Isospora bertoi n. sp. of the saffron finch, Sicalis flaveola (Aves: Passeriformes) from Brazil

Abstract A new species of coccidia (Protozoa: Apicomplexa: Eimeriidae) is described from the saffron finch, Sicalis flaveola, is reported from Brazil. Sporulated oocysts of Isospora bertoi n. sp. are spherical to subspherical; 23.6 (21.1-26.5) x 22.0 (19.4-24.6) µm; shape Index (L/W ratio) 1.1 (1.0-1.2) µm; with bilayer smooth walls, ~1.1 μm. Micropyle and oocyst residuum are absent, but polar granules are present. Sporocysts are elongated ellipsoidal, 16.2 (13.6-17.9) x 10.1 (8.9-12.4) µm. Stieda body is button-shaped and Sub-Stieda and Para-Stieda body are absent. Sporocyst residuum is compact and composed of hundreds of granules scattered among the sporozoites. The sporozoite is claviform with an elongated posterior refractile body and nucleus.


Introduction
The saffron finch, Sicalis flaveola (Linnaeus, 1766), is a bird belonging to the order Passeriformes.It has tiny dimensions and a more complex and developed syrinx, granting it greater song capacity and ability (Silveira and Méndez, 1999).Mainly a granivorous species, it also includes in its diet, fruits and insects that it obtains from the soil and herbaceous strata, branches and foliage up to five meters in height (Zotta, 1940;La Peña and Pensiero, 2003;La Peña, 2011).Its distribution extends from Central to South America, with introductions in Hawaii, Puerto Rico and Jamaica (Ridgely and Tudor, 1989).
Among the diseases that affect birds, coccidiosis is considered an important cause of enteritis and death of the species (Freitas et al., 2003;Coelho et al., 2011).
According to Gallo et al. (2014) several species of Coccidia occur in the same species of bird, and most of these species are not pathogenic, demonstrating the importance of not only quantitatively assessing its diagnosis, but also qualitatively assessing it (Teixeira et al., 2014).
To date, two species of Isospora have been described in S. flaveola in captivity in the Municipality of Seropedica in the State of Rio de Janeiro, Brazil, and identified as Isospora cetasiensis (Coelho et al., 2011) and Isospora sicalisi (Coelho et al., 2011).The present study describes a new species of Isospora found in free-ranging S. flaveola with habitat in the Municipality of Eugenopolis in the State of Minas Gerais (MG), Brazil.
Federal Rural do Rio de Janeiro (UFRRJ), Campus Seropédica, Rio de Janeiro, Brazil, granted in his honor for describing the two species of Isospora of S. flaveola and several other species of coccidia in birds.

Discussion
Coccidia found in the present study were compared in detail with species reported in S. flaveola and other birds as suggested by Duszynski and Wilber (1997) for the description of a new species.In addition, they were compared with coccidia of New World passerine birds also belonging to the Emberizidae family, since there is a higher probability of transmission of this parasite among sympatric birds (Carvalho Filho et al., 2005;Berto et al., 2009).
Two species of Isospora have been described in S. flaveola in the State of Rio de Janeiro, Brazil previously (Coelho et al., 2011).Isospora cetasiensis differs from I. bertoi n. sp.; it does not possess a polar granule, its sporocyst is ovoidal with a rounded Sub-Stieda body and a diffuse residual body (Table 1).The oocyst of I. sicalisi has the largest size (27.5 x 25.2 µm) among the species described in S. flaveola and does not have a polar granule (Table 1).It has a trapezoidal Sub-Stieda body (Table 1), which was not observed in the new species described in this study, and the sporocyst residuum is diffuse, in contrast to the compact oocyst of I. bertoi n. sp.
In 1985, Upton et al. (1985) described Isospora paroariae (Upton et al., 1985) found in Paroaria coronata (Miller, 1776) (Red-crested cardinal) in Argentina.This species, in addition to having a much thicker oocyst wall than the species described in the present study, does not have a polar granule, and its sporocyst is ovoid with a prominent Sub-Stieda body.

