Isospora similisi recovered from a new host, Saltator aurantiirostris, with supplementary molecular data and notes on its taxonomy and distribution in the Neotropical region

Isospora similisi de um novo hospedeiro, Saltator aurantiirostris, com dados moleculares suplementares e notas sobre sua taxonomia e distribuição na região Neotropical

Abstract

This article reports on a golden-billed saltator Saltator aurantiirostris Vieillot, 1817, kept in captivity outside its natural distribution area, in the proximities of the Itatiaia National Park, as a new host for Isospora similisi Coelho, Berto, Neves, Oliveira, Flausino & Lopes, 2013. Additionally, a supplementary molecular identification is provided through the sequencing of three non-overlapping loci of mitochondrial DNA and one locus of the 18S small subunit ribosomal RNA (18S) gene. All the taxonomic features of the I. similisi oocysts shed by S. aurantiirostris were equivalent to those originally described from Saltator similis d’Orbigny & Lafresnaye, 1837. The new sequenced loci were identical, or showed 99.9% similarity, to the samples of I. similisi from S. similis and S. aurantiirostris, confirming the same species from both hosts. Lastly, I. similisi is estimated as a junior synonym of Isospora formarum McQuistion & Capparella, 1992, due to the morphological similarities and wide distribution of its hosts in the Neotropical region. Therefore, this study encourages future taxonomic inquiries into I. similisi collected from other Saltator spp. in order to establish this synonymization of I. formarum with I. similisi, and hence, its wide distribution and dispersion in the Neotropical region, including across the Andes mountains.

Keywords:
Coccidia; oocysts; taxonomy; sequencing; phylogeny; Itatiaia National Park

Resumo

Este artigo relata um bico-duro Saltator aurantiirostris Vieillot, 1817, mantido em cativeiro fora de sua área de distribuição natural, nas proximidades do Parque Nacional de Itatiaia, como novo hospedeiro de Isospora similisi Coelho, Berto, Neves, Oliveira, Flausino & Lopes, 2013. Adicionalmente, uma identificação molecular suplementar foi fornecida por intermédio do sequenciamento de três loci não sobrepostos de DNA mitocondrial e um locus da subunidade pequena do gene 18S do RNA ribossômico (18S). Todas os caracteres taxonômicos dos oocistos de I. similisi, eliminados por S. aurantiirostris, foram equivalentes àqueles originalmente descritos de Saltator similis d'Orbigny & Lafresnaye, 1837. Os novos loci sequenciados foram idênticos, ou tiveram 99,9% de similaridade, com as amostras de I. similisi de S. similis e S. aurantiirostris, confirmando a mesma espécie de ambos os hospedeiros. Por fim, I. similisi foi estimada como sinonímia júnior de Isospora formarum McQuistion & Capparella, 1992, devido às semelhanças morfológicas e ampla distribuição de seus hospedeiros na região Neotropical. Portanto, este estudo incentiva futuras investigações taxonômicas sobre I. similisi, recuperados de outros Saltator spp., a fim de estabelecer essa sinonimização de I. formarum com I. similisi e, consequentemente, sua ampla distribuição e dispersão na região Neotropical, inclusive Cordilheira dos Andes.

Palavras-chave:
Coccídios; oocistos; taxonomia; sequenciamento; filogenia; Parque Nacional de Itatiaia

Introduction

Saltators (Saltator spp.) are passerines of the family Thraupidae and subfamily Saltatorinae with a diversity of 19 species and an exclusively Neotropical distribution. Of this total, only nine Saltator spp. occur in Brazil (Chaves et al., 2013; BirdLife International, 2024). The green-winged saltator Saltator similis d’Orbigny & Lafresnaye, 1837 is the most well-known and valued Saltator sp. in Brazil for breeding in captivity, due to its vocal repertoire, which is why it is one of the main wild bird species illegally captured and trafficked. The golden-billed saltator Saltator aurantiirostris Vieillot, 1817, in turn, is a passerine uncommonly observed in captivity and rarely related to animal trafficking in Brazil, despite also having a vocal repertoire typical of the Saltarorinae (Nunes et al., 2012; Freitas et al., 2015).

