Construction of an Ostrea edulis database from genomic and expressed sequence tags (ESTs) obtained from Bonamia ostreae infected haemocytes: Development of an immune-enriched oligo-microarray.

The flat oyster, Ostrea edulis, is one of the main farmed oysters, not only in Europe but also in the United States and Canada. Bonamiosis due to the parasite Bonamia ostreae has been associated with high mortality episodes in this species. This parasite is an intracellular protozoan that infects haemocytes, the main cells involved in oyster defence. Due to the economical and ecological importance of flat oyster, genomic data are badly needed for genetic improvement of the species, but they are still very scarce. The objective of this study is to develop a sequence database, OedulisDB, with new genomic and transcriptomic resources, providing new data and convenient tools to improve our knowledge of the oyster's immune mechanisms. Transcriptomic and genomic sequences were obtained using 454 pyrosequencing and compiled into an O. edulis database, OedulisDB, consisting of two sets of 10,318 and 7159 unique sequences that represent the oyster's genome (WG) and de novo haemocyte transcriptome (HT), respectively. The flat oyster transcriptome was obtained from two strains (naïve and tolerant) challenged with B. ostreae, and from their corresponding non-challenged controls. Approximately 78.5% of 5619 HT unique sequences were successfully annotated by Blast search using public databases. A total of 984 sequences were identified as being related to immune response and several key immune genes were identified for the first time in flat oyster. Additionally, transcriptome information was used to design and validate the first oligo-microarray in flat oyster enriched with immune sequences from haemocytes. Our transcriptomic and genomic sequencing and subsequent annotation have largely increased the scarce resources available for this economically important species and have enabled us to develop an OedulisDB database and accompanying tools for gene expression analysis. This study represents the first attempt to characterize in depth the O. edulis haemocyte transcriptome in response to B. ostreae through massively sequencing and has aided to improve our knowledge of the immune mechanisms of flat oyster. The validated oligo-microarray and the establishment of a reference transcriptome will be useful for large-scale gene expression studies in this species.

Role of microRNAs in the immunity process of the flat oyster Ostrea edulis against bonamiosis.

MicroRNAs (miRNAs) are small (∼22nt) non-coding regulatory single strand RNA molecules that reduce stability and/or translation of sequence-complementary target. miRNAs are a key component of gene regulatory networks and have been involved in a wide variety of biological processes, such as signal transduction, cell proliferation and apoptosis. Many miRNAs are broadly conserved among the animal lineages and even between invertebrates and vertebrates. The European flat oyster Ostrea edulis is highly susceptible to infection with Bonamia ostreae, an intracellular parasite able to survive and proliferate within oyster haemocytes. Mollusc haemocytes play a key role in the immune response of molluscs as main cellular effectors. The roles of miRNAs in the immune response of O. edulis to bonamiosis were analysed using a commercial microarray platform (miRCURY LNA™ v2, Exiqon) for miRNAs. Expression of miRNAs in haemocytes from oysters with different bonamiosis intensity was compared. Differential expression was detected in 63 and 76 miRNAs when comparing heavily-affected with non-affected oysters and with lightly-affected ones, respectively. Among them, 19 miRNAs are known to be linked to immune response, being responsible of proliferation and activation of macrophages, inflammation, apoptosis and/or oxidative damage, which is consistent with the modulation of their expression in oyster haemocytes due to bonamiosis.

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.

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.

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.

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 ß-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 ß-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.

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.