Publications by authors named "Paola Venier"

48 Publications

Digging into bivalve miRNAomes: between conservation and innovation.

Philos Trans R Soc Lond B Biol Sci 2021 May 5;376(1825):20200165. Epub 2021 Apr 5.

Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 10691 Stockholm, Sweden.

Bivalves are a diverse mollusc group of economic and ecological importance. An evident resilience to pollution, parasites and extreme environments makes some bivalve species important models for studying adaptation and immunity. Despite substantial progress in sequencing projects of bivalves, information on non-coding genes and gene-regulatory aspects is still lacking. Here, we review the current repertoire of bivalve microRNAs (miRNAs), important regulators of gene expression in Metazoa. We exploited available short non-coding RNA (sncRNA) data for and , and we produced new sncRNA data for two additional bivalves, the Mediterranean mussel and the blood clam . We found substantial heterogeneity and incorrect annotations of miRNAs; hence, we reannotated conserved miRNA families using recently established criteria for microRNA annotation. We found 106 miRNA families missing in the previously published bivalve datasets and 89 and 87 miRNA complements were identified in the two additional species. The overall results provide a homogeneous and evolutionarily consistent picture of miRNAs in bivalves and enable future comparative studies. The identification of two bivalve-specific miRNA families sheds further light on the complexity of transcription and its regulation in bivalve molluscs. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.
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http://dx.doi.org/10.1098/rstb.2020.0165DOI Listing
May 2021

Massive gene presence-absence variation shapes an open pan-genome in the Mediterranean mussel.

Genome Biol 2020 11 10;21(1):275. Epub 2020 Nov 10.

Instituto de Investigaciones Marinas (IIM - CSIC), Eduardo Cabello, 6, 36208, Vigo, Spain.

Background: The Mediterranean mussel Mytilus galloprovincialis is an ecologically and economically relevant edible marine bivalve, highly invasive and resilient to biotic and abiotic stressors causing recurrent massive mortalities in other bivalves. Although these traits have been recently linked with the maintenance of a high genetic variation within natural populations, the factors underlying the evolutionary success of this species remain unclear.

Results: Here, after the assembly of a 1.28-Gb reference genome and the resequencing of 14 individuals from two independent populations, we reveal a complex pan-genomic architecture in M. galloprovincialis, with a core set of 45,000 genes plus a strikingly high number of dispensable genes (20,000) subject to presence-absence variation, which may be entirely missing in several individuals. We show that dispensable genes are associated with hemizygous genomic regions affected by structural variants, which overall account for nearly 580 Mb of DNA sequence not included in the reference genome assembly. As such, this is the first study to report the widespread occurrence of gene presence-absence variation at a whole-genome scale in the animal kingdom.

Conclusions: Dispensable genes usually belong to young and recently expanded gene families enriched in survival functions, which might be the key to explain the resilience and invasiveness of this species. This unique pan-genome architecture is characterized by dispensable genes in accessory genomic regions that exceed by orders of magnitude those observed in other metazoans, including humans, and closely mirror the open pan-genomes found in prokaryotes and in a few non-metazoan eukaryotes.
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http://dx.doi.org/10.1186/s13059-020-02180-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653742PMC
November 2020

Parallel analysis of miRNAs and mRNAs suggests distinct regulatory networks in Crassostrea gigas infected by Ostreid herpesvirus 1.

BMC Genomics 2020 Sep 10;21(1):620. Epub 2020 Sep 10.

Department of Biology, University of Padova, 35121, Padova, Italy.

Background: Since 2008, the aquaculture production of Crassostrea gigas was heavily affected by mass mortalities associated to Ostreid herpesvirus 1 (OsHV-1) microvariants worldwide. Transcriptomic studies revealed the major antiviral pathways of the oyster immune response while other findings suggested that also small non-coding RNAs (sncRNA) such as microRNAs might act as key regulators of the oyster response against OsHV-1. To explore the explicit connection between small non-coding and protein-coding transcripts, we performed paired whole transcriptome analysis of sncRNA and messenger RNA (mRNA) in six oysters selected for different intensities of OsHV-1 infection.

Results: The mRNA profiles of the naturally infected oysters were mostly governed by the transcriptional activity of OsHV-1, with several differentially expressed genes mapping to the interferon, toll, apoptosis, and pro-PO pathways. In contrast, miRNA profiles suggested more complex regulatory mechanisms, with 15 differentially expressed miRNAs (DE-miRNA) pointing to a possible modulation of the host response during OsHV-1 infection. We predicted 68 interactions between DE-miRNAs and oyster 3'-UTRs, but only few of them involved antiviral genes. The sncRNA reads assigned to OsHV-1 rather resembled mRNA degradation products, suggesting the absence of genuine viral miRNAs.

