ISJ 13: 44-55, 2016 ISSN 1824-307X

REPORT OF MEETING

XVIIth scientific meeting of the Italian Association of Developmental and Comparative Immunobiology (IADCI), 11 - 13 February 2016, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy

Organizers: P Pagliara, L Stabili

Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy

Session 1. Chairmen: G Scapigliati, Università highlights the potential weaknesses in host immune della Tuscia, Viterbo, Italy and A Vallesi, defenses. On the other hand, investigating the University of Camerino, Camerino (MC), Italy defence mechanisms which species other than Insights into fish immunity tetrapods have evolved to counter infectious agents may allow to identify novel molecules and strategies Lecture useful to manage an infection in the host’ s favor. Evolution of the immune response to pathogens So far, few attempts have been made at considering host and pathogen as interacting partners into a MR Coscia common evolutionary framework. A short overview Institute of Protein Biochemistry, CNR, Naples, Italy on how the host-pathogen interaction has been shaped by evolution will be given. To survive, organisms must continually adapt to continually evolving invading organisms. Hosts and Analysis of Antarctic Teleosts transcriptomes as pathogens are the key players of a continous a tool to explore adaptive immune responses conflict in which natural selection aids pathogens to increase virulence to escape host surveillance, and F Buonocore1, M Gerdol2, A Pallavicini2, C hosts to acquire adequate defence strategies. In Bernini1, S Mattiucci3, D Lucente4, R Cimmaruta4, both cases, these achievements are limited by G Scapigliati1 several factors such as the genetic fitness and the 1Department for Innovation in Biological, Agro-food number of genes required, much larger than that and Forest Systems, Tuscia University, Largo available in the genome. Moreover, since the dell'Università s/n, Viterbo, Italy pathogen usually has a very shorter life than the 2Department of Life Sciences, University of Trieste, host, it has to fix new mutations favoring virulence Via Giorgieri 5, 34127 Trieste, Italy much faster than the host can evolve effective 3Department of Public Health and Infectious defense mechanisms. Another constrain concerns Diseases, Section of Parasitology, "Sapienza" the host that must avoid adverse effects which may University of Rome, P.le Aldo Moro 5, Rome, Italy derive from the defence system itself. Once 4Department of Ecological and Biological Sciences, resistance and counter resistance are genetically Tuscia University, Largo dell'Università s/n, Viterbo, assessed, both the host and the pathogen evolve in Italy response to mutually exerted pressures. This is generally referred to as the “Red Queen Paradigm”, During the last decade the use of next- that highlights the significance of biotic versus generation sequencing technology has become abiotic factors that lead to constant evolutionary highly widespread to generate massive amounts of changes. Evolution acts at different levels: biotic sequence data mainly due to its reduced costs and factors mainly shape species diversity over short to the possibility of collecting simultaneously time periods, whereas changes in the physical information useful both for transcriptome environment such as climate changes drive characterization and quantification. In the present evolution at a large scale, during much longer time. work we analyzed two transcriptomes from the head Much of our current knowledge of infection biology kidney of the emerald rockcod (Trematomus is based on studies of the immune system in bernacchii) and from gills of the icefish humans and mice. In contrast, much less attention (Chionodraco hamatus). The sequences were has been paid to immune response in lower generated with an Illumina platform and, vertebrates. Since many features of immune successively, de novo assembling procedures were defence mechanisms have been acquired performed to obtain the final transcripts. A first aim throughout evolution, studying the evolution of was to identify new genes related to adaptive successful pathogen virulence mechanisms immune responses by using as a comparison the

44 known genome of the Antarctic fish Notothenia production; phagocytosis; expression of pro and coriiceps, which contains about 30.900 expected anti-inflammatory cytokines. Our results indicate transcripts. We were able to confirm that in the that superoxide anion production and phagocytosis icefish transcriptome about 20400 transcripts were decreased in both the blood and GALT leucocytes present (66,1 %), whereas in the emerald rockcod incubated in vitro with concentrations of chestnut we found about 19800 transcripts (64,2 %). skin polyphenols ranging from 10 to 100 µg/ml. Moreover, orthologous proteins showed about 80 % Higher concentrations (500 and 5000 µg/ml) were amino acid identity considering Chionodraco and instead stimulating both anion superoxide Trematomus transcriptomes. From these production and phagocytosis. Chestnut skin transcriptomes, a relative high number of polyphenols used in our experiments were also able sequences related to adaptive immune responses to modulate the gene expression of immune-related genes have been identified and confirmed by cytokines, such as TNF-α and IL-10. Specifically, it cloning from cDNA, e.g., MHC-I, MHC-II, beta2- was observed an upregulation of the pro- microglobulin, CD4, CD8alpha, IgT, IgD, etc. The inflammatory cytokine TNF-α and a downregulation identification of MHC-II molecules provided the of the anti-inflammatory cytokine IL-10 in blood and opportunity of evaluating the levels of genetic GALT leucocytes. Similar results were obtained with variation at a MHC-II β locus in the icefish Gallic acid and Ellagic acid, although the effects population from the Ross Sea. Preliminary data were less evident, suggesting that the effects of suggest a genetic variability comparable to that chestnut skin polyphenols are depending on the reported for other fish species at both inter- and mixture synergism between the various phenolic intra-individual levels. This finding allows exploring compounds. possible relationships occurring between the levels In the light of these preliminary results, we of genetic variation of the MHC in the icefish with suggest that the addition of polyphenols to standard respect to the parasitic load recorded in this fish diet may improve the immune response of farmed host. fish. Moreover, this study suggests the possible re- use of agri-food industry wastes as feed additives Preliminary data on the effects of chestnut skin for farmed animals. extracted polyphenols on Oncorhynchus mykiss blood and GALT Strategies for detection and vaccination of juveniles european sea bass (Dicentrarchus L Parrillo1, E Coccia1, MG Volpe2, S Costantini3, labrax) against betanodavirus E Varricchio1, M Paolucci1,2 1Dipartimento di Scienze e Tecnologie, Università N Nuñez-Ortiz1, F Pascoli2, A Toffan2, F degli Studi del Sannio, via Port’Arsa 11, 82100 Buonocore1, S Picchetti1, G Scapigliati1 Benevento, Italy 1Dipartimento per l’Innovazione nei Sistemi Biologici 2Istituto di Scienze dell’Alimentazione-CNR, Via Agroalimentari e Forestali, Università della Tuscia, Roma 64, 83100 Avellino, Italy Italy 3CROM: Centro Ricerche Oncologiche, 2Istituto Zooprofilattico Sperimentale delle Venezie, Mercogliano, Italy Centro di Referenza Nazionale (NRL) per le patologie dei pesci, molluschi e crostacei, Legnaro Agricultural by-products are a rich source of (PD), Italy bioactive molecules, including polyphenol compounds or “polyphenols”. The Immunostimulant Encephalopathy and retinopathy virus (VERv) and antioxidant properties of polyphenols may have or betanodavirus causes massive mortalities of the a potential role for animal welfare and for the most important farmed species in Mediterranean production of “ healthy “ feed. Polyphenol-enriched area, the European sea bass. In order to develop feed has been finding application in animal farming, strategies for the control of virus infection and virus thank to polyphenol capability to improve the detection, we have studied the possibilities of productive performance, immune response and vaccinating sea bass at young age (2-10 grams) health of livestock. For this reason, polyphenols through mucosal and intraperitoneal immunization may also represent a valid alternative to antibiotics using VERv inactivated by differents ways. After and medicines currently employed in animal inactivation of VERv by differents protocols: farming. The introduction of natural extracts in formalin, BPL and heat treatment we have animal feed represent a booming business after the performed two control experiments using the ban of auxinic antibiotics and it proves that invest differents inactivated VERv by immersion and resources in the search for plant extracts can deliver intraperitoneal administration. Serum antigen- significant benefits. The Interest in use natural specific IgM titers was determined by Indirect Elisa substances, known and used since ancient times in being specially significant after intraperitoneal the care of man, can certainly be considered immunization with formalin-inactivated VERv. VERv- innovative for the animal diet. The potential free juveniles immunised by immersion in formalin- immunostimulant activity of polyphenols extracted inactivated virus showed the presence and the from chestnut skin has been studied on uptake of VERv in the gills by Immunohistochemistry lymphocytes isolated from blood and lymphocytes (IHC). Quantitative PCR expression on gut and head extracted from GALT of the rainbow trout kidney after intraperitoneal administration with Oncorhynchus mykiss. The assays used to evaluate formalin-inactivated virus showed modulation in the the parameters of both innate immunity and expression of antiviral gene ISG-12 after 48 h in acquired immunity were: superoxide anion both organs and Mx gene in gut after 48 h too but

