Articulating the "stem cell niche" paradigm through the lens of non-model aquatic invertebrates.

Stem cells (SCs) in vertebrates typically reside in "stem cell niches" (SCNs), morphologically restricted tissue microenvironments that are important for SC survival and proliferation. SCNs are broadly defined by properties including physical location, but in contrast to vertebrates and other "model" organisms, aquatic invertebrate SCs do not have clearly documented niche outlines or properties. Life strategies such as regeneration or asexual reproduction may have conditioned the niche architectural variability in aquatic or marine animal groups. By both establishing the invertebrates SCNs as independent types, yet allowing inclusiveness among them, the comparative analysis will allow the future functional characterization of SCNs.

The digestive system of the stony coral Stylophora pistillata.

Because hermatypic species use symbiotic algal photosynthesis, most of the literature in this field focuses on this autotrophic mode and very little research has studied the morphology of the coral's digestive system or the digestion process of particulate food. Using histology and histochemestry, our research reveals that Stylophora pistillata's digestive system is concentrated at the corals' peristome, actinopharynx and mesenterial filaments (MF). We used in-situ hybridization (ISH) of the RNA transcript of the gene that codes for the S. pistillata digestive enzyme, chymotrypsinogen, to shed light on the functionality of the digestive system. Both the histochemistry and the ISH pointed to the MF being specialized digestive organs, equipped with large numbers of acidophilic and basophilic granular gland cells, as well as acidophilic non-granular gland cells, some of which produce chymotrypsinogen. We identified two types of MF: short, trilobed MF and unilobed, long and convoluted MF. Each S. pistillata polyp harbors two long convoluted MF and 10 short MF. While the short MF have neither secreting nor stinging cells, each of the convoluted MF display gradual cytological changes along their longitudinal axis, alternating between stinging and secreting cells and three distinctive types of secretory cells. These observations indicate the important digestive role of the long convoluted MF. They also indicate the existence of novel feeding compartments in the gastric cavity of the polyp, primarily in the nutritionally active peristome, in the actinopharynx and in three regions of the MF that differ from each other in their cellular components, general morphology and chymotrypsinogen excretion.

Integrative approach revises the frequently misidentified species of Sardinella (Clupeidae) of the Indo-West Pacific Ocean.

To deal with the difficulties of species differentiation and delimitation among the commercially important sardines from the genus Sardinella, an integrative approach was adopted, incorporating traditional taxonomy with four DNA markers (coI, cytb, 16s and nuclear rag2). Combining these methodologies has enabled a thorough re-description of three of the most common species of Sardinella of the Indo-west Pacific Ocean: white sardinella Sardinella albella, fringescale sardinella Sardinella fimbriata and the goldstripe sardinella Sardinella gibbosa, as well as a description of a new species, Gon's sardinella Sardinella goni, from the island of Boracay, Philippines. In addition, extensive widespread sampling of S. gibbosa reveals a significant genetic separation between the populations from the western Indian Ocean and the west Pacific Ocean, despite no supporting morphological differentiation. An updated morphological key of the species of Sardinella of the Indo-west Pacific Ocean is also provided in order to minimize future misidentifications within these economically important taxa. Finally, the genetic and morphological variabilities within and between the investigated species are used to discuss their biogeographical distribution and possible processes of speciation.

A conceptual approach for an innovative marine animal forest apparatus that facilitates carbon sequestration and biodiversity enhancement.

Climate change, mainly caused by the indiscriminate usage of fossil fuels, is an urgent global challenge which endangers lives and livelihood of billions of people, the integrity of environmental well-being and the composition and functioning of terrestrial/marine ecosystems alike. To address this pressing concern, climate mitigation and adaptation solutions that target "carbon neutrality by 2050" becomes a crucial global mission. Yet, numerous emerged broad solutions that support biological approaches, such as tree planting, are less stable under enhanced climate change impacts (e.g., forests go on fire). Targeting to achieve the Paris Agreement goals, a wide range of blue carbon sequestering (BCS) approaches have been suggested, since they may contribute considerably to carbon neutrality. Unfortunately, most biological solutions, neglect the employment of marine animal-forests. Here I discuss the potential significance of a novel approach for marine animal forests' BCS, converting the commonly used coral nursery tool into a carbon sequestering floating reef device, a modular device that may accommodate carbon and biodiversity credits.

