Introduction

The Thamnophilidae family is comprised of small passerines often observed capturing ants and other arthropods in dense vegetation. Because of this behavior, they are named “antbirds” (Wiens 1989). The South American forest on the Atlantic coast and the Amazon are two major distribution areas of the Thamnophilidae family (Zimmer and Isler 2003).

The white-shouldered fire-eye Pyriglena leucoptera (Vieillot, 1818) is common in the understory of tropical moist forests, secondary forests, and forest edges and occurs from the state of Bahia to Rio Grande do Sul, in Brazil, as well as the east of Paraguay and Misiones, Argentina (Sick 1997; BirdLife International 2012).

The Parque Nacional do Itatiaia (PNI) is located in the Serra da Mantiqueira on the border of the states of Rio de Janeiro, Minas Gerais, and São Paulo. It is a protected area with a high degree of vulnerability and is considered a “conservation island” of biodiversity (ICMBIO 2015).

To date, only one coccidian species had been reported from Thamnophilidae. Isospora sagittulae McQuistion & Capparella, 1992 was originally described from the spotted antbird Hylophylax naevioides (Lafresnaye, 1847) (McQuistion and Capparella 1992) collected in the rain forests of northwestern Ecuador. Recently, this same isosporoid species was reported from the white-throated antbird Gymnopithys salvini (Berlepsch, 1901) and from the common scale-backed antbird Willisornis poecilinotus (Cabanis, 1847) in the Brazilian Amazon. These reports demonstrate the occurrence of this parasite in non-sympatric birds of the same family but from different genera that inhabit opposite sides of the Andes and of the Amazon River (Berto et al. 2014a).

The aim of this study was to examine the feces from white-shouldered fire-eyes P. leucoptera to determine what coccidian parasites were present. These P. leucoptera specimens were captured in and around the PNI boundaries, in southeastern Brazil.

Materials and methods

A total of four sample periods were conducted in and around the PNI boundaries. Sampling occurred in August (22° 25′ 46″ S, 44° 37′ 17″ W), November (22° 26′ 57″ S, 44° 36′ 25″ W), December 2014 (22° 27′ 20″ S, 44° 36′ 28″ W), and March 2015 (22° 27′ 38″ S, 44° 35′ 34″ W). A total of eight P. leucoptera were captured. The birds were kept in individual boxes and feces collected immediately after defecation. After identification of the species, the bird was released and stool samples were placed in centrifuge tubes containing a potassium dichromate 2.5 % (K2Cr2O7) solution at 1:6 (v/v). Samples were carried to the Laboratório de Coccídios e Coccidioses, Universidade Federal Rural do Rio de Janeiro (UFRRJ). Samples were incubated at room temperature for 10 days or until ~70 % of the oocysts were sporulated. Oocysts were isolated by flotation in Sheather’s sugar solution (specific gravity, 1.20) and examined microscopically using the technique described by Duszynski and Wilber (1997) and Berto et al. (2014b). Morphological observations, line drawings, photomicrographs, and measurements were made using an Olympus BX binocular microscope coupled to a digital camera Eurocam 5.0. Line drawings were edited using two software applications from CorelDRAW® (Corel Draw Graphics Suite, Version 11.0, Corel Corporation, Canada), specifically Corel DRAW and Corel PHOTO-PAINT. All measurements are in micrometers and are given as the range followed by the mean in parentheses.

Results

Eight P. leucoptera were captured and examined, and three were positive for coccidia. All observed oocysts were characteristic of Isospora, which is described and named as follows:

Isospora parnaitatiaiensis n. sp. (Figs. 1 and 2a–c).

Fig. 1
figure 1

Line drawing of Isospora parnaitatiaiensis, a new coccidium species recovered from the white-shouldered fire-eye Pyriglena leucoptera. Scale bar 10 μm

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Fig. 2
figure 2

Photomicrographs (a–c) of sporulated oocysts of Isospora parnaitatiaiensis, a new coccidium species recovered from the white-shouldered fire-eye Pyriglena leucoptera. Scale bar 10 μm

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Type-host: White-shouldered fire-eye Pyriglena leucoptera (Vieillot, 1818) (Aves: Passeriformes: Thamnophilidae).

Type-specimens: Oocysts in 70–100 % ethanol, phototypes and line drawings are deposited and available (http://r1.ufrrj.br/lcc) in the Parasitology Collection of the Laboratório de Coccídios e Coccidioses, at UFRRJ, located in Seropédica, Rio de Janeiro, Brazil. Photographs of the type-host specimens (symbiotypes) are deposited in the same collection. The repository number is P-59/2015.

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

Site of infection: Unknown.

Etymology: The specific epithet is a homage to the Parque Nacional do Itatiaia, which is the first national park in Brazil (founded in 1937).

Description of the sporulated oocyst: Oocyst (n = 23) ellipsoidal, 22–27 × 18–21 (23.8 × 19.4); length/width (L/W) ratio 1.1–1.5 (1.23). Wall bi-layered, 1.0–1.2 (1.1) thick, outer layer smooth, c.2/3 of total thickness. Micropyle and oocyst residuum both absent, but one or two polar granules are present.

Description of the sporocyst and sporozoites: Sporocysts (n = 22) 2, ellipsoidal, 13–16 × 8–10 (14.6 × 9.3); L/W ratio 1.5–1.7 (1.6). Stieda body present, nipple-like to knob-like, 1.0 high × 2.0 wide; sub-Stieda present, rounded to rectangular, 1.5 high × 2.0 wide; para-Stieda body absent; sporocyst residuum present, usually as a distinctly ovoidal to ellipsoidal body consisting of numerous small granules that appear to be membrane-bounded, 5–6 × 4–5 (5.5 × 4.5). Sporozoites 4, vermiform, with anterior and posterior refractile bodies and centrally located nucleus.

