Metagenomic sequencing suggests a diversity of RNA interference-like responses to viruses across multicellular eukaryotes.

RNA interference (RNAi)-related pathways target viruses and transposable element (TE) transcripts in plants, fungi, and ecdysozoans (nematodes and arthropods), giving protection against infection and transmission. In each case, this produces abundant TE and virus-derived 20-30nt small RNAs, which provide a characteristic signature of RNAi-mediated defence. The broad phylogenetic distribution of the Argonaute and Dicer-family genes that mediate these pathways suggests that defensive RNAi is ancient, and probably shared by most animal (metazoan) phyla. Indeed, while vertebrates had been thought an exception, it has recently been argued that mammals also possess an antiviral RNAi pathway, although its immunological relevance is currently uncertain and the viral small RNAs (viRNAs) are not easily detectable. Here we use a metagenomic approach to test for the presence of viRNAs in five species from divergent animal phyla (Porifera, Cnidaria, Echinodermata, Mollusca, and Annelida), and in a brown alga-which represents an independent origin of multicellularity from plants, fungi, and animals. We use metagenomic RNA sequencing to identify around 80 virus-like contigs in these lineages, and small RNA sequencing to identify viRNAs derived from those viruses. We identified 21U small RNAs derived from an RNA virus in the brown alga, reminiscent of plant and fungal viRNAs, despite the deep divergence between these lineages. However, contrary to our expectations, we were unable to identify canonical (i.e. Drosophila- or nematode-like) viRNAs in any of the animals, despite the widespread presence of abundant micro-RNAs, and somatic transposon-derived piwi-interacting RNAs. We did identify a distinctive group of small RNAs derived from RNA viruses in the mollusc. However, unlike ecdysozoan viRNAs, these had a piRNA-like length distribution but lacked key signatures of piRNA biogenesis. We also identified primary piRNAs derived from putatively endogenous copies of DNA viruses in the cnidarian and the echinoderm, and an endogenous RNA virus in the mollusc. The absence of canonical virus-derived small RNAs from our samples may suggest that the majority of animal phyla lack an antiviral RNAi response. Alternatively, these phyla could possess an antiviral RNAi response resembling that reported for vertebrates, with cryptic viRNAs not detectable through simple metagenomic sequencing of wild-type individuals. In either case, our findings show that the antiviral RNAi responses of arthropods and nematodes, which are highly divergent from each other and from that of plants and fungi, are also highly diverged from the most likely ancestral metazoan state.

Evidence of anticipatory immune and hormonal responses to predation risk in an echinoderm.

Recent efforts have been devoted to the link between responses to non-physical stressors and immune states in animals, mostly using human and other vertebrate models. Despite evolutionary relevance, comparatively limited work on the appraisal of predation risk and aspects of cognitive ecology and ecoimmunology has been carried out in non-chordate animals. The present study explored the capacity of holothuroid echinoderms to display an immune response to both reactive and anticipatory predatory stressors. Experimental trials and a mix of behavioural, cellular and hormonal markers were used, with a focus on coelomocytes (analogues of mammalian leukocytes), which are the main components of the echinoderm innate immunity. Findings suggest that holothuroids can not only appraise threatening cues (i.e. scent of a predator or alarm signals from injured conspecifics) but prepare themselves immunologically, presumably to cope more efficiently with potential future injuries. The responses share features with recently defined central emotional states and wane after prolonged stress in a manner akin to habituation, which are traits that have rarely been shown in non-vertebrates, and never in echinoderms. Because echinoderms sit alongside chordates in the deuterostome clade, such findings offer unique insights into the adaptive value and evolution of stress responses in animals.

Current progress on identification of virus pathogens and the antiviral effectors in echinoderms.

Echinoderms are important marine organisms that live in a wide range from the intertidal zone to the abyssal zone. Members of this phylum are prone to dramatic population fluctuations that may trigger dramatic shifts in ecosystem structure. Despite the extremely complex nature of the marine environment, the immune systems of echinoderms induce a complex innate immune response to prokaryotic and eukaryotic pathogens. Previous studies showed that many echinoderm disease outbreaks were associated with specific bacteria, whereas recent scientific investigations using newly developed technologies revealed the amazing diversity of viruses in seawater. Viruses are potential pathogens of several infectious diseases of marine echinoderms. We reviewed the discovery of viruses in echinoderms and discussed the relationship between viruses and diseases for the first time. We further summarized the research progress of the potential immune-related genes and signal pathways induced by viruses and poly (I:C). Additionally, numbers of studies showed that active substances extracted from echinoderms, or the compounds synthesized from these substances, have significant antihuman virus ability. This result suggests that the active substances derived from echinoderms provide potential antiviral protection for the organism, which may provide future research directions for the antiviral immunity of echinoderms. Thus, this review also collected information on the antiviral activities of biologically active substances from echinoderms, which may pave the way for new trends in antiviral immunity for echinoderms and antiviral drugs in humans.

