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Characterization of Actin Genes in Bonamia Ostreae and Their Application to Phylogeny of the Haplosporidia

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Characterization of Actin Genes in Bonamia Ostreae and Their Application to Phylogeny of the Haplosporidia View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library 1941 Characterization of actin genes in Bonamia ostreae and their application to phylogeny of the Haplosporidia I. LO´ PEZ-FLORES1*, V. N. SUA´ REZ-SANTIAGO2,D.LONGET3,D.SAULNIER1, B. CHOLLET1 and I. ARZUL1 1 Laboratoire de Ge´ne´tique et Pathologie. Station La Tremblade, IFREMER. Avenue Mus de Loup, 17390 La Tremblade, France 2 Department of Botany, Faculty of Sciences, University of Granada, Avenida Severo Ochoa s/n, 18071 Granada, Spain 3 Department of Zoology and Animal Biology, University of Geneva, Sciences III, 30 Quai Ernest Ansermet, CH 1211 Geneva 4, Switzerland (Received 18 April 2007; revised 8 May and 14 June 2007; accepted 15 June 2007; first published online 3 August 2007) SUMMARY Bonamia ostreae is a protozoan parasite that infects the European flat oyster Ostrea edulis, causing systemic infections and resulting in massive mortalities in populations of this valuable bivalve species. In this work, we have characterized B. ostreae actin genes and used their sequences for a phylogenetic analysis. Design of different primer sets was necessary to amplify the central coding region of actin genes of B. ostreae. Characterization of the sequences and their amplification in different samples demonstrated the presence of 2 intragenomic actin genes in B. ostreae, without any intron. The phylogenetic analysis placed B. ostreae in a clade with Minchinia tapetis, Minchinia teredinis and Haplosporidium costale as its closest relatives, and demonstrated that the paralogous actin genes found in Bonamia resulted from a duplication of the original actin gene after the Bonamia origin. Key words: Bonamia ostreae, Ostrea edulis parasite, actin gene, molecular phylogeny. INTRODUCTION B. perspora in Ostreola equestris in North Carolina, USA (Carnegie et al. 2006). Bonamia ostreae is a small-sized (2–3 mm) protozoan Recent isolation of the gene coding for the small parasite, responsible for bonamiosis, a haemocytic subunit of the ribosomal RNA in B. ostreae (18S or disease affecting the European flat oyster Ostrea SSU rDNA) has clarified phylogenetic affinities of edulis. In Europe, it was first described in France Bonamia species. Thus, after different taxonomic (Pichot et al. 1980) as the causative agent of serious affiliations, analysis of SSU rRNA placed Bonamia mortalities in this native species. Since then, it has within the Haplosporidia (Carnegie et al. 2000; been responsible for a drastic decrease in O. edulis Reece et al. 2004). Genes coding for proteins provide production in different farming areas along the the possibility of performing phylogenetic analyses European Atlantic coast (see review by Carnegie and under a different evolutionary rate compared to those Cochennec-Laureau, 2004). It has also been detected governing ribosomal genes. In this respect, actin has in O. edulis populations on the Pacific and Atlantic the advantage of being an ubiquitous protein in eu- coasts of North America (Carnegie and Cochennec- karyotic cells and one of the most conserved Laureau, 2004; Marty et al. 2006). Other known and throughout evolution, from yeast to human characterized species of the genus are B. exitiosa from (Sheterline and Sparrow, 1994). Actin is a cytoskel- O. chilensis in New Zealand and Chile, O. angasi in etal protein involved in cellular functions like Australia, O. puelchana in Argentina, and Crassostrea maintaining cell morphology, cell motility and div- ariakensis in North Carolina, USA (Campalans et al. ision, and intracellular transport (Sheterline and 2000; Hine et al. 2001; Burreson et al. 2004; Kroeck Sparrow, 1994). Actin proteins are encoded by a and Montes, 2005; Corbeil et al. 2006); B. roughleyi multigene family in all organisms examined so far, (previously known as Mikrocytos roughleyi; Carnegie except in yeast and in some alveolata where they are and Cochennec-Laureau, 2004) in Saccostrea glo- encoded by one single gene (Hightower and merata in Australia (Farley et al. 1988); and Meagher, 1986; Reece et al. 1997; Zhou et al. 2006). In other protozoans, like Plasmodium falciparum, Entamoeba histolytica, several species of foraminifera and the haplosporidians Haplosporidium louisiana, * Corresponding author: Present address: Laboratoire de Ge´ne´tique et Pathologie, IFREMER. Avenue Mus de Minchinia chitonis, Minchinia teredinis and Minchinia Loup, 17390 La Tremblade, France. Tel: +33 546762647. tapetis, 2–4 actin genes have been described (Huber Fax: +33 546762611. E-mail: [email protected] et al. 1987; Wesseling et al. 1988; Pawlowski et al. Parasitology (2007), 134, 1941–1948. f 2007 Cambridge University Press doi:10.1017/S0031182007003307 Printed in the United Kingdom Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 15:09:20, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0031182007003307 I. Lo´pez-Flores and others 1942 