Detection of Bonamia ostreae in larvae of flat oysters, Ostrea edulis

Transcription

1 Detection of Bonamia ostreae in larvae of flat oysters, Ostrea edulis Isabelle Arzul, Aimé Langlade, Bruno Chollet, Maeva Robert, Emmanuelle Omnes, Sophie Lerond, Yann Couraleau, Jean-Pierre Joly, Cyrille François, Céline Garcia LGP, IFREMER La Tremblade - LER/MPL, IFREMER La Trinité-sur-Mer Annual Meeting of NRLs for Mollusc Diseases, 23 & 24 March 2010, Nantes

2 Parasite life cycle Infection Natural host: Ostrea edulis? Entry and release routes remain uncertain Photo: B. Morga Protozoan parasite : Bonamia ostreae Adult oysters are supposed to be more affected by the disease. However, juveniles can be found infected (Lynch et al. 2005; Lallias et al. 2008) Photo: Ifremer Intrahaemocytic Sometimes extracellular Presence in the gonad suspected (Van Banning 1990)

3 Flat oyster life cycle External fecondation in the palleal cavity of female oysters Incubation of larvae for days Photo: E. Bédier Planctonic phase (~2-3 weeks) Metamorphosis and settlement Close contact between adults and larvae which could favour transmission of Bonamia ostreae?

4 Study site : Quiberon Bay Carnac Penthièvre Le Pô Beaumer Main French site for flat oyster spat collection Men er Roué Larvae fishing Monitoring of flat oyster reproduction (SRC & IFREMER) Spawner sampling Google Earth Site known to be infected with Bonamia ostreae since 1979

5 Sampling Spawners were collected weekly by diving between May and August in 2007, 2008 and 2009 After opening the shell, incubating oysters were selected Ethanol (95 ) fixation for molecular analyses Ethanol (95 ) fixation of gills for molecular analyses Spawners Larvae Davidson fixation for in situ hybridization tests Tissue sections for histology and in situ hybridization

6 Detection methods PCR (Cochennec et al. 2000) + for the detection of the parasite Real time PCR (Robert et al. 2009) for parasite quantification In situ Hybridation (Cochennec et al. 2000) for the confirmation of the infection RFLP (Cochennec et al. 2003; Hine et al. 2001) for the determination of the parasite species Histology (not performed on larvae) for the detection of other pathogens and/or lesions

7 Results (1) Adults Larvae Adult- Larvae 2007 PCR 4/31 1/31 1/31 31 Histology 2/31 incubating R t PCR 5/31 7/31 3/31 spawners Total positives 23% (7) 23% (7) 3/ PCR 13/53 9/53 3/53 53 Histology 9/53 incubating R t PCR 16/53 10/53 4/53 spawners Total positives 47% (25) 24 % (13) 8/ PCR 8/36 5/36 5/36 36 Histology In progress incubating R t PCR 12/36 10/36 4/36 spawners Total positives 36% (13) 30% (11) 6/36

8 Results (1) Result disparity (R t PCR more sensitive) between diagnostic tools probably due to the low infection levels Spawners (23-47%) appear more often positive than larvae (23-30%) Parasite detection in larvae for 32% to 46% of incubating spawners detected positives.

9 Real time PCR Results (2) Parasite loads were not significantly different between larvae and adults RFLP Bonamia ostreae mainly detected. Detection of B. exitiosa in one larvae sample collected in 2008 (confirmed by sequencing)

10 Results (3) In situ hybridation Spawners 2/4 12/19 5/8 Larvae ND 6/9 1/5 Number of oysters (spawners) or samples (larvae) detected positives

11 Results (3) In situ hybridation Specific labelling (=presence of Bonamia sp.) in 19 spawners and 7 larvae samples previously detected positive by PCR. In spawners, the parasite is observed in different organs including the gonad (42% of positive oysters) as well as on larvae sections (16% of positive oysters). In larvae, the parasite is observed in cells surrounding coelomic cavity.

12 Results (3)

13 Results (3)

14 Conclusions Detection by PCR (RFLP) of Bonamia ostreae in incubating flat oysters as well as in incubated larvae In situ hybridization results confirm the infection (and not just carrying) Detection of B. exitiosa in onelarvaesamplecollectedin 2008 At least a third of the infected incubating oysters showed positive results for their larvae Larvae might contribute to the spread of the disease

15 Perspectives Are incubating oysters more susceptible to the disease? In which extent larvae contribute to the parasite spread? What is the impact of the parasite on larvae? Are defense mechanisms developed by larvae similar to adults?

16 Thanks for your attention