Avispora mochogalegoi n . sp . ( Apicomplexa : Sarcocystidae ) in the little owl , Athene noctua ( Strigiformes : Strigidae ) , in mainland Portugal

The little owl Athene noctua (Scopoli, 1769) is a small raptor that is widely distributed from northern to southern Portugal and several other countries in Europe, Asia and North Africa, and which has been introduced into New Zealand. In the current study, 18 fecal samples were collected from little owls kept at the Lisbon Center for Wild Animal Recovery, which is located in Monsanto Forest Park, Lisbon, Portugal. Twelve (67%) of them were found to be passing an undescribed species of Avispora in their feces. The oocysts of Avispora mochogalegoi n. sp. were ellipsoidal with a bilayered wall and measured 38.9 × 32.9 μm, with a shape index of 1.18. No micropyle, oocyst residuum or polar granule was present. The sporocysts were subspherical, measuring 21.1 × 20.1 μm. Stieda, sub-Stieda and para-Stieda bodies were absent. The sporocyst residuum was composed of a compact subspherical mass of granules. This is the fourth species of Avispora reported in Strigiformes.


Introduction
The little owl Athene noctua (Scopoli, 1769) is a small raptor that is often observed in Portugal because of its diurnal and nocturnal habit.It is widely distributed from northern to southern Portugal and in several other countries in Europe, Asia and North Africa, and it has also been introduced into New Zealand.It is common on agricultural land with a few scattered trees and in olive groves.It is often seen on ruins or heaps of stones, which it uses to nest in.It is absent from high-altitude areas and densely forested areas (IUCN, 2016;ELIAS, 2017).
The genus Caryospora Leger, 1904, is traditionally recognized as one of the main genera of coccidian parasites of both raptors and reptiles (UPTON et al., 1990;BERTO et al., 2014).Hence, the species of Caryospora that have been recorded in raptors were recently taxonomically transferred to a new genus, termed Avispora Schuster, Woo, Poon, Lau, Sivakumar, Kinne, 2016.This reclassification was based on the morphological, biological and, finally, molecular differences of Caryospora spp.found in raptors and reptiles (BERTO et al., 2014;SCHUSTER et al., 2016) In this context, the current study describes a new species of Avispora found in little owls (A.noctua) that were being kept for rehabilitation and reintroduction into the wild at the Lisbon Center for Wild Animal Recovery (Centro de Recuperação de Animais Silvestres de Lisboa, LxCRAS), in Monsanto Forest Park, Lisbon, Portugal.

Materials and Methods
Eighteen fecal samples were collected from little owls (A.noctua) that were being kept in individual cages on the premises of the Lisbon Center for Wild Animal Recovery (LxCRAS), which is located in Monsanto Forest Park, Lisbon, Portugal.The samples were collected immediately after defecation and were placed in plastic vials containing 2.5% potassium dichromate (K 2 Cr 2 O 7 ) solution at 1:6 (v/v).In the laboratory, the samples were incubated at room temperature for 10 days or until day three when around 70% of the oocysts had sporulated.The oocysts were recovered by means of flotation in Sheather's sugar solution (specific gravity: 1.20).Morphological observations, line drawings, photomicrographs and measurements were made using an Olympus BX40 microscope equipped with a digital camera (Olympus DP10).This processing for identification and description was made a few days after sporulation of the oocysts to avoid morphological alterations in the oocysts derived from the long storage time.The line drawings were edited using two software applications in CorelDRAW® (Corel Draw Graphics Suite, Version 11.0, Corel Corporation, Canada), specifically Corel DRAW and Corel PHOTO-PAINT.All measurements were made in micrometers and are given as the range followed by the mean in parentheses.The descriptions of oocysts and sporocysts followed the guidelines of Duszynski & Wilber (1997) and Berto et al. (2014), using the following: oocyst (O) length (L) and width (W) and their ranges and ratios (L/W); micropyle (M), oocyst residuum (OR), polar granule (PG) and sporocyst (SP) length (L) and width (W) and their ranges and ratios (L/W); and Stieda body (SB), sub-Stieda body (SSB), para-Stieda body (PSB), sporocyst residuum (SR), sporozoite (SZ), refractile body (RB) and nucleus (N).
Other hosts: None.Type specimens: Phototypes and line drawings have been deposited and are available in the Parasitology Collection of the Laboratório de Biologia de Coccídios, at Universidade Federal Rural do Rio de Janeiro, in the municipality of Seropédica, Rio de Janeiro, Brazil (LABICOC, 2017).Photographs of the host specimens have been deposited in the same collection.The repository number is P-78/2017.
Prevalence: 12 out of 18 specimens (67%).Sporulation: Exogenous.All oocysts were passed in the feces unsporulated and became fully sporulated by day 7 in K 2 Cr 2 O 7 solution at room temperature.
Prepatent and patent periods: Unknown.Site of infection, definitive host: Unknown.Oocysts recovered from feces.

