Sarcocystis scandentiborneensis, P & erez & Wibbelt & Brinkmann & Galindo Puentes & Tuh & Lakim & Nitsche & Wells & J & akel, 2020

P, Paula Ortega, erez, Wibbelt, Gudrun, Brinkmann, Annika, Galindo Puentes, John A., Tuh, Fred Y. Y., Lakim, Maklarin B., Nitsche, Andreas, Wells, Konstans, J, Thomas & akel, 2020, Description of Sarcocystis scandentiborneensis sp. nov. from treeshrews (Tupaia minor, T. tana) in northern Borneo with annotations on the utility of COI and 18 S rDNA sequences for species delineation, International Journal for Parasitology: Parasites and Wildlife 12, pp. 220-231 : 228

Sarcocystis scandentiborneensis sp. nov. ( Figs. 1–5 , Table 1).

Diagnosis: Microscopic sarcocysts (about 500 × 50 μm and smaller in histological sections) in skeletal muscles of treeshrews ( Scandentia ). Cyst wall striated with brush-like appearance, thickness variable (average 4–5 μm, maximal 10 μm), including a very thin layer of GS (about 0.3 μm) and finger-like VP, that are tightly packed, their tip revealing an electron-dense, U-shaped apex at ultrastructural level. VP contain longitudinally oriented microtubules that extend to the GS and show thorn-like structures on the surface. Loosely packed cystozoites in chambers delineated by thin septae have a maximum length of 6.7 μm in H&E-stained sections.

Taxonomic summary

Type (natural) intermediate host: Tupaia minor (lesser treeshrew) and T. tana (large treeshrew).

Definitive host: Unknown.

Type locality: Poring Hot Springs , Northern Borneo, Sabah, Malaysia .

Other locations: Unknown.

Etymology: Species named after the order of the intermediate host ( Scandentia ) and the geographic location of collection.

Specimens deposited: Striated muscle specimens from eleven Tupaia sp. individuals are deposited at the Sabah Parks Museum, Kinabalu Park, Borneo, Malaysia and the Museum für Naturkunde – Leibniz Institute for Evolution and Biodiversity Science (MfN), Berlin, Germany, including: tissue sections stained with H&E and Richardson’ s staining and ethanolfixed, paraffin embedded as well as resin embedded muscle tissue, and extracted DNA stored at – 80 Ǫ C. Sarcocysts in tissue samples from Tupaia minor E357-13 are considered the holotypus of Sarcocystis scandentiborneensis sp. nov. with the MfN registration number ZMB-Protoz 66. The other deposited samples serve as paratypes with the MfN registration number ZMB-Protoz 67.

Sequences deposited: Nuclear 18S rDNA: full-length sequences E357- 13 (MN733816) and E364-13 (MN733817); mitochondrial COI: partial sequences E120-13 (MN732562) and E357-13 (MN732561).

ZooBank registration number (http://zoobank.org/References/): C0DCC101-235B-41E0-97 EB-45ABB74517F6.

3.5. Taxonomic remarks

The ultrastructure of the sarcocyst wall is in many cases a useful criterion for distinguishing between species of Sarcocystis ( Dubey et al., 2016) . The sarcocyst wall of the species under investigation is ultrastructurally as well as histologically distinct from the only other Sarcocystis sp. known to occur in treeshrews: S. tupaia , found in Tupaia belangeri chinensis from Yunnan, China (Xiang et al., 2010). Although our TEM results were not optimal due to ethanol-fixation, the ultrastructural details nevertheless permit comparison and categorization of wall morphology ( Dubey et al., 2016). While S. tupaia possesses a smooth type 1 wall (Xiang et al., 2010), the ultrastructure of the present species clearly reveals a thick striated wall with VP that share structural elements with the tightly packed protrusions of wall type 12. Because our phylogenetic results indicate a sister taxon relationship with three previously described Sarcocystis species that undergo a snake-rodent life cycle ( Slapeta et al., 2003; Hu et al., 2012, 2014, 2015; Wassermann et al., 2017), morphological comparison with those species is implicit: S. singaporensis , S. clethrionomyelaphis , or S. zuoi all possess spatula or finger-like VP, but ultrastructurally they do not match with the new species. There is also no match with another nine, relatively thick-walled snakehost Sarcocystis spp. as accounted for in Dubey et al. (2016) and discussed by Verma et al. (2017): S. acanthocolubri , S. gongyli , S. lacertae , S. podarcicolubris , S. stenodactylicolubris , S. hoarensis , S. murinotechis , S. muriviperae , and S. villivillosi ( S. zamani cannot be included here, since it exhibits a quite thin cyst wall; Beaver and Maleckar, 1981). Furthermore, unlike S. zuoi , S. clethrionomyelaphis , and representatives of the S2 lineage of snakehost Sarcocystis ( Wassermann et al., 2017) such as S. pantherophisi ( Verma et al., 2017) and S. eothenomysi ( Hu et al., 2014) , cystozoites of the Sarcocystis sp. infecting Bornean treeshrews are markedly smaller: similar in size to species with a snake-lizard life cycle, where cystozoites are between 4 and 7 μm long ( Matuschka, 1981; Abdel-Ghaffar et al., 1990; Modry et al., 2000; Morsy et al., 2012). Biologically, the full life cycle of the new species remains unknown, since we could not identify a definitive host. However, our phylogenetic results on the 18S rRNA gene suggest a snake as definitive host (see below). Regarding intermediate host specificity, our trapping results provide some insight: none of the murid rodent species captured in the same location was infected with the new Sarcocystis sp. from tree shews, suggesting that the Scandentia are the preferred hosts. Molecularly, the Sarcocystis sp. described here forms a close sister species relationship with Sarcocystis spp. with colubrid snake-rodent life cycle in 18S rDNA phylogenies. Our phylogenetic and molecular results indicate that the species from Borneo is distinct from S. zuoi ; it also appears likely that S. zuoi reported from Thailand may be a different species, if one considers the topology of the BI tree, Ti/Tv ratios, and differences in evolutionary rates between sequences. Preliminary reconstruction of the COI gene tree suggests a sister-species relationship with S. pantherophisi (KU891603), but due to lack of COI sequences from closely related species this part of the tree can currently not be resolved. The question why the partial COI sequence of S. canis is identical to S. pantherophisi , including the potential taxonomic implications, is beyond the scope of this study. Considering all morphological, biological, and molecular aspects of the taxa compared, we regard S. scandentiborneensis a new species.

