Benjamin Morga

Detection and description of a particular Ostreid herpesvirus 1 genotype associated with massive mortality outbreaks of Pacific oysters, Crassostrea gigas, in France in 2008

Ostreid herpesvirus 1 (OsHV-1) infections have been reported around the world and are associated with high mortalities of the Pacific oyster (Crassostrea gigas). In the summer 2008, abnormal mortality rates ranging from 80% to 100% were reported in France and affected only Pacific oysters. Analyses of oyster samples collected during mortality outbreaks demonstrated a significant detection of OsHV-1 (75% of analysed batches), which appeared stronger than previous years. DNA sequencing based on C and IA regions was carried out on 28 batches of OsHV-1 infected Pacific oysters collected in 2008. Polymorphisms were described in both the C and IA regions and characterized a genotype of OsHV-1 not already reported and termed OsHV-1 microVar. A microsatellite zone present in the C region showed several deletions. Additionally, 44 isolates collected in France and in the USA, from 1995 to 2007 were sequenced and compared to the 2008 sequences. The analyses of 76 sequences showed OsHV-1 microVar detection only in 2008 isolates. These data suggest that OsHV-1 microVar can be assumed as an emergent genotype.

Identification of genes from flat oyster Ostrea edulis as suitable housekeeping genes for quantitative real time PCR

Bonamia ostreae is an intrahaemocytic protozoan affecting Ostrea edulis. The parasite multiplies within haemocytes without being degraded and involves changes in cellular activities. Studies aiming at better understanding host response to a pathogen at the transcriptome levels are frequently based on the use of real time PCR assays, which require some reference genes. However, very few sequence data is available for O. edulis in public databases. Subtracted cDNA libraries were constructed from the O. edulis haemocytes in order to identify genes involved in host reactions against the parasite and quantitative real time PCR assays were developed to study expression of these genes. In this context, identification of reference genes and study of their relative expression stability were required for quantitative real time PCR normalization. The expression of 5 potential candidate reference genes from O. edulis (ie elongation factor 1 alpha (EF1-α), 60S ribosomal protein L5 (L5), glyceraldehyde 3-phosphate-dehydrogenase (GAPDH), polyubiquitin (Ubiq) and β-actin (ACT)) was studied using RNAs extracted from pools of haemocytes in contact with the parasite B. ostreae and haemocytes alone. Gene expression was quantified by real time PCR and expression stability was analysed with two analytical approaches GeNorm and NormFinder. GAPDH and EF1-α were identified as the most stable genes with the GeNorm analysis. Whatever were the tested conditions, EF1-α was also found as the most stable gene using Normfinder. The less stable gene was β-actin although this gene is commonly used as housekeeping gene in many studies. Our results suggest using GAPDH and EF1-α combined as reference genes when studying expression levels in haemocytes of O. edulis. In addition, the complete ORF of these two genes was characterized.

Molecular responses of Ostrea edulis haemocytes to an in vitro infection with Bonamia ostreae

Bonamiosis due to the parasite Bonamia ostreae is a disease affecting the flat oyster Ostrea edulis. B. ostreae is a protozoan, affiliated to the order of haplosporidia and to the cercozoan phylum. This parasite is mainly intracellular, infecting haemocytes, cells notably involved in oyster defence mechanisms. Suppression subtractive hybridisation (SSH) was carried out in order to identify oyster genes differentially expressed during an infection of haemocytes with B. ostreae. Forward and reverse banks allowed obtaining 1104 and 1344 clones respectively, among which 391 and 480 clones showed a differential expression between both tested conditions (haemocytes alone versus haemocytes in contact with parasites). ESTs of interest including genes involved in cytoskeleton, respiratory chain, detoxification membrane receptors, and immune system were identified. The open reading frames of two selected genes (galectin and IRF-like) were completely sequenced and characterized. Real time PCR assays were developed to study the relative expression of candidate ESTs during an in vitro infection of haemocytes by live and dead parasites. Haemocyte infection with B. ostreae induced an increased expression of omega glutathione S-transferase (OGST), superoxide dismutase (SOD), tissue inhibitor of metalloproteinase (TIMP), galectin, interferon regulatory factor (IRF-like) and filamin genes.