45 induced detectable modulation of Mx and ISG12 and Vibrio anguillarum. The results have showed genes was detected in the gills 24 h postimmersion. that IL-4/13B is high responsive to all stimulations, Finally, challenge experiment using live VERv were whereas, in contrast, IL-4/13A1 and IL-4/13A2 are performed after immunization with formalin- less up-regulated. Successively, we have produced inactivated VERv, and we observed a 80 % the three isoforms as recombinant molecules and increase in a relative protection value in we tested their action in vitro on leukocytes from intraperitoneal immunizated fish with respect to head kidney and spleen. This is the first in-depth unimmunized fish. In the other hand, bath analysis of the biological activity of IL-4/13 cytokines immunization resulted in no protection in in vivo in sea bass and it will highly contribute to a broader challenge. In addition, by employing a rabbit understanding of the evolution of type-2 immunity in antiserum against VERv (pAb 283) and a mouse this species. monoclonal against VERv capsid protein (mAb 4C3) we have developed an ELISA system to detect and Session 2. Chairmen: N Parrinello, University of quantitate the presence of VERv in solutions and Palermo, Palermo, Italy and P Luporini, biological fluids. University of Camerino, Camerino (MC), Italy Ascidian immunity Fish cytokines: IL-4/13 in sea bass (Dicentrarchus labrax), molecular New data on the expression of molecular characterization and expression analysis after in markers involved in stemness and vitro and in vivo stimulation differentiation in the colonial ascidian Botryllus schlosseri V Stocchi, E Randelli, M Gerdol1, A Pallavicini1, CJ Secombes2, T Wang2, G Scapigliati, F F Ballin, N Franchi, L Ballarin Buonocore Department of Biology, University of Padua, Padua, Dipartimento DIBAF, Università della Tuscia, Italy Viterbo, Italy 1Dipartimento di Scienze della Vita, Università di Cell types are often identified by determining Trieste. Trieste, Italy which genes they express specifically. The use of 2Scottish Fish Immunology Research Centre, specific antibodies or complementary RNA probes School of Biological Sciences, University of allows the identification of the Aberdeen, Zoology Building, Tillydrone Avenue, translational/transcriptional products: These Aberdeen AB24 2TZ, UK molecules, also called “molecular markers”, show an unique expression pattern, frequently used to identify Interleukin-4 (IL-4) and interleukin-13 (IL-13) and isolate stem cell populations. In B. schlosseri, a are key cytokines in Th2 mediated immune compound ascidian, there are three important responses. These cytokines have been extensively processes that suggest the presence of stem cells studied in mammals where they show different roles during the life cycle: i) embryogenesis, in which an with some overlapping bioactivities exploited embryo develops from a zygote, palleal budding through shared receptors. Usually IL-4 and IL-13 where new buds emerge as thickenings of the are activated at the same time when the immune peribranchial epithelium and vascular budding, i.e., system recognizes the presence of an external the development of new buds within the vasculature pathogen. In Teleost fish the situation seems quite by circulating multipotent or pluripotent cells. During different from mammals, in fact only recently, in the cyclical generation changes, which characterize 2007, a gene with some relation to IL-4 and IL-13 the colonial blastogenetic cycle, an increase in the was found in the pufferfish genome and, number of hemoblasts, i.e., undifferentiated successively, a second IL-4/13 like gene was circulating cells, occurs which will replace, once identified in zebrafish at a different locus. These two differentiated, the hemocytes undergoing apoptotic genes were called IL-4/13A and IL-4/13B depending cell death. Ascidian hematopoiesis occurs in close on their position on fish locus and they have been proximity to the pharyngeal vessels, in the so-called successively identified in other species, like rainbow “hematopoietic nodules” and in the endostyle, the trout, Atlantic salmon and medaka. In our work we cells of which proliferate and migrate to developing have found in a sea bass gills transcriptome three buds. Despite the morphologic suggestions that IL4/13 transcripts that have been mapped on the hemoblasts are the precursors of all the circulating available sea bass genome and therefore identified cell types, immunocytes included, there is a general as IL4/13A1, IL4/13A2 and IL4/13B. The identified lack of biochemical and molecular data supporting sequences have been confirmed by cloning on sea this assumption. Here we report the identification of bass gills cDNA and their expression has been hematopoietic molecular markers in B. schlosseri, studied by real-time PCR. Basal expression analysis very similar to those of vertebrates, their localization revealed a different expression of the IL-4/13 genes and expression profile during the blastogenetic cycle. in the various tested organs and tissues: in particular the IL4/13B expression is very high in Characterization and genetic variability of spleen, while the expression of IL-4/13A1 and IL- Tumor Necrosis Factor alpha (CiTNFα) in Ciona 4/13A2 is high in head kidney. Moreover, we intestinalis investigated the expression of the IL4/13 isoforms in sea bass head kidney and spleen leukocytes after in A Vizzini, MG Parisi, L Cardinale, M Cammarata vitro stimulation with the T cell mitogen agents PHA Dipartimento di Scienze e Tecnologie Biologiche, and PMA and after in vivo infection with nodavirus Chimiche e Farmaceutiche, Palermo, Italy

