Umberto Rosani

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis

BackgroundSessile bivalves of the genus Mytilus are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of M. galloprovincialis, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes.ResultsWe have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in M. galloprovincialis. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with Vibrio splendidus at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the Vibrio-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways.ConclusionsThe Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on Vibrio-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the Mytilus species to an evolving microbial world.

Toll-like receptors and MyD88 adaptors in Mytilus: complete cds and gene expression levels.

TLR- and MyD88-related sequences have been previously investigated in Mytibase and then in new transcript reads obtained by Illumina technology from the mussel, Mytilus galloprovincialis. Based on full cds and domain organizations of virtual translations, we identified 23 Toll-like receptors (TLRs) and 3 MyD88 adaptors. MgTLRs can be arranged in 4 clusters according to extra-cellular LRR domain content. MgTLR-b, -i and -k were the only ones containing a multiple cysteine cluster (mccTLR), a domain composition also found in Drosophila Toll-1 and 18-wheeler. The 3 MyD88 we identified in M. galloprovincialis were also retrieved from Mytilus edulis, as well as MgTLR-b and -i. All MgTLRs were constitutively expressed in digestive gland whereas only 4 of them were also present in hemocytes. On the opposite, the 3 MgMyD88s were constitutively expressed in all the tissues. In vivo challenge of M. galloprovincialis with bacteria caused the up regulation of only MgTLR-i, but of all the 3 MgMyD88s. Highest response was induced by Gram-negative Vibrio anguillarum at 9h p.i. Injection of filamentous fungus, Fusarium oxysporum, resulted in up regulation of MgTLR-i and MgMyD88-c at 9h p.i. Such similar pattern of responses suggested MgMyD88-c represents the intra cytoplasm partner of MgTLR-i. Their interaction constituted the first cellular event revealing the existence of a Toll-signaling pathway in Lophotrochozoa.

DNA Damage and Transcriptional Changes in the Gills of Mytilus galloprovincialis Exposed to Nanomolar Doses of Combined Metal Salts (Cd, Cu, Hg)

Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism Mytilus galloprovincialis, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes). Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programs.

Mortality occurrence and pathogen detection in Crassostrea gigas and Mytilus galloprovincialis close-growing in shallow waters (Goro lagoon, Italy)

The complex interactions occurring between farmed bivalves and their potential pathogens in the circumstances of global climate changes are current matter of study, owing to the recurrent production breakdowns reported in Europe and other regions of the world. In the frame of Project FP7-KBBE-2010-4 BIVALIFE, we investigated the occurrence of mortality and potential pathogens during the Spring-Summer transition in Crassostrea gigas and Mytilus galloprovincialis cohabiting in the shallow waters of one northern Italian lagoon (Sacca di Goro, Adriatic Sea) and regarded as susceptible and resistant species, respectively. In 2011, limited bivalve mortality was detected in the open-field trial performed with 6-12 month old spat whereas subsequent trials with 2-3 month old spat produced almost complete (2012) and considerable (2013) oyster mortality. Macroscopical examination and histology excluded the presence of notifiable pathogens but, in the sampling preceding the massive oyster spat mortality of 2012, a μdeleted variant of OsHV-1 DNA was found in wide-ranging amounts in all analyzed oysters in conjunction with substantial levels of Vibrio splendidus and Vibrio aestuarianus. The large oyster spat mortality with borderline OsHV-1 positivity recorded in 2013 supports the multi-factorial etiology of the syndrome. This is the first report of a OsHV-1 (under a form interpreted as the variant μVar) in the Goro lagoon. Transcriptional host footprints are under investigation to better understand the bivalve response to environmental factors, included viral and bacterial pathogens, in relation to the observed mortalities.

