ŘŚǰȱž••ǯȱž›ǯȱœœǯȱ’œ‘ȱŠ‘˜•ǯǰȱřřǻŗǼȱŘŖŗř

NOTE Putative Ichthyophthiriusȱ’Ž—’ꮍȱ in the amphibian Bufo calamita through molecular screening.

D. J. Harris1,2*, J. Seabra-BaboŚ, J. TavaresŚ and J. P. M. C. Maia1,2,3

1CIBIO-UP, Centro de Investigação em Biodiversidade e Recursos Genéticos da Universidade do Porto, Campus Agrário de Vairão, 4485-661 Vairão, Portugal; 2Departamento de Biologia, Faculdade de Ciências, —’ŸŽ›œ’ŠŽȱ˜ȱ˜›˜ǰȱžŠȱ˜ȱŠ–™˜ȱ•Ž›ŽȱŚǰȱŚŗŜşȬŖŖŝȱ˜›˜ǰȱ˜›žŠ•Dzȱ3Institut de Biologia Ÿ˜•ž’ŸŠȱǻ ȬǼǰȱŠœœŽ’ȱŠ›Ç’–ȱŽȱ•ŠȱŠ›ŒŽ•˜—ŽŠǰȱřŝȬŚşǰȱŖŞŖŖřȱŠ›ŒŽ•˜—Šǰȱ™Š’—Dzȱ4Departamento Žȱ’˜•˜’Šǰȱ—’ŸŽ›œ’ŠŽȱŽȱŸŽ’›˜ǰȱŠ–™žœȱ—’ŸŽ›œ’¤›’˜ȱŽȱŠ—’Š˜ǰȱřŞŗŖȬŗşřȱŸŽ’›˜ǰȱ˜›žŠ•ǯ

Abstract The protozoan parasite Œ‘‘¢˜™‘’›’žœȱ–ž•’ę•’’œȱ’œȱŠ—ȱ’–™˜›Š—ȱ™Š‘˜Ž—ȱ˜ȱ–Š—¢ȱ꜑ȱœ™ŽŒ’Žœǯȱ ž›’—ȱ–˜•ŽŒž•Š›ȱœŒ›ŽŽ—’—ȱ˜ȱŗŞȱ›ȱŽ—Žǰȱ Žȱ’Ž—’ꮍȱŠ—ȱŠ™™Š›Ž—ȱIchthyopthirius from the amphibian Bufo calamitaǰȱ˜ȱ˜ž›ȱ”—˜ •ŽŽȱ‘ŽȱꛜȱœžŒ‘ȱŽŽŒ’˜—ȱ›Ž™˜›Žȱ›˜–ȱŠȱ ’•ȱŠ–™‘’‹’Š—ǯȱ This has important implications for parasite control approaches in aquaculture.

Protistans are one of the most important groups One important, unanswered question is how ˜ȱ™Š‘˜Ž—œȱ˜ȱ꜑ȱǻŒ‘˜•£ǰȱŗşşşǼǯȱȱ‘ŽœŽǰȱ does an obligate parasite survive between out- one of the most pathogenic protozoan parasites breaks? It has been hypothesized that it is most ˜ȱ›Žœ‘ ŠŽ›ȱ꜑ȱ’œȱ‘ŽȱŒ’•’ŠŽȱIchthyopthirius likely that the parasite is maintained through –ž•’ę•’’œ, which causes Ichthyopthiriasis, or low level infections (Dickerson, 2006). “white spot disease” (Dickerson, 2006). This is a major problem in aquaculture, compounded •‘˜ž‘ȱ’Ž—’ęŒŠ’˜—ȱ˜ȱ™Š›Šœ’Žœȱ Šœȱ›Š- by its cosmopolitan distribution, having been ditionally based on morphological characters, ›Ž™˜›Žȱ›˜–ȱŠ••ȱ›Ž’˜—œȱ ‘Ž›Žȱ꜑ȱŠ›ŽȱŒž•’- molecular techniques are a powerful tool to ŸŠŽǯȱž›‘Ž›–˜›Žǰȱ’ȱŠěŽŒœȱŠȱ ’Žȱ›Š—Žȱ˜ȱ identify various parasite groups in freshwater ꜑ȱ’—Œ•ž’—ȱŒŠ›™ǰȱ›˜žǰȱŽŽ•ǰȱŒŠęœ‘ȱŠ—ȱ˜›—Š- environments. Not only can larger numbers –Ž—Š•ȱ꜑ȱǻŒ‘˜•£ǰȱŗşşşǼǯȱŠŽ›ȱŽ–™Ž›Šž›Žȱ of samples be screened quickly, but molecular appears to be critical for disease outbreaks, data can also give information regarding the ‘’Œ‘ȱŠ›Žȱ–˜›ŽȱŒ˜––˜—ȱ ‘Ž—ȱ꜑ȱŠ›Žȱœ›ŽœœŽȱ diversity of parasites and their phylogenetic and water temperature rises (Dickerson, 2006). relationships.

