The Richness of Sarcocystis Species in the Common Gull ( Larus canus ) and Black-Headed Gull ( Larus ridibundus ) from Lithuania

: The common gull ( Larus canus ) and the black-headed gull ( Larus ridibundus ) are common waterbird species in Lithuania. Until now, the composition of Sarcocystis species in these birds was unknown. The current study aimed to identify Sarcocystis spp. by the morphological examination of sarcocysts found in the muscle tissues and by DNA sequence analysis. Between 2011 and 2019, the leg muscles of 42 common gulls and 63 black-headed gulls were tested for Sarcocystis spp. Based on the methylene blue staining of squashed muscle samples, sarcocysts were detected in six common gulls (14.3%) and in six black-headed gulls (9.5%). Under a light microscope, one type of microcyst was observed. Sarcocysts were thread-like (2860–8250 × 40–180 µ m) and had a smooth and thin (0.8–1.4 µ m) cyst wall, while bradyzoites were banana-shaped and 5.0–9.2 × 1.3–2.4 µ m in size. The sequencing of complete ITS1 showed the presence of S . columbae , S . halieti and S . wobeseri in the common gull and S . columbae and S . halieti in the black-headed gull. The highest intraspeciﬁc genetic variability was established for S . halieti , which is characterized by a wide host range. This species is considered to be pathogenic, therefore further histopathological examination of the various organs of gulls is needed.


Introduction
Protozoans belonging to the genus Sarcocystis are intracellular parasites infecting mammals, birds and reptiles [1]. These apicomplexan parasites have an obligatory twohost prey-predator life cycle. Sarcocysts are mainly formed in the muscles or CNS of an intermediate host, while oocysts/sporocysts develop in the intestinal mucosa of a definitive host. Intermediate hosts usually acquire a Sarcocystis infection through the accidental ingestion of sporocysts in contaminated feed or water, whereas definitive hosts become infected through the ingestion of sarcocysts via predation or scavenging [1,2]. Birds serve as intermediate or definitive hosts for many Sarcocystis species [1,[3][4][5]. Some of them are pathogenic for their intermediate host [1,6]. Sarcocystis falcatula and S. calchasi, each with distinct morphologies and biology, are two well-investigated pathogenic Sarcocystis spp. that are hazardous for birds [1,[7][8][9].
The members of the family Laridae are abundant and adaptable birds are distributed worldwide. This family consist of around 100 species arranged into 22 genera [10,11]. Gulls are found mainly on the coast in summer, but some species also nest inland, especially in winter [10][11][12][13]. Gulls are an example of opportunistic birds that have successfully adapted to urban environments, and some species have become more common in urban areas and have even established breeding populations in cities around the world [14,15]. Common gulls (Larus canus) and black-headed gulls (Larus ridibundus) are opportunistic predators of marine invertebrates, fishes, insects and birds, as well as opportunistic scavengers of dead animals, who are capable of roosting in urban landfills and other waste sites [12,13,16,17]. The common gull and black-headed gull are common bird species breeding in various bodies of water and marshes in Lithuania [12,13]. Common gulls are more prevalent near water bodies, while black-headed gulls are also found near human settlements [18]. To date, four Sarcocystis species, S. columbae, S. halieti, S. lari and S. wobeseri, have been identified in the muscles of larids. All four of these species were confirmed in the herring gull (Larus argentatus), while S. lari was described in the great black-backed gull (Larus marinus) [18][19][20]. The richness of Sarcocystis species in common gulls and black-headed gulls is unknown. Therefore, the aim of the present study was to identify Sarcocystis spp. from the leg muscles of common gulls and black-headed gulls by the morphological examination of sarcocysts and the ITS1 (internal transcribed spacer 1) region sequence analysis of isolated parasites.

