UV-B radiation bearings on ephemeral soma in the shallow water tunicate Botryllus schlosseri.

Sedentary shallow water marine organisms acquire numerous protective mechanisms to mitigate the detrimental effects of UV radiation (UV-R). Here we investigated morphological and gene expression outcomes in colonies of the cosmopolitan ascidian Botryllus schlosseri, up to 15-days post UV-B irradiation. Astogeny in Botryllus is characterized by weekly repeating sets of asexual budding, coinciding with apoptotic elimination of functional zooids (blastogenesis). Ten UV-B doses were administered to three clusters: sublethal, enhanced-mortality, lethal (LD50 = 6.048 kJ/m2) which differed in mortality rates, yet reflected similar distorted morphotypes, and arrested blastogenesis, all intensified in the enhanced-mortality/lethal clusters. Even the sub-lethal doses inflicted expression modifications in 8 stress proteins (HSP 90/70 families and NIMA) as well as morphological blastogenesis. The morphological/gene-expression impacts in surviving colonies lasted for 15 days post irradiation (two blastogenic-cycles), where all damaged and arrested zooids/buds were absorbed, after which the colonies returned to their normal blastogenic-cycles and gene expression profiles, and initiated new buds. The above reflects a novel colonial maintenance strategy associated with the disposable-soma tenet, where the ephemeral soma in Botryllus is eliminated without engaging with the costs of repair, whereas other colonial components, primarily the pool of totipotent stem cells, are sustained under yet unknown colonial-level regulatory cues.

Coupling astogenic aging in the colonial tunicate Botryllus schlosseri with the stress protein mortalin.

Botryllus schlosseri, a colonial marine invertebrate, exhibits three generations of short-lived astogenic modules that continuously grow and die throughout the colony's entire lifespan, within week-long repeating budding cycles (blastogenesis), each consisting of four stages (A-D). At stage D, aging is followed by the complete absorption of adult modules (zooids) via a massive apoptotic process. Here we studied in Botryllus the protein mortalin (HSP70s member), a molecule largely known for its association with aging and proliferation. In-situ hybridization and qPCR assays reveal that mortalin follows the cyclic pattern of blastogenesis. Colonies at blastogenic stage D display the highest mortalin levels, and young modules exhibit elevated mortalin levels compared to old modules. Manipulations of mortalin with the specific allosteric inhibitor MKT-077 has led to a decrease in the modules' growth rate and the development of abnormal somatic/germinal morphologies (primarily in vasculature and in organs such as the endostyle, the stomach and gonads). We therefore propose that mortalin plays a significant role in the astogeny and aging of colonial modules in B. schlosseri, by direct involvement in the regulation of blastogenesis.

Histological study on maturation, fertilization and the state of gonadal region following spawning in the model sea anemone, Nematostella vectensis.

The starlet sea-anemone Nematostella vectensis has emerged as a model organism in developmental biology. Still, our understanding of various biological features, including reproductive biology of this model species are in its infancy. Consequently, through histological sections, we study here key stages of the oogenesis (oocyte maturation/fertilization), as the state of the gonad region immediately after natural spawning. Germ cells develop in a secluded mesenterial gastrodermal zone, where the developing oocytes are surrounded by mucoid glandular cells and trophocytes (accessory cells). During vitellogenesis, the germinal vesicle in oocytes migrates towards the animal pole and the large polarized oocytes begin to mature, characterized by karyosphere formation. Then, the karyosphere breaks down, the chromosomes form the metaphase plate I and the eggs are extruded from the animal enclosed in a sticky, jelly-like mucoid mass, along with numerous nematosomes. Fertilization occurs externally at metaphase II via swimming sperm extruded by males during natural spawning. The polar bodies are ejected from the eggs and are situated within a narrow space between the egg's vitelline membrane and the adjacent edge of the jelly coat. The cortical reaction occurs only at the polar bodies' ejection site. Several spermatozoa can penetrate the same egg. Fertilization is accompanied by a strong ooplasmatic segregation. Immediately after spawning, the gonad region holds many previtellogenic and vitellogenic oocytes, though no oocytes with karyosphere. Above are the first histological descriptions for egg maturation, meiotic chromosome's status at fertilization, fertilization and the gonadal region's state following spawning, also documenting for the first time the ejection of the polar body.

Insights into the unique torpor of Botrylloides leachi, a colonial urochordate.

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 3-5day 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 20-50µm "morula-like" opaque balls of cells, and small single-layer epithelial spheres, "blastula-like" structures (50-80µm). 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.

In vitro cultures of ectodermal monolayers from the model sea anemone Nematostella vectensis.