Figures

Fig. 1. Light microscopy of Sarcocystis scandentiborneensis sp. nov. A and B, Haematoxylin & Eosinstained histological sections of striated musculature; C and D, Richardson’s dye-stained 1.0 μm thin sections of sarcocysts. A) Tissue section of laryngeal muscle with various sarcocysts in cross section (asterisks), indicating a relatively high density of cysts in this part of musculature. B) Longitudinal section through a sarcocyst, showing a cigar-shaped appearance; however, isolated native sarcocysts, which were not available, may look different. C) Part of a longitudinal section through the tip of a sarcocyst, note the very thin ground substance (arrows) and the fine septae extending into the interior of the cyst (arrowheads); cystozoites (CZ) were loosely scattered within chambers while metrocytes were rarely seen, indicating maturity of the cyst; bars indicate the variable thickness of the cyst wall: the wall was thinner in regions where the villous protrusions were bent (right bar); note that the intense staining at the interface between host cell (HC) and parasite is part of the host cell. D) Cross-section through a sarcocyst showing cystozoites and the cyst wall (bar) including its thin ground substance (arrows).

Fig. 2. Ultrastructure of S. scandentiborneensis sp. nov. Note, due to ethanol-fixation some ultrastructural details are poorly resolved (e.g. membranes). A) Longitudinal section through the same sample as in Fig. 1C, showing a gross view of the sarcocyst and its villous protrusions (VP) that are sectioned in different orientations. The inset shows a cross section through various VP that reveals the arrangement of microtubules in their inner core; while in this case 16 microtubules are visible (asterisks), sections through more apical portions of the VP showed lower numbers. B) Longitudinal section through the fingerlike VPs that appear to be anchored in the ground substance (arrow) by microtubules (asterisks) that extend into each protrusion; note the electron-dense, U-shaped structure at each tip of the protrusions (arrowheads) and the apparently serrated surface of the VP (flat arrowheads). The inset shows a higher magnification of the apical part of a single VP with the typical U-shaped apex (asterisk), which appears to be connected with the host cell through an electronlucent contact zone (white arrowheads); interestingly, the protrusion appears fenestrated (also visible in the main image) possessing thorn-like structures (black arrows; the white arrow indicates a crosssectional view) that could be responsible for the serration visible at lower magnification. CZ, cystozoites; HC, host cell; VP, villous protrusion.

Fig. 3. Phylogenetic tree of nuclear 18S rDNA sequences of the Sarcocystidae, including the new Sarcocystis sp. from treeshrews (black symbols). Taxa of the Eimeriidae served as outgroup. Bayesian Inference was used for phylogeny reconstruction, whereby the general time-reversible substitution model (GTR + G + I) combined with an assumed among-site variation (‘covariotide’ model) was applied. Values for posterior probability are indicated behind nodes. Note that the new species is part of a monophyletic subclade previously tagged S1 (Wassermann et al., 2017), which includes taxa known to prefer snakes as definitive and rodents as intermediate hosts. Sequences E357-13 and E364-13 are available at GenBank under MN733816 and MN733817, respectively.

Fig. 4. Mapping (to the Toxoplasma gondii reference molecule M97703) of frequencies (%) of base pair changes observed in sequence comparisons of nu clear 18S rDNA within the new Sarcocystis sp. from treeshrews (intraspecific variation: isolates E364–13 versus E357–13) and between the new species and Sarcocystis zuoi and/or S. clethrionomyelaphis (interspecific variation: E364–13 versus S. zuoi/clethrionomyelaphis). Results were combined for the two latter species to simplify the graph. Here, 87.2% of 2118 alignment positions showed moderate to high levels of consistency, while sections of ambiguous alignment did not relate to the species under investigation. Due to gaps in the alignment, not all of the observed nt changes could be mapped to a homologous position of the reference molecule (i.e., 7 out of 24 bp changes in intraspecific comparison; 33 out of 74 bp changes in interspecific comparison), in which case the position of each nt relative to the helix was inferred from neighboring nt for which such position was known. Gaps were mainly due to insertions in helices V2, V4, and V9 rendering E357-13/E364-13 longer than the sequence of T. gondii. The percentage of parsimony-informative (pi) bp changes per helix is shown for helices V1, V2, V4, V7, and V9 above each column. Also shown is the ratio of transitions versus transversions (Ti/Tv) for selected helices.

Fig. 5. Phylogenetic tree based on analysis of mitochondrial COI sequences of the Sarcocystidae including the new Sarcocystis sp. examined in this study (black symbols). Other taxa of the Apicomplexa served as root. Evolutionary history was inferred by the Maximum Likelihood (ML) method based on the Tamura- Nei model, whereby 619 positions were included in the final data set. All positions with less than 95% site coverage were eliminated; that is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Bootstrap percentages (1000 iterations) are shown next to branches. COI sequences E357-13 and E120-13 (not shown in the tree) are available at GenBank (MN732561 and MN732562, respectively).