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Phylogenetic analysis recognizes echinoderms as a key group of deuterostomes, therefore the species in this group are useful for the study of the evolution of innate immunity responses. In addition, this marine invertebrate lives in coastal and estuarine waters that are directly exposed to potentially pathogenic microorganisms and stressful anthropogenic factors. Coelomocyte populations seem to be essential to immune-defence functions such as phagocytosis, ROI production, cytotoxicity, synthesis and release of antimicrobial substances including lectin, cytokine, C3-like expression, prophenoloxidase activity and capsule formation. Holothurians’ coelomocyte populations contain several coelomocyte types, including phagocytes, and can form brown bodies in response to multicellular parasites. Holothuria tubulosa coelomic fluid contains three main coelomocyte categories: amoebocytes, spherulocytes and progenitor cells. The amoebocytes represent about 30 % of the total population, and in living cell preparations exhibit two distinct forms: petaloid and philopodial; spherule cells represent the numerically dominant cell type (about 60 %). Progenitor cells are present in a lesser amount (about 20 %); they are similar to lymphocytes and show a nucleus that is typically prominent with a thin rim of cytoplasm. In the present report we show that coelomocytes of Holothuria tubulosa are able to exert cytotoxic activity against different cellular targets such as rabbit or sheep erythrocytes and the human erythromyeloid leukaemia-derived cell line K562. Moreover, the unseparated coelomocyte supernatant lysate (CLS) exerts a lytic activity, even in the absence of calcium, against the same targets. Analysis of the coelomocyte lysate by overlay assays with sheep and rabbit erythrocytes on PAGE without SDS showed a protein pattern composed by two main hemolytic bands with different electrophoretic mobility. The one with low mobility (I) showed calcium independent haemolytic activity while high mobility band (II) showed calcium dependent activity. The two bands were eluted from the gel and analyzed by SDS-PAGE and stained with silver stain; band I could be separated in three components of different sizes (52, 42 and 41 kDa), and band II had a size of 43 kDa. Spherule cells seemed to be the effector cells, as shown by a plaque lysis assay. Further studies are in progress to identify the lytic proteins and the lytic mechanism at work on both a molecular and cellular level.

Aqueous extracts from Posidonia oceanica’s green and brown (beached) leaves and rhizomes were prepared, submitted to phenolic compound and proteomic analysis, and examined for their potential cytotoxic effect on HepG2 liver cancer cells in culture. The chosen endpoints related to survival and death were cell viability and locomotory behavior, cell-cycle analysis, apoptosis and autophagy, mitochondrial membrane polarization, and cell redox state. Here, we show that 24 h exposure to both green-leaf- and rhizome-derived extracts decreased tumor cell number in a dose–response manner, with a mean half maximal inhibitory concentration (IC50) estimated at 83 and 11.5 μg of dry extract/mL, respectively. Exposure to the IC50 of the extracts appeared to inhibit cell motility and long-term cell replicating capacity, with a more pronounced effect exerted by the rhizome-derived preparation. The underlying death-promoting mechanisms identified involved the down-regulation of autophagy, the onset of apoptosis, the decrease in the generation of reactive oxygen species, and the dissipation of mitochondrial transmembrane potential, although, at the molecular level, the two extracts appeared to elicit partially differentiating effects, conceivably due to their diverse composition. In conclusion, P. oceanica extracts merit further investigation to develop novel promising prevention and/or treatment agents, as well as beneficial supplements for the formulation of functional foods and food-packaging material with antioxidant and anticancer properties.