Morphological Examination of Sarcocystis spp. Detected in Common Gull and Black-Headed Gull
Based on methylene blue staining, sarcocysts were found in 12 of the 105 (11.4%) birds (6/42, 14.3% common gulls and 6/63, 9.5% black-headed gulls) examined in Lithuania. The parasite load ranged from 1 to 107 cysts/g of the muscle samples. In most muscle samples the parasite load was low, since the median parasite load was 2.5 cysts/g of muscle.
In fresh muscle samples, sarcocysts were detected in nine birds (five common gulls and four black-headed gulls). Due to low parasite load sarcocysts were not detected in fresh muscle samples of one common gull (specimen LcLt31) and two black-headed gulls (LrLt24, LrLt35).
An examination of 36 sarcocysts under LM revealed that they were quite similar by light microscope (LM). Sarcocysts were microscopic, thread-like, 2860-8250 × 40-180 µm (5992 ± 1832 × 99 ± 30 µm; n = 36) in size (Figure 1a), with a thin (0.8-1.4 µm; n = 36) and apparently smooth cyst wall (Figure 1b). Septa divided sarcocysts into compartments filled with banana-shaped bradyzoites, which were 5.0-9.2 × 1.3-2.4 µm (7.2 ± 0.9 × 2.0 ± 0.4 µm; n = 150) in size (Figure 1c). Parasitologia 2023, 3, FOR PEER REVIEW 2 scavengers of dead animals, who are capable of roosting in urban landfills and other waste sites [12,13,16,17]. The common gull and black-headed gull are common bird species breeding in various bodies of water and marshes in Lithuania [12,13]. Common gulls are more prevalent near water bodies, while black-headed gulls are also found near human settlements [18]. To date, four Sarcocystis species, S. columbae, S. halieti, S. lari and S. wobeseri, have been identified in the muscles of larids. All four of these species were confirmed in the herring gull (Larus argentatus), while S. lari was described in the great black-backed gull (Larus marinus) [18][19][20]. The richness of Sarcocystis species in common gulls and black-headed gulls is unknown. Therefore, the aim of the present study was to identify Sarcocystis spp. from the leg muscles of common gulls and black-headed gulls by the morphological examination of sarcocysts and the ITS1 (internal transcribed spacer 1) region sequence analysis of isolated parasites.

Morphological Examination of Sarcocystis spp. Detected in Common Gull and Black-Headed Gull
Based on methylene blue staining, sarcocysts were found in 12 of the 105 (11.4%) birds (6/42, 14.3% common gulls and 6/63, 9.5% black-headed gulls) examined in Lithuania. The parasite load ranged from 1 to 107 cysts/g of the muscle samples. In most muscle samples the parasite load was low, since the median parasite load was 2.5 cysts/g of muscle.
In fresh muscle samples, sarcocysts were detected in nine birds (five common gulls and four black-headed gulls). Due to low parasite load sarcocysts were not detected in fresh muscle samples of one common gull (specimen LcLt31) and two black-headed gulls (LrLt24, LrLt35).

Genetic Identification and Intraspecific Variation of Sarcocystis spp.
Based on the comparison of complete ITS1 sequences obtained in this study, S. columbae, S. halieti and S. wobeseri were identified in the leg muscles of two of the examined larid species. All these Sarcocystis species were confirmed in the common gull, while two of them (S. halieti and S. columbae) were detected in the black-headed gull (Table 1).