Complement and complement-like activity in lower vertebrates and invertebrates.

A purified cobra venom factor with C-inhibiting activity also promotes lysis of erythrocytes in fresh mammalian serum. Lysis-inducing activity of purified cobra venom factor was found in sera of lower vertebrates including the cyclostome hagfish and in invertebrates. Lysis-inducing activity was most effective with frog serum. Frog serum was found to be more hemolytic for E(s) in the presence of CVF than when cells were sensitized with hemolysin. The hemolysis induced by CVF with frog serum, as in the higher vertebrates, was inhibited when sera were pretreated with known C inhibitors including heat, chelators, endotoxin, immune complexes, and CVF itself. Complexes formed with CVF and either frog serum or invertebrate hemolymph promoted lysis of indicator cells in the presence of frog serum in EDTA. This lysis was most marked when the starfish-CVF complex was used and was C-dependent. Conversely, complex formed with frog serum and CVF promoted lysis of E in the presence of invertebrate hemolymph (Limulus) in EDTA. Hence, serum components were to some degree at least interchangeable between vertebrate sera and invertebrate hemolymph. Lysis-inducing activity of purified CVF occurs in a wide range of species, has revealed activities resembling those of terminal C-components in lower vertebrates and invertebrates, and provides one means for study of C and C-like activities in primitive species.

Sea urchin response to foreign substances.

An assessment of the response of Strongylocentrotus purpuratus to substances injected into the coelom shows that (i) foreign molecules are cleared from the coelomic fluid more rapidly than native molecules, (ii) coelomocytes can respond selectively to albumins of human and bovine origin, and (iii) immunization attempts fail to elicit accelerated coelomocyte uptake or accelerated clear-with antigen.

Evolution of immune reactions.

The history and evolutionary pathways of defense reactions among various forms of life are reconstructed. The vertebrates evolved step-by-step from their invertebrate ancestors living in the distant past. The ancestry of vertebrate defense mechanisms must be traceable to them because these functions cannot be considered separately from the common evolutionary schema. The first part of this survey is therefore devoted to the description of major defense reactions and achievements in invertebrates. Particular emphasis is given to the taxa of present-day invertebrates most likely to exhibit some relationship to the chordates and thus to show real traces of vertebrate immune patterns. The second part involves three key assemblages of deuterostomate animals--the echinoderms, the nonvertebrate chordates, and the first vertebrates because these animals are believed to have created the prerequisites for transition from the invertebrate type of defense to the vertebrate type of adaptive immunity.

Antimicrobial peptides in echinoderm host defense.

Antimicrobial peptides (AMPs) are important effector molecules in innate immunity. Here we briefly summarize characteristic traits of AMPs and their mechanisms of antimicrobial activity. Echinoderms live in a microbe-rich marine environment and are known to express a wide range of AMPs. We address two novel AMP families from coelomocytes of sea urchins: cysteine-rich AMPs (strongylocins) and heterodimeric AMPs (centrocins). These peptide families have conserved preprosequences, are present in both adults and pluteus stage larvae, have potent antimicrobial properties, and therefore appear to be important innate immune effectors. Strongylocins have a unique cysteine pattern compared to other cysteine-rich peptides, which suggests a novel AMP folding pattern. Centrocins and SdStrongylocin 2 contain brominated tryptophan residues in their native form. This review also includes AMPs isolated from other echinoderms, such as holothuroidins, fragments of beta-thymosin, and fragments of lectin (CEL-III). Echinoderm AMPs are crucial molecules for the understanding of echinoderm immunity, and their potent antimicrobial activity makes them potential precursors of novel drug leads.

Specific allograft reactivity in the sea star Dermasterias imbricata.