Echinoderms are a phylum of deuterostomic invertebrates that play a key role in maintaining the biodiversity of marine ecosystems. They represent a good study model for immunity because their coelomic fluid contains different types of cells involved in the inflammatory response: the coelomocytes. In the case of the sea urchin Paracentrotus lividus, the coelomocyte population is mainly represented by amoebocytes and uncoloured spherulocytes that implement a defence program through phagocytosis, encapsulation, cytotoxicity, and production of antimicrobial agents. The purpose of this study was to find evidence of a possible modulating effect of lipopolysaccharide LPS on the expression of the AIF-1 gene and therefore to determine whether or not there is a correlation between the P. lividus immune response and the expression of this gene when the homeostasis of the animals is disturbed by a bacterial infection which, in this case, was simulated with treatment with LPS. AIF-1 (Allograft Inflammatory Factor 1) is a 17 kDa calcium-binding protein that, in vertebrates, is involved in the activation of macrophages. Similarly, in Echinoids and in general in invertebrates, the expression of this gene increases considerably after a bacterial attack and this suggests that it plays a key role during the immune responses.

Marine crustaceans are influenced by numerous environmental factors. Environmental stress from pollutants seems to affect their metabolism, growth, moulting, survival, and immune defence. Recently, it has become clear that there is an impact of the global increase in sea noise levels due to shipping traffic on crustacean welfare. Considering the ecological and commercial importance of the European spiny lobster (Palinurus elephas (Fabricius, 1787)) in most parts of the Mediterranean coastal area, in the present study we investigated whether shipping noise pollution contribute to changing the immune parameters of stress in the European spiny lobster. The animals were exposed to a mix of noises produced by different typologies of the boat played back in a tank, and the values of cellular and humeral parameters were evaluated. Total haemocyte count (THC), haemolymphatic protein concentration, phenoloxidase (PO) activity in cell-free haemolymph, and heat shock protein 27 (Hsp27) expression in haemocyte lysate were considered potential biomarkers of stress. THC and PO activity decreased significantly, whereas total protein and Hsp27 expression increased significantly. Overall, the results demonstrate that the stressful conditions investigated in this study affect both cellular and biochemical parameters in the European spiny lobster.

In the immune system of vertebrates, gender-specific differences in individual immune competence are well known. In general, females possess more powerful immune response than males. In invertebrates, the situation is much less clear. For this purpose we have chosen to study the immune response of the two sexes of the echinoderm Paracentrotus lividus in pre- and post-spawning phases. The coelomic fluid from the echinoderms contains several coelomocyte types and molecules involved in innate immune defenses. In this article we report that the degree of immune responses in the P. lividus differs according to sex in both pre- and post-spawning phases. We found in all tests that females were more active than males. The results indicate that females possess a significant higher number of immunocytes consisting of phagocytes and uncolored spherulocytes. Since the immunological activity is mainly based on immunocytes, it was not surprising that females possessed the highest values of cytotoxicity and hemolysis activity and showed a greater ability to uptake neutral red and phagocyte yeasts cells, while the average number of ingested particles per active phagocyte was not significantly different. Furthermore, agglutinating activity was more evident in the coelomocyte lysate and coelomic fluid of females than in those of males. Finally we found that the acidic extract of female gonads possessed greater antimicrobial activity than that of male gonads. These results make it very likely that gender differences in the immune response are not restricted to vertebrates; rather, they are a general evolutionary phenomenon.

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 THS was assayed for its PO activity. Analysis of variance of the time-course profiles showed that LPS was more effective in activating proPO. To disclose the PO response at the injured site, an assay with Dopa-MBTH was performed in vitro. Quinones were mainly contained in the tunic matrix enriched with inflammatory cells around the injection site. Microscopic observations and immunohistochemistry with anti-CinPO-2 antibodies showed granulocytes and unilocular refractile granulocytes containing PO, whereas few morula cells were stained. In THS zymograms (SDS-polyacrylamide gel electrophoresis), PO activity linked to 90-kDa and 120-kDa bands was observed as an effect of LPS injection, whereas the density of 170-kDa PO was weak. A third presumptive PO enzyme (CinPO-3) containing the CinPO-2 peptide was identified in the recent Ciona genome version. Presumably, LPS stimulated the production and dimerization (120 kDa) of CinPO-3 (66 kDa). Thus, the activated proPO system includes several POs that are distinguishable by size and that are contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars.