Discussion

The PNI is characterized by mountains and rocky terrain with elevation ranging from 600 to 2791 at the peak of Agulhas Negras (ICMBIO 2015). The specimens of P. leucoptera were captured at all four sample sites at altitudes ranging from 600 to 1,200 m indicating that this antbird species is widely distributed in the PNI and a potential disperser of coccidia to other antbird species (Berto and Lopes 2013).

The results of this study reveal that the oocysts of I. parnaitatiaiensis are noticeably polymorphic in the characteristics of the sporocyst, Stieda and sub-Stieda bodies. In some oocysts, the sporocysts were observed more (40 %) (Fig. 2c) and in others less (60 %) (Fig. 2b) elongated. The Stieda body was observed sometimes as nipple-like (50 %) (Fig. 2a) and in others knob-like (50 %) (Fig. 2b). The sub-Stieda body was observed sometimes as rounded (85 %) (Fig. 2a) and in others rectangular (15 %) (Fig. 2b).

This polymorphism in the oocysts exposes the importance and complexity of identifying coccidian species. Currently, the identification of coccidia from Passeriformes has been based on different criteria by different authors. For example, prior to 1982, more than 100 species of passerines had been reported as hosts of Isospora lacazei (Labbé, 1893). Therefore, based on available descriptions and on the improbability that a single species could parasitize so many hosts, Levine (1982) suggested the names Isospora passeris Levine, 1982 for parasites of Passer domesticus Linnaeus, 1758 and I. lacazei for parasites of Carduelis carduelis Linnaeus, 1758. However, Al-Quraishy and Al-Nasr (2009) discussed whether all coccidia from passerines that were called I. lacazei were the same species. They concluded that diet influenced oocyst size in different passerines and that at least the species described from P. domesticus should be considered junior synonyms of I. lacazei. More recently, Dolnik et al. (2009) used a technique to isolate single oocysts and extract DNA for PCR and sequencing and found six different haplotypes of Isospora from wild birds. They found both the same haplotype in different host individuals and various combinations of haplotypes in the same host individual. When they found oocysts that belonged to four different haplotypes in the feces of one passerine, they concluded that mixed infections with different haplotypes are rather common in wild birds. Finally, Duszynski and Wilber (1997) suggest that a new coccidian species should be compared by morphology and morphometry of the oocysts in detail with coccidian species that are feature-similar and belong to the same host family. In this sense, they put forward the concept of intra-familial specificity, which was reaffirmed by Berto et al. (2011) in New World passerines.

We agree with Al-Quraishy and Al-Nasr (2009) that there should be synonyms in some species of coccidia of passerines, especially those described in the same host at different times and conditions and without detailed description. In contrast, the idea that hundreds of Isospora spp. (distinct and identifiable morphologically) belong to a single species is inconsistent and may well be an obstacle to our knowledge of the coccidia of passerines.

At another extreme, the concept that slight genetic variation is sufficient to differentiate species brings confusion and an exponential amplification of the number of existing species. For example, Hafeez et al. (2014) described two new species from starlings Lamprotornis superbus Rüppell, 1845. In their work, they observed the same extra-intestinal stages and the same oocyst morphology. However, they obtained two distinct COI sequences (1.7 % divergence) and two distinct 18S rDNA sequences (0.6 % divergence), which they used as their basis for naming two new species. These findings do not approach with studies of other protozoa, as Toxoplasma gondii Dubey & Beattie, 1988 and Paramecium spp. which have more prudent and consistent criteria and approaches. T. gondii has a genetic divergence of 2–3 % in North American and European genotypes, with the exception of those ones of South America, which have much more genetic diversity due to older and hybridized population (Khan et al. 2014). In a study of the genetic diversity of Paramecium spp. using the COI gene, an intra-specific variation of 11 % was obtained, or rather, specimens with up to 11 % of genetic divergence were considered as the same species (Zhao et al. 2013). In this sense, we understand that small genotypic variations favored the polymorphism observed in oocysts of this work, although these morphological changes have been observed in sporocysts in a single oocyst. Therefore, if genetic variations actually exist, they should not be sufficient to describe other species, but genotypes within a single species. Moreover, Grulet et al. (1982) and Berto et al. (2014b) suggest that small variations in Stieda and sub-Stieda bodies may be a result of the sporulation process, the position of the sporozoite inside the sporocyst, or the position of their oocysts and sporocysts under the coverslip at the time of evaluation.

Thus, we opted to compare our new species with coccidian species that are feature-similar and belong to the same host family (Duszynski and Wilber 1997). The only other coccidian species described in Thamnophilidae is I. sagittulae, which was originally described from H. naevioides in Ecuador and, recently, reported from the Amazonian antbirds G. salvini and W. poecilinotus (McQuistion and Capparella 1992; Berto et al. 2014a).

I. sagittulae (Table 1) differed from I. parnaitatiaiensis in having larger oocysts with sporocyst subspherical to ovoidal and Stieda body thin and flattened. Therefore, this species is easily distinguished from I. parnaitatiaiensis.

Table 1 Comparative morphology of Isospora spp. recorded from New World antbirds (Thamnophilidae)
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This finding emphasizes how little we know about the parasites of New World passerines. There are 186 Thamnophilidae species in Brazil, and so far, only three antbird species have been examined for coccidia and all have been found infected with at least one Isospora. Antbirds have wide geographic ranges (Berto et al. 2014a). Thus, our work encourages further studies to examine all other species in the family to determine the true coccidian fauna of this most interesting family of birds.

Based on the morphological features described above, I. parnaitatiaiensis is considered as new to science and the second isosporoid coccidian reported from a thamnophilid bird.