Syst Parasitol (2019) 96:423–431 https://doi.org/10.1007/s11230-019-09859-7 (0123456789().,-volV)( 0123456789().,-volV) The vulnerable Sporophila frontalis (Verreaux) and Haplospiza unicolor Cabanis as new hosts for Isospora sporophilae Carvalho-Filho, Meireles, Ribeiro & Lopes, 2005 (Eimeriidae) in Brazil Mariana Borges Rodrigues . Jhon Lennon Genovez de Oliveira . Lidiane Maria da Silva-Carvalho . Danilo Giovanni Narciso Pastura . Jennifer Vieira Gomes . Mariana de Souza Oliveira . Priscyanne Barreto Siqueira . A´ guida Aparecida de Oliveira . Viviane Moreira de Lima . Ildemar Ferreira . Bruno Pereira Berto Received: 18 September 2018 / Accepted: 22 February 2019 / Published online: 10 May 2019 Ó Springer Nature B.V. 2019 Abstract Isospora sporophilae Carvalho-Filho, described from double-collared seedeaters Sporophila Meireles, Ribeiro & Lopes, 2005 was morphologically caerulescens (Vieillot) in a center screening of wild and molecularly identified from the double-collared animals; therefore, this new report emphasises a potential seedeater Sporophila frontalis (Verreaux), which is occurrence of anthropomorphic dispersion of coccidia categorised as ‘vulnerable’ by the International Union through illegal trade, seizures and reintroductions in the for Conservation of Nature and Natural Resources wild. (IUCN), and from the uniform finch Haplospiza unicolor Cabanis in conserved and anthropomorphic/fragmented areas of Atlantic Forest in the southeastern Brazil. The oo¨cysts recovered from S. frontalis and H. unicolor had Introduction small morphological and genotypic differences that were not considered sufficient for the description of new Thraupidae Cabanis comprises the second largest species, but only different genotypes of I. sporophilae family of the order Passeriformes with 408 species related to each host. This coccidian species was originally distributed predominantly in the Neotropical region M. B. Rodrigues Á L. M. da Silva-Carvalho A´ . A. de Oliveira Á V. M. de Lima Á I. Ferreira Á Programa de Po´s-Graduac¸a˜o em Cieˆncias Veterina´rias, B. P. Berto (&) Instituto de Veterina´ria, Universidade Federal Rural do Departamento de Biologia Animal, Instituto de Cieˆncias Rio de Janeiro, BR-465 km 7, Serope´dica, Biolo´gicas e da Sau´de, Universidade Federal Rural do Rio Rio de Janeiro 23897-000, Brazil de Janeiro, BR-465 km 7, Serope´dica, Rio de Janeiro 23897-000, Brazil J. L. G. de Oliveira Á M. de Souza Oliveira e-mail: [email protected] Programa de Po´s-Graduac¸a˜o em Biologia Animal, Instituto de Cieˆncias Biolo´gicas e da Sau´de, Universidade Federal Rural do Rio de Janeiro, BR-465 km 7, Serope´dica, Rio de Janeiro 23897-000, Brazil D. G. N. Pastura Á J. V. Gomes Á P. B. Siqueira Curso de Graduac¸a˜o em Cieˆncias Biolo´gicas, Instituto de Cieˆncias Biolo´gicas e da Sau´de, Universidade Federal Rural do Rio de Janeiro, BR-465 km 7, Serope´dica, Rio de Janeiro 23897-000, Brazil 123 424 Syst Parasitol (2019) 96:423–431 (BirdLife International, 2016). This family comprises categorised as ‘vulnerable’ by the International Union passerines with varied colors, vocalizations, foraging for Conservation of Nature and Natural Resources behaviors, ecotypes and habitat preferences (Sick, (IUCN) (BirdLife International, 2016), in a preserved 1997; Burns et al., 2014). Atlantic Forest area corresponding to the Itatiaia Recently there was a redistribution of genera in the National Park (Parque Nacional do Itatiaia); and (ii) families of Passeriformes, which considerably the uniform finch Haplospiza unicolor Cabanis in a expanded the number of genera/species in the Thraup- fragmented Atlantic Forest area in the district of idae. In this redistribution, seedeaters of the genus Cacaria, Piraı´, all in the southeastern Brazil. Sporophila and several other genera previously clas- sified in the families Emberizidae and Cardinalidae were included in the Thraupidae (Burns et al., 2014; Materials and methods CBRO, 2014; BirdLife International, 2016; del Hoyo et al., 2016; Brands, 2018). Sample collection The beauty and vocal repertoire of the tanagers and seedeaters makes them valuable as companion ani- A total of eight expeditions were conducted in two mals, thereby stimulating captive breeding for legal different localities in southeastern Brazil: (i) Itatiaia trade, but also illegal wildlife trade. For this reason, National Park, a protected area with a high degree of the thraupids are one of the main wild birds, victims of vulnerability, located in the Serra da Mantiqueira on the illegal trade in countries of the Neotropical region. the border of the States of Rio de Janeiro, Minas Gerais This can be observed in the centers screening of wild and Sa˜o Paulo (ICMBIO, 2016), in August and animals, which has as one of the functions