Coral Reefs (2002) 21: 143–146 DOI 10.1007/s00338-002-0220-2 NOTE W.W. Toller Æ R. Rowan Æ N. Knowlton Genetic evidence for a protozoan (phylum Apicomplexa) associated with corals of the Montastraea annularis species complex Received: 8 June 2001 / Accepted: 13 March 2002 / Published online: 8 June 2002 Ó Springer-Verlag 2002 Keywords Coccidia Æ Apicomplexa Æ Coral disease Æ a coccidian protozoan (phylum Apicomplexa) which srDNA Æ Symbiodinium Æ Montastraea commonly associates with corals of the Montastraea annularis species complex. Introduction Methods Scleractinian corals associate with diverse microbial communities (e.g., Rowan 1998; Rohwer et al. 2001). Genotype N was identified by an RFLP that was distinct from Some eukaryotic microbes are common, well-known those of Symbiodinium taxa known to associate with M. annularis associates of corals; notably dinoflagellates of the genus (Fig. 1a). Here, we report only those methods specific to the identification and characterization of genotype N; methods Symbiodinium (e.g., Trench 1987), endolithic algae (e.g., for routine RFLP analysis of Symbiodinium srDNA, as well as Lukas 1974), and fungi (e.g., Bentis et al. 2000). Other methods for experimental manipulation of corals, are given eukaryotic associates have not received detailed study. elsewhere (Toller et al. 2001a, 2001b). Descriptions of these normal associations are a neces- Initially, the srDNA of genotype N was detected following polymerase chain reaction (PCR) with a ‘‘host-excluding’’ primer sary first step towards identifying abnormal microbial combination (Rowan and Powers 1991): the primers were ss5 (a associations (i.e., pathogens) which lead to coral disease universal primer) and ss3Z (a primer designed toexclude cnidarian (Richardson 1998). srDNA). We first observed genotype N in samples where Symbio- 5 2 Methods for molecular genetic analysis have revolu- dinium cell numbers were extremely low (<0.4·10 cells/cm ) following experimental bleaching of host corals (100-fold reduc- tionized the study of marine microbes (e.g., Brinkmeyer tion in Symbiodinium numbers relative to controls). These samples et al. 2000) and have proven useful in the study of coral yielded little srDNA when PCR-amplified in the usual (Toller et al. microbial communities (e.g., Rowan 1998; Rohwer et al. 2001b) manner. To obtain sufficient srDNA, we used two rounds of 2001). Here we give a brief account of a coral-associated amplification as follows: srDNA was amplified from samples with eukaryote which was identified by molecular methods host-excluding primers for 34 PCR cycles. Aliquots (10 ll) of those amplifications were electrophoresed on agarose gels (1.0% Nuseive during a bleaching experiment with the Caribbean coral GTG; FMC BioProducts, Rockland, ME), and faint bands of Montastraea annularis (Toller et al. 2001a). This sym- srDNA were excised and added to100 ll water. The gel-purified biont, provisionally named genotype N, was identified srDNA was heated to65 °C for 2 min, and then 1 ll of each was during routine screening of Symbiodinium samples by PCR-amplified with host-excluding primers in the same manner. The resulting re-amplified srDNA was then analyzed by RFLP as restriction fragment length polymorphism (RFLP) described elsewhere (Toller et al. 2001b). analysis of small subunit ribosomal RNA genes To further characterize genotype N, srDNA was cloned and (srDNA). Our data indicate that genotype N represents sequenced, as described previously (Toller et al. 2001b). Samples came from three different colonies of M. annularis in experiment I of Toller et al. (2001a). srDNA was amplified with universal PCR primers (clone N0–1) and with host-excluding primers (clones N0–2 0–3 W.W. Toller (&) Æ N. Knowlton and N ). For each sample, a minimum of 12 recombinants was Marine Biology Research Division, 0202, Scripps Institution examined by RFLP with Dpn II and with Taq I. DNA sequences of Oceanography, University of California San Diego, were deposited in GenBank (http://www.ncbi.nlm.nih.gov/) under accession numbers AF238264 (N0–1), AF238265 (N0–2), and La Jolla, California 92093-0202, USA 0–3 E-mail: [email protected] AF238266 (N ). Tel.: +1-858-8222486 srDNAsequences fromgenotype N,SymbiodiniumA,B,C,andE, Fax: +1-858-5347313 and the coral M. annularis were used todesign ‘‘N-specific’’ PCR primers that amplify only srDNA of genotype N from samples that R. Rowan alsocontainedcoralandzooxanthellarsrDNAs:The5¢endsofprimer University of Guam Marine Laboratory, 18N-F2 (5¢-TAGGAATCTAAACCTCTTCCA-3¢; forward primer) Mangilao, Guam 96923, USA and of primer 18N-R1 (5¢-CAGGAACAAGGGTTCCCGACC-3¢; 144 normally be present at undetectable levels, and ‘‘revealed’’ when Symbiodinium numbers fall. The design of that experiment enabled us to return to archived samples taken from the same corals, but prior to bleaching (i.e., prior to reduction of Symbiodinium numbers). Re-analysis of these samples with N-specific PCR primers confirmed that genotype N was present (in all cases) prior toexperimental treatment, when Symbiodinium were abundant. Using