Sarcocystis muricoelognathis, Qin & Ortega-Perez & Wibbelt & Lakim & Ginting & Khoprasert & Wells & Hu & Jäkel, 2024

Qin, Tao, Ortega-Perez, Paula, Wibbelt, Gudrun, Lakim, Maklarin B., Ginting, Sulaiman, Khoprasert, Yuvaluk, Wells, Konstans, Hu, Junjie & Jäkel, Thomas, 2024, A cyst-forming coccidian with large geographical range infecting forest and commensal rodents: Sarcocystis muricoelognathis sp. nov., Parasites & Vectors (135) 17 (1), pp. 1-19 : 13-14

Taxonomic summary of Sarcocystis muricoelognathis sp. nov.

( Sarcocystis sp. : Figs. 1 , 2 , 3 , 4 , 5 , 6 , Additional file 1: Table S1 ).

Diagnosis

Oocyst containing two sporocysts with four sporozoites each; sporocysts measuring on average 10.1–10.3 × 7.2– 7.5 µm; sarcocysts microscopic with striated cyst wall in fresh preparations (similar to S. singaporensis but cyst wall protrusions not resting on stalks), cigar-shaped with slightly pointed ends in older cysts; in skeletal musculature except heart; native sarcocysts in rats infected experimentally for 5 months measured 400–700 × 70–90 µm, in wild-caught rats 770–1580 × 87–123 μm; cyst wall with palisade-like, villar protrusions, 8 to 11 µm long and 2–4 µm wide at their base; protrusions at the tips of the sarcocyst often longer, 10–16 µm; banana-shaped, live cystozoites on average 5.9–6.5 µm long and 2.1–2.2 µm in diameter; ultrastructurally, villar protrusions palisade-like, attached upright to the cyst (if not bent by preparation), sometimes slightly broadened towards the apical ends, delimited by a knob-like lining and filled with fine, electron-light granular material; cystozoites with only two rhoptries throughout distribution range.

Type natural definitive hosts

Coelognathus radiatus (holotype), Coelognathus flavolineatus (paratype).

Type experimental intermediate host

Rattus norvegicus (holotype)

Type natural intermediate hosts

Maxomys whiteheadi (paratype); Rattus norvegicus (paratype)

Type localities

Holotype: central Bangkok, Tailand ; paratypes: Chumphon, southern Tailand ; Poring Hot Springs, Northern Borneo, Sabah, Malaysia; Anning and Nanjian Prefectures, China.

Other locations and natural hosts not included in type series Sarcocysts with the same ultrastructure recorded from Rattus argentiventer and Bandicota indica in Tailand [ 2] and reported from Rattus rattus in Singapore [ 15].

Etymology

Species name in reference to the intermediate and definitive hosts.

Type specimens deposited

Holotype deposited at the Department of Parasitology, University of Hohenheim , Emil-Wolff-Str. 34, 70599 Stuttgart-Hohenheim, Germany, accession number DP/ R41C961764: Resin-embedded sarcocyst from Rattus norvegicus infected with sporocysts from Coelognathus radiatus ; paratype from Borneo deposited at the Sabah Parks Museum, Kinabalu Park, Sabah, Malaysia, accession number SP/EAC/00102: sarcocyst in musculature from Maxomys whiteheadi ; paratypes from China deposited at the Zoological Specimen Museum of Yunnan University, Kunming, accession number Prot202302: formalin-fixed tissue containing sarcocysts as well as photomicrographs .

Sequences deposited at GenBank

18S rRNA: OR976520, OR976521; 28S rRNA: isolates from China OR979639, OR979640, OR979641, OR979642, and Borneo OR979643; ITS1 - 5.8S- ITS2: OR977565, OR977566, OR977567, OR977568; cox1: PP033596.

ZooBank Registration

Tis article has been submitted to ZooBank under the Life Science Identifier (LSID) urn:lsid:zoobank. o r g: p u b: E 8 6 A 1 F E F - 6 1 8 9 - 4 E 2 8 - A 6 2 7 - 1 1 A B - FAA635AC. Te LSID for the new species name Sarcocystis muricoelognathis is urn:lsid:zoobank.org:act:000AF647-D4AC-4609-914D-61C1449A2DAA .

