Molecular diversity of antimicrobial effectors in the oyster Crassostrea gigas

Background: To gain insight into the molecular diversity of antimicrobial peptides and proteins in the oyster Crassostrea gigas, we characterized and compared the sequence polymorphism of the antimicrobial peptides (AMPs), Cg-Defensins (Cg-Defs) and Cg-Proline Rich peptide (Cg-Prp), and of the bactericidal permeability increasing protein, Cg-BPI. For that, we analyzed genomic and transcript sequences obtained by specific PCR amplification and in silico searches. Results: High diversification among the three antimicrobial effectors was evidenced by this polymorphism survey. On the basis of sequence phylogenies, each AMP aggregates into clearly defined groups of variants and is the product of a multigenic family displaying a variety of gene structures. In contrast, Cg-bpi forms a single group and is encoded by a single gene copy. Moreover, we identified for both AMPs several genetic mechanisms of diversification such as recombination, parallel mutations leading to phylogenetic homoplasy and indel events. In addition, the non synonymous to synonymous substitutions ratio by codon (dN/dS) revealed several negatively and positively selected sites for both AMPs, suggesting that directional selection pressures have shaped their sequence variations. Conclusions: This study shows for the first time in a mollusc that antimicrobial peptides and proteins have been subject to distinct patterns of diversification and we evidence the existence of different evolutionary routes leading to such sequence variability

Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas

Background: Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. Findings: Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate beta-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CS alpha beta-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. Conclusions: We provide here the first report showing that big defensins form a family of antimicrobial peptides diverse not only in terms of sequences but also in terms of genomic organization and regulation of gene expression

Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase

Background: Although bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide polymorphisms and microsatellites in coding regions, and for identifying genes when the entire genome sequence of C. gigas becomes available. Description: In the present paper, we report the production of 40,845 high-quality ESTs that identify 29,745 unique transcribed sequences consisting of 7,940 contigs and 21,805 singletons. All of these new sequences, together with existing public sequence data, have been compiled into a publicly-available Website http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.htm l. Approximately 43% of the unique ESTs had significant matches against the SwissProt database and 27% were annotated using Gene Ontology terms. In addition, we identified a total of 208 in silico microsatellites from the ESTs, with 173 having sufficient flanking sequence for primer design. We also identified a total of 7,530 putative in silico, single-nucleotide polymorphisms using existing and newly-generated EST resources for the Pacific oyster. Conclusion: A publicly-available database has been populated with 29,745 unique sequences for the Pacific oyster Crassostrea gigas. The database provides many tools to search cleaned and assembled ESTs. The user may input and submit several filters, such as protein or nucleotide hits, to select and download relevant elements. This database constitutes one of the most developed genomic resources accessible among Lophotrochozoans, an orphan clade of bilateral animals. These data will accelerate the development of both genomics and genetics in a commercially-important species with the highest annual, commercial production of any aquatic organism

Whole Transcriptome Profiling of Successful Immune Response to Vibrio Infections in the Oyster Crassostrea gigas by Digital Gene Expression Analysis

The cultivated Pacific oyster Crassostrea gigas has suffered for decades large scale summer mortality phenomenon resulting from the interaction between the environment parameters, the oyster physiological and/or genetic status and the presence of pathogenic microorganisms including Vibrio species. To obtain a general picture of the molecular mechanisms implicated in C. gigas immune responsiveness to circumvent Vibrio infections, we have developed the first deep sequencing study of the transcriptome of hemocytes, the immunocompetent cells. Using Digital Gene Expression (DGE), we generated a transcript catalog of up-regulated genes from oysters surviving infection with virulent Vibrio strains (Vibrio splendidus LGP32 and V. aestuarianus LPi 02/41) compared to an avirulent one, V. tasmaniensis LMG 20012T. For that an original experimental infection protocol was developed in which only animals that were able to survive infections were considered for the DGE approach. We report the identification of cellular and immune functions that characterize the oyster capability to survive pathogenic Vibrio infections. Functional annotations highlight genes related to signal transduction of immune response, cell adhesion and communication as well as cellular processes and defence mechanisms of phagocytosis, actin cytosqueleton reorganization, cell trafficking and autophagy, but also antioxidant and anti-apoptotic reactions. In addition, quantitative PCR analysis reveals the first identification of pathogen-specific signatures in oyster gene regulation, which opens the way for in depth molecular studies of oyster-pathogen interaction and pathogenesis. This work is a prerequisite for the identification of those physiological traits controlling oyster capacity to survive a Vibrio infection and, subsequently, for a better understanding of the phenomenon of summer mortality

Genotoxic and epigenetic effects of diuron in the Pacific oyster: in vitro evidence of interaction between DNA damage and DNA methylation

