Perkinsus olseni and P. chesapeaki, sympatric in clams Ruditapes decussatus from Leucate lagoon, France

Transcription

1 Perkinsus olseni and P. chesapeaki, sympatric in clams Ruditapes decussatus from Leucate lagoon, France lfremer I. Arzul, B. Chollet, J. Michel, M. Robert, L. Miossec, J-P. Joly, C. François and C. Garcia IFREMER Laboratory of Genetics and Pathology, La Tremblade, France

2 Introduction CONTEXT 42% Clam marketing tons (2001) Cultured : tons (60%) Harvested : tons (40%) Perkinsosis distribution and prevalence 2% 31% 37% 12% 92% 87% Clams = third most important bivalve production in France Two main species : Ruditapes philippinarum and R. decussatus 76% Present in all sampling sites Variable prevalence 96% Production sites and prevalence of perkinsosis in France (2005)

3 Introduction PERKINSUS CHARACTERIZATION Clams collected in 2005 detected infected by RFTM culture for which cultures were available Gulf of Morbihan PCR ( Casas et al. 2002) RFLP (Abollo et al. 2006) Arcachon bay Cloning Thau lagoon PCR RFLP (10-20 clones / cloning) Leucate lagoon Sequencing Only Perkinsus olseni detected in these samples

4 Introduction BUT SOME NO EXPECTED ADDITIONAL RESULTS were subsequently obtained for two clams collected from Leucate Lagoon 413pb 193pb 74pb 363pb 160pb Rsa I DIGESTION Hinf I DIGESTION PCR-RFLP profiles different from Perkinsus olseni ones and similar to P. chesapeaki ones

5 Objective Confirmation of these results and characterization of the «French Perkinsus chesapeaki» Photo: Ifremer

6 Study site and biological material Leucate lagoon: ha October clams collected, 29 found infected by RFTM (parasite burden mean : par/g of gills) October clams collected, 51 found infected by PCR Crassostrea gigas 800 t Mytilus galloprovincialis 200 t Ruditapes decussatus 7t

7 Methods APPROACH Cultures from clams collected in 2005 Cultures from clams collected in 2008 DNA extraction (QIAamp DNA easy kit (QIAGEN) PCR ( Casas et al. 2002) RFLP (Abollo et al. 2006) Photo: Ifremer Cloning (TA cloning kit, Invitrogen) PCR RFLP (10-20 clones / cloning) Sequencing (Applied Biosystems- ABI PRISM 3100 Avant) Clonal cultures PCR RFLP Sequencing (Applied Biosystems- ABI PRISM 3100 Avant) Culture observation Paraffin blocks from clams collected in 2005 and 2008 In situ hybridization (Reece et al.2008 ) (Moss et al. 2006) Cultures = parasites maintained in DMEM:HAM S F-12 (Invitrogen) according to Gauthier et al. (1995) Clonal cultures according to Robledo et al. (2002)

8 Results-Molecular characterization Individuals Cultures PCR-RFLP PCR-RFLP clones Sequencing ITS PC 2 PO 2 PC 10 PO 2 PC 1 PO PO 0 1 PC 9 PC 2 PC PO 6 PO 1 PO PC 1 PO 10 PC 1 PO 3 PC 1 PO PO 8 PO + 1? 5 PO PO 12 PO 2 PO PO: P. olseni PC: P. chesapeaki RsaI HinfI PO PC PO PC RsaI

9 Results-Molecular characterization Cultures PCR-RFLP Sequencing ITS Clonal cultures Sequencing ITS and LSU PO 4 PO 2 PC 2 PC 1 PC 2PO/PC? - 4 PO RsaI HinfI PO: P. olseni PC: P. chesapeaki

10 Sequencing In addition LSUA and B (provided by Reece) 97-99% of identities with P. chesapeaki % of identities with P. olseni P. gugwadi AF AY AY AY AY AY AY AY AY AY AY DQ DQ DQ DQ P2cl10 AF cl p2cl10 AF p3Cl p1cl p3cl P2/1cl P2/1cl cl p3cl cl p3Cl cl P1cl p1Cl12 AF cl p2/1cl16 AF P2/1cl cl p3cl cl4 AF P. marinus AF AF P1cl P1cl P3cl P3cl P1cl5 AY AF AY P. mediterraneus P. honshuensis P. olseni P. chesapeaki

11 Results- Cell observation Pictures: B. Chollet a b c d P. chesapeaki P. olseni Size (µm) Number Size (µm) number Trophozoites (a & b) Schizonts (c) Zoosporangiums (d) Mean size (µm) of the different parasite stages observed in clonal cultures after 16 days of culture

12 Results In situ hybridization - P. chesapeaki Positive signals in some individuals (8/17 tested ones) Signal in tested clams fainter than in control Control provided by R. Carnegie Pictures: B. Chollet

13 Results In situ hybridization - P. olseni Positive signals in all the tested individuals (17/17) Control from Arcachon bay Pictures: B. Chollet

14 Summary -Conclusions Perkinsus chesapeaki and P. olseni PCR-RFLP profiles were obtained from clams collected in Leucate lagoon in 2005 and PCR-RFLP results were confirmed by sequencing ITS and LSU fragments. According to the phylogenetic analysis, Perkinsus parasites isolated from Leucate lagoon in France belong to the species P. chesapeaki and P. olseni. Cultures obtained from a same individual could present different PCR- RFLP profiles suggesting that co infection can occur. In situ hybridization confirmed that P. chesapeaki and P. olseni detection by PCR-RFLP translates a true infection and not only carrying. Examination of clonal cultures from P. chesapeaki and P. olseni showed differences in size especially for schizonts and zoosporangiums. First detection of Perkinsus chesapeaki in Europe and in Ruditapes decussatus P. chesapeaki and P. olseni appear as sympatric species in clams from Leucate lagoon

15 Discussion The presence of Perkinsus chesapeaki in France could be explained by previous imports of susceptible species including Mya arenaria and Mercenaria mercenaria from North America This work should be completed by more data on other geographic places in order assess P. chesapeaki distribution in Europe Impact of Perkinsus olseni and P. chesapeaki on clam production is not clear and despite their detection in Leucate lagoon in 2005 and then 2008, no mortality was reported P. olseni seems to be majority compared to P. chesapeaki in tested clams. However relationships between both species need further investigations

16 Thanks for your attention

17 Methods PCR-RFLP 18S ITS1 5.8S ITS2 26S PerkITS750 PerkITS85 PCR ( Casas et al. 2002) RFLP (Abollo et al. 2006) Casas et al Diseases of Aquatic Organisms, 50: Abollo et al Molecular and Cellular Probes, 20:

18 Methods APPROACH In situ hybridization Perkinsus chesapeaki-specific LSU-rRNA gene probe (Reece et al. 2008): PchesLSU-485DIG (5 -CAG GAA ACA CCA CGC ACK AG-3 ) Perkinsus olseni specific LSU-rRNA gene probe (Moss et al. 2006): PolsLSU-464DIG (5 -CTCACAAGTGCCAAACAACTG-3 ) Moss et al Journal of Shellfish Research, 25: Reece et al Diseases of Aquatic Organisms, 82: