D.P14 In vitro cytotoxic activity against erythrocytes by ascidians hemocytes: Target/effector interactions

The cell-mediated, calcium-dependent cytotoxic activity against mammalian erythrocytes by the hemocytes of the ascidians Ciona intestinalis and Stye/a plicata was investigated by in vitro cytotoxic assay (HCA). Hemocytes/erythrocytes mixtures were performed in artificial sea water or TBS enriched with 10 mM CaC12, at 25° or 3rC, and the released haemoglobin was estimated by reading the absorbance at 541 nm (Parrinello et al., D.C.I., 17:19, 1993). With the aim to examine the cytotoxic mechanism involved, inhibition experiments with lipidic components of the erythrocyte membrane have been carried out in TBS-Ca. Among the phospholipids tested sphingomyelin is an inhibitor of the Ciona intesti…

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 …

FACIT collagen (1alpha-chain) is expressed by hemocytes and epidermis during the inflammatory response of the ascidian Ciona intestinalis

Based on previous cloning and sequencing study, real-time PCR and in situ hybridization assays of the inflamed body wall of LPS-injected Ciona intestinalis showed the enhanced gene expression of a collagen with FACIT structural features (Ci-type IX-Col 1a-chain). By using specific antibodies raised against an opportunely chosen Ci-type IX-Col synthetic peptide, the fibroblast property of hemocytes challenged in vitro with LPS (at 4 h) was displayed by flow cytometry, while immunocytochemistry identified hemocytes with large granules (morula cells) as collagen-producing cells. Hemocyte lysate supernatant analyzed in immunoblotting contained a 60 kDa band identifiable as 1a-chain-Ci-type IX-C…

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…

Genetic Variability of Mediterranean Sea red mullet Mullus barbatus (Pisces, Mullidae)

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

Preliminary evidence for the cytotoxic molecule in hemocyte supernatant lysate from Ciona intestinalis unilocular hemocytes

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…

Spontaneous cytotoxic activity of eosinophilic granule cells separated from the normal peritoneal cavity ofDicentrarchus labrax

Abstract In this study the spontaneous in vitro cytotoxic activity to tumour cell lines, (K562), by unstimulated sea bass ( Dicentrarchus labrax ) leukocytes was examined by trypan blue exclusion test and lactate dehydrogenase release assay. A high anti-tumour cell line activity of resident peritoneal leukocytes was found at an effector to target ratio (E:T) of 25:1 after incubation for 2 h at 18° C. Rabbit and sheep erythrocytes were not lysed. A low activity was displayed by head kidney and spleen cell populations whereas blood leukocytes revealed no significant activity. The effect of E:T ratio on cytotoxicity as well as microscopy observations suggested that the cytotoxic reaction requi…

Galectine nella risposta infiammatoria di Ciona intestinalis cDNA ed espressione genica

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

Expression and distribution of DIGR1 in the ontogeny of Dicentrarchus labrax

Prophenoloxidase activating system in tunicate hemolymph

The activation sequence and related factors of the prophenoloxidase activating system in crustaceans was compared with the equivalent system in tunicates. Both solitary and colonial ascidians present in their hemolymph a copper-dependent phenoloxidase activity that may be inhibited by tropolone and phenylthiourea. Carbohydrates are able, to various extents, to trigger proPO system which requires serine protease cleavage for activation to phenoloxidase (PO). In some ascidians, hemocytes called ≪morula cells≫ show PO activity, while in Ciona intestinalis the ≪univacuolar refractile granulocytes≫ are positive after cytochemical staining with L-dopa. The relationships between proPO system and d…

BIO-PULITURA: PROTEASI DA ORGANISMI MARINI.

Isolation and characterization of a fish F-type lectin from gilt head bream (Sparus aurata) serum.

A novel fucose-binding lectin, designated SauFBP32, was purified by affinity chromatography on fucose-agarose, from the serum of the gilt head bream Sparus aurata. Electrophoretic mobility of the subunit revealed apparent molecular weights of 35 and 30 kDa under reducing and non-reducing conditions, respectively. Size exclusion analysis suggests that the native lectin is a monomer under the selected experimental conditions. Agglutinating activity towards rabbit erythrocytes was not significantly modified by addition of calcium or EDTA; activity was optimal at 37 degrees C, retained partial activity by treatment at 70 degrees C, and was fully inactivated at 90 degrees C. On western blot anal…

Antimicrobial response in Anemonia sulcata (Cnidaria)

Effetti della cattività  su alcuni parametri ematologici del grifone (Gyps fulvus)

Haematological analysis is an essential field of veterinary medicine that provides inexpensive and reliable support to determinate animal health. The knowledge of how different factors affect the normal mean values of blood parameters is key to understand and improve animal health. In order to investigate how captivity can affect the haematological profile of birds of prey, the erythrocyte count, haemoglobin concentration, haematocrit, mean corpuscular volume, total leukocytes count of 123 griffon vultures (Gyps fulvus) were analysed. The birds were divided into 4 groups according to their life conditions: a control group of free-living griffons, 2 semi-captive groups held in an aviary for …

Seabream (Sparus aurata) hierarchy among alfa and beta subordinates and dominant interplay affects stress responses and phagocytic activity by peritoneal cavity cells

Putative rhamnose-binding lectin in the solitary ascidian Ciona intestinalis.

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

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

Titolo: Novel proteases from marine organisms with potential interest in restoration procedure

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…

Marcatori genetici molecolari e caratterizzazione delle cultivar

Isolation of a novel LPS-induced component of the ML superfamily in Ciona intestinalis

ML superfamily represents a group of proteins playing important roles in lipid metabolism and innate immune response. In this study, we report the identification of the first component of the ML superfamily in the invertebrate Ciona intestinalis by means of a subtractive hybridization strategy. Sequence homology and phylogenetic analysis showed that this protein forms a specific clade with vertebrate components of the Niemann-Pick type C2 protein and, for this reason, it has been named Ci-NPC2. The putative Ci-NPC2 is a 150 amino acids long protein with a short signal peptide, seven cysteine residues, three putative lipid binding site and a three-dimensional model showing a characteristic b…

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

Physiological stress indicators in the plasma of Scyliorhinus canicula captured by botto trawling at two depths.

The quantification of capture-related physiological stress is an important factor when assessing the potential for post-release survival in sharks that are incidentally captured. In the absence of these biological data and when the post-release fate is unknown, effective management plans cannot be formulated and may lead to highly susceptible shark populations being overfished. Here, we measured the levels of lactate, glucose, alanine amino transferase (ALT), aspartate amino transferase (AST), Ca2+, Na+ K+,Cl - Mg 2+ and Pi in the plasma of mature and immature lesser spotted dogfish (Scyliorhinus canicula, herein dogfish) which were incidentally captured at two depths (shallow: 50-200 m, an…

LPS injection reprograms the expression and the 3′ UTR of a CAP gene by alternative polyadenylation and the formation of a GAIT element in Ciona intestinalis

The diversification of cellular functions is one of the major characteristics of multicellular organisms which allow cells to modulate their gene expression, leading to the formation of transcripts and proteins with different functions and concentrations in response to different stimuli. CAP genes represent a widespread family of proteins belonging to the cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 superfamily which, it has been proposed, play key roles in the infection process and the modulation of immune responses in host animals. The ascidian Ciona intestinalis represents a group of proto-chordates with an exclusively innate immune system that has been widely st…

Effects of Social Hierarchy Establishment on Stress Response and Cell Phagocytosis in Gilt-Head Sea Bream (Sparus aurata)

Social stress can affect the ability of fish to respond to various stressors, such as pathogens or environmental variations. In this paper, the effects of social stress on gilt-head bream (Sparus aurata) were investigated. To study the effects of physiological stress, we evaluated biochemical and cellular parameters, such as cortisol, glucose, lactate, osmolarity, and phagocytosis, 24 h after the establishment of social hierarchy in a group of three fish. Social hierarchy was determined and characterized by behavioral observation (aggressive acts and feeding order) of the specimens (dominant: “α”; subordinate: “β” and “γ”). After the esta…

Components of hemocyte extracts from marine invertebrates exert antimicrobial activity

Co-inertia multivariate approach for the evaluation of anthropogenic impact on two commercial fish along Tyrrhenian coasts

Abstract Aliphatic hydrocarbon levels were determined by the GC/MS technique in fish livers of Engraulis encrasicolus (Ee) and Trachurus trachurus (Tt), collected from a particular area of the Mediterranean Sea, called GSA 10, which is located exactly in Tyrrhenian Sea between Campania coast and North Sicily coast. The aim was to evaluate their potential use as specific bioindicators towards this class of contaminants. Both Tt and Ee are considered to be pollution monitoring bioindicators, due to their dominance in marine communities and economic fishing interest. Ee showed a higher tendency to bioaccumulate TAHs, due to the lower quantity of fatty acids in liver tissues with respect to Tt.…

Ciona intestinalis galectin (CiLgals-a and CiLgals-b) genes are differentially expressed in endostyle zones and challenged by LPS

Abstract Immunohistochemical and in situ hybridization assays were performed to answer the question whether the endostyle, that is the initial gastro-intestinal trait of Ciona intestinalis pharynx, is involved in galectin (CiLgals-a and CiLgals-b) production during the pharynx inflammatory response to LPS inoculation. Specific anti-CiLgal-a and anti-CiLgals-b antibodies, and oligonucleotide probes, that mark inflammatory hemocytes inside the pharynx vessels and vessel epithelium as shown by a previous paper, were assayed on endostyle histological sections. For the first time, we show that galectins are produced by endostyle zones, and both CiLgals-a and –b genes are upregulated by LPS. CiLg…

Cellule che producono ciTNFalfa sono attive nell'infiammazione e nello sviluppo larvale di Ciona intestinalis

A D-galactose specific lectin is an inducible inflammatory IL-1-like opsonin in the hemolymph of the ascidian Ciona intestinalis challegend with LPS.

