Host responses to infection with Bonamia ostreae : comparison between resistant and wild flat oysters

Transcription

1 Host responses to infection with Bonamia ostreae : comparison between resistant and wild flat oysters B. Morga, I. Arzul, N. Faury, C. Garcia, S. Lerond, M. Robert, B. Chollet, S. Lapegue and T. Renault. Laboratory of Genetic and Pathology- IFREMER- La Tremblade- France

2 Bonamiosis: an interesting model to study host/parasite interactions Wild population Resistant population Infection Host Parasite Ostrea edulis Bonamia ostreae Defence Bonamia ostreae is an intra-haemocytic parasite. Haemocyte infiltration is associated with B. ostreae infection. Phagocytosis is one of the main response against pathogens. Parasite is engulfed in haemocytes and not destroyed after in vitro infection. Resistance is observed in natural conditions and has been used for genetic improvement.

3 Objectives of the present study 1-Identification of genes potentially involved in susceptibility or resistance to bonamiosis. -Production of SSH libraries 2-Comparison of the response of resistant and wild flat oysters in the context of a cohabitation challenge. -Measure of expression levels of genes of interest by real time PCR -Measure of haemocyte activities by flow cytometry (ROS, esterase and phagocytosis)

4 Introduction//Objectives//Biological material and methods//results//conclusios Biological material Wild susceptible oysters (18 months old) were collected from Quiberon bay Resistant oysters were produced in 2007 at IFREMER Argenton Haemolymph was withdrawn from the adductor muscle and filtered at 75 µm. Parasite Bonamia ostreae was obtained by purification from highly infected oysters according to Mialhe et al. (1988).

5 SSH libraries design Haemocytes from resistant oysters+ parasites Haemocytes from susceptible oysters+ parasites Haemolymph samples were pooled Haemocytes were maintained with parasites (1:5) for 2 hours in flasks Total RNA was extracted using trizol reagent Libraries were prepared using BD Clontech kit Forward bank: identification of genes expressed by haemocytes from resistant oysters Haemocytes from susceptible oysters+ parasites Haemocytes from resistant oysters+ parasites Reverse bank: identification of genes expressed by haemocytes from wild oysters

6 SSH results Forward Bank Reverse Bank sequenced clones -246 show an homology > 10-4 in NCBI data base sequenced clones -145 show an homology > 10-4 in NCBI data base IAP, OGST, Metallothionein, SOD,Ec-SOD and Fas-ligand

7 Experimental infection design Tank 1 Batch 1 Batch Resistant oysters +60 injected oysters 100 Resistant oysters +60 injected oysters Tank 3 Tank Wild oysters +60 injected oysters 100 Wild oysters +60 injected oysters Tank 4 6 months of challenge -3 sampling times (4, 5 and 6 months) -4 months (n=25 per tank) -5 months (n=25 per tank) -6 months (remaining oysters)

8 Sample analysis: 1.Individual ponction of haemolymph placed on ice 2. Tissue imprint and examination Evaluation of infection level 3. Construction of haemolymph pools according to the infection statut (- or +) and the type of population Wild (W) or Resistant (R) 4. Analysis of pools by: (-) free of parasite (+) infected oyster (+) < 5 parasites (++) < 20 parasites (+++) > 50 parasites Flow cytometry analysis including ROS, esterases and phagocytosis Real-time PCR some candidate gene identified throught SSH

9 Infection monitoring 4 months 5 months 6 months Infected Mortality Infected Mortality Infected Mortality R Batch 1 7/25 11 (2/6) 2/25 2 (0/2) 1/37 0 Batch 2 6/25 14 (1/6) 2/25 3 (1/3) 6/23 5 (1/1) W Batch 1 2/25 24 (7/18) 4/25 8 (1/2) 5/17 0 Batch 2 6/25 15 (5/8) 8/25 9 (2/4) 2/17 0 W infected oyster after 6months 42/166 (25%) R infected oyster after 6months 29/178 (16%)

10 Infection monitoring Cumulative mortality Wild mortality (30%) > Resistant mortality (17%) Nb of oyster W R Ranking of oyster in function of the level 4 months 5 months 6 months Number of infected wild oyster 2+ (15) > number of infected resistant oyster 2+ (5)

11 Haemocyte activities Esterase activities ROS production N=4 pools W-=80 oysters W+=20 oysters R-=83 oysters R+=17 oysters No significant difference between W- and W+ No significant difference between R- and R+

12 Haemocyte activities Haemocyte activities Phagocytosis N=4 pools W-=80 oysters W+=20 oysters R-=83 oysters R+=17 oysters No significant difference between W- and W+ Significant difference between R- and R+, R+< R-

13 Molecular responses Normalisator: W- /W+ and R-/R+ Calibrator:EF1-α

14 Molecular responses Normalisator: W- /W+ and R-/R+ Calibrator:EF1-α

15 Conclusions New data concerning flat oyster genome. 6 genes of interest were selected from the SSH libraries. Infection by cohabitation induced mortality in both wild and resistant populations, however mortality rate and infection frequency were higher in wild oysters compared to resistant ones. Phagocytosis capacity decreased in resistant infected oysters. Similar results were observed during a previous injection experiment. ROS production and esterase activities did not show significant modulation while a reduced ROS production was observed in resistant oysters injected with parasites compared to wild oysters.

16 Conclusions Significant differences could be observed between gene expression levels in infected resistant and wild oysters Down regulation of Ec-SOD in infected resistant oysters compared to non infected ones Up regulation of metalothionein in infected resistant oysters compared to non infected ones Up regulation of two genes involved in apoptosis pathway (IAP and Fas ligand in wild infected oyster and infected resistant oyster. Up regulation of Fas ligand has already been observed in other intracellular parasite models like Trypanosoma cruzy, Cryptosporidium parvum and Plasmodium sp.

17 Perspectives Study the apoptosis by flow cytometry with specific labelled and completed the apopotosis pathway by molecular approches. Molecular data obtained by SSH libraries allow us to complete the genetic card of Ostrea edulis and to identify some markers potentialy involved in resistance. (Phd Student of Estelle Harrang IFREMER )

18 Thanks for your attention LGP team Ifremer Région Poitou Charente