Molecular identification of Taenia mustelae cysts in subterranean rodent plateau zokors (Eospalax baileyi)

Fang ZHAO1,#, Jun-Ying MA2,#, Hui-Xia CAI2, Jian-Ping SU1, Zhi-Bin HOU3, Tong-Zuo ZHANG1,Gong-Hua LIN1,*

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Molecular identification ofcysts in subterranean rodent plateau zokors ()

Fang ZHAO, Jun-Ying MA, Hui-Xia CAI, Jian-Ping SU, Zhi-Bin HOU, Tong-Zuo ZHANG,Gong-Hua LIN

1.Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China 2.Qinghai Institute for Endemic Disease Prevention and Control, Xining 811602, China 3.Xining No.2 People’s Hospital of Qinghai Province, Xining 810003, China

Cestode larvae spend one phase of their two-phase life cycle in the viscera of rodents, but cases of cestodes infecting subterranean rodents have only been rarely observed. To experimentally gain some insight into this phenomenon, we captured approximately 300 plateau zokors (), a typical subterranean rodent inhabiting the Qinghai-Tibet Plateau, and examined their livers for the presence of cysts. Totally, we collected five cysts, and using a mitochondrial gene () and two nuclear genes (and) as genetic markers, we were able to analyze the taxonomy of the cysts. Both the maximum likelihood and Bayesian methods showed that the cysts share a monophyly with, while Kimura 2-parameter distances and number of different sites between our sequences andwere far less than those found between the examined sequences and other Taeniidae species. These results, alongside supporting paraffin section histology, imply that the cysts found in plateau zokors can be regarded as larvae of, illustrating that zokors are a newly discovered intermediate host record of this parasite.

Endoparasites; New host record; Phylogenetic relationships; Subterranean rodent

Larval taeniid cestodes (Taeniidae, Cyclophyllidea, Cestoda) are known to require either human or other herbivorous mammals—generally rodents—to serve as intermediate hosts during their larval stage (Knapp et al, 2011). While there are many parasites within humans and rodents, larval cestodes are a special epidemiological focus, because they can cause serious pathological changes in viscera and tissues, and even death of the host (Eckert et al, 2001; Hoberg, 2002). While humans and other herbivorous mammals are often used as hosts, subterranean rodents are generally not thought to be viable host options. As a widely distributed group of species that live primarily underground and are highly adapted to that environment (Lacey et al, 2000; Nevo, 1999), most major activities—foraging, mating, and breeding, etc. —take place underground. Consequently, these rodents have rare contact with predators (Begall et al, 2007), but more importantly, since subterranean rodents generally forage underground parts of plants, they have a markedly smaller probability of encountering food contaminated by cestode eggs. As a result, it is commonly believed that these animals have a compar-atively rare chance of becoming infected by cestodes, but that preconception may be, at best, flawed, or even incorrect, because these animals are typically hidden, the presence of cestodiasis is not well empirically studied.

Boev et al (1971) first identifiedfrom cysts isolated from a zokor species (sp.) in Kazakhstan. Li et al (1985) andHong & Lin (1987) reported the same identification of cysts from the viscera of Chinese zokors () in Ningxia, China. Because of the paucity of adult cestode phenotypic characteristics and the great plasticity of larvae, ide-ntifying the Taeniidae species using traditional histologic examination of paraffin slices is prone to errors (Nakao et al, 2010). Thankfully, modern molecular technologies provide far more accurate methods of identification. In this study, we sought to use both approaches to gain a more complete picture of cestode infections. To that end, we collected several cysts from the livers of captured plateau zokors () (Figure 1A) and used molecular phylogenetic methods to determine the phylo-geny of the larval cysts.

Figure 1 Photographs of Samples

A: Example of plateau zokor; B: Cyst in zokor liver tissue; C and D: Histopathological slices of a cyst.

