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Allorecognition, the ability of an organism to distinguish kin from non-kin, or self from non-self, has been studied extensively in a group of invertebrate chordates, the colonial ascidians called botryllids (Subphylum Tunicata, Class Ascidiacea, Family Styelidae). When two conspecific botryllid colonies come in contact, there are two potential outcomes to an allorecognition reaction: fusion or rejection. The rejection outcome of allorecognition varies by species, and has been classified by type (referred to as R-Type). R-Type is defined according to how far the fusion process progresses before the rejection begins, since the rejection reaction appears as an interference of the fusion process. Here, we map the evolution of R-Types onto an extended and robust phylogeny of the botryllids. In this study, we have reconstructed the largest phylogenomic tree of botryllids, including 97 samples and more than 40 different species, and mapped on it nine of the 13 species for which the R-Type is known. Based on the R-Type known in a single outgroup species (Symplegma reptans), we infer that at least R-Type B and E-like could be ancestral to the Botrylloides/Botryllus group. However, the application of ancestral character state reconstructions does not provide conclusive results since several clades show more than one equiparsimonious R-Type state. Notably, all R-Type A species are clustered together and certainly evolved later than other R-Types. Our phylogenomic tree has been built on 177 nuclear loci and nearly all clades are well supported. Moreover, our phylogenetic analyses also take into account the results of species delimitation analyses based on the mitochondrial COI gene and of careful morphological analyses of the samples. The implementation of this integrated taxonomic approach, combining morphological as well as nuclear and mitochondrial data, has allowed the description of six new species, and the identification of a number of putative unnamed taxa. Thus, our results also demonstrate the existence of an unexplored hidden diversity within botryllids. [ABSTRACT FROM AUTHOR]

Allorecognition, the ability of an organism to distinguish kin from non-kin, or self from non-self, has been studied extensively in a group of invertebrate chordates, the colonial ascidians called botryllids (Subphylum Tunicata, Class Ascidiacea, Family Styelidae). When two conspecific botryllid colonies come in contact, there are two potential outcomes to an allorecognition reaction: fusion or rejection. The rejection outcome of allorecognition varies by species, and has been classified by type (referred to as R-Type). R-Type is defined according to how far the fusion process progresses before the rejection begins, since the rejection reaction appears as an interference of the fusion process. Here, we map the evolution of R-Types onto an extended and robust phylogeny of the botryllids. In this study, we have reconstructed the largest phylogenomic tree of botryllids, including 97 samples and more than 40 different species, and mapped on it nine of the 13 species for which the R-Type is known. Based on the R-Type known in a single outgroup species (Symplegma reptans), we infer that at least R-Type B and E-like could be ancestral to the Botrylloides/Botryllus group. However, the application of ancestral character state reconstructions does not provide conclusive results since several clades show more than one equiparsimonious R-Type state. Notably, all R-Type A species are clustered together and certainly evolved later than other R-Types. Our phylogenomic tree has been built on 177 nuclear loci and nearly all clades are well supported. Moreover, our phylogenetic analyses also take into account the results of species delimitation analyses based on the mitochondrial COI gene and of careful morphological analyses of the samples. The implementation of this integrated taxonomic approach, combining morphological as well as nuclear and mitochondrial data, has allowed the description of six new species, and the identification of a number of putative unnamed taxa. Thus, our results also demonstrate the existence of an unexplored hidden diversity within botryllids.