Discussion

Taxonomic remarks

Since the ultrastructure of the sarcocyst wall is in many cases a useful criterion for distinguishing between species of Sarcocystis [ 40], we show here that S. muricoelognathis is clearly distinct from two similar species, S. zuoi and S. murinotechis . In the case of S. zuoi , its villar protrusions of the cyst wall are bent by a certain angle, which is also visible in live sarcocysts when the surrounding host tissue has been removed, and exhibit a reticulate base [ 11]. In contrast, S. muricoelognathis shows upright protrusions with a largely non-reticulate base. Compared to S. murinotechis from Australia [ 7], the two former species have markedly longer protrusions (roughly twice as long). Although only two electron micrographs of the sarcocysts of S. murinotechis exist in the original species description, the accompanying text characterizes them as possessing “short, broad protrusions” that were about 6 µm in length [ 7]. Relatively short protrusions are also visible in a histological section of a sarcocyst of S. cf. murinotechis from Rattus lutreolus [ 18] supporting the observations on S. murinotechis published by these authors 2 years later. All in all, we propose that the sarcocyst ultrastructure of S. muricoelognathis could be categorized under group 10a of the common cyst wall classification system for Sarcocystis [ 40]. Additionally, the sizes of the transmission stages, sporocysts and cystozoites, were significantly different among the three species discussed above. Notably, sporocysts of S. murinotechis exhibited a shape index different from S. muricoelognathis and S. zuoi : Size differences of sporocysts can be regarded a suitable indicator for the potential presence of different Sarcocystis species [ 41]. Regarding intraspecific variability, cystozoites of S. muricoelognathis were remarkably constant in size independent of geographic location and intermediate host and consistently showed two rhoptries at the ultrastructural level. Tese characteristics underscore the uniqueness of S. muricoelognathis compared to other species with striated cyst walls, which we have discussed previously [ 20]. At the light microscopic level, sarcocysts of S. muricoelognathis may be easily confused with S. singaporensis , because both species are microscopic, show a striated cyst wall with relatively long protrusions, exhibit pointed sarcocyst ends and share similar geographic distributions and rodent hosts. However, the cyst wall protrusions of the latter species rest on typical stalks, which appear as a thin, basal layer of the striated wall at lower magnification in live and histological preparations [ 2]. Molecularly, S. muricoelognathis can be unequivocally identified using its 28S rRNA and ITS1 sequences, whereby the partial 28S rRNA gene (≥ 600 bp) is already sufficient for differential diagnosis if the variable expansion segments D1 and D2 are included (as discussed below).

In a taxonomic context, we think we provided substantial molecular evidence for the proposition that the rRNA sequences from Tailand deposited under accession number KU341120 [ 19] do not belong to S. zuoi but represent S. muricoelognathis . Although a high degree of sequence identity between partial sequences alone may be insufficient for species discrimination, we must add here that the 28S rRNA gene sequence of S. muricoelognathis from Borneo included the variable expansion segments D1 and D2 of the rRNA molecule according to the predicted secondary structure of the reference sequence of T. gondii (L25635, [ 27]). Especially, domain D2 has been shown to be highly variable [ 42]. Moreover, the overlapping parts of the alignments of partial sequence KU341120 with the (almost) full-length 28S rRNA sequences of S. muricoelognathis from China included expansion segments D1–D6, which, in view of an identity score of 99%, strongly suggested that these sequences belong to the same species. Furthermore, both taxa clustered together in the ITS1 phylogenetic tree, which is increasingly used for discriminating between closely related Sarcocystis spp. (e.g. [ 43]) and shows much greater evolutionary rates than other rRNA markers [ 28]. Given that the sequences under KU341120 have been included in major rRNA databases under the species name ‘ Sarcocystis zuoi ’ and are frequently cited in the context of Sarcocystis of rodents (e.g. [ 44, 45]), it will probably take some time until any annotation in line with our proposal could be expected. Moreover, based on the observations here and a previous study [ 6], it can be safely assumed that the sequences KU341118/19/21 also do not represent S. zuoi but belong to a different species, because they clustered in a clade apart from the original S. zuoi sequences.

Figures

Fig. 1 a–f Light microscopic and ultrastructural morphology of sarcocysts of Sarcocystis sp. in SD rats 5 months after inoculation with sporocysts isolated from rat snakes in Thailand.a Typical sporocyst from a fecal sample of Coelognathus radiatus; sporocysts from C.flavolineatus were identical in size and appearance; asterisk indicates single sporozoite. b Live sarcocyst, freed from muscle tissue; note the broad, palisade-like villar protrusions that could at times resemble those of Sarcocystis singaporensis with which this species can co-occur; however, the protrusions lack the basal stalks typical for the former species; the arrow highlights the septated compartments in the interior of the sarcocyst, and the inset shows a micrograph of live cystozoites freshly released from a cyst (arrowheads). c Typical structure of a cyst wall protrusion (isolate from C.flavolineatus); the arrows point to the electron-dense, knob-like structures of the primary cyst wall, whereby the knobs could apparently fuse to form an electron-dense borderline in larger protrusions (inset: arrowhead); also note the electron-light, thin layer of ground substance (GS) underneath the protrusions.d Typical cystozoites of the new species, which contained only two rhoptries (arrowheads) among relatively few micronemes (asterisks); additionally, the cystozoites exhibited vesicle-like structures in the anterior third of the cell containing electron-light,reticulate matter (arrow); the inset shows such a vesicle-like compartment at higher magnification, which was apparently not bound by a membrane (white arrow) and often located near micronemes (white asterisk); dense granules were present but rarely observed. e Interior and cyst wall of a mature sarcocyst (isolate from C. radiatus); metrocytes (asterisks) exclusively divided by endodyogeny, producing only two cystozoites (CZ). f Full-length section through a 15-µm-long protrusion of the sarcocyst wall; note that larger protrusions often occurred close to the tips of a cyst and showed a base with folds. AP,apicoplast; MI, mitochondrion; NU, nucleus; PT,villar protrusions

