Sarcocystis blastoceris Berra, Gast, Mor, e, Helman, Argibay & Orozco, 2023, n. sp.
Creators: name: Berra, Yanina
name: Gast
name: Mor, on
name: e
name: Helman, Elisa
name: Argibay, Hernan D.
name: Orozco, M. Marcela
Publisher: name: Zenodo
Year: 2023
Subjects: Biodiversity
Taxonomy
Protozoa
Microsporidia
Microsporea
Meiodihaplophasida
Amblyosporidae
Sarcocystis
Sarcocystis blastoceris

description: Sarcocystis blastoceris n. sp.
Named after the record in marsh deer muscles (Blastocerus dichotomus).
Sarcocysts morphology: thin-walled (≤1 μm) without visible protrusions at optical microscopy. Measured up to 650 μm long and 70 μm wide. Cyst wall ultrastructure by TEM formed by a layer of ground substance and the outer unit membrane which form small invaginations into the ground substance giving an undulating appearance, from which arise bent ribbon-like protrusions folded over the cyst surface. Smooth ground substance layer measured 0.4 μm thick.
Intermediate hosts: Marsh deer (Blastocerus dichotomus).
Distribution: Argentina and probably South America following distribution of intermediate host.
Definitive host: based on the phylogenetic results and predator-prey geographical distribution, highly probable to be wild native canids (i.e. Chrysocyon brachyurus, Lycalopex gymnocercus, Cerdocyon thous) and domestic dogs.
Molecular characteristics: sequences registered in GenBank from 18S rRNA gene fragment (Accession numbers ON911503-ON911505) and cytochrome oxidase subunit I gene (COI) fragment (Accession numbers ON932790-ON932794).
4. Discussion
The current study presents a morphological and molecular characterization of an undescribed Sarcocystis sp. found in muscles from marsh deer from Argentina. A moderate frequency was observed by optical microscopy, observing sarcocysts in muscles from 6/14 animals. Some of the samples were in advanced autolysis. Therefore, the true prevalence of Sarcocystis spp. in marsh deer may be even higher than reported here due to cyst disruption prior to or during homogenization (More´et al., 2011, 2013). Sarcocystis sp. infected animals were observed in both sampled regions, being 5/ 10 in Ibera´and 1/ 4 in Delta. In Brazil, a frequency of Sarcocystis spp. infection of 3/31 was observed in musculoskeletal system of marsh deer, also by optical microscopy (Navas-Suarez´et al., 2018). This lower proportion observed in Brazil could be due to a lower interspecific contact or deer predation. However, to confirm such a hypothesis, more representative samplings are required.
All the sarcocysts observed were microscopic and thin-walled. At optical microscopy, no evident protrusions were observed, similar to features from other Sarcocystis spp. detected in cervids in Argentina (Chang Reissig et al., 2016, 2020). The cyst wall ultrastructure from four cysts obtained from two marsh deer from Ibera´Wetlands showing ribbon-like protrusions resembled other species in cervids muscles like S. cervicanis S. grueneri, S. linearis, S. morae, S. taeniata and S. wapiti (Gjerde, 2014a; Dubey et al., 2016; Gjerde et al., 2017a, b; Cerqueira-C´ezar et al., 2018; Delgado de Las Cuevas et al., 2019; Rudaityte-Luko ˙ˇsiene˙et al., 2021). A similar image was obtained previously from cysts in pudu muscles from Argentina (Chang Reissig et al., 2020). Altogether, the morphological information suggested that marsh deer are frequently infected with a single species, similar to other Sarcocystis spp. affecting cervids worldwide and the Sarcocystis sp. identified in pudu muscles in Argentina.