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 lines appeared to be suppressed. Plasma levels of cortisol, glucose, osmolarity were correlated with cellular immunity, by the linear regression method. The suppression of cytotoxic activity was found to be significantly correlated with high plasma cortisol and glucose levels. These parameters could affect the eosinophilic granule cells of the peritoneal cavity.

Wound repair is a key event in the regeneration mechanisms of echinoderms. We studied, at the behavioural, cellular and molecular levels, the wound healing processes in Holothuria tubulosa after injuries to the body wall. The experiments were performed for periods of up to 72 h, and various coelomocyte counts, as well as the expression of heat shock proteins (HS27, HSP70 and HSP90), were recorded. Dermal wound healing was nearly complete within 72 h. In the early stages, we observed the injured animals twisting their bodies to keep their injuries on the surface of the water for the extrusion of the buccal pedicles. At the cellular level, we found time-dependent variations in the circulating coelomocyte counts. After injury, in particular, we observed a significant increase in spherule cells at 2.5 h post-injury. Using the western blot method, we observed and reported that the wounds produced, compared with controls, a significant increase in HSP27 and HSP70 expression in coelomocytes, whereas HSP70 was increased in scar tissue and HSP90 was increased only in cell-free coelomic fluid. These results highlight that the wounds were responsible for the stress condition with the induction of cellular and biochemical responses.

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. The CL enhancement observed at 1 h after the treatment with xenobiotic was followed by a decreased response at 24 h and appeared to be lower at 1-4 weeks when compared to the CL response of the control, suggesting a protracted effect of PCBs on the peritoneal cavity. Since PCCs incubated in vitro for 1 h with 0.05 and 0.1 mu g ml(-1) Aroclor showed an enhanced CL, the effect of the xenobiotic could be exerted on the cell responsiveness to zymosan. It is known that fish CL response of PCCs can be imputed to phagocyte (macrophages and neutrophils) activation, these cells and their responsiveness to zymosan can be used in immunotoxicology assay to monitor the fish health in polluted environment. (C) 2014 Elsevier Ltd. All rights reserved.

The immunotoxic effects of heavy metals on blood leukocytes of sea bass (Dicentrarchus labrax) were examined. The cells, separated by a discontinuous Percoll-gradients, were exposed in vitro to various sublethal concentrations of cadmium and copper (10(-7) M, 10(-5) M, and 10(-3) M) and their immunotoxic effect was then evaluated by measuring neutral red uptake, MU assay, DNA fragmentation and Hsp70 gene expression. First of all, we demonstrated that the cells treated in vitro could incorporate Cd and Cu. A relationship between heavy metal exposure and dose-time-dependent alterations in responses of leukocytes from blood was found for both metals, but copper was more immunotoxic than cadmium in all assays performed. A significant reduction in the cells' ability to uptake neutral red and viability by MU assay was recorded, indicating that both cadmium and copper could change the membrane permeability, inducing cellular apoptosis when the concentration of metals reached 10(-3) M. The apoptotic effect may also explain the high level of cytotoxicity found when the leukocytes were exposed to higher concentration of metals. These results demonstrated that toxic effect of copper and cadmium affect on the mechanisms of cell-mediated immunity reducing the immune defences of the organism. (C) 2014 Elsevier Inc. All rights reserved.

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 proposed. The Ca(2+)-independent fucose-binding specificity, a significant amino acid sequence homology of the N-terminal trait, and cross-reaction of eel fucolectin with antibodies to DLL2 suggest that this lectin may be included in the recently identified fucolectin family.

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 required effector-target cell contact. Eosinophilic granule cells, isolated on a Percoll density gradient from a peritoneal wash, appeared to be responsible for the in vitro cytotoxic activity.

