INTRODUCTION
Bonamia ostreae is a small-sized (2–3 μm) protozoan parasite, responsible for bonamiosis, a haemocytic disease affecting the European flat oyster Ostrea edulis. In Europe, it was first described in France (Pichot et al. Reference Pichot, Comps, Tigé, Grizel and Rabouin1980) as the causative agent of serious mortalities in this native species. Since then, it has been responsible for a drastic decrease in O. edulis production in different farming areas along the European Atlantic coast (see review by Carnegie and Cochennec-Laureau, Reference Carnegie and Cochennec-Laureau2004). It has also been detected in O. edulis populations on the Pacific and Atlantic coasts of North America (Carnegie and Cochennec-Laureau, Reference Carnegie and Cochennec-Laureau2004; Marty et al. Reference Marty, Bower, Clarke, Meyer, Lowe, Osborn, Chow, Hannah, Byrne, Sojonky and Robinson2006). Other known and characterized species of the genus are B. exitiosa from O. chilensis in New Zealand and Chile, O. angasi in Australia, O. puelchana in Argentina, and Crassostrea ariakensis in North Carolina, USA (Campalans et al. Reference Campalans, Rojas and González2000; Hine et al. Reference Hine, Cochennec-Laureau and Berthe2001; Burreson et al. Reference Burreson, Stokes, Carnegie and Bishop2004; Kroeck and Montes, Reference Kroeck and Montes2005; Corbeil et al. Reference Corbeil, Arzul, Robert, Berthe, Besnard-Cochennec and Crane2006); B. roughleyi (previously known as Mikrocytos roughleyi; Carnegie and Cochennec-Laureau, Reference Carnegie and Cochennec-Laureau2004) in Saccostrea glomerata in Australia (Farley et al. Reference Farley, Wolf and Elston1988); and B. perspora in Ostreola equestris in North Carolina, USA (Carnegie et al. Reference Carnegie, Burreson, Hine, Stokes, Audemard, Bishop and Peterson2006).
Recent isolation of the gene coding for the small subunit of the ribosomal RNA in B. ostreae (18S or SSU rDNA) has clarified phylogenetic affinities of Bonamia species. Thus, after different taxonomic affiliations, analysis of SSU rRNA placed Bonamia within the Haplosporidia (Carnegie et al. Reference Carnegie, Barber, Culloty, Figueras and Distel2000; Reece et al. Reference Reece, Siddall, Stokes and Burreson2004). Genes coding for proteins provide the possibility of performing phylogenetic analyses under a different evolutionary rate compared to those governing ribosomal genes. In this respect, actin has the advantage of being an ubiquitous protein in eukaryotic cells and one of the most conserved throughout evolution, from yeast to human (Sheterline and Sparrow, Reference Sheterline and Sparrow1994). Actin is a cytoskeletal protein involved in cellular functions like maintaining cell morphology, cell motility and division, and intracellular transport (Sheterline and Sparrow, Reference Sheterline and Sparrow1994). Actin proteins are encoded by a multigene family in all organisms examined so far, except in yeast and in some alveolata where they are encoded by one single gene (Hightower and Meagher, Reference Hightower and Meagher1986; Reece et al. Reference Reece, Siddall, Burreson and Graves1997; Zhou et al. Reference Zhou, Huang, Suzuki, Fujisaki, Igarashi and Xuan2006). In other protozoans, like Plasmodium falciparum, Entamoeba histolytica, several species of foraminifera and the haplosporidians Haplosporidium louisiana, Minchinia chitonis, Minchinia teredinis and Minchinia tapetis, 2–4 actin genes have been described (Huber et al. Reference Huber, Garfinkel, Gitler, Mirelman, Revel and Rozenblatt1987; Wesseling et al. Reference Wesseling, Smits and Schoenmakers1988; Pawlowski et al. Reference Pawlowski, Bolivar, Fahrni, de Vargas and Bowser1999; Reece et al. Reference Reece, Siddall, Stokes and Burreson2004). The gene family differs in size among these organisms, and the number and location of introns within actin genes are variable (Hightower and Meagher, Reference Hightower and Meagher1986; Reece et al. Reference Reece, Siddall, Stokes and Burreson2004; Flakowski et al. Reference Flakowski, Bolivar, Fahrni and Pawlowski2006).
