residents affect settlement, we exposed experimental substrata with three densities of adults of a single species at a site in eastern Long Island Sound, USA for a 24-h period. Four species of common ascidians, Botryllus schlosseri (Pallas), Botrylloides diegensis Ritter and Forsyth, Diplosoma macdonaldi Herdman, and Molgula manhattensis (De Kay), were used in 11 separate experiments. Few individuals of any species settling attached to the surfaces of these species and this resulted in the main effect of these residents being the usurpation of space and the restricting of settlement to unoccupied areas. A model is also presented to explain the apparent aggregated settlement of several species in open areas adjacent to the resident ascidians. From this model we suggest that the aggregated settlement can result from limited larval mobility such that some larvae that contact and reject the resident species as settlement sites may subsequently contact open surfaces of the same substratum and increase settlement densities there over those observed on control substrata. Finally, settlement data for several species indicate that Molgula may influence settlement by preying on larvae.

The importance of larval dispersal in determining the distribution and abundance of benthic marine organisms is well recognized; however, the contribution of post-larval dispersal has not been measured. I compared the dispersal of swimming larvae with that of rafting colonies in a population of compound ascidians, Botrylloides sp., living attached to leaves of the eelgrass Zostera marina in Tomales Bay, California, USA in 1990–1992. Colonies rafting on broken eelgrass traveled over 200 times farther and had comparable recruitment success relative to swimming larvae. The recruitment of rafting colonies into new habitats was facilitated by the ability of thesecolonial animals to grow asexually onto surrounding substrata. Rafting colonies brooded larvae that were subsequently relased after the colony settled into a new habitat. These results suggest that colonization of new habitats can occur by post-larvae as well as larvae, and that long-range dispersal by species with short-lived larvae may occur by post-larval rafting.

Summary The initiation of the first embryo-derived cell cultures and the establishment of in vitro metamorphosis from aseptic tadpole larvae are described in the cosmopolitan, shallow water colonial tunicate, Botryllus schlosseri. A total of more than 1,600 embryos, collected from different colonies at various stages of blastogenic cycles, were used in ten experiments (up to 350 embryos/experiment). Embryos were fully dissociated by mechanical or chemical treatments. In three experiments we succeeded in initiating continuous cell lines from embryonic tissue (designated NIO-BSE-1 to −3). Using different substrates and supplements, we found that embryonic cells cultured in botryllid cell culture medium (Rinkevich and Rabinowitz, 1993) supplemented with either chick embryo extract or heat inactivated Botrylloides hemolymph and on plastic or gelatin coated substrates may acquire cell-line characteristics. Tunic cells of maternal origin were found in all wells of primary cultures but subsequently disappeared. ...

Previous work on the distribution of historecognition alleles in botrylloid ascidians has revealed extensive polymorphism at this locus, but has yielded little information on the distri- bution of alleles among populations and the rate of allele proliferation within populations. We first present preliminary data on the genetics of historecognition in Botrylloides diegense, a West Coast native that was introduced to the East Coast in 1972. We then compare the distribution of alleles in 2 populations of different size in a Maine estuary. Finally, we compare the alleles in those 2 popula- tions to the alleles in a population at Woods Hole, the site of the original introduction. Our results indicate that a small, peripheral population contains fewer alleles than a large population and sug- gest that alleles have proliferated rapidly since this species was introduced to the East Coast. The rapid proliferation of alleles reported here provides additional support for the hypothesis that historecognition systems are subject to frequency dependent selection in natural populations. 0 1992 Wiley-Liss, Inc. Colonial marine invertebrates typically possess a genetically based historecognition system that regulates fusion among conspecifics, and these sys- tems are generally characterized by high allotype diversity (reviewed in Grosberg, '88a). Unfortu- nately, a lack of information on the genetics of historecognition in most of the subject species pre- vents direct interpretation of allele frequencies from allotype data. However, in the few species for which the genetic mechanism underlying historecognition is known (all botrylloid ascidians), high levels of allele polymorphism are common (Karakashian and Milkman, '67; Tanaka and Watanabe, '73; Mukai and Watanabe, '75a,b; Scofield et al., '82). Work to date has focused mainly on assaying the number of alleles in a given population. However, further progress on understanding the processes that generate these high levels of polymorphism requires consideration of patterns of allele distri- butions both within and among populations (Gros- berg, '88a). Of particular interest is information on the effects of population size and age on the level of polymorphism. This problem has received little empirical attention due to the difficulties inher- ent in obtaining historical information on allele distributions. We here explore patterns of historecognition allele distributions in the colonial ascidian Botryl- loides diegense Van Name. Botrylloides diegense is native to southern California (Van Name, '45) but