Transplantation studies with the sea star Dermasterias imbricata revealed that this echinoderm rejected most first-set allografts after prolonged chronic reactions. All control autografts remained intact and fully viable. The greatly accelerated rejection of subsequent second- and third-set allografts demonstrated that these reactions were not only highly specific, but that these animals possess at least short-term immunological memory. The specific nature of the allograft response was confirmed by the prolonged survival of unrelated third party allografts placed at the same time as second- or third-set allografts. Animals in these experiments were maintained in sea water at temperatures of 14-16 C. Since this low temperature is required for successful maintenance of this species, temperature-dependence studies could not be undertaken. The rejection reaction was characterized by loss of pigmentation, edematous swelling, and necrosis. Histologically, the allograft tissue was heavily infiltrated by lymphocyte-like cells and phagocytic cells, causing disruption of the normal cytoarchitecture of the dermis. Deposits of fibrous material were also in evidence. Autografts, on the other hand, remained fully viable with no signs of rejection. We conclude that this asteroid, a representative of the echinoderms, has a well-developed, cell-mediated immune response analogous to that of vertebrates.

Studies on Holothuria polii (Echinodermata) coelomocyte lysate. II. Isolation of coelomocyte hemolysins.

The lytic activity of the Holothuria polii coelomocyte lysate resides in two electrophoretically distinct hemolysins identified as He1 and He2. He1 represents the calcium dependent, heat-labile component whereas He2 is calcium independent and heat-stable. The two hemolysins share serological identity. Both hemolysins appear as single protein molecules of 80KDa molecular weight by SDS-PAGE and transblotting analysis under non-reducing conditions. However under reducing conditions, they are doublets of 76 and 80KDa molecular weight. The hypothesis that the two hemolysins could be isoforms is discussed.

The hemolysin-producer coelomocytes in Holothuria polii.

Using sodium metrizoate discontinuous gradients, two hemolysin-producer amebocyte populations have been separated from total circulating Holothuria polii coelomocytes. The amebocytes of population 1 are responsible for the production of the calcium-dependent and temperature-labile hemolysin, whereas those of population 2 produce the calcium-independent and temperature-stable one. The intracytoplasmic hemolysins were evidenced also by immunofluorescence. Petaloid and filipodial amebocytes were the only positive cell types.

Echinoderm immunity and the evolution of the complement system.

Our understanding of inflammatory responses in humans has its roots in the comparative approach to immunology. In the late 1900s, research on echinoderms provided the initial evidence for the importance of phagocytic cells in reactions to foreign material. Studies of allograft rejection kinetics have shown that echinoderms have a non-adaptive, activation type of immune response. Coelomocytes mediate the cellular responses to immune challenges through phagocytosis, encapsulation, cytotoxicity, and the production of antimicrobial agents. In addition, a variety of humoral factors found in the coelomic fluid, including lectins, agglutinins, and lysins, are important in host defense against pathogens and other foreign substances. Recently, a simple complement system has been identified in the purple sea urchin that is homologous to the alternative pathway in vertebrates. The sea urchin [corrected] homologue of C3, is inducible by challenge with lipopolysaccharide, which is known to activate coelomocytes. Complement components have been identified in all vertebrate classes, and now have been characterized in protochordates and echinoderms indicating the primordial nature of the complement system. Because it is thought that the complement system evolved from a few primordial genes by gene duplication and divergence, the origin of this system appears to have occurred within the common ancestor of the deuterostomes.

Inhibitory activity of sphingomyelin on hemolytic activity of coelomic fluid of Holothuria polii (Echinodermata).

The hemolytic activity of coelomic fluid from Holothuria polii is specifically inhibited by sphingomyelin. This phospholipid is the constituent of the membrane which probably interacts with the hemolysin thereby leading to the lysis.

Evolution of the acute phase response: iron release by echinoderm (Asterias forbesi) coelomocytes, and cloning of an echinoderm ferritin molecule.

That the plasma concentration of certain divalent cations change during an inflammatory insult provides a major host defense response in vertebrate animals. This study was designed to investigate the involvement of iron sequestration in invertebrate immune responses. A ferritin molecule was cloned from an echinoderm coelomocyte cDNA library. The amino acid sequence showed sequence homology with vertebrate ferritin. The cDNA contained a conserved iron responsive element sequence. Studies showed that stimulated coelomocytes released iron into in vitro culture supernatants. The amount of iron in the supernatants decreased over time when the amebocytes were stimulated with LPS or PMA. Coelomocytes increased expression of ferritin mRNA after stimulation. In vertebrates, cytokines can cause changes in iron levels in macrophages. Similarly, echinoderm macrokines produced decreases in iron levels in coelomocyte supernatant fluids. These results suggest that echinoderm ferritin is an acute phase protein and suggest that sequestration of iron is an ancient host defense response in animals.