the December 2014, April and May 2015, July 2017, and rehabilitation and release of wild animals seized from April and July 2017; and (ii) Cacaria, a district of the the illegal trade. In these centers, the thraupids are Municipality of Piraı´ in the State of the Rio de Janeiro, often the most abundant animals (Carvalho-Filho in September 2016. A total of eight S. frontalis (all et al., 2005; Berto & Lopes, 2013; Lopes et al., 2013). from Itatiaia National Park) and nine H. unicolor (eight In addition to the direct impact of the illegal trade from Itatiaia National Park and one from Cacaria) were on the wild birds, this activity enables and/or enhances captured with mist nets. The birds were kept in the dispersion of parasites in an unnatural (anthropo- individual boxes and faeces collected immediately morphic) way. Coccidia are one of the principal after defecation. After identification of the species, the parasite groups of thraupids, which can be easily bird was photographed and released and stool samples transmitted via the oral-faecal route, i.e. a thraupid were placed in centrifuge tubes containing a potassium transported from one environment to the other carries dichromate 2.5% (K2Cr2O7) solution at 1:6 (v/v). its coccidia which could be transmitted to another susceptible thraupid, in both natural and captive Morphological analyses environments. This understanding makes the function of the centers screening of wild animals more impor- Samples were taken to the Laborato´rio de Biologia de tant, since the failure to identify a parasite of a thraupid Coccı´dios, Universidade Federal Rural do Rio de seized, followed by its release in the wild, different Janeiro (UFRRJ). Samples were incubated at room from its original locality, would provide the introduc- temperature (25°C) for 10 days or until c.70% of the tion of a new parasite to susceptible hosts in a new oo¨cysts were sporulated. Oo¨cysts were isolated by locality (Berto & Lopes, 2013; Lopes et al., 2013). flotation in Sheather’s sugar saturated solution (speci- In the present study Isospora sporophilae Car- fic gravity: 1.20) and examined microscopically using valho-Filho, Meireles, Ribeiro & Lopes, 2005, which the technique described by Duszynski & Wilber was originally described from double-collared see- (1997) and Berto et al. (2014). Morphological obser- deaters Sporophila caerulescens (Vieillot) in a center vations, line drawings, photomicrographs and mea- screening of wild animals (Carvalho-Filho et al., surements were made using an Olympus BX binocular 2005), was morphologically and molecularly identi- microscope (Olympus Optical, Tokyo, Japan) coupled fied from two new hosts: (i) the buffy-fronted to a digital camera Eurekam 5.0 (BEL Photonics, seedeater Sporophila frontalis (Verreaux), which is Monza, Italy). Line drawings were edited using two 123 Syst Parasitol (2019) 96:423–431 425 software applications from CorelDRAWÒ (Corel GoTaqÒ DNA polymerase, 3 ll of DNA (for primary Draw Graphics Suite, Version 11.0, Corel Corporation, reaction) or 3ll primary PCR product (for the secondary Canada), i.e. Corel DRAW and Corel PHOTO-PAINT. reaction). Both primary and secondary PCR were All measurements are in micrometres and are given as conducted using the same cycling conditions: 1 cycle the range followed by the mean in parentheses. of 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 47°C for 45 s, and 72°C for 1 min and a final extension Morphometric analyses of 72°C for 5 min. The amplicons from the second round of PCR were purified using the Qiagen MinElute PCR Two parametric statistical methods were employed in the Purfication (Qiagen, Sa˜o Paulo, Brazil). All PCR morphometric data of the oo¨cysts after previous evalu- products were sequenced using the PCR forward and ation of the data by DAgostino& s& test of normality. reverse primers by Ludwig Biotecnology, were an ABI- Analysis of variance (ANOVA) was used to compare Prism 3500 Genetic Analyzer (Applied Biosystems, measurements of the length, width and length/width (L/ Foster City, California) was used for Sanger sequencing. W) ratio of the oo¨cysts and sporocysts recovered from S. The results of the sequencing reactions were analysed frontalis and H. unicolor. The statistical package Bioestat andeditedusingtheprogramChromas2.6. 5.0 (Ayres et al., 2007) was used to calculate the mean, variance, degrees of freedom and P-value (Sampaio, DNA sequence analyses 2002; Berto et al., 2014). Linear regression was used to determine the distribution of oo¨cysts recovered from S. Sequences were compared to each other and with other frontalis and H. unicolor using methods proposed by coccidian parasites available on the GenBank database Norton & Joyner (1981) and subsequently modified by using the Basic