Attivazione del sistema della profenolossidasi nella risposta infiammatoria della tunica di Ciona intestinalis (L.)

Attività emolitica dell’estratto da nematocisti di Pelagia noctiluca (Cnidaria, Scyphozoa)

Identification of antimicrobial peptides in the gonad of European sea bass males and females

In vertebrates, the gonad is considered an immunologically privileged site as it triggers lower immune responses aiming to avoid germ cell damage. In fish,several studies reported that leucocytes show conditioned immune response and modulate some reproductive functions, allowing the pathogen to establish chronic and latent infections into reproductive organs. In mammals, antimicrobial peptides (AMPs) have recently been recognised as important effectors in male reproductive tract immunity. In fish, AMPs are increasingly recognized as a critical first line of defence against many pathogens as bacteria, fungi, virus, protozoa and even tumour cells. We have recently determined that the European…

The Expression of an Immune-Related Phenoloxidase Gene is Modulated in Ciona Intestinalis Ovary, Test Cells, Embryos and Larva

Two distinct Ciona intestinalis phenoloxidases (CinPO1, 2) had previously been cloned and sequenced. The CinPO2 is involved in innate immunity and is expressed by inflammatory hemocytes that populate the tunic and pharynx vessels as a response to LPS inoculation. In situ hybridization and immunohistochemistry assays on histological section, showed that the expression of this gene and the produced protein are shared with oogenesis, embryogenesis and larval morphogenesis. Intriguingly, upregulation of gene transcription was found in the test cell layer that envelopes the ovary follicle, ovulated egg, and gastrula, as well as it was modulated in the zygotic nucleus of outer balstomers of 32-ce…

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…

Structural and functional diversity of the lectin repertoire in teleost fish: Relevance to innate and adaptive immunity

Protein–carbohydrate interactions mediated by lectins have been recognized as key components of innate immunity in vertebrates and invertebrates, not only for recognition of potential pathogens, but also for participating in downstream effector functions, such as their agglutination, immobilization, and complement-mediated opsonization and killing. More recently, lectins have been identified as critical regulators of mammalian adaptive immune responses. Fish are endowed with virtually all components of the mammalian adaptive immunity, and are equipped with a complex lectin repertoire. In this review, we discuss evidence suggesting that: (a) lectin repertoires in teleost fish are highly dive…

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…

RIFLESSIONI SULL'EDUCAZIONE SCIENTIFICA MEDIANTE L'INSEGNAMENTO DELLE SCIENZE DELLA VITA

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…

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.

seabream(Sparus aurata)hierarchies of social behaviour affects stress responses and immunity

Biochemical genetic differentiation between Pomatoschistus marmoratus and P. tortonesei

Several diagnostic genetic markers were identified in Pomatoschistus marmoratus and P. tortonesei using polyacrylamide gel electrophoresis (PAGE) of allozymes. Twenty-one loci were resolved, including the electrophoretic pattern of muscle proteins. The MDH*, PGM1,2*, EST-1,2*, FUM* and PGI-2* loci exhibited different alleles which were fixed for the two species being analysed. Genetic distance, as calculated by Nei's index, showed a value of 0.413. Environmental hypersalinity, could have influenced the geographical distribution of P. tortonesei.

IDENTIFICAZIONE SIMULTANEA DEI PATOGENI DI PESCI: AEROMONAS SALMONICIDA, YERSINIA RUCKERI E FLAVOBACTERIUM PSYCROPHILUM, TRAMITE PCR

The Inflammatory Response of Urochordata: The Basic Process of the Ascidians’ Innate Immunity

Ascidians form a widespread marine invertebrate group and are heterogeneous in terms of the taxonomic groups’ evolutionary lineages. The ascidian genomes lack significant homologies for rearranging genes of the vertebrate adoptive immunity. Genome analysis, gene sequencing, and transcriptional profiling have allowed us to disclose upregulation of innate immunity genes and cell labeling with riboprobes and antibodies has identified hemocyte types in tunic and pharynx inflammatory responses. Lymphocyte-like cells are stem cells and their immunocompetence has been proposed. Granulocyte types (compartment/morula cells) and hemocytes with large granules/vacuoles (compartment/morula cells) are ma…

Presence of thyroid hormones in ascidian larvae and their involvement in metamorphosis

In this study we investigated the presence and localization of thyroxine in Ciona intestinalis larvae and its involvement in metamorphosis. To date, the mechanisms regulating the metamorphosis of ascidians remain largely unknown. In vivo treatment of swimming larvae with exogenous L-thyroxine and thiourea, and in vitro experiments utilizing high performance liquid chromatography, radioimmunoassay, and immunoperoxidase staining demonstrate the presence of thyroxine at the larval stage. This suggests that this hormone may participate in the control of metamorphosis and thus play a different role from that observed in adults.

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 …

Natural immunity in Paracentrotus lividus: coelomocyte cooperation

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

Biochemical taxonomic differentiation between Mullus barbatus and mullus surmuletus (Pisces, mullidae)

Abstract 1. 1. The electrophoretic data from seven enzymatic systems, codifying for 20 loci, and the patterns of general proteins from some tissues of Mullus barbatus and Mullus surmuletus were examined. 2. 2. The value of the genetic distance index as per Nei is 0.068, this criterium showing a high similarity between the two species. 3. 3. Species-specific patterns were found for the SOD enzyme and general proteins of the muscle.

Purification and characterization of D-galactose binding lectin involved in the inflammatory response in Ciona intestinalis

Ciona intestinalis interleukin 17-like genes expression is upregulated by LPS challenge

In humans, IL-17 is a proinflammatory cytokine that plays a key role in the clearance of extracellular bacteria promoting cell infiltration and production of several cytokines and chemokines. Here, we report on three Ciona intestinalis IL-17 homologues (CiIL17-1, CiIL17-2, CiIL17-3). The gene organisation, phylogenetic tree and modelling supported the close relationship with the mammalian IL-17A and IL-17F suggesting that the C. intestinalis IL-17 genes share a common ancestor in the chordate lineages. Real time PCR analysis showed a prompt expression induced by LPS inoculation suggesting that they are involved in the first phase of inflammatory response. In situ hybridization assays disclo…

Upregulated transcription of phenoloxidase genes in the pharynx and endostyle of Ciona intestinalis in response to LPS

We investigated the role of phenoloxidases (POs) in ascidians inflammatory reaction, a components of a copper-containing protein family involved in invertebrate immune system. In Ciona intestinalis two phenoloxidases (CinPO-1, CinPO-2) have been sequenced. In the present study, real time PCR analysis showed that both CinPO-1 and CinPO-2 genes were modulated by LPS inoculation suggesting that they are inducible and highly expressed in the inflamed pharynx. In situ hybridization disclosed CinPO-1 and CinPO-2 transcripts in pharynx hemocytes (granulocytes) and, mainly, in unilocular refractile granulocytes (URG) which mainly populated the inflamed tunic matrix. Interestingly, the genes are als…

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…

Further insight on Ciona intestinalis prophenoloxidase system activated during the LPS induced inflammatory response

DIVERSITY OF LECTIN-SUGAR RECOGNITION SYSTEMS IN THE EVOLUTION OF FISH INNATE IMMUNITY

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. Fish are equipped with a complex lectin repertoire that, like mammals, are involved almost all the immune reactions. On the basis of our results on the isolation, cDNA cloning, structural analysis, tissue expression and localization, and opsonic activity of F-type and RBL lectins from Dicentrarchus labrax and Sparus aurata we showed that: lectin repertoires in fish are highly diversified and include not only representatives of the lectin fami…

A lytic mechanism based on soluble phospholypases A2 (sPLA2) and b-galactoside specific lectins is exerted by Ciona intestinalis (ascidian) unilocular refractile hemocytes against K562 cell line and mammalian erythrocytes

Abstract Hemocytes from the ascidian Ciona intestinalis exert in vitro Ca 2+ -dependent cytotoxic activity toward mammalian erythrocytes and K562 cells. To examine the lytic mechanism, hemocyte populations were separated (B1–B6 bands) through a Percoll discontinuous density gradient, the hemocyte cytotoxic activity (HCA) and the lytic activity of the hemocyte lysate supernatant (HLS) were assayed. In addition the separated hemocytes were cultured and the cell-free culture medium (CFM) assayed after 3 h culture. Results support that unilocular refractile hemocytes (URGs), enriched in B5, are cytotoxic. The B5-HLS contains lysins and the activity of B5-CFM shows that lysins can be released in…

A glucocorticoid receptor in sea bass leukocytes

Seabream (Sparus aurata) long-term dominant-subordinate interplay affects phagocytosis by peritoneal cavity cells.