Tunicates are highly diverse marine invertebrate filter-feeders that are vertebrates' closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually by budding, typically leading to the formation of colonies where animals, called zooids, are interconnected through an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions. To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates. Herein we present our success in breeding Botrylloides diegensis , a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 ​km away from their natural habitat. We show that colonies adapt well to in-lab culturing provided that a suitable marine microbiome is present, and that a specific strain can be isolated, propagated and efficiently used for research over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species, such as Botryllus schlosseri , as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals. [Display omitted] • First artificial seawater based recirculating aquaculture for colonial ascidians. • Over 3 years of continuous breeding. • Semi-automated setup with minimized maintenance. • Good biomass production for strain propagation. • 4 different culture conditions for optimized breeding for species of interest. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rough environmental conditions make the survival of many multi-cellular organisms almost impossible, enforcing behavioral, morphological, physiological and reproductive rejoinders that can cope with harsh times and hostile environments, frequently through down-regulation of metabolism into basal states of dormancy, or torpor. This study examines one of the most unique torpor strategies seen within the phylum Chordata, exhibited by the colonial urochordate Botrylloides leachi, which enters a state of hibernation or aestivation in response to thermal stress, during which all of its functional colonial units (zooids) are entirely absorbed and the colony survives as small remnants of the vasculature, lacking both feeding and reproduction organs. Tissue vestiges then regenerate fully functional colony when re-exposed to milder environmental conditions. The whole metamorphic cycle of hibernation and arousal was studied here and divided into seven major stages, during which the anatomical characteristics of the zooids, the blood cell populations and the expression patterns of some “stem cell” markers were monitored. The first two phases are associated with the shortening of the blastogenic cycles from the typical 7-day cycle to 3–5 day long cycles and with the significant diminution of zooids, leaving a carpet of vasculature. During hibernation this colonial carpet is made of a twisted, opaque and condensed mass of vasculature, loaded with condensed masses of blood cells that possess two types of multicellular structures, the 20–50 µm “morula-like” opaque balls of cells, and small single-layer epithelial spheres, “blastula-like” structures (50–80 µm). Arousal from hibernation starts with the emergence of several clear tunic areas among the vasculature lacunae, which then turn into transparent buds that become progressively larger and opaque. This is followed by sluggish, newfangled cell movement within the vasculature, which increases in intensity and rate over time. A closer examination of the vasculature revealed dramatic vicissitudes in the blood cell constituency as hibernation progressed, which is manifested by the appearance of two novel cell types not recorded in regular colonies, the multinucleate cells ( MNC ) and storage cells, each with 2–3 distinct cell morphs. Using mixtures of pre-labeled where half stained with a florescent marker for membranes and half stained for DNA we recorded within 2–3 days from onset new MNC stained by both staining, attesting for the de novo formation of MNC through cells fusion. At the outset of hibernation we documented high expression levels of PIWI, PL-10 and PCNA in cells residing in cell islands ( CIs ), which are the specific stem cell niches found along the endostyle at the ventral side of the zooids. During hibernation, most of the PIWI + / PL-10 + /PCNA + cells were the MNCs , now located in the newly shaped and dilated vasculature, where they increased in numbers. Also, most of the PCNA + cells were identified as MNCs . We further documented that the Bl–PIWI RNA ( in situ hybridization) and protein (immunohistochemistry) expressions documented during the hibernation/arousal processes diverged significantly from normal blastogenesis expressions. Counting PIWI + blood cells at various blastogenic stages revealed a significant increase as the hibernation progressed, peaking in aroused colonies at an average of 30 PIWI + cells/ampulla. The Pl-10 protein expression patterns in the zooids and buds changed as the hibernation progressed, similar to the PIWI and PCNA expressions. Considering the evolutionary perspectives to hibernation we propose linkages to the disposable-soma theory. [ABSTRACT FROM AUTHOR]

Whole-bodyregeneration(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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Colonial ascidians are common occupants of hard surfaces in coastal marinas as fouling organisms on pier pilings and ship hulls and have significant financial impact. Methods to inhibit fouling often are time consuming and labor intensive, thus treatments that prevent settlement for long time periods are warranted. Trybutyltin (TBT) appeared to be an effective antifouling agent, but is highly toxic to non-target species and now globally prohibited. Medetomidine is the active ingredient in the anti-fouling paint, Selektope, that shows promise to prevent attachment of marine fouling species. Here, we assessed the potential of medetomidine on larval metamorphosis of the invasive ascidian Botrylloides violaceus in static bioassays. We exposed newly released tadpole larvae to eight concentrations (0.5 mg/L, 1 mg/L, 5 mg/L, 10 mg/L, 20 mg/L, 40 mg/L, 50 mg/L) of medetomidine and recorded the number of larvae that were mobile, attached, metamorphosed or deceased after 1 h, 2 h, 4 h, 6 h, 24 h and 48 h. Larvae exposed to the highest concentrations of medetomidine quickly attached to the surface of Petri dishes, but failed to metamorphose. Larvae were mobile over the first 2 to 4 h of the experiment in the filtered sea water controls and low medetomidine concentrations. Larvae metamorphosed in all treatments except the two highest concentrations of medetomidine (40 and 50 mg/L), but ultimately all larvae exposed to medetomidine died. Medetomidine appears to have potential as a mechanism to control populations of B. violaceus , but we caution that further testing of non-target species is warranted. • The invasive ascidian Botrylloides violaceus can dominate benthic/fouling communities. • Medetomidine exposure enhances settlement of Botrylloides larvae. • Metamorphosis of larvae is inhibited by exposure to medetomidine. • Medetomidine has potential to be an effective antifouling treatment. [ABSTRACT FROM AUTHOR]