Materials and Methods
Fecal samples were collected from 30 birds captured in a mist net in the Municipality of Eugenopolis (MG) Brazil.After capture, the birds were individually housed for 24 h in cages with water and food ad libitum.Feces found at the bottom of the cage were placed in 15 mL tubes, identified, placed in an isothermal box with ice and immediately transported to the Núcleo de Pesquisas Avançadas em Parasitologia (NUPAP) at the Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF) in the Municipality of Campos dos Goytacazes, RJ.All methods were performed in accordance with the relevant guidelines and regulations.Fecal samples were filtered through double gauze, mixed with 2.5% potassium dichromate (K 2 Cr 2 O 7 ), placed in each Petri dish and incubated at 23-28 °C until 70% of the oocysts were sporulated.Oocysts were retrieved by the flotation method with Sheather's sugar solution and examined microscopically using the method described by Duszynski and Wilber (1997).
Morphological observations and measurements obtained in micrometers were made by using an Eclipse i80 optical microscope (Nikon, Japan).Photomicrographs were taken using a Coolpix 5700 digital camera (Nikon, Japan) and drawings were made by superimposing images at different focuses using CorelDRAW software version 2020.Size ranges are given in parentheses, followed by the mean and Shape Index (SI) = Length/Width ratio.

Results
Thirty free-ranging saffron finches were captured, and oocysts with different morphological characteristics of I. sicalisi and I. cetasiensis were observed in the fecal samples of two (7%) of 30 birds.After the fourth day, more than 70% of these oocysts were fully sporulated under the conditions recommended in this study.

Host type: Sicalis flaveola (Aves: Passeriformes: Emberizidae)
Type material: Photosyntypes and linear drawings of sporulated oocysts are deposited and available in the Parasitology Collection at the Laboratório de Biologia de Coccídios in the Universidade Federal Rural do Rio de Janeiro, Campus Seropédica, Rio de Janeiro, Brazil.Photographs of the host specimens (symbiotypes) are deposited in the same collection.The repository number is P-126/2021.
Etymology: The specific name is derived from the last name of Professor Bruno Pereira Berto, eminent protozoologist of the Departamento de Biologia Animal, Instituto de Ciências Biológicas e da Saúde Universidade Brazilian Journal of Biology, 2023, vol.83, e270649 3/9 New Isospora from saffron-finch All the described species have simple walls and sub-Stieda bodies, thus distinguishing them from I. bertoi n. sp.Isospora exigua has no polar granules, and its average size (20.4 x 20.1 µm) is smaller than that of I. bertoi n. sp.Isospora fragmenta and I. temeraria have polar granules of different shapes and in greater quantities when compared to the new species Isospora found in S. flaveola and described in this study.
The following year, the same authors described Isospora geospizae (McQuiston and Wilson, 1989) in Geospiza fuliginosa Gould, 1837 (Small ground finch) and Geospiza fortis Gould, 1837 (Medium ground finch) in the Galapagos Islands too.The oocyst (15.5 x 14.5 µm) and sporocyst (10 x 7.5 µm) of this species have a much smaller average size than I. bertoi n. sp., in addition to the oocyst having a simple wall and the sporocyst having a sub-Stieda body.

Table 1.
Comparative morphology and morphometry of Isospora spp. of American birds of the order Passeriformes, family Emberizidae.The oocysts of this species are considerably larger (32.1 x 28.9 µm) than those described in I. bertoi n. sp. in addition to having a thicker wall (~1.5 µm).The sporocysts have a different shape from that observed in I. bertoi n. sp. and a prominent Sub-Stieda body, which was not observed in the species described in this study.

Species
In 2014, the oocysts of Isospora nigricollis (Barreto, 2014) were described in Sporophila nigricollis (Yellow-bellied Seedeater) at the Municipality of Belo Horizonte in the State of Minas Gerais, Brazil by Barreto (2014).Both oocysts (18 x 17 µm) and sporocysts (12 x 8 µm) have much smaller dimensions than those observed in I. bertoi n. sp.In addition, the oocysts do not have a polar granule, and the sporocyst is not composed of residuum.
Based on morphological and morphometric characteristics, Isospora bertoi is considered a new species not previously described.It is the third species of Isospora described in S. flaveola.