High prevalence of Sarcocystis funereus sp. nov. (Apicomplexa, Sarcocystidae) in offspring of Tengmalm’s owls Aegolius funereus (Aves, Strigidae)

Birds act as intermediate or denitive hosts of cyst-forming coccidia parasites of the genus Sarcocystis Lankester, 1882. However, the spectrum of species of Sarcocystis in birds and the role of the latter in the transmission of coccidia are still incomplete for many avian species including Tengmalm’s owl Aegolius funereus (Linnaeus, 1758). During a research of Tengmalm’s owls in Finland some edglings were found dead and subsequently parasitologically examined. Therefore, this study is focused on the morphological and molecular description of a new Sarcocystis species found in the intestine of the Tengmalm’s owl and its possible role as denitive host.


Conclusions
This work provided the rst and the most comprehensive record on Sarcocystis from owls obtained in Finland, thus highlighting the importance of molecular data in the species identi cation.

Background
Cyst-forming coccidia parasites of the genus Sarcocystis Lankester, 1882 can infect a wide variety of vertebrates, including birds, which could act as de nitive and intermediate hosts in the life cycle of these parasites. However, the spectrum of species of Sarcocystis in birds and the role of the latter in the transmission of coccidia are still incomplete for many avian species including Tengmalm's owl Aegolius funereus (Linnaeus, 1758). This species is a small nocturnal cavity-nesting owl living in coniferous forests in the boreal zone and in alpine forests further south in the Holarctic region [1,2]. It feeds mainly on small mammals, among which voles of the genera Myodes Pallas, 1779 (= Clethrionomys Tilesius, 1850) and Microtus Schrank, 1798 are their main preys, while shrews of the genus Sorex and small forest birds are their most important alternative prey items [3 − 5].
To date, relatively few studies have been conducted with the Sarcocystis species in A. funereus in wild; in fact, only Wiesner [6], in a scienti c meeting, reported sporocysts of Sarcocystis sp. in the Tengmalm's owl, which were experimentally developed in the bank vole Myodes glareolus (= Clethrionomys glareolus) Schreber, 1780. Whereas Zuo et al. [7] and Zuo and Yang [8] were unsuccessful in experimentally infecting A. funereus with Sarcocystis sinensis Zuo, Zhang et Yie, 1990 from China.
During radio-telemetry research of Tengmalm's owls in Finland, where decreasing densities of main prey (voles) occurred, some edglings were found dead and subsequently parasitologically examined. Since this owl species has practically no records of species of Sarcocystis and the role of owl in the life cycle of parasite is partially known, this study is focused on the morphological and molecular description of a new Sarcocystis species found in the intestine of the Tengmalm's owl and its possible role as de nitive host.

