Introduction

The parvorder Thamnophilida (Passeriformes: Tyranni) is divided into three families: Thamnophilidae, Melanopareiidae and Conopophagidae. The family Conopophagidae brings together two genera: Conopophaga Vieillot, 1816 and Pittasoma Cassin, 1860. In Brazil, seven Conopophaga spp. are reported; however, the two Pittasoma spp. are restricted to Colombia, Costa Rica, Panama and Ecuador [4, 12].

The gnateaters Conopophaga spp. are small, with a long tarsus, short tail and rounded wing. Most species have an elongated, usually white, post-ocular stripe. They are terrestrial insectivores commonly observed in high and humid forests, where they remain lodged in thin branches and, sometimes, they fly to the ground to catch insects [13].

The insectivorous feeding habit was previously related to the low prevalence and density of Isospora spp. in passerines [7]; however, several species of coccidia are recorded for families of insectivorous passerines [1]. In this context, the aim of this study was to examine the feces from gnateaters Conopophaga spp. captured in different localities in the Médio Paraíba region of the State of Rio de Janeiro, Southeastern Brazil, to determine what coccidian parasites were present.

Materials and Methods

Sample Collection

A total of five expeditions were conducted in two different localities in the Médio Paraíba Region in the State of the Rio de Janeiro, Southeastern Brazil: (1) Itatiaia National Park (Parque Nacional do Itatiaia), a protected area with a high degree of vulnerability, located in the Serra da Mantiqueira [9]; and (2) an Atlantic forest fragment area at the Municipality of Barra Mansa. A total of five black-cheeked gnateater Conopophaga melanops (Vieillot, 1818) (all from Itatiaia National Park) and four rufous gnateater Conopophaga lineata (Wied, 1831) (two from Itatiaia National Park and two from Barra Mansa) were captured with mist nets. The birds were kept in individual boxes and feces collected immediately after defecation. After identification of the species, the birds were photographed and released and stool samples were placed in centrifuge tubes containing a potassium dichromate 2.5% (K2Cr2O7) solution at 1:6 (v/v).

Morphological Analyses

Samples were taken to the Laboratório de Biologia de Coccídios, Universidade Federal Rural do Rio de Janeiro (UFRRJ). Samples were incubated at room temperature (25 °C) for 10 days or until ~ 70% of the oocysts were sporulated. Oocysts were isolated by flotation in Sheather’s sugar saturated solution (specific gravity: 1.20) and examined microscopically using the technique described by Duszynski and Wilber [8] and Berto et al. [2]. Morphological observations, line drawings, photomicrographs and measurements were made using an Olympus BX binocular microscope (Olympus Optical, Tokyo, Japan) coupled to a digital camera Eurekam 5.0 (BEL Photonics, Monza, Italy). Line drawings were edited using two software applications from CorelDRAW® (Corel Draw Graphics Suite, Version 11.0, Corel Corporation, Canada), i.e., Corel DRAW and Corel PHOTO-PAINT. All measurements are in micrometres and are given as the range followed by the mean in parentheses.

Molecular Analyses

An individual oocyst identified with the characteristic features of the new species under light microscopy was isolated and resuspended in PBS [5]. DNA was extracted from the oocyst using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, São Paulo, Brazil) according to the manufacturer’s instructions. To fully lyse the oocyst, four freeze–thaw cycles were applied prior to the DNA extraction. The PCR amplification for the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was carried out using a nested PCR, as previously described by Dolnik et al. [5] and Yang et al. [19]. The external primers COIbF1 (5′-GWT CAT TAG TAT GGG CAC ATC A-3′) and COIbR1 (5′-CCA AGA GAT AAT ACR AAR TGG AA-3′) produced a PCR product of c.302 bp in size. The internal primes COIbF2 (5′-GGG CAC ATC ATA TGA TGA C-3′) and COIbR2 (5′-ATA GTA TGT ATC ATG TAR WGC AA-3′) produced an amplicon of 257 bp in size. The PCR contained 10 µl of 5 × Green GoTaq® Flexi Buffer, 3 µl of 25 mM MgCl2, 1 µl of 10 mM dNTPs, 0.4 µM of each primer, 1.25 units of GoTaq® DNA polymerase, 3 µl of DNA (for primary reaction) or 3 µl primary PCR product (for the secondary reaction). Both primary and secondary PCR were conducted using the same cycling conditions: one cycle 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 of 72 °C for 5 min. The amplicons from the second round of PCR were purified using the Qiagen MinElute PCR Purification (Qiagen, São Paulo, Brazil). All PCR products were sequenced using the PCR forward and reverse primers by Ludwig Biotechnology, were an ABI-Prism 3500 Genetic Analyzer (Applied Biosystems, Foster City, California) was used for Sanger sequencing. The results of the sequencing reactions were analysed and edited using the program Chromas 2.6.