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The tumor necrosis factor superfamily (TNFSF) Nonetheless, no complement genes are present in is involved in a lot of cellular signaling pathways like the genome of the sponge Amphimedon inflammation, apoptosis, lymphocyte homeostasis queenslandica or the choanoflagellate Monosiga and tissue development. TNFSF ligands are bound brevicollis and, although their presence in to membrane. About half of different ligands encode invertebrate protostomes is generally accepted, proteolytic cleavage sites that can generate soluble complement genes are missing also in Drosophila forms holding biological activity. The study of and Caenorhabditis, probably due to a secondary evolution of genes and genomes allows to loss. As regards deuterostome complement system, understand the role of different evolutionary forces, it is well studied in mammals, where more than 30 including natural selection. The structure and different proteins have been described, involved in composition of a gene are inherited from the the activation and regulation of this fundamental organism’s ancestors, so various driving forces humoral system able to modulate immunocytes could cause the change of structural and functional behaviour, belonging to both innate and adaptive aspects of a gene, for a best adaptation to immune response. However, the mere report of the environment. In Ciona intestinalis, a Tumor Necrosis presence of C3, factor B and lectin pathway in a Factor-alpha (TNFα)-like gene challenged with species cannot help in elucidating the evolution of bacterial lipopolysaccharide (LPS) was cloned and the complement system. Since adaptive immunity sequenced. It encodes a propeptide of 312 amino evolved in the presence of a functioning acids (35.4 kDa), shows a transmembrane domain complement system, the presence of considerable from positions 7 to 29, a TACE cleavage site and a and important interaction between complement and mature peptide domain of 185 amino acids (20.9 adaptive responses is not surprising. In particular, kDa). The diversity of mRNAs from CiTNFα has referring to the invertebrate-vertebrate transition, the been investigated in 30 Ascidians, collected from description of the complement-mediated immune Termini Imerese (Italy), by denaturing gradient gel modulation requires the identification and electrophoresis (DGGE). DGGE migration revealed characterization of additional factors, such as different molecular patterns. All patterns observed complement control proteins (e.g., factor H, C4bp, were sequenced and variants were identified in 30 CR1, CD46, CD 55) and receptors in invertebrates CiTNF-α. Several comparisons have been made: (i) chordates. for each DGGE pattern, all the sequences were Here we report on the identification and aligned and clustered according to nucleotide analysis of transcripts for CR1 (C3b receptor), C3aR sequences, (ii) the different cds were translated into and two factors H in the colonial ascidian Botryllus pro-peptides and (iii) the resulting aminoacid schlosseri that, for the first time are described in sequences were compared. The evolutionary tunicates, the sister group of vertebrates. The relationships were inferred using the Neighbor- localization of CR1 and C3aR on phagocytes and Joining algorithm (MEGA-6). Site-by-site frequency morula cells, respectively, open the possibility to spectrum-based statistical assays were used to test use such molecules as molecular markers for the hypothesis of polymorphisms within loci being immunocytes. In addition, the presence of a neutral. All three used tests were in agreement with complement regulator, such as factor H, in tunicates the hypothesis of negative selection pressure linked suggests a higher level of complexity than that to transmembrane domain, propeptide and mature expected in an invertebrate. peptide. Fu and Li’s D and F tests suggested possible positive selection pressure linked to Session 3. Chairmen: L Ballarin, University of COOH-terminus region only. All statistical tests Padua, Padua, Italy and P Pagliara, Università indicated possible negative selection pressure when del Salento, Lecce, Italy applied to full cds sequences. TNF mRNAs from invertebrate and vertebrate animals were used to A spotlight on Echinoderms construct a phylogenetic tree to study the evolutionary dynamic of TNF family gene. These Lecture results show that the TNF family gene has The Sp185/333 system in the Sea Urchin encountered remarkable changes in invertebrates, but conserved in vertebrates during history of LC Smith evolution. George Washington University, Department of Biological Sciences, Washington, USA Growing complexity of the invertebrate complement system: evidences from colonial The arms race between a host immune system tunicates and its pathogens drives diversity on both sides of the conflict. Pathogens change their approaches for N Franchi, L Ballarin infection and virulence, and the host changes the Department of Biology, University of Padua, Padua, diversity of the immune detection and effector Italy proteins. The purple sea urchin, Strongylocentrotus purpuratus, survives in the marine habitat with an The evolutionary history of the complement innate immune system of the is both complex and system is not yet fully elucidated. Evolutionary sophisticated. There are several large gene families studies suggest that the origin of the complement in the sea urchin genome that encode pathogen system can be traced back to the common ancestor detection and immune response proteins. One of of the Eumetazoa as the genes for the C3, factor B these is the Sp185/333 gene family, which is and MASP have been identified in sea anemones. composed of ~50 small genes that are tightly linked