MATERIALS AND METHODS

Totally, 300 plateau zokors were captured for study from Datong County (N37°7.5′, E101°48.7′), in the east of Qinghai Province, China. After euthanizing the speci-mens and dissecting them, liver tissues were extract and tissues that appeared to be infected (Figure 1B) were fixed in 4% formalin and embedded in paraffin wax, with 3-5μm sections prepared for histopathological observat-ion. The samples were stained overnight with Delafield’s haematoxylin, destained with 70% ethanol containing 1% hydrochloric acid, dehydrated in ethanol, and then cleared with xylene and mounted in Canada balsam. The found cysts that were to be used for molecular analyses were fixed in 95% ethanol before total genomic DNA was extracted using a spin column kit (DNeasy tissue kit, Qiagen, Germany) and then used as a template for PCR. Here, two cysts were randomly selected for molecular analyses. Partial fragments of the mitochondrial gene for cytochrome c oxidase subunit 1 gene () (approxi-mately 880 bp)were amplified using previously publis-hed primers cox1/F and cox1/R by Nakao et al(2000). We also selected genes for phosphoenolpyruvate carbox-yk-inase () (approximately 1 650 bp)and DNA poly-m-erase delta () (approximately 2 000 bp) to serve as targets for nuclear markers in the cyst DNA (Knapp et al, 2011).

PCR was performed using a 40 μL final reaction volume, with 40 to 60 ng of genomic DNA, 0.6 mmol/L dNTPs, 0.2 μmol/L of each primer, 1U Taq polymerase and the manufacturer-supplied reaction buffer. Therm-ocycling was conducted in a T-Gradient Thermoblock PCR mach-ine (Biometra, Gottingen, Germany). After initial denat-ur-ation at 94 °C for 7 min, the reaction proceeded for 35 cycles as follows: 30 s at 94 °C, 30 s at 54 °C to 56 °C and 90 s at 72C and terminated with a final extension step of 72 °C for 5 min. Resulting PCR produ-cts were purified using a CASpure PCR Purific-ation Kit following the manufacturer’s recomm-ended protocols (Casarray, Shanghai, China), and directly sequenced using the same primers that were previously used for amplifying the sequences mentioned above. Sequencing reactions were conducted in a Biometra therm-ocycler using a DYEn-amic ET Termin-ator Cycle Sequencing Kit (Amersham Biosciences, UK) following the manufact-urer’s protocols. Sequencing products were later separated and analyzed on an ABI 3730 DNA Analysis System (Applied Biosy-stems, USA). Putative exon regions for both theandgenes were extracted from each of the respe-ctive sequence align-ments under the guidance of previ-ously published exon–intron maps for(Knapp et al, 2011).

We obtained the sequences of,andgenes from the available species in the family (9and 15taxa) from GenBank to serve as a basis for identifying taxonomic status of the cystic larvae using phylogenetic methods. The additional taxaforandfor the two nuclear genes (and) were included as outgroups for reconstructing maximum likelihood (ML) and Bayesian trees (accession numbers of these sequences are in Table 1). The coding sequences for each gene were aligned using ClustalW in MEGA 5.0 (Tamura et al, 2011), cut to the length of the shortest sequence. Maximum likelihood trees were generated in PAUP 4b10 (Swofford, 2002) and the Bayesian trees in MrBayes 3.2 (Ronquist et al, 2012), each with 1000 bootstrap replicates. Nucleotide substitution models were selected using the Akaike information criterion (AIC) within Modeltest 3.7 (Posada & Crandall, 1998) running on PAUP 4b10 and in MrModeltest 2.3 (Posada & Crandall, 2001) running on MrBayes3.2. The Kimura 2-parameter pairwise divergence (K2P) and the number of different nucleotide substitutions (N.diff) among seque-nces was calculated using MEGA 5.0.

Table 1 GenBank accession numbers for all reference sequences (9 Echinococcus and 15 Taenia taxaand 2 outgroups Dipylidium caninum and Hymenolepis diminuta) for each gene used in this study

RESULTS

Totally, 300 plateau zokors were captured and then examined for the presence of cestode parasites. Among these five separate cysts were detected in five of the examined zokors, each ranging from 3-5 mm in diameter (Figure 1B). The cysts were found deeply embedded, and partly or fully covered by liver tissue. Physically, they are thin-walled and either transparent to translucent. Slices of the tissues (depicted in Figure 1C, D)showed that only one larva attached to the inner face of the cyst. The larva was about 3.5 mm in major diameter with no sign of scolex formation. Together, these physical chara-c-te-ristics suggest a close relationship of the observed cysts withGmelin 1790 (Freeman, 1956).

Three genes each from two cysts were sequenced and the resulting sequences submitted to GenBank(accession numbers: KC898934-KC898939). The,, and(partial sequences) were 820 bp, 1041 bp, and 1873 bp in length, respectively. The putative exon regions forandgenes were likewise 921 bp and 867 bp, respectively. Finally, three alignments in lengths of 384 bp, 921 bp, and 867 bp respectively for the,, andgenes were used for molecular analyses. Testing showed that the three genes differed at only one or two sites betw-een the DNA sequences from the larvae from the two cysts.