DNA Sequence Analyses

The newly generated sequences were compared to those for Isospora spp. and other coccidian parasites available on the GenBank database using the Basic Local Alignment Search Tool (BLAST). Phylogenetic trees were constructed for Isospora spp. at the cox1 sequences for additional isolates from GenBank. Alignment and parsimony analyses were conducted using MEGA version 7 [18]. The evolutionary history was inferred using the neighbor-joining (NJ) and maximum likelihood (ML) methods and the distances were computed using the Tamura-Nei method based on model selection using ModelTest in MEGA 7. Bootstrap analyses were conducted using 1000 replicates to assess the reliability of inferred tree topologies.

Results

Nine Conopophaga spp. were examined and four were positive for coccidia (two C. melanops and one C. lineata from Itatiaia National Park; and one C. lineata from Barra Mansa). All observed oocysts were characteristic of Isospora. This material is described below.

Family Eimeriidae Minchin, 1903.

Genus Isospora Schneider, 1881.

Isospora borbaiSilva-Carvalho et Berto n. sp. (Figures 1, 2).

Fig. 1
figure 1

Composite line drawing of the sporulated oocyst of Isospora borbai n. sp. from Conopophaga spp. Scale-bar: 10 µm

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Fig. 2
figure 2

Photomicrographs of sporulated oocysts of Isospora borbai n. sp. from Conopophaga spp. Inner (il) and outer (ol) layers of the oocyst wall, m micropyle, pg polar granule, sb Stieda, ssb sub-Stieda bodies, sr sporocyst residuum, sz sporozoite, rb refractile body. All to same scale. Scale-bar: 10 µm

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Oocysts (n = 32) subspheroidal, 17–22 × 15–22 (20.2 × 19.1); length/width (L/W) ratio 1.0–1.1 (1.06). Wall bi-layered, 1.5–2.1 (1.7) thick, outer layer rough, c.2/3 of total thickness. Micropyle present, without micropyle cap or wrinkles; however, generally with slight invagination of the inner layer. Oocyst residuum absent, but one or two (frequently one subspheroidal) polar granules are present. Sporocysts (n = 25) ellipsoidal, 12–15 × 8–11 (14.1 × 9.1); L/W ratio 1.4–1.7 (1.56). Stieda body present, knob-like to half-moon-shaped, 1.0 × 2.5; sub-Stieda present, rounded, 2.0 × 3.5; para-Stieda body absent; sporocyst residuum present, composed of scattered spherules of different sizes. Sporozoites vermiform, with posterior refractile body and centrally located nucleus.

Type-host Conopophaga lineata (Wied, 1831) (Aves: Passeriformes: Tyranni: Conopophagidae), rufous gnateater.

Other host Conopophaga melanops (Vieillot, 1818) (Aves: Passeriformes: Tyranni: Conopophagidae), black-cheeked gnateater.

Type locality Parque Nacional do Itatiaia (22°27′S, 44°35′W), southeastern Brazil.

Other locality Barra Mansa (22°29′S, 44°09′W), southeastern Brazil.

Type specimens Photosyntypes, line drawing, and oocysts in 70% ethanol are deposited at the Museu de Zoologia at the Universidade Federal Rural do Rio de Janeiro, Brazil, under the accession number MZURPTZ2018008. Phototypes and line drawings are also deposited and available (http://r1.ufrrj.br/labicoc/colecao.html) in the Parasitology Collection of the Laboratório de Biologia de Coccídios, at UFRRJ, under the repository number P-91/2018. Photographs of the type-host specimen (symbiotype) are deposited in the same collection.

Site in host Unknown.

Prevalence 44% (four out of nine birds infected).

Representative DNA sequence One representative cox1 sequence was deposited in the GenBank database under the accession number MK057528.

ZooBank registration To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature [10], details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) for Isospora borbai is urn:lsid:zoobank.org:act:D3BE104D-300F-4251-981C-393CD86800F7.

