Syst Parasitol https://doi.org/10.1007/s11230-021-09981-5 (0123456789().,-volV)( 0123456789().,-volV) Redescription and molecular identification of Isospora feroxis Berto, Luz, Flausino, Ferreira & Lopes, 2009 (Eimeriidae) from tyrant-flycatchers (Tyrannoidea) in South America Carlos Nei Ortu´zar-Ferreira . Ericson Ramos de Mello . Jennifer Oliveira Melo . Mariana de Souza Oliveira . Sergio Thode-Filho . Sergian Vianna Cardozo . A´ guida Aparecida de Oliveira . Viviane Moreira de Lima . Ildemar Ferreira . Bruno Pereira Berto Received: 5 June 2020 / Accepted: 23 April 2021 Ó The Author(s), under exclusive licence to Springer Nature B.V. 2021 Abstract In the present study Isospora feroxis the interior of Southeastern Brazil, providing a Berto, Luz, Flausino, Ferreira & Lopes, 2009 is preliminary genotypic characterization via sequencing redescribed from the photosyntypes and from new of the mitochondrial cytochrome c oxidase subunit 1 samples from a short-crested flycatcher Myiarchus (COI) gene. Micropyle and rough oo¨cyst wall are ferox (Gmelin), which is the type-host in the type- added to the description of I. feroxis, in addition to locality, the Marambaia Island in Southeastern Brazil. other details. This is the sixth species identified from In addition, the yellow-olive flycatcher Tolmomyias suboscine birds (Tyranni) to have a COI gene sulphurescens Spix is recorded as a new host for this sequence deposited in GenBank and, although it is species, in a new locality, the Itatiaia National Park, in not yet possible to make conclusions on the phylogeny C. N. Ortu´zar-Ferreira Á J. O. Melo S. V. Cardozo Curso de Graduac¸a˜o em Medicina Veterina´ria, Instituto de Programa de Po´s-Graduac¸a˜o em Biomedicina Veterina´ria, Universidade Federal Rural do Rio de Translacional, Universidade do Grande Rio, Rua Janeiro, BR-465 km 7, Professor Jose´ de Souza Herdy 1160, 23897-000 Serope´dica, Rio de Janeiro, Brazil 25071-202 Duque de Caxias, RJ, Brazil E. R. de Mello A´ . A. de Oliveira Programa de Po´s-Graduac¸a˜o em Cieˆncias Veterina´rias, Departamento de Microbiologia e Imunologia Veterina´ria, Instituto de Veterina´ria, Universidade Federal Rural do Instituto de Veterina´ria, Universidade Federal Rural do Rio de Janeiro, BR-465 km 7, Rio de Janeiro, BR-465 km 7, 23897-000 Serope´dica, Rio de Janeiro, Brazil 23897-000 Serope´dica, Rio de Janeiro, Brazil M. de Souza Oliveira V. M. de Lima Á I. Ferreira Á B. P. Berto (&) Programa de Po´s-Graduac¸a˜o em Biologia Animal, Departamento de Biologia Animal, Instituto de Cieˆncias Instituto de Cieˆncias Biolo´gicas e da Sau´de, Universidade Biolo´gicas e da Sau´de, Universidade Federal Rural do Rio Federal Rural do Rio de Janeiro, BR-465 km 7, de Janeiro, BR-465 km 7, 23897-000 Serope´dica, Rio de Janeiro, Brazil 23897-000 Serope´dica, Rio de Janeiro, Brazil e-mail: [email protected] S. Thode-Filho Instituto Federal de Educac¸a˜o, Cieˆncia e Tecnologia do Rio de Janeiro, Av. Repu´blica do Paraguai 120, 25050-100 Duque de Caxias, Brazil 123 Syst Parasitol of Isospora spp. from Passeriformes by the COI gene, Southeastern Brazil, providing a preliminary geno- the molecular analysis confirmed the differences typic characterization via sequencing of the mito- between coccidian species from tyrant-flycatchers. chondrial cytochrome c oxidase subunit 1 (COI) gene. Materials and methods Introduction Sample collection Biodiversity in the Neotropical Region is very relevant for researchers around the world, among various Between August 2014 and August 2018, 19 expedi- aspects, due to its potential and recurring new tions were conducted in different locations in the discoveries (Tundisi & Matsumura-Tundisi, 2008). Itatiaia National Park (22°270S, 44°360W), a protected In this context, the class Aves stands out for its great area with a high degree of vulnerability, located in the diversity and exuberance, attracting not only scien- Serra da Mantiqueira on the border of the States of Rio tists, but also ecotourists, especially birdwatchers de Janeiro, Minas Gerais and Sa˜o Paulo (ICMBIO, (Stotz et al., 1996). Brazil is the second country in the 2016), to capture wild birds with mist nets and collect Neotropical region with the largest number of bird faecal samples. A total of seven yellow-olive flycatch- species; where, currently, there are 1919 species listed ers T. sulphurescens were captured. In addition to the by the Ornithological Records Committee of Brazil Itatiaia National Park, an expedition in September (Piacentini et al., 2015). In the context of research on 2014 was conducted on Maramabia Island Neotropical birds, there is the study of their parasites, (23°3038.86’’S, 43°58047.56’’W), on the coast of the which has been increasingly related to ecology, state of Rio de Janeiro, Southeastern Brazil, where one physiology and conservation of wild species. Among short-crested flycatcher M. ferox was captured. The bird parasites, coccidian protozoa