Polar Constituents of the Tunicate Botryllus schlosseri

Eighteen compounds were identified by GC–MS of their trimethylsilyl derivatives in n-butanolic extract from the biomass of Botryllus schlosseri. Three of them, 5-oxoproline, 5-hydroxyhydantoin, and kinurenic acid, were found in marine invertebrates for the first time. In addition to cellulose, the biomass was also shown to contain complex water-soluble sulfated polysaccharides. These were extracted and fractionated, and sulfate content and monosaccharide composition were determined in the fractions; fucose, xylose, galactose, mannose, glucose, glucosamine, galactosamine, and uronic acids were found. Unlike several other tunicate species, Botryllus schlosseri does not seem to contain any simple galactan sulfate.     

Chemical composition of the lipophylic extract from the tunicate Botryllus schlosseri

Eighteen sterols were identified in Botryllus schlosseri, stanols being the main sterols. The sterol composition is in agreement with our recent paper on taxonomic separation of tunicates into three groups. Again we found in invertebrates of the Black Sea sterols with a (22Z)-double bond. This confirms the presence of such sterols in nature. The composition of the phospholipids appeared to be complex, and twelve groups of them were identified in Botryllus schlosseri. The main representatives of phospholipids appeared to be phosphatidylcholine and phosphatidylethanolamine. Only four volatile compounds were identified, which is unusual for marine invertebrates.     

Development of monoclonal antibodies specific to urochordate intracellular epitopes

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.     

Comparative studies on the structure of reproductive organs of four botryllid ascidians

Reproductive organs of four botryllid ascidians, Botryllus primigenus, Botryllus schlosseri, Botrylloides violaceus and Botrylloides leachi, were studied histologically. In every species, the egg follicle consisting of an egg and its inner and outer follicles, is attached to the follicle stalk, the vesicle being composed of a flat epithelium, which in its turn is connected to the atrial epithelium or to the brood pouch specialized from it. In B. schlosseri, the egg is ovulated into the atrial cavity and remains there held by the brood cup, of which the inner epithelium is derived from the follicle stalk and the outer one from the atrial epithelium. In B. primigenus, the brood pouch develops as a diverticulum of the atrial cavity, around the entrance of which a fold differentiates from the atrial epithelium and closes the pouch during embryogenesis. In both species of Botrylloides, the brood pouch is formed by the outgrowth of the thickened atrial epithelium into the blood space, the entrance of which is closed during embryogenesis. The discarded outer follicle completely disintegrates soon after ovulation in B. schlosseri, but part of it remains throughout embryogenesis in the blood space in B. primigenus or projecting into the interior of the brood pouch in Botrylloides. In primigenus, the testis, when it accompanies the egg follicle, is placed at the bottom of the brood pouch and the sperm is shed through the pouch prior to ovulation. In B. schlosseri and the Botrylloides species, the testis is located independently from the egg follicle and the sperm matures after ovulation.     

T system in ascidian muscle: organization of the sarcotubular system in the caudal muscle cells of Botryllus schlosseri tadpole larvae.

The organization of the sarcotubular system has been examined in the caudal muscle cells of the ascidian. Botryllus schlosseri. At variance with striated muscle of other protochordates. Botryllus muscle cells are endowed with a well-developed T system, which has a peculiar laminar structure. The thin T laminae are in continuity with the plasma membrane and extend longitudinally in the intermyofibrillar spaces. At the level of the I-band the T laminae are focally associated with SR cisternae in dyad junctions similar to those observed in invertebrate muscles. These findings are discussed in relation to the origin of the sarcotubular system in vertebrate muscle.     