Fish are sensitive to stressful conditions that affect their innate immune systems and increase their susceptibility to diseases. We examined the social stress of paired gilthead seabream (Sparus aurata). Social hierarchies (dominant/subordinate) were characterised by behavioural changes, such as “aggressiveness” and “feeding order”; hierarchical positions were established within an hour of exposure to social stress and remained unchanged for approximately 1 year. To characterise physiological stress, we measured blood plasma levels of cortisol, glucose, and lactate as well as osmolarity and observed that the levels of these stress markers were higher in subordinate individuals than in domi…

Aroclor 1254 inhibits the chemiluminescence response of peritoneal cavity cells from sharpsnout sea bream (Diplodus puntazzo)

Chronic exposure to polychlorinated biphenyls (PCBs) affect the immune system of fish and could lead to a decreased disease resistance. The effects of Aroclor 1254, PCB mixtures, on the Diplodus puntazzo innate immunity were examined by assaying the zymosan stimulated chemiluminescence response (CL) of peritoneal cavity cells (PCCs) at various times (1, 24, 48 h and 1-4 weeks) from intraperitoneal injection of the xenobiotic (1 mg kg(-1) body weight). Controls were performed by assaying cells from medium-treated fish. Since the kinetic of the chemiluminescence response showed the highest peak at 25 min after the zymosan stimulation of the cells, the values found at that time were considered…

In vitro cytokine profile revealed differences from dorsal and ventral skin susceptibility to pathogen-probiotic interaction in gilthead seabream

Skin is the first barrier of defense on fish, which is crucial to protection against different stressors, including pathogens. Skin samples obtained from dorsal and ventral part of Sparus aurata specimens were incubated with Photobacterium damselae subsp. piscicida (a pathogen for this fish species), with Shewanella putrefaciens Pdp11 (a probiotic bacteria isolated from healthy gilthead seabream skin) or with both bacteria. The gene expression profile of nine cytokines (il1b, tnfa, il6, il7, il8, il15, il18, il10 and tgfb) was studied by qPCR in all the skin samples. The present findings revealed different patterns of cytokine profile in dorsal and ventral skin of gilthead seabream, which c…

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)

Immunological Activity of Ascidian Hemocytes

In ascidians, various hemocyte types and their differentiation stages may be responsible for several immune functions. A central role in the immune effector mechanisms can be assigned to PO-containing hemocytes and prophenoloxidase system. Morula cell in Styela plicata and univacuolar refringent granulocyte in Ciona intestinalis, a probable intermediate stages in the differentiation pathway of morula cell, may be cytotoxic cells. They are involved in immune reactions of solitary and colonial ascidians. We have shown that they are provided with a PO-linked spontaneous cytotoxic activity and recognize mammalian erythrocytes or tumor cells markers. The toxic molecules can be radical oxygen int…

First evidences of Toll like receptor on Paracentrotus livisua coelomocytes.

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…

Amino acid composition in eyes from zebrafish (Danio rerio) and sardine (Sardina pilchardus) at the larval stage

A comparative study was performed to identify differences in the amino acid composition of the eyes from zebrafish (Danio rerio) and sardine (Sardina pilchardus) larvae and their link to the environmental adaption of the species. Amino acids in the acidic hydrolysates of eyes from 11 zebrafish and 12 sardine were determined with the use of high-performance liquid chromatography involving precolumn derivatization with ortho-phthalaldehyde. Differences in the content of most amino acids were detected between zebrafish and sardine. These amino acids were aspartate, glutamate, serine, glycine, threonine, arginine, methionine, valine, phenylalanine, isoleucine, leucine and lysine. Of particular …

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

The prophenoloxidase system is activated during the tunic inflammatory reaction of Ciona intestinalis

Phenoloxidase (PO) activity was examined in the tunic tissue of Ciona intestinalis following lipopolysaccharide (LPS) intratunic injection. Tunic homogenate supernatant (THS), assayed with the Dopa-MBTH reaction, displayed Ca(2+)-independent PO activity that was raised by LPS and further enhanced by proteases. Specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. Assay with soybean trypsin inhibitor showed that, in the tunic, PO activation with trypsin was not significantly inhibited suggesting that proteases diverse from serine proteases were involved. In vivo experiments were carried out by injecting isosmotic medium or LPS, and T…

The Anemonia viridis Venom: Coupling Biochemical Purification and RNA-Seq for Translational Research

Blue biotechnologies implement marine bio-resources for addressing practical concerns. The isolation of biologically active molecules from marine animals is one of the main ways this field develops. Strikingly, cnidaria are considered as sustainable resources for this purpose, as they possess unique cells for attack and protection, producing an articulated cocktail of bioactive substances. The Mediterranean sea anemone Anemonia viridis has been studied extensively for years. In this short review, we summarize advances in bioprospecting of the A. viridis toxin arsenal. A. viridis RNA datasets and toxin data mining approaches are briefly described. Analysis reveals the major pool of neurotoxi…

Phenoloxidases in ascidian hemocytes: characterization of the pro-phenoloxidase activating system.

The phenoloxidase (PO) activity of the hemocytes lysate supernatant from three ascidians species, assayed by means of 3-methyl-2-benzothiazolinone hydrazone hydrochloride, have been compared. PO-containing hemocytes were identified by a cytochemical reaction and the enzymatic activity measured by a spectrophotometric assay of lysate supernatant from hemocyte populations separated on a discontinuous Percoll density gradient. In Styela plicata, the enzyme appeared to be contained in morula cells only. In Ciona intestinalis, PO activity was shown in univacuolar refractile granulocyte and granular hemocyte. In Phallusia mammillata both compartment cell and granular hemocytes were positive. Enzy…

Invertebrate lectins present cytokine properties.

Inflamed adult pharynx tissues and swimming larva of Ciona intestinalis share CiTNFalfa-producing cells

Invertebrate lectins present cytockine properties.

Biological activity, tissue distribution and preliminary moleular characterization of a serum fucolectin from the sea bass (Dicentrarchus labrax)

Peritoneal cavity phagocytes from the teleost sea bass express a glucocorticoid receptor (cloned and sequenced) involved in genomic modulation of the in vitro chemiluminescence response to zymosan

Abstract To gain further insight into the role of cortisol in Wsh innate immune responses, we cloned and sequenced a 2592 bp cDNA from sea bass (Dicentrarchus labrax) peritoneal leukocytes (PCLs) encoding a glucocorticoid receptor (DlGR1). The deduced aminoacid sequence displayed that DlGR1 belong to a multigenic family of steroid hormone receptors, and exhibited high homology (80%) to the Burton’s mouth breeder (Haplochromis burtoni) HbGR1. The DlGR1 functional domains presented homologies with those of several vertebrate species. In situ hybridization assay revealed that DlGR1 was expressed in macrophages and neutrophils from the peritoneal cavity. Since in a previous paper, sea bass PCL …

Compendio di Immunobiologia Comparata: Poriferi e Cnidari

In Vitro Release of Lectins From Phallusia mamillata Hemocytes After Their Fractionation on a Density Gradient

Hemocytes were fractionated by centrifugation on a discontinuous Percoll density gradient from the hemolymph of Phallusia mamillata. Results obtained from microcultures of the fractionated hemocytes, sugar-inhibition experiments, SDS-PAGE, and immunoblotting indicate that “compartment cells” release cellular-type (CL) lectins that are specific for α-lactose and lactulose. The released lectins have the same properties as the CL lectins that were previously isolated from sonicated unfractionated hemocytes, but they differ in terms of some molecular and immunological properties from the lectins (SL) purified from the serum. SLs were never found in the supernatants from microcultures of the fra…

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…

Unità operativa "biotassonomia"

Enhanced expression of a cloned and sequenced Ciona intestinalis TNFa-like (CiTNFa) gene during the LPS-induced inflammatory response.