Site of infection, secondary host: Unknown.
Endogenous stages, definitive host: Unknown.Endogenous stages, secondary host: Unknown.Cross-transmission: None to date.Pathology, definitive host: Unknown.Pathology, secondary host: Unknown.Etymology: The specific epitaph is derived from the common local name for the host, which is 'mocho-galego' (= galician owl).

Remarks
Table 1 shows some characteristic features of Caryospora spp., described from Strigiformes.Among the Avispora species listed in Table 1, only Avispora bubonis (CAWTHORN & STOCKDALE, 1981) and Avispora henryae (YAKIMOFF & MATIKASCHWILI, 1932) have oocyst measurements similar to those of A. mochogalegoi.The main distinctive feature of A. mochogalegoi is the irregularsubspherical body consisting of numerous granules that appear to be membrane-bounded.In contrast, A. bubonis and A. henryae have scattered sporocyst residuum.In fact, as emphasized by Upton et al. (1986), A. henryae was described from several hosts of different forms and, therefore, it is possible that more than one species may be involved; however, in any case, none of these descriptions of A. henryae contain sub-spherical sporocysts with an irregular-subspherical body consisting of numerous granules that appear to be membrane-bounded (UPTON et al., 1986;1990).
Thus, the type-host of A. mochogalegoi recorded in this study is only sympatric with A. brama in Asia.Therefore, A. brama becomes a potential host for this coccidium, since it is congeneric with A. noctua.At the same time, there is a well-established consensus of intra-familial specificity for coccidia of Aves, which was suggested by Duszynski & Wilber (1997) and has been reaffirmed in several subsequent studies on coccidians in birds (BERTO et al., 2011).In this context, more than 100 species of Strigidae distributed in the Old World would be susceptible to A. mochogalegoi (IUCN, 2016).
Avispora henryae, which was originally described from Eurasian eagle-owls Bubo bubo (LINNAEUS, 1758), was subsequently reported in several raptors of the orders Accipitriformes and Falconiformes (HOARE, 1933).However, Upton et al. (1986) contested this transmission between different orders of Aves, taking the view that these subsequent reports should be of other coccidian species.Bubo bubo is sympatric with A. noctua in the Russian region (KONTORSHIKOV et al., 1996); therefore, it would be possible for these owls to share the same coccidian species, but as mentioned above, the morphology identified in the oocysts of the current work is incompatible with all descriptions of A. henryae.
The genus Avispora was recently introduced after observation of the wide phylogenetic distance between species of the genus Caryospora in raptors and species of Caryospora in reptiles, through comparison of DNA sequencing of the cox1, 18S ribosomal ribonucleic acid (rRNA) and 28S rRNA genes (SCHUSTER et al., 2016).In fact, previous studies had already highlighted the evident morphological and biological differences of Caryospora spp.found in raptors and reptiles, such as the presence of a Stieda body and developmental details in the intermediate host, especially regarding caryocysts, along with the evident host specificity (CAWTHORN & STOCKDALE, 1981;WACHA & CHRISTIANSEN, 1982;UPTON et al., 1986;BERTO et al., 2014).
The molecular phylogenetic analysis provided conclusive evidence for transferring the species of the genus Caryospora in raptors to the new genus Avispora.Moreover, it placed these species closer to another coccidian family: Sarcocystidae, which comprises the cyst-forming coccidians.This inclusion in Sarcocystidae is consistent with the morphological characteristics of the oocysts, given that like Avispora, sarcocystids do not have a Stieda body.
Therefore, based on the morphological features described above, A. mochogalegoi is considered to be new to science and to be the fourth species reported in Strigiformes.