Mounting of erratic histoincompatible responses in hermatypic corals: a multi-year interval comparison.

Studies on allorecognition in the phylum Cnidaria have disclosed complex arrays of effector mechanisms, specificity and competency to distinguish precisely between self and non-self attributes, and have revealed the existence of allogeneic maturity. Here we studied allo-responses between young Stylophora pistillata colonies by following 517 allogeneic interactions between naturally settled kin aggregates and by establishing 417 forced allogeneic and autogeneic assays made of solitarily settled spat that were cut into two similar size subclones, of which one had been challenged allogeneically. Fused assays were exposed to a second allorecognition challenge, made of three allogeneic types. Whereas about half of the kin allogeneic interactions led to tissue fusions and chimera formations, none of the 83 non-sibling pair combinations were histocompatible. In contrast to previous results we recorded rejections between siblings at the age of less than two months. More challenging, we documented cases of fusions between interacting siblings at ages older than one-year-old partners, all differing from a previous study made on the same coral population more than a decade ago. Similar erratic histoincompatible responses were recorded in other pocilloporid species. We suggest that these results reflect reduced genetic heterogeneity caused by chronic anthropogenic impacts on shallow water coral populations where planulae originating from the same mother colony or from different mother colonies that are genetically related share increasing parts of their genomes. Offspring born to related parents may also reveal an increase in genomic homozygosity, and altogether impose erratic alloimmunity.

The DDX3 subfamily of the DEAD box helicases: divergent roles as unveiled by studying different organisms and in vitro assays.

DDX3 (or Ded1p), the highly conserved subfamily of the DEAD-box RNA helicase family (40 members in humans), plays important roles in RNA metabolism. DDX3X and DDX3Y, the two human paralogous genes of this subfamily of proteins, have orthologous candidates in a diverse range of eukaryotes, from yeast and plants to animals. While DDX3Y, which is essential for normal spermatogenesis, is translated only in the testes, DDX3X protein is ubiquitously expressed, involved in RNA transcription, RNA splicing, mRNA transport, translation initiation and cell cycle regulation. Studies of recent years have revealed that DDX3X participates in HIV and hepatitis C viral infections, and in hepatocellular carcinoma, a complication of hepatitis B and hepatitis C infections. In the urochordates (i.e., Botryllus schlosseri) and in diverse invertebrate phyla (represented by model organisms such as: Drosophila, Hydra, Planaria), DDX3 proteins (termed also PL10) are involved in developmental pathways, highly expressed in adult undifferentiated soma and germ cells and in some adult and embryo's differentiating tissues. As the mechanistic and functional knowledge of DDX3 proteins is limited, we suggest assembling the available data on DDX3 proteins, from all studied organisms and in vitro assays, depicting a unified mechanistic scheme for DDX3 proteins' functions. Understanding the diverse functions of DDX3 in multicellular organisms may be particularly important for effective strategies of drug design.

Cloning and characterization of BS-cadherin, a novel cadherin from the colonial urochordate Botryllus schlosseri.

The genomic DNA for a novel member of the cadherin family (BS-cadherin) was cloned and characterized from the colonial marine invertebrate, Botryllus schlosseri. Using a differential display of mRNA by means of PCR, a small cDNA fragment of 380 nucleotides was found to be specifically expressed in a colony undergoing allogeneic rejection processes, as compared with naive parts of the same genotype. This cDNA fragment was used as a probe to screen a genomic library of Botryllus schlosseri. A genomic fragment containing an ORF of 2718 nucleotides, with no introns, was isolated. The encoded protein exhibits a typical structure of cadherins; an extracellular domain with conserved repeated sequences (cadherin signatures), a single transmembrane domain and a conserved cytoplasmic tail region. The BS-cadherin amino-acid sequence shows 32-35% identity to mature classical cadherins type I, e.g., N-, P- and E-cadherin as well as mature classical cadherins type II, e.g., human cadherin-6, -8 and OB-cadherin. This cadherin represents a new cadherin gene family, evolutionarily distant to all other known classical cadherins.

Molecular evidence for the presence of a developmental gene in the lowest animals: identification of a homeobox-like gene in the marine sponge Geodia cydonium.

