www.ifremer.fr 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
Introduction CONTEXT 42% Clam marketing 3 442 tons (2001) Cultured : 2 034 tons (60%) Harvested : 1 408 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)
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
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
Objective Confirmation of these results and characterization of the «French Perkinsus chesapeaki» Photo: Ifremer
Study site and biological material Leucate lagoon: 5 400 ha October 2005 30 clams collected, 29 found infected by RFTM (parasite burden mean : 84 422 par/g of gills) October 2008 60 clams collected, 51 found infected by PCR Crassostrea gigas 800 t Mytilus galloprovincialis 200 t Ruditapes decussatus 7t
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)
Results-Molecular characterization Individuals Cultures PCR-RFLP PCR-RFLP clones Sequencing ITS 1 3 1 PC 2 PO 2 PC 10 PO 2 PC 1 PO 2005 2 2 1 PO 0 1 PC 9 PC 2 PC 3 2 2 PO 6 PO 1 PO 4 2 1 PC 1 PO 10 PC 1 PO 3 PC 1 PO 5 2 2 PO 8 PO + 1? 5 PO 6 1 1 PO 12 PO 2 PO PO: P. olseni PC: P. chesapeaki RsaI HinfI PO PC PO PC RsaI
Results-Molecular characterization Cultures PCR-RFLP Sequencing ITS Clonal cultures Sequencing ITS and LSU 2008 16 9 PO 4 PO 2 PC 2 PC 1 PC 2PO/PC? - 4 PO RsaI HinfI PO: P. olseni PC: P. chesapeaki
Sequencing In addition LSUA and B (provided by Reece) 97-99% of identities with P. chesapeaki 99-100% of identities with P. olseni P. gugwadi AF151528 AY295185 AY295177 AY295183 AY295179 AY295181 AY487834 AY487838 AY487842 AY487836 AY487840 DQ516696 DQ516697 DQ516698 DQ516699 05-673P2cl10 AF441209 06-02111cl1 05-0642p2cl10 AF441207 05-0685p3Cl13 05-0686p1cl2 05-0685p3cl9 05-0673P2/1cl12 05-0673P2/1cl13 05-0691cl8 05-0685p3cl5 06-02111cl5 05-0685p3Cl11 06-02111cl8 05-0694P1cl10 05-0686p1Cl12 AF441211 05-0632cl5 05-0673p2/1cl16 AF441214 05-0673P2/1cl6 06-02111cl14 05-0685p3cl4 05-0691cl4 AF441216 P. marinus AF440468 AF091541 0684P1cl1 05-0684P1cl8 05-0681P3cl1 05-0681P3cl13 05-0684P1cl5 AY876302 AF440466 AY876304 P. mediterraneus P. honshuensis P. olseni P. chesapeaki
Results- Cell observation Pictures: B. Chollet a b c d P. chesapeaki P. olseni Size (µm) Number Size (µm) number Trophozoites (a & b) 10.7 936 9.7 1048 Schizonts (c) 34.5 277 18.2 752 Zoosporangiums (d) 39 265 24.4 3 Mean size (µm) of the different parasite stages observed in clonal cultures after 16 days of culture
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
Results In situ hybridization - P. olseni Positive signals in all the tested individuals (17/17) Control from Arcachon bay Pictures: B. Chollet
Summary -Conclusions Perkinsus chesapeaki and P. olseni PCR-RFLP profiles were obtained from clams collected in Leucate lagoon in 2005 and 2008- 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
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
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Methods PCR-RFLP 18S ITS1 5.8S ITS2 26S PerkITS750 PerkITS85 PCR ( Casas et al. 2002) RFLP (Abollo et al. 2006) Casas et al. 2002- Diseases of Aquatic Organisms, 50:51 62. Abollo et al. 2006. Molecular and Cellular Probes, 20:323-329
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. 2006 - Journal of Shellfish Research, 25: 65 72 Reece et al. 2008 Diseases of Aquatic Organisms, 82: 237 248