Coccidiosis stands out among the parasitic diseases of passerines, including Saltator spp. There have been a few reports of symptomatic and/or severe coccidiosis in captive green-winged saltators and buff-throated saltators Saltator maximus (Müller, 1776) (Coelho et al., 2012; Vasconcellos et al., 2013; Maronezi et al., 2022), and also in the wild (Maronezi et al., 2024). However, scientific literature contains no reports of coccidians collected from other Saltator spp. in Brazil, which are wild species difficult to capture and/or less valued for commercial breeding in captivity (Nunes et al., 2012; Freitas et al., 2015; Maronezi et al., 2022).

In this context, the aim of the current study was to record a golden-billed saltator S. aurantiirostris, living in captivity outside its natural distribution area, in the municipality of Resende, in the proximities of Itatiaia National Park (Parque Nacional de Itatiaia) in the state of Rio de Janeiro, as a new host for Isospora similisi Coelho, Berto, Neves, Oliveira, Flausino & Lopes, 2013, which is a coccidian species so far recorded only on green-winged saltators S. similis. In addition, a supplementary molecular identification is provided through the sequencing of three non-overlapping loci of mitochondrial DNA and one locus of the 18S small subunit ribosomal RNA (18S) gene. Also included are taxonomic notes on the strong evidence of synonyms between Isospora spp. recorded from Saltator spp., based on the current new record, morphological equivalence and the wide distribution areas of host Saltator spp. in the Neotropical region.

Materials and Methods

Sample collection

Fecal samples were collected from an adult male specimen of golden-billed saltator S. aurantiirostris owned by a breeder located near the Itatiaia National Park (22°28’0”S, 44°27’56”W), which includes part of the municipalities of Itatiaia and Resende in the state of Rio de Janeiro, southeastern Brazil. This park is a Brazilian federal conservation unit with an extremely high priority for biodiversity conservation (ICMBIO, 2016). The golden-billed saltator was already banded with metal ring associated with the Registration System of Passeriformes (Sistema de Cadastramento de Passeriformes - SISPASS) of the Brazilian Institute for the Environment and Renewable Natural Resources (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis - IBAMA), which, in principle, confers an origin of legalized captive breeding; however, the life history of this passerine was inaccessible and unknown to the owner who claimed to have recently received the passerine as a donation. The bottom of the cage where the golden-billed saltator was kept was lined with clean paper for a period of 5 hours (2 pm to 7 pm) to collect its droppings. During this period, the paper was examined and replaced several times in order to obtain fresh fecal droplets separately. Each fecal droplet was placed individually in a centrifuge tube with a solution of 2.5% potassium dichromate (K₂Cr₂O₇) (Dolnik, 2006).

Morphological analysis

Samples were examined at the Laboratory of Coccidia Biology (Laboratório de Biologia de Coccídios - LABICOC) of the Federal Rural University of Rio de Janeiro (Universidade Federal Rural do Rio de Janeiro - UFRRJ). All the samples were incubated at room temperature (25 °C) for 7 days. Oocysts were isolated by flotation in Sheather’s sugar saturated solution (specific gravity: 1.20) (Duszynski & Wilber, 1997). Sample density was calculated as the number of oocysts per fecal droplet (Dolnik, 2006; Bush et al., 1997). Morphological observations and measurements were taken following the guidelines of Duszynski & Wilber (1997) and Berto et al. (2014a), using an Olympus BX binocular microscope (Olympus Optical, Tokyo, Japan) equipped with a Eurekam 5.0 digital camera (BEL Photonics, Monza, Italy). Photomicrographs and other figures were edited using two software programs (Corel DRAW and Corel PHOTO-PAINT) from CorelDRAW® (Corel Draw Graphics Suite, Version 2020, Corel Corporation, Canada). All the measurements are shown in micrometers and are given as the range, followed by the mean in parentheses.

Obtaining representative specimens of Isospora similisi

Some of the representative specimens of I. similisi collected from S. similis identified in Maronezi et al. (2022), which are deposited in the Parasitology Collection of the LABICOC (UFRRJ, 2024) at UFRRJ under Repository No. 118/2021, were requested for supplementary molecular identification and comparison.