A tumor necrosis factor-alpha (TNFalpha)-like gene from Ciona intestinalis (CiTNF alpha-like) body wall challenged with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation. An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster…

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…

cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax

A 483-bp cDNA was isolated from sea bass (Dicentrarchus labrax) head kidney leukocytes, dicentracin, using PCR primers designed from conserved moronecidin domains. Gene bank analysis revealed that dicentracin cDNA belongs to the moronecidin family. As deduced from alignment with Morone chrysops moronecidin, the precursor of 79 aa appeared to be composed of a signal peptide of 22 aa, followed by the mature AMP (antimicrobial peptide) of 22 aa named dicentracin, and a C-terminal extension of 35 aa. Dicentracin precursor displayed 3 aa substitutions with other moronecidin sequence but none in the mature peptide sequence. Using in situ hybridization assay, dicentracin gene expression was observ…

Nuovi dati sulla risposta infiammatoria dell’ascidia Ciona intestinalis

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…

Tunicate immunocytes can be cytotoxic toward foreign cells

Sphingomyelin as well as carbohydrates are involved in the mechanism of cytotoxic molecules contained and released in vitro by Ciona intestinalis granulocytes.

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…

EFFECTS OF THE ENRICHED-ORGANIC DIETS COMPOSITION ON EUROPEAN SEA BASS (Dicentrarchus labrax) NEW TOOLS FOR FISH WELFARE EVALUATION IN AQUACULTURE

In recent years, concern for the welfare of animals, including fish, has a strong impact on public opinion, putting pressure not only on producers and the scientific community, but also on institution to guarantee adequate "standards” for animal welfare. The market today challenges the production capacity of aquaculture by asking for products with consistently high quality. Unlike other farming methods, there is a lack of scientific information on the welfare of fish reared under intensive aquaculture conditions. Among the various aspects that characterize aquaculture, the increase in fish welfare has an impact on public opinion also for economic reasons whereby is very important to keep an…

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…

Espressione di recettori glucocorticoidei in cellule dell’immunità innata di Dicentrarchus labrax

Espressione e distribuzione del recettore DLGR1 nell’ontogenesi di Dicentrarchus labrax.

Cytotoxic activity of Ciona intestinalis (Tunicata) hemocytes: Properties of the in vitro reaction against erythrocyte targets

Hemocytes (effectors) of Ciona intestinalis showed a natural cytotoxic capacity (HCA) when assayed in vitro against erythrocytes (targets). Cytotoxic cells lysed, to a variable extent, rabbit (RE), human (A, B, O), guinea pig, and sheep (SE) erythrocytes. Hemocyte cytotoxic activity (HCA) assayed against SE is a calcium-dependent reaction, occurs rapidly (15-30 min), at 25-37 degrees C over a wide range of pH (5.4-8.0). Assays were carried out using: 1) the medium in which hemocytes were maintained, 2) the soluble portion of hemocyte lysates, and 3) debris prepared from hemocyte lysates. Results suggest that HCA is a cell-mediated process that requires effector-target cell contacts. Anti-SE…

Il sistema della profenolossidasi in Ciona intestinalis. Caratterizzazione e interazione tra fenolossidasi, perossinectina e superossido dismutasi Cu-Zn dipendente nella risposta infiammatoria indotta da LPS

Effects of dietary vitamin D3 administration on innate immune response of sea bass (Dicentrarchus labrax)

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

The expression of an immune-related phenoloxidase gene is modulated inCiona intestinalisovary, test cells, embryos and larva

Two distinct Ciona intestinalis phenoloxidases (CinPO1, 2) had previously been cloned and sequenced. The CinPO2 is involved in innate immunity and is expressed by inflammatory hemocytes that populate the tunic and pharynx vessels as a response to LPS inoculation. In situ hybridization and immunohistochemistry assays on histological section, showed that the expression of this gene and the produced protein are shared with oogenesis, embryogenesis and larval morphogenesis. Intriguingly, upregulation of gene transcription was found in the test cell layer that envelopes the ovary follicle, ovulated egg, and gastrula, as well as it was modulated in the zygotic nucleus of outer balstomers of 32-ce…

Evaluation of the Effects of the Enriched-Organic Diets Composition on European Sea Bass Welfare through a Multi-Parametric Approach

Three groups of European sea bass (Dicentrarchus labrax) were fed for seven months, with either a conventional diet or two different organic diets, which contain organic vegetables and a natural antioxidant compound. The two organic diets differed themselves in terms of raw proteins, fish oil, and lipid contents. Sea bass welfare condition was assessed in relation to these three diets, using 16 different indicators. These were: swimming activity (recovery test, muscle activity), haematological and serological stress indicators (haematocrit, haemoglobin, red-blood-cell count, cortisol, glucose, lactate), aspecific immunity parameter (lysozyme), indicators of exposure to organic contaminants …

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…

Routes in Innate Immunity Evolution: Galectins and Rhamnose-binding Lectins in Ascidians

Modulation of prophenoloxidase activity in Ciona intestinalis inflammatory response

Allozymic variation in Mediterranean hake, Merluccius merluccius (Gadidae)

Abstract Four hundred and twenty individual hake from 10 sample sites in the Mediterranean Sea were analysed in order to study genetic variability and identify genetic stock structure. Twenty loci were identified, four of which were polymorphic at the 95% level: ADH*, PGI‐1*, PGI‐2* and SOD‐1*. Average observed and expected heterozygosity were 0.084 and 0.090, respectively. PG1–1* deviated from Hardy‐Weinberg expectations due to an excess of heterozygotes and F‐statistic analysis showed also a significant excess of heterozygosity at SOD‐1*. FST was not significant for each locus except for PGI‐2*, where a single sample from the Channel of Sicily (C5) showed a different pattern in allelic fr…

Sphingomyelin inhibition of Ciona intestinalis (Tunicata) cytotoxic hemocytes assayed against sheep erythrocytes

Hemocytes from the ascidian, Ciona intestinalis, are capable of lysing erythrocytes in vitro following cell membrane contact. With the aim of examining the mechanism of cytotoxicity, we performed inhibition experiments with lipid components of erythrocyte membranes. Cholesterol is not an inhibitor, whereas, among the phospholipids tested, (sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine) sphingomyelin inhibits the hemolytic activity of hemocytes. However, thin layer chromatography showed that sphingomyelinase activity was not contained in the chloroform-methanol extracts from hemocyte debris. The inhibition capacity of the components ceramide and phosphorylc…

Identification of Type I and IX Collagens in the Ascidian Ciona intestinalis

Immunohistochemical methods showed that a type I collagen is a component of the tunic of Ciona intestinalis, involved in the encapsulation process. Since the fibril-forming collagen types are characterized by triple helical domain with a highly preserved Gly-Xaa-Yaa repeated sequence, a probe coding the fibril-forming type I collagen of the echinoderm Paracentrotus lividus was used to identify ascidian cDNA clones. Northern blot hybridization established that P. lividus probe cross-hybridizes with a 6 Kb C. intestinalis mRNA isolated from the pharynx. Using the echinodermal type I collagen cDNA as a probe several positive clones were identified. Analysis of sequence and the deduced amino ac…

Vitamin D3 affects innate immune status of European sea bass (Dicentrarchus labrax L.)

The effects of vitamin D3 dietary administration on certain innate immune parameters on the expression of immune-related genes in head-kidney (HK) and gut were investigated in European sea bass Dicentrarchus labrax. Vitamin D3 (vD3) was orally administered to fish in a commercial pellet food supplemented with 0 (control); 3750; 18,750; or 37,500 U kg−1. Furthermore, gut histology was considered. This study showed a modulation in the activities examined in fish fed with the addition of vD3. After just 2 weeks of administration, diet supplementation with the vitamin resulted in increased phagocytic ability, while serum peroxidase content was increased in fish fed with all experimental diets a…

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…

RAPD (Random Amplified Polymorphic DNA) profiles distinguish Paracentrotus lividus populations living in a stressing environment (Amvrakikos Gulf, Greece)

Random amplified polymorphic DNA (RAPD) analysis was performed to assess genetic markers of Paracentrotus lividus populations living in stressing environment in the Amvrakikos Gulf (Western Greece, Ionian Sea) where two populations distinguishable in body size, smaller than the open sea ones, were detected. The UPGMA dendrogram, constructed from pairwise . st values among population nuclear DNA markers, revealed that the small and medium-sized populations living inside the Amvrakikos presented a lower polymorphism, and form a cluster that shows the genetic distance with normal-sized populations (Ionian and Tyrrhenian Seas) living in open sea. AMOVA analysis indicated a genetic distance amon…

Immune-Endocrine Interactions in the Fish Gonad during Infection: An Open Door to Vertical Transmission