Infection with the protozoan parasite Bonamia ostreae modifies in vitro haemocyte activities of flat oyster Ostrea edulis

Bonamia ostreae is an intracellular protozoan parasite, infecting haemocytes of the European flat oyster Ostrea edulis. Oyster defence mechanisms mainly rely on haemocytes. In the present study in vitro interactions between parasites and flat oyster haemocytes were investigated using flow cytometry and light microscopy. Haemocyte parameters including: non specific esterase activity, reactive oxygen species (ROS) production and phagocytosis were monitored using flow cytometry after 2 h cell incubation with live and dead B. ostreae. Two ratios of parasites per haemocyte were tested (5:1 and 10:1), haemocytes alone were used as controls and the experiment was carried out three times. Flow cytometry revealed a decrease of non specific esterase activities and ROS production by haemocytes after incubation with live parasites, while there was little difference in phagocytosis activity when compared with controls. Similarly, dead parasites induced a decrease in haemocyte activities but to a lesser extent compared to live parasites. These results suggest that B. ostreae actively contributes to the modification of haemocyte activities in order to ensure its own intracellular survival.

Cellular and molecular responses of haemocytes from Ostrea edulis during in vitro infection by the parasite Bonamia ostreae

Bonamia ostreae is a protozoan, affiliated to the order Haplosporidia and to the phylum Cercozoa. This parasite is intracellular and infects haemocytes, cells notably involved in oyster defence mechanisms. Bonamiosis due to the parasite B. ostreae is a disease affecting the flat oyster, Ostrea edulis. The strategies used by protozoan parasites to circumvent host defence mechanisms remain largely unknown in marine bivalve molluscs. In the present work, in vitro experiments were carried out in order to study the interactions between haemocytes from O. edulis and purified parasite, B. ostreae. We monitored cellular and molecular responses of oyster haemocytes by light microscopy, flow cytometry and real-time PCR 1, 2, 4 and 8h p.i. Light microscopy was used to measure parasite phagocytosis by oyster haemocytes. Parasites were observed inside haemocytes 1h p.i. and the parasite number increased during the time course of the experiment. Moreover, some bi-nucleated and tri-nucleated parasites were found within haemocytes 2 and 4h p.i., respectively, suggesting that the parasite can divide inside haemocytes. Host responses to B. ostreae were investigated at the cellular and molecular levels using flow cytometry and real-time PCR. Phagocytosis capacity of haemocytes, esterase activity and production of radical oxygen species appeared modulated during the infection with B. ostreae. Expression levels of expressed sequence tags selected in this study showed variations during the experiment as soon as 1h p.i. An up-regulation of galectin (OeGal), cytochrome p450 (CYP450), lysozyme, omega GST (OGST), super oxide dismutase Cu/Zn (Oe-SOD Cu/Zn) and a down-regulation of the extracellular super oxide dismutase SOD (Oe-EcSOD) were observed in the presence of the parasite. Finally, the open reading frames of both SODs (Oe-SOD Cu/Zn and Oe-EcSOD) were completely sequenced. These findings provide new insights into the cellular and molecular bases of the host-parasite interactions between the flat oyster, O. edulis, and the parasite, B. ostreae.

Effects of temperature and salinity on the survival of Bonamia ostreae, a parasite infecting flat oysters Ostrea edulis.

