A new species of Isospora (Apicomplexa: Eimeriidae) from the yellow warbler, Setophaga petechia (L.) (Passeriformes: Parulidae: Parulinae), in Oklahoma, USA

Isospora fitzpatricki n. sp. is described from a yellow warbler, Setophaga petechia (L.), from McCurtain County, Oklahoma, USA. Oöcysts of the new species are subspheroidal to ovoidal with a smooth bi-layered wall, measure (L × W) 24.2 × 20.4 μm, and have a length/width (L/W) ratio of 1.2; a micropyle and oöcyst residuum are both absent, but polar granule(s) are present. Sporocysts are ovoidal to ellipsoidal and measure 14.5 × 9.2 μm, L/W ratio of 1.6; a knob-like Stieda body is present as well as a distinct rounded sub-Stieda body. The sporocyst residuum is composed of various-sized granules in a compact rounded or irregular mass or dispersed between and across the sporozoite. The oöcysts of I. fitzpatricki n. sp. can be differentiated from five other congeners reported from members of the New World warbler family Parulidae from either Mexico, Costa Rica, or Brazil, by being larger on average as well as by possessing a prominent rounded sub-Stieda body, sporozoite striations, and an posterior refractile body. Information is also presented on an Isospora sp. being passed by S. petechia that we choose not to describe as a putative novel species of Isospora at this time. These two isosporans represent the first coccidians reported from S. petechia and, more importantly, the first known from the members of the Parulidae in the USA.


Introduction
The yellow warbler, Setophaga petechia (L.) (Passeriformes: Parulidae: Parulinae) is a long-distance migrant that ranges from northern Alaska and northern Canada to Labrador south through the central plateau of Mexico to Panama and central Peru, northern Bolivia, and Amazonian Brazil, and east through the West Indies (Pashley, 1988a, b;Pashley & Hamilton, 1990). The species breeds in most of North America from Alaska and far northern Canada south to northwest Mexico and winters from Mexico to Peru (Cornell Lab of Ornithology, 2019). In Oklahoma, it is widespread in the state but is more common in the eastern portion (Tekiela, 2002;Kuhnert, 2004). It inhabits a variety of habitat including open scrub, thickets, second-growth woodland, farmland, and gardens, riparian woodlands, swamps, and along other watercourses. This bird is a copious feeder of insects, especially caterpillars and will occasionally take small fruits/berries or probes in flowers (Lack, 1976;Tekiela, 2002).
Phylogenetic analyses of sequences of nuclear and mitochondrial DNA (Lovette et al., 2010) indicated that all species formerly placed in the genus Dendroica G.R. Gray, one species formerly placed in Wilsonia Bonaparte (W. citrina Boddaert), and two species formerly placed in Parula Bonaparte (P. americana (L.) and P. pitiayumi (Vieillot)) form a clade with the single species customarily placed in the genus Setophaga Swainson (S. ruticilla (L.)). Thus, the generic name Setophaga has priority for this clade (Chesser et al., 2011). There are 34 recognized subspecies of S. petechia (Gill et al., 2022).
The New World warbler family Parulidae includes 111 species of New World or wood-warblers (Winkler et al., 2020). To date, only five species (4.5%) have been reported to harbor isosporan coccidians. All of these coccidians have been previously described from birds collected in Mexico, Costa Rica, or Brazil (Berto et al., 2010(Berto et al., , 2014Keeler et al., 2014;Salgado-Miranda et al., 2016;Mello et al., 2022). Nothing, however, is known of the coccidian parasites of S. petechia. Here, we describe a new species of Isospora as well as document another Isospora sp. from S. petechia from Oklahoma, USA.

Sample collection
Faecal samples were collected during March 2022 from the rectum of two adult S. petechia found dead in McCurtain County, Oklahoma, USA, and examined for coccidian parasites. Briefly, a mid-ventral incision was made to expose the gastrointestinal tract; faeces were taken from the rectum of each bird. Samples were placed in individual vials containing 2.5% (K 2 Cr 2 O 7 ) aqueous potassium dichromate at 1:6 (v/v).