During the development of higher animals, morphogenetic programs are switched on which are frequently controlled by homeotic genes. Until now these genes have not been identified in the lowest animals, the marine sponges. Since sponges show (i) an antero-posterior and/or dorso-ventral axis during embryogenesis and (ii) a complex differentiation pattern during spicula formation, we hypothesized that in sponges homeotic genes--if present--are also involved in the control of these processes. Therefore, we searched for homeobox or homeobox-like sequences in the marine sponge Geodia cydonium. Here we describe a homeobox-like sequence from these animals; it was isolated from a cDNA library of an adult specimen. The deduced amino acid sequence of the complete homeodomain shares over 70% similarity with other homeodomain sequences, including those from hydra, insects and vertebrates. These data indicate that the sponge homeodomain-like sequence is similar with respect to structure to those of other animals and may suggest that the sponge homeodomain-like sequence(s) might function during developmental processes and/or during spiculogenesis in a similar manner to that known for higher animals.

A novel galactose- and arabinose-specific lectin from the sponge Pellina semitubulosa: isolation, characterization and immunobiological properties.

A new lectin from the sponge Pellina semitubulosa is derived which was extracted and purified to homogeneity. The purified lectin is probably a hexamer of polypeptide chains (each M(r) 34,000) which are covalently linked via disulfide linkages; the isoelectric point is 6.1. The lectin displays the following specificities: D-galactose (50% inhibition of hemagglutination at 0.2 mM) = L-arabinose (0.2 mM) greater than D-fucose (1.5 mM) greater than D-glucose (3.0 mM). It precipitates human erythrocytes (A1, A2, A1B, B, and O) with a titer between 2(8) and 2(11) and erythrocytes from sheep and rabbits with a titer between 2(5) and 2(10). The Pellina lectin displays a strong mitogenic effect on spleen lymphocytes from mice. Immunochemical analyses revealed that both murine T- and B-lymphocytes display a capping of the lectin receptors on their cell surfaces after lectin treatment. Murine macrophages were found to endocytose the lectin. Pellina lectin at concentrations between 0.3 and 10.0 micrograms/ml potently enhances interleukin 1 (IL-1) release from mouse peritoneal macrophages and interleukin 2 (IL-2) production in mixed murine lymphocyte cultures.

Human natural chimerism: an acquired character or a vestige of evolution?

Analysis on five common classes of human natural chimeras (cytomictical, whole body, fetal-maternal, germ cell, and tumor chimeras) reveals that (1) they initiate only during pregnancy, (2) the most common class are chimeras which contain maternal cells, and (3) the primary mechanisms that are involved in their formation and establishment are still elusive. These classes of natural chimerism, are involved only with maladaptive phenomena such as malignancy and autoimmune diseases and without any documented benefit. A recent review has challenged the accepted dogma that the evolution of immunity is pathogen-directed and asserted that preserving individuality from littering the soma and the germline by conspecific alien cells might have been the original function of the innate immunity. Following this tenet, I propose here that human natural chimerism is a by-product of the new role evolved from primitive components of immunity to "educate" the developing embryo with the armamentarium of effector mechanisms, dedicated to purge the individual from pervasive somatic and germline variants, and is not a vestige of evolution.

Characteristics of allogeneic resorption in Botrylloides from the Mediterranean coast of Israel.

Some of the characteristics of allogeneic resorption have been revealed in studies of chimeras of two Botrylloides subpopulations (SP1 and SP3) inhabiting the Mediterranean coast of Israel. Both Botrylloides SPs exhibited the same outcomes, which differ significantly from the resorption documented in the well-studied botryllid ascidian, Botryllus schlosseri: 1. Resorption in Botrylloides chimeras was usually a very fast process starting 1 to 26 days after fusion and terminating in < 9 days thereafter. 2. Resorption occurs at any blastogenic stage. 3. Resorption is complete, including zooids, buds, blood vessels and ampullae. 4. Either one of the genotypes can be resorbed in multipair chimeras of any specific combination. 5. Resorption starts simultaneously in all zooids of the partner to be resorbed. 6. Time for resorption and directionality were not affected by the size ratios between the partners (0.3-1.8). As in B. schlosseri, other phenomena (disconnection, chimeric death and retreat growth) interrupt resorption in ca. 60% of the chimeras, under laboratory conditions. We propose that external stress conditions also have a significant role in a nonimmunological expression of resorption in Botrylloides.

Allogeneic resorption in colonial protochordates: consequences of nonself recognition.