The echinoderm immune system. Characters shared with vertebrate immune systems and characters arising later in deuterostome phylogeny.

In summary, the characters of the echinoderm immune system that we review here can be considered to illuminate the baseline nonadaptive immune systems that were our original deuterostome heritage. We still retain--and greatly rely upon--similarly functioning, nonadaptive cellular defense systems. It is worth stressing that sea urchins are long lived, normally healthy animals that display remarkable abilities to heal wounds and combat major infections. From an external point of view, their immune systems obviously work very well. Thus, their cellular defense systems are extremely sensitive, and they respond rapidly to minor perturbations, all without any specific adaptive capabilities. These systems probably function through the transduction of signals conveying information on injury and infection, just as do the equivalent systems that underlie and back up our own adaptive immune systems, and that provide the initial series of defenses against pathogenic invasions. Many extremely interesting questions remain regarding the evolution of the deuterostome immune response. Are the echinoderm and tunicate systems the same, or have the protochordates augmented the basic phagocyte system with an as yet unidentified chordate-like character? Do the jawless fishes produce Igs that would make them similar to the sharks, or are they vertebrates without an Ig system that essentially rely on an invertebrate-like, nonspecific, activated phagocyte type of immune system? How do sharks regulate their immune system without T cells and MHC class I? How do they avoid producing autoantibodies? Future research will not only answer these questions, but those answers will also be enlightening with regard to the origins of the mammalian immune system in which ancient functions and subsystems remain.

Immune phenomena in echinoderms.

Advances in biochemistry and molecular biology have made it possible to identify a number of mechanisms active in the immune phenomena of echinoderms. It is obvious that echinoderms have the ability to distinguish between different foreign objects (pathologically changed tissues, microorganisms, parasites, grafts) and to express variable effector mechanisms which are elicited specifically and repeatably after a variety of non-self challenges. The molecular and biochemical basis for the expression of these variable defense mechanisms and the specific signals which elicit one type of effector mechanism are not, however, yet well known. The high capacity of coelomocytes to phagocytose, entrap and encapsulate invading microorganisms is a valid immune cell-mediated mechanism of echinoderms. The entrapped bacteria, discharged cellular materials and disintegrating granular cells are compacted and provoke the cellular encapsulation reaction. Moreover, humoral-based reactions form an integral part of the echinoderm defense system against microbial invaders. Factors such as lysozyme, perforins (hemolysins) vitellogenin and lectins are normal constituents of hemolymph, while cytokines are synthesized by echinoderms in response to infection.

[Comparative immunological analysis of echinoderm cellular and humoral defense factors]. / Sravnitel'no-immunologicheskii analiz kletochnykh i gumoral'nykh zashchitnykh faktorov iglokozhikh.

Coelomocyte are found in the fluid filling coelomic cavity of echinoderms and depending on species can be a mixture of several morphologically different types. There are among them: granular and agranular amoebocytes, morula cells, vibratile and lymphocyte-like cells. All these cells take part in cellular response to immune challenges through phagocytosis, clotting, encapsulation of foreign particles, cytotoxicity, and the production of antimicrobial agents, such as reactive oxygen and nitric oxide. The data are given on a variety of humoral factors found in the coelomic fluid, including different types of lectines, agglutinins, hemolysins, acute phase proteins and antimicrobial factors. The discussion on cooperation between cellular and humoral arms of defense reactions during inflammation reveals the crucial role of coelomocytes in immune response. It is suggested that the sea urchin complement system (that is homologous to the alternative pathway in vertebrates) is appeared initially in echinoderms as a protein cascade that points to opsonization of foreign cells and particles, augmenting their phagocytosis and subsequent destruction by coelomocytes. So the identification of a simple complement system as a part of the echinoderm immune response shows that these animals as well as all invertebrate deuterostomes share innate immune system homologies with vertebrates. Studying the simpler immune response demonstrated by echinoderms is important for understanding the ancestral deuterostome defense system and reconstructing the evolution of immune system in higher vertebrates.

Comparative DNA damage and repair in echinoderm coelomocytes exposed to genotoxicants.

