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Humans have intensively sailed the Mediterranean and European Atlantic waters throughout history, from the upper Paleolithic until today and centuries of human seafaring have established complex coastal and cross-seas navigation networks. Historical literature revealed three major long-lasting maritime routes (eastern, western, northern) with four commencing locations (Alexandria, Venice, Genoa, Gibraltar) and a fourth route (circum-Italian) that connected between them. Due to oceangoing and technological constraints, most voyages were coastal, lasted weeks to months, with extended resting periods, allowing the development of fouling organisms on ship hulls. One of the abiding travellers in maritime routes is the colonial ascidian Botryllus schlosseri already known since the eighteenth century in European and Mediterranean ports. This species, was almost certainly one of the common hull fouling travellers in all trade routes for centuries. Employing COI haplotypes (1008 samples) and microsatellite alleles (995 samples) on colonies sampled from 64 pan-European sites, present-day Botryllus populations in the Mediterranean Sea/European Atlantic revealed significant segregation between all four maritime routes with a conspicuous partition of the northern route. These results reveal that past anthropogenic transports of sedentary marine species throughout millennia long seafaring have left their footprint on contemporary seascape genetics of marine organisms., (© 2023. The Author(s).)

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.

The wide distribution of the ascidian Botryllus schlosseri along the Mediterranean coasts has been documented since the eighteenth century. However, despite copious documentation, analyses of dispersal modes and genetic profiles were limited to local populations or restricted regions. In order to get a pan-Mediterranean overview, 288 specimens from 11 populations of B. schlosseri from the western and eastern Mediterranean basins were sampled and analyzed using five microsatellite loci and COI sequences. Both molecular markers revealed high polymorphisms, with 182 microsatellites alleles and 54 COI haplotypes. Overall, F st , D est , and COI Ф pt values were 0.146, 0.635 and 0.322, respectively, reflecting a high genetic diversity and a significant genetic structure as compared to other B. schlosseri populations worldwide, reflected by substantially higher values for effective number of alleles (N e ) in the Mediterranean. A phylogenetic analysis of the COI sequences resulted in four distinct clades and two molecular operational taxonomic units (OTUs). We recorded a stronger genetic structure among the populations of the eastern basin compared to the western basin (microsatellites F st =0.217 versus 0.082; COI Ф pt =0.416 versus 0.171), suggesting either a restricted connectivity between the basins or a stronger genetic drift in each basin. The occurrence of two OTUs and different ecological conditions may also contribute to this finding. Mean Nei's genetic distance in the eastern Mediterranean populations was more than three times higher compared to the western basin. No correlation was observed between geographic and genetic distances (Mantel test), suggesting that maritime transport is the main dispersal vector of B. schlosseri colonies. The possibility that the Mediterranean is a center of diversity for B. schlosseri, and probably its site of origin, is further discussed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The colonial tunicate Botryllus schlosseri is a globally distributed, invasive ascidian that has colonized the Californian coasts of the USA during the mid-late 1940s and has, since the late 1980s, spread north to Washington. This study analyzes the population genetic characteristics of transient populations residing at the Elkhorn Yacht-Club (EYC), in central California (seven sessions, 1996-2008), which suffered periodic catastrophes caused by episodic fresh-water floods and a single sampling session (in the year 2001) of five West-Coast populations using the mtDNA COI gene and five microsatellite markers. EYC microsatellite results were further compared with the closely situated but persistent population of the Santa Cruz Harbor (SCH) to understand the impact on EYC population regeneration processes after the 2005-flood catastrophe. All microsatellites were highly polymorphic, revealing a large number of unique alleles at different sampling dates. Whereas pairwise θ did not reveal significant differences between the EYC time-series samplings, the overall θ was significant, as it was between all the 2001 West Coast populations. The most likely cluster number was 3 for the EYC samples whereas two K values were obtained (2 and 5) for the 2001 samples. Tajima's D and Fu's/Fs tests did not reject the null hypothesis for COI neutral evolution, except for in the EYC-2000, 2007 and two 2001 samplings. The wide geographical range of the analyses has indicated that following the EYC 2005-flood catastrophe, newcomers could have originated from neighboring populations, from deep-water colonies that may have escaped the 2005 low salinity event, or less expectedly, from far away West-Coast populations, while revealing that the SCH population is the most probable source for the EYC population., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The patterning of the modular body plan in colonial organisms is termed astogeny, as distinct from ontogeny, the development of an individual organism from embryo to adult. Evolutionarily conserved signaling pathways suggest shared roots and common uses for both ontogeny and astogeny. Botryllid ascidians, a widely dispersed group of colonial tunicates, exhibit an intricate modular life form, in which astogeny develops as weekly, highly synchronized growth/death cycles termed blastogenesis, abiding by a strictly regulated plan. In these organisms both astogeny and ontogeny form similar body structures. Working on Botryllus schlosseri, and choosing a representative gene from each of three key Signal Transduction Pathways (STPs: Wnt/β-catenin; TGF-β, MAPK/ERK), we explored and compared gene expression at different stages of ontogeny and blastogenesis. Protein expression was studied via immunohistochemistry, ELISA and Western blotting. Five specific inhibitors and an activator for the selected pathways were used and followed to assess their impact during the blastogenic cycle and the development of distinctive phenotypes. Outcomes show that STPs are activated and function (while not necessarily co-localized) during both ontogeny and astogeny. Cellular patterns in blastogenesis, such as colony architecture, are shaped by these STPs. These results are further supported by administering Wnt agonist and anatagonist, TGF-β receptor antagonists and inhibitors of Mek1/Mek2. Independent of their expression during ontogeny, some of the spatiotemporal patterns of STPs developed within short blastogenic windows. The results support the notion that while the same molecular machinery is functioning in Botryllus schlosseri astogeny and ontogeny, astogenic development is not an ontogenic replicate.