Colonial tunicates (protochordates of the subfamily Botryllinae) have the property of forming natural chimeras in the wild or in the laboratory with other members of the same species if both individuals share at least one allele at a single highly polymorphic locus, termed the fusibility/histocompatibility (Fu/HC) locus. Laboratory studies revealed that after the establishment of a common blood circulatory system between a fusible pair one of the colonies in the chimera is usually resorbed, a phenomenon that occurs after an interval of 1 week or extends to up to 8 months. In the present article, we review this allogeneic resorption in Botryllus schlosseri, a cosmopolitan protochordate. The studies on allogeneic resorption in B. schlosseri revealed 13 typical characteristics. However, several processes interrupt successful resorption, such as the retreat growth phenomenon, chimeric death, and others. It was also found that allogeneic resorption is not only genetically controlled but is also controlled by a polymorphic hierarchial phenomenon, including the Fu/HC locus and additional unrelated loci. One basic rule for this genetic system is that colonies heterozygotic on the resorption elements will resorb more homozygotic partners. This colony resorption, expressed by a morphological elimination of one partner, is probably manifested by cellular elements circulating in the tunicate blood system. However, it was also shown that some blood-borne cells of the inferior partner, such as the stem cells, may escape resorption, a phenomenon that raises the threat of germ/somatic cell parasitism.

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.

Intraspecific competition in a reef coral: effects on growth and reproduction.

Growth rates and reproduction of a branching coral (Stylophora pistillata) were compared in the presence and in the absence of intraspecific competition. Field experiments demonstrated a significant decline in the growth rate of competing colonies compared to noncompeting control colonies; the growth rate slowed in all of the interacting individuals, irrespective of their place in the hierarchy of the intraspecific dominance or of their color morph. In case of immediate killing of the subordinate, the dominant colony grew at a normal rate. In addition to the marked decrease in the growth rate of interacting colonies, the typical symmetry shape of these colonies was changed to an abnormal growth form. The number of female gonads per polyp was significantly reduced in colonies competing intraspecifically, and the typical synchrony in reproduction among different branches of a given colony was changed and desynchronized. Again, these results did not correlate with the hierarchy of dominance. We conclude that intraspecific competition in reef corals involves great investment of energy. The ecological significance and the different pathways of this competition are discussed.

A simple, reliable, and fast protocol for thraustochytrid DNA extraction.

DNA extraction of thraustochytrids, common marine unicellular organisms, is usually accomplished by either the cetyltrimethylammonium bromide (CTAB) or proteinase K protocols. A novel lysis buffer protocol for thraustochytrid total DNA extraction is described. The average isolated total DNA is 20 to 40 kb, and DNA samples are suitable for a variety of uses including 18S-ribosomal DNA polymerase chain reaction, restriction enzyme digestions, and amplified fragment length polymorphism analyses. The new protocol is also faster than the other protocols.

In vitro application of the comet assay for aquatic genotoxicity: considering a primary culture versus a cell line.

The comet assay, one of the most widely used techniques for the evaluation and detection of DNA strand breaks, is frequently employed in vivo. In vitro assays are usually performed with mammalian cell lines, clearly not the best choice for tests on aquatic genotoxicity. Here we evaluated a fish hepatoma cell line (RTH-149) and a primary blood cell culture from the intertidal colonial tunicate Botryllus schlosseri as possible model targets for comet assays using the genotoxic agent H2O2. We found that DNA strand break levels in RTH-149 fitted dose-dependent responses better than the tunicate cells. Moreover, in B. schlosseri controls, 34% of the cells were already ranked as severely damaged. Assays were then performed on water samples from the polluted Kishon river (Israel) on three different dates, using RTH-149 cells (50% dilutions, 2-h exposures). In all cases, high genotoxicity of the river water was revealed by evaluating comet percentages, average tail lengths and DNA damage levels. This assay was found to be fast and sensitive, appropriate to be employed as a part of a monitoring program. The use of B. schlosseri blood cells should be validated in additional work.

Cloning of a urochordate cDNA featuring mammalian short consensus repeats (SCR) of complement-control protein superfamily.

