Gene expression specificity of the mussel antifungal mytimycin (MytM)

Abstract We previously reported the nucleotide sequences and diversity of mytimycin (MytM) from the Mediterranean mussel, Mytilus galloprovincialis. Using real-time PCR (q-PCR), we observed that the MytM gene was mainly expressed in circulating hemocytes and to a less extent in the mantle. In vivo challenge with bacteria or with the yeast, Candida albicans, did not increase the expression as measured by q-PCR in hemocytes. By contrast, injection of the filamentous fungus, Fusarium oxysporum, induced a sudden and strong increase of expression at 9h p.i. (stimulation index of 25.7 ± 2.1). Optimum stimulating dose was 104 spores of F. oxysporum per mussel. In the same samples, AMP mytilin and …

Le Cycle des Myxozoa et les Cnidaires: comparaison des formes planctoniques

Les études moléculaires rapprochent désormais les Myxozoaires des Cnidaires. Les deux groupes sont présents exclusivement en milieu aquatique et utilisent l’eau comme vecteur de prolifération et de dissémination Au cours de leur cycle de vie, les Cnidaires passent d’une phase sessile fixée sur des substrats, la forme Polype, à une phase libre autonome ou planctonique, la forme méduse et planula. Chez les Myxozoa on observe aussi une phase planctonique à morphologie actinomyxidie issue de la reproduction sexuée chez un annélide et une phase non planctonique à morphologie myxosporidie produite par reproduction sexuée chez le poisson. Toutefois, les connaissances sur la sexualité des Cnidaires…

A rhamnose-binding lectin from sea bass (Dicentrarchus labrax) plasma agglutinates and opsonizes pathogenic bacteria

Abstract The discovery of rhamnose-binding lectins (RBLs) in teleost fish eggs led to the identification of a novel lectin family characterized by a unique sequence motif and a structural fold, and initially proposed to modulate fertilization. Further studies of the RBL tissue localization and gene organization were also suggestive of role(s) in innate immunity. Here we describe the purification, and biochemical and functional characterization of a novel RBL (DlRBL) from sea bass (Dicentrarchus labrax) serum. The purified DlRBL had electrophoretic mobilities corresponding to 24 kDa and 100 kDa under reducing and non-reducing conditions, respectively, suggesting that in plasma the DlRBL is p…

Haemolytic activity and characterization of nematocyst venom fromPelagia noctiluca(Cnidaria: Scyphozoa)

We investigated the haemolytic capacity of the crude venom extracted from isolated nematocysts of Pelagia noctiluca (Cnidaria: Scyphozoa), and evidenced the proteic fractions responsible for this activity. The nematocyst venom was used at various concentrations to evaluate the haemolytic activity and the lysosomal membrane stability of red blood cells of two teleostean species treated with the extract. The nematocyst extract was assayed against erythrocytes of the two teleostean species living in different environments, Carassius auratus as a common freshwater species, and Liza aurata as a representative of seawater species. Experiments on the haemolytic activity of P. noctiluca in the pres…

Purification and characterization of an f-type lectin from small-spotted catshark (Scyliorhinus canicula)serum

METHYLMERCURY EFFECTS ON Mytilus galloprovincialis HAEMOCYTES ACTIVITY

Bivalves, filter-feeding organisms, due to their wide distri-bution, are used by many countries in biomonitoring, particu-larly in the assessment of xenobiotics in the marine ecosystem.These sentinel organisms are able to accumulate trace metalsand other substances in their tissues. Haemocytes are effectorsof cellular immunity in bivalves and are capable of respond-ing to stressors through activities such as phagocytosis or cyto-toxicity. In order to identify cellular markers to investigate pol-lution, the effect of different concentrations of organic mercury(CH3HgCl) on the morphology and responses of Mytilus gal-loprovincialishaemocytes was studied in this work. Sublethalconcentrations of…

Inflammatory-like reaction following bacterial injection and antimicrobial peptide isolation from Anemonia sulcata (Cnidaria)

BIO-PULITURA: PROTEASI DA ORGANISMI MARINI.

Granulocytes of sea anemone Actinia equina (Linnaeus, 1758) body fluid contain and release cytolysins forming plaques of lysis

The Cnidaria phylum includes organisms that are among the most poisonous animals. The exact composition of cnidarian bioactive molecules is not known in detail, but little is known on the cells that produce the toxins. Here we have shown that the presence of cytolysins is not exclusive of nematocysts. A plaque-forming assay was carried out with cell populations extracted from the percoled body fluid showed for the first time that anthozoan granulocytes are able to form plaque of lysis. We have partitioned the total population of free cells into three distinct discrete bands by discontinuous Percoll gradient, and we have identified six small different types cells: morular granulocytes; cells…

Antimicrobial response in Anemonia sulcata (Cnidaria)

L’uso della biodiversita’ nella zonizzazione delle aree marine protette: un’applicazione nelle Isole Egadi.

Attività lisozima-simile e citotossica nel muco di Actina equina (Cnidaria, Anthozoa)

INFLAMMATION AND TENTACLE REGENERATION RESPONSES IN ANEMONIA VIRIDIS (ANTHOZOA, CNIDARIA)

Isolamento, caratterizzazione e distribuzione cellulare di una neurotossina ad attività litica di Actinia equina (Anthozoa, Cnidaria

Components of hemocyte extracts from marine invertebrates exert antimicrobial activity

Role of mucosal immune response and histopathological study in European eel (Anguilla anguilla L.) intraperitoneal challenged by Vibrio anguillarum or Tenacibaculum soleae.

