Genetic diversity in Echinococcus shiquicus from the plateau pika (Ochotona curzoniae) in Darlag County, Qinghai, China

    Genetic diversity in Echinococcus shiquicus from the plateau pika (Ochotona curzoniae) in Darlag County, Qinghai, China Yan-Lei Fan, Zhong-Zi Lou, Li Li, Hong-Bin Yan, Quan-Yuan Liu, Fang Zhan, Jian-Qiu Li, Cong-Nuan Liu, Jin-Zhong Cai, Meng-Tong Lei, Wan-Gui Shi, Yu-Rong Yang, Donald P. McManus, Wan-Zhong Jia PII: DOI: Reference:

S1567-1348(16)30240-4 doi: 10.1016/j.meegid.2016.06.016 MEEGID 2791

To appear in: Received date: Revised date: Accepted date:

27 August 2015 2 May 2016 5 June 2016

Please cite this article as: Fan, Yan-Lei, Lou, Zhong-Zi, Li, Li, Yan, Hong-Bin, Liu, Quan-Yuan, Zhan, Fang, Li, Jian-Qiu, Liu, Cong-Nuan, Cai, Jin-Zhong, Lei, MengTong, Shi, Wan-Gui, Yang, Yu-Rong, McManus, Donald P., Jia, Wan-Zhong, Genetic diversity in Echinococcus shiquicus from the plateau pika (Ochotona curzoniae) in Darlag County, Qinghai, China, (2016), doi: 10.1016/j.meegid.2016.06.016

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ACCEPTED MANUSCRIPT Genetic diversity in Echinococcus shiquicus from the plateau pika

Yan-Lei Fan

a,bǂ ,

Zhong-Zi Lou

a

aǂ ,

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(Ochotona curzoniae) in Darlag County, Qinghai, China

a

c

c

Li Li , Hong-Bin Yan , Quan-Yuan Liu , Fang Zhan ,

a

d

d

c

Jian-Qiu Li , Cong-Nuan Liu , Jin-Zhong Cai , Meng-Tong Lei , Wan-Gui Shi , Yu-Rong

State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary

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a

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Yang e*, Donald P. McManus e* and Wan-Zhong Jia a, f*

Parasitology of Gansu Province, Key Laboratory of Veterinary Public Health of Agriculture Ministry, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, Gansu Province, P. R. China.

Institute of Biomechanics and Medical Engineering, School of Aerospace, Tsinghua University,

Beijing, 100084, P.R. China.

Gansu Provincial Center for Animal Disease Control and Prevention, Lanzhou 730046, Gansu

Province, P. R. China. d

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c

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b

Laboratory of Plateau Veterinary Parasitology, Veterinary Research Institute, Qinghai Academy

e

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of Animal Science and Veterinary Medicine, Xining 810016, Qinghai Province, P. R. China. Molecular Parasitology Laboratory, Infectious Diseases Division, Queensland Institute of

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Medical Research, Brisbane, QLD 4006, Australia. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease,

Yangzhou 225009, Jiangsu Province, P. R. China.

*Corresponding authors: Wan-Zhong Jia ([email protected]), Donald P. McManus ([email protected]) Yu-Rong Yang ([email protected]).

ǂ These authors contributed equally to this work.

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ACCEPTED MANUSCRIPT ABSTRACT The metacestode of Echinococcus shiquicus has been recorded previously in the

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lung and liver of its intermediate host, the plateau pika (Ochotona curzoniae), but there is limited information regarding other organ sites. There is also limited evidence

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of intra-specific genetic variation within E. shiquicus. A PCR-amplified mitochondrial (mt) nad1 gene fragment (approximately 1400 bp in size), with unique EcoRI and

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SspI restriction sites, was used to distinguish cysts or cyst-like lesions of E. shiquicus from E. multilocularis. Then, the complete mt nad1 and cox1 genes for the E. shiquicus isolates were amplified and sequenced. Phylogenetic tree and haplotype

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network analyses for the isolates were then generated based on a concatenated dataset

