Spatial distribution of conspecific genotypes within chimeras of the branching coral Stylophora pistillata.

Chimerism is a coalescence of conspecific genotypes. Although common in nature, fundamental knowledge, such as the spatial distribution of the genotypes within chimeras, is lacking. Hence, we investigated the spatial distribution of conspecific genotypes within the brooding coral Stylophora pistillata, a common species throughout the Indo-Pacific and Red Sea. From eight gravid colonies, we collected planula larvae that settled in aggregates, forming 2-3 partner chimeras. Coral chimeras grew in situ for up to 25 months. Nine chimeras (8 kin, 1 non-related genotypes) were sectioned into 7-17 fragments (6-26 polyps/fragment), and genotyped using eight microsatellite loci. The discrimination power of each microsatellite-locus was evaluated with 330 'artificial chimeras,' made by mixing DNA from three different S. pistillata genotypes in pairwise combinations. In 68% of 'artificial chimeras,' the second genotype was detected if it constituted 5-30% of the chimera. Analyses of S. pistillata chimeras revealed that: (a) chimerism is a long-term state; (b) conspecifics were intermixed (not separate from one another); (c) disproportionate distribution of the conspecifics occurred; (d) cryptic chimerism (chimerism not detected via a given microsatellite) existed, alluding to the underestimation of chimerism in nature. Mixed chimerism may affect ecological/physiological outcomes for a chimera, especially in clonal organisms, and challenges the concept of individuality, affecting our understanding of the unit of selection.

Morphometric and allometric rules of polyp's landscape in regular and chimeric coral colonies of the branching species Stylophora pistillata.

BACKGROUND: Most studies on architectural rules in corals have focused on the branch and the colony level, unveiling a variety of allometric rules. Working on the branching coral Stylophora pistillata, here we further extend the astogenic directives of this species at the polyp level, to reveal allometric and morphometric rules dictating polyps' arrangement. RESULTS: We identified a basic morphometric landscape as a six-polyp circlet developed around a founder polyp, with established distances between polyps (six equilateral triangles), reflecting a strong genetic-based background vs high plasticity on the population level. Testing these rules in regular and chimeric S. pistillata colonies, we revealed similar morphometric/allometric rules developed via a single astogenic pathway. In regular colonies, this pathway was driven by the presence/absence of intra-circlet budding polyps, while in chimeras, by the distances between the two founder polyps. In addition, we identified the intra-circlet budding as the origin of first branching, if BPC distances are kept <1.09 ± 0.25 mm. CONCLUSIONS: The emerged allometric/morphometric rules indicate the existence of a positional information paradigm for polyps' landscape distribution, where each polyp creates its own positional field of morphogen gradients through six inductive sites, thus forming six positional fields for the development of the archetypal "six-polyp crown".

Cell Communication-mediated Nonself-Recognition and -Intolerance in Representative Species of the Animal Kingdom.

Why has histo-incompatibility arisen in evolution and can cause self-intolerance? Compatible/incompatible reactions following natural contacts between genetically-different (allogeneic) colonies of marine organisms have inspired the conception that self-nonself discrimination has developed to reduce invasion threats by migratory foreign germ/somatic stem cells, in extreme cases resulting in conquest of the whole body by a foreign genome. Two prominent model species for allogeneic discrimination are the marine invertebrates Hydractinia (Cnidaria) and Botryllus (Ascidiacea). In Hydractinia, self-nonself recognition is based on polymorphic surface markers encoded by two genes (alr1, alr2), with self recognition enabled by homophilic binding of identical ALR molecules. Variable expression patterns of alr alleles presumably account for the first paradigm of autoaggression in an invertebrate. In Botryllus, self-nonself recognition is controlled by a single polymorphic gene locus (BHF) with hundreds of codominantly expressed alleles. Fusion occurs when both partners share at least one BHF allele while rejection develops when no allele is shared. Molecules involved in allorecognition frequently contain immunoglobulin or Ig-like motifs, case-by-case supplemented by additional molecules enabling homophilic interaction, while the mechanisms applied to destroy allogeneic grafts or neighbors include taxon-specific tools besides common facilities of natural immunity. The review encompasses comparison with allorecognition in mammals based on MHC-polymorphism in transplantation and following feto-maternal cell trafficking.

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.

IAP genes partake weighty roles in the astogeny and whole body regeneration in the colonial urochordate Botryllus schlosseri.