Mammalian tumor necrosis factor (TNF)-alpha degenerate polymerase chain reaction (PCR) primers were used to amplify a probe from Botryllus schlosseri (colonial ascidian) allogeneic rejection-cDNA library. A PCR product (269 bp) was cloned and sequenced encoding an open reading frame (ORF) of 89 amino acids (aa). This clone, which revealed no similarity to TNF-alpha, but a substantial similarity to mammalian proteins featuring short consensus repeats (SCRs) of the complement control superfamily, was used to probe the rejection-cDNA library. Two partial cDNA clones were isolated and sequenced (Bs.1, 846 bp; Bs.2, 712 bp). The longest ORF in clone Bs.1 (which lacks the 5' end of the cDNA) predicts a protein of 251 aa, which differs from Bs.2 at six nucleotides and four aa. We compare the aa similarity (up to 50.5%) of Bs.1 with the SCR-region of mammalian complement factor H, apolipoprotein H, selectins, and complement receptors type 1 and type 2. A somatomedin B-like domain at the C-terminus of Bs.1 deduced protein was also recorded. We propose that this mosaic and polymorphic botryllid sequence, featuring mammalian-like SCRs, might be an ancestral molecule in the evolution of the chordate's complement-control protein superfamily.

A critical approach to the definition of Darwinian units of selection.

What are the biological units of selection? In fact, the notion of "unit of selection" (UOS) is blurred by ambiguity and controversy. To further evaluate the biological entities that are the objects of natural selection, three novel conceptual criteria (holism, minimalism, functionalism) are critically applied; they reveal, in addition to the self-evident case of the "individual," at least six distinct types of UOSs. These UOSs do not always have a defined structural organization; they can be parts of a living organism, a cohesive group of conspecifics, a multiunit entity, a totipotent cell, a DNA fragment, or a whole organism. UOS types diversify by amalgamation or parcelation processes of apparent entities. Therefore, previous attempts to characterize the UOSs solely on some morphological levels (gene, individual, group) without applying stringent criteria have failed to cope with the structural variations of natural phenomena and have led to the ambiguity of terms used.

A morphological study of nonrandom senescence in a colonial urochordate.

Botryllus schlosseri is a clonally modular ascidian, in which individuals (zooids) have a finite life span that is intimately associated with a weekly budding process called blastogenesis. Every blastogenic cycle concludes with a synchronized phase of regression called takeover, during which all zooids in a colony die, primarily by apoptosis, and are replaced by a new generation of asexually derived zooids. We have previously documented that, in addition to this cyclical death phase, entire colonies undergo senescence during which all asexually derived individuals in a colony, buds and zooids, die in concert. In addition, when a specific parent colony (genet) is experimentally separated into a number of clonal replicates (ramets), ramets frequently undergo senescence simultaneously, indicating that mortality can manifest itself in nonrandom fashion. Here, we document a morphological portrait of senescence in laboratory-maintained colonies from Monterey Bay, California, that exhibit nonrandom mortality. Nonrandom senescence proceeded according to a series of characteristic changes within the colony over a period of about one week. These changes included systemic constriction and congestion of the vasculature accompanied by massive accumulation of pigment cells in the zooid body wall (mantle), blood vessels, and ampullae; gradual shrinkage of individual zooids; loss of colonial architecture, and ultimately death. At the ultrastructural level, individual cells exhibited changes typical of ischemic cell death, culminating in necrotic cell lysis rather than apoptosis. Collectively, these observations indicate that senescence is accompanied by unique morphological changes that occur systemically, and which are distinct from those occurring during takeover. We discuss our findings in relation to current experimental models of aging and the possible role of a humoral factor in bringing about the onset of senescence.

Transplantation of Fu/HC-incompatible zooids in Botryllus schlosseri results in chimerism.

The colonial urochordate Botryllus schlosseri undergoes a genetically defined, natural transplantation reaction that is controlled by a single Mendelian locus (called the Fu/HC). This Fu/HC-based allorecognition system is initiated when peripheral elements of the vasculature interact on the edges of two asexually expanding colonies. To better understand the spatial organization of the cellular elements responsible for Fu/HC-based allorecognition, we bypassed the normal site of interaction (the ampullae) and experimentally transplanted zooids between Fu/HC-noncompatible Botryllus schlosseri pairs. The results show that (1) instead of the expected rejections (tissue necroses) that develop after natural contacts between peripheral blood vessels, the transplanted organs are morphologically eliminated within a few days in conjunction with the normal blastogenic cycle; and (2) donor-recipient chimerism is established after complete morphological elimination of transplanted tissues. These results suggest that Fu/HC-based allorecognition responses in Botryllus schlosseri occur exclusively at the ampullae and that once cells have crossed this barrier, they are able to survive and proliferate in the new host colony.