The capacity to withstand and repair DNA damage differs among species and plays a role in determining an organism's resistance to genotoxicity, life history, and susceptibility to disease. Environmental stressors that affect organisms at the genetic level are of particular concern in ecotoxicology due to the potential for chronic effects and trans-generational impacts on populations. Echinoderms are valuable organisms to study the relationship between DNA repair and resistance to genotoxic stress due to their history and use as ecotoxicological models, little evidence of senescence, and few reported cases of neoplasia. Coelomocytes (immune cells) have been proposed to serve as sensitive bioindicators of environmental stress and are often used to assess genotoxicity; however, little is known about how coelomocytes from different echinoderm species respond to genotoxic stress. In this study, DNA damage was assessed (by Fast Micromethod) in coelomocytes of four echinoderm species (sea urchins Lytechinus variegatus, Echinometra lucunter lucunter, and Tripneustes ventricosus, and a sea cucumber Isostichopus badionotus) after acute exposure to H2O2 (0-100 mM) and UV-C (0-9999 J/m2), and DNA repair was analyzed over a 24-hour period of recovery. Results show that coelomocytes from all four echinoderm species have the capacity to repair both UV-C and H2O2-induced DNA damage; however, there were differences in repair capacity between species. At 24 hours following exposure to the highest concentration of H2O2 (100 mM) and highest dose of UV-C (9999 J/m2) cell viability remained high (>94.6 ± 1.2%) but DNA repair ranged from 18.2 ± 9.2% to 70.8 ± 16.0% for H2O2 and 8.4 ± 3.2% to 79.8 ± 9.0% for UV-C exposure. Species-specific differences in genotoxic susceptibility and capacity for DNA repair are important to consider when evaluating ecogenotoxicological model organisms and assessing overall impacts of genotoxicants in the environment.

Immune response of the Antarctic sea urchin Sterechinus neumayeri: cellular, molecular and physiological approach / Respuesta inmune del erizo de mar Antártico Sterechinus neumayeri: un enfoque celular, molecular y fisiológico

Abstract In the Antarctic marine environment, the water temperature is usually between 2 and - 1.9 °C. Consequently, some Antarctic species have lost the capacity to adapt to sudden changes in temperature. The study of the immune response in Antarctic sea urchin (Sterechinus neumayeri) could help us understand the future impacts of global warming on endemic animals in the Antarctic Peninsula. In this study, the Antarctic sea urchins were challenged with lipopolysaccharides and Vibrio alginolitycus. The cellular response was evaluated by the number of coelomocytes and phagocytosis. A significant increase was observed in red sphere cells and total coelomocytes in animals exposed to LPS. A significant rise in phagocytosis in animals stimulated by LPS was also evidenced. Moreover, the gene expression of three immune related genes was measured by qPCR, showing a rapid increase in their expression levels. By contrast, these immune genes showed a depression in their expression by a thermal effect at 5 and 10 °C. In addition, during bacterial injection, the oxygen consumption was higher in challenged animals. Our results showed that the immune response in the Antarctic sea urchin may be affected by acute thermal stress and that this immune response has a metabolic cost. Rev. Biol. Trop. 63 (Suppl. 2): 309-320. Epub 2015 June 01.

Resumen En el medio ambiente de la Antártica la temperatura del agua es de entre 2 y - 1.9 °C. Por consecuencia ciertas especies han perdido la capacidad de adaptarse a los cambios repentinos de la temperatura del agua. El estudio de la respuesta inmune del erizo antártico (Sterechinus neumayeri) podría ayudar a comprender los futuros impactos en los animales endémicos del cambio climático en la Península Antártica. En este estudio nosotros hemos evaluado la respuesta inmunitaria de S. neumayeri respecto de estimulaciones con bacterias (Lipopolisacáridos y Vibrio alginolitycus) asi como durante el estrés térmico a 5 y 10 °C. La respuesta del erizo fue evaluada en relación al número de celomocitos circulantes, capacidad fagocítica de estos y por el análisis de la expresión de tres genes inmunitarios. Después de la estimulación con LPS un aumento significativo de células esferoidales rojas, de amebocitos fagocíticos y de celomocitos totales fue observado después de las primeras horas de estimulación, de la misma manera que la capacidad fagocítica. Por otra parte los tres genes inmunes medidos mostraron un aumento significativo de su expresión por qPCR después de la estimulación con LPS. El estrés térmico de 5 °C produjo un aumento de la expresión de estos tres genes inmunitarios, por el contrario a una temperatura más alta (10 °C) se produce la reducción de dos de entre ellos. Adicionalmente un aumento del consumo de oxígeno fue observado durante la estimulación bacteriana. Nuestros resultados muestran que la respuesta inmunitaria en el erizo antártico puede ser afectada por el estrés térmico agudo y que la respuesta inmune en invertebrados antárticos tendría un costo metabólico.