Oyster hemolymph is a complex and dynamic ecosystem hosting bacteria, protists and viruses.

BACKGROUND: The impact of the microbiota on host fitness has so far mainly been demonstrated for the bacterial microbiome. We know much less about host-associated protist and viral communities, largely due to technical issues. However, all microorganisms within a microbiome potentially interact with each other as well as with the host and the environment, therefore likely affecting the host health. RESULTS: We set out to explore how environmental and host factors shape the composition and diversity of bacterial, protist and viral microbial communities in the Pacific oyster hemolymph, both in health and disease. To do so, five oyster families differing in susceptibility to the Pacific oyster mortality syndrome were reared in hatchery and transplanted into a natural environment either before or during a disease outbreak. Using metabarcoding and shotgun metagenomics, we demonstrate that hemolymph can be considered as an ecological niche hosting bacterial, protist and viral communities, each of them shaped by different factors and distinct from the corresponding communities in the surrounding seawater. Overall, we found that hemolymph microbiota is more strongly shaped by the environment than by host genetic background. Co-occurrence network analyses suggest a disruption of the microbial network after transplantation into natural environment during both non-infectious and infectious periods. Whereas we could not identify a common microbial community signature for healthy animals, OsHV-1 µVar virus dominated the hemolymph virome during the disease outbreak, without significant modifications of other microbiota components. CONCLUSION: Our study shows that oyster hemolymph is a complex ecosystem containing diverse bacteria, protists and viruses, whose composition and dynamics are primarily determined by the environment. However, all of these are also shaped by oyster genetic backgrounds, indicating they indeed interact with the oyster host and are therefore not only of transient character. Although it seems that the three microbiome components respond independently to environmental conditions, better characterization of hemolymph-associated viruses could change this picture.

A European epidemiological survey of Vibrio splendidus clade shows unexplored diversity and massive exchange of virulence factors.

The Vibrio splendidus clade has previously been associated with epidemic outbreaks of various aquatic animals, as in the case of the cupped oyster, Crassostrea gigas. To investigate whether involved strains could present a clonal origin and to identify possible alternative background carriage animals or zooplankton, a large epidemiological survey was conducted on isolates of the splendidus clade. For this purpose, Vibrio strains were isolated from various samples including oysters, mussels, sediments, zooplankton, and sea water on the basis of a North/South gradient of the European sea water zone (Ireland, The Netherlands, France, Italy, and Spain). A total of 435 isolates were successfully associated to the V. splendidus clade using real time polymerase chain reaction with 16S specific primers and probes. A multiple-locus variable-number tandem-repeat analysis (VNTR) was conducted on all isolates based on a multiplex PCR-VNTR with a set of primer pairs designed from the V. tasmaniensis LGP32 genome. Preliminary validation of the primers on a set of collection strains from the V. splendidus clade confirmed that the former V. splendidus-related LGP32 and relative strains were related to V. tasmaniensis rather than to the type strain V. splendidus LMG 4042. The VNTR analysis was then successfully conducted on 335 isolates which led to the characterization of 87 different profiles. Our results showed that (1) the high diversity of VNTR did not enlighten significant correlation between a specific pattern and the origin of collected samples. However, populations isolated from animal samples tend to differ from those of the background environment; (2) oyster mortality events could not be linked to the clonal proliferation of a particular VNTR type. However, few different patterns seemed successively associated with samples collected during peaks of oyster's mortality. (3) Finally, no correlation could be seen between specific VNTR patterns and sequence phylogeny of the virulence factors vsm and ompU that were detected among strains isolated during as well as outside mortality events. These results, combined with incongruence observed between the ompU and vsm phylogenetic trees, suggested both large diffusion of strains and massive lateral gene transfer within the V. splendidus clade.

The effect of environmental salinity on the proteome of the sea bass (Dicentrarchus labrax L.).

The European sea bass, Dicentrarchus labrax L., tolerates a range of salinities from freshwater to hyper-saline. To study differences in protein expression, fish were reared in both freshwater and seawater. After 3-month acclimation, gill and intestine epithelia were collected and the soluble protein extracted. In all, 362 spots were differentially expressed in the gills and intestines of fishes reared in seawater compared to those from freshwater. Fifty differential protein spots were excised from a colloidal Coomassie-stained gel. Nine separate protein spots were identified unambiguously by mass spectrometry and database searching. Among the six proteins over-expressed in gill cells in seawater, five were cytoskeleton proteins and one was the aromatase cytochrome P450. In gill cells under freshwater conditions, the two over-expressed proteins identified were the prolactin receptor and the major histocompatibility complex class II beta-antigen. In intestinal cells under freshwater conditions, the Iroquois homeobox protein Ziro5 was upregulated over ninefold. The expression of these proteins, their possible direct or indirect roles in the adaptation of D. labrax to salinity, and their correspondences with a previous study are discussed.

Growth hormone and Prolactin-1 gene transcription in natural populations of the black-chinned tilapia Sarotherodon melanotheron acclimatised to different salinities.