To study the pathogen-host relationship, we used the model of the entomopathogenic bacterium Bacillus thuringiensis (Bt) and Rhynchophorus ferrugineus, a quarantine pest that attacks palm trees. In particular, we focused on the Bt stress-induced infections. We studied the effect of Bt on larval and adult growth, and on the expression of the heat shock proteins (HSPs), rapidly synthesized in the cell after exposure to stress including pathogens. Bt has negative effects on larval and adult growth, on total hemocytes counts and on the hemocyte type. HSP70 was evaluated in the supernatant of the brain lysate obtained from larvae and adults fed with sublethal doses of Bt. HSP70 expression was modulated in time (3h, 6h, 12h, 24h) in response to Bt ingestion, highlighting that Bt is a stress factor for the R. ferrugineus. Further investigation is needed to understand the possible correlation between the reduction of hemocytes and HSP70 modulation. The potential costs of the different bioassay used will be compared.

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 dominant ones. The discriminant analysis revealed a separation of the subordinate fish groups, and at 15 days, a significant separation among groups was observed. Moreover, diminished phagocytic and respiratory burst activities revealed that social stress appeared to affect the cellular innate immune response of the subordinate specimens. Finally, to examine the effect of cortisol on phagocytosis, peritoneal cavity cells were treated in vitro, and an inhibitory effect was observed.

Diversi tipi di stress ambientali possono indurre l’espressione delle Hsps vengono rapidamente sintetizzate all'interno delle cellule dopo esposizione ad uno stressor. Le HSPs sono raggruppate in diverse famiglie in base alle dimensioni delle proteine. Nella maggior parte degli organismi ci sono diversi geni che codificano i membri di questa famiglia HSP. In studi precedenti è stata valutata l'interazione tra Bacillus thuringiensis e gli emociti circolanti di Rhynchophorus ferrugineus il punteruolo rosso delle palme. Questo coleottero è grande, da due a cinque centimetri di lunghezza, ed è di colore rosso ruggine, le sue larve scavano buche fino a un metro lungo il tronco delle palme, e può uccidere la pianta ospite. Bacillus thuringiensis (Bt), un batterio presente in natura, è un insetticida utile per il controllo dei parassiti in determinate situazioni. Diversi ceppi possono infettare e uccidere gli insetti. Attualmente, il Bt è l'unico "insetticida microbico" in uso. Negli ultimi anni c'è stato molto interesse per l'attività Bt e sono stati sviluppati nuovi prodotti. Il Bt agisce producendo proteine ("cristalli tossici") che reagiscono con le cellule del rivestimento intestinale degli insetti sensibili. Queste proteine paralizzano il sistema digestivo dell’insetto che muore di fame. E’ stato già dimostrato che il Bt interagisce negativamente con gli emociti circolanti di R. ferrugineus che diminuiscono drasticamente, in particolare il numero dei plasmatociti significativamente si abbassa dopo 19 ore di trattamento. In questo studio, si esamina per la prima volta l'espressione di Hsp70 nelle larve di R. ferrugineus. Le analisi di Western Blot con anticorpi monoclonali anti-HSP 70 hanno mostrato che l’ epressione di Hsp70 negli emociti aumenta di circa sette volte dopo 3 ore di trattamento con BT ritornando al valore controllo dopo 6 ore di trattamento.

To study the pathogen-host relationship, we used the model of the entomopathogenic bacterium Bacillus thuringiensis (Bt) and Rhynchophorus ferrugineus, a quarantine pest that attacks the palm trees. In particular, we focused on the Bt stress-induced infections. We studied the effect of Bt on larval growth, on hemocytes and on the expression of the heat shock proteins (Hsp70). HSPs are rapidly synthesized in the cells after a stress exposition including pathogens. The Hsp70 was evaluated in the supernatant of the hemocyte lysate (HLS) obtained from larvae fed with Bt. This is the first time that the presence of Hsp70 has been recorded in R. ferrugineus. Bt has negative effects on larval growth, on total hemocytes counts and on the hemocyte type. In addition Hsp70 expression was modulated in the time (3h, 6h, 12h, 24h) in the response to Bt ingestion, highlighting that Bt is a stress factor for the R. ferrugineus. Further investigation is needed to understand the possible correlation between the reduction of hemocytes and Hsp70 modulation.

