Oyster RNA-seq data support the development of Malacoherpesviridae genomics

The family of double-stranded DNA (dsDNA) Malacoherpesviridae 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 Malacoherpesviridae 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 Malacoherpesviridae 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 Malacoherpesviridae. However, the presence of Malacoherpesviridae-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 Crassostrea gigas provide further support for the Malacoherpesviridae 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

An updated molecular basis for mussel immunity

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

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

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

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

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

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

MgC1q, a novel C1q-domain-containing protein involved in the immune response of Mytilus galloprovincialis

9 páginas, 6 figuras, 1 tablaIn this study, we present the characterization of a newly identified gene, MgC1q, revealed in suppression subtractive hybridization and cDNA libraries from immunostimulated mussels. Based on sequence homology, molecular architecture and domain similarity, this new C1q-domain-containing gene may be classified as a member of the C1q family and, therefore, part of the C1q–TNF superfamily. The expression of MgC1q was detected all along the mussel ontogeny, being detectable within 2 h post-fertilization, with a notable increase after 1 month and continuing to increase until 3 months. Measurable transcript levels were also evident in all analyzed tissues of naïve adult mussels, and the hemocytes showed the highest expression levels. Experimental infection of adult mussels with Gram positive or Gram negative bacteria significantly modulated the MgC1q expression, and confirmed it as an immune-related gene. Intra- and inter-individual sequence analyses revealed extraordinary diversity of MgC1q at both the DNA and cDNA levels. While further research is needed to define its function, our data indicate that MgC1q is a pattern recognition molecule able to recognize pathogens during innate immune responses in Myitilus galloprovincialis. The high sequence variability suggests that somatic diversification of these nonself recognition molecules could have occurred.This work has been funded by the EU Integrated Project FOOD-CT-2005-007103 and AGL2008-05111/ACU from the Spanish Ministerio de Ciencia e Innovación. Camino Gestal wishes to acknowledge additional funding from the Spanish Ministerio de Educación y Ciencia through the “Ramón y Cajal” Contract.Peer reviewe

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

[ENG]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 programsWork granted by MIUR and Co.Ri.La. to PV. LV is currently supported by the FP7-KBBE-2010-4-266157 Bivalife project. Work in the laboratory of MPC is funded by grants to consolidated research groups (ref GIC07/26-IT-393-07, Basque Government), and to the unit of formation and research (UFI ref 11/37, University of the Basque Country) and projects Nanoretox (ref CP-FP 214478-2, UE 7th FP) and Nanocancer (ref CTM2009-13477, Spanish Ministry of Science and Innovation

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

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

Study of diseases and the immune system of bivalves using molecular biology and genomics

24 páginas, 3 tablasEnvironmental chemico-physical factors, pathogens, and biological interactions constantly affect organism physiology and behavior. Invertebrates, including bivalve mollusks do not possess acquired immunity. Their defense mechanisms rely on an innate, non-adaptive immune system employing circulating cells and a large variety of molecular effectors. The mechanisms underlying host defense depend on the presence of functional proteins in appropriate quantities, within a crucial time window. These proteins are encoded by genes whose transcription is tightly coordinated by complex programs of gene expression. Currently available advanced techniques allow the evaluation of this gene expression, expanding our understanding of the behavior and function of cells and tissues under varying conditions. In particular, DNA microarray technology enables measurement of a large predetermined set of known genes or sequences. Expressed sequence tag sequencing from redundant, normalized, subtractive hybridization libraries is a robust method for sampling the protein encoding genes that are expressed within a tissue. The elimination of microorganisms by defense cells is a dynamic process that involves integrating synthesis of granule proteins during differentiation, migration onto sites of infection, phagocytosis and killing of microorganisms, modulation of their effector cells, and finally apoptosis. Understanding how this complex biological process is regulated can best be addressed using a systems biology approach to the study of organisms and populations in order to more effectively decipher the continuous challenge between two genomes, i.e., evolving host-pathogen interactionsPeer reviewe

Myticalins: A novel multigenic family of linear, cationic antimicrobial peptides from marine mussels (Mytilus spp.)

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 de novo 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 Mytilus galloprovincialis, and a similar family of AMPs from Modiolus 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 de novo discovery of bioactive peptides in non-model organisms