Further phylogenetic analysis yielded both ML and Bayesian trees, (Figure 2) both of which clearly demo-nstrated that the sequences of the cysts andwere closely related. Further analysis between the sequences of the cysts and other sequences yielded K2P and N.diff values (Table 2), and the values for both measurements between the studied sequences and earlier reported sequences ofwere far less than those between our sequences and other Taeniidae species.

Figure 2 Phylogenetic trees of cyst tissues found in Plateau Zokors using theand exon data sets ofand

Upper tree is generated by maximum likelihood and the lower by Bayesian analysis. Each tree was rooted withfor cox1 andforand. Values at each node are bootstrap proportions (%).

DISCUSSION

Both the histopathological and molecular analyses we conducted indicated that the cysts can be regarded as larvae ofmeaning that the zokors are a newly identified host forinfection. A recent DNA barcoding of taeniids using the samegene segment as we used in this study found that the optimum threshold for distinguishing aspecies is 3.6% of K2P distance (Galimberti et al, 2012), which is higher than the value (2.7%) found between our samples and. Given this threshold, we can conclude that our speculation on the nature of the cysts as beinglarvae is likely accurate. Unfortunately, the “gold standard” of determining a taeniid species relies on using both molecular data of adult specimens as well as morpho-logical observations. Though our present findings are intriguing, further studies, such as survey of potential hosts for adult taeniids, are still necessary to make a definitive taxonomic review of the cysts.

Combined with former reports, our results make it clear that that zokors can harbor two Taeniidae species with two different genuses. Taeniid parasites require two m-am-malian hosts to perpetuate their life cycles.

Table 2 Mean values of different sites (N) and Kimura 2-parameter distances (K2P) between the sampled sequences and earlier published sequences for cox1, pepck, and pold genes

Terres-trial carnivorous predators act as hosts for the adult worms, while their prey, such as the zokors and other rodents, act as intermediate hosts for the cystic larvae. Zokors are commonly thought to have only rare contact with predators because of their absolute underground habitat. An earlier study noted that the numbers of plateau zokors in the pellets and food remains ofandonly consisted ~5% of total prey individuals (Cui et al, 2003). Likewise, the average feeding intensity by predators of the zokor, such as the red fox (), polecat () and weasel () of plateau zokor was much lower than that of the sympatric distributed plateau pika () (Yang et al, 2007). Moreover, as a typical subterranean rodent, plateau zokors mainly feed on underground roots and shoots of plants (Zhang, 1999), both of which are less likely to be contaminated by cestode eggs contained in the feces of carnivorous hosts. Collectively, these characteristics make the zokors somewhat unlikely infectious targets for taeniid cestodes. The reality that zokors can indeed be infected by bothandlarvae shows that an underg-round habitat alone cannot prevent infection by such parasites, which may then hold some interesting implications for studies of similar parasitic organisms

One possibility that may explain the infection is that when zokors collect food items from underground, they may also pull down the aboveground parts of plants. In fact, recent studies on winter caches of plateau zokors showed that they collected considerable amount of aboveground plant parts, some of which are even positively selected as food sources (Xie et al, 2013). This novel observation gives some credence to our observed infection of zokors by parasites, because if zokors actually harvest aboveground sources of food, they may have more chances to become infected by cestodes as expected. Another possibility is that while zokors constitute only a small part of food resources of predat-ors, some carnivores, such as the polecat, frequently invade to their burrows in search of food (Zheng et al, 1983) Even when unsuccessful, such forays into the underground zokor burrows may increase the probability of contamination of cestodes eggs (from feces) in the zokor habitats and consequently increase their infection rate. Since there are many predators and other herbiv-orous mammals such as plateau pika, root vole (), hamster (), and marmot () that are sympatrically distributed with plateau zokors, the taeniid parasites that infect zokors may complete their life cycles locally, placing many of these wild mammals of the plateau at risk for cestodiasis, though this clearly needs further detailed follow-up. Accordingly, we suggest that the health challenges of endoparasites from zokors (and probably other subterranean rodent species) should not be neglected and indeed warrant greater attention and observation.

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01 July 2013; Accepted: 27 February 2014

This work was supported by the West Light Found-ation of the Chinese Academy of Sciences and the Chinese Academy of Sciences President Scholarship (to G. Lin)

These authors contributed equally to this work

, E-mail:lingonghua@gmail.com