We continued our study on fusion and non-fusion reactions in the compound ascidian Botrylloides leachi with particular attention to the role of hemocytes in the induction of the strong cytotoxic reaction observed when genetically incompatible colonies encounter each other in cut surface assays. We used antibodies raised against Botryllus phenoloxidase (PO) and pro-inflammatory vertebrate cytokines for immunocytochemical and immunohistochemical analysis on contacting incompatible colonies in order to investigate cellular events during the non-fusion reaction. Results indicate that, analogously to Botryllus schlosseri, morula cells play a key role in the above reaction as they crowded inside the facing ampullae and entered the tunic where they degranulated and induced PO-driven cytotoxicity. Similarly to Botryllus, they also expressed molecules recognised by anti-IL-1-α antibodies, whereas the immunopositivity to anti-TNF-α was unexpectedly restricted to granular cells.

We studied colony specificity in the colonial ascidian Botrylloides leachi which, as in other botryllid ascidians, leads to either fusion or non-fusion between contacting colonies. Fusion requires the prior disappearance of contacting tunic cuticles and contact between facing ampullar epithelia. The epithelial cells of the ampullar tip show “pad regions” rich in ribosomes, which contribute to the synthesis of new tunic and cuticle. Blood cells, mainly phagocytes and pigment cells, increase their concentrations inside the ampullar lumen and phagocytes can cross the ampullar epithelium and enter the tunic, where they can contribute to the digestion of tunic cuticles and cells of the ampullar epithelium in order to establish a common circulation. Non-fusion reaction, as studied in the colony allorecognition assay, resembles the subcuticular rejection described in Japanese Botrylloides, characterised by limited tunic fusion, hemocyte leakage, and necrotic spots. Conversely, in the cut surface assay, a more intense cytotoxic reaction is observed along the contact border. In this case, morula cells crowd massively inside the facing ampullae, enter the tunic, and release their vacuolar contents which are probably required for the formation of necrotic spots.

Marine fouling communities on artificial structures are invasion hotspots for non-indigenous species (NIS). Yet, little is known about NIS in fouling communities of British Columbia (BC), Canada. To determine NIS identity and richness in BC fouling communities, we deployed settlement plates at 108 sites along the coast of BC between 2006 and 2012. Of the 295 invertebrate taxa identified to species, 20 were NIS while an additional 14 were cryptogenic, including several global invaders. This study documents the range expansion of tunicates Botrylloides violaceus Oka, 1927 and Botryllus schlosseri (Pallas, 1766), including the first known records on Haida Gwaii. NIS were detected within each of the six distinct geographic regions with the southern, more populated regions of BC (Straits of Georgia and Juan De Fuca) having the highest NIS richness and frequency of occurrence compared to the less populated northern regions. This study provides a contemporary baseline of invertebrate NIS identity and richness in fouling communities that will allow comparisons through time and a means to focus research and prioritize management efforts. [ABSTRACT FROM AUTHOR]