47 in clusters. The genes share blocks of sequence capacity) based on a hydrogen transfer mechanism called elements that are present in mosaic patterns (HAT). Both the antioxidant assays showed a higher and have a variety of repeats within the second antioxidant activity in the coelomocyte lysate exon, and each gene is surrounded by one or two compared to coelomic fluid for all the Echinoderm types of microsatellites. These characteristics species studied. Moreover A. lixula cell lysate had suggest that the genomic region may be unstable, the highest antioxidand activity both with TEAC and which may drive sequence diversification of the ORAC assay. These antioxidant values are genes, a benefit for keeping up in the arms race comparable with those reported in the literature for with pathogens. Gene sequence diversity and various high antioxidant fruit and spice extracts. expression patterns suggest that the encoded Among the investigated species, the coelomocyte proteins may have effector functions. To test this, lysate of A. lixula showed a bacteriostatic activity we have evaluated one Sp185/333 recombinant against two emerging pathogenic bacteria protein (rSp0032), which binds specifically to Vibrio Pseudomonas aerugionsa and Staphylococcus and yeast, but not to Bacillus. It also binds LPS, β- aureus and against the yeast Candida famata. 1,3-glucan, and flagellin with specificity and high These results are noteworthy considering the affinity, but does not bind peptidoglycan. rSp0032 resistance against antibiotics developed by bacteria also binds phosphatidic acid (PA) and can deform and the need to control human infections. The liposomes composed of 10 % PA. rSp0032 is antioxidant activity was also of interest since it is the intrinsically disordered, however, when bound to first record for the investigated species and LPS or PA, it transforms to α helical suggesting represents a potential for applicative purposes. “Shapeshifter” activities for binding lipids, sugars and proteins. Given that single sea urchins are Bacterial challenge induces variation of the capable of expressing up to 260 Sp185/333 protein Allograft Inflammatory Factor I (AIF-1) gene variants, and if each one has a range of overlapping expression in Paracentrotus lividus binding activities that target simultaneously multiple F Vacca1, A Barca1, J Vizioli2, F Drago2, T Verri1, PAMPs, this may facilitate highly effective and P Pagliara1 flexible host protection against a broad array of 1Dipartimento di Scienze e Tecnologie Biologiche e potential pathogens encountered in the marine Ambientali, Università del Salento, Lecce, Italy environment. 2Laboratoire PRISM-UMR Inserm U1192. Université de Lille, Villeneuve d’Ascq France Antimicrobial and antioxidant activity in some Echinoderm species The phylum Echinodermata is a very L Stabili1,2, MI Acquaviva2, RA Cavallo2, C heterogeneous group of marine invertebrate Gerardi3, M Narracci2, P Pagliara1 species. After Chordata, it represents the second 1Dipartimento di Scienze e Tecnologie Biologiche e largest group of deuterostomes. The interest for Ambientali, Università del Salento, Lecce, Italy echinoderm immune defence system is steadily 2CNR-IAMC, U.O.S. Taranto, Italy increasing in the last years, due to their 3CNR-ISPA, UOS Lecce, via Prov.le Lecce phylogenetic, ecological, biotechnological and Monteroni, Lecce, Italy economical relevance. Noteworthy, their basal position within the deuterostome lineage makes the Echinoderms represent one interesting marine analysis of their defense mechanisms highly renewable resource and produce bioactive relevant to understand the evolution of the compounds related to their innate immune system. deuterostome immune system as it now appears in These invertebrates indeed are able to differentiate Vertebrata. Suggestions derive from the systematic self from non-self through the production of soluble analysis of the immune genes and of their products, molecules and coelomocytes response playing an such as cytokines, i.e., a specific group of effectors important role in the resistance to disease. molecules that play a variety of roles in the immune Therefore, they appear as a promising alternative response. The Allograft Inflammatory Factor-1 (AIF- valuable source of new compounds for drug 1), a calcium-binding cytokine, has been identified development. In particular, the application of new as i) a key regulator of the immune response; ii) a marine antioxidants in foods, food supplements, central player of the self-intensifying cycle of nutraceuticals and medicine is recently considered inflammation; and iii) an important pathogenic factor from the perspective of benefits to human health. In in multiple inflammatory diseases in Vertebrata. this study, the antimicrobial and antioxidant activity Recent literature evidences that proteins of the AIF- of several echinoderm species was investigated. 1 superfamily are present in several We focused our attention on the two sea urchins phylogenetically distant species, all showing high Sphaerechinus granularis and Arbacia lixula and on similarity at the primary protein sequence level. the sea star Echinaster sepositus. Coelomic fluid These data suggest a significant conservation of the and coelomocyte lysate of each species were functional properties of AIF-1 in the immune utilized for antimicrobial activity assay using the response. In the present work, we report on the Kirby Bauer method (1966). The antioxidant activity immune response of the sea urchin Paracentrotus of the samples was measured by two in vitro lividus after challenge with Micrococcus assays: the TEAC (Trolox Equivalent Antioxidant lysodeikticus, and we estimate the variation of Capacity) assay based on a single electron transfer cellular and humoral responses after bacterial (SET) reaction, using ABTS [2-2’-azino-bis (3- injection. In particular, AIF-1-like protein constitutive ethylbenz-thazoline-6-sulfonic acid)] as chromogen expression has been evidenced in P. lividus and the ORAC (oxygen radical absorbance coelomocytes by immunocytochemistry performed

48 using a human anti-AIF-1 antibody. Subsequent Invertebrate models suitable for the study of mining of expressed nucleotide sequences (ESTs) mammalian diseases databases allowed us to identify an AIF-1-like mRNA fragment sequence and to study its Lecture expression. Notably, mRNA levels were found to be Bivalves as a translational model in up-regulated in coelomocytes at 24 h post-bacterial inflammation and cancer research injection. Overall, our data suggest novel hints on the AIF-1 involvement and responsiveness to G De Vico and F Carella immune activation by bacteria in cells and tissues of Dipartimento di Biologia, Complesso Universitario di P. lividus. Monte S. Angelo, Edificio VII, Università degli studi di Napoli Federico II, Naples, Italy X-ray photoelectron spectroscopy as a non- conventional analytical technique for bio- Historically, animal models have been used in organic materials characterization: the sea the biomedical research to characterize disease urchin coelomocytes and the purple progressions, evaluate the action of drugs and also photosynthetic bacteria examination discover new biomarkers. Invertebrate models of human disease first appeared in the scientific MR Guascito1, D Chirizzi2, P Pagliara1, L Giotta1, literature in the late 19th century. Currently, model S Rella1, D Mastrogiacomo1, F Milano3 species range from terrestrial invertebrates such as 1Dipartimento di Scienze e Tecnologie Biologiche e nematodes and insects, to freshwater and marine Ambientali, Università del Salento, Lecce, Italy life including planarians, crustaceans and molluscs. 2Dipartimento di Beni Culturali, Università del Among new invertebrate translational models in Salento, Lecce, Italy biomedical research, mollusc are emerging as a 3Istituto per i Processi Chimico Fisici, UOS Bari, promising one in many areas, such as those Bari, Italy concerning immune-neuro-endocrine system, neurodegenerative disorders, inflammatory lesions X-ray photoelectron spectroscopy is a surface and cancer. Along with an overview of the main technique (depth profiling analysis 5 - 10 nm) that features concerning both inflammation and cancer allows to analyze, in terms of chemical speciation, pathogenesis in bivalve molluscs, we will also stress all elements, with the exception of H and He, the rationale concerning the potential use of present in different typologies of solid samples bivalves as a translational model for early validation (inorganic and organic), as long as their atomic of new therapeutic approaches in both human percent concentrations (At.%) are not below 0.01 - inflammation and cancer. At the same time, we 0.l %, depending on the specific element. This highlight the need for standardization of scientific technique potentially can also allow to obtain terminology in this new field of investigation. chemical imaging of surfaces with lateral resolution of 3 m. However, at the state of the art, the Amyloidogenesis in animal models employment of this analytical technique as a non- conventional tool for the investigation of bio-organic M de Eguileor, A Grimaldi, G Tettamanti materials (i.e., microorganisms and their related Department of Biotechnology and Life Sciences, systems such as biofilms, extracellular polymeric University of Insubria, Varese, Italy substances (EPS) and bio-adhesions), has been reported only in a limited number of papers. In this The biomedical research depends on the use of study, we report some preliminary results on the animal models that can be utilised to understand, at XPS characterization of sea urchins coelomocytes different levels, the pathogenesis of human and photosynthetic bacteria. The sea urchin Arbacia diseases. The ultimate goal of this approach is to lixula coelomocyte population is characterized by develop and test new therapies to eradicate the the presence of red cells, which number may examined diseases. Many researchers interested in increase in response to different stress. As red embriologically and genetically tractable disease spherula cells accumulate around injuries and sites models have opted for zebrafish. This animal can of infection, this analysis may help to understand provide a variety of human diseases due to what is the role of cell surface interacting with sophisticated mutagenesis and supply economic bacteria in addition to the action of echinochrome A, screening strategies on a large scale. Furthermore, present inside the cells. In particular, here we despite the advantages and the pre-eminence of the compare the red and withe coelomocytes surface. mouse in modeling human diseases there is a lot of Furthermore, the characterization of purple aspects limiting the use of this animal in large-scale photosynthetic bacteria (Rhodobacter sphaeroides) genetic and therapeutic screening. Even if able to interact with detrimental heavy metal ions, invertebrates lack structures and organs that can be such as nickel and chromium has been performed, involved in human pathologies it is important to bear successfully highlighting both the immobilization of in mind that there is a surprising degree of metals and surface modifications induced by the functional conservation in basic cellular biological environmental stress. processes between mammals and invertebrates (such as worms and insects). Thus when there is a slow down or a total block in one of complex and Session 4. Chairmen: E Ottaviani, University of basic processes, pathological events take place. Modena and Reggio Emilia, Modena, Italy and L Amyloidogenesis represents a primitive, simple Stabili Università del Salento, Lecce, Italy response, widespread in invertebrates and