1999; Reece et al. 2004). The gene family differs in Accession numbers: AY450412, AY450407, size among these organisms, and the number and AY450414, AY450416, AJ132374, AJ32375, J01016, location of introns within actin genes are variable D50839, AF057161, M86241, U84287, TGU10429, (Hightower and Meagher, 1986; Reece et al. 2004; M19871, M19871). An available oyster actin se- Flakowski et al. 2006). quence (AAB81845) was also included to prevent This study reports, through the use of nucleotide amplification of the host gene. DNA from a non- and amino acid sequence analyses, the identification infected O. edulis was used as control. of at least 2 actin genes within Bonamia ostreae. Amplification products were separated on 1% Phylogenetic analyses were carried out to assess agarose gels stained with ethidium bromide. Testing orthologous/paralogous relationships of the se- of all primer combinations showed that one forward quences and to infer the phylogenetic position of primer (F) and 2 reverse primers (R1 and R2) al- B. ostreae within the Haplosporidia based on the lowed amplification of 2 putative B. ostreae se- actin gene sequences. quences (Fig. 1A). The reaction was carried out in a . volume of 50 ml with 2 mM Mg2SO4,02mM of each dNTP, 1 mM of each primer, 1.5UofTaq polymerase MATERIALS AND METHODS (New England Biolabs) and 5–10 ng of DNA. Samples and DNA extraction Thermal cycling was 95 xC for 1 min, 40 cycles of 95 xC for 1 min, 50 xC for 1 min and 65 xC for 1 min, Infected oysters (Ostrea edulis) were obtained by followed by 65 xC for 10 min. experimental infections. Healthy animals were in- Amplified products were cloned using TOPO jected with Bonamia ostreae cells previously purified vector system (Invitrogen) and the nucleotide se- (see below) from naturally infected oysters from quences were determined using an ABI Prism Quiberon bay, Brittany (France) and maintained in a Dye Terminator Cycle Sequencing kit (Applied laboratory tank. This procedure allowed us to obtain Biosystems) following the manufacturer’s re- highly infected oysters that were used for parasite commendations. Sequences were identified by com- purification. In parallel, non-injected oysters were parison with those included in the GenBank and used as negative control. Infections were confirmed EMBL databases using BLAST algorithm (Altschul by microscopical examination of tissue imprints and et al. 1997). by PCR assay based on the 18S rDNA gene of the Primer pairs BostAct1F/BostAct1R and parasite, using primers Bo and BoAs (Cochennec BostAct2F/BostAct2R were designed for indepen- et al. 2000). dent amplification of Actin1 and Actin2 genes of Parasites were purified as described previously B. ostreae based on the cloned sequences (Fig. 1A). 6 (Cochennec et al. 2000). About 50r10 cells could be Actin gene sequences from Ostrea edulis and Ostrea obtained from each highly infected oyster. Cells were chilensis obtained during this work (recorded in centrifuged at 12 000 g and pellets from several GenBank with Accession numbers AM410916, x purifications were pooled and conserved at x20 C. AM410917 and AM410918) were aligned with In addition, 2 samples (gill tissues fixed in 95% B. ostreae sequences to ensure specificity of the pri- ethanol) of O. chilensis from New Zealand infected mers for B. ostreae actins. DNA from a non-infected with B. exitiosa were used as controls. Ostrea edulis and DNA from an O. chilensis highly Genomic DNA from purified parasites and oyster infected with Bonamia exitiosa were used as controls. gills was extracted according to the method of Amplification reactions were performed as described 8 Winnepenninckx et al. (1993) using 2r10 parasite previously but increasing annealing and extension cells or 100 mg of oyster tissue. Integrity and quan- temperatures from 50 to 55 xC and 65 to 72 xC re- tity of DNA was measured by electrophoresis on spectively. Identification of each sequence type, agarose gel stained with ethidium bromide and Actin1 and Actin2, was done by digestion of PCR spectrophotometry, respectively. product (PCR-RFLP) with the restriction enzyme BstUI (Promega). The reactions consisted of 16 mlof PCR product, 2 ml10 Buffer and 5 U of enzyme in a Amplification of actin genes by PCR r final volume of 20 ml. Different samples and sets of degenerate primers (4 forward primers were combined with 5 reverse Sequence and phylogenetic analyses ones) were used to amplify B. ostreae actin gene fragments. Two of these primer pairs were used Phylogenetic analyses of the actin genes were per- previously for the amplification of actin genes in formed using both nucleotide and amino acid dinoflagellata and haplosporidia species (Reece sequences. Sequences used in the phylogenetic et al. 1997, 2004), and in a rhizopod species analyses from haplosporidian species were down- (Longet et al. 2004). The remaining primers tested loaded from GenBank (Haplosporidium costale, have been designed for this work based on alignment AY450407; H. louisiana, AY450411; H. nelsoni, of conserved regions of actin protein (GenBank AY450412; Minchinia chitonis, AY450414; Downloaded from https:/www.cambridge.org/core.
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