Abstract The external mucus layer that covers fish skin contains numerous immune substances scarcely studied that act as the first line of defence against a broad spectrum of pathogens. This study aimed to characterize and describe for the first time several humoral immune defence parameters in the skin mucus of the European eel (Anguilla anguilla) after intraperitoneal injection with Vibrio anguillarum or Tenacibaculum soleae. This study evaluated several immune-related enzymes and bactericidal activity against fish pathogenic bacteria in the skin mucus of European eels at 24, 48, and 72 h post-challenge. The results demonstrated that European eel skin mucus showed significant increments i…

Evolution and Immune Function of Fish Lectins

Abstract Lectins are sugar-binding proteins widely distributed among animals, plants, and microbial taxon, involved in diverse biological processes. In both invertebrates and vertebrates, they play key roles in nonself recognition and immune responses, such as nonself recognition, inflammatory processes, and immunomodulation. In fish, many lectin families have been identified, and their tissue-specific expression and localization of the various lectin repertoires and their ligands are consistent with their distinct biological roles in innate and adaptive immunity. Here, we discuss the involvement of F-type lectins, rhamnose-binding lectins, galectins, and C-type lectins in pathogen recognit…

Attività biologica e caratterizzazione molecolare di un peptide neurotossico ad attività litica di Actinia equina (Anthozoa, Cnidaria)

Individual variability of mytimycin gene expression in mussel

The antifungal peptide mytimycin (MytM) is synthesized by hemocytes of the Mediterranean mussel, Mytilus galloprovincialis. In addition to sequence and gene structure diversities previously reported from pooled hemocytes, the present report focused on the expression of mytm gene in individual M. galloprovincialis, before and after challenge. Within untreated mussel, MytM mRNA was observed by ISH in about 42% of circulating hemocytes, characterized by large, diffuse nucleus. Injection with Fusarium oxysporum increased such percentage, but in only some of the mussels. Similarly, MytM gene expression increased after injection in only some of the mussels, as measured by qPCR. Responders and not…

Cnidarian Interaction with Microbial Communities: From Aid to Animal’s Health to Rejection Responses

The phylum Cnidaria is an ancient branch in the tree of metazoans. Several species exert a remarkable longevity, suggesting the existence of a developed and consistent defense mechanism of the innate immunity capable to overcome the potential repeated exposure to microbial pathogenic agents. Increasing evidence indicates that the innate immune system in Cnidarians is not only involved in the disruption of harmful microorganisms, but also is crucial in structuring tissue-associated microbial communities that are essential components of the Cnidarian holobiont and useful to the animal’s health for several functions, including metabolism, immune defense, development, and behavior. Someti…

Mesoglea Extracellular Matrix Reorganization during Regenerative Process in Anemonia viridis (Forskål, 1775)

Given the anatomical simplicity and the extraordinary ability to regenerate missing parts of the body, Cnidaria represent an excellent model for the study of the mechanisms regulating regenerative processes. They possess the mesoglea, an amorphous and practically acellular extracellular matrix (ECM) located between the epidermis and the gastrodermis of the body and tentacles and consists of the same molecules present in the ECM of vertebrates, such as collagen, laminin, fibronectin and proteoglycans. This feature makes cnidarians anthozoans valid models for understanding the ECM role during regenerative processes. Indeed, it is now clear that its role in animal tissues is not just tissue su…

Study of immunotoxicity responses of Sabella spallanzanii exposed to copper sulphate

Effetti dell'esposizione in vitro al cloruro di metilmercurio Sulle risposte immunitarie di Dicentrarchus labrax.

This study shows that high methylmercury concentrations are cytotoxic for Dicentrarchus labrax leucocytes, whereas subletal concentrations affect leucocyte phagocytosis and cells morphology in a dose dependent fashion. Although the serum hemoagglutinating activity was not inhibited by the metal, the activity of purified serum F-lectin fraction and mucus was significantly decreased by relevant methylmercury concentrations.

Evolution of Ciona intestinalis Tumor necrosis factor alpha ( Ci TNFα): Polymorphism, tissues expression, and 3D modeling

Although the Tumor necrosis factor gene superfamily seems to be very conserved in vertebrates, phylogeny, tissue expression, genomic and gene organization, protein domains and polymorphism analyses showed that a strong change has happened mostly in invertebrates in which protochordates were a constraint during the immune-molecules history and evolution. RT PCR was used to investigate differential gene expression in different tissues. The expression shown was greater in the pharynx. Single-nucleotide polymorphism has been investigated in Ciona intestinalis Tumor necrosis factor alpha (CiTNFα) mRNA isolated from the pharynx of 30 ascidians collected from Licata, Sicily (Italy), by denaturing …

Lectine sieriche di "tipo F" nell'immunità innata dei pesci: aspetti molecolari e funzionali.

