Revista Brasileira de Parasitologia Veterinária ISSN: 0103-846X [email protected] Colégio Brasileiro de Parasitologia Veterinária Brasil Klein Sercundes, Michelle; Oshiro Branco Valadas, Samantha Yuri; Borges Keid, Lara; Ferreira Souza Oliveira, Tricia Maria; Lage Ferreira, Helena; Wagner de Almeida Vitor, Ricardo; Gregori, Fábio; Martins Soares, Rodrigo Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences Revista Brasileira de Parasitologia Veterinária, vol. 25, núm. 1, enero-marzo, 2016, pp. 82 -89 Colégio Brasileiro de Parasitologia Veterinária Jaboticabal, Brasil Available in: http://www.redalyc.org/articulo.oa?id=397844775009 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Original Article Braz. J. Vet. Parasitol., Jaboticabal, v. 25, n. 1, p. 82-89, jan.-mar. 2016 ISSN 0103-846X (Print) / ISSN 1984-2961 (Electronic) Doi: http://dx.doi.org/10.1590/S1984-29612016015 Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences Filogenia molecular de Toxoplasmatinae: comparação entre inferências baseadas em sequências genéticas mitocondriais e de apicoplasto Michelle Klein Sercundes1; Samantha Yuri Oshiro Branco Valadas1; Lara Borges Keid2; Tricia Maria Ferreira Souza Oliveira2; Helena Lage Ferreira2; Ricardo Wagner de Almeida Vitor3; Fábio Gregori1; Rodrigo Martins Soares1* 1 Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo – USP, São Paulo, SP, Brasil 2 Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo – USP, Pirassununga, SP, Brasil 3 Departamento de Parasitologia, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG, Brasil Received November 24, 2015 Accepted February 16, 2016 Abstract Phylogenies within Toxoplasmatinae have been widely investigated with different molecular markers. Here, we studied molecular phylogenies of the Toxoplasmatinae subfamily based on apicoplast and mitochondrial genes. Partial sequences of apicoplast genes coding for caseinolytic protease (clpC) and beta subunit of RNA polymerase (rpoB), and mitochondrial gene coding for cytochrome B (cytB) were analyzed. Laboratory-adapted strains of the closely related parasites Sarcocystis falcatula and Sarcocystis neurona were investigated, along with Neospora caninum, Neospora hughesi, Toxoplasma gondii (strains RH, CTG and PTG), Besnoitia akodoni, Hammondia hammondi and two genetically divergent lineages of Hammondia heydorni. The molecular analysis based on organellar genes did not clearly differentiate between N. caninum and N. hughesi, but the two lineages of H. heydorni were confirmed. Slight differences between the strains of S. falcatula and S. neurona were encountered in all markers. In conclusion, congruent phylogenies were inferred from the three different genes and they might be used for screening undescribed sarcocystid parasites in order to ascertain their phylogenetic relationships with organisms of the family Sarcocystidae. The evolutionary studies based on organelar genes confirm that the genus Hammondia is paraphyletic. The primers used for amplification of clpC and rpoB were able to amplify genetic sequences of organisms of the genus Sarcocystis and organisms of the subfamily Toxoplasmatinae as well. Keywords: Sarcocystidae, molecular characterization, phylogeny, Toxoplasmatinae, apicoplast gene, mitochondrial gene. Resumo A filogenia da subfamília Toxoplasmatinae tem sido amplamente investigada com diversos marcadores moleculares. Neste estudo, a filogenia molecular da subfamília Toxoplasmatinae foi analisada através de genes de apicoplasto e mitocondriais. Foram analisadas sequências parciais de genes de apicoplasto codificadores da protease caseinolítica (clpC), e da subunidade beta da RNA polimerase (rpoB) e de gene mitocondrial codificador de citocromo B cytB( ). Foram investigadas cepas adaptadas em laboratório de Sarcocystis neurona e Sarcocystis falcatula, parasitos estreitamente relacionados, além de Neospora caninum, Neospora hughesi, Toxoplasma gondii (cepas RH, CTG e PTG), Besnoitia akodoni, Hammondia hammondi e duas linhagens geneticamente divergentes de Hammondia heydorni. A análise molecular, baseada em genes de organelas, não diferenciou claramente N. caninum de N. hughesi, porém foi possível confirmar as duas linhagens de H. heydorni. Foram encontradas pequenas diferenças entre as cepas adaptadas em laboratório de *Corresponding author: Rodrigo Martins Soares. Departamento de Medicina Veterinária Preventiva e Saúde Animal – VPS, Faculdade de Medicina Veterinária e Zootecnia – FMVZ, Universidade de São Paulo – USP, Av. Prof. Dr. Orlando Marques de Paiva, 87, Cidade Universitária, CEP 05508-270, São Paulo, SP, Brasil. e-mail: [email protected] www.cbpv.org.br/rbpv v. 25, n. 1, jan.