Bonamiosis due to the intrahaemocytic protistan parasite Bonamia ostreae is a European endemic disease affecting the flat oyster Ostrea edulis. The parasite has been described in various ecosystems from estuaries to open sea, but no clear correlation has yet been demonstrated between disease development and environmental parameters. In this study, the effect of temperature and salinity on the survival of purified parasites maintained in vitro in seawater was investigated by flow cytometry. Purified parasites were incubated in various seawater media (artificial seawater, natural seawater, seabed borewater) at various temperatures (4, 15 and 25 degrees C) and subjected to a range of salinities from 5 to 45 g l(-1). Parasites were collected after 12, 24 and 48 h of incubation for flow cytometry analyses including estimation of parasite mortality and parasite viability through detection of non-specific esterase activities. Artificial seawater appeared unsuitable for parasite survival, and results for all media showed a significantly lower survival at 25 degrees C compared to 4 degrees C and 15 degrees C. Moreover, high salinities (> or = 35 g l(-1)) favoured parasite survival and detection of esterase activities. Flow cytometry appears to be a suitable technique to investigate survival and activities of unicellular parasites like B. ostreae under varied conditions. Although these results contribute to a better understanding of existing interactions between the parasite B. ostreae and its environment, validation through epidemiological surveys in the field is also needed.

A High Load of Non-neutral Amino-Acid Polymorphisms Explains High Protein Diversity Despite Moderate Effective Population Size in a Marine Bivalve With Sweepstakes Reproduction

Marine bivalves show among the greatest allozyme diversity ever reported in Eukaryotes, putting them historically at the heart of the neutralist−selectionist controversy on the maintenance of genetic variation. Although it is now acknowledged that this high diversity is most probably a simple consequence of a large population size, convincing support for this explanation would require a rigorous assessment of the silent nucleotide diversity in natural populations of marine bivalves, which has not yet been done. This study investigated DNA sequence polymorphism in a set of 37 nuclear loci in wild samples of the flat oyster Ostrea edulis. Silent diversity was found to be only moderate (0.7%), and there was no departure from demographic equilibrium under the Wright-Fisher model, suggesting that the effective population size might not be as large as might have been expected. In accordance with allozyme heterozygosity, nonsynonymous diversity was comparatively very high (0.3%), so that the nonsynonymous to silent diversity ratio reached a value rarely observed in any other organism. We estimated that one-quarter of amino acid-changing mutations behave as neutral in O. edulis, and as many as one-third are sufficiently weakly selected to segregate at low frequency in the polymorphism. Finally, we inferred that one oyster is expected to carry more than 4800 non-neutral alleles (or 4.2 cM−1). We conclude that a high load of segregating non-neutral amino-acid polymorphisms contributes to high protein diversity in O. edulis. The high fecundity of marine bivalves together with an unpredictable and highly variable success of reproduction and recruitment (sweepstakes reproduction) might produce a greater decoupling between Ne and N than in other organisms with lower fecundities, and we suggest this could explain why a higher segregating load could be maintained for a given silent mutation effective size.

Comparison of haemocytic parameters among flat oyster Ostrea edulis stocks with different susceptibility to bonamiosis and the Pacific oyster Crassostrea gigas.

Farming of the flat oyster Ostrea edulis in Europe is severely constrained by the protozoan Bonamia ostreae. The introduction of the resistant species Crassostrea gigas has been a relief for the farmers, while the pilot programmes to select O. edulis strains resistant to bonamiosis performed in various countries can be seen as a promising strategy to minimise the effects of bonamiosis. However, the physiological bases of this differential susceptibility remain unknown. A search for an explanation of the intra and interspecific differences in oyster susceptibility to bonamiosis was accomplished by comparing some immune parameters among various O. edulis stocks and C. gigas. On December 2003, naïve and Bonamia-relatively resistant flat oysters from Ireland, Galician flat oysters and Pacific oysters C. gigas were deployed in a Galician area affected by bonamiosis; haemolymph samples were taken in February and May 2004. A new oyster deployment at the same place was carried out on June 2004 and haemolymph sampling was performed on April 2005. On November 2004, new sets of Irish flat oysters and C. gigas were deployed in Ireland and haemolymph sampling was performed in June 2005. Various haemocytic parameters were measured: total and differential haemocyte count, phagocytic ability, respiratory burst (superoxide anion [O(2)(-)] and hydrogen peroxide [H(2)O(2)]) and nitric oxide [NO] production. The comparison of the parameters was carried out at 3 levels: (1) between O. edulis and C. gigas, (2) among O. edulis stocks with different susceptibility to bonamiosis, and (3) between Bonamia-infected and non infected O. edulis. In addition, haemocyte-B. ostreaein vitro encounters were performed to analyse interspecific differences in the haemocytic respiratory burst, using flow cytometry. Significant differences associated with total and differential haemocyte count, and respiratory burst between O. edulis and C. gigas were detected, which could be linked to differences in susceptibility to bonamiosis between both species. Additionally, significant changes in total and differential haemocyte count, and respiratory burst of O. edulis associated with B. ostreae infection were found. However, no consistent difference in any haemocyte parameter between the O. edulis stocks involved in the study was recorded.