Mollusk antimicrobial peptides A review from activity-based evidences to computer-assisted sequences

Antimicrobial peptides (AMPs) represent the most universal immune effectors. Molluscs constitute the second largest animal phylum, after Arthropods, in term of number of species. Only a negligible number has been investigated regarding AMPs. The choice of the species to be studied relied on their economical importance and availability. First studies on molluscan AMPs dated from 1996 and were based on biological activities of biochemical-purified fractions. Such approach released all the original structures we know, with biological activity sometimes different from one isoform to another. Then, molecular biology techniques were applied to molluscan AMPs starting in 1999. Complete screening o…

Inflammation events occurring upon bacterial infection in Mytilus galloprovincialis

Bivalves, and in particular the Mediterranean Mytilus galloprovincialis are important sources of food in several countries in the world. Because of that, mussels farming has a strong economic impact. Due to their status as sessile and filter-feeding animals, bivalves accumulate in their tissues environmental pollutants and a larger amount of microorganisms and between these, a multitude of infective bacteria for higher vertebrates and humans, such as Vibrio species. Several immunological responses of M. galloprovincialis were investigated and described after Vibrio infection both, in vitro and in vivo conditions, such as hemocytes count and different cellular subpopulations. Particularly, i…

Serum lectins in fish innate immunity: molecular and functional aspects

Fucose-binding lectins (FBL) are present in tissues and fluids from invertebrates and vertebrates. The lectin repertoires in teleost fish are highly diversified and recently has been described the structure of the fucose-binding agglutinin that revealed a novel lectin fold (the “F-type” eel (Anguilla anguilla) fold), which shared a unique fucose-binding sequence motif contained both in carbohydrate-binding proteins and unrelated proteins. In this report, we describe serum FBL from sea bass Dicentrarchus labrax and sea bream Sparus aurata. These lectins were purified, characterized, cloned and sequenced. Studies on structural aspects, biological activity, tissue distribution as well as ontog…

Purificazione di una lectina calcio dipendente dal muco di Sabella spallanzanii (Polychaeta: Sabellidae)

From Cnidarian immunobiology to cultural heritage applications

The study of cnidarians immunity, as model systems of metazoans, lead additional informations on the first steps of the immunity evolution. The functions of the genes and cellular pathways in higher vertebrates are conserved during the evolution of metazoans, as shown by the discovery of homologues in cnidarians. These basal metazoans in fact, are far from "simples" in the range of methods at their disposal to deal with potential prey but also invading microbes and pathogens. They can give informations about the invertebrates innate immune repertoire. We investigated the immunobiology starting from the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of sub…

Molecular characterization of macrophage migration inhibitory factor (MIF) from mussel, Mytilus galloprovincialis

GE.B.EC.SUD: STUDIO GEOLOGICO, BIONOMICO ED ECOLOGICO DI AREE MARINE PROTETTE DELL’ITALIA MERIDIONALE.RISULTATI

αβ defensin antimicrobial peptide (BPDEF) from the invasive red sea mussel Brachidontes pharaonis (P. Fischer, 1870)

The immune system that plays a major role in determining host fitness in the wild, i.e. under the constraints imposed by ecology and life history. Permanent conflict interactions with the environment are the natural situation for a living creature. To partially resolve this, the immune system evolved and is characterized by an enormous variety of mechanisms and effectors, including the AMPs, although their specificity is poor. In fact, AMPs are universal and extremely successful in dealing with a huge range of pathogens, including bacteria, fungi, protozoa and viruses Amps are oligopeptides composed of varying number of amino acids with a broad spectrum of targeted organisms ranging from vi…

Evaluation and comparison of trace metal accumulation in different tissues of potential bioindicator organisms: Macrobenthic filter feedersStyela plicata, Sabella spallanzanii, andMytilus galloprovincialis

Trace metal concentrations were measured in different tissues of Sabella spallanzanii, Styela plicata, and Mytilus galloprovincialis collected in the Termini Imerese Harbor (Sicily, Italy) to evaluate the potential use of these species as bioindicators. Higher bioaccumulation factors (BAFs) were calculated in the tube of S. spallanzanii, except for As, which had a higher BAF in the branchial crown of the same species. Regarding the other species analyzed, higher BAFs were found in the digestive gland of M. galloprovincialis. An exception was Pb, which was significantly more concentrated in the branchial basket and tunic of S. plicata. The BAFs calculated in the present study show that all t…

The Mucus of Actinia equina (Anthozoa, Cnidaria): An Unexplored Resource for Potential Applicative Purposes

The mucus produced by many marine organisms is a complex mixture of proteins and polysaccharides forming a weak watery gel. It is essential for vital processes including locomotion, navigation, structural support, heterotrophic feeding and defence against a multitude of environmental stresses, predators, parasites, and pathogens. In the present study we focused on mucus produced by a benthic cnidarian, the sea anemone Actinia equina (Linnaeus, 1758) for preventing burial by excess sedimentation and for protection. We investigated some of the physico-chemical properties of this matrix such as viscosity, osmolarity, electrical conductivity, protein, carbohydrate, and total lipid contents. Som…

In the ovary of Ciona intestinalis (Type A), immune-related galectin and phenoloxidase genes are differentially expressed by the follicle accessory cells

Abstract Riboprobes (in situ hybridization) and antibodies (immunohistochemistry), previously used to show the upregulation of Ciona intestinalis (Type A) galectins (CiLgals-a, CiLgals-b) and phenoloxidase (CinPO2) immune-related genes, were tested on histological sections of the ovary. The ovarian follicles are composed of oocytes encased by follicular cells (FCs) and test cells (TCs). Results show the transcription upregulation of both CiLgals and CinPO2 genes in the vitellogenic FCs, conversely distinct cytolocalization of the proteins are shown. At vitellogenic stage, the CiLgals are localized in the FCs, in the oocyte cytoplasm, and close to the germinal vesicle (GV), whereas the CinPO…

ANALISI DELL’ETEROGENEITA’ BIOCENOTICA LUNGO LA FASCIA COSTIERA DELLE ISOLE DI USTICA E MARETTIMO

Evaluation and comparison of trace metals accumulation in different tissues of potential bioindicator organisms: Macrobenthic filter feeders Styela plicata, sabella spallanzanii and Mytilus galloprovincialis