Conclusions: We provided data describing the miRNAome during OsHV-1 infection in C. gigas. This information can be used to understand the role of miRNAs in healthy and diseased oysters, to identify new targets for functional studies and, eventually to disentangle cause and effect relationships during viral infections in marine mollusks.
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http://dx.doi.org/10.1186/s12864-020-07026-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7488030PMC
September 2020

Functional Insights From the Evolutionary Diversification of Big Defensins.

Front Immunol 2020 30;11:758. Epub 2020 Apr 30.

IHPE, Université de Montpellier, CNRS, Ifremer, Université de Perpignan Via Domitia, Montpellier, France.

Big defensins are antimicrobial polypeptides believed to be the ancestors of β-defensins, the most evolutionary conserved family of host defense peptides (HDPs) in vertebrates. Nevertheless, big defensins underwent several independent gene loss events during animal evolution, being only retained in a limited number of phylogenetically distant invertebrates. Here, we explore the evolutionary history of this fascinating HDP family and investigate its patchy distribution in extant metazoans. We highlight the presence of big defensins in various classes of lophotrochozoans, as well as in a few arthropods and basal chordates (amphioxus), mostly adapted to life in marine environments. Bivalve mollusks often display an expanded repertoire of big defensin sequences, which appear to be the product of independent lineage-specific gene tandem duplications, followed by a rapid molecular diversification of newly acquired gene copies. This ongoing evolutionary process could underpin the simultaneous presence of canonical big defensins and non-canonical (β-defensin-like) sequences in some species. The big defensin genes of mussels and oysters, two species target of in-depth studies, are subjected to gene presence/absence variation (PAV), i.e., they can be present or absent in the genomes of different individuals. Moreover, big defensins follow different patterns of gene expression within a given species and respond differently to microbial challenges, suggesting functional divergence. Consistently, current structural data show that big defensin sequence diversity affects the 3D structure and biophysical properties of these polypeptides. We discuss here the role of the N-terminal hydrophobic domain, lost during evolution toward β-defensins, in the big defensin stability to high salt concentrations and its mechanism of action. Finally, we discuss the potential of big defensins as markers for animal health and for the nature-based design of novel therapeutics active at high salt concentrations.
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http://dx.doi.org/10.3389/fimmu.2020.00758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203481PMC
March 2021

A-to-I editing of Malacoherpesviridae RNAs supports the antiviral role of ADAR1 in mollusks.

BMC Evol Biol 2019 07 23;19(1):149. Epub 2019 Jul 23.

Department of Biology, University of Padova, 32121, Padova, Italy.

Background: Adenosine deaminase enzymes of the ADAR family are conserved in metazoans. They convert adenine into inosine in dsRNAs and thus alter both structural properties and the coding potential of their substrates. Acting on exogenous dsRNAs, ADAR1 exerts a pro- or anti-viral role in vertebrates and Drosophila.

Results: We traced 4 ADAR homologs in 14 lophotrochozoan genomes and we classified them into ADAD, ADAR1 or ADAR2, based on phylogenetic and structural analyses of the enzymatic domain. Using RNA-seq and quantitative real time PCR we demonstrated the upregulation of one ADAR1 homolog in the bivalve Crassostrea gigas and in the gastropod Haliotis diversicolor supertexta during Ostreid herpesvirus-1 or Haliotid herpesvirus-1 infection. Accordingly, we demonstrated an extensive ADAR-mediated editing of viral RNAs. Single nucleotide variation (SNV) profiles obtained by pairing RNA- and DNA-seq data from the viral infected individuals resulted to be mostly compatible with ADAR-mediated A-to-I editing (up to 97%). SNVs occurred at low frequency in genomic hotspots, denoted by the overlapping of viral genes encoded on opposite DNA strands. The SNV sites and their upstream neighbor nucleotide indicated the targeting of selected adenosines. The analysis of viral sequences suggested that, under the pressure of the ADAR editing, the two Malacoherpesviridae genomes have evolved to reduce the number of deamination targets.

Conclusions: We report, for the first time, evidence of an extensive editing of Malacoherpesviridae RNAs attributable to host ADAR1 enzymes. The analysis of base neighbor preferences, structural features and expression profiles of molluscan ADAR1 supports the conservation of the enzyme function among metazoans and further suggested that ADAR1 exerts an antiviral role in mollusks.
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http://dx.doi.org/10.1186/s12862-019-1472-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6651903PMC
July 2019

Expansion and loss events characterized the occurrence of MIF-like genes in bivalves.

Fish Shellfish Immunol 2019 Oct 12;93:39-49. Epub 2019 Jul 12.