Methods
The carcasses of 11 specimens (29 − 47 days old from hatching, 98 − 136 g in weight) from 7 different nests (10 died due to starvation and infection, 1 per marten Martes martes Linnaeus, 1758 predation) (MK and EK unpublished data) were examined in this study. They were collected in the Kauhava region of west-central Finland (63ºN, 23ºE) during a radio-tracking study of Tengmalm's owl edglings during the post-edging dependence period in 2019. The study area is located 30 − 120 m above sea level and mostly covered by forest [for detailed description of the study area (see [4,9,10]). Aerial distances (n = 21) between involved nest boxes from which the edglings originated and later subjected to necropsy were 19.2 ± 9.3 km on average (range 3.8-38.7 km).
Necropsies were carried out in the State Veterinary Institute (SVI) Prague, Czech Republic, where the intestinal content and muscular samples (breast, legs, heart) of thawed birds were parasitologically examined in wet mouth using water or glycerine for orientation purposes. After parasite nding, intestinal mucosa scrapings were used for otation-centrifugation coprological method under light microscopy for the nal evaluation and presence of oocysts/sporocysts using a Leica DMLB optical microscope with a Leica DFC420 digital camera (Leica Microsystems, Wetzlar, Germany), and isolation to Eppendorf tubes for DNA extraction. All measurements are given in micrometres, unless otherwise mentioned.
Genomic DNA was extracted by glass bead disruption from 22 isolates (two from each owl) of oocysts/sporocysts using the QIAamp® Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. DNA was stored at -20°C until further use.
Polymerase chain reaction (PCR) and nested-PCR were carried out by using primers for 18S rRNA (ERIB1/A2R, A1F/S2r, A2F/Primer BSarc and Fext/Rext, Fint/Rint, respectively) [11 − 15], 28S rRNA (KL_P1R/KL_P1F) [16], ITS1 region (ITS-F/ITS-R) [16] and cox1 genes (SF1/SR10) [17,18]. The PCR mixture (20 µl of reaction mixture and 5 µl of DNA extract) comprising of GoTaq® G2 Green Master Mix (Promega, Madison, Wisconsin, USA), 0.4 µM of each primer, DNA template and nuclease-free water to obtain a nal volume of 25 µl. PCR conditions consisted of initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 52-60°C for 30 s, 72°C for 1 min, and then a nal extension step at 72°C for 10 min. The ampli cation products were resolved on 1.5% agarose gels and visualized by ethidium bromide staining. The PCR products were puri ed using the ExoSAP-IT™ Express PCR Product Cleanup Reagent kit (Thermo Fisher Scienti c) according to the manufacturer's protocol. Puri ed PCR products were directly sequenced in both forward and reverse directions using the same primers as for PCR through the commercial company Euro ns Genomics (Ebersberg, Germany). Nucleotide sequences of 18S rRNA, 28S rRNA, ITS1 and cox1 loci of S. funereus sp. nov. have been deposited in GenBank under the accession numbers MW349706, MW349707, MW373964, MW489293, respectively. Sequences from both forward and reverse strands were assembled and manually edited using FinchTV software (Geospiza Inc., Seattle, Washington) followed by BLAST (Basic Local Alignment Search Tool) program at the NCBI (National Center for Biotechnology Information) server, searches were conducted on obtained sequences for genus/species identi cation. Sequence chromatograms obtained in this study were aligned using MAFFT software version 7 [19].
Phylogenetic trees for all datasets were generated form nucleotide sequences of the selected Sarcocystis species using the MEGA X [20] and reconstructed by using the Neighbor-Joining (NJ) and Maximum Likelihood (ML) methods. A NJ phylogenetic tree for 18S rRNA gene (dataset with 25 nucleotide sequences with a total of 1644 aligned nucleotide positions) was computed according to the Tamura-Nei model with a gamma distribution (TN93 + G). Other phylogenetic trees were generated using ML analyses based on the Kimura 2-parameter model with a gamma distribution rate and a proportion of invariant sites (K2 + G + I) for 28S rRNA gene (25 sequences with 1442 positions); for cox1 gene (18 sequences with 1013 positions) the Hasegawa-Kishino-Yano model with a gamma distribution (HKY + G) was used, while the Tamura-Nei model with a gamma distribution rate and a proportion of invariant sites (TN93 + G + I) was used for ITS1 region (24 sequences with 1426 positions). All four phylogenetic trees were rooted using Toxoplasma gondii sequence. Consensus trees were obtained after bootstrap analysis with 1000 replications.

Etymology
The speci c epithet is derived from the species name of its de nitive host, i.e., funereus.
Genetic sequences of 20 Sarcocystis isolates (10 birds) were obtained for the 18S rRNA, 28S rRNA, ITS1 and cox1 loci. All obtained 18S rRNA sequences were identical; therefore, only one of 1773 bp was submitted to GenBank (MW349706 The phylogenetic trees showed different topologies and relationships between the new species with its congeners according to the availability of sequences. Phylogenetic trees based on 18S rRNA, 28S rRNA and cox1 genes showed a clade formed by the new species and S. strixi, as well as Sarcocystis sp. 5 (in the case of 18S rRNA) (Fig. 2a, b, d), while tree of ITS1 region showed S. funereus sp. nov. lonely in a single clade since the ITS1 sequence of S. strixi was not available, although the new species formed a group with other Sarcocystis spp. (Fig. 2c).