This study reports, through the use of nucleotide and amino acid sequence analyses, the identification of at least 2 actin genes within Bonamia ostreae. Phylogenetic analyses were carried out to assess orthologous/paralogous relationships of the sequences and to infer the phylogenetic position of B. ostreae within the Haplosporidia based on the actin gene sequences.
MATERIALS AND METHODS
Samples and DNA extraction
Infected oysters (Ostrea edulis) were obtained by experimental infections. Healthy animals were injected with Bonamia ostreae cells previously purified (see below) from naturally infected oysters from Quiberon bay, Brittany (France) and maintained in a laboratory tank. This procedure allowed us to obtain highly infected oysters that were used for parasite purification. In parallel, non-injected oysters were used as negative control. Infections were confirmed by microscopical examination of tissue imprints and by PCR assay based on the 18S rDNA gene of the parasite, using primers Bo and BoAs (Cochennec et al. Reference Cochennec, Le Roux, Berthe and Gerard2000).
Parasites were purified as described previously (Cochennec et al. Reference Cochennec, Le Roux, Berthe and Gerard2000). About 50×106 cells could be obtained from each highly infected oyster. Cells were centrifuged at 12 000 g and pellets from several purifications were pooled and conserved at −20°C.
In addition, 2 samples (gill tissues fixed in 95% ethanol) of O. chilensis from New Zealand infected with B. exitiosa were used as controls.
Genomic DNA from purified parasites and oyster gills was extracted according to the method of Winnepenninckx et al. (Reference Winnepenninckx, Backeljau and de Wachter1993) using 2×108 parasite cells or 100 mg of oyster tissue. Integrity and quantity of DNA was measured by electrophoresis on agarose gel stained with ethidium bromide and spectrophotometry, respectively.
Amplification of actin genes by PCR
Different samples and sets of degenerate primers (4 forward primers were combined with 5 reverse ones) were used to amplify B. ostreae actin gene fragments. Two of these primer pairs were used previously for the amplification of actin genes in dinoflagellata and haplosporidia species (Reece et al. Reference Reece, Siddall, Burreson and Graves1997, Reference Reece, Siddall, Stokes and Burreson2004), and in a rhizopod species (Longet et al. Reference Longet, Burki, Flakowski, Berney, Polet, Fahrni and Pawlowski2004). The remaining primers tested have been designed for this work based on alignment of conserved regions of actin protein (GenBank Accession numbers: AY450412, AY450407, AY450414, AY450416, AJ132374, AJ32375, J01016, D50839, AF057161, M86241, U84287, TGU10429, M19871, M19871). An available oyster actin sequence (AAB81845) was also included to prevent amplification of the host gene. DNA from a non-infected O. edulis was used as control.
Amplification products were separated on 1% agarose gels stained with ethidium bromide. Testing of all primer combinations showed that one forward primer (F) and 2 reverse primers (R1 and R2) allowed amplification of 2 putative B. ostreae sequences (Fig. 1A). The reaction was carried out in a volume of 50 μl with 2 mm Mg2SO4, 0·2 mm of each dNTP, 1 μm of each primer, 1·5 U of Taq polymerase (New England Biolabs) and 5–10 ng of DNA. Thermal cycling was 95°C for 1 min, 40 cycles of 95°C for 1 min, 50°C for 1 min and 65°C for 1 min, followed by 65°C for 10 min.