11 Páginas ; 3 Figuras ; 3 Tablas, The Order Stolidobranchiata comprises the families Pyuridae, Styelidae and Molgulidae. Early molecular data was consistent with monophyly of the Stolidobranchiata and also the Molgulidae. Internal phylogeny and relationships between Styelidae and Pyuridae were inconclusive however. In order to clarify these points we used mitochondrial and nuclear sequences from 31 species of Styelidae and 25 of Pyuridae. Phylogenetic trees recovered the Pyuridae as a monophyletic clade, and their genera appeared as monophyletic with the exception of Pyura. The Styelidae, on the other hand, appeared as a paraphyletic group split into several clades. One of them was formed by solitary oviparous species, of which the Pyuridae were a sister group. A second clade included the colonial genera Botryllus, Botrylloides and Symplegma. The remaining colonial and solitary genera formed several poorly resolved clades. One of the more species genus, Polycarpa, was shown to be polyphyletic, and the species Styela plicata grouped into two genetically distant clades suggesting the existence of two cryptic species. The internal phylogeny of Styelidae has bearings on the origin of coloniality in this family. We suggest to abandon the traditional division of colonial forms into social and compound species and use instead the categories of aggregated colonies that do not have common vascular systems, and integrated colonies, that do possess such systems. Our molecular results indicate that there have been several independent acquisitions of coloniality in the Styelidae, and that viviparity may be a pre-adaptation for a colonial life-style., This research was supported by a postdoctoral grant from the Spanish foundation ‘‘Fundación Alfonso Martín Escudero (FUNDAME)”, a FCT postdoctoral fellowship from the Portuguese Government (SFRH/BPD/41055/2007) and by project CTM2007-66635 of the Spanish Government and project 2007301026 (CSIC). This represents contribution no. 284 from the Ecology and Genetics Group at the University of Wollongong.

To examine the possible importance of limiting factors other than space for fouling organisms, a fouling panel study was carried out in Eel Pond, Woods Hole, Massachusetts, USA. The growth rate of the colonial tunicate Botrylloides leachii (Savigny) alone on fouling plates was significantly higher (P

Regeneration in adult chordates is confined to a few model cases and terminates in restoration of restricted tissues and organs. Here, we study the unique phenomenon of whole body regeneration (WBR) in the colonial urochordate Botrylloides leachi in which an entire adult zooid is restored from a miniscule blood vessel fragment. In contrast to all other documented cases, regeneration is induced systemically in blood vessels. Multiple buds appear simultaneously in newly established regeneration niches within vasculature fragments, stemming from composites of pluripotent blood cells and terminating in one functional zooid. We found that retinoic acid (RA) regulates diverse developmental aspects in WBR. The homologue of the RA receptor and a retinaldehyde dehydrogenase-related gene were expressed specifically in blood cells within regeneration niches and throughout bud development. The addition of RA inhibitors as well as RNA interference knockdown experiments resulted in WBR arrest and bud malformations. The administration of all-trans RA to blood vessel fragments resulted in doubly accelerated regeneration and multibud formation, leading to restored colonies with multiple zooids. The Botrylloides system differs from known regeneration model systems by several fundamental criteria, including epimorphosis without the formation of blastema and the induction of a “multifocal regeneration niche” system. This is also to our knowledge the first documented case of WBR from circulating blood cells that restores not only the soma, but also the germ line. This unique Botrylloides WBR process could serve as a new in vivo model system for regeneration, suggesting that RA signaling may have had ancestral roles in body restoration events., Author Summary Whole body regeneration (WBR) in Animalia is rare, confined to morphologically less complex taxa such as sponges, cnidarians, and flatworms. In the chordate phylum, only colonial ascidians (invertebrate chordates also known as sea squirts) have the documented ability to wholly regenerate. Once separated from the colony, any minute fragment of peripheral blood vessel (about 1 mm in length, containing 100–300 blood cells) of the colonial ascidian Botrylloides leachi regenerates an entire functional adult within one to three weeks. By following cellular and molecular events in Botrylloides WBR, we revealed that this system proceeds differently from regeneration events in other model organisms by several fundamental criteria. This is, for example, to our knowledge the first documented case of WBR initiating from circulating blood cells that restore not only the body tissue, but also the germ line. We found that retinoic acid (RA) signaling, previously reported in the regeneration of specific vertebrate tissues and organs, plays a major role in WBR via RA receptor expression throughout the entire regenerating animal. This suggests that RA signaling may have had ancestral roles in body restoration events. Elucidating the processes involved in this WBR system will improve our understanding of the nature of regeneration and the reduced regeneration capabilities represented in so many vertebrates., The uniqueBotrylloides whole body regeneration process could serve as a new in vivo model system for regeneration, suggesting that retinoic acid signaling may have had ancestral roles in body restoration events.

