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Various key economic sectors of the National and Sicilian economy, in addition to contributing to the economic growth also cause a considerable production of waste which today are a problem both for companies then for the environment. For this reason, there is considerable interest in this waste and in the possibility of their reuse to extract bioactive molecules with added value, all in accordance with the objectives of the Blue Economy. In this regard, it has already been demonstrated that from the production waste of various economic sectors it is possible to obtain bioactive molecules with anticancer, antimicrobial, cosmetic, antioxidant and nutraceutical properties (Mauro et al., 2022). If we focus on farm animals and the feed used, it is known that the latter can be a source of antioxidants which help the body to prevent the harmful effects of free radicals and metabolic products. In this context, polyphenolic compounds (e.g. flavonoids) showed important antioxidant activities in vitro, although it seems that these are poorly absorbed in the intestine and their tissue concentrations are too low to contribute to the antioxidant defense (Surai, 2013). In light of all this, within the SMILING Project a study concerned the possibility of reusing wine production waste (pomace and grape seeds, the cause of the production of considerable quantities of waste) rich in fatty acids, triglycerides and polyphenols (Di Stefano et al., 2021; 2022) in the feeding of broiler chickens by evaluating the possi- ble effects of these diets on the animal’s health status also in terms of antioxidant capacity. Three experimental sets were carried out and concerned the administration of three different types of diets: Grape marc (0%, 3% and 6%), Grape seeds (0%, 3% and 6%) and a mix of grape marc and grape seed (0 and 3%). At the end of the experimental times, biomarkers typical of the evaluation of the state of health of the organisms were used for the preliminary analyzes of the blood and meat samples. Significant effects were observed in all diets depending on the concentration administered. Probably the phenolic content of the flours used contributes to the improvement of the health of the farmed animals.

Marine species are a rich source of bioactive molecules and among them, it is known that marine algae produce different secondary metabolites for which different biological activities such as: immunomodulatory (Raposo et al., 2016), antioxidant (Fisch et al., 2003) and antimicrobial (Pinteus et al., 2015) were demostrated. The aim of this study was chemical charac- terize the extracts of three macroalgae species: Carpodesmia crinite (Duby, Orellana & Sansón, 2019), Carpodesmia brachy- carpa (J. Agardh, Orellana & Sansón 2019, WoRMS, 2023), Ericaria brachycarpa (J. Agardh, Molinari & Guiry, 2020), Asparagopsis taxiformis (Delile) Trevisan 1845) to evaluate also their biological activities. The characterization of the secondary metabolites was performed by HPLC-MS and showed higher meroterpenoids levels. Then, the extracts were tested on the Arbacia lixula sea urchin and against the bacterial strains Listeria monocytogenes and Staphylococcus aureus. In the first case results showed a modulation in total and differential cell count demonstrating their involvement in immunity responses. In the second case important antimicrobial activities were observed against both bacterial strains tested. The results obtained, although preliminary, are certainly encouraging to understand better the biological potentiality of these metabolites.

Biodiversity is my main research focus and that of my research group. We study a range of different aspects concerning biodiversity, such as the impact anthropic activities have on biodiversity and how to improve its preservation, its use as a valuable resource for the extraction of bioactive molecules, and the study of its distribution. The research group has over twenty years of experience in the study of zoology, and, more in particular, the immune and behavioural responses of aquatic invertebrates and fish. For many years, the group has observed the effects of various anthropogenic activities, such as the acoustic and chemical impacts on the physiological and behavioral responses of invertebrate organisms, such as Arbacia lixula and Mytilus galloprovincialis, and of fishes, such as Sparus aurata, evaluating not only adult stage but also embryonic stage [1,2]. From a sustainable development perspective, we have recently turned our attention to an evaluation of the nutritional potential of aquaculture species which are recent additions to farming in Sicily: Cherax quadricarinatus and Cherax destructor [3]. However, biodiversity, as well as needing our protection, is also a resource which we can seek to harness in an eco-sustainable way. In recent years, research in our group has focused on the extraction of bioactive molecules from invertebrates and vertebrate living organisms (including from waste from the processing industry), with evident antimicrobial, anticancer and food preservation potential [4]. Last but not least, the regenerative capacities of invertebrate organisms such as Holothuria tubulosa [5] were evaluated with the possibility of transferring this knowledge to the biomedical sector. In addition to carrying out the aforementioned research, we are currently working on a nationally funded PON research project to conduct a freshwater biodiversity census in Palermo, including environmental DNA evaluation.

