LM and TEM of sarcocysts
Spindle-shaped sarcocysts were found in 17 of the 42 (40.5%) Asian gray shrews and were located in skeletal muscle, the esophagus, the diaphragm, the tongue and the heart. Only one form of sarcocyst was observed. The examination of fresh samples under LM revealed that the cyst walls of the sarcocysts had numerous 3.3- to 4.5-μm-long [mean = 4.0 ± 0.26 μm (± SD); n = 25 measurements taken from ten sarcocysts] saw- or tooth-like protrusions (Fig. 1a). Mature sarcocysts were 740–1355 × 117–250 μm [average = 1020 (± 176) × 175 (± 44) μm; n = 20 isolated from four Asian gray shrews] in size; they were septate and contained bradyzoites measuring 8.2–10.4 × 2.0–3.0 μm [average = 9.2 (± 0.7) × 2.5 (± 0.3) μm; n = 40 measurements taken from two sarcocysts] in size.
Ultrastructurally, the sarcocysts had lancet- or leaf-like villous protrusions measuring 2.8–6.4 μm [mean = 4.5 (± 1.1) μm; n = 15], which contained numerous electron-dense granules in their core; microtubules and fibrils were absent. The primary cyst wall had minute undulations over its entire surface, and was lined with an electron-dense layer. The distances between the protrusions varied. A layer of ground substance with a thickness of 1.2–1.6 μm (mean = 1.3 ± 0.1 μm) was located immediately beneath the primary sarcocyst wall. This layer gave rise to thin septa, which divided the cyst interior into a series of compartments. The compartments were filled with numerous bradyzoites and several undifferentiated metrocytes (Fig. 1b, c).
Infection of the definitive host
The two beauty rat snakes fed muscle tissue from an Asian gray shrew containing sarcocysts excreted sporulated oocysts/sporocysts (Fig. 1d, e) in their feces, one beginning on day 10 PI and the other on day 11 PI. Upon the death of the snakes at day 29 PI, numerous oocysts/sporocysts were also observed in the small intestine. Under LM, the sporulated oocysts measured 11.9–16.7 × 9.2–10.6 μm [average = 13.5 (± 1.3) × 9.9 (± 0.4) μm; n = 27], with two elliptical sporocysts measuring 9.2–10.6 × 6.3–6.8 μm [average 9.9 (± 0.4) × 6.6 (± 0.2) μm; (n = 30)]. No oocysts/sporocysts were found in the feces or small intestine of the control snake.
Molecular analysis
Seven genes (18S rDNA, ITS1, cox1, cox3, cytb, rpoB and clpC) were successfully amplified from ten individual sarcocysts and oocysts/sporocysts. A total of 16 clones, comprising 10 from 10 individual sarcocysts and six from oocysts/sporocysts, were sequenced, assembled, and submitted to GenBank. The ten 18S rDNA sequences obtained from the sarcocysts were 1863–1867 base pairs (bp) in length and shared 99.7–100% identity (average 99.8%). The six 18S rDNA sequences obtained from the oocysts/sporocysts were 1865–1868 bp in length and shared 99.7–100% identity (average 99.8%). Therefore, only three sequences from sarcocysts (accession numbers MZ826981–MZ826983) and two sequences from oocysts/sporocysts (MZ826984 and MZ826985) were deposited in GenBank. The sequence identity between the sarcocysts and oocysts/sporocysts was 99.7–100%, with an average of 99.8%. The most similar sequences in GenBank were those of Sarcocystis scandentiborneensis (MN733816 and MN733817) obtained from the tree shrews Tupaia minor and Tupaia tana (97.6–98.3% identity, average 97.9%), followed by those of S. zuoi (JQ029112 and JQ029113) from the Norway rat R. norvegicus (97.1–97.4% identity, average 97.3%), S. cf. zuoi (KU341118–KU341121) from the Malayan field rat R. tiomanicus (96.7–98.0% identity, average 97.2%), Sarcocystis sp. (AB251613) from the raccoon Procyon lotor (95.9–96.9% identity, average 96.3%), and Sarcocystis clethrionomyelaphis (KP057504, KF309700, and KF309701) from the large oriental vole Eothenomys miletus (95.9–96.4% identity, average 96.2%).