Morphological analyses
Samples were further examined for coccidia via flotation in 15-ml conical centrifuge tubes (with centrifugation) containing Sheather's sugar solution (specific gravity: 1.30) using an Olympus BX43 light microscope (Olympus Corporation, Center Valley, Pennsylvania, USA). A positive faecal sample was placed in a Petri dish containing a small layer of K 2 Cr 2 O 7 for 48-72 h to allow sporulation. All morphological measurements are reported in micrometres (μm) and are given as the range followed by the mean in parentheses. Photographs were taken using Nomarski interference-contrast optics at Â1000 magnification. O€ ocysts were c.45 days-old when measured and photographed. Descriptions of o€ ocysts and sporocysts follow the standard guidelines of Wilber et al. (1998) including o€ ocyst length (L) and width (W), their ranges and ratios (L/W), micropyle (M), o€ ocyst residuum (OR), polar granule(s) (PG), sporocyst length (L) and width (W), their ranges and ratio (L/W), sporocyst (SP), Stieda body (SB), sub-Stieda body (SSB), para-Stieda body (PSB), sporocyst residuum (SR), sporozoites (SZ) anterior (ARB) and posterior (PRB) refractile bodies, and nucleus (N).

Remarks
Five species of Isospora have been previously described from parulid birds (Berto et al., 2010(Berto et al., , 2014Keeler et al., 2014;Salgado-Miranda et al., 2016;Mello et al., 2022) (Table 1). None, however, have been reported from any member of the family in the USA. When the new species is compared to these isosporans, it is most similar to three of them, including Isospora basileuterusi Mello & Berto, 2022  The new species differs from these as follows: (i) o€ ocysts of Isospora fitzpatricki n. sp. are larger (on average) than each of them (see Table 1); (ii) although the knob-like Stieda body is similar among all of them, when compared to the new species, the sub-Stieda body of all three previously described congeners is large and trapezoidal versus the prominent rounded sub-Stieda body of I. fitzpatricki n. sp.; (iii) sporozoites of the new species have striations (see Fig. 1C) and no striations were mentioned in the species description or delineated on the line drawing for the sporozoites of I. basileuterusi; and (iv) sporozoites of I. basileuterusi are described having both an anterior refractile body (ARB) and posterior  refractile body (PRB), whereas those of I. fitzpatricki n. sp. possess only the PRB. Based on these notable metrical and morphological differences, we believe that I. fitzpatricki is a genuine new species.

Remarks
Unfortunately, only five o€ ocysts were recovered from this faecal sample. This form does, however, represent a species of Isospora possessing one of the largest o€ ocysts (on average) of any parulid coccidian, except for Isospora celata Berto, Medina, Zepeda-Vel azquez, García-Conejo, Galindo-S anchez, Janczur & Soriano-Vargas, 2016 (see Table 1). Nevertheless, I. celata does not possess a polar granule and its sub-Stieda body is barely discernible which differs considerably from the Isospora sp. described here. In addition, the o€ ocysts of Isospora orbisreinitas (Table 1) are equivalent in the range of measurements as well as the possession of a compartmentalized sub-Stieda to Isospora sp. However, because few o€ ocysts were found, we choose to take the conservative approach and, until additional samples are available, we decline to name this species at this time.

Discussion
Yellow warblers are one of the most numerous warblers in North America but their populations have been slowly declining, and have decreased by 25% between 1966 and 2014, according to the North American Breeding Bird Survey (Sauer et al., 2014). Populations are jeopardized in some areas by loss of riparian habitat in combination with heavy brood parasitism by brown-headed cowbirds, Molothrus ater (Boddaert), and shiny cowbirds, Molothrus bonariensis (Gmelin) (Ehrlich et al., 1988(Ehrlich et al., , 1992Weatherhead, 1989)

Conclusion
Our comparison of o€ ocysts of I. fitzpatricki n. sp. with o€ ocysts of Isospora spp. previously described from other Western Hemisphere parulid birds supports our designation of a novel species. The new species is the first known from members of its family in the USA. As such, we consider I. fitzpatricki new to science, which, to date, becomes the sixth species of Isospora described from a host in this family of passeriform birds. We also document an unidentified species of Isospora from this host that requires additional samples. Additional parulid birds should be examined for coccidia, particularly those that occur in North America.

Funding
Official funding for this study was not available.

Ethical approval
Ethical Approval for collecting was granted per the Oklahoma Department of Wildlife Conservation Scientific Collecting Permit No. 1551646 to CTM. All institutional, national, and international guidelines for the care and use of animals were followed.

CRediT author statement
This study was designed by both authors. Field collections were performed by CTM. Laboratory procedures for recovery of o€ ocysts were performed by CTM. Laboratory procedures for measurements, photomicrographs, and isolation of o€ ocysts were performed by JAH. CTM drew the coccidian o€ ocyst. The manuscript was written by both authors and subsequently revised by both authors. Both authors read and approved the final manuscript.

Data availability
Photosyntypes and a photovoucher are accessioned into the Harold W. Manter Laboratory of Parasitology (HWML), University of Nebraska, Lincoln, Nebraska, USA. The host voucher was accessioned into the Eastern Oklahoma State College (EOSC) Collection, Idabel, Oklahoma, USA.

Declaration of competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.