Phenotypic plasticity of reproductive effort in a colonial ascidian,Botryllus schlosseri

Phenotypic plasticity is the capability of a genotype to produce different phenotypes in different environments. Previous studies have indicated phenotypic variability in asexual, male, and female reproduction in Botryllus schlosseri, a hermaphroditic, colonial ascidian, but not explicitly tested for genotype by environment interactions that indicate genetic variation in plastic responses. Consequently, clones derived from an estuarine population were deployed at their native site and a warmer, higher productivity site 10 km up-river. Male reproduction was assayed by testis size, female reproduction by the number of eggs produced, and asexual reproduction by colony growth rate. To test for ontogenetic effects, data were collected from two different generations of zooids born in the field. Analyses of variance indicated plasticity in asexual and female reproduction during the first zooid generation and plasticity in all three traits during the third zooid generation. Reaction norms varied significantly among genotypes in direction and magnitude for asexual reproduction at both times, implying that selection on asexual reproduction is weak. Sperm production during the third zooid generation was significantly lower at the nonnative site, but there was no genotype by environment interaction. The reaction norms for female reproduction varied significantly among genotypes in direction and magnitude during the first zooid generation, but only varied in magnitude during the third generation, with egg production being higher in all genotypes at the nonnative site. Comparisons of weighted frequency distributions between sites demonstrated that differences in egg production in the third generation were due to increases in the proportion of reproductive zooids within a colony. The greater emphasis on female reproduction at a site associated with higher food availability and temperature, and the greater emphasis on male reproduction at a colder, food-limited site, supports predictions from sex allocation theory.     

Aging in the colonial chordate,Botryllus schlosseri

What mechanisms underlie aging? One theory, the wear-and-tear model, attributes aging to progressive deterioration in the molecular and cellular machinery which eventually lead to death through the disruption of physiological homeostasis. The second suggests that life span is genetically programmed, and aging may be derived from intrinsic processes which enforce a non-random, terminal time interval for the survivability of the organism. We are studying an organism that demonstrates both properties: the colonial ascidian, Botryllus schlosseri. Botryllus is a member of the Tunicata, the sister group to the vertebrates, and has a number of life history traits which make it an excellent model for studies on aging. First, Botryllus has a colonial life history, and grows by a process of asexual reproduction during which entire bodies, including all somatic and germline lineages, regenerate every week, resulting in a colony of genetically identical individuals. Second, previous studies of lifespan in genetically distinct Botryllus lineages suggest that a direct, heritable basis underlying mortality exists that is unlinked to reproductive effort and other life history traits. Here we will review recent efforts to take advantage of the unique life history traits of B. schlosseri and develop it into a robust model for aging research.     

'Cup cell disease' in the colonial tunicate Botryllus schlosseri

A new progressive, fatal disease called 'cup cell disease' was characterized in ex situ cultures of Botryllus schlosseri, a colonial tunicate. The disease originated as a few dark spots growing within zooids. The infected colonies then started to deteriorate, morphologically diagnosed by ampullar retraction, lethargic blood circulation and by a swollen and soft tunic matrix. In later stages of the disease, developed buds were also affected. Many large black dots were scattered within the tunic matrix, and zooids were transformed to opaque, dilated, sac-like structures, signaling impending death. Colonies were infected periodically, even without direct tissue contact. The time course from first appearance to colony death ranged between 30 and 45 d. Histological studies, in vitro culturing of blood cells and blood smears revealed the existence of numerous cup-like cells (up to 4.8 microm diameter on average) with a yellowish cell wall and transparent cytoplasm that was not stained by various dyes (except azocarmine-G). Cells were refractive under bright-field illumination and revealed a flattened wall with flanges, characteristic of species of the phylum Haplosporidia. Cup cells aggregated in blood vessels and in internal parts of zooids and buds and were phagocytosed by blood cells. In a single case, plasmodia-like structures were found only in the tunic matrix of an infected colony. This is the first record in botryllid ascidians of an infectious lethal disease associated with haplosporidian protists.     