Naturally occurring histocompatibility responses, following tissue-to-tissue allogeneic contacts, are common among numerous colonial marine invertebrate taxa, including sponges, cnidarians, bryozoans and ascidians. These responses, often culminating in either tissue fusions or rejections, activate a wide array of innate immune components. By comparing two allorejection EST libraries, developed from alloincompatible challenged colonies of the stony coral Stylophora pistillata and the ascidian Botryllus schlosseri, we revealed a common basis for innate immunity in these two evolutionary distant species. Two prominent genes within this common basis were the immunophilins, Cyclophilin A (CypA) and FK506-binding protein (FKBP). In situ hybridizations revealed that mRNA expression of the coral and ascidian immunophilins was restricted to specific allorecognition effector cell populations (nematoblasts and nematocytes in the coral and morula cells in the ascidian). The expressions were limited to only some of the effector cells within a population, disclosing disparities in numbers and location between naïve colonies and their immune challenged counterparts. Administration of the immunosuppression drug Cyclosporine-A during ascidian's allogeneic assays inhibited both fusion and rejection reactions, probably through the inhibition of ascidian's immunocytes (morula cells) movement and activation. Our results, together with previous published data, depict an immunophilins-based immune mechanism, which is similarly activated in allogeneic responses of distantly related animals from sponges to humans., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The mechanisms that sustain stem cells are fundamental to tissue maintenance. Here, we identify "cell islands" (CIs) as a niche for putative germ and somatic stem cells in Botryllus schlosseri, a colonial chordate that undergoes weekly cycles of death and regeneration. Cells within CIs express markers associated with germ and somatic stem cells and gene products that implicate CIs as signaling centers for stem cells. Transplantation of CIs induced long-term germline and somatic chimerism, demonstrating self-renewal and pluripotency of CI cells. Cell labeling and in vivo time-lapse imaging of CI cells reveal waves of migrations from degrading CIs into developing buds, contributing to soma and germline development. Knockdown of cadherin, which is highly expressed within CIs, elicited the migration of CI cells to circulation. Piwi knockdown resulted in regeneration arrest. We suggest that repeated trafficking of stem cells allows them to escape constraints imposed by the niche, enabling self-preservation throughout life., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Allorecognition, distinguishing self from non-self allogeneic tissues is the underlying basis of innate immunity. In the colonial tunicate Botryllus schlosseri this historecognition is governed at a single genetic locus, Fu/HC (for fusibility/histocompatibility), with hundreds of co-dominantly expressed alleles. Several years ago, De Tomaso et al. (2005) have revolutionized the discipline of invertebrate allorecognition by describing a novel form of immune recognition in B. schlosseri, a non-vertebrate candidate histocompatibility gene (cFu/HC), revealing that allorecognition machinery in urochordates has nothing in common with the vertebrates' MHC-based histocompatibility. The authors reported absolute concordance of fusibility and cFu/HC genotype, predicted fusion/rejection outcomes in allorecognition settings from allelic polymorphism at the cFu/HC, also claiming cFu/HC gene expressions only in tissues directly engaged in histocompatibility. Here, we raise