Department of Biology, University of Padova, via U. Bassi 58/b, 35121, Padova, Italy. Electronic address:

Macrophage migration inhibitory factor (MIF) dynamically connects innate and adaptive immune systems in vertebrate animals, allowing highly orchestrated systemic responses to various insults. The occurrence of MIF-like genes in non-vertebrate organisms suggests its origin from an ancestral metazoan gene, whose function is still a matter of debate. In the present work, by analyzing available genomic and transcriptomic data from bivalve mollusks, we identified 137 MIF-like sequences, which were classified into three types, based on phylogeny and conservation of key residues: MIF, D-DT, and the lineage-specific type MDL. Comparative genomics revealed syntenic conservation of homologous genes at the family level, the loss of D-DT in the Ostreidae family as well as the expansion of MIF-like genes in the Mytilidae family, possibly underpinning the neofunctionalization of duplicated gene copies. In M. galloprovincialis, MIF and one D-DT were mostly expressed in haemocytes and mantle rim of untreated animals, while D-DT paralogs often showed very limited expression, suggesting an accessory role or their persistence as relict genes.
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http://dx.doi.org/10.1016/j.fsi.2019.07.019DOI Listing
October 2019

An Evolutionary Perspective of Dopachrome Tautomerase Enzymes in Metazoans.

Genes (Basel) 2019 06 28;10(7). Epub 2019 Jun 28.

Department of Biology, University of Padova, Padova, 35121, Italy.

Melanin plays a pivotal role in the cellular processes of several metazoans. The final step of the enzymically-regulated melanin biogenesis is the conversion of dopachrome into dihydroxyindoles, a reaction catalyzed by a class of enzymes called dopachrome tautomerases. We traced (DCT) and (DCE) genes throughout metazoans and we could show that only one class is present in most of the phyla. While DCTs are typically found in deuterostomes, DCEs are present in several protostome phyla, including arthropods and mollusks. The respective DCEs belong to the gene family, previously reported to be taxonomically restricted to insects, bacteria and fungi. Mining genomic and transcriptomic data of metazoans, we updated the distribution of DCE/ genes, demonstrating their presence and active expression in most of the lophotrochozoan phyla as well as in copepods (Crustacea). We have traced one intronless DCE/ gene through most of the analyzed lophotrochozoan genomes and we could show that it was subjected to genomic diversification in some species, while it is conserved in other species. DCE/ was expressed in most phyla, although it showed tissue specific expression patterns. In the parasitic copepod DCE/ even belonged to the 100 most expressed genes. Both tissue specificity and high expression suggests that diverse functions of this gene family also evolved in other phyla apart from insects.
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http://dx.doi.org/10.3390/genes10070495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678240PMC
June 2019

A Needle in A Haystack: Tracing Bivalve-Associated Viruses in High-Throughput Transcriptomic Data.

Viruses 2019 03 1;11(3). Epub 2019 Mar 1.

School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794-5000, USA.

Bivalve mollusks thrive in environments rich in microorganisms, such as estuarine and coastal waters, and they tend to accumulate various particles, including viruses. However, the current knowledge on mollusk viruses is mainly centered on few pathogenic viruses, whereas a general view of bivalve-associated viromes is lacking. This study was designed to explore the viral abundance and diversity in bivalve mollusks using transcriptomic datasets. From analyzing RNA-seq data of 58 bivalve species, we have reconstructed 26 nearly complete and over 413 partial RNA virus genomes. Although 96.4% of the predicted viral proteins refer to new viruses, some sequences belong to viruses associated with bivalve species or other marine invertebrates. We considered short non-coding RNAs (sncRNA) and post-transcriptional modifications occurring specifically on viral RNAs as tools for virus host-assignment. We could not identify virus-derived small RNAs in sncRNA reads obtained from the oyster sample richest in viral reads. Single Nucleotide Polymorphism (SNP) analysis revealed 938 A-to-G substitutions occurring on the 26 identified RNA viruses, preferentially impacting the AA di-nucleotide motif. Under-representation analysis revealed that the AA motif is under-represented in these bivalve-associated viruses. These findings improve our understanding of bivalve viromes, and set the stage for targeted investigations on the specificity and dynamics of identified viruses.
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http://dx.doi.org/10.3390/v11030205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466128PMC
March 2019

Dual Analysis of Virus-Host Interactions: The Case of 1 and the Cupped Oyster .

Evol Bioinform Online 2019 22;15:1176934319831305. Epub 2019 Feb 22.

Department of Biology, University of Padova, Padova, Italy.

Dual analyses of the interactions between 1 (OsHV-1) and the bivalve during infection can unveil events critical to the onset and progression of this viral disease and can provide novel strategies for mitigating and preventing oyster mortality. Among the currently used "omics" technologies, dual transcriptomics (dual RNA-seq) coupled with the analysis of viral DNA in the host tissues has greatly advanced the knowledge of genes and pathways mostly contributing to host defense responses, expression profiles of annotated and unknown OsHV-1 open reading frames (ORFs), and viral genome variability. In addition to dual RNA-seq, proteomics and metabolomics analyses have the potential to add complementary information, needed to understand how a malacoherpesvirus can redirect and exploit the vital processes of its host. This review explores our current knowledge of "omics" technologies in the study of host-pathogen interactions and highlights relevant applications of these fields of expertise to the complex case of infections by OsHV-1, which currently threaten the mollusk production sector worldwide.
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http://dx.doi.org/10.1177/1176934319831305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388457PMC
February 2019

Induced expression of cathelicidins in trout (Oncorhynchus mykiss) challenged with four different bacterial pathogens.