Inhibitors of Apoptosis Protein (IAP) genes participate in processes like apoptosis, proliferation, innate immunity, inflammation, cell motility, differentiation and in malignancies. Here we reveal 25 IAP genes in the tunicate Botryllus schlosseri's genome and their functions in two developmental biology phenomena, a new mode of whole body regeneration (WBR) induced by budectomy, and blastogenesis, the four-staged cycles of botryllid ascidian astogeny. IAP genes that were specifically upregulated during these developmental phenomena were identified, and protein expression patterns of one of these genes, IAP28, were followed. Most of the IAP genes upregulation recorded at blastogenetic stages C/D was in concert with the upregulation at 100 µM H2O2 apoptotic-induced treatment and in parallel to expressions of AIF1, Bax, Mcl1, caspase 2 and two orthologues of caspase 7. Wnt agonist altered the takeover duration along with reduced IAP expressions, and displacement of IAP28+ phagocytes. WBR was initiated solely at blastogenetic stage D, where zooidal absorption was attenuated and regeneration centers were formed either from remains of partially absorbed zooids or from deformed ampullae. Subsequently, bud-bearing zooids developed, in concert with a massive IAP28-dependent phagocytic wave that eliminated the old zooids, then proceeded with the establishment of morphologically normal-looking colonies. IAP4, IAP14 and IAP28 were also involved in WBR, in conjunction with the expression of the pro-survival PI3K-Akt pathway. IAPs function deregulation by Smac mimetics resulted in severe morphological damages, attenuation in bud growth and differentiation, and in destabilization of colonial coordination. Longtime knockdown of IAP functions prior to the budectomy, resulted in colonial death.

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.

Cell cultures from marine invertebrates: new insights for capturing endless stemness.

Despite several decades of extensive research efforts, there is yet no single permanent cell line available from marine invertebrates as these cells stop dividing in vitro within 24-72 h after their isolation, starting cellular quiescence. This ubiquitous quiescent state should be modified in a way that at least some of the quiescent cells will become pluripotent, so they will have the ability to divide and become immortal. Following the above need, this essay introduces the rationale that the discipline of marine invertebrates' cell culture should gain from applying of two research routes, relevant to mammalian systems but less explored in the marine arena. The first is the use of adult stem cells (ASC) from marine organisms. Many marine invertebrate taxa maintain large pools of ASC in adulthood. Ample evidence attests that these cells from sponges, cnidarians, flatworms, crustaceans, mollusks, echinoderms, and ascidians play important roles in maintenance, regeneration, and asexual cloning, actively proliferating in vivo, resembling the vertebrates' cancer stem cells features. The second route is to target resting somatic cell constituents, manipulating them in the same way as has recently been performed on mammalian induced pluripotent stem (iPS) cells. While "iPS cells" are the outcome of an experimental manipulation, ASC are natural and rather frequent in a number of marine invertebrates. Above two cell categories reveal that there are more than a few types of seeds (cells) waiting to be sowed in the right soil (in vitro environmental conditions) for acquiring stemness and immortality. This rationale carries the potential to revolutionize the discipline of marine invertebrate cell cultures. When cultured "correctly," ASC and "iPS cells" from marine invertebrates may stay in their primitive stage and proliferate without differentiating into cells lineages, harnessing the stem cell's inherent abilities of self-replication versus differentiated progenies, toward the development of immortal cell lines.

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.

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.

Vasa and the germ line lineage in a colonial urochordate.

Germ cell sequestering in Animalia is enlightened by either, launching true germ line along epigenetic or preformistic modes of development, or by somatic embryogenesis, where no true germ line is set aside. The research on germ line-somatic tissue segregation is of special relevancy to colonial organisms like botryllid ascidians that reconstruct, on a weekly basis, completely new sets of male and female gonads in newly formed somatic tissues. By sequencing and evaluating expression patterns of BS-Vasa, the Botryllus schlosseri orthologue of Vasa, in sexually mature and asexual colonies during blastogenesis, we have demonstrated that the BS-Vasa mRNA and protein are not expressed exclusively in germ cell lineages, but appeared in cells repeatedly emerging de novo in the colony, independently of its sexual state. In addition, we recorded an immediate Vasa response to cellular stress (UV irradiation) indicating additional functions to its germ line assignments. To confirm germ lineage exclusivity, we examined the expression of three more stem cell markers (BS-Pl10, Bl-piwi and Oct4). Vasa co-expression with Pl10 and Oct4 was detected in germ line derivatives and with Bl-piwi in somatic tissues. Presumptive primordial germ cells (PGC-like cells), that are Vasa(+)/Pl10(+)/Oct4(+) and 6-12 microm in diameter, were first detected in wrapped-tail embryos, in oozooids, in sexual/asexual colonies, within a newly identified PGC niche termed as 'budlet niche', and in circulating blood borne cells, indicating epigenetic embryogenesis. Alternatively, BS-Vasa co-expression with piwi orthologue, an omnipresent bona fide stemness flag, in non germ line cell populations, may indicate germ cell neogenesis (somatic embryogenesis) in B. schlosseri. Both alternatives are not necessarily mutually exclusive.