Molecular analysis

Individual oocysts morphologically identified as I. similisi, both from the original collection (representative specimens of S. similis) deposited by Maronezi et al. (2022), and from S. aurantiirostris of the current study, were photomicrographed under light microscopy, isolated, resuspended in 0.9% phosphate buffered saline (PBS), and washed by centrifuging until the supernatant became clear (Dolnik et al., 2009). DNA was extracted from the individual oocysts, using a Quick-DNA Micropep kit from Zymo Research Corporation according to the manufacturer’s instructions. Four freeze-thaw cycles were applied prior to DNA extraction in order to ensure complete lysis of the oocysts. PCR amplification was performed for three non-overlapping loci in cox1 (MAVCOXI), cox3 (MACOIII) genes and fragments of small and large subunit rDNA (MARI) of mitochondrial DNA (Ortúzar-Ferreira et al., 2023), and one locus of the 18S small subunit ribosomal RNA (1NF/18S) (Andrade et al., 2024). For amplification, a 25 µl PCR reaction was prepared using 3 µl of genomic DNA (<1 µg), 12.5 µl of GoTaq® G2 Hot Start Colorless Master Mix (Promega Labs) (1X), 0.25 µl of each Primer (0.2 µM) and 9µL of Nuclease Free Water. PCR amplifications were conducted using the cycling conditions originally described by Ortúzar-Ferreira et al. (2023) and Andrade et al. (2024).

DNA sequence analysis

All the PCR products were sequenced with PCR forward and reverse primers by Ludwig Biotechnology, using an ABI-Prism 3500 Genetic Analyzer (Applied Biosystems, Foster City, California) for Sanger sequencing. The results of the sequencing reactions were analyzed and edited in the Chromas 2.6 program. Sequences were compared with other eimeriid coccidia available in the GenBank database, using the Basic Local Alignment Search Tool (BLAST). Alignments were created in MEGA v10.2.6 using Clustal W. Phylogenetic relationships were reconstructed using Bayesian Inference in MrBayes v3.2.7 software (Ronquist et al., 2012) and the Maximum likelihood method in the MEGA software (Kumar et al., 2018). The evolutionary model that best fit all the phylogenetic analyses was selected using the Model Selection tool of MEGA software. Bayesian Inference analysis was conducted under the GTR+G evolutionary model for 1,000,000 generations, and the trees were summarized after removing 25% of burn-in. Maximum likelihood analysis was conducted under the TN93+G evolutionary model, and the bootstrap values were calculated using 1,000 replicates. The resulting phylogenetic trees were viewed in the MrBayes and MEGA software programs and exported in FigTree v1.4.4.

Results

Examination of samples and identification of species

Four samples (fecal droplets) from the golden-billed saltator S. aurantiirostris were examined and found positive for coccidian oocysts, which were morphologically identified as I. similisi. The taxonomic features observed are described below:

Isospora similisi Coelho, Berto, Neves, Oliveira, Flausino & Lopes, 2013 (Figure 1)

Figure 1
Photomicrographs of sporulated oocysts of Isospora similisi from a golden-billed saltator Saltator aurantiirostris, highlighting the main taxonomic characters (A-F). Note the inner (il) and outer (ol) layers of the oocyst wall; polar granules (pg); Stieda (sb) and sub-Stieda bodies (ssb); sporocyst residuum (sr); and refractile body (rb). Scale-bar: 10 µm.

Oocyst (n = 22) subspherical to ovoid, 20-25 × 20-22 (22.8 × 21.3); length/width (L/W) ratio 1.0-1.2 (1.09). Wall bi-layered, 1.3-2.0 (1.6) thick, outer layer smooth, c.2/3 of total thickness. Micropyle and oocyst residuum are absent, but splinter-like or comma-like polar granules are present. Sporocyst (n = 16) ovoid to slightly piriform, 14-17 × 10-11 (15.7 × 10.6); L/W ratio 1.4-1.6 (1.50). Stieda body present, half-moon-shaped to knob-like, 0.8-1.2 high × 2.0-2.4 wide (1.0 × 2.2). Sub-Stieda body present, rounded to trapezoidal, 1.5-2.4 high × 3.9-4.4 wide (2.0 × 4.1), frequently with density variations resembling a compartmentalized sub-Stieda. Para-Stieda body absent. Sporocyst residuum present, consisting of spherules scattered among the sporozoites. Sporozoites vermiform, with posterior refractile body and a central nucleus.

Taxonomic summary

Type host: Saltator similis d’Orbigny & Lafresnaye, 1837 (Aves: Passeriformes: Thraupidae: Saltatorinae), green-winged saltator (Coelho et al., 2013; Maronezi et al., 2022, 2024).

Other host: Saltator aurantiirostris Vieillot, 1817 (Aves: Passeriformes: Thraupidae: Saltatorinae), golden-billed saltator (current study).

Type locality: Centro de Triagem de Animais Silvestres – CETAS (Wild Animal Screening Center) in the municipality of Seropédica, state of Rio de Janeiro, southeastern Brazil (22°43’24”S, 43°42’37”W) (Coelho et al., 2013).