the same method, we then screened samples from other corals. Approximately 90% of samples taken from apparently healthy, unmanipulated colonies of M. annularis (45 colonies) and M. faveolata (7 colonies) tested positive for genotype N, yielding a PCR product of ca. 860 bp which, by digestion with Dpn II and with Taq I, was indistinguishable from the PCR product obtained from clone N0–1 (Fig. 1b). These data indicate that genotype N commonly associates with M. annularis and M. faveolata, and that its presence was previously masked in our analyses by the much greater abundance of Symbiodinium. Sequence data confirm that the srDNA of genotype N is quite divergent (12–17%) from those of Symbiodinium A, B, C, and E – the zooxanthellae known to associate with M. annularis (see Toller et al. 2001a, 2001b). Sequence comparisons also explain why genotype N was amplified with host-excluding PCR primers – the primer ss3Z has an extensive match to the srDNA of genotype N (clone N0–1; not shown; see also McNally et al. 1994). We performed experiments with mixtures of srDNA clones from genotype N (clone N0–1) and Symbiodinium C (clone C0; Toller et al. 2001b) and confirmed that our host- Fig. 1. a RFLP analysis of srDNA from genotype N and excluding protocol amplifies both srDNA with approxi- Symbiodinium. srDNA was amplified with host-excluding PCR mately equal efficiency (not shown). Thus, our method primers from a coral sample (N) and from different srDNA clones apparently detects both genotype N and Symbiodinium in representing Symbiodinium A (A), Symbiodinium B (B), Symbiodi- nium C (C), and Symbiodinium E (E). srDNA was digested with proportion to the relative abundance of their srDNA. Dpn II (left)orTaq I(right). The sample containing RFLP Phylogenetic analysis (Fig. 2) indicates that genotype genotype N was taken from Montastraea annularis following N is a member of the protozoan phylum Apicomplexa, a experimental bleaching (see text). Lanes marked M contain size sister group to the dinoflagellates (Gajadhar et al. 1991; standards of (from top to bottom) 1,500 bp, 1,200 bp, and then McNally et al. 1994). As far as is known, all apicom- 1,000 bp to100 bp in 100-bp increments. b RFLP analysis of srDNA obtained by PCR with N-specific primers. srDNA was amplified plexans are animal parasites (Levine 1982). Some, for from coral samples (lanes 1–3) and an srDNA clone (N0–1) and example the apicomplexans Plasmodium and Toxoplas- digested with Dpn II (left)orTaq I(right). Samples came from three ma, are notorious pathogens in humans and domestic different colonies of M. annularis. Size markers (M) are as above animals. In our analysis, genotype N shared greatest similarity to Toxoplasma, Neospora, Isospora,andSar- reverse primer) correspond to nucleotides 515 and 1374 (respectively) cocystis, representative taxa from the apicomplexan 0–1 in clone N . These primers were used in PCR amplifications as class Coccidia (Levine 1982). From these data, we con- before, except that DNA synthesis steps were reduced to 1 min. PCR products were analyzed by RFLP with Dpn II and with Taq I. clude that genotype N represents a coccidian protozoan For phylogenetic analysis, we aligned srDNA sequences with that associates with corals. Clustal X software (Thompson et al. 1997) and used neighbor- Our three sequences from genotype N (from three joining reconstruction (Saitou and Nei 1987) with 1,000 bootstrap different colonies of M. annularis) suggest that consid- replications. We included srDNA sequences from representative erable srDNA nucleotide variation (1.0–1.7%) exists alveolate taxa (apicomplexans, dinoflagellates, ciliates) which were used previously in similar phylogenetic analyses (McNally et al. between samples obtained from different colonies. These 1994; Escalante and Ayala 1995). srDNA sequences differ as much as comparisons be- tween different species of coccidians (e.g., srDNA from Toxoplasma gondii and Neospora caninum differ by only Results and discussion 0.4%). However, srDNA heterogeneity is common in Apicomplexa in general (e.g., Li et al. 1997), and the Our initial observations of genotype N in experimentally accuracy and/or taxonomic significance of individual bleached corals (see Methods) suggested that N might sequences may be suspect in such mixtures (see Wintz- 145 Fig. 2. Phylogenetic analysis of srDNA sequences from geno- type N and representative pro- tozoa. srDNA sequences were aligned and neighbor-joining reconstruction was used to ex- plore the relationship of RFLP genotype N to members of the protozoan phyla Apicomplexa (apicomplexans, light gray box), Dinozoa (dinoflagellates, medi- um-gray box), and Ciliophora (ciliates, dark gray box). Boot- strap values (percent of 1,000 replicates) are shown where support for grouping exceeded 70% ingerode et al. 1997; Toller et al. 2001b). In our limited associated with host coral pathology (Upton and Peters examination of srDNA from genotype N, we found 1986). The use of in situ hybridization methods (e.g., evidence of within-sample heterogeneity – approxi- Distel et al.