We report here a novel approach for the extraction, isolation and culturing of intact ectodermal tissue layers from a model marine invertebrate, the sea anemone Nematostella vectensis. A methodology is described in which a brief exposure of the animal to the mucolytic agent N-acetyl-L-cysteine (NAC) solution triggers the dislodging of the ectodermis from its underlying basement membrane and mesoglea. These extracted fragments of cell sheets adherent to culture-dish substrates, initially form 2D monolayers that are transformed within 24 h post-isolation into 3D structures. These ectodermal tissues were sustained in vitro for several months, retaining their 3D structure while continuously releasing cells into the surrounding media. Cultures were then used for cell type characterizations and, additionally, the underlying organization of actin filaments in the 3D structures are demonstrated. Incorporation of BrdU and immunohistochemical labeling using p-histone H3 primary antibody were performed to compare mitotic activities of ectodermal cells originating from intact and from in vivo regenerating animals. Results revealed no change in mitotic activities at 2 h after bisection and a 1.67-, 1.71- and 3.74-fold increase over 24, 48 and 72 h of regeneration, respectively, depicting a significant correlation coefficient (p < 0.05; R 2 = 0.74). A significant difference was found only between the control and 3-day regenerations (p = 0.016). Cell proliferation was demonstrated in the 3D ectodermis after 6 culturing days. Moreover, monolayers that were subjected to Ca++/Mg++ free medium for the first 2 h after isolation and then replaced by standard medium, showed, at 6 days of culturing, profuse appearance of positive p-histone H3-labeled nuclei in the 3D tissues. Cytochalasin administered throughout the culturing period abolished all p-histone H3 labeling. This study thus depicts novel in vitro tissue culturing of ectodermal layers from a model marine invertebrate, demonstrating the ease with which experiments can be performed and cellular and molecular pathways can be revealed, thus opening studies on 2D tissue organizations and morphogenesis as well as the roles of cellular components in the formation of tissues in this organism.

The involvement of three signal transduction pathways in botryllid ascidian astogeny, as revealed by expression patterns of representative genes.

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

Germ lineage properties in the urochordate Botryllus schlosseri - from markers to temporal niches.

The primordial germ cells (PGCs) in the colonial urochordate Botryllus schlosseri are sequestered in late embryonic stage. PGC-like populations, located at any blastogenic stage in specific niches, inside modules with curtailed lifespan, survive throughout the life of the colony by repeated weekly migration to newly formed buds. This cyclical migration and the lack of specific markers for PGC-like populations are obstacles to the study on PGCs. For that purpose, we isolated the Botryllus DDX1 (BS-DDX1) and characterized it by normal expression patterns and by specific siRNA knockdown experiments. Expression of BS-DDX1 concurrent with BS-Vasa, γ-H2AX, BS-cadherin and phospho-Smad1/5/8, demarcate PGC cells from soma cells and from more differentiated germ cells lineages, which enabled the detection of additional putative transient niches in zooids. Employing BS-cadherin siRNA knockdown, retinoic acid (RA) administration or ß-estradiol administration affirmed the BS-Vasa(+)BS-DDX1(+)BS-cadherin(+)γ-H2AX(+)phospho-Smad1/5/8(+) population as the B. schlosseri PGC-like cells. By striving to understand the PGC-like cells trafficking between transient niches along blastogenic cycles, CM-DiI-stained PGC-like enriched populations from late blastogenic stage D zooids were injected into genetically matched colonial ramets at blastogenic stages A or C and their fates were observed for 9 days. Based on the accumulated data, we conceived a novel network of several transient and short lived 'germ line niches' that preserve PGCs homeostasis, protecting these cells from the weekly astogenic senescence processes, thus enabling the survival of the PGCs throughout the organism's life.

Repeated, long-term cycling of putative stem cells between niches in a basal chordate.

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

The candidate Fu/HC gene in Botryllusschlosseri (Urochordata) and ascidians' historecognition--an oxymoron?

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

De novo emerged stemness signatures in epithelial monolayers developed from extirpated palleal buds.

In botryllid ascidians, astogeny is executed through blastogenesis, a weekly, highly synchronized phenomenon of growth and death cycles, each constitutes four major developmental stages (A-D), operating simultaneously on three coexisting asexually derived generations, including primary and secondary palleal buds. This study documents the de novo expression of Piwi transcript and protein in extirpated blastogenic stage "D" buds isolated from Botryllus schlosseri colonies that are maintained in vitro, days after the disappearance of corresponding intact zooids in control colonies. Under in vitro conditions, floating buds attach to substrates and develop monolayers of epithelial sheets that live for long periods (compared to intact colonial buds) prior to their deterioration. Here, we further demonstrate that various cell types within floating blastogenic stage "D" buds are labeled as Piwi +, as do other cells that are dispersed over the epithelial sheets (that are Piwi -), representing highly differentiated state), all revealing a surprising new flag for stemness in these tissue fragments that developed exclusively under in vitro conditions. No single permanent cell-line is currently available from colonial tunicates or from other marine invertebrates, since cells stop dividing in vitro within 24-72 h after their isolation and start cellular quiescence. The development of epithelial sheets from isolated Botryllus palleal buds and the recorded molecular stemness flag of various cells, remaining for long periods under in vitro conditions, may pave the way for establishing cell cultures from Botryllus epithelial cells.