stand out as the birds were kept in individual boxes and faeces cause of morbidity and mortality in unfavorable collected immediately after defecation. After identi- conditions of host and environment, to a commensal- fication of the species, the bird was photographed and ism-like in bird populations in conserved and balanced released and faecal samples were placed in centrifuge environments (Berto & Lopes, 2020). tubes containing a potassium dichromate 2.5% Tyrant-flycatchers represent a superfamily, Tyran- (K2Cr2O7) solution at 1:6 (v/v). noidea (Piacentini et al., 2015), or simply the family Tyrannidae, according to BirdLife International (Del Morphological analyses Hoyo & Collar, 2016), which distribution extends across North, Central and South America, being more Samples were taken to the Laborato´rio de Biologia de concentrated in the Neotropical Region. It is currently Coccı´dios, Universidade Federal Rural do Rio de the largest superfamily/family of the class Aves Janeiro (UFRRJ). Samples were incubated at room worldwide, with 450 species recorded. In Brazil, 218 temperature (25°C) for 10 days or until *70% of the species are listed (Piacentini et al., 2015; Del Hoyo & oo¨cysts were sporulated. Oo¨cysts were isolated by Collar, 2016). Even with this great diversity, there are flotation in Sheather’s sugar saturated solution (speci- few reports of coccidian species from tyrant-flycatch- fic gravity: 1.20) and examined microscopically using ers, when compared to other bird families. In this the technique described by Duszynski & Wilber context, the current study aimed to redescribe Isospora (1997) and Berto et al. (2014). Morphological obser- feroxis Berto, Luz, Flausino, Ferreira & Lopes, 2009 vations, line drawings, photomicrographs and mea- from the photosyntypes and from samples of a short- surements were made using an Olympus BX binocular crested flycatcher Myiarchus ferox (Gmelin), which is microscope (Olympus Optical, Tokyo, Japan) coupled the type-host in the type-locality, the Marambaia to a digital camera Eurekam 5.0 (BEL Photonics, Island in Southeastern Brazil. In addition, the yellow- Monza, Italy). Line drawings were edited using Corel olive flycatcher Tolmomyias sulphurescens Spix is DRAWÒ and Corel PHOTO-PAINT (Corel Draw recorded as a new host for this species, in a new Graphics Suite, Version 11.0, Corel Corporation, locality, the Itatiaia National Park, in the interior of Canada). All measurements are in micrometres and 123 Syst Parasitol are given as the range followed by the mean in DNA sequence analyses parentheses. The newly generated sequence was compared to those Molecular analyses for Isospora spp. and other coccidian parasites avail- able on the GenBank database using the Basic Local An individual oo¨cyst from a fecal sample of T. Alignment Search Tool (BLAST). Phylogenetic trees sulphurescens was isolated from serial dilutions of the were constructed for Isospora spp. at the COI oo¨cysts in drops on a microscope slide using a sterile sequences for additional isolates from GenBank. micropipette. This isolated oo¨cyst was resuspended in Alignment and parsimony analyses were conducted PBS and washed by centrifuging until the supernatant using MEGA version 7, using ClustalW as alignment became clear (Dolnik et al., 2009). DNA was extracted algorithm (Tamura et al., 2007). The evolutionary from the purified oo¨cysts using the Qiagen DNeasy history was inferred using the Neighbor-Joining (NJ) Blood and Tissue Kit (Qiagen, Sa˜o Paulo, Brazil) and Maximum Likelihood (ML) methods and the according to the manufacturers& instructions. In order distances were computed using the Tamura-Nei to fully lyse the oo¨cyst, four freeze-thaw cycles were method based on model selection using ModelTest applied prior to the DNA extraction. The PCR in MEGA 7. Bootstrap analyses were conducted using amplification for the COI gene was carried out using 1,000 replicates to assess the reliability of inferred tree a nested PCR, as previously described by Dolnik et al. topologies. (2009) and Yang et al. (2015). The external primers COIbF1 (50-GWT CAT TAG TAT GGG CAC ATC Obtaining the photosyntypes of Isospora feroxis A-30) and COIbR1 (50-CCA AGA GAT AAT ACR Berto, Luz, Flausino, Ferreira & Lopes, 2009 AAR TGG AA-30) produced a PCR product of 302 bp in size. The internal primes COIbF2 (50-GGG CAC The photosyntypes of I. feroxis from M. ferox ATC ATA TGA TGA C-30) and COIbR2 (50-ATA identified in Berto et al. (2009a), which were deposited GTA TGT ATC ATG TAR WGC AA-30) produced an in the Parasitology Collection of the Laborato´rio de amplicon of 257 bp in size. The PCR reaction Biologia de Coccı´dios (http://r1.ufrrj.br/labicoc/ contained 10 llof59 Green GoTaqÒ Flexi Buffer, colecao.html) at UFRRJ under repository number 3 ll of 25 mM MgCl2,1ll of 10 mM dNTPs, 0.4 lM P-30/2009, were required for morphological of each primer, 1.25 units of GoTaqÒ DNA poly- comparison.