Programmed cell death in vegetative development: apoptosis during the colonial life cycle of the ascidian Botryllus schlosseri

Programmed cell death (PCD) by apoptosis is a physiological mechanism by which cells are eliminated during embryonic and post-embryonic stages of animal life cycle. During asexual reproduction, the zooids of colonial ascidians originate from an assorted cell population instead of a single zygote, so that we assume that regulation of the equilibrium among proliferation, differentiation and cell death may follow different pathways in comparison to the embryonic development. Here we investigate the presence of apoptotic events throughout the blastogenetic life cycle of the colonial ascidian Botryllus schlosseri, by means of terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) coupled with histochemical and electron microscopy techniques. The occurrence of low levels of morphogenetic cell death suggests that, in contrast to what happens during sexual development (embryogenesis and metamorphosis), apoptosis does not play a pivotal role during asexual propagation in botryllid ascidian. Nevertheless, PCD emerges as a key force to regulate homeostasis in adult zooids and to shape and modulate the growth of the whole colony.     

Colony specificity and chemotaxis in the compound ascidian Botryllus schlosseri

We re-investigated the behavior of hemocytes during the non-fusion (rejection) reaction between genetically incompatible colonies of the ascidian Botryllus schlosseri. In the course of the reaction, hemocytes - mainly morula cells - crowd inside the blind ends of marginal vascular vessels (known as ampullae) of the colonial leading edge (LE) facing the foreign colony which suggests the occurrence of chemotactic attraction of circulating hemocytes towards the ampullar lumen. Then, cells migrate, through the ampullar tips, into the partially fused tunics and contribute to the formation of the necrotic spots along the contact borders which characterize the reaction. Studies on histological sections clearly indicate that, although morula cell concentration is always higher in ampullae of the LE than in those of the lateral (L) part of the colony, their frequency significantly increases in LE ampullae of rejecting colonies with respect to LE ampullae of both fusing and isolated colonies. In addition, in vitro chemotaxis experiments demonstrated that blood plasma from incompatible colonies can stimulate morula cell migration through polycarbonate filters and this passage is inhibited by antibodies raised against mammalian pro-inflammatory cytokines. The possible nature and role of molecules recognized by anti-cytokine antibodies in hemocyte migration are discussed.     

Relationships among hemocytes, tunic cells, germ cells, and accessory cells in the colonial ascidian Botryllus schlosseri

Monoclonal antibodies were raised against hemocytes of the colonial ascidian Botryllus schlosseri as possible tools to study hemocyte differentiation. In this species, blood cells are involved in various biological functions, such as immunosurveillance, encapsulation of foreign bodies, metal accumulation, and allorecognition. The latter process drives the fusion or rejection of contacting colonies, according to whether they do or do not share at least one allele at the fusibility/histocompatibility (Fu/HC) locus. Hemocytes take part in the rejection reaction, which suggests that they express molecules, coded by the Fu/HC locus, on their surface. A homozygous colony at the Fu/HC locus was used to produce the antibodies, which were screened by immunocytochemistry on hemocyte monolayers, immunohistochemistry on colony paraffin sections, and immunoblotting on colony homogenates. Here, we report on one of the obtained antibodies (1D8), which recognized a surface epitope on hemocytes of the donor colony and other colonies, apparently in a manner specific to the Fu/HC genotype. It also labeled a single 80-kDa band in colony homogenates. In addition, it specifically recognized tunic cells, germ cells, and their accessory cells. These results strengthen the assumption of a close relationship among these types of cells and blood cells, and suggest a close relationship among the above cells, probably deriving from undifferentiated blood cells.     

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.     

Pattern of settlement and natural chimerism in the colonial urochordate Botryllus schlosseri

Colonies of the cosmopolitan urochordate Botryllus schlosseri that share one or both alleles at a single allorecognition locus (Fu/HC) and come into tissue contacts, may fuse and form a mixed entity, a chimera. Botryllus populations worldwide exhibit unprecedented extensive polymorphism at this locus, a result that restricts fusions to kin encounters. This study aims to compare spatiotemporal configurations in source and introduced B. schlosseri populations, residing on natural and man-made substrata, respectively. By using four microsatellite loci, we tested genetic consanguinity of colonies settled naturally along spatial vectors on both, natural (native populations) and man-made (introduced) substrates. Four populations were studied. Results revealed that B. schlosseri colonies, on both substrate types, assemble in groups of relatives that share similar microsatellite profiles. We suggest that this pattern of settlement promotes the formation of chimeras, which evoke conflicting interactions: cooperation between different somatic cell lines that constitute the colonial soma and competition between germ cells that inhabit the chimera gonads. Under natural conditions, the chimera may allow genetic flexibility that depends on joint genomic fitness of its partners. This is probably one of the life history characteristics that led to the worldwide distribution success of this species.     

