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PubMed2000

Tributyltin induces cytoskeletal alterations in the colonial ascidian Botryllus schlosseri phagocytes via interaction with calmodulin.

In the colonial ascidian Botryllus schlosseri, tributyltin (TBT), a powerful antifouling biocide, acts as immunotoxic xenobiotic since, at a sublethal concentration (10 µM), it causes an irreversible and significant decrease in in vitro yeast phagocytosis, associated with considerable changes in the shape of phagocytes, which withdraw their pseudopodia and become spherical, due to structural damage of cytoskeletal components. The addition of TBT to the culture medium causes a significant decrease in the amoebocytic index, i.e. the percentage of amoeboid-shaped haemocytes, and prolonged washing in sea water never succeeds in restoring amoeboid shape. In these cytoskeletal alterations, F-actin undergoes extensive depolymerisation, resulting in the absence of FITC-phalloidin fluorescence. Microtubules are not recognisable as single filaments with anti-alpha-tubulin immunofluorescence, although the centrosome is not affected. The addition of increasing exogenous calmodulin (CaM) concentrations (from 20 to 120 µM) after incubation in TBT determines a significant increase in the amoebocytic index, although it is not able to bring it to that of controls, suggesting that CaM in the medium in any case externally exerts an influence on haemocytes pretreated with TBT. The copresence of TBT and exogenous CaM at concentrations higher than 80 µg/ml restores the amoebocytic index and cytoskeletal morphology. The latter appears complete for microtubules and partial for microfilaments. Experiments with isodynamic mixtures of TBT and specific CaM inhibitors, i.e. chlorpromazine (CPZ) and N-(6-aminohexyl)-5-chloronaphtalene-1-sulfonamide (W-7), reveal the synergistic effect of antagonism, indicating competition for the same site - a Ca(2+)-CaM hydrophobic region - by both interacting substances and, therefore, the formation of a TBT-CaM complex. Instead, isodynamic mixtures with thapsigargin, an inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum, have an effect of potentiation, suggesting that TBT indirectly interacts with this Ca(2+)-ATPase activity. We hypothesise that the main mechanism of action of TBT in B. schlosseri phagocytes is alteration of Ca(2+) homeostasis by means of direct interaction with endogenous CaM, which induces a conformational change preventing the regulative activity of CaM on Ca(2+)-ATPase. Consequently, an excess of cytosolic Ca(2+) accumulates which, together with the inhibition of CaM-dependent kinases and Ca(2+)-regulated proteins, produces extensive cytoskeletal disorganisation.

Journal Article↗

PubMed1997

Highly polymorphic microsatellite loci in the colonial ascidian Botryllus schlosseri.

Five mircosatellite loci are characterized for the colonial ascidian Botryllus schlosseri. Within one population from Monterey, California, these loci have 3 to 17 alleles, observed heterozygosities from 0.40 to 0.63, expected heterozygosities from 0.52 to 0.84, and an overall paternity exclusion rate (QT) of 0.78. Three of the five loci demonstrated Mendelian patterns of inheritance in laboratory crosses. The size distribution of alleles suggests that most allelic diversity within these loci is generated by single-step and less frequently multistep mutations. However, several alleles may also have been generated by single based insertions or deletions. Mutation rates for the five microsatellite loci are less than 1 x 10(-2) per generation. Because or their highly polymorphic nature, these loci should prove useful for exploring issues of identity, kinship, population structure, and phylogenetics.

Alleles↗

PubMedDate not supplied

Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri.

The vacuoles of morula cells (MC) of the colonial ascidian Botryllus schlosseri contain phenoloxidase (PO). As the release of their vacuolar content at the border of incompatible contacting colonies is associated with the formation of necrotic masses which characterize the rejection reaction, the role of PO in Botryllus cytotoxicity was investigated. When hemocytes are incubated with blood plasma from incompatible (heterologous) colonies, MC degranulate and, after 60 min, the cytotoxicity index becomes significantly greater than that observed in controls incubated with autologous plasma. The rise in cell mortality is completely inhibited by the addition of PO inhibitors sodium benzoate, tropolone and phenylthiourea, and serine protease inhibitors phenylmethylsulfonyl fluoride, benzamidine, N-tosyl-L-phenylalanine chloromethyl ketone and N-tosyl-L-lysine chloromethyl ketone. The addition of either reducing agents L-cysteine and ascorbic acid or reactive oxygen species scavenger enzymes superoxide dismutase and catalase has a similar effect. Significant inhibition of cytotoxicity is also observed with the quinone scavenger, 3-methyl-2-benzothiazolinone hydrazone. In the presence of sodium benzoate and phenylthiourea, there is a significant reduction in the number, size and color intensity of necrotic masses along the contact border of incompatible colonies. A significant increase in superoxide anion production, completely inhibited by sodium benzoate, is observed when hemocytes are incubated with heterologous blood plasma. These results indicate that: (i) PO is the enzyme responsible for the cytotoxicity observed in both hemocyte cultures and rejection reactions; (ii) PO is present inside MC vacuoles as a proenzyme which is activated, upon release, by humoral proteases; (iii) cytotoxicity appears to be mainly due to oxidative stress generated by PO during oxidation of polyphenols to quinones without the involvement of other oxidases such as NADPH oxidase and peroxidase.