-mar. 2016 Molecular phylogeny of Toxoplasmatinae 83 S. falcatula e S. neurona em todos os marcadores moleculares avaliados. Concluindo, filogenias congruentes foram reconstruídas com os três diferentes genes que podem ser úteis em triagem de parasitos sarcocistídeos não identificados, para identificar sua relação com organismos da família Sarcocystidae. Os estudos evolutivos com genes organelares confirmam que o gêneroHammondia é parafilético. Osprimers utilizados para amplificação declpC e rpoB foram capazes de amplificar sequências genéticas de organismos do gêneroSarcocystis e da subfamília Toxoplasmatinae. Palavras-chave: Sarcocystidae, caracterização molecular, filogenia, Toxoplasmatinae, gene apicoplasto, gene mitocondrial. Introduction opossum (Didelphis virginiana) that had been fed with skeletal muscles from a raccoon (Procyon lotor) that in turn had been fed Mitochondrial genes are useful for phylogenetic analysis at with sporocysts that had been isolated in Ohio, United States different taxonomic levels of apicomplexan parasites (LIN et al., (LINDSAY et al., 2004). Sarcocystis falcatula SF1 was obtained 2011; HE et al., 2014). Cytochrome oxidase II and cytochrome from tissues of budgerigars (Melopsittacus undulatus) that had B (cytB) coding sequences have been used for this purpose been orally inoculated with sporocysts from the intestines of an (GJERDE, 2013a, b). Similar to mitochondria, apicoplasts are opossum (D. virginiana) that had been isolated in California, organelles of maternal inheritance (FERGUSON et al., 2005) that United States (MARSH et al., 1997). Dr. David Lindsay kindly contain conserved genes that are commonly used to reconstruct provided both the S. neurona and the S. falcatula strain. evolutionary histories (FEAGIN, 1994; KOHLER et al., 1997). DNA from Besnoitia akodoni was also used. This is a parasite Genes encoded in the apicoplast genome are useful markers for isolated from a rodent (Akodon montensis) that is native to Brazil resolving the phylogeny of Plasmodium species, because these genes and was obtained as previously described (DUBEY et al., 2003b). have greater phylogenetic signal than the mitochondrial genome for DNA from the Neospora hughesi Oregon isolate (DUBEY et al., Plasmodium phylogeny (reviewed in ARISUE & HASHIMOTO, 2001), Hammondia hammondi (isolate 300) and two genetically 2015). Among other genes within the plastid genome, two genes divergent lineages of Hammondia heydorni (isolates 376 and BR) are highlighted. The gene coding for caseinolytic protease (clpC) were obtained as described elsewhere (MONTEIRO et al., 2007). (a member of the chaperone family) is a conserved apicoplast gene that is widely applied in phylogenetic reconstructions of apicomplexan organisms (RATHORE et al., 2001; MARTINSEN et al., 2008; PCR and primers LAU et al., 2009). The plastid-encoded beta subunit of RNA polymerase (rpoB), which is responsible for processing polymerase The polymerase chain reaction was performed in accordance activity, has also been used in phylogenetic reconstructions among with the manufacturer’s recommendations for platinum Taq DNA organisms of the Sarcocystidae family (DUBEY et al., 2003a; polymerase (Invitrogen, Carlsbad, CA, USA), using the primers WENDTE et al., 2010). listed in Table 1. The annealing temperature of each primer pair Here, we studied molecular phylogenies of the Toxoplasmatinae is given in Table 1. subfamily in order to target mitochondrial and apicoplast genetic Primers for clpC were designed from a consensus sequence of sequences. The known genus forming the Toxoplasmatinae subfamily clpC from EST database sequences of N. caninum and S. neurona are Besnoitia, Toxoplasma, Neospora, and Hammondia, closely related and RNAm sequences of T. gondii. Primers for cytochrome B coding coccidians with similarly sized oocysts (DUBEY, 1993). The aim sequences (cytB) targeting Sarcocystis spp. were designed from of this paper was to demonstrate the suitability of these molecular genomic sequences of S. neurona and S. falcatula (VALADAS et al., markers for studying the genetic variability and phylogenetic 2016). A CytB primer targeting members of the Toxoplasmatinae relationships among members of the Toxoplasmatinae subfamily. subfamily was designed from EST database sequences of N. caninum Phylogenies within Toxoplasmatinae have been greatly