Fig. 1. Primer description (A) and schematic representation of the genes that encode for actin in Bonamia ostreae (B). Arrows indicate approximate locations of the primers used for PCR amplifications. Primers BostAct1F/R and BostAct2F/R, designed after amplification of Actin1 with primers F/R1 and Actin 2 with primers F/R2, amplify a 264 bp fragment of Actin1 and Actin2 genes.
Amplified products were cloned using TOPO vector system (Invitrogen) and the nucleotide sequences were determined using an ABI Prism Dye Terminator Cycle Sequencing kit (Applied Biosystems) following the manufacturer's recommendations. Sequences were identified by comparison with those included in the GenBank and EMBL databases using BLAST algorithm (Altschul et al. Reference Altschul, Madden, Schaffer, Zhang, Zhang, Miller and Lipman1997).
Primer pairs BostAct1F/BostAct1R and BostAct2F/BostAct2R were designed for independent amplification of Actin1 and Actin2 genes of B. ostreae based on the cloned sequences (Fig. 1A). Actin gene sequences from Ostrea edulis and Ostrea chilensis obtained during this work (recorded in GenBank with Accession numbers AM410916, AM410917 and AM410918) were aligned with B. ostreae sequences to ensure specificity of the primers for B. ostreae actins. DNA from a non-infected Ostrea edulis and DNA from an O. chilensis highly infected with Bonamia exitiosa were used as controls. Amplification reactions were performed as described previously but increasing annealing and extension temperatures from 50 to 55°C and 65 to 72°C respectively. Identification of each sequence type, Actin1 and Actin2, was done by digestion of PCR product (PCR-RFLP) with the restriction enzyme BstUI (Promega). The reactions consisted of 16 μl of PCR product, 2 μl 10× Buffer and 5 U of enzyme in a final volume of 20 μl.
Sequence and phylogenetic analyses
Phylogenetic analyses of the actin genes were performed using both nucleotide and amino acid sequences. Sequences used in the phylogenetic analyses from haplosporidian species were downloaded from GenBank (Haplosporidium costale, AY450407; H. louisiana, AY450411; H. nelsoni, AY450412; Minchinia chitonis, AY450414; M. tapetis, AY450417; M. terenidis, AY450420; Urosporidium crescens, AY450422). Sequences from foraminiferan species Reticulomyxa filosa (AJ132374), Ammonia sp. (AJ132372) and Allogromia sp. (AJ132370) and the Cercozoa Cercomonas sp. (AF363534) were chosen as outgroup according to the results obtained by Reece et al. (Reference Reece, Siddall, Stokes and Burreson2004). Multiple sequence alignment was done using the program MEGALIN of the DNAstar package (LASERGEN) by Clustal method and revised by eye. Phylogenetic analyses were performed with the program PAUP* version 4.0b10 (Swofford, Reference Swofford2003) using the maximum parsimony criterion. Parsimony analysis involved heuristic searches. Data matrices were subjected to 1000 replicates of random sequence additions using tree bisection-reconnection branch-swapping. In the protein analysis the characters were considered as unordered states with equal weight, while in the nucleotide analysis the third nucleotide of each codon was downweighted. Gaps were treated as missing data and the ambiguous regions in the alignment were deleted from the analysis. The starting tree was obtained by stepwise addition. The characters were optimized by accelerated transformation. Finally, 1000 bootstrap replicates (Felsenstein, Reference Felsenstein1985) with 10 heuristic searches were performed to assess internal support for nodes.
RESULTS
Sequence characterization
Two major actin gene fragments, different in size, could be obtained by combining 1 forward with 2 reverse primers in different PCR reactions using DNA of purified B. ostreae. They were 869 bp and 784 bp in length. The shorter one was similar to the larger one except on the 3′ end 85 nucleotides (Fig. 1B). Comparison with sequences included in GenBank and EMBL data-bases by BLAST allowed identification of partial actin DNA fragments, corresponding to the central region of the gene. These sequences were named Actin1 (BoAct1, 869 bp) and Actin2 (BoAct2, 784 bp).