49 vertebrates where innate immune signalling considered two different molecules involved in pathways is linked to stress responses. The critical peripheral tissues as well as neural immune role played by amyloid synthesis and deposition in response: the Macrophage Migration Inhibitory several pathologies, could explain the structural Factor (MIF), a chemotactic cytokine which mediate resistance of these scaffolds and could provide the LPS-induced responses, and the Glia Maturation basis for developing new diagnostic and therapeutic Factor Gamma (GMFG), a member of ADF-gelsolin approaches in all diseases in which the innate superfamily, which seems to be involved in actin branch of the immune system has a pivotal role. cytoskeleton remodelling and TLR4 endocytic pathway in response to LPS. We identified in H. Serum Amyloid A in marine bivalves: an acute medicinalis two genes coding for products showing phase protein of innate immunity high similarity with MIF and GMFG of Vertebrates, respectively. Immunolocalization experiments show S Domeneghetti1, U Rosani1, M Gerdol 2, M a weak expression of both these proteins in the Franzoi1, A Pallavicini2 , P Venier1 leech CNS whereas a stronger signal is detected in 1Department of Biology, University of Padua, Padua, peripheral tissues macrophages. Further studies are Italy needed to assess the expression levels of these 2Department of Life Sciences, University of Trieste, molecules in leech tissues. However, this work Trieste, Italy shows that these molecules are good selective markers of activated macrophages in H. medicinalis, Serum amyloid a (SAA) is an evolutionarily confirming the close correlation between the leech conserved acute-phase protein, involved in many and vertebrates. Moreover, these results suggest vertebrate biological processes, such as lipid the possible presence of more well-conserved metabolism and immunity. The rapid increase of molecules across evolution and represent an SAA can activate the immune system and promote interesting starting point to analyze the complex inflammatory responses after injuries, infections or crosstalk occurring during the innate immune stress. Although SAA homologs are widespread in response as well as the neuroimmunity processes. vertebrates, to date they have only been identified in limited number of invertebrate species. We traced Francisella-like endosymbionts, potentially the presence of SAA genes along metazoan harmful to human health, are transported by the evolution by screening available genomic and universally distributed species of the ciliate transcriptomic data, finally retrieving 51 SAA-like Euplotes proteins in several protostome taxa. In detail, we identified SAA homologs in 21 marine bivalves and A Vallesi1, D Petrelli1, G Di Giuseppe2, A we investigated the gene structure and expression Sjödin3,4, J Thelaus3, E Nilsson3, C Öhrman3, G patterns of mussel and oyster SAAs. Although Gutierrez Pozo5, E Villalobo5 phylogenetic and structural analyses support a 1School of Biosciences and Veterinary Medicine, certain degree of conservation between vertebrate University of Camerino, Camerino (MC), Italy and invertebrate SAA sequences, vertebrate SAAs 2Department of Biology, University of Pisa, Pisa, are mainly expressed in liver, whereas invertebrate Italy SAAs appear to be expressed in various tissues. 3Division of CBRN Defence and Security, Swedish Using both qPCR and RNA-seq approaches, we Defence Research Agency, FOI, Umea, Sweden observed that the two mussel SAA genes are mainly 4Department of Chemistry, Computational Life expressed in gills (MgSAAa), mantle and posterior Science Cluster (CLiC), Umea University, Umea, adductor muscle (MgSAAb), whereas C. gigas SAA Sweden is expressed in significant amounts in mantle and 5Departamento de Microbiología, Universidad de gonads. We also confirmed the inducible nature of Sevilla, Av Reina Mercedes 6 41012 Seville, Spain bivalve SAA transcripts, observing the over- expression of mussel SAAs after challenge with Genome analyses of wild-type strains of two pathogenic bacteria, although timing and extent of ecologically separated Euplotes species, E. raikovi the induction were different for the two mussel SAA living in temperate sea waters and E. petzi living in genes. The overall results provide new insights into the polar seas, revealed that both host bacteria in the evolution of these ancient immune-related their cytoplasm. These bacteria have been identified proteins in invertebrates. with facultative intracellular gamma-proteobacteria of the genus Francisella, which includes a number Research of inflammatory markers in the of closely related species well known as extremely medicinal leech, Hirudo medicinalis infectious to a great variety of organisms. Francisella tularensis, with its four subspecies, is a R Girardello1, F Drago2, M de Eguileor1, R specialized intracellular pathogen capable of Valvassori1, J Vizioli2, G Tettamanti1, A Grimaldi1 infecting both invertebrate and vertebrate hosts, 1Department of Biotechnology and Life Sciences, humans included; F. noatunensis is the etiological University of Insubria, Varese, Italy agent of the fish disease known as francisellosis, 2Laboratoire PRISM-UMR Inserm U1192. Université and its two subspecies well adapt to different de Lille, Villeneuve d’Ascq France temperatures of their hosts; the Francisella-like endosymbionts Wolbachia persica, together with the The leech Hirudo medicinalis is an interesting freely living generalists F. philomiragia and F. model to study inflammatory processes both in novicida cause diseases in humans with a nervous system and in peripheral tissues. Here we compromised immune system. The Francisella