J Pept Sci 2018 Jul 28;24(7):e3089. Epub 2018 May 28.

Department of Life Sciences, University of Trieste, Via L. Giorgieri 5, 34127, Trieste, Italy.

Cathelicidins are an important family of antimicrobial peptide effectors of innate immunity in vertebrates. Two members of this group, CATH-1 and CATH-2, have been identified and characterized in teleosts (ray-finned fish). In this study, we investigated the expression of these genes in different tissues of rainbow trout challenged with 4 different inactivated pathogens. By using qPCR, we detected a strong induction of both cath-1 and cath-2 genes within 24 hours after intraperitoneal inoculation with Lactococcus garvieae, Yersinia ruckeri, Aeromonas salmonicida, or Flavobacterium psychrophilum cells. Up to 700-fold induction of cath-2 was observed in the spleen of animals challenged with Y. ruckeri. Moreover, we found differences in the intensity and timing of gene up-regulation in the analyzed tissues. The overall results highlight the importance of cathelicidins in the immune response mechanisms of salmonids.
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http://dx.doi.org/10.1002/psc.3089DOI Listing
July 2018

Identification of a newly described OsHV-1 µvar from the North Adriatic Sea (Italy).

J Gen Virol 2018 05 26;99(5):693-703. Epub 2018 Mar 26.

Department of Biology, University of Padova, Padova (PD), Italy.

The surveillance activities for abnormal bivalve mortality events in Italy include the diagnosis of ostreid herpesvirus type 1 (OsHV-1) in symptomatic oysters. OsHV-1-positive oysters (Crassostrea gigas) were used as a source for in vivo virus propagation and a virus-rich sample was selected to perform shotgun sequencing based on Illumina technology. Starting from this unpurified supernatant sample from gills and mantle, we generated 3.5 million reads (2×300 bp) and de novo assembled the whole genome of an Italian OsHV-1 microvariant (OsHV-1-PT). The OsHV-1-PT genome encodes 125 putative ORFs, 7 of which had not previously been predicted in other sequenced Malacoherpesviridae. Overall, OsHV-1-PT displays typical microvariant OsHV-1 genome features, while few polymorphisms (0.08 %) determine its uniqueness. As little is known about the genetic determinants of OsHV-1 virulence, comparing complete OsHV-1 genomes supports a better understanding of the virus pathogenicity and provides new insights into virus-host interactions.
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http://dx.doi.org/10.1099/jgv.0.001042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994699PMC
May 2018

Oyster RNA-seq Data Support the Development of Genomics.

Front Microbiol 2017 9;8:1515. Epub 2017 Aug 9.

Department of Biology, University of PaduaPadua, Italy.

The family of double-stranded DNA (dsDNA) includes viruses able to infect marine mollusks and detrimental for worldwide aquaculture production. Due to fast-occurring mortality and a lack of permissive cell lines, the available data on the few known provide only partial support for the study of molecular virus features, life cycle, and evolutionary history. Following thorough data mining of bivalve and gastropod RNA-seq experiments, we used more than five million reads to improve the annotation of viral genomes and to characterize viral InDels, nucleotide stretches, and SNPs. Both genome and protein domain analyses confirmed the evolutionary diversification and gene uniqueness of known . However, the presence of -like sequences integrated within genomes of phylogenetically distant invertebrates indicates broad diffusion of these viruses and indicates the need for confirmatory investigations. The manifest co-occurrence of OsHV-1 genotype variants in single RNA-seq samples of provide further support for the diversification. In addition to simple sequence motifs inter-punctuating viral ORFs, recombination-inducing sequences were found to be enriched in the OsHV-1 and AbHV1-AUS genomes. Finally, the highly correlated expression of most viral ORFs in multiple oyster samples is consistent with the burst of viral proteins during the lytic phase.
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http://dx.doi.org/10.3389/fmicb.2017.01515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552708PMC
August 2017

Myticalins: A Novel Multigenic Family of Linear, Cationic Antimicrobial Peptides from Marine Mussels (Mytilus spp.).

Mar Drugs 2017 Aug 22;15(8). Epub 2017 Aug 22.

Department of Life Sciences, University of Trieste, Via Giorgieri 5, Trieste 34127, Italy.