Other localities: Five sites of captivity in the proximities of Itatiaia National Park (22°30’S, 44°34’W), southeastern Brazil (Maronezi et al., 2022). Kilometer 5 of the ‘Travessia Ruy Braga’ (Ruy Braga Crossing) in Itatiaia National Park (free-living S. similis captured) (22°25’39.9”S; 44°37’49.4”W), southeastern Brazil (Maronezi et al., 2024). Site of captivity in the municipality of Resende, adjacent to Itatiaia National Park, in the state of Rio de Janeiro (22°28’0”S, 44°27’56”W), southeastern Brazil (current study).

Type-material: Photos of syntypes and line drawings are deposited and available (UFRRJ, 2024) in the Parasitology Collection of the Laboratory of Coccidia Biology at UFRRJ, under Repository No. P-41/2011 (Coelho et al., 2013). Physical material (preserved oocysts) has been lost or is unavailable.

Representative specimens: Photomicrographs, line drawing and oocysts in 2.5% K₂Cr₂O₇ solution are deposited and available (UFRRJ, 2024) in the Parasitology Collection of the Laboratory of Coccidia Biology at UFRRJ, under Repository Nos. 118/2021 (Maronezi et al., 2022) and 137/2024 (current study). Photographs of the host specimens are deposited in the same collection.

Representative DNA sequences: DNA amplification of the oocysts from S. similis by Maronezi et al. (2022) and S. aurantiirostris in the current study, at the MAVCOXI, MACOIII, MARI and 1NF/18S loci, showed clear bands around ~653 bp, ~632 bp, ~824 bp and ~444 bp, respectively. Representative sequences are deposited in the GenBank database under Accession Nos. PP723065 (MAVCOXI, S. similis); PP723066 (MACOIII, S. similis); PP723067 (MACOIII, S. aurantiirostris); PP723068 (MARI, S. similis); PP723069 (MARI, S. aurantiirostris); PP716360 (1NF/18S; S. similis); and PP716361 (1NF/18S; S. aurantiirostris).

Site of infection: Unknown, oocysts were recovered from feces.

Prevalence: 100% (1/1).

Density: Mean of 5,786 (ranging from 3,358 to 9,000) oocysts in 4 fecal droplets collected from the golden-billed saltator S. aurantiirostris during the 3-hour period of 2 pm to 7 pm.

Molecular analysis

The sequences at the four gene loci obtained from oocysts morphologically identified as I. similisi differed by several nucleotides when compared with sequences from eimeriid coccidians deposited in GenBank. The sequences from the oocysts of I. similisi recovered from S. similis by Maronezi et al. (2022), in comparison with those obtained from S. aurantiirostris, were identical (1NF/18S) or showed 99.9% similarity (MACOIII and MARI), depending on the locus analyzed.

Only the representative specimens of I. similisi from S. similis recovered from the collection deposit amplified in the MAVCOXI locus; therefore, it was not possible to compare them with samples from S. aurantiirostris for this locus. In any case, this sequence for the MAVCOXI locus of I. similisi was maintained and phylogenetically analyzed in the current study, as it is unprecedented for any host. In comparison with sequences deposited in GenBank, I. similisi showed the highest similarities, i.e., approximately 98%, to Isospora spp. collected from superb starlings Lamprotornis superbus Rüppell, 1845 in Canada (Hafeez & Barta, 2017).

In the MACOIII locus, the sequences of I. similisi from S. similis and S. aurantiirostris were dissimilar in the substitution of only two nucleotides. In GenBank, these sequences were approximately 98% similar to Isospora spp. described/reported from island canaries Serinus canaria (Linnaeus, 1758) (Ogedengbe et al., 2016), superb starlings L. superbus (Hafeez & Barta, 2017) and indigo buntings Passerina cyanea (Linnaeus, 1766) (unpublished data; GenBank Accession No. MW645337).

In the MARI locus, a comparison of I. similisi from the two hosts indicated that the sequences differed by a single nucleotide. In GenBank, the highest similarities between Isospora spp. described/reported from S. canaria (Ogedengbe et al., 2016) and L. superbus (Hafeez & Barta, 2017) in this locus were approximately 99%.