50 endosymbionts of E. raikovi and E. petzi have been The leech Hirudo medicinalis is a well-studied successfully isolated and their genomes completely model in neurobiology because its Central Nervous sequenced. They are genetically distant from one System (CNS) spontaneously repairs after a another and form two different clades in the mechanical lesion. This process, leading in a few Francisella phylogenetic tree, which are distinct weeks to the synapse regeneration and a complete from the all other well-established Francisella functional recovery, is linked to the activity of clades. The finding that Francisella has equally microglial cells migrating to the injured area. In colonized polar and temperate-water species leech, a few hours after the injury the activated provides evidence that this bacterium is more microglia release an impressive amount of common and widespread than previously extracellular vesicles (EVs) that appear to constitute hypothesized, and confirms that free-living Euplotes an important element in the cross-talk between species and ciliates in general, with their worldwide microglia and lesioned neurons. By differential distribution, may represent a natural reservoir of centrifugation we separately isolated small (10 - 100 Francisella in every aquatic environment. nm, exosomes) and bigger (100 - 1000 nm, ectosomes) EVs. We also investigated the amount Lymnaea stagnalis ganglia transcriptional of exosomes and ectosomes released by naïve vs. activity after LPS induced immune activation ATP-stimulated microglia. In order to assess the function of these microglia-released EVs we F Tascedda1, C Benatti1, D Malagoli1, JMC Blom2, characterized their proteomic and RNA content and E Ottaviani1 we started investigating their potential in neurite 1Department of Life Sciences, University of Modena outgrowth. Proteomic analyses of leech vesicles and Reggio Emilia, Modena, Italy revealed the presence of many proteins typically 2Department of Education and Human Sciences and present in mammalian EVs, including several Paediatric Oncology Unit, University of Modena and surface molecules, and the presence of specific Reggio Emilia, Italy elements in differentially-stimulated samples. Functional assays were performed to assess the The mechanisms by which the neuroendocrine neurothrophic role of microglia-released EVs on a and immune systems communicate and influence mammalian neuron-like cell line (PC12). Neurite each other from invertebrates to vertebrates are well outgrowth was measured upon incubation with known and are among the most exiting areas of extracellular vesicles issued from leech microglial research in biology. Moreover, environmental cells. Results show a significant increase in neurites influences, such as inflammation or stress, play a outgrowth indicating that both leech exosomes and key role in determining susceptibility to disease and ectosomes can exert a neurothrophic effect on in particular to nervous system linked diseases. mammalian cells. The association of a specific Until now, studies regarding the genetic neurothrophic phenotype with its protein and RNA mechanisms underlying neuroendocrine and signatures would help to understand the role of immune interactions have used rodent models, these microglial EVs in promoting CNS repair. while invertebrate models have been used to a much lesser extent. Among gastropods, the Session 5. Chairmen: P Venier, University of freshwater snail Lymnaea stagnalis is emerging as Padua, Padua, Italy and L Abelli University of an important model to study immune- Ferrara, Ferrara, Italy neuroendocrine functions from an ecological, Immune response in Molluscs and Cnidarians parasitological and immunological point of view. In the present research, L. stagnalis, was used as an Structure and distribution of Astakine in the invertebrate model to study neuronal responses to organs of the freshwater snail Pomacea LPS induced immune activation. More precisely, we canaliculata tested the hypothesis that transcriptional changes occur in molluscan neural cells in response to LPS. A Accorsi1,2, S Benatti3, K Gotting1, E Ross1,2, A Adult snails were exposed to LPS after which L. Sánchez Alvarado1,2, D Malagoli3 stagnalis ganglia were dissected, RNA extracted 1Stowers Institute for Medical Research, Kansas and analyzed for expression levels of genes related City, MO, USA to neural and immune plasticity, such as, AIF-1and 2Howard Hughes Medical Institute, Stowers Institute HSP70. Preliminary data suggest that LPS induced for Medical Research, Kansas City, MO, USA immune and neural activity alters plasticity related 3Department of Life Sciences, University of Modena gene expression. and Reggio Emilia, Modena, Italy

Characterization and neurothrophic effects of The freshwater snail Pomacea canaliculata is leech microglia-released Extracellular Vesicles an emerging pest in EU, and its immune system is a (EVs) potential target for developing strategies of pest control. Circulating hemocytes represent the cellular T Arab1, F Drago1, I. Prada2, C Van Camp1, F Le component of the P. canaliculata immune system. Marrec-Croq1, J Franck1, M Wisztorski1, J-P P. canaliculata hemocytes originate in the Gimeno1, M Salzet1, P-E Sautière1, C Lefebvre1, pericardial fluid, and are maintained in the ampulla, C Verderio2, J Vizioli1 which may act as a hemocyte reservoir. Astakine-1 1Laboratoire PRISM-UMR Inserm U1192. Université is a hematopoietic cytokine first described in the de Lille, Villeneuve d’Ascq France crayfish Pacifastacus leniusculus, and recently 2Institute of Neuroscience (IN), CNR, Milano, Italy described also in the insect Lygus lineolaris and in

51 the bivalve mollusc Crassostrea gigas. Bioinformatic transcription rate. Similarly, in the bivalve analyses of a comprehensive P. canaliculata Crassostrea gigas, it has been suggested that transcriptome demonstrated the presence of an accumulated Cg-astakine transcripts are largely Astakine 1-like molecule (Pc-Astakine) also in this translated under some environment stress, including organism. Pc-Astakine is 121 aa and contains a immune stimuli. On the whole, our results indicate domain characterized by 8 cysteins with a that the expression of Pc-astakine and the conserved distribution pattern homologous to the translation rate of its mRNA may be influenced by vertebrate domain prokineticin. Further bioinformatic immune stimuli, and support the hypothesis that Pc- predictions suggest that the structure of Pc-Astakine Astakine may be involved in Pomacea may be similar to that one of P. leniusculus hematopoiesis and/or may have immune-related Astakine-1. The distribution of Pc-astakine gene functions, as well. expression was evaluated by qPCR. We have observed that all organs analyzed express Pc- Myticalins: a novel family of linear cationic astakine at detectable levels. However, high AMPs from Mytilus galloprovincialis identified expression levels were detected in the ampulla, by de novo bioinformatics analysis pericardial fluid and circulating hemocytes. The data suggest that Pc-Astakine may have a M Gerdol1, G Leoni2, P Venier3, A Tossi1, A wide range of functions, including the regulation of Pallavicini1 hematopoiesis and the modulation of inflammatory 1Department of Life Sciences, University of Trieste, responses, as previously reported for the human Trieste, Italy Prokineticin-1. 2International School for Advanced Studies (SISSA), Trieste, Italy Effects of LPS injection on Pc-astakine 3Department of Biology, University of Padua, Padua, expression in the gastropod Pomacea Italy canaliculata The de novo discovery of bioactive peptides by S Benatti1, A Accorsi2,3, M Nasi4, E Ottaviani1, D bioinformatics screening methods is undoubtedly Malagoli1 experiencing a renewed impulse due to the 1Department of Life Sciences, University of Modena increasing availability of genomic and transcriptomic and Reggio Emilia, Modena, Italy data from non-model organisms. Marine 2Stowers Institute for Medical Research, Kansas invertebrates in particular appear to be a vast and City, MO, USA still largely unexplored resource due to their 3Howard Hughes Medical Institute, Stowers Institute adaptation to diverse challenging environments. All for Medical Research, Kansas City, MO, USA the antimicrobial peptides (AMPs) described so far 4Department of Surgery, Medicine, Dentistry and in mussels (Mytilus spp.) display a structure Morphological Sciences, University of Modena and stabilized by intra-molecular disulphide bridges. No Reggio Emilia, Modena, Italy linear AMPs, relatively widespread in other invertebrates, have been identified so far, arguably Astakine-1 is a prokineticin-containing factor due to the fact that they are encoded by and the first hematopoietic cytokine described in taxonomically-restricted genes with no sequence invertebrates. Astakine-1 was firstly retrieved in the homology to other known AMPs. Here we describe freshwater crayfish Pacifastacus leniusculus, and myticalins (Mytilus cationic linear AMPs), a novel recent experiments have demonstrated the family of AMPs produced as pre-propeptides and presence of astakine-like molecules also in insects characterized by the absence of cysteine residues and molluscs, including the freshwater snail, and by a positive net charge. Myticalins have been Pomacea canaliculata. In control conditions Pc- fully identified through a bioinformatics approach astakine is expressed in several organs, especially with no prior knowledge concerning their biological in the ampulla (reservoir of hemocytes and potential activity, by scanning the M. galloprovicialis district of hemocyte maturation) and in the transcriptome for potential AMP sequences, based pericardial fluid (i.e. the hematopoietic tissue). By on isoelectric point and amino acid composition. mean of qPCR experiments, we have analyzed the Although these AMPs appear to be largely effects of the injection of 50 µg LPS on the diversified, they can be categorized into 4 main expression of the gene Pc-astakine. Our subclasses (A, B, C and D). We selected myticalin observations indicate that 24 h after the injection, A-5, whose mature peptide sequence presents the major modification of the Pc-astakine expression several Pro-Arg-X repeats, and chemically was evident in the anterior kidney, a potential synthetized the predited mature peptide sequence. hemocyte reservoir, in which the expression of the The synthetic peptide displayed a significant gene decreased to almost undetectable level. In the antimicrobial activity both against Gram+ and Gram- pericardial fluid, ampulla and circulating hemocytes, bacteria, validating the effectiveness of our de novo the expression of Pc-astakine dropped to less than approach. Differently from other mussel AMPs, 50 % with respect to the sham-injected control myticalins are mainly expressed in the gills tissue. snails. The drop in the amount of mRNA detected Although many aspects concerning the biological by qPCR could reflect an increased rate of activity of myticalins remain to be clarified, they translation and consequent degradation of the represent a promising class of peptides with available mRNA, rather than a decrease of the potential biotechnological applications.