Genetic Identification and Intraspecific Variation of Sarcocystis spp.
Based on the comparison of complete ITS1 sequences obtained in this study, S. columbae, S. halieti and S. wobeseri were identified in the leg muscles of two of the examined larid species. All these Sarcocystis species were confirmed in the common gull, while two of them (S. halieti and S. columbae) were detected in the black-headed gull (Table 1). Furthermore, of the 16 sarcocysts isolates excreted from the black-headed gulls, 14 were attributed to S. halieti and only 2 isolates were identified as S. columbae. Whereas the distribution of the identified Sarcocystis species in common gulls was more uniform. In general, four, seven and nine sequences of S. columbae, S. wobeseri and S. halieti were obtained, respectively.
Co-infections with two Sarcocystis species were detected in a single common gull (isolate LcLt34) and in a single black-headed gull (isolate LrLt53).  .40%), and less than 93% similarity compared to other named Sarcocystis spp. In total, 6 sequences of the S. columbae determined in the present study were 100% identical, demonstrating 99.40-100% identity to S. columbae isolated from other hosts, and displaying the greatest similarity to Sarcocystis sp. ex Accipiter cooperii (96.04%) and S. columbae (93.80%). Finally, 4 haplotypes differing up to 0.47% were defined by 7 ITS1 sequences of S. wobeseri. These 4 haplotypes shared 99.41-100% identity with those of S. wobeseri deposited in GenBank and showed the greatest similarity (92.54-93.30%) to S. calchasi.
The comparison of ITS1 sequences of Sarcocystis spp. employing birds as intermediate hosts showed that S. halieti, which was also determined in the current study, was distinguished by the highest intraspecific variation reaching almost 3% ( Figure 2). The intraspecific genetic differences of other avian Sarcocystis spp. did not exceed 1.2%. The lowest interspecific differences slightly less than 4% were determined comparing S. columbae, S. halieti, Sarcocystis sp. ex Accipiter cooperii and Sarcocystis sp. isolate Skua-2016-CH with other Sarcocystis spp. However, for all examined Sarcocystis spp., interspecific and intraspecific genetic differences did not overlap, showing that ITS1 is a useful genetic marker for the discrimination of avian Sarcocystis spp. region. The X axis shows genetic differences expressed in percentages, which was calculated 100% minus genetic similarity values obtained by BLAST analysis. Notably, S. falcatula was not included into the analysis, since it is assumed that this parasite is speciescomplex [21,22]. Based on sequence comparison, Sarcocystis sp. isolate MP9 and Sarcocystis sp. isolate 471 should belong to the same species.  [1]. Since studies on the ultrastructure of sarcocyst walls are expensive and time consuming, usually the morphology of the cyst wall is evaluated in fresh preparations or histological sections under a LM. The morphology of the sarcocyst wall becomes of little significance if morphologically indistinguishable sarcocysts occur in more than one taxonomically closely related intermediate hosts. Furthermore, the structure of the sarcocyst wall is a non-informative feature in case more than one Sarcocystis species having the same type of sarcocysts wall is detected in a single animal [18,[23][24][25][26].

