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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.

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.

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.

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.

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.

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.

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.