Tunicates are useful models for exploring microbiomes because they have an innate immune system resembling that of chordates. Automated ribosomal RNA intergenic spacer analysis and High-Throughput Sequencing were used to compare the tunic microbiomes of Ciona robusta (formerly Ciona intestinalis type A), Ciona savignyi, Botrylloides leachi and Botryllus schlosserisampled from three distinct locations with limited genetic connectivity. Bacterial phylotype profiles were conserved within each species, and there were no detectable differences between tunic and tunic 1 cuticle subsamples from an individual. Bacterial operational taxonomic unit (OTU) diversity was lowest for C. savignyi (320 ± 190 OTUs) and highest for B. schlosseri (1260 ± 190 OTUs). Each species had a distinct set of bacterial OTUs (pseudo-F = 3.0, p > 0.001), with the exception of B. leachi and B. schlosseri from one sampling location (t = 1.2, p = 0.09). Of note were OTUs assigned to Alphaproteobacteria from C. robusta plus Phyllobacteriaceae and Endozoicomonas from C. savignyi. These OTUs contributed 51, 22 and 10% of sequence reads, respectively, and are related to known bacterial symbionts. The within-species conservation of core OTUs across three distinct and co-occurring populations of tunicates provides compelling evidence that these tunicates foster defined microbiomes. [ABSTRACT FROM AUTHOR]

In order to evaluate the potential risk posed by transporting invasive tunicates away from infested sites it is important to be able to measure the viability of these organisms. A Neutral Red (NR) viability assay was developed using a colonial invasive tunicate, Botrylloides violaceus (Oka, 1927) that could be applied to other species. Unlike some colonial tunicates B. violaceus' mortality can be determined under light microscopy by observing changes in the physiological parameters of filtration and reaction to tactile stimulus making them a good study organism. A total of 32 B. violaceus segments (3 cm2 ) were allocated to treatment and control groups. The treatment group was subjected to acetic acid which resulted in 100 % mortality. Viability was then assessed by comparing treatment and control group responses to NR uptake. There was no mortality in the control that demonstrated a response to tactile stimulus and a noticeable amount of stain uptake inside zooids. In contrast, the treatment groups did not respond to tactile stimulus and no stain was taken in by the zooids. In this study the viability of Didemnum vexillum (Kott, 2002) was assessed under controlled experimental conditions by simulating the air exposure encountered during the normal transportation and processing of tunicate fouled Pacific oysters, Crassostrea gigas (Thunberg, 1793). Clusters containing C. gigas and D. vexillum were collected from an aquaculture lease, in Lemmens Inlet, BC, for an air exposure trial comprised of 9 equally divided treatments (0, 0.5, 1.5, 3, 6, 12, 24, 48 and 72 hours). Three segments of D. vexillum on each cluster were removed and evaluated with the NR protocol developed for B. violaceus. Preserved tissues were analyzed and no sign of stain was noticeable, even with the control segments. The NR protocol developed worked efficiently with B. violaceus, but appeared to fail with D. vexillum. The findings are consistent with D. vexillum demonstrating a high tolerance in situations they are not accustomed to reflecting their ability to enter a dormant state during adverse environmental conditions (hence lack of response to clinical or NR markers via lack of filtering). Further studies are required to determine the viability of D. vexillum and potential risk movements pose. [ABSTRACT FROM AUTHOR]