Three cloned sequences per actin sequence type were obtained (Fig. 2A). The GenBank Accession numbers are AM410919-AM410921 and AM410922-AM410924. Excluding primer sites, the sequence to be compared was 745 bp in length. Intra-type variability between clones was low, and estimated at 5 nucleotides between Actin1 sequences and 1 between Actin2 sequences (Fig. 2A). Three of the 5 sites corresponded to non-synonymous changes with respect to deduced Actin1 amino acid sequence (TCT coding S to CCT coding P, GAT coding D to GAA coding E, and ATG coding M to TTG coding L at positions 451, 474 and 700 respectively). The variable site of Actin2 partial sequences corresponded also to a non-synonymous change (ATC coding I to GTC coding V at position 403) (Fig. 2B). Nucleotide sequence type 1 and type 2 (Actin1 and Actin2) demonstrated 83·7% identity. Variable positions were mainly located in the second half of the sequences (Fig. 2A). These variable positions (108 in total) usually corresponded to the third position of the codons (58) while 33 and 17 variable sites corresponded to the second and the first codon positions respectively. No gene intron was identified when sequences were aligned with homologous actin genes.
Fig. 2. Sequences alignment of Bonamia ostreae actin genes 1 and 2. (A) Nucleotide sequences corresponding to cloned actin sequences Type 1 (BostAct1) and Type 2 (BostAct2). Last numeric values indicate the recombinant E. coli clones. Dots (.) indicate identical bases. Locations of degenerate forward primer (position 1–17) and reverse ones (763–784 and 850–869) are underlined. Locations of specific primers for each actin gene type (BostAct1F and 2F, position 454–472, and BostAct1R and 2R, position 697–717) are highlighted in grey. Recognition sites for restriction enzyme BstUI (CG∧CG) in Type 1 sequences are showed in bold and underlined (cut sites: positions 537 and 705). (B) Inferred amino acid sequences corresponding to protein Actin 1 and Actin 2. Dots (.) indicate identical residue.
Encoded amino acid sequences of these 2 actin gene fragments (289 and 261 amino acid residues for Actin1 and Actin2 respectively) showed a full length open reading frame and demonstrated 87·2% sequence identity. The first 121 amino acid residues were identical between the sequences and corresponded to identical nucleotide sequences (Fig. 2B).
In order to determine if sequence types, Actin1 and Actin2, corresponding to actin genes belong to B. ostreae genome, a variable region of nucleotide sequences was used to design primers for amplification of Actin1 and Actin2 sequences in different isolates (BostAct1F/BostAct1R and BostAct2F/BostAct2R respectively, see Fig. 2A). Both primer pairs amplified the expected 264 bp product when DNA of the parasite purified from 4 different naturally infected oysters (O. edulis) was used as template. The different sequence types were determined by digestion of PCR products with the restriction enzyme BstUI (PCR-RFLP). Actin2 sequences had no recognition site for this enzyme while Actin1 sequences contained 2 of them, yielding, after digestion of the 264 bp amplicon, 3 fragments of 13, 83 and 168 bp. Figure 3 shows differential band patterns obtained by PCR-RFLP. Both types of sequences could be easily identified in the 4 different DNAs from B. ostreae purified cells, while no amplification was obtained for oyster DNA either healthy Ostrea edulis or O. chilensis infected with B. exitiosa.
Fig. 3. Detection of a polymorphism involving BstUI restriction sites in Bonamia ostreae actin gene sequences. DNA from 4 different B. ostreae samples (purified parasite) independently amplified with primers BostAct1F/R (Actin Type 1) and BostAct2F/R (Actin Type 2) yielded amplicons 264 bp in length. After digestion with BstUI, 2 RFLP profiles were observed: a no-digested 264 bp band in Actin2 sequences (lanes 6 to 9) and bands of 168+83 bp for Actin1 sequences (lanes 2 to 5; the third expected band of 13 bp can not be observed in the gel). Lanes 1 and 10 correspond to the molecular weight marker (Smart Ladder SF, Eurogentec). Negative controls were non-infected Ostrea edulis DNA (lane 11), DNA from O. chilensis highly infected with B. exitiosa (lane 12) and no-DNA water control (lane 13).