Similar Morphology of Saroccystis spp. in Gulls
In the present study, three Sarcocystis species, S. columbae, S. halieti and S. wobeseri, were identified in common gulls and black-headed gulls from Lithuania. The abovementioned Sarcocystis species detected had thread-like sarcocysts with smooth cyst walls, banana-shaped bradyzoites and were indistinguishable from one another under LM ( Figure  1). Furthermore, by LM, two inseparable Sarcocystis species were found in the two gulls examined ( Table 1). Sarcocysts of Sarcocystis spp. detected in the common gull and in the black-headed gull in the present study were morphologically indistinguishable from those of parasite species identified in the herring gull from Lithuania [18]. Furthermore, sarcocysts observed in gulls from Lithuania were morphologically quite similar to those recorded in Canada in the muscle of the California gull (Larus californicus). Sarcocysts isolated from the California gull were characterised by a thin (0.8 µm) and smooth cyst wall [27]. Sarcocysts described as having smooth and thin cyst walls were also found in Kazakhstan in the muscles of the black-headed gull and the common gull [28]. Thus, it is likely that the Sarcocystis spp. identified in gulls from Lithuania may be present in Asia and America; however, molecular examinations are needed to confirm such an assumption. In conclusion, LM analysis of sarcocysts isolated from gulls has little diagnostic value. On the contrary, four Sarcocystis species forming microcysts in the muscles of birds region. The X axis shows genetic differences expressed in percentages, which was calculated 100% minus genetic similarity values obtained by BLAST analysis. Notably, S. falcatula was not included into the analysis, since it is assumed that this parasite is speciescomplex [21,22]. Based on sequence comparison, Sarcocystis sp. isolate MP9 and Sarcocystis sp. isolate 471 should belong to the same species.  [1]. Since studies on the ultrastructure of sarcocyst walls are expensive and time consuming, usually the morphology of the cyst wall is evaluated in fresh preparations or histological sections under a LM. The morphology of the sarcocyst wall becomes of little significance if morphologically indistinguishable sarcocysts occur in more than one taxonomically closely related intermediate hosts. Furthermore, the structure of the sarcocyst wall is a non-informative feature in case more than one Sarcocystis species having the same type of sarcocysts wall is detected in a single animal [18,[23][24][25][26].
In the present study, three Sarcocystis species, S. columbae, S. halieti and S. wobeseri, were identified in common gulls and black-headed gulls from Lithuania. The abovementioned Sarcocystis species detected had thread-like sarcocysts with smooth cyst walls, bananashaped bradyzoites and were indistinguishable from one another under LM (Figure 1). Furthermore, by LM, two inseparable Sarcocystis species were found in the two gulls examined ( Table 1). Sarcocysts of Sarcocystis spp. detected in the common gull and in the black-headed gull in the present study were morphologically indistinguishable from those of parasite species identified in the herring gull from Lithuania [18]. Furthermore, sarcocysts observed in gulls from Lithuania were morphologically quite similar to those recorded in Canada in the muscle of the California gull (Larus californicus). Sarcocysts isolated from the California gull were characterised by a thin (0.8 µm) and smooth cyst wall [27]. Sarcocysts described as having smooth and thin cyst walls were also found in Kazakhstan in the muscles of the black-headed gull and the common gull [28]. Thus, it is likely that the Sarcocystis spp. identified in gulls from Lithuania may be present in Asia and America; however, molecular examinations are needed to confirm such an assumption. In conclusion, LM analysis of sarcocysts isolated from gulls has little diagnostic value.
On the contrary, four Sarcocystis species forming microcysts in the muscles of birds of the order Anseriformes (S. anasi, S. albifronsi, S. platyrhynchosi and S. wobeseri) can be discerned on the basis of the sarcocyst wall appearance and morphometric parameters of bradyzoites [29][30][31].
Under TEM, the sarcocyst walls of the three Sarcocystis identified in this study are attributed to the same type, but different subtypes (1a S. columbae) [32,33], (1d S. wobeseri) [29] and (1e S. halieti) [34]. However, morphological differences between these three species are minor, somewhat ambiguous and require extensive scientific research. Further studies on the morphological differences in the apical complex or other structures of bradyzoites could help to distinguish Sarcocystis species, which are very similar by LM.

The Richness and Possible Pathogenicity of Sarcocystis Species in Gulls
In the current work, S. halieti was the most commonly identified species in the leg muscles of common gulls and black-headed gulls ( Table 1). This species was also distinguished by the highest intraspecific variation among Sarcocystis spp. employing birds as intermediate hosts (Figure 2). The high intraspecific variability of S. halieti could be explained by a wide range of intermediate hosts [18,22,25,[34][35][36][37][38][39][40] and the broad geographical distribution of this Sarcocystis species [22]. As reported by Maier-Sam et al. [36], S. halieti might be pathogenic for birds, as it has been associated with encephalitis in a small owl (Athene noctua).
There is a gap in the knowledge regarding the pathogenicity of Sarcocystis spp. in birds in Europe. Previously, Sarcocystis species detected in the muscles of birds in Europe were thought to be non-pathogenic. The first pathogenic Sarcocystis species, S. calchasi, was described in Germany in 2010 [8]. Currently, it is known that S. calchasi may parasitise birds belonging to at least five different orders, Columbiformes, Galliformes, Piciformes, Psittaciformes and Suliformes [1,8,9,38,[41][42][43][44][45][46][47]. Meanwhile, the pathogenicity of S. halieti in birds was only revealed in 2021 with the detection of granulomatous cerebral encephalitis [36]. Notably, mainly the leg muscles of birds were examined in the studies during which S. halieti have been identified [18,25,34,37]. Extending research on the pathogenicity of Sarcocystis spp. requires the analysis of internal organs such as the brain, liver, lungs and spleen.
Presently, 28 Sarcocystis spp. have been identified using birds as intermediate hosts [5,31,48]. Gulls (Charadriiformes: Laridae) and corvids (Passeriformes: Corvidae) serve as intermediate hosts for four Sarcocystis spp. [18,25]. The great Sarcocystis spp. diversity determined in gulls can be related to the different habitats and ecosystems of the common gull and black-headed gull [11][12][13][14][15][16][17]. The ability of gulls to adapt to the human impacts associated with cities leads to the establishment and growth of urban bird populations worldwide. Urban ecosystems can provide abundant food resources for gull species, as well as a stable urban microclimate and fewer natural predators than natural environments [14,15,49]. Naturally, both the common gull and black-headed gull breed colonially near water or in marshes, or on islands in lakes, nesting on the ground [12,13]. Conjointly, gulls are omnivorous scavengers of small prey and they are capable of roosting in urban landfills and other waste sites [12,16,17].