* Corresponding author’s e-mail: [email protected] Bull. Eur. Ass. Fish Pathol., 33(1) 2013, 25

During routine screening of the amphibian Bufo using the most closely related sequences avail- calamita from the Iberian Peninsula a standard able on GenBank. Maximum Likelihood (ML) protocol was followed. DNA was extracted analysis included random sequence addition from toe-clips using standard high-salt methods (100 replicate heuristic searches), and support (Sambrook et al., 1989). Primers used were for nodes was estimated using the bootstrap Ž™řŖŖȱŠ—ȱ Ž™şŖŖȱǻ“ŸŠ›’ȱŽȱŠ•ǯǰȱŘŖŖŚǼǰȱ technique (Felsenstein, 1985) with 500 replicates, that target a part of the 18S rRNA gene. The using PhyML 3.0 (Guindon et al., 2010). The AIC are known to amplify Apicomplexan parasites criteria carried out in jModeltest 0.1.1 (Posada, (Harris et al., 2012) as well as various other 2008) were used to choose the model of evolu- groups including Stramenopiles (Maia et al., tion employed (TrN+G). Bayesian analysis was 2012) and some fungi (Tomé et al., 2012). PCR implemented using Mr. Bayes 3.1 (Huelsenbeck Œ˜—’’˜—œȱŒ˜—œ’œŽȱ˜ȱřśȱŒ¢Œ•Žœȱ˜ȱşŚķȱǻřŖȱ and Ronquist, 2001) with parameters estimated œŽŒ˜—œǼǰȱŜŖķȱǻřŖȱœŽŒ˜—œǼȱŠ—ȱŝŘķȱǻŗȱ–’—žŽǼȱ as part of the analysis. The analysis was run for – see Harris et al. (2011) for more details. Nega- 5x106 generations, saving one tree each 1000 tive and positive controls were run with each generations. The log-likelihood values of the reaction. All positive PCRs were sequenced by œŠ–™•Žȱ™˜’—ȱ Ž›Žȱ™•˜ĴŽȱŠŠ’—œȱ‘ŽȱŽ—- a commercial service (Macrogen Inc.). Resulting eration time and all the trees prior to reaching chromatograms were checked manually and stationary were discarded. Remaining trees assembled in BioEdit (Hall, 1999). BLAST was were combined in a 50% majority consensus used to identify closest matches in GenBank. tree. Following Wright and Lynn (1995), Para- mecium tetraureliaȱǻŖřŝŝŘǼȱ ŠœȱŽœ’—ŠŽȱŠœȱ Out of the 56 samples of B. calamita screened an outgroup (Figure 1). (10 from Castelo Branco, 5 from Mindelo and 19 from Aveiro in Portugal, and 22 from Tra- œȱŽ¡™ŽŒŽǰȱ‘ŽȱŒ’•’ŠŽȱ’Ž—’ꮍȱ›˜–ȱB. ca- bazos in Spain), two positives (DB16921 and lamita was sister taxa to ǯȱ–ž•’ę•’’œȱwith high DB16925 from Trabazos) matched ciliates in the support. Sister taxa to this was Ophryoglena BLAST search (GenBank Accession numbers catenulaȱǻŗŝřśśǰȱ›’‘ȱŠ—ȱ¢——ǰȱŗşşśǼǰȱ‘Ž—ȱ śŗŘŝŜŝȱŠ—ȱ śŗŘŝŜŞȱ›Žœ™ŽŒ’ŸŽ•¢Ǽǯȱ‘Žȱ two uncultured clones (EF586162 and EF586110, ꛜȱŠŸŽȱ‘ŽȱŒ•˜œŽœȱ–ŠŒ‘ȱ ’‘ȱHausmann- Dopheide et al., 2008), and then Tetrahymena iella discoidea (Accession number EU039900, paravoraxȱǻŖŝŖŘśřǰȱ‘Š—Š—œ’ȱŽȱŠ•ǯǰȱŘŖŖŝǼǰȱ Dunthorne et al., 2008). The match was 98% relationships similar to those previously pro- ’‘ȱŝȱ’Ž—’ꮍȱ’쎛Ž—ŒŽœȱ˜ŸŽ›ȱśřŗ‹™ǯȱHaus- posed (Wright and Lynn, 1995). manniellaȱŠ›Žȱ ’Žœ™›ŽŠȱ™›˜˜£˜Š—œǰȱ˜ĞŽ—ȱ ’Ž—’ꮍȱ’—ȱŽ—Ÿ’›˜—–Ž—Š•ȱŠœœŽœœ–Ž—œȱǻŽȱ This result is important for several reasons. Š›˜œåŸŠȱŠ—ȱ’›“Š”˜ŸŠǰȱŘŖŖŞǼǯȱ ˜ ŽŸŽ›ǰȱ‘Žȱ First, ǯȱ–ž•’ę•’’œȱis currently considered to ex- second ciliate gave a closest match with I. mul- Œ•žœ’ŸŽ•¢ȱ™Š›Šœ’’œŽȱ›Žœ‘ ŠŽ›ȱ꜑ǰȱŠ•‘˜ž‘ȱ ’ę•’’œȱǻŒŒŽœœ’˜—ȱ—ž–‹Ž›ȱŗŝřśŚǰȱ›’‘ȱŠ—ȱ tadpole stages of the marsh frog Limnodynastes ¢——ǰȱŗşşśǼǯȱ˜ȱŒ˜—ę›–ȱ‘’œȱŠœœŽœœ–Ž—ǰȱ ˜ȱ peronii were successfully infected experimen- ’쎛Ž—ȱ™‘¢•˜Ž—Ž’ŒȱŠ—Š•¢œŽœȱ Ž›ŽȱŒŠ››’Žȱ˜žȱ tally (Gleeson, 1999). Our results indicate that (Maximum Likelihood and Bayesian Inference) the parasite DNA could be detected from the 26, Bull. Eur. Ass. Fish Pathol., 33(1) 2013