The ten ITS1 sequences obtained from sarcocysts were 874–978 bp in length and shared 97.9–100% identity (average 99.0%). The six ITS1 sequences obtained from oocysts/sporocysts were 875–877 bp in length and shared 98.6–100% identity (average 99.0%). Therefore, only 14 sequences, comprising nine from sarcocysts (MZ826986–MZ826994) and five (MZ826995–MZ826999) from oocysts/sporocysts, were deposited in GenBank. The similarity between the sarcocysts and oocysts/sporocysts was 97.4–100%, with an average of 98.7%. The most similar sequences in GenBank were those of S. cf. zuoi (KU341118–KU341121) from the Malayan field rat, but the sequence identity was only 72.4–86.0% (average 76.4%).
The ten cox1 sequences obtained from sarcocysts were 1333 bp in length and shared 99.8–100% identity (average 99.9%). The six cox1 sequences obtained from oocysts/sporocysts were 1333 bp in length and shared 99.9–100% identity (average 99.9%). Therefore, only five cox1 sequences, comprising three from sarcocysts (MZ889669–MZ889671) and two from oocysts/sporocysts (MZ889672 and MZ889673), were deposited in GenBank. The similarity between the sarcocysts and the oocysts/sporocysts was 99.8–100%, with an average of 99.9%. The most similar sequences in GenBank were those of Sarcocystis scandentiborneensis (MN732561 and MN732562, 100% identity), followed by those of Sarcocystis sp. (MT411016, 99.8% identity) from the greater white-toothed shrew Crocidura russula, and Sarcocystis canis (KX721496 and KX721497) from the Indo-Pacific bottlenose dolphin Tursiops aduncus (95.0–95.4% identity, average 95.2%).
The 16 cox3 sequences from the sarcocysts and oocysts/sporocysts were 675 bp in length and identical. Therefore, only one sarcocyst sequence (OK001462) and one oocysts/sporocyst sequence (OK001463) were deposited in GenBank. No sequences with significant similarity to these sequences were found in GenBank.
The 16 cytb sequences from the sarcocysts and the oocysts/sporocysts were 1080 bp in length and identical. Therefore, only one sarcocyst sequence (OK001464) and one oocysts/sporocysts sequence (OK001465) were deposited in GenBank. The most similar sequences in GenBank were those of Sarcocystis falcatula (MF034168–MF034187) from the budgerigar Melopsittacus undulatus, the identity of which was 96.4%.
The 16 rpoB sequences obtained from the sarcocysts and the oocysts/sporocysts were 511 bp in length and shared 100% identity. Therefore, only one sarcocyst sequence (OK001466) and one oocysts/sporocysts sequence (OK001467) were deposited in GenBank. The most similar sequence in GenBank was that of Sarcocystis neurona (GQ851961) obtained from the southern sea otter Enhydra lutris nereis, for which the identity was 91.9%.
The 16 clpC sequences from the sarcocysts and the oocysts/sporocysts were 534 bp in length and shared 100% identity. Therefore, only one sarcocyst sequence (OK001468) and one oocysts/sporocysts sequence (OK001469) were deposited in GenBank. The most similar sequences were those of S. falcatula (KP871717) and S. neurona (KP871716), for which the identity was 92.5%.
Phylogenetic analysis
Phylogenetic analysis based on 18S rDNA or ITS1 sequences confirmed that the parasite found in the present study belonged to Sarcocystis (Figs. 2, 3). In the phylogenetic tree inferred from 18S rDNA sequences (Fig. 2), the parasite formed an individual clade and clustered with Sarcocystis spp. that use colubrids as their definitive or presumed definitive hosts [i.e., Sarcocystis scandentiborneensis (MN733816), Sarcocystis zuoi from R. norvegicus (JQ029113), Sarcocystis cf. zuoi from R. tiomanicus (KU341120), Sarcocystis clethrionomyelaphis (KP057504), Sarcocystis sp. from Procyon lotor (AB251613), Sarcocystis sp. from Morelia viridis (KC201639), Sarcocystis zamani (KU244524) and Sarcocystis singaporensis (AF434054)]. In the phylogenetic tree inferred from the ITS1 sequences, the parasite formed an individual group and clustered with Sarcocystis spp., with snakes as the definitive or presumed definitive hosts [i.e., S. cf. zuoi from R. tiomanicus (KU341120), S. zamani (KU244525), Sarcocystis sp. (MH590230) and S. singaporensis (KU341123)].