Animals↗

PubMed1999

Purification and characterization of a humoral opsonin, with specificity for D-galactose, in the colonial ascidian Botryllus schlosseri.

A humoral agglutinin from the hemolysate of the colonial ascidian Botryllus schlosseri was purified by affinity chromatography. This agglutinin does not require metal cations for its activity and is specific for derivatives of D-galactose. On SDS-PAGE analysis, it was resolved in two bands, of 17 and 19 kDa in reducing conditions and 15 and 16 kDa in non-reducing conditions. This behavior is due to the establishment of disulfide bridge between the thiols of cysteine, well represented in the molecule as revealed by amino acid analysis. The latter also indicated high percentages of hydrophilic residues, probably involved in sugar recognition. The lectin is an opsonin, as it increases both the phagocytic index and the number of phagocytized yeast cells. The hypothesis that this Botryllus agglutinin belongs to the galectin family of lectins is discussed.

Agglutinins↗

PubMed1984

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

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.

Animals↗

PubMedDate not supplied

Construction and characterization of large-insert genomic libraries (BAC and fosmid) from the Ascidian Botryllus schlosseri and initial physical mapping of a histocompatibility locus.

The colonial protochordate Botryllus schlosseri is genetically manipulable and represents a potential model organism for a variety of biological disciplines, including immunology, stem cell biology and development. This article presents the construction and characterization of both BAC and fosmid genomic libraries of the 725-Mbp B. schlosseri genome. The BAC library currently consists of 2x genome coverage with an average insert size of 80 kb. The fosmid library is at 11x genome coverage with an average insert of 40 kb. B. schlosseri is a small organism containing a large number of compounds that hinder DNA purification. Thus a number of protocols had to be modified in order to make purified, high molecular weight inserts for cloning, including both gel purification and insert concentration techniques. Both libraries were characterized by using them in initial physical mapping of a single histocompatibility locus, and were found to be representative and functional. These libraries are important tools for physical mapping and positional cloning in the B. schlosseri genome, and the techniques adapted to make them are suitable for use on other organisms in which high molecular weight DNA is difficult to purify.

Animals↗

PubMed1994

Molecular cloning and localization of a novel serine protease from the colonial tunicate Botryllus schlosseri.

A cDNA encoding a putative serine protease was isolated and characterized from Botryllus schlosseri, a colonial protochordate. The open-reading frame of the 846 bp cDNA is 744-nt long. The deduced aa sequence predicts a protein of 26,134 D, with the signature of chymotrypsin. The mRNA of the protease is expressed only in the zooids as a single 0.9-kb transcript. No transcript was found in the tunic matrix (test). Surprisingly, a 3.7-fold higher enzyme activity was found in the test, compared with the zooids. We propose that this serine protease may have a role as a biologically active compound in the defense mechanisms against epibionts or as part of the effector mechanisms expressed in allogeneic and xenogeneic interactions.

Amino Acid Sequence↗

PubMed1976

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.

Animals↗

PubMed1995

Comparison of the fatty acids of the tunicate Botryllus schlosseri from the Black Sea with two associated bacterial strains.

The fatty acid composition of the tunicate Botryllus schlosseri and of two bacterial strains found within the tunicate, namely Vibrio parahaemolyticus and of an associated but previously unreported gram positive cocci were studied. The polyunsaturated fatty acids 6,9,12-octadecatrienoic acid, 5,8,11,14,17-eicosapentaenoic acid, and 4,7,10,13,16,19-docosahexaenoic acid were particularly abundant in B. schlosseri and were not detected in the two bacterial strains found in the tunicate. The iso/anteiso pair, 13-methyltetradecanoic acid and 12-methyltetradecanoic acid, were the principal fatty acids in the gram positive cocci, and the 9- and 11-hexadecenoic acids were particularly abundant in V. parahaemolyticus. The diunsaturated fatty acid 9,12-octadecadienoic acid was also shown to be present in V. parahaemolyticus. The fatty acid composition of a third bacterial strain, characterized as either a Pseudomonas or an Alteromonas species, and shown to be present only in the sea water from the Black Sea and not in B. schlosseri, is also reported. This is the first investigation on fatty acids from Black Sea bacteria.