Phylogeny based on actin genes
Alignment of all 17 actin gene nucleotide sequences (all B. ostreae actin gene sequences, 7 of haplosporidian, and the 4 outgroup sequences) resulted in a 1113 bp matrix (after excluding ambiguous regions). Of the 1113 characters 416 were parsimony-informative (285 in the ingroup). Phylogenetic analysis based on nucleotide sequences produced 3 most-parsimonious trees of 3377 steps in length (CI: 0·585; RI: 0·595; HI: 0·415), which differ only in the relationships between the Actin2 sequences of B. ostreae. Figure 4A shows the strict consensus tree that coincides with one of the most-parsimonious trees. The tree shows that Bonamia ostreae Actin1 and Actin2 were paralogous sequences to each other, and orthologous to the remaining haplosporidian actin gene sequences (Fig. 4A). B. ostreae actin genes clustered together with 100% bootstrap support (Fig. 4A), and this grouping remained when the multiple haplosporidian paralogous sequences described by Reece and co-workers (Reece et al. Reference Reece, Siddall, Stokes and Burreson2004) were included in an overall analysis (data not shown). B. ostreae appeared to be a sister clade to that formed by Minchinia tapetis and M. teredinis and Haplosporidium costale. Both Haplosporidium and Minchinia genera were paraphyletic. Urosporidum crecens was the basal species of the haplosporidians.
Fig. 4. Parsimony analysis based on actin gene sequences showing the paralogy of Bonamia ostreae sequences obtained in this study. Numbers at nodes indicate percentage support of 1000 bootstrap replicates (bootstrap support values below 50 are not presented). Phylogeny was performed using (A) nucleotide sequences and (B) translated amino acid sequences.
Alignment of all 17 protein sequences resulted in a 370 amino acid matrix (excluding an ambiguous region), from which 84 were parsimony-informative (65 in the ingroup). Phylogenetic analysis using the protein sequences yielded 4 most-parsimonious trees of 329 steps in length (CI: 0·796; RI: 0·765; HI: 0·204), which differ only in the relationships between the Actin2 sequences of B. ostreae, and also in the relationships between H. costale, M. tapetis and M. teredinis. The strict consensus tree is shown in Fig. 4B. The main difference of this tree with respect to the nucleotide tree (Fig. 4A) was the closer relationship of B. ostreae with H. nelsoni instead of with the clade consisting of M. tapetis, M. teredinis and H. costale.
DISCUSSION
Two actin genes in the genome of Bonamia ostreae
Amplification by PCR using ‘universal’ primers of unknown genes from non-cultivable eukaryotic parasites like Bonamia ostreae is a difficult aim. Conserved or ‘universal’ primers are mostly designed from higher organism sequences and frequently amplified host DNA (always present in the samples) instead of parasite DNA. Combination of conserved primers and primers designed using sequences from related organisms, and the use of DNA from Ostrea edulis and from O. chilensis infected with B. exitiosa as separate controls, allowed us to amplify actin genes in B. ostreae. Characterization of amplified products allowed the identification of 2 sequence types encoding 2 proteins. Both types were identical at the amino terminal end. Variable positions were mainly located at third codon positions with decreasing proportions at second and first codon positions. Deduced amino acid sequences of Actin1 and Actin2 were about 13% divergent, a relatively high value for actin proteins encoded by genes from the same species (Hightower and Meagher, Reference Hightower and Meagher1986). Nevertheless, higher divergence values could be observed between both actin paralogues described in species of the related foraminiferan phylum, 16% in Reticulomyxa filosa, 17% in Ammonia sp. and 18% in Allogromia sp. (personal observations. based on sequences analysed by Pawlowski et al. Reference Pawlowski, Bolivar, Fahrni, de Vargas and Bowser1999). In the apicomplexa Plasmodium falciparum, actin protein sequences showed 21% sequence divergence and were considered extremely divergent (Wesseling et al. Reference Wesseling, Smits and Schoenmakers1988).