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Antiviral immunity in oysters infected by Ostreid acting on voltage-gated sodium channels, 3.5 to 6.5 herpesvirus-1 kDa neurotoxins acting on voltage-gated Kv1 potassium channels, 20 kDa pore-forming cytolysins U Rosani1, S Domeneghetti1, M Gerdol2, G inhibited by sphingomyelin and serine protease Arcangeli3, A Pallavicini2, P Venier1 inhibitors belonging to the Kunitz-type family. Type 1 1Department of Biology, University of Padova, and 2 Na+ channel toxins are composed of 46 to 49 Padua, Italy amino acid residues, except for A. equina toxin AeI 2Department of Life Sciences, University of Trieste, of 54 residues and cross-linked by three disulfide Trieste, Italy bridges. In this study toxic components from acid 3Istituto Zooprofilattico Sperimentale delle Venezie, tissue tentacles extracts of sea anemone A. equina Adria, Italy were investigated by size exclusion separation to characterize cytolytic molecules of low molecular Marine bivalves do not possess an acquired weight. Tentacles extracts with low molecular mass immune system that allows selective responses were filtered through a membrane (10 kDa Nanosep based on immunological memory. Conversely, device). Sample was subjected to size exclusion bivalves are evolutionary successful species that chromatography using BioSuite 250, 10 μm SEC, live in an environment rich of viruses and bacteria, 7.5×300 mm column on a HPLC system and thus meaning that they have effective defense collected fractions were concentrated through systems against pathogens. Ostreid herpesvirus micro-concentrators to be tested for lytic activity. type 1 (OsHV-1) is characterized by a 207 kb After electrophoretical analysis of lytic fractions, dsDNA genome and has been associated to bands were excised and sent to sequencing by N- sporadic mortalities of several bivalve species since gel digestion and MALDI TOF analysis. Database mid ’60. In 2008, a new OsHV-1 genotype called searching and blast analysis allowed to identify a μVAR was reported to be the causative agent of protein of 5,5 kDa molecular weight correspondent massive mortalities of young oysters in France and, to the Ae1 toxin from A. equina. Then, the peptide in the following years, its presence was reported AeI was synthesized from GenScript (Chemical worldwide. Today, OsHV-1 has become a peptide Synthesis service) and it was assayed for its problematic infective agent for the Pacific oyster hemolytic activity using the sheep erythrocytes Crassostrea gigas. Genetic susceptibility and critical (RBCs) by a spectrophotometric quantization assay chemico-physical conditions could facilitate the of percentage of hemolysis. Lysis was recorded overwhelming of the oyster immune system and after 1 h of incubation in polyethylene tubes and 58 result in an uncontrolled viral replication (lytic % and hemolysis was found when the targets were phase). Parallel sequencing of host and pathogen put in contact with the neurotoxin AeI. Current RNAs (DualRNA-seq) has the potential to reveal the studies concerning the mechanism of action of the host-pathogen interactions during infection. In detail, molecule and its structure are carrying out. The the availability of a C. gigas sample (Goro lagoon, discovery of cytolytic activity in addition to neuro- North Adriatic Sea, Italy) highly infected by OsHV-1 inhibitory effect shows the evolutionary trend to μVAR (up to 8.4×104 copies/ng DNA) allowed us to combine two functions in one compound: ion analyze both oyster and viral transcriptomes. Owing channel inhibitor and membranolytic activity. to the high sequence coverage we were able to describe the complete genome of an Italian virus From cnidarian immunobiology to cultural genotype with strong pathogenicity. In the analyzed heritage applications oyster sample, we found genes with similarity to elements of the vertebrate interferon pathway and MR Trapani, MG Parisi, D Parrinello, MA several other defense genes. Overall, these facts Sanfratello, G Benenati, G Barresi, F Palla, M indicate that oyster possesses an antiviral-specific Cammarata immunity, mainly based on an interferon-like Department of Biological, Chemical and pathway, RNA interference and programmed cell Pharmaceutical Science and Technology, University death. of Palermo, Palermo, Italy

Voltage-gated sodium channels neurotoxin from The study of cnidarians immunity, as model tentacles of sea anemone Actinia equina systems of metazoans, lead additional information exhibits cytolityc activity on the first steps of the immunity evolution. The functions of the genes and cellular pathways in MG Parisi, MR Trapani, M Cammarata higher vertebrates are conserved during the Animal biology section, Department of Biological, evolution of metazoans, as shown by the discovery Chemical and Pharmaceutical Science and of homologues in cnidarians. These basal Technology, University of Palermo, Palermo, Italy metazoans in fact, are far from "simples" in the range of methods at their disposal to deal with Sea anemones are sessile benthic organisms potential prey but also invading microbes and and their evolution has led to a high specific pathogens. They can give information about the richness in the course of millions of years. The invertebrates innate immune repertoire. We presence, diversification and multi-functionality of investigated the immunobiology starting from the the toxins may have played an important role in the inflammatory response in Anemonia sulcata ability of colonization and adaptation to ecological (Cnidaria: Anthozoa) following injection of niches that change over time. Venoms include substances different in type and dimension, to peptide and protein toxins as 3 to 5 kDa neurotoxins understand the effector mechanisms involved in this