Milne Edwards (1841) introduced Botrylloides listing species with both feminine and neuter word endings. The International Code on Zoological Nomenclature, however, indicates that generic names ending -oides should be masculine unless the introducing author unambiguously indicated a different gender. The resulting uncertainty has caused prolonged confusion over the correct gender of Botrylloides. It is here affirmed that Milne Edwards did not provide a clear indication of gender and the general rule of the International Code applies: Botrylloides has masculine gender. The type species is B. rotifer Milne Edwards (its ending corrected from the feminine B. rotifera).

Two novel quinoline-2-carboxylic acid derivatives, perspicamides A (2) and B (3), were isolated from the Australian ascidian Botrylloides perspicuum. Extraction of the freeze-dried ascidian with methanol and purification of the extract by C18 MPLC followed by repeated C18 HPLC yielded the new compounds 2 and 3 as well as the known compounds botryllamides A-C (4-6). The structures of 2 and 3 were determined from analysis of 2D NMR spectra.

Sixteen monoclonal antibodies (MAbs) specific to 2 urochordate genera (Botryllus schlosseri and Botrylloides) intracellular epitopes were generated in mice immunized with a mixture of fresh and paraformaldehyde-fixed cells obtained from animal's blood and cells from dissociated organs. Hybridoma clones were selected by ELISA tests and immunohistochemistry assays on paraffin-embedded animal tissues. Five MAbs were tested for reactions with different zooidal organs and cell compartments; 7 MAbs were tested, separately, on 5 different botryllid colonies (3 Botryllus and 2 Botrylloides). The results revealed high polymorphism. Whereas some of the MAbs recognized, specifically, only part of the botryllid genotypes tested, others recognized only part of the cellular compartments. These MAbs will be used as an important tool in the study of botryllid ascidian immunology and developmental biology, revealing the first wide panel of MAbs specific to urochordate intracellular antigens.

Two new marine metabolites, 3Z, 6Z, 9Z-dodecatrien-1-ol (1) from the ascidian Botrylloides giganteum and 4H-pyran-2ol acetate from the sponge Ircinia felix (4) are herein reported. The known bromotyrosine compounds, 2-(3,5-dibromo-4-methoxyphenyl)-N,N,N-dimethylethanammonium (2) and 2,6-dibromo-4-(2-(trimethylammonium)ethyl)phenol (3), have been isolated from the sponge Verongula gigantea. Serotonin (5) is reported for the first time from the sponge Cliona delitrix, and tambjamines A (15) and D (16) isolated as their respective salts from the nudibranch Tambja eliora. Only tambjamine D presented cytotoxicity against CEM (IC50 12.2 µg/mL) and HL60 (IC50 13.2 µg/mL) human leukemya cells, MCF-7 breast cancer cells (IC50 13.2 µg/mL), colon HCT-8 cancer cells (IC50 10.1 µg/mL) and murine melanoma B16 cancer cells (IC50 6.7 µg/mL).