Sarcocystis Attenuati N. Sp. (Apicomplexa: Sarcocystidae) Infecting the Asian Gray Shrew, Crocidura Attenuata (Insectivora: Soricidae), in China

Background: There are limited data on Sarcocystis in insectivores. The Asian gray shrew, Crocidura attenuata, is one of the most common species of insectivores in the family Soricidae distributed in South Asia and Southeast Asia. To date, Sarcocystis has never been recorded in this host. Methods: Tissues from 42 Asian gray shrews were collected in China in 2017 and 2018. Sarcocysts were observed using light (LM) and transmission electron microscopy (TEM). To complete the parasite life cycle, muscle tissues of the host infected with sarcocysts were force-fed to two beauty rat snakes, Elaphe taeniura. Individual sarcocysts from different Asian gray shrews and oocysts/sporocysts isolated from the small intestines and feces of the experimental snakes were selected for DNA extraction, and seven genetic markers, including two nuclear loci (18S rDNA and ITS1), three mitochondrial genes (cox1, cox3 and cytb), and two apicoplastic genes (rpoB and clpC), were amplied, sequenced and analyzed. Results: Sarcocysts were found in 17 of 42 (40.5%) Asian gray shrews. Under LM, the microscopic sarcocysts were exhibited saw-tooth-like protrusions measuring 3.3–4.5 μm. Ultrastructurally, the sarcocyst wall contained numerous lancet- or leaf-like villous protrusions, similar to type 9h. The experimental beauty rat snakes shed oocysts/sporcysts measuring 11.9–16.7 × 9.2–10.6 μm with a prepatent period of 10 to 11 days. Comparing these sequences with those previously deposited in GenBank revealed that the 18S rDNA sequences and cox1 sequences shared the highest similarity with those of S. scandentiborneensis recorded in tree shrews, Tuaia minor and T. tana (i.e., 97.6–98.3% and 100% identity, respectively). Phylogenetic analysis based on 18S rDNA, ITS1 or cox1 sequences revealed that this parasite formed an independent clade with Sarcocystis

Conclusions: Sarcocysts were recorded in Asian gray shrews for the rst time. The sarcocysts were characterized morphologically and molecularly. The 18S rDNA and cox1 sequences of S. attenuati, named in the present study, shared the highest identities with those of S. scandentiborneensis. However, the sarcocysts of the two species of Sarcocystis were quite different under LM and TEM. Based on experimental infection, beauty rat snakes have been proven to be a de nitive host of S. attenuati. As more species of Sarcocystis from insectivores and other small mammals are properly morphologically and molecularly characterized, we may gain a better understanding of the biodiversity, host speci city and evolution of Sarcocystis in the future.

Background
Species of the genus Sarcocystis (Apicomplexa: Sarcocystidae) exhibit an obligate two-host life cycle, with sexual development in the small intestine of the de nitive host and asexual development in different tissues of the intermediate host, which are usually herbivores. More than 200 species of Sarcocystis have been described in a variety of wild or domesticated animals [1]. However, only two species, S. booliati and S. russuli, have been described and proposed to use insectivorous animals as intermediate hosts [1,2,3]. Insectivores are a group of mammals including 441 species and constitute almost 10% of extant mammal species. Asian gray shrew Crocidura attenuata, a species of insectivore in the family Soricidae, is one of the most common species throughout South and Southeast Asia [4]. To date, no species of Sarcocystis has been found and recorded in the host.
The present study describes the morphological and molecular characteristics of a new species of Sarcocystis detected in Asian gray shrews. Additionally, the life cycle of the new species was completed using animal experiments in the laboratory based on a speculation arising from phylogenetic analysis.

Materials And Methods
Microscopic examination of sarcocysts from Asian gray shrews A total of 42 Asian gray shrews were captured by live trapping on farmland from July 2017 to December 2018 in Anning Prefecture, located in the mountainous zone of the central part of Yunnan Province, China. All hosts were killed with ether and transported to the zoological laboratory of Yunnan University, Kunming, China, on the same day.
Fresh preparations of the esophagus, diaphragm, tongue, skeletal muscles (thigh, loin, rump, and ribs) and heart of each animal were pressed and squeezed between two glass slides and inspected for sarcocysts using a stereomicroscope. Sarcocysts were extracted from muscular bers using dissection needles and processed for light (LM) and transmission electron microscopy (TEM) and DNA analysis.
For TEM, the sarcocysts were xed in 2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4) at 4°C and post xed in 1% osmium tetroxide in the same buffer, dehydrated in a graded alcohol series, and embedded in Durcupan. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a JEM100-CX transmission electron microscope at 80 kV.