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 into a culture medium. The B5 activity was blocked by d -Galactose, α-Lactose, Lactulose, LacNAc, thiodigalactoside (TDG), l -Fucose, d -Mannose, d -Glucose, sphingomyelin (SM), and soluble phospholipase A2 (sPLA2) inhibitors (dibucain, quinacrine). Accordingly, HLS chemico-physical properties (alkaline medium, high thermostability, Ca 2+ -dependence, trypsin treatment, protease inhibitors) and SEM observations of the affected targets suggested that sPLA2 could be responsible for changes and large alterations of the target cell membrane. An apoptotic activity, as recorded by a caspase 3, 7 assay, was found by treating K562 cells with very diluted HLS. A lytic mechanism involving sPLA2 and lectins promptly released by URGs and morula cells respectively is suggested, whereas target cell membrane SM could be a modulator of the enzyme activity.

Stressful conditions include a prompt release of corticosteroid hormones which can mediate gene expression through glucocorticoid receptors (GR). Since two seabass (Dicentrarchus labrax) GRs have been cloned and sequenced from peritoneal cavity cells (DlGR1) and liver (DlGR2), a comparative amino acid sequence analysis that included Haplochromis burtoni HbGRs, was carried out and homologies disclosed. The DlGR1 and DlGR2 deduced aminoacid sequences showed 61% identity (I) and 70% similarity (S). Moreover, DlGR2 was similar to HbGR2b (69% I, 73% S), and the DlGR1 to HbGR1 (72% I, 78% S). In addition, we examined the expression of the DlGRs after exogeneous cortisol inoculation into the peritoneal cavity, mimicking stress effects. At various times after the administration (3 h, 24 h, 1 week), gene expressions was evaluated in head kidney by real-time PCR. In addition, immunoblotting and densitometry analyses were performed with anti- DlGR1 antibodies. Although sea bass head kidney expressed both DlGR1 and DlGR2 they were differentially modulated by intraperitoneal implant of exogeneous cortisol.

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 differentiation of inflammatory haemocytes. Specific antibodies against a CiTNFalpha peptide have identified a 43-kDa cell-bound form of the protein. Observations of pharynx histological sections (at 4 and 8 h post-LPS inoculation) from naive and medium-inoculated ascidians have confirmed the CiTNFalpha-positive tissue response. Larval histological sections and whole-mount preparations have revealed that CiTNFalpha is expressed by trunk mesenchyme, preoral lobe and tunic cells, indicating CiTNFalpha-expressing cell immigration events and an ontogenetic role.

Numerosi studi hanno accertato che nel fluido celomatico degli echinodermi sono presenti cellule con attività diretta verso agenti estranei. In particolare i celomociti svolgono un repertorio di funzioni immunitarie tra cui la fagocitosi, la citotossicità, l’attività antibatterica, la formazione di capsule e il rigetto dei trapianti. Nel presente lavoro abbiamo studiato l’attività citotossica dei celomociti di Holothuria tubulosa (Oloturoideo) non separati ed abbiamo utilizzato cellule di mammifero come target. I celomociti prelevati dal fluido celomatico lisano gli eritrociti di coniglio o di montone e le cellule tumorali della linea eritromieloide umana K562. Inoltre anche il supernatante del lisato dei celomociti (CLS) ha mostrato attività litica contro gli stessi target cellulari. L’analisi del CLS attraverso un “overlay” di eritrociti di montone su un gel di polyacrilamide? in assenza di SDS, ha evidenziato la presenza di almeno due bande con attività litica e differente mobilità elettroforetica. Le bande litiche eluite ed analizzate in SDS-PAGE mostrano una taglia molecolare di 79 e 44 kDa. Questi dati preludono ad ulteriori ricerche per l’identificazione dei tipi cellulari responsabili dell’attività, per l’analisi di un più ampio spettro di target e per la caratterizzazione delle due componenti proteiche apparentemente dotate di attività litica.