Background: Regenerative capacity differs greatly between animals. In vertebrates regenerative abilities are highly limited and tissue or organ specific. However the closest related chordate to the vertebrate clade, Botrylloides leachi, can undergo whole body regeneration (WBR). Therefore, research on WBR in B. leachi has focused on pathways known to be important for regeneration in vertebrates. To obtain a comprehensive vision of this unique process we have carried out the first de novo transcriptome sequencing for multiple stages of WBR occurring in B. leachi. The identified changes in gene expression during B. leachi WBR offer novel insights into this remarkable ability to regenerate. Results: The transcriptome of B. leachi tissue undergoing WBR were analysed using differential gene expression, gene ontology and pathway analyses. We observed up-regulation in the expression of genes involved in wound healing and known developmental pathways including WNT, TGF-β and Notch, during the earliest stages of WBR. Later in WBR, the expression patterns in several pathways required for protein synthesis, biogenesis and the organisation of cellular components were up-regulated. Conclusions: While the genes expressed early on are characteristic of a necessary wound healing response to an otherwise lethal injury, the subsequent vast increase in protein synthesis conceivably sustains the reestablishment of the tissue complexity and body axis polarity within the regenerating zooid. We have, for the first time, provided a global overview of the genes and their corresponding pathways that are modulated during WBR in B. leachi. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A number of challenges have hindered the development of a unified theory for metazoan regeneration. To describe the full range of complex regeneration phenomena in Animalia, we suggest that metazoans that regenerate missing body parts exhibit biological attributes that are tailored along a morpho-spatial regeneration continuum, illustrated in its polar scenarios by the USA "stars and stripes" flag. Type 1 organisms ("T1, 'stars'") are typical colonial organisms (but contain unitary taxa) that are able to regenerate "whole new stars", namely, whole bodies and colonial modules, through systemic induction and sometimes multiple regeneration foci (hollow regeneration spheres, resembling the blastula) that compete for dominance. They regenerate soma and germ constituents with pluripotent adult stem cells and exhibit somatic-embryogenesis mode of ontogeny. Type 2 organisms ("T2, 'stripes'") are capable of limited regeneration of somatic constituents via fate-restricted stem cells, and regenerate through centralized inductions that lead to a single regeneration front. T2 organisms are unitary and use preformistic mode of ontogeny. T1 and T2 organisms also differ in interpretation of what constitutes positional information. T2 organisms also execute alternative, less effective, regeneration designs (i.e., scar formation). We assigned 15 characteristics that distinguish between T1/T2 strategies: those involving specific regeneration features and those operating on biological features at the whole-organism level. Two model organisms are discussed, representing the two strategies of T1/T2 along the regeneration continuum, the Botrylloides whole body regeneration (T1) and the mouse digit-tip regeneration (T2) phenomena. The above working hypothesis also postulates that regeneration is a primeval attribute of metazoans. As specified, the "stars and stripes" paradigm allows various combinations of the biological features assigned to T1 and T2 regeneration strategies. It does not consider any concentration gradient or thresholds and does not refer to the "epimorphosis" and "morphallaxis" terms, regeneration types across phyla or across body plans. The "stars and stripes" paradigm also ignores, at this stage of analysis, cases of regeneration loss that may obscure biological trajectories. The main advantage of the "stars and stripes" paradigm is that it allows us to compare T1/T2 regeneration, as well as other modes of regeneration, through critical determining characteristics. [ABSTRACT FROM AUTHOR]

The development of effective mitigation techniques against Botryllus schlosseri and Botrylloides violaceus colonizing blue mussel aquaculture operations has not been well studied. The objectives of our research were to determine the efficacy of using pressurized seawater in the mitigation of colonial tunicate fouling and to identify optimal treatment timing and frequencies in reducing tunicate biomass. Treatment trials using high- (∼700 psi) and low-pressure (∼40 psi) seawater spraying were conducted in St. Peters Bay and Savage Harbour, PEI, from May to November 2009. The use of high-pressure seawater was an effective anti-fouling measure for these species, causing significant reductions in tunicate biomass. In contrast, low-pressure seawater had no discernable effect. The timing of treatment was found to be the most important factor affecting efficacy, with reductions in tunicate biomass increasing in magnitude the closer the treatment occurred to harvest. Treatment frequency did not affect tunicate biomass. In addition, fewer treatments also resulted in less nuisance mussel spat fouling the mussel socks. Colonial tunicate fouling did not affect adult mussel growth and productivity, and no evidence of smothering or crop loss was observed. [ABSTRACT FROM AUTHOR]