The effects of salinity on the expression of genes coding for growth hormone (GH) and prolactin-1 (PRL1) were studied in various natural populations of the black-chinned tilapia Sarotherodon melanotheron from West Africa. Individuals were sampled in June 2005 in six locations in Senegal and the Gambia, at various salinities between 0 and 101. The poorest condition factors were recorded in the most saline sampling site and the best growth in the fish from a marine environment. The pituitary GH mRNA levels were significantly higher in fish adapted to seawater, whereas the PRL1 mRNA levels were highest in fish adapted to fresh- and brackish water. These results show that the PRL1 mRNA levels seem to reflect relatively well the differences in environmental salinity, in contrast to those of GH, which would tend instead to reflect the individual growth in each environment. However, no relation could be found between growth in the hypersaline areas and the expression profile of GH. Although the fish analysed were morphologically identical, the expression of genes coding for GH and PRL1 showed large differences between individuals. This inter-individual variation in gene expression remains poorly understood.

First evidence of the activation of Cg-timp, an immune response component of Pacific oysters, through a damage-associated molecular pattern pathway.

In a previous work, we characterized a Crassostrea gigas cDNA (Cg-timp) encoding a protein which presents all the features of vertebrate tissue inhibitor of metalloproteinase (TIMP). The expression pattern of this gene led us to propose that Cg-timp is an important factor in oyster wound healing and defense mechanisms. Here we describe the analysis of Cg-timp expression in oysters challenged by live or dead bacteria as well as by bacterial secretory/excretory products and metalloproteinase. Surprisingly, bacterial secretory/excretory products activate Cg-timp gene expression whereas heat-inactivated ones do not. To address the question of the signal transduction pathway involved in Cg-timp gene activation, we isolated and sequenced Cg-timp promoter and upstream region. A 1-kb genomic DNA fragment flanking the 5'-end of the gene contains several regulatory elements and notably three NF-kappaB binding sites. The potential involvement of these motifs in Cg-timp gene regulation is discussed.

Molecular characterization of two isoforms of defensin from hemocytes of the oyster Crassostrea gigas.

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. We previously characterized the first AMP from an oyster, a defensin, that was shown to be continuously expressed in the mantle of Crassostrea gigas. In this study, we report the cDNA cloning of two new isoforms of the defensin AMP family (Cg-defh1 and Cg-defh2) from the hemocytes of the oyster. The deduced amino acid sequences reveal two peptides of 73 amino acid residues with a mature portion consisting of 43 amino acid residues. Cg-Defh1 and Cg-Defh2 share 86% amino acid identity and belong to the "arthropod-molluscs defensin family". qRT-PCR analyses indicate that Cg-defh2 is continuously expressed in the hemocytes of C. gigas. In addition, after a bacterial challenge, the level of Cg-defh2 transcripts decreases dramatically in the circulating hemocyte population and this decrease can be correlated with an increase of Cg-defh2 transcripts in the gill and the mantle tissue, suggesting a possible migration of the hemocytes expressing Cg-defh2 towards the tissues implicated in the first defense barrier of the oyster. These results would suggest an important role of Cg-Defh2 in the oyster response to a microbial challenge.

Molecular characterisation of prolactin and analysis of extrapituitary expression in the European sea bass Dicentrarchus labrax under various salinity conditions.

Although prolactin has been demonstrated to be the main hormone involved in adaptation to dilute media in several freshwater teleosts, few studies have been conducted in marine teleosts. In the Mediterranean, the sea bass Dicentrarchus labrax inhabits environments ranging from the open sea to coastal lagoons, where salinity varies greatly. We characterised the prolactin (prl) gene and analysed its expression in two organs (gill and intestine) in D. labrax acclimated to either freshwater or seawater. A 2819 bp long sequence encompassing the prl gene and a part (282 bp) of the promoter were identified, and these comprised 5 coding exons separated by 4 introns. Prolactin was similarly expressed in fresh- and seawater adapted fish, although expression in gills was significantly greater than in the intestine. Nonetheless, individuals unable to successfully regulate osmotic balance in freshwater presented overall low expression rates. Results are discussed according to the mechanism of sea bass adaptation in the wild and to their life cycle between open sea and lagoons. Finally, a phylogenetic analysis indicated that teleosts are not branched according to their life-history features (e.g. seawater vs. freshwater habitats), and no signature of positive selection was detected across the phylogeny of the prl gene in teleosts.

Field evidence for density-dependent effects in the trematode Microphallus papillorobustus in its manipulated host, Gammarus insensibilis.

Numerous studies have demonstrated that parasites with complex life cycles frequently manipulate the phenotype of their hosts to increase their transmission rate. Little is known, however, concerning density-dependent processes within infrapopulations of manipulative parasites--whether parasites cooperate to manipulate the host, whether competition counteracts with these potential cooperative benefits, or both. Here we explored these ideas, focusing on the association between the manipulative trematode Microphallus papillorobustus and its second intermediate host, the gammarid Gammarus insensibilis. From the data collected in the field, we found no evidence that co-occurring M. papillorobustus individuals benefit from the presence of conspecifics; instead, individuals in larger infrapopulations suffered reduced size and fecundity. Thus, the net effect of increasing density suggests that competition rather than cooperation is the dominant force in infrapopulations of M. papillorobustus.