Heat shock proteins (Hsps) are rapidly synthesized within stressed cells after exposure to an environmental stressor. A variety of environmental stresses, including heat, cold, trace-metal exposure, xenobiotics have been reported to modulate Hsps expression in various organisms. Hsps are grouped into several families based on their protein size. Most organisms have several genes encoding members of this Hsp family. In particularly Hsp70 can be induced quickly under stressful conditions, but return to a normal expression level under non-stressful conditions. Few studies have been done to detect the Hsp70 expression in phytophagous insects towards pathogens. Since a preliminary research disclosed that Bacillus thuringiensis (Bt) negatively interacts with R. ferrugineus circulating hemocytes so that their number was dramatically decreased. In the present research we examine the expression of Hsp70 in hemocytes from R. ferrugineus larvae feed with a commercial product based on Bt. Western blot analyses using monoclonal anti-HSP 70 antibody showed that the expression of Hsp 70 was modulated reaching the highest value, seven times highest to the control, after 3h from the treatment. The Hsp70 values had the same value of the control at 6 hours. Monitoring the Hsp70 for 48 hours we notice a further decrement. This result highlights a stress condition, caused by Bt, as showed also by the reduction of the larval weight. So Hsp70 may be a suitable tool to detect rapidly stress condition induced by potential entomopathogens.

The development of an assay to determine insecticidal properties for either biological and conventional plant protection products plays an important role on the early screening of potential pathogens or derived toxins candidates. The standard methods for the evaluation it has been by bioassay, especially determination of LD50 or LC 50 requiring the use of relatively large numbers of insects and toxin tests. There are several problems connected with these bioassays: availability of insects and in the right life stage, mass producing the candidate species, preparation, reproducibly and costs relative to intensive manpower. These aspects are really important especially when bio-insectides should be tested against new target eg. invasive species in a new country. We present the results of the use of alternative systems in a screening study of Bacillus thuringensis (Bt) strain as potential pathogen for the Red Palm weevil, Rhynchophorous ferrugineus. We propose the use of different techniques such as growth inhibition, effect of pathogen on total number of hemocytes, ratio of selected hemocytes, espression of HSP 70 protein. In particular this protein is involved in the biochemical pathways response in stressed animals. Tests were carried out against the potential less sensitive stage to have a new predictive parameter for screening the reaction of the host to the pathogen. The preliminary results obtained with the proposed tests allows to enlarge the information of the potential pathogenicity of the entomopathogen bacteria and highlight the potential of this set of tests as screening methods to select new potential entomopathogens or bioinsecticides.

Negli echinodermi la difesa dell’organismo dalle invasioni microbiche avviene tramite meccanismi cellulari e umorali dell’immunità innata. Una componente fondamentale dei fattori umorali è costituita dai peptidi antimicrobici (AMPs) molto conservati con un ampio spettro di attività antimicrobica contro batteri, virus e funghi. Solitamente gli AMPs sono costituiti da meno di 100 amminoacidi, sono cationici e formano strutture anfipatiche. In questo lavoro dimostriamo che i celomociti dell’Echinoideo Paracentrotus lividus e dell’Oloturoideo Holothuria tubulosa contengono sostanze con attività antimicrobica verso diversi ceppi batterici patogeni umani. In entrambe le specie, nel precipitato acido del supernatante del lisato dei celomociti è presente una frazione peptidica a basso peso molecolare (Low Molecular Peptides. LMP) con massa compresa tra i 3 e i 5 kDa che mostra attività antibatterica, valutata come concentrazione minima inibente (MIC). In particolare, in P. lividus la frazione peptidica con massa molecolare inferiore a 5 kDa è stata valutata contro un gruppo di batteri di riferimento, Gram-positivi e Gram-negativi e contro funghi patogeni dell’uomo: Staphylococcus. aureus ATCC 29213, ATCC 25923, ATCC 43866, Staphylococcus. epidermis DSM 3269, 1457, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231 e Candida tropicalis ATCC 13803. Il 5-CC risulta attivo contro tutti i ceppi microbici testati in vitro, con valori di MIC compresi fra 15.8 mg ml-1e 253.7 mg ml-1. Inoltre, nella specie Paracentrotus lividus, sono stati caratterizzati gli AMPs presenti nella frazione peptidica, attraverso l’utilizzo di un RP-HPLC/nESI-MSMS. I risultati hanno individuato tre principali peptidi (Paracentrina I, II, III) con una taglia molecolare rispettivamente di 1251.7, 2088.1 e 2292.2 d. L’analisi MSMS ha evidenziato che i peptidi comprendono rispettivamente le sequenze peptidiche 9-19, 12-31 e 24-41 della β-timosina di P. lividus (AN AJ439718). La β-timosina è uno dei peptidi antibatterici presenti negli invertebrati. In H. tubulosa dal precipitato acido del supernatante del lisato dei celomociti è stata isolata una frazione di 3 kDa che ha mostrato attività verso diversi ceppi batterici: Enterococcus fecalis, Pseudomonas aeruginosa, Escherichia coli e Staphylococcus aureus con valori di MIC compresi fra 16,5 µg ml-1e 22.5 µg ml-1. Inoltre in entrambe le specie, le LMPs sono capaci di inibire l’attività dei biofilm da stafilococco, come dimostrato da osservazioni al microscopio fluorescenza e confocale. Ulteriori studi sono necessari per caratterizzare meglio queste componenti antimicrobiche, sia da un punto di vista biochimico che molecolare, sia per valutarne la localizzazione cellulare e tissutale e la modulazione dell’espressione genica in presenza di microorganismi. In conclusione i celomociti degli echinodermi possono rappresentare un’importante fonte di agenti antimicrobici utili per lo sviluppo di nuove strategie per il trattamento dei biofilm.