Abstract: The colonial tunicate Botrylloides leachi can regenerate functional adults from minute vasculature fragments, in a poorly understood phenomenon termed Whole Body Regeneration (WBR). Using Piwi expression (Bl-Piwi), blood cell labeling and electron microscopy, we show that WBR develops through activation, mobilization and expansion of ‘dormant’ cells which normally line the internal vasculature epithelium of blood vessels. Following a mechanical insult, these cells express Bl-Piwi de novo, change morphology and invade niches of the vasculature lumen, where they proliferate and differentiate, regenerating a functional organism. Mitomycin C treatments and siRNA knockdown of Bl-Piwi result in deficient cells incapable of expanding or differentiating and to subsequent regeneration arrest. Last, we find similar transient mobilization of Piwi + cells recurring every week, as part of normal colony development, and also during acute environmental stress. This recurrent activation of Piwi + cells in response to developmental, physiological and environmental insults may have enabled the adaptation of colonial tunicates to the imposed varied conditions in the marine, shallow water environment. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Natural chimeras are commonly distributed in the wild, challenging the traditional paradigm for the advantages of genetically homogenous entities, where uniclonality prevents within-organism conflicts. This essay focuses on the last two-decade studies on chimerism in the cosmopolitan urochordate Botryllus schlosseri, enlightening and focusing the idea of multichimeras as a primary tool for fending off the pervasiveness of super parasitic germ lines. Interacting Botryllus colonies may fuse or reject each other based on allelic compatibility on a single highly polymorphic gene locus. After fusion and establishment of a chimera, a second tier of allorecognition is developed, expressed as genetically controlled morphological resorption of one of the chimeric partners. This is followed by the third tier of allorecognition where somatic and germ cell lineages parasitism are developed. Studies revealed a complex network of costs and few suggested benefits for the state of chimerism in botryllid ascidians. Two life history traits (diversification of allorecognition allele repertoire, colonial programmed lifespan) were considered as selected to combat the major cost of chimeric associated germ cell parasitism. Three other ecological traits (heterosis, settlement of kin larvae in aggregates, multichimerism) have been suggested as selected to enhance the existence of chimerism in botryllid ascidians. Recent results revealing a fine-tuning of the chimerical somatic genetic components in response to changes in environmental conditions are discussed. Results further elucidate the possible existence of multichimeras, each made of several genotypes. It is proposed that natural multichimeras form more stable and vigorous entities, depicting a unique way for domesticating consortia of selfish cells that may otherwise seriously threaten survivorship of the entity. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Recruitment is an important process in regulating many marine benthic communities and many studies have examined factors controlling the dispersal and distribution of larval immigrants. However, benthic species also have early post-settlement life-stages that are dramatically different from adult and larval stages. Predation on these stages potentially impacts measured recruitment and the benthic populations and communities that ultimately develop.We examined the consequences of post-settlement predation on 1-day-old to 1-month-old recruits of sessile invertebrates at two field sites in southern New England. One site (Breakwater) was in a protected area with few predators and the other (Pine Island) was <1 km away in an open coast area with three different predator guilds: small and large invertebrates and fish. The Breakwater site had been dominated for >10 years by colonial and solitary ascidians. These species were absent from the Pine Island site which was dominated by bryozoans. Our goal was to examine whether post-settlement predation influenced the development and subsequent structure of the epifaunal community.Here we examine long-term changes in community development resulting from post-settlement predation, and contrast these results to those of earlier experiments examining the reductions in observed recruitment by post-settlement predation. Our first long-term experiment examined natural community development at the two sites and whether transplanted communities changed when exposed to the different levels of predation at these sites. The communities that developed at both sites were consistently different from each other and similar to resident communities at their respective sites. On panels transplanted from the Breakwater to Pine Island, solitary ascidians and the colonial ascidian, Botryllus schlosseri, suffered high mortalities on both caged and uncaged treatments, indicative of predation by small predators that could enter cages. Some solitary ascidians did survive inside cages and the colonial ascidian, Botrylloides violaceus, became dominant on all transplanted treatments. On panels transplanted from Pine Island to the Breakwater, ascidians invaded and dominated all treatments except those that were originally caged at Pine Island.In the second long-term experiment, natural communities were allowed to develop on panels exposed at the Breakwater for 1, 2, 3, and 4 weeks. Each set was transplanted to three treatments at Pine Island: open uncaged pilings, caged pilings to exclude fish and large invertebrates, and racks suspended above the bottom to exclude all predators. When 1-week-old communities were transplanted, after 2–3 weeks only bryozoans were found on the open and caged pilings, while colonial ascidians dominated the suspended rack treatment. When older 2-week-old communities were transplanted, colonial ascidians also became dominant in the caged piling treatment and when 3- and 4-week-old communities were transplanted colonial ascidians dominated all three treatments. Solitary ascidians were never abundant on open pilings exposed to fish and large benthic invertebrate predators.Post-settlement predator–prey interactions involved newly settled and juvenile life-stages of a variety of prey species and many invertebrate and vertebrate predator species. The effects of these interactions on recruitment did result in differences in the development and eventual species composition of the communities, even though predators had little if any effect on the adults of the prey species. [Copyright &y& Elsevier]