The sequences of I. similisi from the two hosts were identical in the 1NF/18S locus, and showed the highest similarities, i.e., 99.7%, with Isospora spp. described/reported from red-browed finches Neochmia temporalis (Latham, 1801) (Yang et al., 2016), superb starlings L. superbus (Hafeez & Barta, 2017), common starlings Sturnus vulgaris Linnaeus, 1758 (unpublished data; GenBank Accession No. MW667591), yellow-shafted flickers Colaptes auratus (Linnaeus, 1758) (unpublished data; GenBank Accession No. MW618926), rock doves Columba livia Gmelin, 1789 (Matsubara et al., 2017) and austral thrushes Turdus falcklandii Quoy & Gaimard, 1824 (Martínez et al., 2015).

Phylogenetic analysis

Phylogenetic analysis based on the MAVCOXI, MACOIII, MARI and 1NF/18S loci included sequences from eimeriid coccidia available in GenBank (Figures 2-5). An unnamed Choleoeimeria sp. (GenBank Accession No. KT203395) was used as the outgroup in the phylogenies of the mitochondrial loci (Figures 2-4), and Toxoplasma gondii (Nicolle & Manceaux, 1908) (GenBank Accession No. L24381) was used as the outgroup in the phylogenetic analysis of the 1NF/18S locus (Figure 5).

Figure 2
Phylogenetic relationship of Isospora similisi from the green-winged saltator Saltator similis inferred by Bayesian analysis for a locus (MAVCOXI) within cox1 gene of the mitochondrial genome. Branch lengths correspond to mean posterior estimates of evolutionary distances (scale-bar: 0.05). Branch labels at the nodes show posterior probabilities under the Bayesian Inference analysis and bootstrap values derived from Maximum likelihood analysis. Only posterior probabilities higher than 0.5 are displayed. The phylograms were outgrouped using an unnamed Choleoeimeria sp. (GenBank accession number: KT203395).

Figure 5
Phylogenetic relationship of Isospora similisi from the green-winged saltator Saltator similis and golden-billed saltator Saltator aurantiirostris inferred by Bayesian analysis for a locus (1NF) within 18S small subunit ribosomal RNA. Branch lengths correspond to mean posterior estimates of evolutionary distances (scale-bar: 0.003). Branch labels at the nodes show posterior probabilities under the Bayesian Inference analysis and bootstrap values derived from Maximum likelihood analysis. Only posterior probabilities higher than 0.5 are displayed. The phylograms were outgrouped using Toxoplasma gondii (GenBank accession number: L24381).

Figure 4
Phylogenetic relationship of Isospora similisi from the green-winged saltator Saltator similis and golden-billed saltator Saltator aurantiirostris inferred by Bayesian analysis for a locus (MARI) within fragments of small and large subunit rDNA of the mitochondrial genome. Branch lengths correspond to mean posterior estimates of evolutionary distances (scale-bar: 0.03). Branch labels at the nodes show posterior probabilities under the Bayesian Inference analysis and bootstrap values derived from Maximum likelihood analysis. Only posterior probabilities higher than 0.5 are displayed. The phylograms were outgrouped using an unnamed Choleoeimeria sp. (GenBank accession number: KT203395).

In the cladogram produced by the phylogenetic analysis of the MAVCOXI locus (Figure 2), I. similisi exhibited monophyly with the two Isospora spp. from the superb starling L. superbus and, in a larger monophyletic group, with other Isospora spp. from passerines and Eimeria spp. from rabbits. The cladograms of the MACOIII and MARI loci (Figures 3 and 4) resulted in coherent monophyletic groups for Eimeria spp. from Galliformes and Eimeria spp. from rabbits, in addition to other groups of Isospora spp. from passerines, including I. similisi from S. similis and S. aurantiirostris. Lastly, the phylogenetic analysis of the 1NF/18S locus (Figure 5) resulted in coherent monophyletic groups for Cyclospora spp., Eimeria spp. from cattle, Eimeria spp. from rodents, Isospora spp. from passerines and Lankesterella sp. and Caryospora sp., which are related genera, in addition to Eimeria spp. from bats and squirrels that were not monophyletically grouped.

Figure 3
Phylogenetic relationship of Isospora similisi from the green-winged saltator Saltator similis and golden-billed saltator Saltator aurantiirostris inferred by Bayesian analysis for a locus (MACOIII) within cox3 gene of the mitochondrial genome. Branch lengths correspond to mean posterior estimates of evolutionary distances (scale-bar: 0.2). Branch labels at the nodes show posterior probabilities under the Bayesian Inference analysis and bootstrap values derived from Maximum likelihood analysis. Only posterior probabilities higher than 0.5 are displayed. The phylograms were outgrouped using an unnamed Choleoeimeria sp. (GenBank accession number: KT203395).