Marine species represent a rich source of biologically active products that can be used in various fields. Among them, marine algae produce numerous secondary metabolites responsible for different biological activities such as: immunomodulatory [1], antioxidant [2], and antimicrobial [3]. The aim of this study was chemically characterizing the extracts of three macroalgae species: Carpodesmia crinite (Duby) Orellana & Sansón, 2019, Carpodesmia brachycarpa (J. Agardh) Orellana & Sansón 2019, Asparagopsis taxiformis (Delile) Trevisan 1845 and evaluate their biological activities. The characterization of the secondary metabolites was performed by HPLC-MS and the results obtained showed higher meroterpenoids levels. Moreover, the extracts tested against the Arbacia lixula sea urchin modulate the total and differential cellular count demonstrating their involvement in immunity responses. Furthermore, important antimicrobial activities were observed by testing these extracts against the bacterial strains Listeria monocytogenes and Staphylococcus aureus. For the first time our study shows the effects of macroalgae extracts on the immunomodulatory activity in Arbacia lixula sea urchin and important antimicrobial activity. The results obtained, although preliminary, are certainly encouraging and our purpose is also improved this information performing biochemical and molecular assays of extracts obtained to understand better the potential that these metabolites have towards the sea urchin Arbacia lixula that graze on these macroalga.

To date, drugs released into the aquatic environment are a real problem, and among antibiotics, sulfamethoxazole is the one most widely found in wastewater; thus, the evaluation of its toxicity on marine organisms is very important. This study, for the first time, investigates the in vitro effects of 4 concentrations of sulfamethoxazole (0.05 mg/L, 0.5 mg/L, 5 mg/L, 50 mg/L) on the fertilization and development of the sea urchin Arbacia lixula. The gametes were exposed to drugs in three different stages: simultaneously with, prior to, and post-fertilization. The results show a significant reduction in the percentage of fertilized oocytes at the highest drug concentrations. Moreover, an increase in anomalies and delays in embryo development following the treatment with the drug was demonstrated. Therefore, the data suggest that this antibiotic can alter the development of marine organisms, making it urgent to act to reduce their release and to determine the concentration range with the greatest impact.

Pentraxins (PTXs) are a superfamily of conserved proteins which are components of the humoral arm of innate immunity. They are considered to be functional ancestors of antibodies and are classified into short and long types. In this study, we show that a pentraxin-like component (Ptx-like) with a C-terminal PTX domain, highly homologous to the short PTX of H. sapiens CRP, and a long N-terminal domain typical of long PTXs, is involved in the inflammatory response of Ciona robusta under LPS exposure in vivo. Analyses of protein domains as well as 3D modelling and phylogenetic tree supported the close relationship of Ptx-like with mammalian CRP, suggesting that C. robusta Ptx-like shares a common ancestor in the chordate lineages. qRT-PCR analysis showed that Ptx-like was transcriptionally upregulated during the inflammatory process induced by LPS inoculation and that it is involved in the initial phase as well as the secondary phase of the inflammatory response in which matrix remodelling and the achievement of homeostasis occur. In situ hybridisation assays revealed that gene transcription was upregulated in the pharynx post-LPS challenge in vivo, and that Ptx-like was expressed by clusters of haemocytes, mainly granulocytes, inside the pharynx vessels. We also found transcript-expressing granulocytes flowing in the musculature and in the lacunae of the circulatory system. These data supported that Ptx-like is a potential molecule of the acute-phase response in C. robusta immune defence systems against bacterial infection.

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.

Matrix metalloproteinases (MMPs) are a family of endopeptidases collectively able to degrade the components of the extracellular matrix (ECM), with important roles in many biological processes, such as embryogenesis, normal tissue remodelling, angiogenesis and wound healing. New views on the function of MMPs reveal that they regulate inflammatory response and therefore might represent an early step in the evolution of the immune system. MMPs can affect the activity of cytokines involved in inflammation including TGF-β and TNF-α. MMPs are widely distributed in all kingdoms of life and have likely evolved from a single-domain protein which underwent successive rounds of duplications. In this study, we focused on the Ciona robusta (formerly known as Ciona intestinalis) MMP gelatinase homologue. Gene organization, phylogenetic analysis and 3D modeling supported the closest correlation of C. robusta gelatinase with the human MMP-9. Real-time PCR analysis and zymographic assay showed a prompt expression induced by LPS inoculation and an upregulation of enzymatic activity. Furthermore, we showed that before of the well-known increase of TGF-β and TNF-α levels, a MMP- 9like boost occurred, suggesting a possible involvement of MMP-9like in regulating inflammatory response in C. robusta.

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 gradient gel electrophoresis (DGGE). For this analysis, CiTNFα nucleotide sequence was separated into two fragments, TNF-1 and -2, respectively, of 630 and 540 bp. We defined 23 individual DGGE patterns (named 1 to 10 for TNF-1 and 1 to 13 for TNF-2). Five patterns for TNF-1 accounted for 20% of the individuals. All the patterns were verified by direct sequencing. Single base-pair mutations were observed mainly within COOH-terminus, leading to 30 nucleotide sequence variants and 30 different coding sequences segregating in two main different clusters. Although most of the base mutations were silent, four propeptide variants were detected and six amino acid replacements occurred within COOH-terminus. Statistical tests for neutrality indicated negative selection pressure on signal and mature peptide domains, but possible positive selection pressure on COOH-terminus domain. Lastly we displayed the in silico 3D structure analysis including the CiTNFα variable region.