Discussion
The rst published nding and description of the oocysts/sporocysts of Sarcocystis sp. in the Tengmalm's owl was made by Wiesner [34]. Most of these were solely morphologically studied, while only sporocysts of S. dispersa in Tyto alba (18S rRNA) [35] and S. strixi (18S rRNA, 28S rRNA and cox1) [31] were morphological and molecularly characterized.  Wiesner [6] were not described, so it is impossible to say if it belongs to S. funereus sp. nov., although they could be conspeci c.
On the other hand, of those species molecularly characterized, as S. dispersa (18S rRNA) and S. strixi (18S rRNA, 28S rRNA and cox1), the rst formed a different clade than that of S. funereus sp. nov., while the second grouped together with the new species in the three genes. Apparently, S. strixi and Sarcocystis sp. 5 are closely related (sister) to S. funereus sp. nov., but with genetic differences to be still considered as separated species. The ITS1 region is more sensitive to the genetic differences among Sarcocystis species (see [36 − 38]), while the 18S rRNA gene is now considered of limited taxonomic help [28]. Unfortunately, ITS1 region was not used previously in S. dispersa nor S. strixi, thus making their comparison with the new species limited. This is the rst time that the ITS1 region was sequenced for a Sarcocystis from owls as de nitive hosts and clearly revealed the differences among species.
The intermediate host of S. funereus sp. nov. is unknown, but apparently rodents (different species of mice and voles inhabiting the study area) (see [4,39]) play that important role. Experimentally, Wiesner [6] observed that the bank vole M. glareolus acts as intermediate host and it could also be the potential host for the new species, while the northern saw-whet owl A. acadicus, a congeneric owl species from the USA, used the deer mice (Peromyscus maniculatus) (see [22]). According to Mikkola [1], König [4,39]. In the study area, the main preys of Tengmalm's owl are bank voles, eld voles and sibling voles, whose abundances regularly uctuate in high-amplitude (100 − 200-fold) three-year cycle [40 − 43]. Accordingly, the abundances of individual vole species vary strongly. The overall prey abundance could be 0.2-13.1 and 0.6-28.2 vole individuals per 100 trap-nights as revealed by regular long-term snap-trapping in the study area during spring and autumn, respectively, thus differing up to 65-fold times between different years/phases of the vole cycle [4,10,42].
It has been mentioned that species of Sarcocystis are more speci c to their intermediate than de nitive hosts, especially those using rodents as intermediate hosts (see [28]). In the case of M. glareolus, it has been found as intermediate host of several types and unnamed species of Sarcocystis from the Czech Republic [44], Baltic region [45] and Lithuania [46], as well as of S. clethrionomyelaphis Matuschka, 1986 in Germany, which uses canids, mustelids, snakes or birds of prey as de nitive hosts (see [47,48]). One of the forms from Lithuania showed similar features (dense hair-like projections on cyst wall) than that of Sarcocystis sp. described by Wiesner [6] (see [46]), thus corroborating the role of bank vole in the life cycle of the parasite.
The cause of death of Tengmalm's owls was undetermined, but the occurrence of Sarcocystis in these birds should be monitored, since other taxa of this genus (S. falcatula, Sarcocystis sp. isolate from chicken) have been reported as causing encephalitis in free-ranging great owls (B. virginianus) and meningoencephalitis in chickens, respectively (see [33,49]).
In the last decades, the integrative taxonomy by using morphological features and molecular analysis has uncovered the huge diversity of species in various groups of organisms, including protists [28]. Additionally, it particularly improved the recognition of the speci city of Sarcocystis in their intermediate and de nitive hosts around the world. According to the high infection prevalence of the present study, the Tengmalm's owl acted as natural de nitive host for S. funereus sp. nov., thus representing the rst host record in A. funereus and the nineth owl species with a Sarcocystis species. Apparently, S. funereus sp. nov. is speci c to A. funereus, since the latter was experimentally infected with S. sinensis and sporocysts and oocysts were no found after some days of infection (see [8]). However, birds of prey might be infected by more than one Sarcocystis species, as S. halieti and S. lari in the white-tailed sea eagle (H. albicilla) (see [15]); therefore, more Tengmalm's owls, other owl species and birds of prey should be examined to determine the presence of other species or forms.
Tengmalm's owls are nomadic and the natal dispersal movements of those juveniles hatched in Finland could extend more than 1000 km [50,51], while adult females show long distance breeding dispersal up to > 600 km in Finland [4,50] and adult males are usually resident after their rst breeding attempt [4].
They can also move over long distances and are widely distributed in North and Central Europe including the Italian Alps and Pyrenees in North Spain [1]. Therefore, it is highly probable that Tengmalm's owls could spread S. funereus sp. nov. out of Finland to various other locations within their distribution range. For instance, during a long-term study of Tengmalm's owl in the Czech Republic (years 2010-12 and 2015) a prevalence of 40% was found for a Sarcocystis sp. in 10 edglings [52,53]. Thus, these parasites seem to be present in that country, although the species identi cation should be con rmed to determine the real distribution of S. funereus sp. nov. However, Svobodová [54] examined two Tengmalm's owls in the Czech Republic, which were negative to the presence of oocysts/sporocysts of Sarcocystis.
If we considered that family Strigidae comprises 223 species of owls reported around the world, more studies are needed to elucidate the parasite fauna and involvement of these birds in the life cycles of parasites. Thus, new ndings will help in increasing the knowledge about this interesting group of predators, as well as their role as predators of rodents, which also act as intermediate hosts of several Sarcocystis.

Conclusions
This work provided the rst and the most comprehensive record on Sarcocystis from owls, thus highlighting the importance of molecular data in the species identi cation. This work contributes to the better understanding of species diversity and current taxonomic status of the new species within the genus Sarcocystis. Further works including examinations of owl populations and particularly their preys in Finland, the Czech Republic and worldwide are required to elucidate the life cycle of the parasite.