The application of high-throughput sequencing technologies to non-model organisms has brought new opportunities for the identification of bioactive peptides from genomes and transcriptomes. From this point of view, marine invertebrates represent a potentially rich, yet largely unexplored resource for discovery due to their adaptation to diverse challenging habitats. Bioinformatics analyses of available genomic and transcriptomic data allowed us to identify myticalins, a novel family of antimicrobial peptides (AMPs) from the mussel , and a similar family of AMPs from spp., named modiocalins. Their coding sequence encompasses two conserved N-terminal (signal peptide) and C-terminal (propeptide) regions and a hypervariable central cationic region corresponding to the mature peptide. Myticalins are taxonomically restricted to Mytiloida and they can be classified into four subfamilies. These AMPs are subject to considerable interindividual sequence variability and possibly to presence/absence variation. Functional assays performed on selected members of this family indicate a remarkable tissue-specific expression (in gills) and broad spectrum of activity against both Gram-positive and Gram-negative bacteria. Overall, we present the first linear AMPs ever described in marine mussels and confirm the great potential of bioinformatics tools for the discovery of bioactive peptides in non-model organisms.
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http://dx.doi.org/10.3390/md15080261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577615PMC
August 2017

Structural Insights into the Mode of Action of the Peptide Antibiotic Copsin.

Biochemistry 2017 09 6;56(37):4992-5001. Epub 2017 Sep 6.

Institute of Microbiology, Swiss Federal Institute of Technology, ETH Zurich , CH-8093 Zurich, Switzerland.

Defensins make up a class of cysteine-rich antimicrobial peptides, expressed by virtually all eukaryotes as part of their innate immune response. Because of their unique mode of action and rapid killing of pathogenic microbes, defensins are considered promising alternatives to clinically applied antibiotics. Copsin is a defensin-like peptide, previously identified in the mushroom Coprinopsis cinerea. It exerts its activity against a range of Gram-positive bacteria by binding to the peptidoglycan precursor lipid II and prevention of proper cell wall formation. In this study, we present a new workflow for the generation, production, and activity-driven selection of copsin derivatives, based on their expression in Pichia pastoris. One hundred fifty-two single-amino acid mutants and combinations thereof allowed the identification of k-copsin, a peptide variant exhibiting significantly enhanced activity against Bacillus subtilis and Staphylococcus aureus. Furthermore, we performed in silico characterizations of membrane interactions of copsin and k-copsin, in the presence and absence of lipid II. The molecular dynamics data highlighted a high variability in lipid II binding, with a preference for the MurNAc moiety with 47 and 35% of the total contacts for copsin and k-copsin, respectively. Mutated amino acids were located in loop regions of k-copsin and shown to be crucial in the perturbation of the bacterial membrane. These structural studies provide a better understanding of how defensins can be developed toward antibacterial therapies less prone to resistance issues.
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http://dx.doi.org/10.1021/acs.biochem.7b00697DOI Listing
September 2017

Diversity and evolution of TIR-domain-containing proteins in bivalves and Metazoa: New insights from comparative genomics.

Dev Comp Immunol 2017 05 18;70:145-164. Epub 2017 Jan 18.

University of Trieste, Department of Life Sciences, Via Licio Giorgieri 5, 34127 Trieste, Italy. Electronic address:

The Toll/interleukin-1 receptor (TIR) domain has a fundamental role in the innate defence response of plants, vertebrate and invertebrate animals. Mostly found in the cytosolic side of membrane-bound receptor proteins, it mediates the intracellular signalling upon pathogen recognition via heterotypic interactions. Although a number of TIR-domain-containing (TIR-DC) proteins have been characterized in vertebrates, their evolutionary relationships and functional role in protostomes are still largely unknown. Due to the high abundance and diversity of TIR-DC proteins in bivalve molluscs, we investigated this class of marine invertebrates as a case study. The analysis of the available genomic and transcriptomic data allowed the identification of over 400 full-length sequences and their classification in protein families based on sequence homology and domain organization. In addition to TLRs and MyD88 adaptors, bivalves possess a surprisingly large repertoire of intracellular TIR-DC proteins, which are conserved across a broad range of metazoan taxa. Overall, we report the expansion and diversification of TIR-DC proteins in several invertebrate lineages and the identification of many novel protein families possibly involved in both immune-related signalling and embryonic development.
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http://dx.doi.org/10.1016/j.dci.2017.01.014DOI Listing
May 2017

Colloidal Surface Active Maghemite Nanoparticles for Biologically Safe Cr(VI) Remediation: from Core-Shell Nanostructures to Pilot Plant Development.

Chemistry 2016 Sep 16;22(40):14219-26. Epub 2016 Aug 16.

Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro, 35020, Italy.