queries for the validity of the results and conclusions of De Tomaso et al. (2005) publication. Our reservations include discrepancies in the paper's results, including the perplexing absence of key sequencing material from public domains and above all, our own impugning outcomes. These include cloning efforts, in situ hybridization results, semi quantitative PCR outcomes, and the incongruence emerged between fusion/rejection profiles and cFu/HC segregated polymorphism that separately and cumulatively contradict the original publication. We conclude that Botryllus histocompatibility properties are not signaled in the claimed cFu/HC and that cFu/HC gene is unlikely the allodeterminant for Botryllus histocompatibility locus. Hence, the molecular nature of the Fu/HC locus in botryllid ascidians is still awaiting elucidation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In botryllid ascidians, allogeneic contacts between histoincompatible colonies lead to inflammatory rejection responses, which eventually separate the interacting colonies. In order to elucidate the molecular background of allogeneic rejection in the colonial ascidian Botryllus schlosseri, we performed microarray assays verified by qPCR, and employed bioinformatic analyses of the results, revealing disparate transcription profiles of the rejecting partners. While only minor expression changes were documented during rejection when both interacting genotypes were pooled together, analyses performed on each genotype separately portrayed disparate transcriptome responses. Allogeneic interacting genotypes that developed the morphological markers of rejection (points of rejection; PORs), termed 'rejected' genotypes, showed transcription inhibition of key functional gene groups, including protein biosynthesis, cell structure and motility and stress response genes. In contrast, the allogeneic partners that did not show PORs, termed 'rejecting' genotypes, showed minor expression changes that were different from those of the 'rejected' genotypes. This data demonstrates that the observed morphological changes in the 'rejected' genotypes are not due to active transcriptional response to the immune challenge but reflect transcription inhibition of response elements. Based on the morphological and molecular outcomes we suggest that the 'rejected' colony activates an injurious self-destructive mechanism in order to disconnect itself from its histoincompatible neighboring colony., ((c) 2010 Elsevier Ltd. All rights reserved.)

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.

The colonial ascidian Botryllus schlosseri manifests a unique allorecognition system that is controlled by a single histocompatibility haplotype, the Fu/HC locus. When two allogeneic incompatible colonies come into direct contact, they develop inflammatory-like rejection lesions, called points of rejection (POR). While screening for differentially expressed genes during POR formation, we developed and analyzed a cDNA library of expressed sequence tags (ESTs) with 1693 unique ESTs that were clustered and assembled into 217 contigs and 1476 singlets. About 51% of these ESTs showed high similarity (E-value 0.005) to known database sequences, of which 123 matches were identified as immune-relevant genes encoding for stress proteins, pattern recognition receptors and complement proteins, proteases and protease inhibitors, cell adhesion and coagulation proteins, cytokine-related proteins, programmed cell death and proteasome-associated proteins. This first EST wide-screening analysis of the Botryllus allorecognition effector arm reveals a complex innate immune system, hallmarked by a whole genome response to allorecognition challenge.