X03772 - Paramecium tetrawelia

EF070253 - Tetrahymena paravorax

EF586162 - uncultured clone 99 100 EF586110 - uncultured clone

70 72 U17355 - Ophryoglena catenula

0.01 subs/site 79 57 U17354 - Ichthyophthirius multifiliis 88 79 DB16925 - ex. Bufo calamita

Figure 1. ML tree of the apparent Ichthyophthirius from a Bufo calamita, and closest available comparative sequences from GenBank. Support for the Bayesian and for ML analysis are given above and below the nodes, respectively. The branch of Paramecium tetraweliaȱ Šœȱœ‘˜›Ž—ŽȱŝśƖǯ clipped toe of a wild amphibian. This does not ascertain if other amphibian species can also per se demonstrate infection - during the tomont host similar parasites, and whether these can stage of the life cycle of ǯȱ–ž•’ę•’’œȱit produces Œ›˜œœȱ’—ŽŒȱ‹Ž ŽŽ—ȱŠ–™‘’‹’Š—œȱŠ—ȱ꜑ǯȱ ȱŠ–- Šȱœ’Œ”¢ȱŒŠ™œž•Žȱ‘ŠȱŒŠ—ȱŠĴŠŒ‘ȱ˜ȱŸŠ›’˜žœȱœž‹- phibians are acting as sinks for these parasites, strates. From this capsule theronts are released it will be important to take this into account ‘’Œ‘ȱŒŠ—ȱœž›Ÿ’ŸŽȱ˜›ȱŘȬŚȱŠ¢œȱ ‘’•ŽȱŠŒ’ŸŽ•¢ȱ when trying to control disease outbreaks. searching for a host. Whether detection indicates ž›‘Ž›ȱ–˜•ŽŒž•Š›ȱœŒ›ŽŽ—’—ȱ˜ȱŠ’’˜—Š•ȱ꜑ȱ an infection or not, clearly needs further inves- and amphibian species, either using general tigation. Secondly the sequence shows limited ™›’–Ž›œȱŠœȱ’—ȱ‘’œȱœž¢ȱ˜›ȱ˜—Žœȱ–˜›Žȱœ™ŽŒ’ęŒȱ ’쎛Ž—’Š’˜—ȱ›˜–ȱ‘Žȱ™ž‹•’œ‘Žȱ ǯȱ–ž•’ę•’’œȱ for Ichthyopthirius, will be essential to provide œŽšžŽ—ŒŽǰȱ ’‘ȱŗŘȱ’쎛Ž—ŒŽœȱ˜ŸŽ›ȱśŚŚȱ‹™ǯȱ ȱ further information on this possibility. has previously been proposed that multiple strains or even cryptic species may occur within ȱ’œȱœž™™˜›Žȱ‹¢ȱŠȱž—Š³¨˜ȱ™Š›ŠȱŠȱ ǯȱ–ž•’ę•’’œȱǻ’›Ž••’ȱŽȱŠ•ǯǰȱŗşŝŜǼǰȱŠ—ȱ˜ž›ȱ›Žœž•œȱ ’¹—Œ’ŠȱŽȱŠȱŽŒ—˜•˜’ŠȱǻǼȱ‘ȱ›Š—ȱǻ Ȧ seem to be further evidence for this with the ȦŝŚřŖśȦŘŖŗŖǼȱŠ—ȱŒ˜Ȭꗊ—ŒŽȱ‹¢ȱȱŠ—ȱ two sequences showing some divergence whilst POPH and EU. Thanks to the anonymous re- clearly belonging to the same clade. viewer for their useful comments on an earlier version. Given the importance of ǯȱ–ž•’ę•’’œȱas a parasite ˜ȱ›Žœ‘ ŠŽ›ȱ꜑ǰȱ’ȱ‹ŽŒ˜–Žœȱ’–™Ž›Š’ŸŽȱ˜ȱ ž••ǯȱž›ǯȱœœǯȱ’œ‘ȱŠ‘˜•ǯǰȱřřǻŗǼȱŘŖŗřǰȱŘŝ

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