Collection of Samples
A total of 105 birds (42 common gulls and 63 black-headed gulls) from Lithuania (54-55 • N, 21-24 • W) were sampled in the period of 2011-2019. All birds were found dead (killed by motor vehicles on highways, as a result of collisions with power lines, etc.) and received from Kaunas Tadas Ivanauskas Zoology Museum (the national authority responsible for the monitoring of wild birds found dead). The leg muscle samples were kept frozen (−20 • C) until a microscopical examination was conducted.

Morphological Examination of Sarcocysts
The leg muscles of birds were examined for the presence of Sarcocystis spp. sarcocysts. The prevalence and intensity of Sarcocystis infections were evaluated in methylene-bluestained preparations. For this purpose, 1 g (±0.1) of the leg muscle was cut into 28 oat-sized fragments, stained with 0.2% methylene blue solution, clarified with 1.5% acetic acid solution, pressed into a glass compressor and examined under a light microscope (LM). Eventually, sarcocysts were morphologically characterised in squashed preparations after the cysts had been isolated from the muscle fibres with the help of preparation needles. Overall, 36 sarcocysts were excreted from the leg muscle tissues from 4 individual common gulls and 5 black-headed gulls (isolates LcLt7.

Molecular Analysis of Sarcocystis spp.
Genomic DNA was extracted from individual sarcocysts using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) according to the manufacturer's instructions. The complete ITS1 region was amplified using the SU1F/5.8SR2 primer pair [50]. Each PCR mixture consisted of 25 µL containing 12.5 µL of Dream Taq PCR Master Mix (Thermo Fisher Scientific, Vilnius, Lithuania), 0.5 µM of each primer, 4-µL template DNA and nuclease-free water. The cycling conditions began with one cycle at 95 • C for 5 min followed by 35 cycles of 94 • C for 45 s, 60 • C for 60 s and 72 • C for 80 s, and a final extension step at 72 • C for 7 min. The visualisation, purification, and sequencing of PCR products were carried out using the previously described protocol [51]. In order to detect essentially similar DNA sequences and evaluate the intraspecific and interspecific genetic variability of avian Sarcocystis spp., the ITS1 sequences generated in this study were compared with those of various Sarcocystis spp. using the nucleotide BLAST program with the megablast option (http://blast.ncbi.nlm.nih.gov/, accessed on 10 March 2023). The 830-844 bp-long ITS1 sequences for Sarcocystis spp. from gulls generated in the present study are deposited in the GenBank database under the following accession numbers OP419605-OP419641.

Conclusions
Based on PCR targeting ITS1 and subsequent sequencing, for the first time, three Sarcocystis spp., S. columbae, S. halieti and S. wobeseri were identified in the leg muscles of two larid species, the common gull and the black-headed gull. The relatively high richness of Sarcocystis species in gulls is related to the abundance of habitats and ecosystems occupied by these birds. Notably, the Sarcocystis species detected in the current work were indistinguishable under LM. Therefore, for the morphological discrimination of Sarcocystis spp. found in the muscles of the bird family Laridae, detailed TEM analysis, including examination of the sarcocyst wall, bradyzoites and apical complex, are required. Furthermore, for a comprehensive genetic characterisation of the identified Sarcocystis species in gulls, more genetic markers should be developed.

Data Availability Statement:
The ITS1 sequences of the three Sarcocystis species (S. columbae, S. halieti and S. wobeseri) generated in the current work are available in the GenBank database under accession numbers OP419605-OP419641.