Trace metal concentrations were measured in different tissues of Sabella spallanzanii, Styela plicata and Mytilus galloprovincialis collected in Termini Imerese harbor, located in Sicily (Italy), in order to evaluate the potential use of these species as bioindicators. The higher bioaccumulation factors (BAFs) were calculated in tube of Sabella spallanzanii, except for As which shows the higher BAF in branchial crown of the same specie. Regarding the other species analyzed, higher BAFs were found in the digestive gland of Mytilus galloprovincialis. An exception is represented by Pb that is significantly more concentrated in branchial basket and tunic of Styela plicata. The BAFs here calcula…

Il “problem posing” come metodologia innovativa per lo studio delle Scienze della Vita

Cold-active molecules for a sustainable preservation and restoration of historic-artistic manufacts

In the last decades biotechnology research provides sustainable alternatives to traditional procedures for preventive preservation of cultural assets. Recently, bioactive molecules (BMs) isolated from marine invertebrate organisms have been isolated and tested for bioremoval of protein layers (BMP) or to controlling microbial colonization (BMA), acting at temperature lower than 30°C. The Protease or Antimicrobial activity was tested on ad hoc assembled specimens and on different historic-artistic manufacts. In bio-removing protocol BMP molecules were applied as gelled solutions, in order to guarantees a selective action, respectful of constitutive materials and manufact integrity. Peculiari…

Functional differential hemocytes behaviour in the clearance of bacteria and humoral defense factors variability in Mytilus galloprovincialis

EVOLUTION, ADAPTATION AND IMMUNE FUNCTIONS OF FISH LECTINS

Fish are equipped with a complex lectin repertoire that, like mammals, are involved almost all the immune reactions. Carbohydrate recognition and interactions mediated by lectins have been recognized involved in vertebrate innate immunity, not only for recognition of potential pathogens, but also acting in the agglutination, immobilization and other functional steps. In fish, C, F types galectins, Rhamnose-bind- ing lectin (RBL) and pentraxin have been identified in both car- tilaginous and bony fish. In addition, selectins and other genes have been found in the currently available fish genomes. On the basis of our results about F-type and RBL lectins we showed that: lectin repertoires in f…

MIF from mussel: coding sequence, phylogeny, polymorphism, 3D model and regulation of expression.

Abstract Three macrophage migration inhibitory factor (MIF)-related sequences were identified from a Mytilus galloprovincialis EST library. The consensus sequence included a 5′-UTR of 32 nucleotides, the complete ORF of 345 nucleotides, and a 3′-UTR of 349 nucleotides. As for other MIFs, M. galloprovincialis ORF does not include any signal or C-terminus extensions. The translated sequence of 115 amino acids possesses a molecular mass of 12,681.4, a pI of 6.27 and a stability index of 21.48. Its 3D structure resembles human MIF except for one shorter α-helix. Although evolutionary separated from ticks and vertebrates, Mg-MIF appeared to be closely related to Pinctada fucata and Haliotis, but…

Ciona robusta hemocyte populational dynamics and PO-dependent cytotoxic activity

Hemocyte populations from the ascidian Ciona robusta, separated through a Percoll discontinuous density gradient, are further characterized by May-Grünwald-Giemsa staining and a cytochemical reaction for phenoloxidase. Variability in cell density, acidophilic property and phenoloxidase activity suggest multiple hemocyte type populations, cell lineages and morphotypes that may be involved in distinct cellular responses. Therefore, unilocular refractile granulocytes, typical of this ascidian species, enriched in a fraction separated from the hemolymph show in vitro phenoloxidase-dependent cytotoxic activity against mammalian erythrocytes and a tumor cell lineage, in addition the properties li…

L'uso della biodiversità nella zonizzazione della aree marine protette: un'applicazione nelle Isole Egadi.

Effects of organic mercury on Mytilus galloprovincialis hemocyte function and morphology

Abstract Filter-feeding organisms accumulate xenobiotics and other substances in their tissues. They can be useful as sentinel organisms in biomonitoring of the marine compartment. Bivalve cellular immunity is ensured by phagocytosis and cytotoxic reactions carried out by hemocytes in a network with humoral responses. These can be affected by chemical contaminants in water that can be immunosuppressors also at a low concentration increasing the sensibility to pathogens. This work is an attempt to individuate cellular markers for pollution detection, investigating the effect of methylmercury (CH3HgCl) at different concentrations on the activity and hemocyte morphology of the Mediterranean m…

Bivalve genomics used for characterization of the immune defense against microbial infections in mussel

The conservation and diversity of ascidian cells and molecules involved in the inflammatory reaction: The Ciona robusta model

Ascidians are marine invertebrate chordates belonging to the earliest branch (Tunicata) in the chordate phylum, therefore, they are of interest for studying the evolution of immune systems. Due to the known genome, the non-colonial Ciona robusta, previously considered to be C. intestinalis type A, is a model species for the study of inflammatory response. The internal defense of ascidians mainly relies on hemocytes circulating in the hemolymph and pharynx. Hemocytes can be in vivo challenged by LPS injection and various granulocyte and vacuolated cell populations differentiated to produce and release inflammatory factors. Molecular biology and gene expression studies revealed complex defens…

F-type lectin from the sea bass (Dicentrarchus labrax): purification, cDNA cloning, tissue expression and localization, and opsonic activity.