The interaction between the immune and endocrine systems has long been recognized in vertebrates. In fish, it is known that the prevalence and intensity of such infections are higher in males than in females and probably related to sex steroid hormone levels. In addition, the immune response in the fish gonad tissues is specifically regulated to prevent infertility. This condition is used by some pathogens to colonize the fish gonad, evade the systemic immune response, and so spread to the progeny. This review brings up to date our knowledge concerning fish gonad immunity and its regulation, immune-endocrine interactions, and how some pathogens use this tissue to spread to the progeny throu…

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…

Inducible galectins are expressed in the inflamed pharynx of the ascidian Ciona intestinalis

Although ascidians belong to a key group in chordate phylogenesis, amino acid sequences of Ciona intestinalis galectin-CRDs (CiLgals-a and -b) have been retained too divergent from vertebrate galectins. In the present paper, to contribute in disclosing Bi-CRD galectin evolution a novel attempt was carried out on CiLgals-a and -b CRDs phylogenetic analysis, and their involvement in ascidian inflammatory responses was shown. CiLgals resulted aligned with Bi-CRD galectins from vertebrates (Xenopus tropicalis, Gallus gallus, Mus musculus, Homo sapiens), cephalochordates (Branchiostoma floridae), echinoderms (Strongylocentrotus purpuratus) and a mono-CRD galectin from the ascidian Clavelina pict…

The ascidian prophenoloxidase activating system

Phenoloxidases/tyrosinases initiate melanin synthesis in almost all organisms, and are involved in different biological activities such as the colour change of human hair and the browning or blackening of fruit skin etc. In many invertebrates, defence reactions are linked to phenoloxidase activity and/or melanization. Contacts with foreign molecules are able to trigger the prophenoloxidase (proPO) system that requires serine protease cleavage for activating the zymogen to phenoloxidase (PO). It is generally accepted that the proPO system is fully expressed in arthropods, and, recently, progress in the regulation of crustacean and insect proPO activation steps have been achieved. After cells…

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…

The cytotoxic activity of Ciona intestinalis (ascidian) unilocular refractile hemocytes versus K562 tumor cells and mammalian erythrocytes involves phospholypase A2 and lectins

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…

Engraulis encrasicolus larvae from two different environmental spawning areas of the Central Mediterranean Sea: first data on amino acid profiles and biochemical evaluations

Early life stages of marine fish populations may be strongly affected by environmental factors. Changes in the physical environment or the availability of food resources could lead to stress-related physiological responses affecting larval fitness, growth and survival. In the present study, we determined, for the first time, amino acid composition (AAC), lipid, and carbohydrate content, as well as alkaline phosphatase and peroxidase activities in larvae from the European anchovy Engraulis encrasicolus. Fishes were caught in two different spawning areas of the Strait of Sicily, characterized by different environmental conditions, including a coastal upwelling with a lower temperature (Advent…

Cloning and expression of a type IX-like collagen in tissues of the ascidian Ciona intestinalis

Collagens are highly preserved proteins in invertebrates and vertebrates. To identify the collagens in urochordates, the total RNA extracted from the pharynx of the ascidian Ciona intestinalis was hybridized with a heterologous probe specific for the echinoderm Paracentrotus lividus fibrillar type I-like larval collagen. Using this probe, two main bands (i.e. 6 and 2.8 kb mRNA) were observed on Northern blot hybridization. The cDNA library prepared from poly(A)+RNA extracted from pharyngeal tissue was screened and a cDNA that specifies a type IX-like collagen was identified. This molecule presents a conceptual open reading frame for a protein containing 734 amino acids. In particular, we sh…

Inflamed adult pharynx tissues and swimming larva of Ciona intestinalis share CiTNFalpha-producing cells.

In situ hybridisation and immunohistochemistry analyses have shown that the Ciona intestinalis tumour necrosis factor alpha gene (CiTNFalpha), which has been previously cloned and sequenced, is expressed either during the inflammatory pharynx response to lipopolysaccharide (LPS) or during the swimming larval phase of development. Granulocytes with large granules and compartment/morula cells are CiTNFalpha-producing cells in both inflamed pharynx and larvae. Pharynx vessel endothelium also takes part in the inflammatory response. Haemocyte nodules in the vessel lumen or associated with the endothelium suggest the involvement of CiTNFalpha in recruiting lymphocyte-like cells and promoting the…

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…

Phenoloxidase characterization in vacuolar hemocytes from the solitary ascidian Styela plicata

Phenoloxidase (PO) activity was shown in lysates of Styela plicata hemocytes assayed spectrophotometrically by means of L-Dopa oxidation without divalent cations. Trypsin and chymotrypsin pretreatment and preincubation with microbial lipopolysaccharides significantly activated PO, whereas laminarin or zymosan were ineffective. Soybean trypsin inhibitor, tropolone, and phenylthiourea, but not benzamidine, were inhibitors. Finally, hemocytes were separated by a discontinuous Percoll density gradient to determine which cells were active. PO activity was demonstrated, by both biochemical and cytochemical assays, in the separated fraction enriched mainly with the globular granulocytes called mor…

Zoologia. Cordati invertebrati

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Ancient Immunity. Phylogenetic Emergence of Recognition-Defense Mechanisms.

Although still scarcely considered by the majority of the biomedical world, invertebrates have greatly contributed to the elucidation of fundamental biological problems [...]

Cnidarian inflammatory reaction and bioactive molecules: from multifunctional role to cultural heritage applications

The renewed interest in the study of genes of immunity in Cnidaria has led to additional information to the scenario of the first stages of immunity evolution revealing the cellular processes involved in symbiosis, in the regulation of homeostasis and in the fight against infections. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa), following injection of various substances different in type and dimension, and observed clear, strong and specific reactions especially after injection of bacteria. The enzymes evaluation (protease, phosphatase and esterase), showing how the injection of different bacterial strains alters the expression of these enzymes suggesti…

Molecular cloning, characterization and expression analysis of peroxinectin from Ciona intestinalis

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…

Novel protese from marine organisms with potential interest in restoration procedures

In the last decades molecular biology allowed the development of innovative protocols in the field of conservation/restoration of cultural assets. In this work we presents novel hydrolyses isolated from marine invertebrate organisms, whose protease activity was previously tested in laboratory by zymography on polyacrilamide gel (in presence of 0.1 of gelatin) Very interesting these enzymes are activated/work in a range of temperatures between 4 ° C and 37 ° C. In this study two sets of proteases were applied, to bio-clean works of art surfaces, at the environments temperature (19° to 25.5° C). Before remove, the water-soluble components of the layers have been analyzed by high-pressure size…

Blue Biotechnology and Cultural Heritage: case studies

In order to promote innovative methodologies for conservation and restoration of historic-artistic manufacts, the efforts are focalized on the development of “sustainable” applications as alternatives to traditional restoration procedures, which can sometimes be detrimental for the artworks, humans and environment [1]. The role of Biotechnology in this field showed very useful applications for diagnosis of bio-deterioration of cultural assets, by an integrated methodology based on molecular and microbiological skills, and in bio-cleaning / bio-removing of organic/inorganic layers from artwork surfaces by enzymes or viable bacteria cells [2, 3]. In this work, bioactive molecules isolated fro…

Characterization and metal-induced gene transcription of two new copper zinc superoxide dismutases in the solitary ascidian Ciona intestinalis

Antioxidant enzymes are known to protect living organisms against the oxidative stress risk, also induced by metals. In the present study, we describe the purification and molecular characterization of two Cu,Zn superoxide dismutases (SODs), referred to as Ci-SODa and Ci-SODb, from Ciona intestinalis, a basal chordate widely distributed in temperate shallow seawater. The putative amino acid sequences were compared with Cu,Zn SODs from other metazoans and phylogenetic analyses indicate that the two putative Ci-SODs are more related to invertebrate SODs than vertebrate ones. Both phylogenetic and preliminary homology modeling analyses suggest that Ci-SODa and Ci-SODb are extracellular and int…

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…

Ciona intestinalis peroxinectin is a novel component of the peroxidase–cyclooxygenase gene superfamily upregulated by LPS

Peroxinectins function as hemoperoxidase and cell adhesion factor involved in invertebrate immune reaction. In this study, the ascidian (Ciona intestinalis) peroxinectin gene (CiPxt) and its expression during the inflammatory response have been examined. CiPxt is a new member of the peroxidase-cyclooxygenase gene superfamily that contains both the peroxidase domain and the integrin KGD (Lys-Gly-Asp) binding motif. A phylogenetic tree showed that CiPxt is very close to the chordate group and appears to be the outgroup of mammalian MPO, EPO and TPO clades. The CiPxt molecular structure model resulted superimposable to the human myeloperoxidase. The CiPxt mRNA expression is upregulated by LPS …

Chemiluminescence response of ß-glucan stimulated leukocytes isolated from different tissues and peritoneal cavity of Dicentrarchus labrax

Abstract The respiratory burst of leukocytes isolated from sea bass ( Dicentrarchus labrax ) pronephros, peritoneal cavity (P.C.), spleen and blood, was measured by a chemiluminescence (CL) assay after stimulation with β-glucan. The CL response by P.C. and pronephros leukocytes was significantly higher than that expressed by a similar number of cells separated from spleen and blood. This probably reflects the observation that the proportion of macrophages and neutrophils was highest in the populations of leukocytes from peritoneal cavity and pronephros. Comparative observations showed a higher degree of yeast phagocytosis by leukocytes taken from peritoneal cavity than the pronephros. Moreo…

Transforming growth factor β (CiTGF-β) gene expression is induced in the inflammatory reaction of Ciona intestinalis.