First description of French V. tubiashii strains pathogenic to mollusk: I. Characterization of isolates and detection during mortality events.

Nine dominant bacterial isolates were obtained from different batches of Crassostrea gigas spat experiencing high mortality rates in a French experimental hatchery/nursery in 2007. Using phenotypic analysis combined with multilocus sequence analysis, the isolates were shown to be genetically close to the Vibrio tubiashii type strain. Based on (1) analyses of the recA gene sequences; (2) the results of DNA-DNA hybridization assays between 07/118 T2 (LMG 27884=CECT 8426), which is a representative strain, and the V. tubiashii type strain (69%); and (3) phenotypic traits, the bacteria were classified in a group close to American V. tubiashii strain. Its virulence (70% of mortalities) and the toxicity of the extracellular products of 07/118 T2 was demonstrated (41% of mortalities). Moreover, a QPCR diagnostic tool targeting the gyrB gene was developed to investigate the epidemiological significance of V. tubiashii in French oyster mortality outbreaks recorded by the national surveillance network. Of the 21 batches originating from hatcheries, only two were positive, whereas V. tubiashii DNA could not be detected in any of the batches of moribund animals collected in field/outdoor facilities. These results demonstrate the existence of a group of virulent V. tubiashii in France that episodically infect C. gigas.

Complete mitochondrial DNA sequence of the European flat oyster Ostrea edulis confirms Ostreidae classification

BackgroundBecause of its typical architecture, inheritance and small size, mitochondrial (mt) DNA is widely used for phylogenetic studies. Gene order is generally conserved in most taxa although some groups show considerable variation. This is particularly true in the phylum Mollusca, especially in the Bivalvia. During the last few years, there have been significant increases in the number of complete mitochondrial sequences available. For bivalves, 35 complete mitochondrial genomes are now available in GenBank, a number that has more than doubled in the last three years, representing 6 families and 23 genera. In the current study, we determined the complete mtDNA sequence of O. edulis, the European flat oyster. We present an analysis of features of its gene content and genome organization in comparison with other Ostrea, Saccostrea and Crassostrea species.ResultsThe Ostrea edulis mt genome is 16 320 bp in length and codes for 37 genes (12 protein-coding genes, 2 rRNAs and 23 tRNAs) on the same strand. As in other Ostreidae, O. edulis mt genome contains a split of the rrnL gene and a duplication of trnM. The tRNA gene set of O. edulis, Ostrea denselamellosa and Crassostrea virginica are identical in having 23 tRNA genes, in contrast to Asian oysters, which have 25 tRNA genes (except for C. ariakensis with 24). O. edulis and O. denselamellosa share the same gene order, but differ from other Ostreidae and are closer to Crassostrea than to Saccostrea. Phylogenetic analyses reinforce the taxonomic classification of the 3 families Ostreidae, Mytilidae and Pectinidae. Within the Ostreidae family the results also reveal a closer relationship between Ostrea and Saccostrea than between Ostrea and Crassostrea.ConclusionsOstrea edulis mitogenomic analyses show a high level of conservation within the genus Ostrea, whereas they show a high level of variation within the Ostreidae family. These features provide useful information for further evolutionary analysis of oyster mitogenomes.