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

Based on protein domain structure and organization deduced from mRNA contigs, 15 transcripts of the Toll signaling pathway have been identified in the bivalve, Mytilus galloprovincialis. Identical searches performed on publicly available Mytilus edulis ESTs revealed 11 transcripts, whereas searches performed in genomic and new transcriptome sequences of the Pacific oyster, Crassostrea gigas, identified 21 Toll-related transcripts. The remarkable molecular diversity of TRAF and IKK coding sequences of C. gigas, suggests that the sequence data inferred from Mytilus cDNAs may not be exhaustive. Most of the Toll pathway genes were constitutively and ubiquitously expressed in M. galloprovincialis, although at different levels, and clearly induced after in vivo injection with bacteria. Such over-transcription was more rapid and intense with Gram-negative than with Gram-positive bacteria. Injection of a fungus modulated the transcription of few Toll pathway genes, with the induction levels of TLR/MyD88 complex being always less intense. Purified LPS and β-glucans had marginal effect whereas peptidoglycans were ineffective. At the moment, we found no evidence of an IMD transcript in bivalves. In conclusion, mussels possess a complete Toll pathway which can be triggered either by Gram-positive or Gram-negative bacteria.

Dual Analysis of Host and Pathogen Transcriptomes in Ostreid Herpesvirus 1 - Positive Crassostrea gigas

Ostreid herpesvirus type 1 (OsHV-1) has become a problematic infective agent for the Pacific oyster Crassostrea gigas. In particular, the OsHV-1 μVar subtype has been associated with severe mortality episodes in oyster spat and juvenile oysters in France and other regions of the world. Factors enhancing the infectivity of the virus and its interactions with susceptible and resistant bivalve hosts are still to be understood, and only few studies have explored the expression of oyster or viral genes during productive infections. In this work, we have performed a dual RNA sequencing analysis on an oyster sample with a high viral load. High sequence coverage allowed us to thoroughly explore the OsHV-1 transcriptome and identify the activated molecular pathways in C. gigas. The identification of several highly induced and defence-related oyster transcripts supports the crucial role played by the innate immune system against the virus and opportunistic microbes possibly contributing to subsequent spat mortality.

Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis

Background Effective innate responses against potential pathogens are essential in the living world and possibly contributed to the evolutionary success of invertebrates. Taken together, antimicrobial peptide (AMP) precursors of defensin, mytilin, myticin and mytimycin can represent about 40% of the hemocyte transcriptome in mussels injected with viral-like and bacterial preparations, and unique profiles of myticin C variants are expressed in single mussels. Based on amplicon pyrosequencing, we have ascertained and compared the natural and Vibrio-induced diversity of AMP transcripts in mussel hemocytes from three European regions. Methodology/Principal Findings Hemolymph was collected from mussels farmed in the coastal regions of Palavas (France), Vigo (Spain) and Venice (Italy). To represent the AMP families known in M. galloprovincialis, nine transcript sequences have been selected, amplified from hemocyte RNA and subjected to pyrosequencing. Hemolymph from farmed (offshore) and wild (lagoon) Venice mussels, both injected with 107 Vibrio cells, were similarly processed. Amplicon pyrosequencing emphasized the AMP transcript diversity, with Single Nucleotide Changes (SNC) minimal for mytilin B/C and maximal for arthropod-like defensin and myticin C. Ratio of non-synonymous vs. synonymous changes also greatly differed between AMP isotypes. Overall, each amplicon revealed similar levels of nucleotidic variation across geographical regions, with two main sequence patterns confirmed for mytimycin and no substantial changes after immunostimulation. Conclusions/Significance Barcoding and bidirectional pyrosequencing allowed us to map and compare the transcript diversity of known mussel AMPs. Though most of the genuine cds variation was common to the analyzed samples we could estimate from 9 to 106 peptide variants in hemolymph pools representing 100 mussels, depending on the AMP isoform and sampling site. In this study, no prevailing SNC patterns related to geographical origin or Vibrio injection emerged. Whether or not the contact with potential pathogens can increase the amount of AMP transcript variants in mussels requires additional study.

IL-17 signaling components in bivalves: Comparative sequence analysis and involvement in the immune responses.