Recently described biochemical and structural aspects of fucose-binding lectins from the European eel (Anguilla anguilla) and striped bass (Morone saxatilis) led to the identification of a novel lectin family ("F-type" lectins) characterized by a unique sequence motif and a characteristic structural fold. The F-type fold is shared not only with other members of this lectin family, but also with apparently unrelated proteins ranging from prokaryotes to vertebrates. Here we describe the purification, biochemical and molecular properties, and the opsonic activity of an F-type lectin (DlFBL) isolated from sea bass (Dicentrarchus labrax) serum. DlFBL exhibits two tandemly arranged carbohydrate-r…

A posteriori application of the "Marep" to the zonation of the Egadi Island.

Step-by-Step Regeneration of Tentacles after Injury in Anemonia viridis—Morphological and Structural Cell Analyses

Benthic marine invertebrates, such as corals, are often subjected to injury caused by several sources. Here, the differences and characteristics in injured and health tissues in terms of cellular components are shown through a histological investigation of the soft coral Anemonia viridis at 0 h, 6 h, 24 h, and 7 days after injury caused by tentacle amputation. In addition, a new tool was used for the first time in invertebrates, positron emission tomography, in order to investigate the events that occur during regeneration within a longer time period (0 h, 24 h, and 14 days after the tentacles were cut). Higher integrated density values were measured through a densitometric analysis in sect…

Localizzazione di una lectina di “tipo F” nell’adulto e nell’ontogenesi di Dicentrarchus labrax

The purification, cloning, sequencing, molecular properties and expression of a fucose-binding lectin from the serum of Dicentrarchus labrax (DlFBL) have been previously reported. We now describe the distribution and expression of DlFBL during fish ontogeny. Immunohistochemistry and in situ hybridization assays were carried out at various developmental stages (from 10 days posthatching larvae to juveniles). Another fucose-binding lectin, similar to DlFBL in biochemical, immunochemical and agglutinating properties, was extracted and purified from eggs and appeared to be localized in the embryo yolk sack residual. DlFBL was found in columnar and goblet cells of the intestinal epithelium of la…

Temperature increases, hypoxia, and changes in food availability affect immunological biomarkers in the marine mussel Mytilus galloprovincialis

Temperature increases, hypoxia, and changes in food availability are predicted to occur in the future. There is growing concern for the health status of wild and farmed organisms, since environmental stressors alter organism functions, and elicit coordinated physiological responses for homeostasis. Mussels are good bioindicators of environmental conditions. Their ability to maintain unaltered immunosurveillance under adverse environmental conditions may enhance their survival capability. Few studies are currently concerned with the relationships and feedback among multiple stressors. Here, food concentration, temperature, and oxygenation treatments were evaluated for their effects on immune…

Responses of marine mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) after infection with the pathogen Vibrio splendidus

International audience; Bivalve molluscs possess effective cellular and humoral defence mechanisms against bacterial infection. Although the immune responses of mussels to challenge with pathogenic vibrios have been largely investigated, the effects at the site of injection at the tissue level have not been so far evaluated. To this aim, mussels Mytilus galloprovincialis were herein in vivo challenged with Vibrio splendidus to assess the responses induced in hemolymph and posterior adductor muscle (PAM), being the site of bacterial infection. The number of living intra-hemocyte bacteria increased after the first hour post-injection (p.i.), suggesting the occurrence of an intense phagocytosi…

Hemocytes and humoral defense factors variability from different Mediterranean populations of Mytilus galloprovincialis

Variabilità dei peptidi antimicrobici negli invertebrati: esempio della mitilina da Mytilus galloprovincialis

Molluscan antimicrobial peptides, a review from activity-based evidences to computer-assisted sequences

Antimicrobial peptides (AMPs) represent the most universal immune effectors. Molluscs constitute the second largest animal phylum, after Arthropods, in term of number of species. Only a negligible number has been investigated regarding AMPs. The choice of the species to be studied relied on their economical importance and availability. First studies on molluscan AMPs dated from 1996 and were based on biological activities of biochemical-purified fractions. Such approach released all the original structures we know, with biological activity sometimes different from one isoform to another. Then, molecular biology techniques were applied to molluscan AMPs starting in 1999. Complete screening o…

F-type lectin from serum of the Antarctic teleost fish Trematomus bernacchii (Boulenger, 1902): Purification, structural characterization, and bacterial agglutinating activity.

Abstract The increasing availability of sequenced genomes has enabled a deeper understanding of the complexity of fish lectin repertoires involved in early development and immune recognition. The teleost fucose-type lectin (FTL) family includes proteins that preferentially bind fucose and display tandemly arrayed carbohydrate-recognition domains (CRDs) or are found in mosaic combinations with other domains. They function as opsonins, promoting phagocytosis and the clearance of microbial pathogens. The Antarctic fish Trematomus bernacchii is a Perciforme living at extremely low temperatures (−1.68 °C) which is considered a model for studying adaptability to the variability of environmental w…

Biotecnology and Cultural Heritage: bioactive molecules applied in restoration projects

Effetti tossici del metilmercurio sulle risposte immunitarie dell'ascidia Styela plicata

Cnidarian Immunity and the Repertoire of Defense Mechanisms in Anthozoans

Anthozoa is the most specious class of the phylum Cnidaria that is phylogenetically basal within the Metazoa. It is an interesting group for studying the evolution of mutualisms and immunity, for despite their morphological simplicity, Anthozoans are unexpectedly immunologically complex, with large genomes and gene families similar to those of the Bilateria. Evidence indicates that the Anthozoan innate immune system is not only involved in the disruption of harmful microorganisms, but is also crucial in structuring tissue-associated microbial communities that are essential components of the cnidarian holobiont and useful to the animal’s health for several functions including metabolism, imm…

specific inflammatory response of Anemonia sulcata (cnidaria) after bacterial injection cause tissue rejection and enzymatic activity alteration