Discussion

Wild birds are recognized as bioindicators of environmental conservation, since the presence of certain specialist species and/or those with high forest dependence dictates preserved/conserved environments. Coccidians from wild birds, in turn, can be considered biomarkers, as the increase in their densities and/or clinical signs in wild birds determine early changes in the environment (Berto & Lopes, 2020; Maronezi et al., 2024). This association is easily observed in captive birds or birds rescued from wildlife trafficking, which often show very high densities of coccidians associated with severe coccidiosis (Coelho et al., 2012, 2013; Vasconcellos et al., 2013; Maronezi et al., 2022; Berto & Lopes, 2020). In this context, Maronezi et al. (2022) highlight the risk of captivity of wild birds around conservation units, such as Itatiaia National Park, since captive birds shed high densities of coccidian oocysts in their feces, which can easily be transmitted directly or indirectly to free-living birds. Subsequently, Maronezi et al. (2024) highlighted this possibility in the first report of a free-living green-winged saltator in Itatiaia National Park showing clinical signs of coccidiosis and high density of coccidian oocysts. Lastly, the current study reports a golden-billed saltator living in captivity in the proximities of Itatiaia National Park as a new host for one of the three Isospora spp. recorded from green-winged saltators, thus expanding the host specificity of I. similisi, and consequently, its distribution in the Neotropical region (Figure 6) (BirdLife International, 2024).

Figure 6
Geographic range of several Saltator spp. in the Neotropical region (based on data from BirdLife International (2024)). The type host of Isospora formarum is the slate-colored grosbeak Saltator grossus, and the type host of Isospora similisi is the green-winged saltator Saltator similis. The new host for I. similisi is the golden-billed saltator Saltator aurantiirostris. Saltator grossus has trans-Andean and cis-Andean populations. Saltator similis and S. aurantiirostris are sympatric with each other in south-central South America, but both are allopatric with S. grossus. The asterisk and sharp indicate approximately the type localities of I. formarum (trans-Andean) and I. similisi (cis-Andean), respectively.

The golden-billed saltator S. aurantiirostris is naturally distributed throughout the Chaco region, Pampas, and central portion of the Andes in the Neotropical region, through Argentina, Bolivia, Brazil, Chile, Paraguay, Peru and Uruguay. In Brazil, it is only observed in Rio Grande do Sul and southwestern Mato Grosso do Sul (Figure 6) (BirdLife International, 2024). The captivity site of the golden-billed saltator of the current study is located in southeastern Brazil; in other words, quite far from its natural area of geographic distribution (Figure 6). This finding therefore highlights the importance of the anthropomorphic dispersion of coccidians (Berto & Lopes, 2020), i.e., the role of trafficking, sale and breeding of wild birds in the transmission of coccidia among allopatric birds. Furthermore, the possibility of transmission between captive birds or from them to susceptible free-living birds is exponentially increased considering the high density of coccidian oocysts in these birds; in other words, in addition to trafficking and captivity enabling transmissions that are very unlikely, if not impossible, under natural conditions, the large numbers of oocysts shed by these birds under these conditions intensifies the successful transmission/dispersion of coccidians (Berto & Lopes, 2020; Maronezi et al., 2022, 2024). Accordingly, the parasite density in the captive golden-billed saltator of the current study was very high, although it appeared to be healthy and showed no observable clinical signs of coccidiosis. In this regard, this finding reveals the potential for transmission and dispersion of I. similisi by this S. aurantiirostris specimen.

The maximum number of 9,000 oocysts per fecal droplet from the golden-billed saltator was even greater than the maximum number (3,668) obtained from S. similis recovered from illegal trafficking and kept in quarantine in a wild animal rehabilitation center, where the highest densities are often observed in weakened birds (Coelho et al., 2013). It is possible that Saltator aurantiirostris is an evolutionarily more recent host species, which is in an early period of parasite-host adaptation (Odum, 1998; Gardner & Duszynski, 1990; Berto & Lopes, 2020). On the other hand, perhaps this specimen is undergoing a primary infection where the absence of immunological memory allows many merogonies and gametogonies with the generation and shedding of a large number of oocysts (Krautwald-Junghanns et al., 2009; Merino, 2010). Additionally, this high density of oocysts associated with the absence of observable clinical signs leads to the assumption that I. similisi, which was the only species or at least the majority species observed in fecal droplets, has very low pathogenicity. This assumption, in association with the clinical signs observed in the juvenile S. similis positive for the three Isospora spp. reported in Maronezi et al. (2024), indicates that Isospora saltatori Berto, Balthazar, Flausino, Lopes, 2008, more abundant in this report, and that Isospora trincaferri Berto, Balthazar, Flausino, Lopes, 2008, should be more pathogenic (causing severe disease). Be that as it may, these occasional findings of coccidiosis and/or high density of coccidian oocysts in a single or several specimens of Saltator spp. do not allow for definitive conclusions.