In invertebrates, cellular and humoral components are evolved to maintain their homeostais and integrity. Both these components respond to different non-self such as microorganisms, vertebrate erythrocytes and foreign proteins. The aim of this research was to study the possible modulating effects of Bacillus thuringiensis (Bt) in a commercial and vegetative form on the immune response of the echinoderm, sea cucumber Holothuria tubulosa. Bt is one of the most used biopesticides worldwide, covering the 90% of the organic market. Sicily is a region vocated to biological control and agricultural area are often closed to the coastal area. Investigate the effect of biopesticides on other non target organisms is important in a more exhaustive environmental risk assessment. Moreover correlative studies indicate that the immune surveillance is affected by both Bt forms in vivo challenge. In particularly we have evaluated the phagocitosys vs Saccharomyces cerevisiae, hemagglutination using rabbit erythrocytes and coelomocyte cytotoxic activity against rabbit erythrocytes and K562 tumor target cells. These results can contribute to improve the knowledge about the use of Bt like biological insecticidal on non target organisms.

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 chemiluminescence response (CL) has been related to increased respiratory burst of phagocytes stimulated with zymosan, PCLs, preincubated in vitro with cortisol at various concentrations, were assayed for their CL response. Dose-dependent cortisol inhibitory eVects, and signiWcant competitive activity of a low concentration of mifepristone (RU486), a glucocorticoid-receptor blocker, supported that cortisol–GR interaction was involved in modulating CL response via a genomic pathway. Results also indicated that cortisol could be eVective through an additional not-genomic way, and showed that high doses of RU486 exerted an inhibitory eVect on PCL chemiluminescence activity.

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 Eiger A and B TNF-like sequences were distinctly separated from the chordate TNFs. Thus, the ascidian TNFalpha-like cytokine was upregulated by in vivo LPS challenge supporting its pro-inflammatory role. In the pharynx, increased expression levels were found following analysis by real-time polymerase chain reaction, whereas in situ hybridization assay showed positive hemocytes both in the tissue and in circulating hemocytes. Finally, Western blot with monoclonal antibodies disclosed human TNFalpha epitopes in a 15-kDa protein component of the hemolymph serum and in a 43-kDa protein contained in the hemocyte lysate supernatant prepared in the presence of detergents. Both soluble and hemocyte-bound CiTNF alpha-like protein therefore appeared to be modulated by the LPS challenge.