Fig. 2 Light microscopic and ultrastructural morphology of sarcocysts from Maxomys whiteheadi in Borneo (a–c) and wild Rattus norvegicus in China (d, e). Note, due to ethanol fixation some ultrastructural details of the samples from Borneo are poorly resolved. a Richardsen’s dye-stained 1.0-µm thin section through a mature sarcocyst showing the villar protrusions (PT) of the cyst wall and numerous relatively small cystozoites (CZ). b Same sample as before under the electron microscope; note the thin layer of ground substance underneath the protrusions. c Enlarged part of the interior of the sarcocyst showing cystozoites—although with limited resolution—that possess a pair of rhoptries each, which is characteristic for this Sarcocystis species (black and white arrows; compare with Fig. 1d). d Live sarcocyst isolated from striated muscle tissue of a wild Norway rat in China; the inset shows live cystozoites that were freshly released from a cyst.e Ultrastructure of the same sarcocyst as before; note that the villar protrusions are highly similar to the samples from Borneo and Thailand regarding size and shape (Fig. 1e); again, cystozoites only exhibit one pair of rhoptries (arrows) and relatively few micronemes

Fig. 3 Graph showing the size of sporocysts (length plotted against diameter, in μm; error bars indicate s.e.) of the Sarcocystis isolates from the colubrid snakes Coelognathus flavolineatus and C. radiatus in Thailand and closely related Sarcocystis. Every isolate/ species is indicated by a different symbol (legend), whereby sporocyst samples with the same shape index (=length/diameter) share the same background shading: white= 1.3; dark= 1.5; Sarcocystis pantherophisi =1.2. Here, S.pantherophisi is included as reference for the snake host Sarcocystis lineage S2,while all other species belong to lineage S1 (except for S. murinotechis, for which no genetic information is available)

Fig. 4 Two separate phylogenies of the 28S rRNA gene (longer and shorter sequence fragments) of the new Sarcocystis sp. sampled in China, newly sequenced S. zuoi from China and novel Sarcocystis isolates from Borneo. Symbols indicate the new sequences of this study, whereby taxa considered conspecific are grouped by shape. GenBank accession numbers are given behind each taxon name. a Maximum likelihood (ML) analysis of an alignment of 29 sequences and 1383 homologous positions. Branch support by bootstrapping (1000 replicate trees) is shown next to the branches, whereby the results of three independent analyses based on independent alignments are shown.The scale bar indicates the number of substitutions per site. All positions with <85% site coverage were eliminated, i.e. fewer than 15% alignment gaps, missing data and ambiguous bases were allowed at any position (partial deletion option). Selected eimeriid coccidia served as outgroup. b ML analysis of a trimmed alignment including five shorter sequences of Sarcocystis sampled in Borneo compared with the samples of Sarcocystis sp. from China; a total of 16 sequences and 366 homologous positions with site coverage of 95% were compared. Sarcocystis pantherophisi served as outgroup.The corresponding natural intermediate hosts are also indicated

Fig. 5 Phylogenetic reconstruction of the 18S rRNA gene tree of the novel Sarcocystis species and other tissue cyst-forming coccidia based on 1465 homologous positions of 40 aligned nucleotide sequences under the minimum evolution (ME) criterion; selected eimeriid coccidia served as outgroup.The new sequences of Sarcocystis sp. from China are highlighted by black symbols. Branch support values are shown for 1000 bootstrap replicates of three independent alignments with a site coverage of 95%.The shaded box highlights the taxa included in the so-called S. zuoi complex

Fig. 6 ML analysis of the ITS1 region of Sarcocystis sp., S.zuoi and other species of the Sarcocystidae; members of the Toxoplasmatinae served as outgroup. Bootstrap branch support values are shown in triplicate, indicating results from three independent alignments and analyses.The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 204 homologous positions of 33 nucleotide sequences