The present study is aimed at the exploration of achievable improvements for Cr(VI) ex situ and in situ water remediation by using novel naked colloidal maghemite (γ-Fe2 O3 ) nanoparticles (surface active maghemite nanoparticles, SAMNs). The reliability of SAMNs for Cr(VI) binding and removal was demonstrated, and SAMN@Cr(VI) complex was characterized, as well as the covalent nature of the absorption was unequivocally proved. SAMNs were structurally and magnetically well conserved after Cr(VI) binding. Thus, in consideration of their affinity for Cr(VI) , SAMNs were exploited in a biological model system, mimicking a real in situ application. The assay evidenced a progressive reduction of revertant colonies of Salmonella typhimurium TA100 strain, as maghemite nanoparticles concentration increased, till the complete suppression of Cr(VI) mutagen effect. Finally, an automatic modular pilot system for continuous magnetic removal and recovery of Cr(VI) from water is proposed. SAMNs, thanks to their colloidal, binding, and catalytic properties, represent a promising tool as a reliable nanomaterial for water remediation by Cr(VI) .
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http://dx.doi.org/10.1002/chem.201600544DOI Listing
September 2016

The miRNA biogenesis in marine bivalves.

PeerJ 2016 7;4:e1763. Epub 2016 Mar 7.

Department of Biology, University of Padova , Padova , Italy.

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.
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http://dx.doi.org/10.7717/peerj.1763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4793324PMC
March 2016

Analysis of synonymous codon usage patterns in sixty-four different bivalve species.

PeerJ 2015 14;3:e1520. Epub 2015 Dec 14.

Department of Life Sciences, University of Trieste , Trieste , Italy.

Synonymous codon usage bias (CUB) is a defined as the non-random usage of codons encoding the same amino acid across different genomes. This phenomenon is common to all organisms and the real weight of the many factors involved in its shaping still remains to be fully determined. So far, relatively little attention has been put in the analysis of CUB in bivalve mollusks due to the limited genomic data available. Taking advantage of the massive sequence data generated from next generation sequencing projects, we explored codon preferences in 64 different species pertaining to the six major evolutionary lineages in Bivalvia. We detected remarkable differences across species, which are only partially dependent on phylogeny. While the intensity of CUB is mild in most organisms, a heterogeneous group of species (including Arcida and Mytilida, among the others) display higher bias and a strong preference for AT-ending codons. We show that the relative strength and direction of mutational bias, selection for translational efficiency and for translational accuracy contribute to the establishment of synonymous codon usage in bivalves. Although many aspects underlying bivalve CUB still remain obscure, we provide for the first time an overview of this phenomenon in this large, commercially and environmentally important, class of marine invertebrates.
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http://dx.doi.org/10.7717/peerj.1520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690358PMC
December 2015

Structural and Antimicrobial Features of Peptides Related to Myticin C, a Special Defense Molecule from the Mediterranean Mussel Mytilus galloprovincialis.

J Agric Food Chem 2015 Oct 16;63(42):9251-9. Epub 2015 Oct 16.

Department of Biology, University of Padova , Via Ugo Bassi 58/B, 35131 Padova, Italy.

Mussels (Mytilus spp.) have a large repertoire of cysteine-stabilized α,β peptides, and myticin C (MytC) was identified in some hundreds of transcript variants after in vivo immunostimulation. Using a sequence expressed in Italian mussels, we computed the MytC structure and synthesized the mature MytC and related peptide fragments (some of them also prepared in oxidized form) to accurately assess their antibacterial and antifungal activity. Only when tested at pH 5 was the reduced MytC as well as reduced and oxidized fragments including structural β-elements able to inhibit Gram-positive and -negative bacteria (MIC ranges of 4-32 and 8-32 μM, respectively). Such fragments caused selective Escherichia coli killing (MBC of 8-32 μM) but scarcely inhibited two fungal strains. In detail, the antimicrobial β-hairpin MytC[19-40]SOX caused membrane-disrupting effects in E. coli despite its partially ordered conformation in membrane-mimetic environments. In perspective, MytC-derived peptides could be employed to protect acidic mucosal tissues, in cosmetic and food products, and, possibly, as adjuvants in aquaculture.
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http://dx.doi.org/10.1021/acs.jafc.5b03491DOI Listing
October 2015

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

PLoS One 2015 23;10(7):e0133173. Epub 2015 Jul 23.

Department of Molecular Medicine, Histology Unit, University of Padova, Padova, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133173PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4512711PMC
May 2016

Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis.

Genome Biol Evol 2015 Jul 21;7(8):2203-19. Epub 2015 Jul 21.

Department of Life Sciences, University of Trieste, Italy

We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling.
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http://dx.doi.org/10.1093/gbe/evv133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4558851PMC
July 2015

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

Dev Comp Immunol 2015 Oct 27;52(2):255-68. Epub 2015 May 27.

Department of Biology, University of Padua, via U. Bassi 58/b, 35121 Padua, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.dci.2015.05.001DOI Listing
October 2015

An updated molecular basis for mussel immunity.

Fish Shellfish Immunol 2015 Sep 18;46(1):17-38. Epub 2015 Feb 18.