Piwi positive cells that line the vasculature epithelium, underlie whole body regeneration in a basal chordate.

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.

Telomere shortening sensitizes cancer cells to selected cytotoxic agents: in vitro and in vivo studies and putative mechanisms.

BACKGROUND: Telomere/telomerase system has been recently recognized as an attractive target for anticancer therapy. Telomerase inhibition results in tumor regression and increased sensitivity to various cytotoxic drugs. However, it has not been fully established yet whether the mediator of these effects is telomerase inhibition per se or telomere shortening resulting from inhibition of telomerase activity. In addition, the characteristics and mechanisms of sensitization to cytotoxic drugs caused by telomerase inhibition has not been elucidated in a systematic manner. METHODOLOGY/PRINCIPAL FINDINGS: In this study we characterized the relative importance of telomerase inhibition versus telomere shortening in cancer cells. Sensitization of cancer cells to cytotoxic drugs was achieved by telomere shortening in a length dependent manner and not by telomerase inhibition per se. In our system this sensitization was related to the mechanism of action of the cytotoxic drug. In addition, telomere shortening affected also other cancer cell functions such as migration. Telomere shortening induced DNA damage whose repair was impaired after administration of cisplatinum while doxorubicin or vincristine did not affect the DNA repair. These findings were verified also in in vivo mouse model. The putative explanation underlying the phenotype induced by telomere shortening may be related to changes in expression of various microRNAs triggered by telomere shortening. CONCLUSIONS/SIGNIFICANCE: To our best knowledge this is the first study characterizing the relative impact of telomerase inhibition and telomere shortening on several aspects of cancer cell phenotype, especially related to sensitivity to cytotoxic drugs and its putative mechanisms. The microRNA changes in cancer cells upon telomere shortening are novel information. These findings may facilitate the development of telomere based approaches in treatment of cancer.

Further portrayal of epithelial monolayers emergent de novo from extirpated ascidians palleal buds.

Astogeny in botryllid ascidians is executed by highly synchronized, repeated development and death cycles operating simultaneously on three coexisting asexually derived generations: zooids, primary buds, and secondary buds. In this study, we validated the fact that surgically removed blastogenic stage "D" primary buds cultured under in vitro conditions, away from any discrete colonial regulatory cues, exhibit intrinsic phenomena that are probably masked by astogenic controls. They produce de novo epithelial monolayers (EM), extending their lifespan from a few days to 1 mo and up to 5 mo when floating in the medium. Enhanced EM formation was documented when fibroblast growth factor (FGF) was added after at least 24 h incubation in FGF-free medium. Surprisingly, with no FGF administration, while intact isolated buds did not develop any EM, injured buds developed EM in half of the cases. Working on actin, PL10, FGF-R, P-MEK, MAP-kinase, and cadherin expressions, we documented that extirpated buds and monolayers are very active on the molecular/biochemical levels, revealing various cells and cellular organelle stains and rapid changes in the protein levels along a daily basis. Cells situated in the center of the monolayers stained differently for some proteins than peripheral cells. Cumulatively, results showed that flattened attached monolayers, as well as free-floating stage "D" buds, are highly active, not only exhibiting differential expressions of various proteins along incubation, but are also highly responsive to physical damages. These results establish a novel in vitro model system for epithelial cell development and senescence, revealing surprising rejuvenation and extended lifespan phenomena.

Employing of the amplified fragment length polymorphism (AFLP) methodology as an efficient population genetic tool for symbiotic cnidarians.