Five Isospora spp. are recorded to date from Saltator spp. (Maronezi et al., 2022). Isospora pityli McQuistion & Capparella, 1992 and Isospora formarum McQuistion & Capparella, 1992 were the first two species described from the slate-colored grosbeak Saltator grossus (Linnaeus, 1766) in the Esmeraldas Province of Ecuador (Figure 6). These two Isospora spp. have not been reported since their original descriptions by McQuistion & Capparella (1992). The other three Isospora spp. from Saltator spp. were identified in S. similis living in captivity or in a rehabilitation center in southeastern Brazil, and were later reported in other locations, also in southeastern Brazil, collected from captive and free-living birds, including from the buff-throated saltator S. maximus infected with I. trincaferri (Coelho et al., 2013; Lopes et al., 2013; Maronezi et al., 2022, 2024).

As previously pointed out by Maronezi et al. (2022), I. pityli and I. formarum are morphologically similar to I. saltatori and I. similisi, respectively, with the exception of some minor differences in a few taxonomic features. In this regard, the main criterion for differentiating these Isospora spp. is ecological, since I. pityli and I. formarum were described in Ecuador, in the trans-Andean region; while I. saltatori and I. similisi have so far been reported in southeastern Brazil, in the cis-Andean region (Figure 6). In other words, in a conservative approach, the strong geographic barrier of the Andes mountain range that separates the locations where these Isospora spp. have been reported and described precludes the establishment of junior synonyms for these species. On the other hand, the wide geographic distribution of Saltator spp., such as S. grossus which has cis-Andean and trans-Andean populations, in addition to the distribution of S. similis and S. aurantiirostris, the latter being a new host for I. similisi in the current study, enables the distribution and dispersion of their coccidian parasites throughout most of the Neotropical region (Figure 6) (Berto & Lopes, 2020). It is worth mentioning that S. similis and S. aurantiirostris are not sympatric with S. grossus, although the distribution of S. aurantiirostris borders the cis-Andean population of S. grossus in the Andes mountains and the distribution of S. similis borders that of S. grossus in central-west Brazil (Figure 6).

In this context, the findings of the current study reinforce the possibility of I. similisi being established as a junior synonym of I. formarum. Coccidia dispersion across the Andes mountains, facilitated by the natural distribution and dispersion of their hosts, has been made in the identification of Isospora sagittulae McQuistion & Capparella, 1992 from antbirds (Berto et al., 2014b; Silva-Carvalho et al., 2018) and Isospora bellicosa Upton, Stamper & Whitaker, 1995 from icterid birds (Silva et al., 2017). In contrast, the current study only demonstrated the susceptibility of S. aurantiirostris to I. similisi in captivity, albeit without identifying it from free-living golden-billed saltators in their natural distribution area. In a conservative approach, this does not favor the establishment of I. similisi as a junior synonym of I. formarum. The ideal strategy for this purpose would be the morphological and molecular identification of oocysts of I. pityli and I. formarum from their type host S. grossus in Ecuador, in direct comparison with oocysts identified as I. saltatori and I. similisi from S. similis in southeastern Brazil. In addition, the finding of these Isospora spp. from the buff-throated saltator S. maximus, which is one of the Saltator spp. with the widest distribution in the Neotropical realm from the trans-Andean region to the Atlantic coast of South America, would confirm this assumption of coccidia dispersion across trans- and cis-Andean saltators.

The molecular analyses performed in the current study supplemented the molecular characterization of I. similisi, and confirmed the finding of this coccidia species from the new host S. aurantiirostris. The very slight differences in sequences of I. similisi from S. similis and S. aurantiirostris observed in the MACOIII (2 nucleotides) and MARI (1 nucleotide) loci should be considered as intraspecific and possibly resulting from adaptation to another host (Berto & Lopes, 2020; Ortúzar-Ferreira et al., 2023). This conclusion can be explained by the fact that these differences compared to other Isospora spp. from passerines deposited in GenBank are considerably higher, in addition to these exhibiting monophyly in the phylogenetic analyses for these loci (Figures 3 and 4).