Glucocorticoid hormone receptors (GR), members of the nuclear hormone receptor superfamily, are ligand-dependent transcription factors expressed in various tissues by binding to specific DNA sequences. Since glucocorticoids have a role in maintaining the homeostatic status in fish, we previously cloned and sequenced a GR (DlGR1) of adult Dicentrarchus labrax; we also showed mRNA expression (in situ hybridization) and tissue immunohistochemical localization of DlGR1 in several organs. This work has now been extended to the examination of the expression, tissue distribution, and cytolocalization of DlGR1 in larval developmental stages by similar methods to those used for the adult organs. The riboprobe included the DlGR1 cDNA transcriptional activation domain (1.0–1,300 nucleotide sequence) showing no significant similarity with a known second GR cDNA sequence of sea bass. The antibody was specific for an opportunely selected peptide sequence of the DlGR1 transcriptional domain. In histological sections of brain, head kidney, gills, liver, anterior intestine, and spleen cells, the riboprobe was mainly located in the cell nucleus. The antibody identified DlGR1 in the head kidney, gills, liver, and anterior intestine, mainly located in the cytosol. These results are in agreement with the receptor location in adult tissues. The greater presence of both the transcript and protein of DlGR1 in the late developmental stages suggests an increasing expression of this receptor. The cytolocalization (nuclear-cytosolic) and presumptive roles of DlGR1-containing tissues are discussed.

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. Moreover phagocytic index evaluated by microscopical observations, indicated that peritoneal macrophages internalised more yeast cells than neutrophils (identified by the peroxidase reaction). Scanning electron microscopy observations were also carried out. Inhibition experiments by a myeloperoxidase inhibitor sodium azide, iodonium-diphenyl-chloride which inhibits NADPH-oxidase, and exogenous superoxide dismutase, which catalyses O−2 dismutation to H 2 O 2 , supported the correlation between CL and respiratory burst. Treatment with ouabain and DNP suggested that in this response, Ca ++ pump channels and calmodulin are involved in a metabolic energy-dependent pathway.

Cortisol is the main corticosteroid secreted by the interrenal cells of the head kidney and it exerts a role in mantaining the omeostatic status in fish. In teleosts its effects are mediated through intracellular receptors expressed in several tissues, that are ligand-dependent transcription factors by binding to specific tissue DNA sequences. In Dicentrarchus labrax we previously cloned and sequenced a glucocorticoid receptor, DlGR1, isolated from leukocytes of peritoneal cavity. In this work we showed mRNA expression and tissue immunohistochemical localization of brain DlGR1 by in situ hybridization assays, with a riboprobe with DlGR1 cDNA trascriptional activation domain, and by immunohistochemical methods, using a specific antibody for a selected sequence of the receptor tran- scriptional domain. The mRNA and the protein are expressed in pyramidal cells of the optic lobe and in the small globular neurons of the diencephalon.

Marine environments are continuously being threatened by a large number of xenobi- otics from anthropogenic sources. The effect of chemical pollution on living organisms are numerous and may impair reproductive success of adults species of marine invertebrate and vertebrate through effects on gamete quality. Echinoderms are characterized by external fertilizzation and gametes, free of any type of protection, may be in contact with toxic substances so the reproductive success depends largely on the environment conditions. The purpose of this work is to assess the effects on the in vitro fertiliza- tion of exposure of sea urchin Paracentrotus lividus gametes to xenobiotic substances as CuSO4, CdCl2 and TBTCl. The effect of contaminant were assessed by two experimental set in which gametes were treated with different concentration (0, 10-3, 10-5 10-7 10-9 M) of different substances as CdCl2, CuSO4 and TBTCl. The effects were evaluated as percentage of fertilization. The results showed that the gametes exposure to xenobiotic decreased the percentage of fertilization and that more sensitive to treatment were the sperm cells, propably because the toxic effect affected the motility of the sperm. In conclusion, the absence of fertilization (spermiotoxicity) may submit the toxic effects of these substances to the level of body and may candidate the sea urchis as biosensors for the evaluation of environmental quality.