Involution of the caudal musculature during metamorphosis in the ascidian,Botryllus schlosseri

Summary The caudal musculature of the free-swimming tadpole of the ascidian, B. schlosseri consists of cylindrical mononucleated cells connected in longitudinal rows flanking the axial notochord. During resorption of the larval tail, which is apparently induced by the contraction of the epidermis, muscle cells are dissociated and pushed into the body cavity where most of them are rapidly engulfed by phagocytes. In the initial stages of tail withdrawal muscle cells display surface alterations due to the disruption of intercellular junctions and disarrangement of myofibrils. Extensive degenerative changes, with shrinkage of mitochondria and disintegration of the contractile material are subsequently observed. Lysosomes and autophagic vacuoles are rarely seen and appear to play a secondary role in the degradation of the muscle cells, which occurs predominantly within the phagocytes. Myofilaments and myofibrils have never been observed within autophagic vacuoles. Clumps of muscle fragments and degenerated phagocytes undergo eventual dissolution in the blood lacunae, concomitantly with the differentiation of the young oozooid.This investigation was supported in part by a grant from the Muscular Dystrophy Associations of America and by CNR contract No. 7100396/04115542 from the Istituto di Biologia del Mare, Venice. We gratefully acknowledge the skillful assistance of Mr. G. Gallian, Mr. M. Fabbri and Mr. G. Tognon. We also thank the staff of the Stazione Idrobiologica at Chioggia for collecting the colonies.     

Immunomodulatory molecules in the compound ascidian Botryllus schlosseri: evidence from conditioned media

The supernatant from cultures of haemocytes of the compound ascidian Botryllus schlosseri incubated with zymosan (conditioned medium; CM) can enhance yeast phagocytosis by Botryllus blood cells. It contains molecules recognised by antibodies raised against the mammalian pro-inflammatory cytokines IL-1-α and TNF-α which appear as a single band of 60 kDa in immunoblot analysis. The effects on phagocytosis are abolished by the presence of sugars, such as galactose and rhamnose, sharing the same hydroxyl group configuration at C2 and C4. The same sugars also inhibit the haemagglutinating activity of the CM, suggesting the presence of lectins with opsonic activity. With immunoblot analysis, we confirmed the presence, in CM, of B. schlosseri rhamnose-binding lectins (BsRBLs), recently identified and characterised by our team, as a single electrophoretic band of 37 kDa. We had already demonstrated that these molecules are synthesised and secreted by activated phagocytes. Since previous studies have demonstrated that cytotoxic morula cells, and not phagocytes, are the haemocytes responsible for the release of molecules recognised by anti-cytokine antibodies, we propose a new scenario in which morula cells act as sentinels, able to sense foreign molecules and release immunomodulatory factors which induce phagocytes to secrete lectins able to enhance phagocytosis by acting as bridges between foreign particles and phagocyte surfaces.     

Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri

When genetically incompatible colonies of the compound ascidian Botryllus schlosseri contact each other, a rejection reaction occurs, characterised by the appearance of cytotoxic foci along the touching borders. In the course of this reaction, morula cells, a common haemocyte-type in ascidians, release their vacuolar content, mainly phenoloxidase and its polyphenol substrata, upon the recognition of soluble factors diffusing from the alien colony through the partially fused tunic. In a previous paper, we demonstrated the relationship between phenoloxidase and cytotoxicity. Here, we investigated the effects of superoxide dismutase, catalase and sorbitol (scavengers of superoxide anions, peroxides and hydroxyl radicals, respectively) on the cytotoxicity observed in haemocyte cultures incubated with heterologous blood plasma. Although the above compounds have no effects on morula cell degranulation and phenoloxidase activity, they suppress cell death, suggesting that oxidative stress plays a key role in in vitro cytotoxicity. In addition, sorbitol reduces the extent of the cytotoxicity occurring in the rejection reaction between incompatible colonies, which stresses the important role of hydroxyl radicals in this process. The observation of a decrease in total and non-protein thiols in haemocytes previously incubated with heterologous blood plasma fits the hypothesis of oxidative stress as the main cause of phenoloxidase-related cytotoxicity.     