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.

Cell signaling and transcription factor genes expressed during whole body regeneration in a colonial chordate.

BACKGROUND: The restoration of adults from fragments of blood vessels in botryllid ascidians (termed whole body regeneration [WBR]) represents an inimitable event in the chordates, which is poorly understood on the mechanistic level. RESULTS: To elucidate mechanisms underlying this phenomenon, a subtracted EST library for early WBR stages was previously assembled, revealing 76 putative genes belonging to major signaling pathways, including Notch/Delta, JAK/STAT, protein kinases, nuclear receptors, Ras oncogene family members, G-Protein coupled receptor (GPCR) and transforming growth factor beta (TGF-beta) signaling. RT-PCR on selected transcripts documented specific up-regulation in only regenerating fragments, pointing to a broad activation of these signaling pathways at onset of WBR. The followed-up expression pattern of seven representative transcripts from JAK/STAT signaling (Bl-STAT), the Ras oncogene family (Bl-Rap1A, Bl-Rab-33), the protein kinase family (Bl-Mnk), Bl-Cnot, Bl-Slit and Bl-Bax inhibitor, revealed systemic and site specific activations during WBR in a sub-population of circulatory cells. CONCLUSION: WBR in the non-vertebrate chordate Botrylloides leachi is a multifaceted phenomenon, presided by a complex array of cell signaling and transcription factors. Above results, provide a first insight into the whole genome molecular machinery of this unique regeneration process, and reveal the broad participation of cell signaling and transcription factors in the process. While regeneration involves the participation of specific cell populations, WBR signals are systemically expressed at the organism level.

Short and long term toxicity of crude oil and oil dispersants to two representative coral species.

Oil dispersants, the tool of choice for treating oil spills in tropical marine environments, is potentially harmful to marine life, including reef corals. In a previous study, we found that dispersed oil and oil dispersants are harmful to soft and hard coral species at early life stages. In this broader study, we employed a "nubbin assay" on more than 10 000 coral fragments to evaluate the short- and long-term impacts of dispersed oil fractions (DOFs) from six commercial dispersants, the dispersants and water-soluble-fractions (WSFs) of Egyptian crude oil, on two Indo Pacific branching coral species, Stylophora pistillata and Pocillopora damicornis. Survivorship and growth of nubbins were recorded for up to 50 days following a single, short (24 h) exposure to toxicants in various concentrations. Manufacturer-recommended dispersant concentrations proved to be highly toxic and resulted in mortality for all nubbins. The dispersed oil and the dispersants were significantly more toxic than crude oil WSFs. As corals are particularly susceptible to oil detergents and dispersed oil, the results of these assays rules out the use of any oil dispersant in coral reefs and in their vicinity. The ecotoxicological impacts of the various dispersants on the corals could be rated on a scale from the least to the most harmful agent, as follows: Slickgone > Petrotech > Inipol = Biorieco > Emulgal > Dispolen.

Identification of immune-relevant genes in histoincompatible rejecting colonies of the tunicate Botryllus schlosseri.

The colonial ascidian Botryllus schlosseri manifests a unique allorecognition system that is controlled by a single histocompatibility haplotype, the Fu/HC locus. When two allogeneic incompatible colonies come into direct contact, they develop inflammatory-like rejection lesions, called points of rejection (POR). While screening for differentially expressed genes during POR formation, we developed and analyzed a cDNA library of expressed sequence tags (ESTs) with 1693 unique ESTs that were clustered and assembled into 217 contigs and 1476 singlets. About 51% of these ESTs showed high similarity (E-value 0.005) to known database sequences, of which 123 matches were identified as immune-relevant genes encoding for stress proteins, pattern recognition receptors and complement proteins, proteases and protease inhibitors, cell adhesion and coagulation proteins, cytokine-related proteins, programmed cell death and proteasome-associated proteins. This first EST wide-screening analysis of the Botryllus allorecognition effector arm reveals a complex innate immune system, hallmarked by a whole genome response to allorecognition challenge.

