Stefania Domeneghetti

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.

Colloidal Surface Active Maghemite Nanoparticles for Biologically Safe CrVI Remediation: from Core‐Shell Nanostructures to Pilot Plant Development

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) .

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

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.

Target Capture and Massive Sequencing of Genes Transcribed in Mytilus galloprovincialis

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.

Serum amyloid A in marine bivalves: An acute phase and innate immunity protein

Serum amyloid A (SAA) is among the most potent acute phase proteins (APP) in vertebrates. After injury, its early expression can dramatically increase to promote the recruitment of immuno-competent cells, expression of pro-inflammatory proteins and the activation of the innate immune defences. Although APP have been studied in many vertebrates, only recently their search was extended to invertebrates and the finding of SAA-like molecules has opened new questions on the immune-regulatory functions of these soluble proteins in the animal kingdom. Taking advantage of the considerable amount of genomic and transcriptomic data currently available, we retrieved 51 SAA-like proteins in several protostome taxa comprising 21 marine bivalve species and basal metazoans. In addition to vertebrate-like SAAs, we identified a second protein type with peculiar features. In the bivalves Crassostrea gigas and Mytilus galloprovincialis, both digital expression analysis and qPCR data indicated an induction of the classical SAA after bacterial challenge.

Immune-Related Signaling in Mussel and Bivalves

Abstract A variety of signals and regulatory molecules coming from the exterior and interior environment significantly influences the vital cell processes and the overall organism responses in health and disease. The immune systems of invertebrates are amazingly diverse and, considering economically relevant bivalve spp., only a few pathways of intracellular signal transduction have been clearly and specifically associated to the innate immune responses (other signaling pathways are still fragmentary or not exclusive to the immune defense). Mainly based on DNA sequencing technologies and supported by functional analyses, not only have the Toll/NF- k B, MAPKs, and IL-17 signaling been demonstrated to be present in Mytilus spp. and other bivalves, but also the identification of gene-encoded molecules expected to work as cytokines, hormones, and neuropeptides reinforces the hypothesis of the ancestry of the immune-neuroendocrine network. Although not exhaustive, this chapter aims to outline the current knowledge and research trends on immune-related bivalve signaling.