Animals↗

PubMed2004

'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.

Animals↗

PubMed1990

Botryllus schlosseri (Tunicata) whole colony irradiation: do senescent zooid resorption and immunological resorption involve similar recognition events?

The colonial tunicate Botryllus schlosseri undergoes cyclic blastogenesis where feeding zooids are senescened and resorbed and a new generation of zooids takes over the colony. When non-identical colonies come into direct contact, they either reject each other or fuse. Fusion is usually followed by the resorption of one of the partners in the chimera (immunological resorption). The striking morphological similarities between the two resorption phenomena suggest that both may involve tissue destruction following self-nonself recognition events. Here we attempt to modify these two events by whole colony gamma irradiation assays. Three sets of experiments were performed: 1) different doses of whole colony irradiation for determination of irradiation effects (110 colonies, up to 8,000 rads); 2) pairs of irradiated-nonirradiated isografts of clonal replicates for the potential of reconstruction of the irradiated partners (23 pairs); 3) chimeras of irradiated-nonirradiated partners for analysis of resorption hierarchy. Mortality increased with the irradiation dose. All colonies exposed to more than 5,000 rads died within 19 days, while no colony died below 2,000 rads. The average mortality periods, in days, for doses of 6,000-8,000, 5,000, and 2,500-4,000 rads were 14.4 +/- 3.1 (n = 24), 19.8 +/- 6.0 (n = 15), and 19.6 + 5.1 (n = 22), respectively. Younger colonies (3-6 months old) may survive radiation better than older ones (more than 13 months). Many morphological alterations were recorded in irradiated colonies: ampullar contraction and/or dilation, accumulation of pigment cells within ampullae, abnormal bleeding from blood vessels, sluggish blood circulation, necrotic zones, reduction in bud number, and irregularities in zooid and system structures. With doses of 3,000-4,000 rads and above, irradiation arrested the formation of new buds and interrupted normal takeover, turning the colony into a chaotic bulk of vessels, buds, and zooid segments. Death supervened after a period of up to 1 month of poor condition, which was also characterized by loss of organization in systems. In isografts of irradiated-nonirradiated parts, the normal subclone resorbed all zooids and buds of the irradiated one within less than 1 week, even if it was up to 13 times smaller, without showing any sign of harmful effects. Thus, the irradiated subclone is not reconstituted by sharing blood circulation with a syngeneic part. Under 2,000 rads some of the irradiated zooids within this type of union started to regenerate, and at 1,000 rads no resorption was recorded, even though the number of zooids decreased in the irradiated part.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1996

Sequence and characterization of two HSP70 genes in the colonial protochordate Botryllus schlosseri.

Two genes belonging to the heat shock protein 70 gene family have been cloned from the colonial protochordate Botryllus schlosseri. The two intronless genes (HSP70.1 and HSP70.2) exhibit 93.6% sequence identity within the predicted coding region, and 83.3% and 81.7% sequence identity in the 5' and 3' flanking regions, respectively. The predicted amino acid sequences are 95% identical and contain several signatures characteristic of cytoplasmic eukaryotic HSP70 genes (Gupta et al. 1994; Rensing and Maier 1994). Northern blotting and sequence analysis suggest that both genes are heat-inducible members of the HSP70 gene family. Given these characteristics, HSP70.1 and HSP70.2 appear to be good candidates for protochordate homologues of the major histocompatibility complex-linked HSP70 genes of human, mouse, and rat (Milner and Campbell 1990; Walter et al. 1994). Further experiments to determine whether there is functional evidence for such similarity are in progress.

Amino Acid Sequence↗

PubMed2004

Macrophage involvement for successful degeneration of apoptotic organs in the colonial urochordate Botryllus schlosseri.

Apoptosis is an important tool for shaping developing organs and for maintaining cellular homeostasis. In the colonial urochordate Botryllus schlosseri, apoptosis is also the hallmark end point in blastogenesis, a cyclical and weekly developmental phenomenon. Then the entire old generation of zooids are eliminated (resorbed) by a process that lasts 24-36 h. Administration of the antioxidant butylated hydroxytoluene (BHT) resulted in resorption being arrested by 1-8 days on average. At high doses (2.5-15.0 mg BHT l(-1)) resorption was completed only after removal of BHT. Colonies that were not removed in time, died. In treated colonies, although DNA fragmentation was high, tissues and organs that would normally have died, survived, and the general oxidative levels of lipids were reduced. Blood vessels were widened, containing aggregates of blood cells with a significantly increased proportion of empty macrophage-like cells without inclusion. In colonies rescued from BHT treatment, resorption of zooids started immediately and was completed within a few days. We propose three possible mechanisms as to how BHT may affect macrophage activity: (1) by interrupting signals that further promote apoptosis; (2) through the respiratory burst initiated following a phagocytic stimulus; and (3) by reducing lipid oxidation and changing cell surface markers of target cells. Our results point, for the first time, to the role of phagocytic cells in the coordination of death and clearance signals in blastogenesis.