Morula cells as the major immunomodulatory hemocytes in ascidians: evidences from the colonial species Botryllus schlosseri

Immunocytochemical methods were used to study the presence and distribution of IL-1-alpha- and TNF-alpha-like molecules in the hemocytes of the colonial ascidian Botryllus schlosseri. Only a few unstimulated hemocytes were positive to both the antibodies used. When the hemocytes were stimulated with either mannan or phorbol 12-mono-myristate, the phagocytes were not significantly changed in their number, staining intensity, or cell morphology. In contrast, stimulated morula cells were intensely labeled, indicating that these cells play an important immunomodulatory role.     

The colonial urochordate Botryllus schlosseri: from stem cells and natural tissue transplantation to issues in evolutionary ecology

The urochordates, whose stem groups may have included the direct predecessors of the chordate line, serve as an excellent model group of organisms for a variety of scientific disciplines. One taxon, the botryllid ascidian, has emerged as the model system for studying allorecognition; this work has concentrated on the cosmopolitan species Botryllus schlosseri. Studies analyzing self-nonself recognition in this colonial marine organism point to three levels of allorecognition, each associated with different outcomes. The first level controls natural allogeneic rejections and fusions, in which blood-shared chimeras are formed. The second level leads to morphological resorption of partners within chimeras while the third allows the development of somatic and germ cell parasitic events. Recent studies on multi-chimeric entities formed in allogeneic fusions reveal evolutionary links between allorecognition, stem cell biology and ecology. Thus, the Botryllus system generates perspectives from different biological disciplines to yield a unique life history portrait.     

Raft fouling in the Menai Strait, 1963–1971

Panels were immersed in the Menai Strait for periods of 14 days and for overlapping periods of 28 days and of 56 days from 1963 to the end of 1971. This system provided data whose interpretation permits the detection of annual and seasonal variations in settlement intensities, duration of settlement seasons, growth and progressive fouling, and competition on fouled surfaces within and between the plant and animal forms Balanus balanoides, Eliminius modestus, Tubularia larynx, Diplosoma listerianum, Botryllus schlosseri, jassa, Giffordia, Ulothrix, Enteromorpha and Ectocarpus.     

Incidents of rejection and indifference in Fu/HC incompatible protochordate colonies.

We test here for the existence of specific alloimmune memory in the rejection responses of the colonial tunicate Botryllus schlosseri. Colony specificity in these organisms is controlled by the Fu/HC locus. Rejection occurs only between colonies that do not share any allelic determinant at this locus. Two sets of experiments were conducted: 1) Forty-nine pairs of nonfusible oozooids were interacted naturally along a period of several months. They rejected, disconnected, and in 20% of the cases, interacted again. 2) Repeated colony allorecognition assays were done on 15 pairs of interacting subclones (up to 5 consecutive tests/pair). Major results indicate: 1) Not all ampulla-ampulla interactions resulted in necrotic areas, points of rejection (PORs). 2) A full repertoire of PORs was attained within the first 10 days. Thereafter, no more PORs were added. 3) The outcome of indifference (cases where ampulla-ampulla contacts did not result in rejection) was repeatedly recorded in multiple tests, and its frequency increased in the secondary and tertiary tests along a set of 5 consecutive tests. It is concluded that allospecific memory, as measured by an accelerated production of PORs and amplification in their number, was not characteristic of the Botryllus rejection phenomenon, which, however, reveals the qualities of a low responder. These results are discussed in the light of some aspects of tolerance in the mammalian system.     

Variation in multiple paternity in natural populations of a free-spawning marine invertebrate

For free-spawning marine invertebrates, fertilization processes control the genetic diversity of offspring. Each egg can potentially be fertilized by a sperm from a different male, and hence genetic diversity within a brood varies with levels of multiple paternity. Yet, few studies have characterized the frequency of multiple paternity in natural spawns. We analysed patterns of multiple paternity in two populations of the colonial ascidian Botryllus schlosseri using microsatellites. Because previous studies have shown that at moderate to high population densities, competition among male-phase B. schlosseri colonies results in the nearest male dominating the paternity of a brood, we specifically tested the effect of population density on patterns of paternity. Paternity was estimated using three multilocus indices: minimum number of fathers, counts of sperm haplotypes, and effective paternity (K(E)). Multiple paternity was evident in more than 92% of the broods analysed, but highly variable, with a few broods displaying unequal contributions of different males. We found no effect of population density on multiple paternity, suggesting that other factors may control paternity levels. Indirect benefits from increasing the genetic diversity of broods are a possible explanation for the high level of multiple paternity in this species.     