Transforming growth factor (TGF-β) is a well-known component of a regulatory cytokines superfamily that has pleiotropic functions in a broad range of cell types and is involved, in vertebrates, in numerous physiological and pathological processes. In the current study, we report on Ciona intestinalis molecular characterisation and expression of a transforming growth factor β homologue (CiTGF-β). The gene organisation, phylogenetic tree and modelling supported the close relationship with the mammalian TGF suggesting that the C. intestinalis TGF-β gene shares a common ancestor in the chordate lineages. Functionally, real-time PCR analysis showed that CiTGF-β was transcriptionally upregulated …

Surgical implantation of electronic tags does not induce medium-term effect: insights from growth and stress physiological profile in two marine fish species

Abstract Background Telemetry applied to aquatic organisms has recently developed greatly. Physiological sensors have been increasingly used as tools for fish welfare monitoring. However, for the technology to be used as a reliable welfare indicator, it is important that the tagging procedure does not disrupt fish physiology, behaviour and performance. In this communication, we share our medium-term data on stress physiological profile and growth performance after surgical tag implantation in two important marine fish species for European aquaculture, the sea bream (Sparus aurata) and the European sea bass (Dicentrarchus labrax). Results Blood samples after surgical tag implantation (46 day…

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

Protein differences among the Mediterranean species of the genus Spicara.

Protein electrophoresis (PAGE) was used to study the three morphologically different species of Spicara (S. flexuosa, S. maena, S. smaris). Of the 28 enzymatic and additional myogenic loci, five monomorphic loci (LDH-1*, G6PD-1*, PGI-1* and two PMMs*) were species-specific markers of S. smaris with respect to S. flexuosa and S. maena. Four of the 28 enzymatic loci were polymorphic (EST-1*, GLDH*, PEPD*, PGI-2*). Discriminating genetic markers were not identified between S. flexuosa and S. maena. Genetic distance (D) as calculated by Nei’s index (1978), between S. smaris v. S. maena and S. flexuosa showed a value, respectively of D=0·137 and 0·141. Between S. flexuosa and S. maena the value …

Properties of sea urchin coelomocyte agglutinins

We examined some biological activities of a 200-kDa glycoprotein, referred to as Paracentrotus lividus vitellogenin, contained both in the coelomic fluid and in a subpopulation of coelomocytes called «colourless spherula cells». Cell-free coelomic fluid, coelomocyte lysate and supernatant obtained after coelomocyte washings were assayed for hemagglutinating activity. All samples agglutinated rabbit erythrocytes in a calcium-dependent way. The comparison between the electrophoretic patterns of erythrocyte lysates, before and after incubation with the coelomic fluid, revealed that a 200-kDa band was obtained from membranes of agglutinated erythrocytes. In addition, polyclonal antibodies again…

Neuronal factors secreted through unusual pathways are probably involved in blood brain barrier (BBB) formation

Edible Insects an Alternative Nutritional Source of Bioactive Compounds: A Review

Edible insects have the potential to become one of the major future foods. In fact, they can be considered cheap, highly nutritious, and healthy food sources. International agencies, such as the Food and Agriculture Organization (FAO), have focused their attention on the consumption of edible insects, in particular, regarding their nutritional value and possible biological, toxicological, and allergenic risks, wishing the development of analytical methods to verify the authenticity, quality, and safety of insect-based products. Edible insects are rich in proteins, fats, fiber, vitamins, and minerals but also seem to contain large amounts of polyphenols able to have a key role in specific bi…

Inferring Population Structure from Early Life Stage: The Case of the European Anchovy in the Sicilian and Maltese Shelves

The European anchovy is an important fishing resource in the Sicilian Channel that supports a high recruitment success variability. The presence of two spawning areas, the drifting of the larvae along the currents and the different oceanographic conditions within the region suggest the presence of different larvae subpopulations. Morphometric and biochemical approaches have been used to analyze the differences among larvae collected. The amino acid composition discriminates two larval groups closely related to the spawning regions: Adventure Bank and the shelf between the South of Sicily and Malta. In addition, there are morphometric and growth differences between recently hatched larvae in…

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…

Expression of a type IX-like collagen in tissue injury of the ascidian Ciona intestinalis durino inflammatory process

Risposta allo stress ambientale di Dentex dentex, Pagrus major ed il loro ibrido “Pantice”.

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…

Il sistema della profenolossidasi nella risposta infiammatoria della tunica di Ciona intestinalis (L.)

Univacuolar refractile hemocytes from the tunicate Ciona intestinalis are cytotoxic for mammalian erythrocytes in vitro

A discontinuous, Percoll density gradient was used to separate hemocyte populations from the hemolymph of Ciona intestinalis. Hemocytes from each band were examined for their frequency, morphology, and cytotoxic activity against rabbit and sheep erythrocytes; results were expressed as a percentage of hemolysis. Statistical analysis revealed that only the "univacuolar" granulocytes from Band 5, which contain a vacuole of refractile material, were cytotoxic. Cytotoxic activity was inhibited by sphingomyelin. For the first time in tunicates, lytic activity against erythrocytes was assessed by an assay based on plaque-forming cells. Plaques of lysis were revealed against rabbit erythrocytes but…

Molecular characterisation, evolution and expression analysis of g-type lysozymes in Ciona intestinalis

Lysozyme is an important defense molecule of the innate immune system. Known for its bactericidal properties, lysozyme catalyzes the hydrolysis of b-(1,4)-glycosidic bonds between the N-acetyl glucosamine and N-acetyl muramic acid in the peptidoglycan layer of bacterial cell walls. In this study, the complete coding sequence of four g-type lysozymes were identified in Ciona intestinalis. Phylogenetic analysis and modelling supported the hypothesis of a close relationship with the vertebrate g-type lysozymes suggesting that the C. intestinalis g-type lysozyme genes (CiLys-g1, Cilys-g2, CiLys-g3, CiLys-g4) share a common ancestor in the chordate lineage. Protein motif searches indicated that …

The Role of Lectins in Finfish: A Review

The immune system of vertebrates involves both innate and acquired immune responses. The innate immunity is more generalized with robust response whereas the other has a highly specific response to infectious pathogens. Because of the lack of specialized lymphatic organs, innate immunity is an important mode of defense in fishes. The less specific innate immune system acts mainly through complement pathway which depends on pattern-based recognition of “self” and “non-self” targets by host lectins and associated proteins. This ultimately results in the clearance of target cells. Lectins are glycoproteins which possess at least one carbohydrate recognition domain (CRD) that specifically and r…

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…

Attività biologica, distribuzione tissutale e caratterizzazione molecolare della fucolectina sierica di spigola (Dicentrarchus labrax).

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

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

A serum fucolectin isolated and characterized from sea bass Dicentrarchus labrax

A lectin specific for fucose and galactose was isolated by affinity chromatography on Sepharose CL-6B from the serum of Dicentrarchus labrax. The hemagglutinating activity against rabbit erythrocytes was calcium-independent, and reached its maximum at 37 degrees C. Two protein components were found in the hemagglutinating fractions eluted from the Sepharose column. Only the 34 kDa component (DLL2) eluted from the polyacrylamide gels (SDS-PAGE) showed agglutinating activity against rabbit erythrocytes. SDS-PAGE, in non-reducing conditions, revealed a single 66 kDa protein that reacted with antibodies to the 34 kDa component. Therefore, a dimeric structure stabilized by disulfide bonds can be…

An Attempt to re-examine the immune role of Ciona intestinalis hemocytes

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 …

Innovative and Integrated Strategies: Case Studies

In this chapter, case studies related to biodeterioration, bioaerosol, biocide and biocleaning are reported. The aim is highlighting the role of biology and biotechnology tools for the preventive conservation of organic and inorganic artifacts, understanding how traditional as well as innovative methods can help the conservationists to develop integrated strategies considering works of art/environment/ humans as a dynamic system. Particularly, based on the experience acquired during the researches of Laboratory of Biology and Biotechnology for Cultural Heritage (LaBBCH), the authors suggest several approaches to reveal and identify biological systems able to induce biodeterioration of cultu…

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

Evoluzione dell’immunità innata. Componenti della reazione infiammatoria indotta da LPS nella parete corporea dell’ascidia Ciona intestinalis.