Analysis of Variance↗

PubMed2003

Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges.

This study focuses on recent improvement in epithelial monolayer cultures originating from whole extirpated Botryllus schlosseri (Urochordata) buds. Buds (n = 2,000) were taken at different ('A' to 'D') blastogenic stages. We tested the suitability of 35 combinations of various substrates and media on attachment, cell spread, epithelial growth frequencies and on monolayer lifespans. Under favorable conditions, cultured buds at blastogenic stages 'B' to 'D' (but not stage 'A') started to attach to the substrates following a 3-day transient period that leads to formation of spheres and attached monolayers. Substrate type is important for the attachment and the development of monolayers. Under various culture conditions, some of stages 'B' and 'C' buds develop (3-20 days) one or more large (1 mm diameter) spheres. Stage 'D' buds develop monolayers (up to 20% of buds) without going through a sphere phase. Neither spheres nor attached monolayers of epithelium were observed in stage 'A' bud cultures. Spheres grew at a rate of 60 microm in diameter per day using specific medium types and did not attach unless the appropriate substrate was present. When attached, epithelial monolayers expanded at a rate of 200 microm in diameter per day, for 3-15 days, and subsequently detached and died. Sixteen types of media were tested. Medium and substrate combinations were found to determine epithelial lifespan. These results revealed significant improvements in the culture of epithelial monolayers from Botryllus palleal buds. However, an early senescence of the developed epithelial sheets (up to two weeks from onset of appearance) may indicate an internal ageing clock that should be taken into consideration in future approaches.

Animals↗

PubMed1998

Linkage analysis of HSP70 genes and historecognition locus in botryllus schlosseri.

The protochordate allorecognition system has long invited comparison with the vertebrate major histocompatibility complex (MHC). In the colonial species Botryllus schlosseri, a rapid fusion or rejection response resembling graft acceptance or rejection in vertebrates is controlled by a single highly polymorphic genetic region. Because linkage between heat shock protein 70 (HSP70) genes and the MHC appears to be conserved within the vertebrate lineage, linkage relationships between two HSP70 genes (HSP70.1 and HSP70.2) and the historecognition locus (FuHC) have been analyzed in B. schlosseri. Segregation patterns of restriction fragment length polymorphisms located in the 3' flanking regions of HSP70.1 and HSP70.2 were determined for progeny of defined crosses. These progeny were also analyzed for fusibility type by an in vivo cut colony assay. No close linkage was detected between any of the three loci. These results do not support the hypothesis that the allorecognition response in B. schlosseri is determined by an MHC homologue. However, it remains a possibility that orthologues of other MHC-linked genes will be linked to the B. schlosseri FuHC.

Animals↗

PubMed1997

A novel tunicate (Botryllus schlosseri) putative C-type lectin features an immunoglobulin domain.

We have cloned a putative C-type lectin of Botryllus schlosseri [Ascidiacea], whose deduced protein of 333 amino acids features three building blocks: (i) a Greek-key motif signature at the amino-terminus, (ii) a C-type lectin domain signature, and (iii) an immunoglobulin (Ig) domain at the carboxyl terminus. This C-type lectin was termed BSCLT. Similarity searches revealed that the Ig domain in BSCLT, which is evidently not polymorphic, is best classified as an Intermediate-type Ig domain. Rabbit antibodies, raised against recombinant BSCLT, cross-reacted in a Western blot with a 38-kD polypeptide in tunicate crude extract. Presumably, this bimodal tunicate protein is the first description of a soluble lectin that features besides the carbohydrate recognition domain also a complete Ig domain.

Amino Acid Sequence↗

PubMedDate not supplied

Monoclonal antibody specific to urochordate Botryllus schlosseri pyloric gland.

We describe here the development of a new hybridoma cell line, CF12F6, that produces a specific antibody to Botryllus schlosseri (a colonial tunicate). The monoclonal antibody was isotyped as IgG1 (by enzyme-linked immunosorbent assay), and the cellular localization of the antigenic epitope that reacts specifically with CF12F6 was confined to the cells of the pyloric gland of the zooid (by immunohistochemistry). The pyloric gland participates in osmoregulation, digestion, glycogen storage, and various other secretion functions that will be studied further by the use of monoclonal antibody CF12F6, the first in botryllid ascidians that recognizes cells of a whole, single organ.

Animals↗

PubMed2006

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.

Animals↗