Experimental infection of potential de nitive hosts
To complete the life cycle of the parasite, three beauty rat snakes, Elaphe taeniura, purchased from a pet market in Kunming city, were housed separately in steel cages at ambient temperature and humidity. The snakes were force-fed quail eggs, and the feces of the snakes were examined for two weeks via the otation technique to con rm that they were coccidian free.
Pieces of muscle from a wild-caught Asian gray shrew were force-fed to two beauty rat snakes, and the remaining snake was kept as a control. Before inoculation, it was con rmed by extensive microscopic examination that the muscles of the Asian gray shrew fed to experimental snakes only contained sarcocysts of the new species. The experimental snakes were each fed muscle pieces containing approximately 300 sarcocysts, and fecal samples of the snakes were examined daily for two weeks postinfection (PI) via the otation method to determine the presence of oocysts/sporocysts. All snakes were killed 29 days PI. The small intestine of each snake was removed and digested (1% trypsin, 37 °C, 3 hours), and the digested tissue was ltered and centrifuged (5000 r/min, 10 mins). The sediments were tested to determine the presence of oocysts/sporocysts using light microscopy. Oocysts/sporocysts from the feces and small intestines of the experimental snakes were collected and stored in sterile water at 4 ℃ for DNA extraction.

Molecular characterization
A total of 11 samples, including ten individual sarcocysts separated from different Asian gray shrews and oocysts/sporocysts (approximately 250) collected from the experimental snakes, were subjected to genetic DNA extraction using the TIANamp Genomic DNA Kit (Tiangen Biotech Ltd., Beijing, China) according to the manufacturer's instructions. The sarcocysts and oocysts/sporocysts were characterized on the basis of seven genes, including two nuclear loci (18S rDNA and ITS1), three mitochondrial genes (cox1, cox3 and cytb), and two apicoplastic genes (rpoB and clpC). The primers used to amplify these genes are shown in Table 1.
PCR was performed in a PCR cocktail with a total volume of 25 µl that included 12.5 µl Green Taq Mix (Vazyme Biotech Co., Ltd, Nanjing, China), 5.5 µl ddH 2 O, 1.0 µl of each primer (10 µM), and 5 µl template DNA. The cycling parameters differed for each gene. For 18S rDNA and ITS1, the cycling parameters started with denaturation at 94 °C for 5 mins, followed by 35 cycles of 94 °C for 1 min, 57 °C for 1 min, and 72 °C for 1.5 mins, with a nal extension at 72 °C for 10 mins. For cox1 and cox3, the cycling parameters started with denaturation at 94 °C for 5 mins, followed by 35 cycles of 94 °C for 1 min, 54°C for 1 min, and 72 °C for 1.5 mins, with a nal extension at 72 °C for 7 mins. For cytb, rpoB and clpC, the cycling parameters started with denaturation at 94 °C for 5 mins, followed by 35 cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 1.5 mins, with a nal extension at 72 °C for 7 minutes. The PCR products were puri ed, cloned, sequenced, and assembled using the methods described in our previous paper [9].
At present, there are limited or few nucleotide sequences of the cox3, cytb, rpoB and clpC genes of Sarcocystis species deposited in GenBank. Therefore, in the present study, only 18S rDNA, ITS1 and cox1 sequences of the new species were used to infer the relationship with other Sarcocystis spp. using MEGAX software [10]. Maximum parsimony (MP) trees for the three genes were created with a treebisection-regrafting (TBR) algorithm. The reliability of the MP phylograms was tested via the bootstrap method using 1,000 replications. The 18S rDNA sequences of Sarcocystis spp. from different hosts were downloaded from GenBank and aligned with the ClustalW program implemented in MEGAX, and the alignment was subsequently checked visually; some sequences were slightly truncated at both ends so that all sequences started and ended at the same nucleotide positions (i.e., 101 and 1,828, respectively) of the sequence of S. cruzi (JX679468). The nal alignment consisted of a total of 43 nucleotide sequences and 1,865 aligned positions of 39 taxa. Cystoisospora ohioensis (GU292304), Besnoitia besnoiti (DQ227418), Hammondia heydorni (GQ984224), and Toxoplasma gondii (U03070) were chosen as outgroups.
A total of 44 ITS1 sequences of 31 taxa used in the analysis were aligned with the ClustalW program integrated in MEGAX. Some sequences were truncated at both ends so that all sequences started and stopped at the same nucleotide positions (i.e., 1,671 and 2,477, respectively) of the sequence of S. zuoi (KU341120). The nal alignment consisted of 1,924 aligned positions. Toxoplasma gondii (KM657806) and H. tri ttae (KJ396594) were used as outgroup species to root the tree.
In total, 31 mitochondrial cox1 sequences of 27 taxa used in the analysis were aligned with the ClustalW program integrated in MEGAX. Some sequences were slightly truncated at both ends so that all sequences started and stopped at the same nucleotide positions (i.e., 48 and 1,020, respectively) of the S. cruzi sequence (KT901095). The nal alignment consisted of 992 aligned positions with no gaps. Toxoplasma gondii (JX473253), H. heydorni (JX473251) and H. tri ttae (JX473247) were used as outgroup species to root the tree.