Altogether these data suggested that 2 paralogous actin genes are present in the genome of B. ostreae. The absence of amplification from non-infected oyster and a sister species (B. exitiosa) DNAs, the comparison with O. edulis sequences and the amplification by PCR of both sequence types in different B. ostreae samples from different origins demonstrated that both types of sequences corresponded to B. ostreae genes and not to actin genes from different species (the host, other parasites, algae, etc.), coding for 1 actin protein each. The 2 actin gene fragments of B. ostreae reported in this study represent the first protein-encoding sequences obtained for any Bonamia species. Isolation and characterization of new genes or loci of its genome is of great interest in order to create a molecular data-base for this parasite, as exists for other protozoans, useful for further research.
Actin phylogeny
PCR amplification and sequencing analysis suggested that there were at least 2 actin genes in the genome of B. ostreae and phylogenetic analysis supported this assumption. Both types of actin gene sequences obtained in this study (Actin1 and Actin2) grouped together on the basis of nucleotide and amino acid sequences when trees were inferred using orthologous sequences from species belonging to the genera Haplosporidium, Minchinia and Urosporidium, as well as when their paralogous sequences were included in the analysis. This result suggests that the two Actin1 and Actin2 nucleotide sequences are the result of a gene duplication event (resulting in 2 paralogous loci) in B. ostreae after differentiation from the common ancestor. Within haplosporidians, multiple paralogues are present in species of the genus Minchinia, while Haplosporidium genus includes species, like H. nelsoni and H. costale in which only a single type of actin gene has been amplified by PCR until now. Many of these sequences were found to contain introns, and genes with introns as well as single intronless actin genes were established to be orthologous (Reece et al. Reference Reece, Siddall, Stokes and Burreson2004). In B. ostreae at least 2 paralogous actin genes without introns exist. Gain of introns in actin sequences throughout evolution has been described in the group of foraminifera (Flakowski et al. Reference Flakowski, Bolivar, Fahrni and Pawlowski2006), and this could be applied to the haplosporidian sequences to explain the existence of paralogous genes in some species containing different numbers of introns.
Actin phylogeny based on amino acid sequences placed Haplosporidium nelsoni as the closest species to B. ostreae, in a sister clade to that formed by Haplosporidium costale, Minchinia tapetis and Minchinia teredinis. In the nucleotide based tree, this topology is reversed since B. ostreae is more closely related to the clade of H. costale-M. tapetis and M. teredinis than to H. nelsoni, although the bootstrap support was low. Minchinia and Haplosporidium resulted as paraphyletic genera in both analyses. Our results are similar to those previously obtained using actin gene sequences (Reece et al. Reference Reece, Siddall, Stokes and Burreson2004). Phylogeny based on a small subunit of ribosomal gene performed by these authors allowed to include more haplosporidian species in the analysis and therefore they described more precisely the ingroup relationships. The genus Bonamia showed closest relationship to the genus Minchinia, which appeared monophyletic on the basis of ribosomal gene sequence. Characterization of actin genes in the remaining haplosporidian species and subsequent analysis may clarify the discrepancies between actin and 18S phylogenies concerning the relationships between Bonamia, Minchinia and Haplosporidium genera, and also provide a wider knowledge of relationships within the group.
This research was supported by the European Community Reference Laboratory for diseases of molluscs and by a postdoctoral fellowship (IFAPA, Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía, Spain) to I. López-Flores. We thank Dr M. A. Garrido-Ramos for critical review of the manuscript. We also thank our colleague S. Corbeil for revising our English text.