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 studied in the field of comparative and developmental immunology. Using this biological system, we describe the identification of a novel APA mechanism by which an intronic polyadenylation signal is activated by LPS injection, leading to the formation of a shorter CAP mRNA capable of expressing the first CAP exon plus 19 amino acid residues whose sequence is contained within the first intron of the annotated gene. Furthermore, such an APA event causes the expression of a translational controlling cis-acting GAIT element which is not present in the previously isolated CAP isoform and identified in the 3'-UTR of other immune-related genes, suggesting an intriguing scenario in which both transcriptional and post-transcriptional control mechanisms are involved in the activation of the CAP gene during inflammatory response in C. intestinalis.

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 inoculation suggesting it is involved in C. intestinalis inflammatory response. The CiPxt was expressed in hemocytes (compartment/morula cells), vessel epithelium, and unilocular refractile granulocytes populating the inflamed tunic matrix and in the zones 7, 8 and 9 of the endostyle, a special pharynx organs homolog to the vertebrate thyroid gland.

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 C. intestinalis g-type lysozymes contain a GEWL domain with a GXXQ signature, typical of goose lysozymes. Quantitative Real-Time PCR analysis results showed that transcripts are expressed in various tissues from C. intestinalis. In order to determine the involvement of C. intestinalis g-type lysozymes in immunity, their expression was analyzed in the pharynx, showing that transcripts were significantly up-regulated in response to a challenge with lipopolysaccharide (LPS). These data support the view that CiLys g-type are molecules with potential for immune defense system against bacterial infection.

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 genes expression and galectin localization in several tissues, including the previous findings on the transcription upregulation, the constitutive expression of these genes by endostylar zones and by the gastric epithelium suggest a potential multifunctional role of these galectins. In this respect, it is of interest to define where the CiLgals are normally found as related to the tissue functions. Such an approach should be a starting point for further investigations.

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.

The Ciona intestinalis inflammatory response to several irritants appears to be composed of a complex reaction set. The cellular reactions in the tunic involve hemocyte infiltration, hemocyte and epidermis activities, vacuolization, and cell disruption, while cell products can contribute to form capsule components and/or cause a tunic wound. In this response, the involvement of the pharynx, as the main immune-competent organ, has been disclosed by a lipopolysaccharide challenge that upregulates innate immunity genes and transcription activation genes. The pharynx responds through hemocyte recruitment into the pharynx vessels, enhancement of galectin-like lectins in the serum hemolymph, an alternative complement pathway, and phenoloxidase system activity. Upregulation involves genes for C-type lectins (CiCD94, CiMBL), galectins, cytokines (CiTNFα, IL17-like), phenoloxidase, FACIT-type IX-like collagen, and a transcription activator gene (CiCAP). The encoded proteins are components of distinct molecular families that support the evolution of several mechanisms, which generate sequence diversity in genes with immune functions. Clusters of similar genes or exons strengthen the concept that inflammatory factors are part of large molecular families originated by a process of divergent evolution from a common ancestor and in some cases of gene co-option during evolution.

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 in the inflammatory process induced by LPS inoculation, suggesting that is involved in the first phase and significant in the secondary phase of the inflammatory response in which cell differentiation occurs. In situ hybridisation assays revealed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by cluster of hemocytes inside the pharynx vessels. These data supported the view that CiTGF-β is a potential molecule in immune defence systems against bacterial infection.

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 also upregulated by LPS in the endostyle (zones 7, 8 and 9) that is considered homolog to the vertebrate thyroid.

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 induced by LPS inoculation showing that they are involved in the first steps of inflammatory response. In situ hybridization assays disclosed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by hemocytes (granulocytes and univacuolar refractile granulocyte) inside the pharynx vessels. As in human, we can assume that CiIL-17-like stimulates the release of CiTNFalpha, which synergizes with CiIL-17 in its effects on cells and molecules of Ciona intestinalis immunity system. In addition, a comparative evaluation with others molecules upregulated by LPS challenge as CiTNF-alpha, phenoloxisases, peroxinectin, galectins and mannan binding lectin, have been evaluated in terms of temporal and quantitative gene expression.

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-strand structure. Gene expression analysis demonstrated that the Ci-NPC2 protein is positively upregulated after LPS inoculum with a peak of expression 1 h after challenge. Finally, in-situ hybridization demonstrated that the Ci-NPC2 protein is preferentially expressed in hemocytes inside the vessel lumen.