Department of Biology, University of Padua, Via U. Bassi 58/b, 35131 Padua, Italy. Electronic address:

Non-self recognition with the consequent tolerance or immune reaction is a crucial process to succeed as living organisms. At the same time the interactions between host species and their microbiome, including potential pathogens and parasites, significantly contribute to animal life diversity. Marine filter-feeding bivalves, mussels in particular, can survive also in heavily anthropized coastal waters despite being constantly surrounded by microorganisms. Based on the first outline of the Mytilus galloprovincialis immunome dated 2011, the continuously growing transcript data and the recent release of a draft mussel genome, we explored the available sequence data and scientific literature to reinforce our knowledge on the main gene-encoded elements of the mussel immune responses, from the pathogen recognition to its clearance. We carefully investigated molecules specialized in the sensing and targeting of potential aggressors, expected to show greater molecular diversification, and outlined, whenever relevant, the interconnected cascades of the intracellular signal transduction. Aiming to explore the diversity of extracellular, membrane-bound and intracellular pattern recognition receptors in mussel, we updated a highly complex immune system, comprising molecules which are described here in detail for the first time (e.g. NOD-like receptors) or which had only been partially characterized in bivalves (e.g. RIG-like receptors). Overall, our comparative sequence analysis supported the identification of over 70 novel full-length immunity-related transcripts in M. galloprovincialis. Nevertheless, the multiplicity of gene functions relevant to immunity, the involvement of part of them in other vital processes, and also the lack of a refined mussel genome make this work still not-exhaustive and support the development of more specific studies.
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http://dx.doi.org/10.1016/j.fsi.2015.02.013DOI Listing
September 2015

The genome of the Pacific oyster Crassostrea gigas brings new insights on the massive expansion of the C1q gene family in Bivalvia.

Dev Comp Immunol 2015 Mar 11;49(1):59-71. Epub 2014 Nov 11.

Department of Life Sciences, University of Trieste, Via Licio Giorgieri 5, 34127 Trieste (TS), Italy. Electronic address:

C1q domain-containing (C1qDC) proteins are regarded as important players in the innate immunity of bivalve mollusks and other invertebrates and their highly adaptive binding properties indicate them as efficient pathogen recognition molecules. Although experimental studies support this view, the molecular data available at the present time are not sufficient to fully explain the great molecular diversification of this family, present in bivalves with hundreds of C1q coding genes. Taking advantage of the fully sequenced genome of the Pacific oyster Crassostrea gigas and more than 100 transcriptomic datasets, we: (i) re-annotated the oyster C1qDC loci, thus identifying the correct genomic organization of 337 C1qDC genes, (ii) explored the expression pattern of oyster C1qDC genes in diverse developmental stages and adult tissues of unchallenged and experimentally treated animals; (iii) investigated the expansion of the C1qDC gene family in all major bivalve subclasses. Overall, we provide a broad description of the functionally relevant features of oyster C1qDC genes, their comparative expression levels and new evidence confirming that a gene family expansion event has occurred during the course of Bivalve evolution, leading to the diversification of hundreds of different C1qDC genes in both the Pteriomorphia and Heterodonta subclasses.
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http://dx.doi.org/10.1016/j.dci.2014.11.007DOI Listing
March 2015

RNA sequencing and de novo assembly of the digestive gland transcriptome in Mytilus galloprovincialis fed with toxinogenic and non-toxic strains of Alexandrium minutum.

BMC Res Notes 2014 Oct 14;7:722. Epub 2014 Oct 14.

Laboratory of Genetics, Department of Life Sciences, University of Trieste, Via Licio Giorgeri 5, Trieste 34126, Italy.

Background: The Mediterranean mussel Mytilus galloprovincialis is marine bivalve with a relevant commercial importance as well as a key sentinel organism for the biomonitoring of environmental pollution. Here we report the RNA sequencing of the mussel digestive gland, performed with the aim: a) to produce a high quality de novo transcriptome assembly, thus improving the genetic and molecular knowledge of this organism b) to provide an initial assessment of the response to paralytic shellfish poisoning (PSP) on a molecular level, in order to identify possible molecular markers of toxin accumulation.

Results: The comprehensive de novo assembly and annotation of the transcriptome yielded a collection of 12,079 non-redundant consensus sequences with an average length of 958 bp, with a high percentage of full-length transcripts. The whole-transcriptome gene expression study indicated that the accumulation of paralytic toxins produced by the dinoflagellate Alexandrium minutum over a time span of 5 days scarcely affected gene expression, but the results need further validation with a greater number of biological samples and naturally contaminated specimens.

Conclusion: The digestive gland reference transcriptome we produced significantly improves the data collected from previous sequencing efforts and provides a basic resource for expanding functional genomics investigations in M. galloprovincialis. Although not conclusive, the results of the RNA-seq gene expression analysis support the classification of mussels as bivalves refractory to paralytic shellfish poisoning and point out that the identification molecular biomarkers of PSP in the digestive gland of this organism is problematic.
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http://dx.doi.org/10.1186/1756-0500-7-722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203926PMC
October 2014

Target capture and massive sequencing of genes transcribed in Mytilus galloprovincialis.