Based on the morphological characteristics of the sarcocysts, molecular analysis and host specificity, the organism found in the Asian gray shrews from Anning Prefecture, China is proposed as a new species with the name Sarcocystis attenuati.
Taxonomic summary of Sarcocystis attenuati n. sp.
Diagnosis
The sarcocysts were microscopic, 740–1355 μm long, and 117–250 μm wide. The sarcocysts had 3.3- to 4.5-μm-long saw- or tooth-like protrusions. The sarcocysts were divided by septa into a series of internal compartments filled with bradyzoites measuring 8.2–10.4 × 2.0–3.0 μm. TEM revealed sarcocysts with lancet- or leaf-like protrusions measuring 2.8–7.4 × 0.4–0.9 μm (n = 15), which contained electron-dense granules in their core. The protrusions were separated from each other at variable distances, similar to wall type 9h in Dubey et al.’s classification [1].
Type (natural) intermediate host
Asian gray shrew Crocidura attenuata.
Type locality
Anning Prefecture (25°24′N, 102°35′E; altitude 1894 m), China.
Site of infection
Muscular tissues throughout the body, including the heart.
Experimental definitive host
Beauty rat snake Elaphe taeniura.
Prevalence
Sarcocysts were found in 17 of the 42 (40.5%) Asian gray shrews examined.
Etymology
The species is named after its intermediate host species.
Molecular characterization
Nucleotide sequences of the 18S rDNA (MZ826981–MZ826985), ITS1 (MZ826986–MZ826999), cox1 (MZ889669–MZ889673), cox3 (OK001462 and OK001463), cytb (OK001464 and OK001465), rpoB (OK001466 and OK001467), and clpC (OK001468 and OK001469) of the new species have been deposited in GenBank. S. attenuati may be differentiated from S. scandentiborneensis, which was found in lesser and large tree shrews, according to 18S rDNA sequences. However, the partial cox1 sequences could not be distinguished from those of S. scandentiborneensis, or even those of Sarcocystis sp. obtained from the greater white-toothed shrew within the covered region of this gene.
Deposited specimens
Formalin-fixed tissues containing cysts of S. attenuati, as well as photomicrographs obtained from the LM and TEM of the sarcocysts, have been deposited at the Zoological Specimen Museum of Yunnan University, Kunming, China (collection no. Pro2018004).
ZooBank registration
To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature [14], details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub: 33DEF1BE-BAC4-4E50-8B12-97D9AB1701D8. The LSID for the new species name Sarcocystis attenuati n. sp. is urn:lsid:zoobank.org:act: 5CE48112-E64D-496D-8386-4FA2C4F6200A.
Remarks
The structure of the sarcocyst wall is a useful taxonomic criterion for differentiating Sarcocystis spp. within a host. Dubey et al. [1] grouped sarcocysts by sarcocyst wall ultrastructure into 42 wall types with several subgroups. To the best of our knowledge, sarcocysts have been observed in only a few species of insectivores, including the shrew mole Urotrichus talpoides in Japan [15], the moonrat Echinosorex gymnurus in Malaysia [2], the short-tailed shrew Blarina brevicauda in the USA [16], the white-toothed shrew Crocidura russula in Russia [3], the common shrew Sorex araneus, and the Eurasian pygmy shrew Sorex minutus in Lithuania [17, 18]. Unfortunately, most of the sarcocysts in these cases were not described in detail. Based on the morphological characterization of their sarcocysts, only two species, Sarcocystis boollati and Sarcocystis russuli, which were found in the moonrat and white-toothed shrew, respectively, have been proposed. Under LM, both species exhibit smooth, thin sarcocyst walls (< 1 μm) [2, 3]. Ultrastructurally, S. booliati sarcocysts show small knob-like protrusions, similar to wall type 1b [19]. Additionally, Grikieniené [18] found large sarcocysts (up to 5.0–10.0 mm) in the common shrew, which could be seen with the naked eye, and the cyst wall of the sarcocysts was smooth and thin (< 1 μm). Here, sarcocysts of S. attenuati n. sp. found in Asian gray shrews were microscopic and exhibited a thick cyst wall (3.3–4.5 μm in length), similar to wall type 9h, and they were unambiguously different from those of S. boollati, S. russuli and Sarcocystis sp. obtained from the moonrat, white-toothed shrews, and the common shrew, respectively.