Etymology The specific name is derived from the family name of the Brazilian parasitologist Dr Hélcio Resende Borba, given in his honor for his contribution to the study of antiparasitic activity of plants.

Remarks To date, only two Isospora spp. are recorded from hosts of the parvorder Thamnophilida (Table 1). Isospora sagittulae McQuistion, Capparella, [11] and Isospora parnaitatiaiensis Silva, Rodrigues, Lopes, Berto, Luz, Ferreira, Lopes, 2015 were recorded from antbirds of the family Thamnophilidae; therefore, no Isospora sp. is recorded from the families Conopophagidae and Melanopareiidae until now. As shown in Table 1, I. borbai is easily differentiated from these two Isospora spp. from Thamnophilidae, due to their smaller size, subspheroidal shape, micropyle and rough outer layer of the oocyst wall.

Table 1 Comparative morphology of Isospora spp. recorded from the parvorder Thamnophilida
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Phylogenetic analysis DNA amplification of an individual oocyst of I. borbai n. sp. recovered from a C. melanops from Itatiaia National Park showed a clear band of c.250 bp. Phylogenetic analysis included 36 sequences for avian Isospora spp. available on GenBank (Fig. 4). Eimeria tenella (Railliet, Lucet, 1891) was used as the outgroup. Isospora borbai sat in a clade with the highest similarity of 99.0–99.5% with Isospora lopesi Silva-Carvalho et Berto, 2018 [15], Isospora sagittulae McQuistion et Capparella, 1992 [16] and Isospora sporophilae Carvalho-Filho, Meireles, Ribeiro et Lopes, 2005 [17] (Fig. 3). In a second analysis, a subset of 215 bp long cox1 gene sequences for 14 Isospora spp. was used (Fig. 4). In this analysis, I. borbai was again grouped with I. lopesi, I. sagittulae and I. sporophilae, next to the other clade with Isospora hypoleucae Dolnik, Rönn et Bensch, [6] (Dolnik et al. [5]) and Isospora isolates from Eurasian blackcaps Sylvia atricapilla (Linnaeus, 1758) (Dolnik et al. [5]) with similarities of 95.7% and 94.8–97.1%, respectively.

Fig. 3
figure 3

Maximum likelihood tree estimated from the cox1 sequences. Numbers at nodes represent bootstrap support (1000 replicates; only values > 50% shown) for Neighbor-Joining and Maximum Likelihood, respectively. The scale-bar represents the number of nucleotide substitutions per site

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Fig. 4
figure 4

Maximum likelihood tree estimated from the 215 bp long cox1 sequence dataset for Isospora spp. Numbers at nodes represent bootstrap support (1000 replicates; only values > 50% shown) for Neighbor-Joining and Maximum Likelihood, respectively. The scale-bar represents the number of nucleotide substitutions per site

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Discussion

Isospora borbai is the first coccidian species to be described from the family Conopophagidae. Duszynski and Wilber [8] advise that new coccidian species should be compared morphologically with all species recorded in the family of the host; therefore, due to the lack of descriptions of coccidians from conopophagids, I. borbai was compared with the coccidians from the parvorder Thamnophilida. In this sense, I. borbai was compared to I. sagittulae and I. parnaitatiaiensis, which are the only coccidian species recorded from the parvorder Thamnophilida, specifically from the family Thamnophilidae (Table 1). In any case, the oocysts of I. borbai are quite distinctive because they have a rough wall with a micropyle, which are unusual characteristic features in Isospora spp.

The phylogenetic analysis (Figs. 3, 4) brings together I. borbai with I. sagittulae, which are also parasites from the parvorder Thamnophilida, and I. lopesi, parasite of eastern white-throated spadebills Platyrinchus mystaceus Vieillot, 1818 that also belong to the suboscines (suborder Tyranni). In contrast, this standard approach related to taxonomic groups of hosts is incompatible with the presence of the genotypes of I. sporophilae in this monophyletic group; since this coccidian is a parasite of buffy-fronted seedeaters Sporophila frontalis (Verreaux, 1869) and uniform finches Haplospiza unicolor Cabanis, 1851, which are passerines of the family Thraupidae and suborder Passeri. Thus, this phylogenetic analysis maintains the assumption raised in Rodrigues et al. [17] that this monophyletic group is related with coccidia of neotropical birds, and not necessarily related to taxonomic groups of hosts.

Finally, based on the morphological and molecular features described above, I. borbai is considered as new to science and the first coccidian species recorded from a gnateater (Conopophagidae).