The Role of Spatial Exploration and Territoriality in Establishing Gilthead Seabream (Sparus aurata) Hierarchies, and Their Effects upon Underlying Stress Physiology

Territoriality, spatial exploration and social hierarchy are strictly related behaviors in gregarious fishes, and are often under-appreciated in farms where the individuals are confined within crowded spaces. In this study, we investigated the role of spatial exploration, elucidating the importance of time upon forming the social organization, and the role of the territoriality in gilthead seabream (Sparus aurata), using two experimental approaches. In the first approach, three fish were placed sequentially in the aquarium with an interval of two days (sequential model), while in the second (simultaneous model), two fish were simultaneously placed in an aquarium divided by a barrier which w…

Molecular characterization of an antimicrobial peptide isolated from leucocytes of the teleostean Dicentrarchus labrax.

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…

The Ciona intestinalis immune-related galectin genes (CiLgals-a and CiLgals-b) are expressed by the gastric epithelium.

The transcription of two Ciona intestinalis galectin genes (CiLgals-a and CiLgalseb) is uparegulated by LPS in the pharynxis (hemocytes, vessel epithelium, endostilar zones) which is retained the main organ of the immunity. In this ascidian, for the first time we show, by immunohistochemistry and in situ hybridization methods, that these two immune-related genes are expressed in the gastric epithelium of naïve ascidians, whereas the galectins appear to be only contained in the intestine columnar epithelium. In addition, according to previous results on the pharynx, the genes are also expressed and galectins produced by hemocytes scattered in the connective tissue surrounding the gut. The ge…

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

Espressione di galactine nell'infiammazione e nella larva natante di Ciona intestinalis

Enhanced CiTNFα expression is an inflammatory response of the ascidian Ciona intestinalis

Il sistema della profenolossidasi in Ciona intestinalis durante il processo infiammatorio indotto da LPS

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…

The Ciona intestinalis prophenoloxidase activating system during LPS inflammatory reaction

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…

FACIT collagen (1α-chain) is expressed by hemocytes and epidermis during the inflammatory response of the ascidian Ciona intestinalis

Based on previous cloning and sequencing study, real-time PCR and in situ hybridization assays of the inflamed body wall of LPS-injected Ciona intestinalis showed the enhanced gene expression of a collagen with FACIT structural features (Ci-type IX-Col 1alpha-chain). By using specific antibodies raised against an opportunely chosen Ci-type IX-Col synthetic peptide, the fibroblast property of hemocytes challenged in vitro with LPS (at 4h) was displayed by flow cytometry, while immunocytochemistry identified hemocytes with large granules (morula cells) as collagen-producing cells. Hemocyte lysate supernatant analyzed in immunoblotting contained a 60 kDa band identifiable as 1alpha-chain-Ci-ty…

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…

cytokines in the inflammatory response of the ascidian ciona intestinalis

The Ciona intestinalis inflammatory response to several irritants have been demonstrated to be composed of a complex set reaction. The cellular reactions involve hemocyte infiltration, hemocyte and epidermis activities, vacuolization, cell disruption, while cell products can contribute to form capsule components and/or cause a wound. In this response the involvement of the pharynx, as the main immune-competent organ, has been disclosed by lipopolysaccharide (LPS) challenge that upregulates innate immunity genes. In vertebrate cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of cell populations that h…

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

Nuovi dati sull'attivazione della profenolossidasi di Ciona intestinalis nel processo infiammatorio

Stress and immunomodulation indicators in gilthead seabream (Sparus aurata): preliminary data concerning the alterations due to the social behaviour.

A Biochemical Genetic Study of Isoenzyme Polymorphism within and between Two Populations of Atherina Boyeri Risso

Allozymic variation of 20 loci was investigated in two samples of Atherina boyeri collected in the Mediterranean Sea from the coast of Trappeto and Marsala Lagoon (Sicily, Italy). Five loci were polymorphic (PGM*, EST-1,2*, SOD-2*, PGI-2*) and appeared to be in Hardy-Weinberg equilibrium. A significant Fst mean value (0.02) (G = 23.77, P < 0.05, df = 5) showed that the samples can be considered as two distinct local populations and supports previous morphometric discrimination.

Toll signal transduction pathway in bivalves: Complete cds of intermediate elements and related gene transcription levels in hemocytes of immune stimulated Mytilus galloprovincialis

Based on protein domain structure and organization deduced from mRNA contigs, 15 transcripts of the Toll signaling pathway have been identified in the bivalve, Mytilus galloprovincialis. Identical searches performed on publicly available Mytilus edulis ESTs revealed 11 transcripts, whereas searches performed in genomic and new transcriptome sequences of the Pacific oyster, Crassostrea gigas, identified 21 Toll-related transcripts. The remarkable molecular diversity of TRAF and IKK coding sequences of C. gigas, suggests that the sequence data inferred from Mytilus cDNAs may not be exhaustive. Most of the Toll pathway genes were constitutively and ubiquitously expressed in M. galloprovinciali…

Prophenoloxidase system is activated in the tunic inflammatory response of ciona intestinalis

Inflammation in ascidians

Seabream(Sparus aurata)hierarchy among alfa and beta subordinates and dominant interplay affects stress responses and phagocytic activity by peritoneal cavity cells

seabream, subordinates, dominant

Cell cooperation in coelomocyte cytotoxic activity of Paracentrotus lividus coelomocytes

The coelomic fluid from the sea urchin Paracentrotus lividus contains several coelomocyte types including amoebocytes and uncoloured spherulocytes involved in immune defences. In the present paper, we show a Ca(2+)-dependent cytotoxic activity for the unfractionated coelomocytes assayed in vitro, with rabbit erythrocytes and the K562 tumour cell line. In a plaque-forming assay, whole coelomocyte preparations as well as density gradient separated coelomocyte populations revealed that cell populations enriched in uncoloured spherulocytes, exerted high cytotoxic activity by releasing lysins in the presence of amoebocytes. This cooperative effect could be dependent on soluble factors released b…

COMPARATIVE ANALYSIS AND DISTRIBUTION OF THYROXINE HORMONE IN ASCIDIANS LARVAE

Indicatori di stress ed immunomodulazione in Sparus aurata: dati preliminari sulle modificazioni indotte dal comportamento sociale di sottomissione.

Basic knowledge of social hierarchies and physiological profile of reared sea bass Dicentrarchus labrax (L.).

The effects of social hierarchies (dominant/subordinate individuals), such as aggressiveness, feeding order, and territoriality, are some of the characteristics used for describing fish behaviour. Social hierarchy patterns are still poorly understood in European-reared sea bass (Dicentrarchus labrax). In this work, we examine the social interactions among captive fish integrating behavioural and physiological profiles. Groups of three fish with EMG (electromyogram) radio transmitters were monitored for two weeks via video recording. Plasma levels of cortisol, glucose, lactate and lysozyme as well as haematological parameters such as haemoglobin, haematocrit and RBCC (red blood cell count) w…

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.…

Molecules and cells in inflammatory responses of the ascidian Ciona intestinalis

Effects of temperature on total phenolic compounds in Cystoseira amentacea (C. Agardh) Bory (Fucales, Phaeophyceae) from southern Mediterranean Sea

The aim of this study was to test the effects of temperature on phenolic content of the brown seaweed Cystoseira amentacea. Phenolic compounds are secondary metabolites involved in different protection mechanisms as, for example, against grazers, epiphytes and UV radiation. Seasonal variations of phenolic content in C. amentacea were analysed and laboratory experiments, in which C. amentacea was exposed to an increase of temperature (25°C and 30°C), were performed. Total phenolic content (TPC) was determined colorimetrically with the Folin–Ciocalteu reagent. In C. amentacea, a seasonal pattern in TPC was observed, with a maximum value in winter-spring. C. amentacea responded significantly t…

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…

Interleukin 17 genes as mediators of inflammatory responses in Ciona intestinalis.