The molecular results reinforce the hypothesis of a single species present in marsh deer from Argentina. However, sequence identities (especially the ones from the COI gene) were below 90% with other Sarcocystis spp. reported sequences. Other cervid species showed a greater diversity of Sarcocysti s spp. in muscles (Gjerde, 2014a; Dubey et al., 2016; Gjerde et al., 2017a, b; Rudaityte-Luko ˙ˇsiene˙et al., 2020a; Rudaityte-Luko ˙ˇsiene˙et al., 2020b; Rudaityte-Luko ˙ˇsiene˙et al., 2021), possibly a restricted predator-prey relationship is occurring with the marsh deer in South America, resulting in a reduced species variety. On the other hand, from 12 individual cysts or cyst portions, only eight resulted positive by 18S rRNA PCR, and from only three samples, a sequence shorter than expected was obtained. Some of the samples were in an advanced autolysis process, which may have resulted in DNA degradation. Regarding the ITS1 fragment, a low level of amplification was also observed, and no consensus sequence was achieved. Similar difficulties in amplifying and sequencing the 18S rRNA and ITS1 fragments from S. wapiti from North American C. elaphus have been mentioned by Cerqueira-C´ezar et al. (2018). Probably, the priming regions from S. wapiti and the species detected in marsh deer are not conserved, as previously assumed from most Sarcocystis spp. (Dahlgren et al., 2008; More´et al., 2013). Additionally, an intraspecific sequence variation (both 18S rRNA and ITS1) could also result in unintelligible sequences when the PCR products are sequenced directly. Despite the morphological similarity, the 18S rRNA gene sequences obtained from Sarcocystis sp. in marsh deer showed a low homology (93.7–96.7%) with sequences from S. cervicanis in red deer in Spain (Gjerde et al., 2017b) S. grueneri from reindeer in Norway (Dahlgren and Gjerde, 2007), S. linearis in roe deer from Spain and Lithuania (Rudaityt´e- Lukoˇsiene ˙ et al., 2020b), S. morae in fallow deer in Lithuania (Rudaityt´e- Lukoˇsiene ˙ et al., 2020a); Sarcocystis sp. from pudu from Argentina (Chang Reissig et al., 2020), S. taeniata in sika deer in Lithuania (Prakas et al., 2016), S. tarandivulpes from reindeer from Iceland (Dahlgren et al., 2007) and S. wapiti from elk (Cerqueira-C´ezar et al., 2018). The differences at this target suggested that the Sarcocystis sp. in marsh deer could be a different species. In addition, the sequences from the present study were phylogenetically positioned in the same branch with only a S. grueneri sequence. Several other sequences from S. cervicanis, S. cruzi, S. hjorti, S. iberica, S. linearis, S. morae, S. rangi, Sarcocystis sp. from huemul and pudu in Argentina, S. taeniata S. tenella, S. venatoria and S. wapiti were distant related, however, according to the positioning it is possible that all these species shared a common ancestor. Since most of these species use canids as definitive hosts, the Sarcocystis sp. in marsh deer may use canids too.
Aiming further characterization, different PCR (using two reverse primers) to obtain COI gene fragments were performed as previously reported for species using cervids as intermediate hosts (Gjerde, 2013; Gjerde et al., 2017a). Both combinations resulted in proper amplicons for sequencing, and good quality sequences were obtained from all three cysts previously analyzed by 18S rRNA. These results reinforce the idea of priming failure or intraspecific sequence variation of 18S rRNA and ITS1 fragments. All the COI sequences obtained in the present study showed a high homology among them and a low homology with others reported in the GenBank, being up to 89.7% with several S. grueneri sequences, with the highest score with the sequence KC209624 from Rangifer tarandus from Norway (Gjerde, 2013).
As far as we know, there are no reports of COI sequences from S. wapiti (Cerqueira-C´ezar et al., 2018), so proper comparisons are not possible. Despite morphological similarities, the high sequence divergence at 18S rRNA and COI allowed the assumption that Sarcocystis sp. from marsh deer is a different species from S. wapiti and other Sarcocystis spp. producing cysts in cervids muscles. Thus, we propose the name Sarcocystis blastoceris n. sp. for the species producing sarcocysts in marsh deer. In the phylogenetic tree, all S. blastoceris COI sequences are positioned along with sequences from S. grueneri from Norway (Gjerde, 2013) and with a sister clade containing sequences from S. cervicanis, S. linearis, S. morae, Sarcocystis sp. from pudu in Argentina and S. taeniata. Several of these species are known to use canids as definitive hosts and suggest a potential common ancestor for these species (Dubey et al., 2016; Tuska-Szalay et al., 2021). Additionally, these native cervids are part of an alimentary chain being consumed (as prey or carrion) by domestic dogs (Canis familiaris) and different native canid species like maned wolf (Chrysocyon brachyurus), pampas fox (Lycalopex gymnocercus) and crab-eating fox (Cerdocyon thous) (Canevari et al., 2007).
In summary, the sarcocyst morphology, the positioning on the phylogenetic trees, and that marsh deer are frequently predated by dogs and foxes, allowed the assumption that S. blastoceris could have canids as definitive hosts.
Further studies should be conducted to properly identify naturally infected definitive hosts for S. blastoceris and its potential role on marsh deer health status.
description: Published as part of Berra, Yanina, Gast, Mor, on, e, Helman, Elisa, Argibay, Hernan D. & Orozco, M. Marcela, 2023, Identification of a new Sarcocystis sp. in marsh deer (Blastocerus dichotomus) from wetlands of Argentina, pp. 39-45 in International Journal for Parasitology: Parasites and Wildlife 20 on pages 42-44, DOI: 10.1016/j.ijppaw.2022.12.007, http://zenodo.org/record/13287653