Identification of the endostyle as a stem cell niche in a colonial chordate

Stem cell populations exist in "niches" that hold them and regulate their fate decisions. Identification and characterization of these niches is essential for understanding stem cell maintenance and tissue regeneration. Here we report on the identification of a novel stem cell niche in Botryllus schlosseri, a colonial urochordate with high stem cell-mediated developmental activities. Using in vivo cell labeling, engraftment, confocal microscopy, and time-lapse imaging, we have identified cells with stemness capabilities in the anterior ventral region of the Botryllus' endostyle. These cells proliferate and migrate to regenerating organs in developing buds and buds of chimeric partners but do not contribute to the germ line. When cells are transplanted from the endostyle region, they contribute to tissue development and induce long-term chimerism in allogeneic tissues. In contrast, cells from other Botryllus' regions do not show comparable stemness capabilities. Cumulatively, these results define the Botryllus' endostyle region as an adult somatic stem cell niche.     

Stem cells are units of natural selection in a colonial ascidian

Stem cells are highly conserved biological units of development and regeneration. Here we formally demonstrate that stem cell lineages are also legitimate units of natural selection. In a colonial ascidian, Botryllus schlosseri, vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues, gametes, or both. We show that genetic hierarchies of somatic and gametic parasitism following fusion can be replicated by transplanting cells between colonies. We prospectively isolate a population of multipotent, self-renewing stem cells that retain their competitive phenotype upon transplantation. Their single-cell contribution to either somatic or germline fates, but not to both, is consistent with separate lineages of somatic and germline stem cells or pluripotent stem cells that differentiate according to the niche in which they land. Since fusion is restricted to individuals that share a fusion/histocompatibility allele, these data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance.     

Cytochemical properties of Botryllus schlosseri haemocytes: indications for morpho-functional characterisation

In the present study, we carried out a detailed light microscopy investigation of the cytochemical properties of the haemocytes of the colonial ascidian Botryllus schlosseri, using new cytochemical stains and enzymatic markers, a panel of antibodies and lectins as probes to characterise Botryllus blood cells further. Results indicate that lymphocyte-like cells are circulating undifferentiated cells recognised by anti-CD34 antibody and there are at least two defined haemocyte differentiation pathways: i) phagocytes, represented by hyaline amoebocytes and macrophage-like cells, which share similar staining properties, the same hydrolytic enzyme content as well as the presence of detectable cytochrome-c-oxidase activity, recognition by anti-CD39 and Narcissus pseudonarcissus agglutinin; ii) cytotoxic cell line, represented by granular amoebocytes and morula cells which have vacuoles stained by Ehrlich's stain and Neutral Red; DOPA-containing protein are present inside morula cell vacuoles. Pigment cells and nephrocytes are involved in catabolite storage but their relationships with other cell types are less clear.     

