Laura Varotto

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.

MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences

BackgroundAlthough Bivalves are among the most studied marine organisms due to their ecological role, economic importance and use in pollution biomonitoring, very little information is available on the genome sequences of mussels. This study reports the functional analysis of a large-scale Expressed Sequence Tag (EST) sequencing from different tissues of Mytilus galloprovincialis (the Mediterranean mussel) challenged with toxic pollutants, temperature and potentially pathogenic bacteria.ResultsWe have constructed and sequenced seventeen cDNA libraries from different Mediterranean mussel tissues: gills, digestive gland, foot, anterior and posterior adductor muscle, mantle and haemocytes. A total of 24,939 clones were sequenced from these libraries generating 18,788 high-quality ESTs which were assembled into 2,446 overlapping clusters and 4,666 singletons resulting in a total of 7,112 non-redundant sequences. In particular, a high-quality normalized cDNA library (Nor01) was constructed as determined by the high rate of gene discovery (65.6%). Bioinformatic screening of the non-redundant M. galloprovincialis sequences identified 159 microsatellite-containing ESTs. Clusters, consensuses, related similarities and gene ontology searches have been organized in a dedicated, searchable database http://mussel.cribi.unipd.it.ConclusionWe defined the first species-specific catalogue of M. galloprovincialis ESTs including 7,112 unique transcribed sequences. Putative microsatellite markers were identified. This annotated catalogue represents a valuable platform for expression studies, marker validation and genetic linkage analysis for investigations in the biology of Mediterranean mussels.

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.

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.

Mediterranean Mussel Gene Expression Profile Induced by Okadaic Acid Exposure

Seasonal seawater temperature increases define optimal growth conditions for Dinoflagellate species which can reach high concentrations in water column and also in filter-feeding organisms like Mytilus galloprovincialis. Commonly produced by Dinophysis and Prorocentrum spp., okadaic acid (OA) and its analogues are responsible for the Diarrheic Shellfish Poisoning (DSP) syndrome in humans. Closure of shellfishing grounds is therefore recommended by the EU when DSP toxin levels in shellfish exceed 16 μg OA 100 g(-1) flesh. Despite not being responsible for casualties either in humans or mussels, DSP outbreaks are considered natural events causing health and economic issues due to the frequency of their occurrence. Since gene expression studies offer a wide range of different solutions, we used a mussel cDNA microarray to evaluate gene expression changes in the digestive gland of mussels fed for five weeks with OA-contaminated nutrient. Among the differentially expressed genes we observed a general up-regulation of transcripts coding for stress proteins, proteins involved in cellular synthesis, and a few not annotated proteins. Overall, at the first time point analyzed we identified 58 candidate transcripts for OA-induced stress in mussels, half of which have unknown function. In this paper we present the first gene expression analysis performed on Mediterranean mussels exposed to okadaic acid. The characterization of these transcripts could be useful for the identification of an early physiological response to OA exposure.

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.