Biomed Res Int 2014 30;2014:538549. Epub 2014 Jun 30.

Department of Biology, University of Padua, Via U. Bassi 58/b, 32121 Padua, Italy.

Next generation sequencing (NGS) allows fast and massive production of both genome and transcriptome sequence datasets. As the genome of the Mediterranean mussel Mytilus galloprovincialis is not available at present, we have explored the possibility of reducing the whole genome sequencing efforts by using capture probes coupled with PCR amplification and high-throughput 454-sequencing to enrich selected genomic regions. The enrichment of DNA target sequences was validated by real-time PCR, whereas the efficacy of the applied strategy was evaluated by mapping the 454-output reads against reference transcript data already available for M. galloprovincialis and by measuring coverage, SNPs, number of de novo sequenced introns, and complete gene sequences. Focusing on a target size of nearly 1.5 Mbp, we obtained a target coverage which allowed the identification of more than 250 complete introns, 10,741 SNPs, and also complete gene sequences. This study confirms the transcriptome-based enrichment of gDNA regions as a good strategy to expand knowledge on specific subsets of genes also in nonmodel organisms.
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http://dx.doi.org/10.1155/2014/538549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4101229PMC
April 2015

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

Fish Shellfish Immunol 2014 Nov 5;41(1):37-44. Epub 2014 Jun 5.

Department of Biology, University of Padova, Padova, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.fsi.2014.05.023DOI Listing
November 2014

Aquatic ecology of the oyster pathogens Vibrio splendidus and Vibrio aestuarianus.

Environ Microbiol 2015 Apr 7;17(4):1065-80. Epub 2014 May 7.

Department of Life, Earth and Environmental Sciences (DISTAV), University of Genoa, Corso Europa, 26, Genoa, 16132, Italy.

The ecology of the oyster pathogens Vibrio splendidus and Vibrio aestuarianus in the brackish aquatic environment was extensively investigated in this study. By conducting laboratory experiments under natural setting conditions, it was shown that V. splendidus LGP32 strain generally exhibits longer persistence in both seawater and sediment than V. aestuarianus 01/32 strain. Both strains maintained viability and culturability for longer times in the sediment, suggesting that this compartment may represent a suitable niche for their persistence in the environment. In addition, both strains attached to chitin particles and copepods, the efficiency of attachment being higher in V. splendidus than in V. aestuarianus. Similarly, LGP32 strain showed a greater capability to form biofilm on poly-vinyl chloride (PVC) surfaces than 01/32 strain. LGP32 and 01/32 strains were also capable of entering a viable but non-culturable state after extended incubation at 5°C, a condition commonly found during cold season in the aquatic brackish environment. These results are consistent with field data collected during a 2-year sampling campaign in the northern Adriatic Sea and provide background information on the mechanisms promoting V. splendidus and V. aestuarianus persistence in coastal water, thus contributing to a better understanding of the epidemiology of the associated diseases.
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http://dx.doi.org/10.1111/1462-2920.12484DOI Listing
April 2015

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

Dev Comp Immunol 2014 Aug 4;45(2):300-12. Epub 2014 Apr 4.

Ecologie des Systèmes Marins Côtiers (EcoSym), CNRS-Université de Montpellier 2-IRD, cc 093, place E. Bataillon, 34095 Montpellier, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.dci.2014.03.021DOI Listing
August 2014

Bivalve omics: state of the art and potential applications for the biomonitoring of harmful marine compounds.

Mar Drugs 2013 Nov 1;11(11):4370-89. Epub 2013 Nov 1.

Chromatin Structure and Evolution (CHROMEVOL) Group, Department of Biological Sciences, Florida International University, North Miami, FL 33181, USA.

The extraordinary progress experienced by sequencing technologies and bioinformatics has made the development of omic studies virtually ubiquitous in all fields of life sciences nowadays. However, scientific attention has been quite unevenly distributed throughout the different branches of the tree of life, leaving molluscs, one of the most diverse animal groups, relatively unexplored and without representation within the narrow collection of well established model organisms. Within this Phylum, bivalve molluscs play a fundamental role in the functioning of the marine ecosystem, constitute very valuable commercial resources in aquaculture, and have been widely used as sentinel organisms in the biomonitoring of marine pollution. Yet, it has only been very recently that this complex group of organisms became a preferential subject for omic studies, posing new challenges for their integrative characterization. The present contribution aims to give a detailed insight into the state of the art of the omic studies and functional information analysis of bivalve molluscs, providing a timely perspective on the available data resources and on the current and prospective applications for the biomonitoring of harmful marine compounds.
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http://dx.doi.org/10.3390/md11114370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853733PMC
November 2013