Effects of pulse versus steady recruitment on sessile marine communities

Variation in patterns of propagule establishment (recruitment) has important effects on population dynamics and the structure of some communities. Most experimental studies have varied recruitment by changing the nature of a single event early in community development, but recruitment can also vary from steady rates of arrival to highly episodic 'pulse' events, causing differences in the temporal spacing of individuals recruiting into patches. We examined whether two different temporal patterns of recruitment of sessile invertebrates affected temperate marine communities in southeastern Australia in two experiments that were run at different times at the same site and that manipulated several different species. Target species entered communities as either a single pulse of recruits within a 2-week period or steady input of the same total number of recruits over a longer time period (5-6 weeks). The pattern of recruitment had variable effects on community structure. The colonial ascidian Botryllus schlosseri did not have a strong influence on community structure whether it recruited in a single pulse or steadily. The cover of B. schlosseri was higher when recruitment occurred as a single pulse. In a second experiment, botryllid ascidians caused changes in the composition of communities when they recruited steadily compared to when they did not recruit or didemnids recruited, but caused no differences in communities when they recruited in a shorter pulse. In contrast, recruitment frequency of didemnid ascidians had little effect, though their presence/absence caused community differences. Though we found that different temporal recruitment patterns can alter community composition, the life history and ecology of particular taxa as well as differences in environmental background processes are likely to influence the strength of these effects.     

Telomerase maintained in self-renewing tissues during serial regeneration of the urochordate Botryllus schlosseri

Telomerase is critical for the protection of germ line and stem cell chromosomes from fatal shortening during replication. In most organisms, telomerase activity is suppressed in progressively committed cells and falls to basal rates in terminally differentiated lineages. The colonial ascidian Botryllus schlosseri propagates asexually and sexually, presumably from pools of stem cells that self-renew throughout the 2- to 5-year colony life span. Asexual budding takes place continuously from the parental body wall. When the colony reaches a critical size, sexual reproduction commences with the generation of gonads. Here, we establish the existence of 6-15 kb telomeres on the ends of Botryllus chromosomes. We develop a real-time quantitative PCR telomeric repeat amplification protocol (TRAP) assay that reliably detects 0.2-100 TPG units in cells and tissues. We find highest levels of enzymatic activity in the gonads, developing embryos, and tissues containing the earliest asexual buds. Telomerase activity appears to be suppressed in later buds during organogenesis and falls to basal rates in mature zooids. We postulate that this pattern reflects maximum telomere restoration in somatic stem cells of early buds and suppression of telomerase activity in progenitors and terminally differentiated cells, indicative of an alternate role for stem cells as repeated body regenerators in colonial life histories.     

Multilocus genetic analyses differentiate between widespread and spatially restricted cryptic species in a model ascidian

Elucidating the factors that shape species distributions has long been a fundamental goal in ecology and evolutionary biology. In spite of significant theoretical advancements, empirical studies of range limits have lagged behind. Specifically, little is known about how the attributes that allow species to expand their ranges and become widespread vary across phylogenies. Here, we studied the ascidian Botryllus schlosseri, a worldwide invasive species that is also characterized by marked genetic subdivision. Our study includes phylogenetic and population genetic data based on mitochondrial and nuclear genes, as well as polymorphic microsatellites for B. schlosseri colonies sampled from the southern and northern coasts of Europe and the eastern and western coasts of North America. We demonstrate that this well-known model organism comprises three highly divergent and probably reproductively isolated cryptic species (A, D and E), with two more (B and C) being suggested by data retrieved from GenBank. Among these, species A, recovered in all of the surveyed regions, is by far the most common and widespread. By contrast, species B-E, occurring mostly in sites from northern Europe, are considerably more geographically restricted. These findings, along with inferences made on transport opportunity, suggest that divergent evolutionary histories promoted differences in invasive potential between B. schlosseri sibling species, indicating that attributes that facilitate dramatic shifts in range limits can evolve more easily and frequently than previously thought. We propose environmental disturbance as a selective force that could have shaped the evolution of invasiveness in the B. schlosseri complex.     