Inflammation is a complex reaction of host defence mechanisms aiming at neutralization of an insult and restoring normal tissue structure and function. In human IL-17 is T-cell derived cytokine plays a key role in the clearance of extracellular bacteria promoting cell infiltration and production of several cytokines and chemokines. Here, we report on three Ciona intestinalis IL-17 homologues (CiIL17-1, CiIL17-2, CiIL17-3). The gene organization, phylogenetic tree and modeling supported the close relationship with the mammalian IL-17A and IL-17F suggesting that the C. intestinalis IL-17 genes share a common ancestor in the chordate lineages. Real time PCR analysis showed a prompt expression …

Confinement stress in sea bass (Dicentrarchus labrax) depresses peritoneal leukocyte cytotoxicity

Fish respond to stressful conditions via neuroendocrine responses (primary response) which result in increased levels of plasma cortisol which is considered immunosuppressive. Sea bass were confined at low (10 kg/m3) and high (60 kg/m3) density for 3-48 h. Plasma cortisol and glucose were evaluated and two principal cellular immune responses were assayed. A significant increase in plasma cortisol and glucose levels, as well as osmolarity, was found following stress. In addition, phagocytic activity, as shown by reactive oxygen species (ROS) production by challenged head kidney phagocytes and cytotoxic activity of eosinophilic granule cells from peritoneal cavity against K562 tumour cell lin…

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

Calibrating accelerometer data, as a promising tool for health and welfare monitoring in aquaculture: Case study in European sea bass (Dicentrarchus labrax) in conventional or organic aquaculture

Abstract Both the conventional and organic aquaculture sectors have grown rapidly over the past few years. Moreover, welfare has attracted increased attention on the part of both consumers and governments. However, fish welfare assessment is complex and thus needs to adapt measurements that are easily applicable to aquaculture conditions. In this study, in addition to classical welfare indicator measurements (physiological stress indicators and growth performance), we recorded the swimming activity data using acoustic transmitters to evaluate the welfare of European sea bass (Dicentrarchus labrax) fed a conventional vs. an organic diet. Prior feeding trial, the swimming activity recorded by…

La risposta infiammatoria in Ciona intestinalis è caratterizzata dall’aumento di lectine specifiche per D-galattosidi e dall’espressione di collagene FACIT di tipo IX

Maristem stem cells of marine/aquatic invertebrates: from basic research to innovative applications

The “stem cells” discipline represents one of the most dynamic areas in biomedicine. While adult marine/aquatic invertebrate stem cell (MISC) biology is of prime research and medical interest, studies on stem cells from organisms outside the classical vertebrate (e.g., human, mouse, and zebrafish) and invertebrate (e.g., Drosophila, Caenorhabditis) models have not been pursued vigorously. Marine/aquatic invertebrates constitute the largest biodiversity and the widest phylogenetic radiation on Earth, from morphologically simple organisms (e.g., sponges, cnidarians), to the more complex mollusks, crustaceans, echinoderms, and protochordates. These organisms contain a kaleidoscope of MISC-type…

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

Allozyme Similarity in Two Morphologically Distinguishable Populations ofParacentrotus Lividus(Echinodermata) From Distinct Areas of the Mediterranean Coast

Allozymes ofParacentrotus lividusfrom Palermo Gulf in the northern Sicilian coast (Italy) and from a small body-size population in the western Greek coast (Ionian Sea) were investigated by PAGE. Five of the twenty examined loci were polymorphic(AAT*, ADH*, ME*, PGI*andPGM*)over each population with a polymorphism value of 0–25. Average heterozygosity was equal to 0081 in the Sicilian sample and 0084 in the Greek. Deviations from Hardy-Weinberg equilibrium were significant inME*andPGI*loci (as calculated byy).Nei's (1978) genetic distance (D=0–0025) index described a close identity between the two samples. FSTvalue of polymorphic loci ranged from 0001 to 0029, its mean value (0–008) resultin…

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…

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

F-Type Lectins: A highly diversified family of fucose-binding proteins with a unique sequence motif and structural fold, involved in self/non-self-recognition

The F-type lectin (FTL) family is one of the most recent to be identified and structurally characterized. Members of the FTL family are characterized by a fucose recognition domain [F-type lectin domain (FTLD)] that displays a novel jellyroll fold (“F-type” fold) and unique carbohydrate- and calcium-binding sequence motifs. This novel lectin family comprises widely distributed proteins exhibiting single, double, or greater multiples of the FTLD, either tandemly arrayed or combined with other structurally and functionally distinct domains, yielding lectin subunits of pleiotropic properties even within a single species. Furthermore, the extraordinary variability of FTL sequences (isoforms) th…

Tributyltin affects phagocytic activity of Ciona intestinalis hemocytes

Organotin compounds have been used in marine anti-fouling paints as biocides. Because tunicates are vulnerable to these compounds in their natural habitats, we used Ciona intestinalis to establish an assay for phagocytosis in vitro of yeast by hemocytes after exposure to different concentrations (0.0015, 0.015, 0.15 and 1.5 microM) of four organotin compounds: tributyltin (TBT), triphenyltin (TPT), dibutyltin (DBT) and diphenyltin (DPT). To evaluate the phagocytic activity, we used a method based on fluorescence excitation of yeast pre-treated with eosin-Y. The percentage of phagocytosis decreased from 45.1 +/- 3.49 to 22.4 +/- 5.14 at 1.5 microM of TBT (P0.001); it was significantly reduce…

Preliminare caratterizzazione di un’attività citotossica nel supernatante del lisato degli emociti con granulo rifrangente di Ciona intestinalis

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

Evolution of Innate Immunity. Components of inflammatory reaction in Ciona intestinalis

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

Are immune responses gender-related in Carabus lefebvrei (Coleoptera: Carabidae)?

The “live hard, die young” theory predicts the evolution of gender differences in immunocompetence, with males having a weaker immune system than females. To test this hypothesis in Carabus lefebvrei, total and basal phenoloxidase (PO) activities and lysozyme-like enzyme activity were compared among males and females of different reproductive status. The sexual dimorphism occurred only in reproductively active adults and for total and basal PO levels, while no significant differences were recorded between sexes in virgin adults. Differences were not recorded for lytic activity between sexes. Basal PO and lytic activities decreased in both males and females after mating, while the total PO v…

Peptidi antimicrobici dei pesci: la famiglia delle piscidine

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…

Elie Metchnikoff and the multidisciplinary link novelty among Zoology, Embryology and Innate Immunity

Elie Metchnikoff was a Russian scientist known as the pioneer of innate immunity. In particular, he was awarded the Nobel Prize for discovering the process of phagocytosis and its significance in the development and disease. Here, we endeavor to demonstrate the enduring fascination of his scientific research, in particular the experiment involving the first observation of a macrophage reaction in the sea star. This applies to both adult and larvae immunity studies. Recent work on sea star larval cellular immunity and adult immune systems using modern expansions of molecular and cellular techniques shows that it is a continually exciting research field that cannot just be consigned to histor…

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…

Removal of old protein layers from surfaces of works of art by new enzymes

The aim of the present project is to set up bio-cleaning protocols in order to easily remove altered protein layers (e.g. animal glues) by enzymatic proteins (proteases). Protein molecules were isolated from marine invertebrate organisms, characterized by size exclusion highpressure liquid chromatography (Waters: SEC-HPLC, BioSuite 250 to 10㎛ SEC 7.5 x 300 mm) and their gelatinase activity analyzed by zymography on polyacrilamide gel. The remarkably proteolytic activity and the reaction temperature range of these enzymes, 4° to 37℃, made it possible to hypothesize their use in bioremediation projects. Tests were performed on protein coating found on some polychrome wooden artifacts exposed …

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 …

Meccanismi dell’immunità naturale in echinodermi. Interazioni tra i celomociti di Paracentrotus lividus

Espression of a type IX-like collagen in tissue injury of the ascidian Ciona intestinalis during inflammatory process

Novel proteases from marine organisms with potential interest in restoration procedure

In the last decades, molecular biology allowed the development of innovative protocols in the field of conservation/restoration of cultural assets. In this work new hydrolyses, isolated from marine invertebrate organisms, are applied to remove protein layers form works of art surface. Proteolytic zymography assay evidenced that these enzymes are active in a broad temperature range, between 4 degrees and 37 degrees C. The enzymatic cleaning by these proteases, tested on wooden furniture of the second half of the eighteenth century showed positive results, without needing to heat the enzyme solution or the surface on which they were applied. The present report proposes novel proteases more ap…

Enhanced expression of a CinTNF gene in the LPS challenged inflammatiry responses of the ascidian Ciona intestinalis

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

Inflammatory response of Ciona intestinalis involve enhanced D-galactoside-specific lectins and FACIT collagen expression.

Diversity of lectin-sugar recognition dystems in the evolution of fish innate immunity

Inducible lectins with galectin properties and human IL1alpha epitopes opsonize yeast during the inflammatory response of the ascidian Ciona intestinalis.

Studies on inducible ascidian lectins may shed light on the evolutionary emergence of cytokine functions. Here, we show that the levels of opsonins, with IL1alpha-epitopes, increase in Ciona intestinalis hemolymph as a response to an inflammatory stimulus and, in particular, to intratunic injection of lipopolysaccharide (LPS). The inflammatory agent promptly (within 4 h) enhances Ca(2+)-independent serum hemagglutinating and opsonizing activities, which are both inhibited by D-galactose and D-galactosides (alpha-lactose, N-acetyl-D-lactosamine, thio-digalactoside), suggesting that anti-rabbit erythrocyte lectins with galectin properties are involved as opsonins. Inducible galectin molecules…

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

Cooperazione cellulare nella reazione citotossica dei celomociti di Paracentrotus lividus

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