The MD2 (Myeloid Differentiation factor-2) protein belongs to the ML superfamily. This group of proteins contain a specific lipid binding domain (ML domain) that plays an important role in lipid recognition and metabolism. In vertebrates, MD-2 is involved in innate immune response as co-receptor in the LPS/TLR4 signaling pathway; MD2 recognizes and binds the bacterial lipid A and drives the TLR4 activation. Two TLR isoforms, CiTLR-1 and CiTLR-2, were identified in Ciona intestinalis with a TIR domain most similar to human TLR4 and TLR 6 respectively. Using a PCR-based subtractive hybridization strategy for isolation of differentially expressed genes between LPS-challenged and naïve C. intestinalis, we identified a full-length cDNA (855 bp) encoding for a 150 a.a. protein (CiMD-2-like). In silico analysis showed that the deduced protein contains a signal peptide (1-19 a.a.) and an E1/Der p2/Der f2/ML domain-MD2 related lipid recognition domain (21-148 a.a) with similarities to ML(MD-2 related Lipid-recognition) domain identified in MD-2 and NPC2 (Niemann-Pick disease type C2). Phylogenetic and structural analysis supported the close relationship with MD-2 and NPC2 suggesting that CiMD-2-like originated from a common ancestor gene. Furthermore, gene expression studies by Real-time PCR demonstrated that this cDNA is up-regulated after LPS injection in the body wall. In situ hybridization performed in controls and LPS-induced animals has shown that this gene is expressed in granular amebocytes, large granules hemocytes and URG (univacuolar refractile granulocyte) in pharynx, the main organ of the ascidian immune system

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. CiLgals-a and CiLgals-b are constitutively expressed in the endostyle zone 2 and 3, respectively, both genes are upregulated by LPS in the zone 2, and CiLgals-b in the zone 3 and 4. The antibody-reacting material contained in intracellular and extracellular large vesicles suggest an unexpected vesicle-dependent transporting mechanism of galectins not provided with signal peptide. Differential expression and gene upregulation in not-treated and LPS-treated specimens, support the role of endostyle galectins both in filter feeding and defense responses.

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 disclosed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by hemocytes (granulocytes and univacuolar refractile granulocyte) inside the pharynx vessels.

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 picta. The CiLgals-a N-terminal and C-terminal CRDs contain the signature sequence involved in carbohydrate binding, whereas the CiLgals-b C-CRD presents only three out of seven key aminoacids and it could not be suitable as sugar binding motif. Sequence similarity between clusters suggests an evolutionary model based on CRD domain gene duplication and sequence diversification. In particular CiLgals-b N-CRD and C-CRD were similar to each other and both grouped with the ascidian C. picta mono-CRD. Homology modeling process shows a CiLgals molecular structure superimposed to chicken and mouse galectins. The CiLgals-a and CiLgals-b genes were upregulated by LPS inoculation suggesting that they are inducible and expressed in the inflamed pharynx as revealed by real-time PCR analysis. Finally, in situ hybridization and immunohistochemical assays showed their localization in the inflamed tissues, while immunoblotting analysis indicated that CiLgals can form oligomers.

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.

The CAP superfamily is a group of proteins that have been linked to several biological functions such as reproduction, cancer, and immune defense. A differential screening between lipopolysaccharide (LPS)-challenged and naive Ciona intestinalis has been performed to identify LPS-induced genes. This strategy has allowed the isolation of a full-length 1471-bp cDNA encoding for a 413-amino-acid protein (CiCAP). In silico analysis has shown that this polypeptide displays a modular structure with similarities to vertebrate CAP-superfamily proteins and to a collagen-binding adhesin of Streptococcus mutans. Domain organization analysis and alignment of CiCAP to other vertebrate CAP proteins have revealed a novel structure suggesting that this protein originated from a common ancestor gene that gave rise to many subfamilies of mosaic proteins with novel functions. Quantitative mRNA expression performed by real-time polymerase chain reaction analysis has demonstrated that this gene is rapidly activated in the pharynx of C. intestinalis a few hours after LPS injection. Moreover, in situ hybridization has shown that CiCAP mRNA is highly expressed by hemocytes with large granules contained inside the pharynx vessels. Thus, CiCAP represents a protein with novel structural domains involved in ascidian immune responses.

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.

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 larvae (from 20 days post-hatching) and juveniles and in parenchymal tissue of juveniles. DlFBL mRNA and protein were detected in the intestinal epithelium and in hepatocytes. An amplification product from degenerate primers indicates that lectin isotypes with DlFBL epitopes are expressed in eggs and embryos. Whether the lectin fraction isolated from eggs and embryos includes DlFBL of maternal origin remains unclear.

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.

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.