Recently, research has contributed to better knowledge on the occurrence of pesticides in coastal water by identifying frequently detected substances, their concentration range and their acute and chronic toxicity for organisms. Pesticide pollution is of particular concern in France due to important agricultural activities and presence of several exoreic catchment areas that vehicle pesticides up to coastal waters, impacting non-target marine species. Several ecotoxicology questions remain to be addressed concerning the long-term effects of chronic pesticide exposure and the mechanisms involved in adaptation to chemical stress. In the present study, we brought new insights on the genetic and epigenetic effects of the herbicide diuron in oyster genitors. During gametogenesis, we exposed Crassostrea gigas to environmentally realistic herbicide concentrations (0.2–0.3 μg L−1 during two 7-day periods at half-course and end of gametogenesis). Diuron exposure was shown to decrease global DNA methylation and total methyltransferase activity in whole oyster tissue; this is consistent with the previous observation of a significant decrease in DNMT1 gene expression. Diuron effect seemed to be tissue-specific; hypermethylation was detected in the digestive gland, whereas diuron exposure had no effect on gill and gonad tissue. The genotoxicity of diuron was confirmed by the detection of one adduct in gonad DNA. By using in vitro approaches and human DNMT1 (DNMT1 has not been purified yet in bivalves), the presence of DNA lesions (adduct, 8-oxodGuo) was shown to interfere with DNMT1 activity, indicating a complex interaction between DNA damage and DNA methylation. Based on our results, we propose mechanisms to explain the effect of diuron exposure on DNA methylation, a widespread epigenetic mark

Quelles stratégies alternatives aux antibiotiques en aquaculture ?

National audienceIn spite of restricting rules, antibiotics are still used in aquaculture for prevention, especially during critical phases (early stages; metamorphosis; transfer), but also during on-growing. Besides improving husbandry techniques and prophylaxis, which is still possible, alternative methods are now available or in progress. Commercial probiotics for land animals are increasingly used for shrimp and fish farming, though every species or rearing condition should require particular experiments to determine the most suitable microbial preparations and doses. The variability in response to probiotics, and the lack of reliable data hinder the use of such practices in routine, indeed. These commercial preparations contain Lactobacillus, Bacillus, or other bacteria belonging to related genera, or yeast. They are not suitable for bivalve molluscs like scallops, oysters, and clams. Only few marine bacteria have been documented to protect efficiently larval bivalves against infection. However, many problems remain unsolved regarding legal authorization, production, preservation, and retailing of such preparations with gram-negative bacteria. Probiotics may display multiple effects, possibly combining bacterial antagonism to some effects on the host, e.g. stimulating immunity, or growth, though the modes of action are not fully understood. Probiotics are not as efficient as antibiotics in case of bacterial infection, however. Prebiotics like fructo-oligosaccharides (FOS) may also improve animal health, since they favor the establishment of commensal or beneficial gut microbiota. For instance, an experiment showed that FOS could increase growth of turbot fry. A third alternative may come from antimicrobial peptides, which have been described in many organisms. These peptides have wide spectra of action. They can kill gram negative and gram positive bacteria, envelop viruses, yeasts, and moulds. Several peptides were recently discovered in shrimp and oysters, where they are essential elements of innate defense, in the absence of acquired immunity. These molecules could advantageously replace antibiotics, since they are less susceptible to cause resistance in the target microorganisms, due to their direct action on membranes, and to their fast degradability, which avoid the accumulation of residues.Malgré des réglementations contraignantes, l’usage des antibiotiques en préventif est encore répandu en aquaculture, particulièrement pendant les phases critiques (stades précoces, métamorphose, transferts d’animaux), mais aussi chez des animaux en croissance. En plus des améliorations que l’on peut encore apporter en matière de zootechnie et de prophylaxie traditionnelle, des méthodes alternatives sont maintenant disponibles ou en développement. Les préparations microbiennes commercialisées pour les élevages terrestres sont de plus en plus utilisées pour les élevages de crevettes et de poissons, mais chaque espèce ou élevage demanderait des expérimentations particulières pour déterminer les produits et les doses les plus efficaces. En effet, les réponses des animaux à l’ajout des probiotiques peuvent être variables et l’absence de données fiables freine leur application en routine. Les probiotiques d’origine terrestre contenant des Lactobacillus, des Bacillus ou d’autres bactéries de genres connexes, ou bien encore des levures, ne conviennent pas pour les mollusques bivalves comme les huîtres, les coquilles St Jacques, les palourdes. Seules quelques bactéries marines sélectionnées protègent les larves de bivalves contre les infections bactériennes. Cependant, l’utilisation pratique de ces bactéries gram (-) pose de nombreux problèmes d’autorisation légale, de production, de conservation et de distribution. L’intérêt des probiotiques réside dans leurs effets multiples, qui associent à des activités antibactériennes, des effets sur l’hôte telles que la stimulation de la réponse immunitaire ou celle de la croissance, bien que les mécanismes d’action ne soient pas clairement identifiés. Par contre ils n’ont pas la même efficacité que les antibiotiques pour stopper une infection. Les prébiotiques comme les fructo-oligosaccharides constituent une autre possibilité d’améliorer la santé des animaux, et des essais ont montré leur efficacité chez les alevins de turbot par exemple. Une troisième alternative est représentée par les peptides antimicrobiens. Ces molécules ont un large spectre d’activité antimicrobienne. Ils peuvent tuer des bactéries gram (-) et gram (+), des champignons ou des virus enveloppés. Plusieurs de ces peptides viennent d’être découverts chez les invertébrés marins (crevettes et huîtres), où ils sont un élément essentiel de la défense de ces animaux sans immunité acquise. Ils pourraient remplacer avantageusement les antibiotiques à terme. Ils devraient générer moins de résistances chez les microorganismes cibles, car ils agissent sur les membranes cellulaires et ils devraient être plus vite dégradés sans produire de résidus