Branch to colony trajectory in a modular organism: pattern formation in the Indo-Pacific coral Stylophora pistillata.

The architecture of the colony in a branching coral is an iterative process in which new layers of calcium carbonate compile atop existing structures that remain unchanged. Colony growth and development, known as astogeny, is believed to be a continuous process, characterized by replication of lower rank unites, polyps, and branches. This study seeks to explore the genetic blueprint of branch-to-colony developmental trajectory in the branching coral Stylophora pistillata, within an astogeny period of 1 year. One hundred small branches (initially 2-4 cm long) were sampled from 10 colonies. A year later, 63 remaining colonies were analyzed for their architectural rules by using 15 morphometric parameters. Multivariate statistical tests were preformed. Cluster and two-dimensional nonmetric Multi-Dimensional Scaling analyses revealed that the 10 genotypes could be divided into two major morphometric groups and two intermediate groups, whereas SIMPER analyses (a similarity percentage test) on within-genet similarities showed high similarity between the ramets developed from each of the 10 genotypes. Although, at first, it seemed that different colonies exhibited variable and different architectural designs (each characterized by specific morphometric parameters), a comprehensive analysis revealed that all 10 coral genotypes exhibited a single common developmental plan that was characterized by a continuum of architectural design with several distinct stages. Each stage is marked by its own characteristic morphometric parameters. Changing of developmental rules during the trajectory from branch to coral colony may help the colony to cope better with environmental constraints.

Conservation of coral reefs through active restoration measures: recent approaches and last decade progress.

The scientific discipline of active restoration of denuded coral reef areas has drawn much attention in the past decade as it became evident that this ecosystem does not often recover naturally from anthropogenic stress without manipulation. Essentially, the choices are eitherthe continuous degradation of the reefs or active restoration to encourage reef development. As a result, worldwide restoration operations during the past decade have been recognized as being a major tool for reef rehabilitation. This situation has also stirred discussions and debates on the various restoration measures suggested as management options, supplementary to the traditional conservation acts. The present essay reviews past decade's (1994-2004) approaches and advances in coral reef restoration. While direct coral transplantation is still the primer vehicle of operations used, the concept of in situ and ex situ coral nurseries (the gardening concept), where coral materials (nubbins, branches, spats) are maricultured to a size suitable for transplantation, has been gaining recognition. The use of nubbins (down to the size of a single or few polyps) has been suggested and employed as a unique technique for mass production of coral colonies. Restoration of ship grounding sites and the use of artificial reefs have become common tools for specific restoration needs. Substrate stabilization, 3-D structural consideration of developing colonies, and the use of molecular/biochemical tools are part of novel technology approaches developed in the past decade. Economic considerations for reef restoration have become an important avenue for evaluating success of restoration activities. It has been suggested that landscape restoration and restoration genetics are important issues to be studied. In the future, as coral reef restoration may become the dominant conservation act, there would be the need not only to develop improved protocols but also to define the conceptual bases.

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.

Protochordate concordant xenotransplantation settings reveal outbreaks of donor cells and divergent life span traits.

If fulminate rejection in allogeneic and xenogeneic engraftments is not an evolutionary relict feature, then any treatment that ablates the host surveillance's effector arms capabilities and eliminates graft vs. host reactivity should induce donor chimerism in transplant settings. We demonstrate here marked proliferative response of Botryllus (Urochordata) blood cells months following their infusions (2x10(4)-10(5) blood cells per host) into the concordant xenogeneic environment of irradiated Botrylloides soma. The state of infused cells was followed by Botryllus specific microsatellite alleles on DNA samples from host zooids and vascular system. Increased growth rates and life spans of engrafted hosts in some cases, and sudden chimerical death following the outbreak of donor cells in others, indicate a 'double-edged sword' expression of concurrent evolutionary selected mechanisms. This DES phenomenon in immunity underlies divergent stem cell competition phenomena in multicellular organisms, leading in mammals, to cases of autoimmune diseases vis-à-vis long-lasting microchimerism events following an iatrogenic transplantation.