Genetic structure of the star sea squirt, Botryllus schlosseri, introduced in southern European harbours

The introduction of new genetic variants or species is often caused by maritime transport between harbours. Botryllus schlosseri is a cosmopolitan ascidian species that is found in both harbours and open shore habitats. In order to determine the influence of ship traffic on the genetic structure and phylogeography of B. schlosseri in southern Europe, we analyzed the variability of a fragment of the mitochondrial gene cytochrome c oxidase subunit I (COI). We sampled seven Atlanto-Mediterranean harbour populations and three open-shore populations. In addition, we sequenced some colonies from the US-Atlantic coast and from other Mediterranean localities to perform phylogenetic analyses. Although the number of polymorphic sites recorded (25.8%) was within the range observed in other population studies based on ascidian COI sequences, the haplotypic diversity (16 haplotypes out of 181 sequences) was much lower. Moreover, a lack of intermediate haplotypes was observed. This pattern of high nucleotide diversity and low haplotype diversity was consistent with introduction events of a few divergent haplotypes. We found a strong genetic structure in the study populations. Gene flow was only appreciable between some harbour populations. Harbour- and open-shore populations were well differentiated, although there was no evidence for isolation by distance. A nested clade analysis pointed to long-distance colonization, possibly coupled with subsequent fragmentation, as the underlying process. Our results suggest that B. schlosseri entered the study area via harbour-hopping, possibly through recurrent introduction events. The haplotypes from North America and most of the European ones were grouped in the same phylogenetic clade. This suggests occasional gene flow between both continents, probably through ship transport.     

Suppression of programmed cell death regulates the cyclical degeneration of organs in a colonial urochordate

The survival of animal tissues and organs is controlled through both activation and suppression of programmed cell death. In the colonial urochordate Botryllus schlosseri, the entire parental generation of zooids in a colony synchronously dies every week as the asexually derived generation of buds reaches functional maturity. This process, called takeover, involves massive programmed cell death (PCD) of zooid organs via apoptosis followed by programmed removal of cell corpses by blood phagocytes within approximately 1 day. We have previously reported that developing buds in conjunction with circulating phagocytes are key effectors of zooid resorption and macromolecular recycling during takeover, and as such engineer the reconstitution of a functional asexual generation every week [Lauzon, R.J., Ishizuka, K.J., Weissman, I.L., 2002. Cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: a model for development and regeneration. Dev. Biol. 249, 333-348]. Here, we demonstrate that zooid lifespan during cyclic blastogenesis is regulated by two independent signals: a bud-independent signal that activates zooid PCD and a bud-dependent, survival signal that acts in short-range fashion via the colonial vasculature. As zooids represent a transient, mass-produced commodity during Botryllus asexual development, PCD regulation in this animal via both activation and suppression enables it to remove and recycle its constituent zooids earlier when intra-colony resources are low, while maintaining the functional filter-feeding state when resources are adequate. We propose that this crosstalk mechanism between bud and parent optimizes survival of a B. schlosseri colony with each round of cyclic blastogenesis.     

An unusual membrane system in the oocyte of the ascidian Botryllus schlosseri

During vitellogenesis, oocytes of Botryllus schlosseri always exhibit an unusual system scattered in the cytoplasm. It consists of an association between a single fenestrated endoplasmic reticulum cisterna and one or a few smooth vesicles (cisterna vesicle association: CVA) containing a dense core facing the cisterna itself. The latter is smooth and perforated by numerous small pores (about 25 nm in diameter) in the area of association; towards the periphery, it extends into several branches with ribosomes bound to their membranes. In the vesicles, fibrillar material radiates from the dense core and is sometimes organized into a long, dense lamina. The membranes of both cisterna and vesicles appear to be coupled, but are in fact separated by a constant narrow space occupied by short densities. The presence in B. schlosseri of this unusual fenestrated membrane system contrasts with the absence of a typical porous cytoplasmic organelle, the annulate lamellae (ALs), which is widely distributed in female gametes. However, as in other animals, B. schlosseri oocytes possess intranuclear annulate lamellae (IALs) and vesicles. Comparative observations extended to the oocytes of the ascidian Ciona intestinalis have shown that the latter species exhibits typical ALs and IALs, but not the CVA. The morphology of the CVA is analysed here in detail, and similarities and differences with ALs are pointed out. Hypotheses regarding CVA function are discussed in terms of possible relations with ALs.