The evolution of multicellular organisms was marked by adaptation to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self” have evolved into a long history of cellular and molecular strategies from damage repair to the co-evolution of host-pathogen interaction. The phylum of Cnidaria is one of the first branches in the tree of animal life to provide crucial insights on the evolution of immunity. Sea anemones (Anthozoa, Cnidaria) are benthic sessile species able to maintain the integrity of the tissues and allorecognition in colonial forms and to differentiate between symbionts and pathogenic intruders. We investigated the inflammatory response in sea ane…

In vitro effects of methylmercury on ascidian (Styela plicata) immunocyte responses

This study shows that high methylmercury concentrations are cytotoxic for Styela plicata hemocytes, whereas sublethal concentrations affect immunocyte responses. Moreover, hemocytes exposed to the xenobiotic present a significantly enhanced phenoloxidase activity as revealed in the hemocyte lysate supernatant compared with the control. Although the cytotoxic activity of S. plicata hemocytes toward rabbit erythrocytes is a PO-dependent cell-target reaction due to quinone products, it was significantly decreased by suitable methylmercury concentrations in the medium. The same xenobiotic concentrations decreased the hemocyte phagocytic activity toward yeast. In both the responses cell-target c…

Characterization of cellular and molecular responses of Actinia equina (Linnaeus, 1758)

Differente ruolo delle subpopolazioni degli emociti di Mytilus galloprovincialis nella clearance batterica

Polymorphism of mytilin B mRNA is not traslated into mature peptide

Diversity of mRNAs from mytilin B, one of the five mytilins identified in the Mediterranean mussel, Mytilus galloprovincialis, has been investigated from circulating hemocytes. One mussel expressed simultaneously two to ten different mytilin B mRNAs as observed in denaturing gradient gel electrophoresis (DGGE), defining 10 individual DGGE patterns (named A to J) within the mussels from Messina, Sicily (Italy). Three patterns accounted for 79% of the individuals whereas other patterns were found in only 2-7% of the 57 analyzed mussels. Base mutations were observed at specific locations, mainly within COOH-terminus and 3'UTR, leading to 36 nucleotide sequence variants and 21 different coding …

Aspetti filogenetici della nuova famiglia di lectine "di tipo F" nei Pesci.

Differential involvement of mussel hemocyte sub-populations in the clearance of bacteria.

Abstract Mussels are filter-feeders living in a bacteria-rich environment. We have previously found that numerous bacterial species are naturally present within the cell-free hemolymph, including several of the Vibrio genus, whereas the intra-cellular content of hemocytes was sterile. When bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12 h post-injection for Micrococcus lysodeikticus, 24 h for Vibrio splendidus and more than 48 h for V ibrio anguillarum. The total hemocyte count (THC) was dramatica…

Reazione infiammatoria ed isolamento di un peptide antimicrobico da Anemonia sulcata (Cnidaria)

Seasonal changes in morpho-functional aspects of two Anemonia sulcata (Pennant, 1777) wild populations

Marine benthic organisms can be used as indicators of the quality of environmental status and as monitoring tools to detect natural or anthropogenic perturbations. In temperate waters, metabolic and biochemical responses may be governed by physiological changes driven by seasonal factors. Gathering baseline information on the mechanisms underlying seasonal acclimation patterns is therefore a critical step towards the understanding of the physiological responses of biological indicators. In poikilothermic metazoans, the production of regulatory metabolic enzymes can be used as tools for deciphering the acclimation potential. The aim of this study was to characterize the natural seasonal vari…

Marine organisms as source of bioactive molecules applied in restoration projects

Abstract In recent decades research in the conservation and restoration field has provided sustainable alternatives to traditional procedures for cleaning or controlling the microbial colonization of works of art. In the present study, for the first time novel bioactive molecules extracted from marine invertebrate organisms (Anthozoa) were tested instead of chemical compounds for removing protein layers or as a biocide for controlling fungal or bacterial colonization. In particular, Bioactive Molecules with Protease activity (BMP), acting in a temperature range of 4- 30°C, were tested for the hydrolysis of protein layers on laboratory specimens. The cleaning protocol provides a selective pr…

Evaluation of waterborne exposure to heavy metals in innate immune defences present on skin mucus of gilthead seabream (Sparus aurata)

Aquatic animals are continuously exposed to chemical pollutants but the effects evoked in skin surfaces, which receive the most direct contact with them, are poorly investigated. Terminal carbohydrate composition and immunological components present in skin mucus of gilthead seabream (Sparus aurata L.) specimens exposed to waterborne sublethal dosages of heavy metals [arsenic (As2O3), cadmium (CdCl2) and mercury (CH3HgCl) at 5, 5 and 0.04 μM, respectively for 2, 10 and 30 days were analysed. Moreover, the presence of a fucose binding lectin (FBL) was evaluated by western blot and the protein profiles were by SDS-PAGE and HPLC. Results showed little effects of heavy metals in the presence of…

Identification of CPE and GAIT elements in 3’UTR of macrophage migration inhibitory factor (MIF) involved in inflammatory response induced by LPS in Ciona robusta