53 process. We observed clear, strong and specific migration, MHC-restricted antigen presentation, reactions especially after injection of bacteria and cytokines release) levels. The best characterized the alteration of the expression of enzymes populations recall conventional DC, but studies in (protease, phosphatase and esterase), showing a Esb additionally detailed IDC occurring in correlation between the appearance of the developing and adult thymus, likely engaged in inflammatory reaction and the modification of thymocyte selection processes. Furthermore, recent enzymatic activities. From cnidarian phylum a large studies in rainbow trout identified MHC II+CD8-α+ number of toxic compounds have been isolated. DC in the skin. This finding prompted to advance Tissues and mucus produced by cnidarians may the hypothesis for a common origin for all have a role in immune defense and contain a variety mammalian DC that may exert antigen cross- of toxins as neurotoxins, cytolysins and presentation. antimicrobial peptides, which can have multifunctional role. The bioactive molecules were Germ line dynamics in bivalve molluscs purified by acid extraction and HPLC purifications and characterized through biological assays, mass L Milani, A Pecci, F Ghiselli, M Passamonti, MG spectroscopy and peptide synthesis. Here, we show Maurizii the cnidarian bioactive molecules as antimicrobial Department of Biological, Geological and peptides and enzymes in order to draw applications Environmental Sciences, University of Bologna, in fields ranging from pharmacology to cultural Bologna, Italy heritage. Particularly, in the control of the microbic growth and especially in the tuning of biocleaning Studies about germ line specification in protocols, bioactive molecules with proteasic and Mollusca have been neglected for years. Available esterasic activity have been used. These novel data based on few species indicate that molluscs enzymes are active at temperature lower than 30°C, exhibit the same cleavage mechanism of other they need a reduced time of application and are spiralians, in which much of the mesoderm comes safety for both operators and environment. Thus form the 4d blastomere, including the germinal they could provide an important contribution to the tissue, whose specification has been identified by development of sustainable innovative protocols. localizing the products of vasa orthologs (VASA is highly conserved in the animal kingdom). Despite Session 6. Chairmen: D Malagoli, University of the relevance of bivalve molluscs in marine Modena and Reggio Emilia, Modena, Italy and M ecosystems and aquaculture, their mechanism of Cammarata, University of Palermo, Palermo, sex determination is still unknown, as well as the Italy details of their seasonal gonad reconstitution (i.e., the gonad is reabsorbed after spawning and Hotspots in metazoan immunity reconstructed at the beginning of the subsequent reproductive season). In order to determine general Lecture features of bivalve germ line development, we Insights into the evolution of dendritic cells employed two antibodies produced against the VASA ortholog of Ruditapes philippinarum S Bisin, L Gagliardi, D Lunardi, G Pavani, L (Subclass Heterodonta, Family Veneridae) to Abelli investigate three additional species. We chose two Department of Life Sciences and Biotechnology, species of the Subclass Pteriomorphia, Scapharca University of Ferrara, Ferrara, Italy inaequivalvis (Family Arcidae) and Crassostrea gigas (Family Ostreidae), and another species of The development in gnathostome vertebrates of the Subclass Heterodonta, Mya arenaria (Family an adaptive immune system, that guarantees fine Myidae). The immunoreactivity of anti-VASA was discrimination of antigenic determinants, also confirmed by Western Blot in which single specific involved main specializations of antigen-presenting bands were obtained in each species, although of cells (APC), which govern presentation to different molecular weight. On the other hand, the lymphocytes of antigens associated to self and non- presence of two different bands in males and self MHC molecules. The dendritic cells (DC), females of the gonochoric R. philippinarum has discovered first in mammals and later classified as been previously related to sex-biased isoforms, conventional, plasmacitoid, interdigitating (IDC), already found in other animal species outside follicular, intra-epithelial (e.g., Langerhans cells), bivalves. The detection of a single band in the two dermal or tissutal, fall into the APC category. Pteriomorphia may be tentatively related to their However, information about their appearance state of proterandric hermaphrodites. M. arenaria is and evolution in vertebrates is still limited and reported to be gonochoric, as R. philippinarum, but fragmentary. DC were indirectly (high levels of MHC the analyzed specimens were all females, so we II molecules) hypothesized in Chondrichthyes, cannot exclude the existence of different sex-related whereas are much better documented in Teleostei isoforms, although no sign of a second band was (Actinopterygii Osteichthyes), paradoxically more present. The immunohistological data obtained than in heteroterm Tetrapods. Studies in rainbow support a conserved mechanism of proliferation of trout, zebrafish, Atlantic salmon and European sea primodial germ cells (showing the VASA labeling at bass (Esb) convincingly reported about DC, at one side of the cell cytoplasm) among the simple cytological, molecular (specific surface markers, columnar epithelium of the gut. Moreover, their cytokines and CpG receptors, lectin-induced seasonal migration to the reconstituting gonad agglutination) and functional (phagocytosis, appears to be a common feature of bivalves. The

54 study of bivalve reproductive biology can clarify beads (LTX) were used as controls. Three types of important aspects of their development, but can also circulating hemocytes were characterized: granular be useful for conservation management and hemocytes (GH), semigranular hemocytes (SH) breeding programs. and hyaline hemocytes (HH). Irradiated animals present highly significantly lower THCs (35357 ± Managing of Procambarus clarkii by X-ray 9643 hemocytes/mL, n = 7) in comparison with sterilisation of males: immunological CTRL (666818 ± 78546 hemocytes/mL, n = 11), competence after the irradiation PBS (1006071 ± 184413 hemocytes/mL, n = 7) and LTX (368437 ± 98895 hemocytes/mL, n = 8) E Simeon, MK Bravin, C Manfrin, F Piazza, S challenged ones. Irradiated animals show Battistella, PG Giulianini significantly higher GH percentages (48.57 ± 6.2) Department of Life Sciences, University of Trieste, in comparison to all controls (CTRL: 17.97 ± 2.1; Trieste, Italy PBS: 29.91 ± 3.2; LTX: 21.89 ± 3.4), but significantly lower HH percentages (17.55 ± 5.2) in Procambarus clarkii (Girard, 1852) is an comparison to controls (CTRL: 59.73 ± 4.9; PBS: invasive alien species spread worldwide. The Sterile 65.83 ± 2.7; LTX: 67.57 ± 4.0). The comparison of Male Release Technique has been chosen in Friuli PO and pPO activities among different groups do Venezia Giulia as part of a strategy to control the not evidence significant differences. The literature red swamp crayfish. This technique consists on the documents a survival of males irradiated with a release into the environment of sterile males which dose of 20 Gy at laboratory conditions of at least are sexually active and able to compete with one year (Aquiloni et al., 2009). The present study untreated males for mating partners. Recently the reports a decrease of about 95 % of circulating gonad damage induced by ionising irradiation were hemocytes and an increase in percentage of GH in described (Piazza et al., 2015), but no data are males irradiated with a dose of 40 Gy after 20 days. available on the immunocompetence of males after Interestingly, the activity of PO and pPO are not irradiation. The present study describes the affected by ionising radiation. The decrease in hemocytes of P. clarkii males 20 days after circulating hemocytes is consistent with the irradiation at a dose of 40 Gy by means of light and cytological damages to the gonads that are transmission electron microscopy. Total (THC) and described as progressive from the day of irradiation differential number of hemocytes (DHC) and activity up to 30 days (Piazza et al., 2015). It remains to be of basal (PO) and total plasmatic phenoloxidase seen how long and how animals are able to offset (pPO) are also evaluated. Untreated animals this hemocyte decline and if this would preclude the (CTRL), unirradiated animals injected with sterile survival in the wild. saline (PBS), and carboxylated polystyrene latex

55