LM and TEM observations of sarcocysts
Spindle-shaped sarcocysts were found in 17 of 42 (40.5%) Asian gray shrews, located in skeletal muscle, the esophagus, the diaphragm, the tongue and the heart. Only one form of sarcocysts was observed. The observation of fresh samples at the light microscopy level showed that the cyst walls of the sarcocysts had numerous, 3.3-4.5 μm-long (n=25) saw-tooth-like protrusions (Fig. 1a). Mature sarcocysts were 740-1,355 × 117-250 μm in size; they were septate and contained bradyzoites measuring 8.2-10.4 × 2.0-3.0 μm (n=40) in size.
Ultrastructurally, the sarcocysts had lancet-or leaf-like villous protrusions measuring 2.8-6.4 × 0.4-0.9 μm (n=15), which contained numerous electron-dense granules in the core; microtubules or brils were absent. The primary cyst wall had minute undulations over the entire sarcocyst surface, lined by an electron-dense layer. The nearly vertical protrusions were separated from each other at inconsistent distances. A layer of ground substance measuring 1.2-1.6 μm (n=15) in thickness was located immediately beneath the primary sarcocyst wall (Fig. 1b).

Infection of the de nitive host
The two beauty rat snakes fed muscle tissue containing sarcocysts in an Asian gray shrew excreted sporulated oocysts/sporocysts (Fig. 1c, d) in their feces, one beginning on Day 10 and another on Day 11 PI. Upon the death of the snakes at Day 29 PI, numerous oocysts/sporocysts were also observed within the small intestines of the experimental animals. Under light microscopy, the sporulated oocysts measured 11.9-16.7 × 9.2-10.6 μm (average 13.5×9.9 μm) (n = 27), with two elliptical sporocysts measuring 9.2-10.6 × 6.3-6.8 μm (average 9.9 × 6.6 μm) (n = 30). No oocysts/sporocysts were found in the feces or small intestine of the control snake.
The 16 cox3 sequences from the sarcocysts and the oocysts/sporocysts were 675 bp in length and were completely identical. Therefore, only one sarcocyst sequence (OK001462) and one oocyst/sporocyst sequence (OK001463) were deposited in GenBank. No sequences with signi cant similarity to these sequences were found in GenBank.
The 16 cytb sequences from the sarcocysts and the oocysts/sporocysts were 1,080 bp in length and were completely identical. Therefore, only one sarcocyst sequence (OK001464) and one oocyst/sporocyst sequence (OK001465) were deposited in GenBank. The most similar sequences in GenBank were those of S. falcatula (MF034168-MF034187) from the budgerigar, Melopstittacus undulatus, and the identity was 96.4%.
The 16 rpoB sequences obtained from the sarcocysts and the oocyst/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 S. neurona (GQ851961) obtained from the southern sea otter, Lutris nereis, and 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 oocyst/sporocyst sequence (OK001469) were deposited in GenBank. The most similar sequences were those of S. falcatula (KP871717) and S. neurona (KP871716), and the identity was 92.5%.
Based on the morphological characteristics of sarcocysts, molecular analysis and host speci city, a new species name, Sarcocystis attenuati, is proposed for the organism found in Asian gray shrews from Anning Prefecture, China. The ultrastructure of sarcocysts has traditionally been used as a reliable indicator for the characterization of different Sarcocystis species in a given host. Nucleotide sequence analysis has now been suggested to be a more useful tool for the delineation or identi cation of Sarcocystis from the same or different hosts. However, different genetic markers have revealed different levels of intra-or interspeci c sequence diversity [15][16][17]. Here, seven molecular markers (18S rDNA, ITS1, cox1, cox3, cytb, rpoB, and clpC) were sequenced and characterized in DNA samples from S. attenuati. The analysis showed that the sequences of these seven genes of the parasite presented high intraspeci c similarities (i.e., 99.7-100%, 97.9-100%, 99.8-100%, 100%, and 100%, and 100%, 100%, respectively). The comparison of these sequences with those previously deposited in GenBank showed that the 18S rDNA and cox1 sequences of S. attenuati shared the highest identity with those of S. scandentiborneensis (i.e., 97.6-98.3% and 100%, respectively). Sarcocystis scandentiborneensis occurs in lesser or large tree shrews belonging to the family Tupaiidae collected from