Innate immune responses face infectious microorganisms by inducing inflammatory responses. Multiple genes within distinct functional categories are coordinately and temporally regulated by transcriptional 'on' and 'off' switches that account for the specificity of gene expression in response to external stimuli. Mechanisms that control transcriptional and post-transcriptional regulation are important in coordinating the initiation and resolution of inflammation. Macrophage migration inhibitory factor (MIF) is an important cytokine that, in Ciona robusta, is related to inflammatory response. It is well known that in C. robusta, formerly known as Ciona intestinalis, the pharynx is involved in…

Sea bass Dicentrarchus labrax (L.) bacterial infection and confinement stress acts on F-type lectin (DlFBL) serum modulation

The F-lectin, a fucose-binding protein found from invertebrates to ectothermic vertebrates, is the last lectin family to be discovered. Here, we describe effects of two different types of stressors, bacterial infection and confinement stress, on the modulation of European sea bass Dicentrarchus labrax (L.) F-lectin (DlFBL), a well-characterized serum opsonin, using a specific antibody. The infection of the Vibrio alginolyticus bacterial strain increased the total haemagglutinating activity during the 16-day testing period. The DlFBL value showed an upward regulation on the first, second and last days and underwent a slight downward regulation 4 days post-challenge. In contrast, the effect o…

Primary structure and opsonic activity of an F-lectin from serum of the gilt head breamSparus aurata(Pisces, Sparidae)

Abstract The recently described fucose-binding agglutinin from the European eel revealed a novel lectin fold (the ‘F-type’ fold) that is shared with other carbohydrate-binding proteins and proteins from prokaryotes to vertebrates clustered under the newly established F-type lectin (FTL) family. We previously reported the purification and biochemical characterization of a fucose-binding protein (FBP) isolated from serum of the gilt head bream (Sparus aurata, SauFBP). In the present article, the complete coding sequence of SauFBP revealed that it is a member of the FTL family, consisting of two tandem carbohydrate recognition domains (CRD) that display the F-type sequence motif. In vitro opso…

Modificazioni indotte dal metilmercurio sugli emociti dell’ascidia Styela plicata

Rare earths, zirconium and hafnium distribution in coastal areas: The example of Sabella spallanzanii (Gmelin, 1791)

The Zr, Hf, Y and lanthanide (REE) distribution in biological tissues of Sabella spallanzanii and Styela plicata species collected from two harbours from the northern Sicily is studied for providing information regarding the Zr, Hf and REE uptake from the environment. Previous studies determined the fractionation of dissolved REE scavenged on binding sites onto biological surfaces. By comparing the recognised shale-normalised REE patterns of studied samples with evidence from reference data, the observed behaviour of these elements in biological tissues of Sabella spallanzanii and Styela plicata is interpreted to result from the preferential uptake of intermediate REE onto carboxylic sites.…

Lysozyme gene expression and hemocyte behaviour in the Mediterranean mussel, Mytilus galloprovincialis, after injection of various bacteria or temperature stresses.

The aim of the present study was to evaluate the expression of the Mytilus galloprovincialis lysozyme gene in different in vivo stress situations, including injection of bacteria Vibrio splendidus LGP32, Vibrio anguillarum or Micrococcus lysodeikticus, as well as heat shock at 30 C and cold stress at 5 C. Injection of V. splendidus LGP32 resulted in: (i) a general down-regulation of lysozyme gene expression, as quantified by Q-PCR; (ii) reduction in the number of circulating hemocytes; (iii) decrease in the percentage of circulating hemocytes expressing lysozyme mRNA which was now restricted to only small cells, as observed by ISH; and (iv) accumulation of hemocytes expressing lysozyme in t…

Invertebrate Models in Innate Immunity and Tissue Remodeling Research

The aim of this Special Issue is to highlight the close functional and highly conserved link between innate immunity, homeostasis maintenance, inflammation, tissue remodeling and regeneration [...]

Variabilità genica dei peptidi antimicrobici: polimorfismo dell’mRNA della mitilina B di M. galloprovincialis

Comparative analysis of fucose binding lectins isolated and characterized from different teleost species, and distribution of a F-Lectin during Dicentrarchus labrax ontogenesis

EFFETTI DELLE VARIAZIONI STAGIONALI IN DUE POPOLAZIONI DI ANEMONIA SULCATA (PENNANT, 1777) E PRIME OSSERVAZIONI SULLA RIGENERAZIONE DEI TENTACOLI

ALESSANDRO LENTINI, MARIA GIOVANNA PARISI, DANIELA PARRINELLO, MATTEO CAMMARATA Dipartimento di Scienze della Terra e del Mare, Viale delle Scienze ed. 16 Università di Palermo EFFETTI DELLE VARIAZIONI STAGIONALI IN DUE POPOLAZIONI DI ANEMONIA SULCATA (PENNANT, 1777) E PRIME OSSERVAZIONI SULLA RIGENERAZIONE DEI TENTACOLI Gli organismi bentonici sono utilizzati sia come indicatori delle condizioni oceanografiche che come strumenti per il monitoraggio delle condizioni della fascia marino costiera su cui insistono fattori antropici. La raccolta di informazioni di base sui meccanismi di acclimatazione stagionale è un passo fondamentale per comprendere le risposte fisiologiche degli indicatori b…

The entangled multi-level responses of Mytilus galloprovincialis (Lamarck, 1819) to environmental stressors as detected by an integrated approach.