The recent discovery of soluble immune-regulatory molecules in invertebrates takes advantage of the rapid growth of next generation sequencing datasets. Following protein domain searches in the transcriptomes of 31 bivalve spp. and in few available mollusk genomes, we retrieved 59 domains uniquely identifying interleukin 17 (IL-17) and 96 SEFIR domains typical of IL-17 receptors and CIKS/ACT1 proteins acting downstream in the IL-17 signaling pathway. Compared to the Chordata IL-17 family members, we confirm a separate clustering of the bivalve domain sequences and a consistent conservation pattern of amino acid residues. Analysis performed at transcript and genome level allowed us to propose an updated view of the components outlining the IL-17 signaling pathway in Mytilus galloprovincialis and Crassostrea gigas (in both species, homology modeling reduced the variety of IL-17 domains to only two 3D structures). Digital expression analysis indicated more heterogeneous expression levels for the mussel and oyster IL-17 ligands than for IL-17 receptors and CIKS/CIKSL proteins. Besides, new qPCR analyses confirmed such gene expression trends in hemocytes and gills of mussels challenged with heat-killed bacteria. These results uphold the involvement of an ancient IL-17 signaling pathway in the bivalve immune responses and, likewise in humans, suggest the possibility of distinctive modulatory roles of individual IL-17s/IL-17 receptors. Overall, the common evidence of pro-inflammatory cytokines and inter-related intracellular signaling pathways in bivalves definitely adds complexity to the invertebrate immunity.

The miRNA biogenesis in marine bivalves

Small non-coding RNAs include powerful regulators of gene expression, transposon mobility and virus activity. Among the various categories, mature microRNAs (miRNAs) guide the translational repression and decay of several targeted mRNAs. The biogenesis of miRNAs depends on few gene products, essentially conserved from basal to higher metazoans, whose protein domains allow specific interactions with dsRNA. Here, we report the identification of key genes responsible of the miRNA biogenesis in 32 bivalves, with particular attention to the aquaculture species Mytilus galloprovincialis and Crassostrea gigas. In detail, we have identified and phylogenetically compared eight evolutionary conserved proteins: DROSHA, DGCR8, EXP5, RAN, DICER TARBP2, AGO and PIWI. In mussels, we recognized several other proteins participating in the miRNA biogenesis or in the subsequent RNA silencing. According to digital expression analysis, these genes display low and not inducible expression levels in adult mussels and oysters whereas they are considerably expressed during development. As miRNAs play an important role also in the antiviral responses, knowledge on their production and regulative effects can shed light on essential molecular processes and provide new hints for disease prevention in bivalves.

Atmospheric-Pressure Cold Plasma Induces Transcriptional Changes in Ex Vivo Human Corneas

Background Atmospheric pressure cold plasma (APCP) might be considered a novel tool for tissue disinfection in medicine since the active chemical species produced by low plasma doses, generated by ionizing helium gas in air, induces reactive oxygen species (ROS) that kill microorganisms without substantially affecting human cells. Objectives In this study, we evaluated morphological and functional changes in human corneas exposed for 2 minutes (min) to APCP and tested if the antioxidant n-acetyl l-cysteine (NAC) was able to inhibit or prevent damage and cell death. Results Immunohistochemistry and western blotting analyses of corneal tissues collected at 6 hours (h) post-APCP treatment demonstrated no morphological tissue changes, but a transient increased expression of OGG1 glycosylase that returned to control levels in 24 h. Transcriptome sequencing and quantitative real time PCR performed on different corneas revealed in the treated corneas many differentially expressed genes: namely, 256 and 304 genes showing expression changes greater than ± 2 folds in the absence and presence of NAC, respectively. At 6 h post-treatment, the most over-expressed gene categories suggested an active or enhanced cell functioning, with only a minority of genes specifically concerning oxidative DNA damage and repair showing slight over-expression values (<2 folds). Moreover, time-related expression analysis of eight genes up-regulated in the APCP-treated corneas overall demonstrated the return to control expression levels after 24 h. Conclusions These findings of transient oxidative stress accompanied by wide-range transcriptome adjustments support the further development of APCP as an ocular disinfectant.