BS-cadherin in the colonial urochordate Botryllus schlosseri: one protein, many functions

Botryllus schlosseri is a colonial urochordate composed of coexisting modules of three asexually derived generations, the zooids and two cohorts of buds, each at disparate developmental stage. Functional zooids are replaced weekly by the older generation of buds through a highly synchronized developmental cycle called blastogenesis (which is, in turn, divided into four major stages, A to D). In this study, we examined the mode of expression of BS-cadherin, a 130-kDa transmembrane protein isolated from this species, during blastogenesis. BS-Cadherin is expressed extensively in internal organs of developing buds, embryos, ampullae and, briefly, in the digestive system of zooids at early blastogenic stage D (in contrast to low mRNA expression at this stage). In vitro trypsin assays on single-cell suspensions prepared from blastogenic stage D zooids, confirmed that BS-cadherin protein is expressed on cell surfaces and is, therefore, functional. BS-Cadherin expression is also upregulated in response to various stress conditions, such as oxidative stress, injury and allorecognition. It plays an important role in colony morphogenesis, because siRNA knockdown during D/A blastogenic transition causes chaotic colonial structures and disrupts oocytes homing onto their bud niches. These results reveal that BS-cadherin protein functions are exerted through a specific spatiotemporal pattern and fluctuating expression levels, in both development/regular homeostasis and in response to various stress conditions.     

Long-term population genetic structure of an invasive urochordate: the ascidian Botryllus schlosseri

The accelerated pace of marine biological invasions raises questions pertaining to genetic traits and dynamics underlying the successful establishment of invasive species. Current research stresses the importance of multiple introductions and prolonged gene flow as the main sources for genetic diversity, which, along with genetic drift, affect invasive species success. We here attempt to determine the relative contribution of gene flow and mutation rates as sources of genetic variability using the invasive tunicate Botryllus schlosseri as a model. The study was performed over a 13-year period in the Santa Cruz Harbor, California. With a characteristic life history of five generations/year, the Santa Cruz Botryllus population has already experienced approximately 155 generations since the onset of its invasion. The results (278 specimens, 127 scored alleles, five microsatellite loci) support limited gene flow rate (2.89?×?10^?3) and relative genetic isolation. Furthermore, the study population was found to be influenced by both, genetic drift and a high mutation rate (2.47?×?10^?2). These findings were supported by high fluctuations in the frequencies of microsatellite alleles, the appearance of new alleles and the loss of others. The balance between genetic drift and a high mutation rate is further elucidated by the high, stable level of genetic variation. We suggest that rapid mutation rates at the microsatellite loci reflect genome-wide phenomena, helping to maintain high genetic variability in relatively isolated populations. The potential adaptability to new environments is discussed.     

Degenerative regression of the digestive tract in the colonial ascidian Botryllus schlossen (Pallas)

Summary Degenerative changes in the digestive tract of zooids of Botryllus schlosseri were studied by light and electron microscopy. Three main processes occurred in the tissues: contraction, involution and phagocytosis. The contraction of epidermis and peribranchial epithelium in which cytoplasmic microfilaments probably participate, seemed to have a special role in compressing the underlying organs. During contraction most of the body cavities collapsed, the branchial walls disintegrated and the fragments were rapidly taken up by large phagocytes. The gut epithelium retained its apparent continuity longer, though isolated phagocytes infiltrated it to eliminate single cells. Cell degeneration came about chiefly either through swelling and lysis of cells or through loss of water and condensation of cytoplasm and nucleus.The fate of all regressed tissues was to be engulfed and digested by wandering phagocytes. However, it was also observed that numerous cells of different epithelia could act as fixed phagocytes by engulfing cell debris and entire cells into heterophagic vacuoles.     

Determination of polarity and bilateral asymmetry in palleal and vascular buds of the ascidian Botryllus schlosseri.

Reversal of the bilateral asymmetry of the zooids was induced in a series of colonies of Botryllus schlosseri. Palleal buds from colonies with normal or reversed bilateral asymmetry were isolated in the early stages from the parental zooids and cultured in the vascularized tunic of the same colony or of another colony with opposite asymmetry. Vascular budding was induced in colonies with either type of asymmetry.The bud polarity was shown to depend on the vascularization; the test vessel entering the isolated palleal bud always causes the entrance point to become the posterior end of the developing zooid. On the contrary, the bilateral asymmetric type is predetermined in the bud primordium; the isolated palleal buds develop the type of asymmetry of their parents, even when grafted in the test of a colony with opposite asymmetry. Since the same was also true of the vascular buds, it is concluded that the information for the kind of bilateral asymmetry to be developed is conveyed by the epidermal envelope of the bud. The epidermis of the parental zooids influences the palleal buds, whereas the wall of the test vessels, epidermal extrusions of the zooids, influences the vascular buds.