Abstract Anthropogenic pressure adds up and interacts with the effects of climate change with a varying magnitude and potential changes depend on species’ Life History (LH) traits, local environmental conditions and co-occurrence of several stressors. Stressors exert negative effects on marine biota when acting as a single factor, but the effects may be amplified when more than one stressor work in combination, producing interacting effects on biodiversity and ecosystem functioning. The impairment of individual functional traits (FT) leads to strong rebounds on LH traits and this may have ecological consequences. No studies actually relate FT and antioxidant enzymes to multiple environmenta…

Biocide

Biodeterioration represents a revealing problem for the conservation of cultural heritage. It can be identified as a complex interaction within the ecosystem of a microbial community and its substrate and involves physical and chemical alterations resulting from biological and metabolic activity. Designing a diagnostic approach for evaluating the extent of the damage, identifying the biological community, and opting for an efficient methodology aimed at eliminating deteriogens is equally complicated. The correct approach would require understanding the nature of the biodeterioration and implementing methodologies respectful of human health which, however, avoid the indiscriminate killing of…

Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration

The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ’’self’’ from ’’non-self” have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different ba…

Isolation and cytotoxic activity of neurotoxin from the mucus of Actinia equina (Anthozoa, Cnidaria).

BIOACTIVE MOLECULES FROM MARINE INVERTEBRATE ORGANISMS AS POTENTIAL TOOLS IN RESTORATION PROCEDURES

In the last decades molecular biology has provided innovative approaches in order to set up specific protocols for the conservation and restoration of cultural assets. In this study, which falls within the so called field of Blue-biotechnology, new bio-reactive peptides isolated from marine invertebrate organisrns (Cnidaria and Molluscs) were tested aim to bio-cleaning (proteolytic- peptides) the surfaces or to control (antimicrobial-peptides) the colonization of historic-artistic manufacts by fungi or bacteria. Particularly, the proteolytic-peptides showed hydrolytic activity, specific for animal-glue, in a range of temperatures of 4-37°C; than acting without heating the surface, by a cont…

Response of Sabella spallanzanii to multiple stressors. The combined effect of infection and copper sulphate

The aim of this work is to study the immune responses of the polychaete Sabella spallanzanii after exposure to copper sulphate, an immunomodulating agent in marine organisms, and the multiple stresses caused by Escherichia coli infection, to validate the species as a model organism in marine-coastal biomonitoring programmes. Polychaetes were housed in laboratory and divided into five experimental groups: 1. Control (no microinjected), 2. filtered seawater + TBS injection (control of point 3), 3. filtered seawater + E. coli injection (control of point 4), 4. CuSO4 + TBS injection (control of point 5), and 5. CuSO4 + E. coli injection. The immune variables, esterase and alkaline phosphatase a…

Purification and molecular characterization of the rhamnose binding lectin from sea bass (Dicentrarchus labrax) that agglutinate Gram positive and negative bacteria

Isolamento di nuovi peptidi citolitici da Actinia equina (Anthozoa, Cnidaria)

Attività citotossica nel muco di Actinia equina (Anthozoa, Cnidaria)

Saggi sull’attività antimicrobica di molecole bioattive in interventi di foderatura di dipinti

In questo studio è stato valutato l’utilizzo di molecole antimicrobiche estratte da organismi marini invertebrati (Anthozoa), al fine di limitare o inibire la crescita microbica su materiali impiegati per il restauro di manufatti storico-artistici. I ceppi batterici e fungini utilizzati nei saggi antimicrobici, sono stati isolati da porzioni di tele, stratificate con colla, impiegate nella foderatura di dipinti. Mediante un approccio integrato che comprende tecniche di microscopia e tecniche molecolari, sono state identificate colonie batteriche appartenenti ai generi Enterobacter e Micrococcus e fungine appartenenti ai generi Aspergillus e Penicillium. L’attività antimicrobica delle moleco…

Attività antibatterica e agglutinante nel muco di Sabella Spallanzanii (Annelida, Polychaeta)

A serum fucose-binding lectin (D1FBL) from adult Didentrarchus labrax is expressed in larva and juvenile tissue and contained in eggs

The purification, cloning, sequencing, molecular properties and expression of a fucose-binding lectin from the serum of Dicentrarchus labrax (DlFBL) have been previously reported. We now describe the distribution and expression of DlFBL during fish ontogeny. Immunohistochemistry and in situ hybridization assays were carried out at various developmental stages (from 10 days posthatching larvae to juveniles). Another fucose-binding lectin, similar to DlFBL in biochemical, immunochemical and agglutinating properties, was extracted and purified from eggs and appeared to be localized in the embryo yolk sack residual. DlFBL was found in columnar and goblet cells of the intestinal epithelium of la…

A posteriori application of the ‘Marep’ to the zonation of the Egadi Islands.

The ascidian Styela plicata hemocytes as a potential biomarker of marine pollution: In vitro effects of seawater and organic mercury.

Toxic metals, such as mercury, contribute substantially to anthropogenic pollution in many estuarine environments. Animals living in those environments, particularly invertebrate filter feeders like tunicates, can be used as bioindicators. In an attempt to identify cellular markers for revealing pollution, this study examined in vitro the effects of different concentrations of methyl mercury on Styela plicata hemocytes. The harvested hemocytes from S. plicata that were exposed to the metal had a significant mortality, cellular count and morphometric alterations. These findings provided evidence of MeHg immunotoxic effects on S. plicata, resulting in hemocyte death and morphological changes …

Individual variability and gene expression specificity of the mussel antifungal mytimycin (MytM)

Primary structure and opsonic activity of an F-lectin from serum of the gilt head bream Sparus aurata (Pisces, Sparidae)