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.

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.

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.

Assembled catalog of immune-related genes from allogeneic challenged corals that unveils the participation of vWF-like transcript.

While reef-building corals portray highly complex and specific allorecognition responses, still, no available synthesis on historecognition at the molecular level exists for this group of organisms. Here, we present the first subtractive library of expressed sequence tags (ESTs) from allogeneic challenged coral (Stylophora pistillata) colonies revealing the differential expression of a wide range of immune-related genes. 1760 unique ESTs were clustered and assembled into 230 contigs and 1530 singlets with 28% that showed homology (E-value < or =0.005) to known database sequences, of which 16% (n=80) homologues were identified as immune-relevant genes, encoding for stress proteins, pattern recognition receptors and complement proteins, proteases, cell adhesion proteins, cytokine related proteins, programmed cell death and proteasome-associated proteins. Transcripts that were subjected to quantitative RT-PCR, further supported the library data. In situ hybridization analyses elucidated specific and enhanced expressions of von Willebrand factor-like transcript during S. pistillata allogeneic rejection. Availability of such genome-wide expression tools may lead to significant advances in the research of coral historecognition and comparative immunology.

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.

Development of monoclonal antibodies specific to urochordate intracellular epitopes.

Sixteen monoclonal antibodies (MAbs) specific to 2 urochordate genera (Botryllus schlosseri and Botrylloides) intracellular epitopes were generated in mice immunized with a mixture of fresh and paraformaldehyde-fixed cells obtained from animal's blood and cells from dissociated organs. Hybridoma clones were selected by ELISA tests and immunohistochemistry assays on paraffin-embedded animal tissues. Five MAbs were tested for reactions with different zooidal organs and cell compartments; 7 MAbs were tested, separately, on 5 different botryllid colonies (3 Botryllus and 2 Botrylloides). The results revealed high polymorphism. Whereas some of the MAbs recognized, specifically, only part of the botryllid genotypes tested, others recognized only part of the cellular compartments. These MAbs will be used as an important tool in the study of botryllid ascidian immunology and developmental biology, revealing the first wide panel of MAbs specific to urochordate intracellular antigens.

Metallothionein induction in the RTH-149 cell line as an indicator for heavy metal bioavailability in a brackish environment: assessment by RT-competitive PCR.

Estuarine and marine near-shore environments are often subjected to heavy metal pollution. We establish a bioassay using the quantitative evaluation of metallothionein (MT) transcript in the fish hepatoma cell line, RTH-149, as a tool for detecting heavy metal pollution in brackish-marine water containing other pollutants in addition to heavy metals. RT-competitive polymerase chain reaction was used for the quantitative evaluation of the transcript in absolute units. Cadmium was used as a model pollutant to optimize two parameters of the assay: exposure periods (24, 96, 144 h) and salinity (0%, 25%, 50%, 75%, 100% sea water). Results revealed that salinity at or below 25% sea water at an exposure period of 144 h are the preferable conditions for detecting MT mRNA levels for in vitro assays employed on water samples from highly polluted brackish habitats.

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.

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.

Alloimmune memory is absent in the Red Sea hydrocoral Millepora dichotoma.

When two allogeneic colonies of the Red Sea hydrocoral Millepora dichotoma (Cnidaria, Hydrozoa) come into tissue contact, one of the genotypes is usually overgrown by the other. The directionality and pace of this alloresponse are thought to be genetically determined. We established tissue contacts between allogeneic colonies in situ in order to elucidate a possible memory component in this response. First-set interactions were established from all possible pairwise combinations between three colonies in eight replicates per combination. Interactions were followed up for 8 weeks. Thereafter, interacting pairs were detached and either regrafted near the original contact area to form second-set assays or challenged by third party grafts. Additional delayed first-set assays was also established. Overgrowth of delayed first-set, second-set, and third-party grafts was followed again for 8 weeks. The mean overgrowths recorded in the second set of the interactions were indistinguishable from the first sets in all three colony combinations. A specific alloimmune memory has not been found in this cnidarian system as opposed to other cases within the phylum.

Interspecific interactions among species of the coral genus Porites from Okinawa, Japan.

Analysis of field established xenogeneic interactions among five Porites species from Sesoko Island, Okinawa, revealed a transitive type of hierarchy as: P. rus >P. cylindrica >P. lobata >P. australiensis>P. lutea. Out of the 111 interspecific encounters studied, in only 5.4% reciprocal interactions were recorded, and in a single case, the opposite directionality of hierarchy was documented. Allogeneic encounters were also observed. A single major effector mechanism, an overgrowth (together with secondary outcomes such as the formation of small points of rejection, bleaching and pink color formation along a narrow peripheral belt of contacting tissues), was the only response in all 10 xenogeneic and 5 allogeneic combinations. In some massive colonies, a long contacting line of up to 50 cm was established. No sign for allelopathy, stand-off or rejection from a distance (i.e., by sweeper tentacles, sweeper polyps) was observed. Results are discussed with the accumulated data on Porites species from different reefs, worldwide, confirming that this genus is commonly lower in the hierarchy of xenogeneic interactions.

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.

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.

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.

Scyphozoan jellyfish's mesoglea supports attachment, spreading and migration of anthozoans' cells in vitro.

Mechanically and enzymatically dissociated cells from five anthozoan species were laid on seven substrates in vitro. Cells were taken from two sea anemones (Aiptasia sp. and Anemonia sulcata), a scleractinian coral (Stylophora pistillata) and two alcyonacean corals (Heteroxenia fuscescence and Nephthea sp). Substrates tested: glass (coverslips), plastic (uncoated tissue culture plates), type IV collagen, gelatin, fibronectin, mesoglea pieces from the scyphozoan jellyfish Rhopilema nomadica and acetic acid extract of jellyfish mesoglea. Except for the mesoglea pieces, cells did not respond to any one of the other substrates, retaining their rounded shape. Following contact with mesoglea pieces, cells attached and spread. Subsequently they migrated into the mesogleal matrix at a rate of 5-10 microm/h during the first 2-5 h. No difference was found between the behavior of cells from the five different cnidarian species.

Invertebrates versus vertebrates innate immunity: In the light of evolution.

Invertebrates use a nonadaptive, innate immunity, the expression of germline encoded receptors, to identify the allogeneic and xenogeneic attributes. Vertebrates also have the capacity to express ontogenically related adaptive immunity which is a somatically selected gene rearrangement process. Several commonly accepted generalizations are utilized to explain the enigmatic lack of the adaptive immunity in invertebrates. All point to the primitive nature of the innate immunity and the primitive organization of the body plan and the life history patterns of invertebrates. Seven of the most common generalizations are reviewed and confuted by virtue of a biased literature presentation. Subsequently, three evolutionary puzzles are raised and the accepted paradigm that the vertebrate immunity is pathogenically directed is further challenged. This leads to an alternative idea suggesting that preserving the individuality against the threat of invading conspecific cells might have been the original function of the immune system. This ancient system has been co-opted later on to serve as a defence mechanism against pathogens. The secondary role arose in the form of a multiplicity of newly developed phenomena, one of them being the vertebrate adaptive immunity. This proposal is supported by the fact that vertebrates still exhibit two distinct but common types of naturally occurring transplantation events (natural chimerism) and by a variety of recent studies, providing evidence for the crucial role of the vertebrate's innate immunity in signalling and triggering the acquired effector mechanisms.