Malaysia. With the aid of LM, the cyst wall of S. scandentiborneensis was shown to present tightly packed gure-like protrusions (2-10 μm in length) that can assume a brushlike appearance; ultrastructurally, the tightly packed gure-like protrusions contain bundled microtubules that extend into the ground substance [18], similar to wall type 11b or 12 according to the classi cation of Dubey et al. (2016) [1]. Additionally, the cox1 sequences of S. attenuata shared a high similarity of 99.8% with that of Sarcocystis sp. obtained from the greater white-toothed shrew collected from Spain. However, sarcocysts of the greater white-toothed shrew have not been described [19].
The phylogenetic relationships among the majority of analyzed Sarcocystis spp. suggest their coevolution with their de nitive hosts rather than their intermediate hosts [20]. The phylogenetic trees based on 18S rDNA sequences and ITS1 sequences revealed that S. attenuati formed an individual clade with Sarcocystis spp. from small mammals using snakes as de nitive or putative de nitive hosts, showing especially close relationships with S. scandentiborneensis, Sarcocystis sp. ex Procyon lotor, S. zuoi, and S. clethrionomyelaphis.
The infection experiment con rmed the speculation arising from the phylogenetic analysis and proved that beauty rat snakes can serve as an experimental de nitive host of S. attenuati. The beauty rat snake is native to eastern and southeastern Asia and feeds mainly on shrews and rodents, although the consumption of amphibians, reptiles, and birds by this species has also been reported [21]. To date, only two species of Sarcocystis, S. zuoi from Norway rat, R. norvegicus, and S. clethrionomyelaphis from large oriental vole, E. miletus, have been proven to use species of Elaphe as de nitive hosts via transmission experiments [22,23]. Morphologically, sarcocysts of S. zuoi exhibit sloping nger-like protrusions, and the base of the protrusions is highly branched [22,24], similar to wall type 17; the sarcocysts of S. clethrionomyelaphis present thin highly folded protrusions, which often bend along the cyst surface [23], similar to type wall type 10f. Therefore, the sarcocysts of S. attenuati can be easily morphologically differentiated from those of S. zuoi and S. clethrionomyelaphis. It is very common that one predator acts as the de nitive host of more than one species of Sarcocystis. For example, Python reticulatus is the de nitive host of three species of Sarcocystis in rats (i.e., S. singaporensis, S. villivillosi, and S. zamani) [25].

Conclusions
In summary, this report demonstrates the presence of Sarcocystis that form microscopic cysts in Asian gray shrews. Based on the observed morphological characteristics and host speci city, a new species name, S. attenuati, is proposed for the new species infecting Asian gray shrews. This is the rst record of Sarcocystis in this host. Seven genetic markers, 18S rDNA, ITS1, cox1, cox3, cytb, rpoB, and clpC, of the species were sequenced and characterized. Interestingly, the 18S rDNA sequences and cox1 sequences of the species shared the highest similarities with those of S. scandentiborneensis obtained from tree shrews, reaching 100% identity for cox1. However, the sarcocysts of the two species presented different morphological characteristics according to LM and TEM observations. Experimental infection revealed that beauty rat snake can serve as the experimental de nitive host of S. attenuati, which was also proven by the high similarities of the nucleotide sequences of the seven genes between the sarcocysts and oocysts/sporocysts of S. attenuati. As more species of Sarcocystis from different insectivores and other small mammals are properly morphologically and molecularly characterized, we may gain a better understanding of the biodiversity, host speci city and evolution of Sarcocystis in the future.

Declarations
Ethics approval and consent to participate The present study was approved by the Animal Ethics Committee of Yunnan.
University (permission number ynucae20180002). All experimental animals were handled following good animal practices according to the regulations for the Care and Use of Laboratory Animals of the National Institutes of Health, China.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

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