Studies on multi-partner chimeras (MC) of the colonial urochordate Botryllus schlosseri have revealed that these chimeras form more stable and vigorous entities than bi-partner chimeras (BC). This outcome has been further studied here on another botryllid species. Botrylloides leachi subpopulation 1, by analysing the morphological consequences of BC, chimeras comprising three partners, tri-chimeras (TC) and chimeras comprising six partners each, hexa-chimeras (HC). For each chimerical type, five different genotypic combinations with three replicates per combination were established. Chimeras were observed for up to 10 months, at which point, all had died. While life spans of BC, TC and HC were the same, the average maximum sizes of HC were higher resulting from more than three times greater daily growth rate. BC and TC reached a maximal chimeric size at a much earlier age. Some morphological resorptions were also expressed differently in HC as compared to BC. When comparing chimerical parameters of bi- vs. multi-chimerism between Botryllus and Botrylloides, these two genera differ in nine out of 13 characteristics tested. However, it seems that irrespective to the species studied and the different characteristics expressed, MC in botryllid ascidians is a commonly developed phenomenon shaped by evolutionary pressures that interact on a ‘group’ level instead of each individual partner. MC reveal modified entities in which contradicting intraspecific interactions are alleviated. [Copyright &y& Elsevier]

Introduced species frequently undergo rapid evolutionary change in ecologically important traits that may strongly influence their performance in their introduced range. But for many introduced species, and particularly those in marine systems, the spatial and temporal dimensions of this evolutionary change are largely unknown. In this study, I conducted a common garden transplant experiment using replicate clones of an introduced tunicate, Botrylloides sp., collected from three sites in the southern Gulf of Maine to test for differential growth and survival in response to local thermal regimes. This species has a restricted larval development period, which may permit significant population differentiation over relatively small spatial scales. I found dramatic among-site differences in survival rates: tunicates collected from both the transplant site and from a distant northern site had high survival, while those collected from a southern site experienced 100% mortality. The most likely cause of this differential mortality was an anomalous upwelling event that produced some of the coldest mid-summer temperatures in the last 10 years based on NOAA weather buoy data. I also found significant differences in growth rates among sites as well as among clones within a site in comparison with growth-rate differences within clones. An estimate of broad-sense heritability based on these data was consistent with a genetic basis for among-site differences in growth rates, which were strongly predictive of subsequent mortality. I conclude that this species has undergone significant evolutionary change that has resulted in population differentiation with respect to temperature tolerances among sites separated by <60 km. [ABSTRACT FROM AUTHOR]

The article discusses the development of tadpole robots (Tadros) by roboticist John Long and colleagues at Vassar College. Topics include Long and colleagues' use of the Tadros to study the ability to evolve, or evolvability, the field of evolutionary robotics, brief biological information on Long, Long's attempts to replicate backbone evolution in sea squirts (Botrylloides), which led to the creation of the Tadro, and the advantages of robots over computer models.

Tunicates are a diverse group of worldwide marine filter-feeders that are vertebrates' closest invertebrate relatives. Colonial tunicates are the only know chordates that have been shown to undergo whole-body regeneration (WBR). Botrylloides in particular can regenerate one fully functional adult from a minute fragment of their vascular system in as little as 10 days. This regenerative process relies on the proliferation of circulating stem cells, likely supported by the activity of some of the 11 identified types of hemocytes. To study and challenge WBR, it is thus important to have the capacity to isolate, analyze, and manipulate hemolymph in regenerating colonies. Here we present a microtransfusion technique that permits the collection of pure hemocytes, the quantification of their purity, their labeling, and reinjection into colonial tunicates. To exemplify our approach, we present in addition a protocol to analyze the isolated hemocytes using flow cytometry. Our approach is minimally invasive, does not induce lethality, and therefore allows repeated transfusion into exactly the same colony with minimal disruption to the process being studied., (© 2022. The Author(s).)