Although the use of molecular markers in population genetics of marine organisms is increasingly employed, methodologic limitations still hampered the research for some taxa, such as symbiotic cnidarians, including scleractinian corals. The development of molecular tools in scleractinian corals' studies is faced with a list of obstacles, such as high cost, labor, time consuming, contamination with foreign DNA, and markers with low resolution. The AFLP (amplified fragment length polymorphism) method, overcomes most of the obstacles listed above except of the difficulty of contamination by algal symbiont DNA. We compared the implication of two pre-DNA extraction treatments to obtain coral DNA free of algal contaminations, termed as CPEM, cell population enriched method, and TTEM, total tissue extraction method. The CPEM process result in pure coral DNA for all samples, but is time consuming, whereas in the TTEM process, approximately 25% to 18% of the samples are still contaminated by algal DNA. However, algal DNA contaminations in the PCR at 2.5 x 10(-1) ng level (approximately 100 algal cells) and below, did not amplify any new AFLP band or peak for neither radioactive nor florescence analyses. Therefore, even the TTEM process may be used because it is faster, easier to handle, and easily employed on a large amount of samples, with minimal contamination artifacts. When correctly employed, both methods are applicable to wide experimental manipulations.

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.

UV incites diverse levels of DNA breaks in different cellular compartments of a branching coral species.

This study evaluates in vitro the effects of UVB irradiation on three cellular compartments of a shallow water coral species. Coral tissues were dissociated by Ca(2+)-Mg(2+)-free artificial seawater. Cell suspensions were divided into the major cellular compartments (animal cells, algal cells, holobiont entities) by sucrose gradient and then by detergent treatments. Cell fractions were irradiated by UVB lamp (4.05, 8.1 and 12.2 kJ m(-2)) and subjected to the comet assay. UVB radiation, at levels that induced a moderate DNA breakage to the non-symbiotic coral and algal cell compartments, caused dramatic increase in DNA breakage to the holobiont entities. After a 1 h repair period, DNA breakage levels in the algal and animal cell fractions were augmented as compared with a reduction in DNA breakage in the holobiont fraction. This discordancy in DNA breakage between the three cellular compartments reveals that the holobiont cell fraction is more vulnerable to increased natural UV irradiation and associated anthropogenic genotoxic impacts, providing another possible explanation for recent increase in worldwide coral bleaching events.

'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.

A 2.5-year genotoxicity profile for a partially restored polluted river.

This study evaluates water genotoxicity of the Kishon River, the most polluted river in Israel that is under restoration. Water samples were collected every other month (January 2001-May 2003) from five sites, and genotoxicity was assayed by the alkaline comet assay using a fish hepatoma cell line (RTH-149). Genotoxicity in the Kishon River was reduced during 2002 as compared to the previous year. The results further revealed fluctuations in genotoxicity levels at all sites throughout the studied period with variations for the same month during consecutive years and seasonality. In general, summer samples were more genotoxic than winter samples. In the vast majority of the 75 water samples, all four parameters for genotoxicity that were employed revealed significant higher genotoxic levels than the controls. Comet percentage values in Kishon River samples were, on average, 1.8-2.4 times higher than controls. Damage score, comet tail length, and cumulative tail length values were 2.2-3.1, 2.4-3.7, and 2.4-3.7 times higher than controls, respectively. The Histadrut Bridge and Haifa fishing harbor (3.0 m depth) emerged as the most polluted sites, whereas Kiryat Haroshet was the least contaminated. Results call for a long-term genotoxicity follow-up plan at the Kishon River in order to assess the possibly evolving chronic genotoxicity state.

In vitro delayed senescence of extirpated buds from zooids of the colonial tunicate Botryllus schlosseri.

In the colonial growth of botryllid ascidians, blastogenesis (bud formation) is a cyclical and synchronized developmental process characterized by a weekly rhythm of budding and apoptotic events. Very little is known about this cycle regulation and its control. In this study, the in vitro fate of developing buds and regressing zooids extirpated from Botryllus schlosseri colonies at different blastogenic stages were examined, revealing that stages 'B' to 'D' buds (but not stage 'A' buds) developed new structures under in vitro conditions. These were mainly spheres (up to 1 mm in diameter) and epithelial monolayers around the attached buds. We also found that: (1) when attached spheres and epithelial monolayers appeared, the life expectancy of an isolated bud in vitro reached 50-60 days, five times the life expectancy of intact, in vivo developing zooids; (2) the life expectancy of in vitro buds that remained unattached to the substrates was at least 150 days; (3) after attaching to the substrates, buds obeyed a newly imposed developmental clock dictating up to 35 survival days for spheres and up to 14 days for epithelial monolayers; (4) the prevailing mode of death in vitro was necrotic, in contrast to the apoptotic mode of zooidal deterioration at the takeover phase of blastogenesis; (5) under in vitro conditions, degenerating zooids surprisingly produced epithelial monolayers within 3 weeks of culturing. Monolayers survived for up to 10 additional days, extending the lifespan of the degenerating zooids from a few hours to up to 1 month. We conclude that under in vitro conditions, not only are the underlying colonial growth mechanisms replaced by different developmental pathways, but also the internal colonial-level clocks programming death, are replaced by a new biological mechanism with different timetables.