Mitochondrial genes are known to be less conserved, and therefore the most suitable for delimiting species, as they indicate even intra-specific differences (Ogedengbe et al., 2011; El-Sherry et al., 2013; Ortúzar-Ferreira et al., 2023). Phylogenetic analyses of mitochondrial loci resulted in monophyletic groups with good support of maximum likelihood bootstrap values and Bayesian posterior probabilities, mainly for Eimeria spp. from rabbits and Eimeria spp. from Galliformes and some Isospora spp. from passerines. However, other Isospora spp. from passerines were ungrouped, or incongruously grouped, in the cladograms of the three mitochondrial loci (Figures 2-4). The significant genotypic variations that were expected for mitochondrial genes, or some other unknown factor for Isospora spp. from passerines, may be associated with the incongruity of these phylogenetic results. The same observations were described by Ogedengbe et al. (2018), who demonstrated the paraphyly of Isospora spp. and Eimeria spp. from different host taxa in a comprehensive phylogenetic analysis involving both mitochondrial and 18S genes. Based on their findings, Ogedengbe et al. (2018) concluded that the necessary taxonomic adjustments aiming at a congruence of phenotypes with the genotypes of Eimeriidae would require the creation of at least 9 new genera not based on the proportion of sporocysts and sporozoites upon which coccidia taxonomy is traditionally based.

The S. similis and S. aurantiirostris samples showed identical the sequences for the 1NF/18S locus. In fact, the 18S gene is known to be more conserved than mitochondrial genes, and hence more suitable for the phylogenetic reconstruction of genera or higher taxa (Ogedengbe et al., 2011; El-Sherry et al., 2013; Andrade et al., 2024). A comparative molecular analysis of the 1NF/18S locus highlights this statement, as very small differences are observed between different coccidia species from large host taxa, including Isospora spp. from different orders of Aves that were 99.7% similar. Along the same vein, the phylogenetic analysis (Figure 5) showed congruent and well-defined monophyletic groups for higher taxa, such as the different genera of Eimeriidae, which were grouped in separate clades. However, Isospora spp., evolutionarily distant because it parasitizes different higher host taxa, belongs to the same monophyletic group.

In conclusion, after analysis and discussion of all the results obtained in the current study, the golden-billed saltator Saltator aurantiirostris is recorded as a new host susceptible to I. similisi, whose molecular characterization is supplemented by the sequencing of three mitochondrial loci and one nuclear locus. In addition, I. similisi is estimated to be a junior synonym of I. formarum due to the broad distribution of its hosts in the Neotropical region. Therefore, the findings of the current study are expected to motivate future taxonomic studies of Isospora spp. from other Saltator spp. in order to establish the synonymization of I. formarum with I. similisi, and thus, its wide distribution and dispersion in the Neotropical region, including across the Andes mountain range.

Acknowledgements

We are thankful to the breeder of the golden-billed saltator located in the Municipality of Resende in the State of Rio de Janeiro, for permitting and assisting with the collection of the material. This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). CM and CNO-F have scholarships from CAPES (Grant/Award Number: 001). MSO has a postdoctoral scholarship from FAPERJ (Grant/Award Number: E-26/204.228/2021). VML has a fellowship from FAPERJ (Grant/Award Number: E-27/211.566/2021). BPB has a fellowship from CNPq (Grant/Award Number: 302345/2022-1) and from FAPERJ (Grant/Award Number: E-26/200.565/2023).

• Ethics declaration

  Collecting permits were issued by the Animal Ethics Committee of the Centro Universitário de Barra Mansa (CEUA/UBM, protocol 007/2018). All applicable institutional, national and international guidelines for the care and use of animals were followed.

• How to cite:

  Maronezi C, Ortúzar-Ferreira CN, Oliveira MS, Cepeda PB, Lima VM, Berto BP. Isospora similisi recovered from a new host, Saltator aurantiirostris, with supplementary molecular data and notes on its taxonomy and distribution in the Neotropical region. Braz J Vet Parasitol 2024; 33(3): e011324. https://doi.org/10.1590/S1984-29612024060

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Publication Dates

• Publication in this collection
  07 Oct 2024
• Date of issue
  2024

History

• Received
  28 May 2024
• Accepted
  12 Aug 2024