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of the nad1 and cox1 genes using the neighbour-joining (NJ) method and TCS1.21 software. Nineteen of eighty trapped pikas were found to harbor cysts (71 in total)

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when dissected at the survey site. Seventeen animals had cysts (fertile) present only in

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the lungs, one animal had fertile cysts in the lungs and spleen, and one individual had an infertile kidney cyst. Restriction endonuclease analysis of a fragment of the nad1 gene indicated all the cysts were due to E. shiquicus. Genetic diversity analysis revealed that the nad1 and cox1 genes varied by 0.1-1.2% and 0.1-1.0%, respectively. Haplotype network analysis of the concatenated nad1 and cox1 sequences of the isolates showed they were classified into at least 6 haplotypes, and different haplotype percentages ranged from 4.2% to 29.6%. Although, high haplotype diversity was evident in the study area, the complete nad1 and cox1 gene sequences obtained indicated that all samples represented isolates of E. shiquicus . The study has also 2

ACCEPTED MANUSCRIPT provided a new PCR-restriction endonuclease-based method to rapidly distinguish E. shiquicus from E. multilocularis which provides a useful tool for epidemiological

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investigations where the two species overlap.

Key words: E. shiquicus; plateau pika; E. multilocularis; restriction endonuclease

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analysis; haplotype analysis; genetic diversity

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1. Introduction

Species of Echinococcus are of major public health significance globally (Cardona and Carmena, 2013; Moro and Schantz, 2009; Schantz, 1977; Wang et al., 2008). To

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date, at least nine species are recognized in the Echinococcus genus, of which four are

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associated with human disease (Nakao et al., 2013). The most common forms are Echinococcus granulosus sensu stricto (genotypes G1-G3) and E. multilocularis,

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responsible for cystic echinococcosis (CE) and alveolar echinococcosis (AE),

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respectively; two other forms, namely E. oligarthrus and E. vogeli cause polycystic echinococcosis (PE), whereas two new species, E. felidis and E. shiquicus, may also contribute to human infection (Huttner et al., 2008; McManus, 2013; Moro and Schantz, 2009; Nakao et al., 2007). E. shiquicus was first recorded on the Qinghai-Tibet Plateau in China, with the Tibetan fox (Vulpes ferrilata) as the main wild definitive host and the plateau pika (Ochotona curzoniae) as intermediate host (Xiao et al., 2005). In some areas, E. multilocularis has a similar transmission cycle to E. shiquicus, involving foxes and O. curzoniae (Carmena and Cardona, 2014; Wang et al., 2008; Xiao et al., 2006). Based on mitochondrial data, E. shiquicus and E. 3

ACCEPTED MANUSCRIPT multilocularis are now considered sister species (Nakao et al., 2007) although the former was mistakenly reported some 20 years ago in Tibetan foxes from western

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Sichuan province as an isolate of E. multilocularis (Qiu et al., 1995). Recent molecular evidence has shown E. shiquicus is a separate species mainly parasitizing

infect other mammals (Xiao et al., 2005).

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the liver of O. curzoniae (McManus, 2013); there is no evidence that the parasite can

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Mitochondrial (mt) DNA has proved invaluable as a genetic marker in molecular taxonomy generally and it has been used extensively for species identification, evolutionary studies and for assessing intra- and interspecies variability in the genus

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Echinococcus (McManus, 2013). In particular, the NADH dehydrogenase subunit 1

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(nad1) gene and the cytochrome c oxidase subunit 1 (cox1) mt gene have been widely used in the identification of isolates and in phylogenic analysis of Echinococcus spp.

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(Nakao et al., 2002; Xiao et al., 2005). E. shiquicus has been shown to have the most

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variable mtDNA with the maximum values of divergence in cox1 sequences when compared with E. granulosus sensu stricto and E. multilocularis (Nakao et al., 2010). There is evidence from genotypic/haplotypic analysis that E. shiquicus has probably evolved with bottleneck effects (Ma et al., 2012) but there is limited information available on the level of genetic diversity within E. shiquicus populations to date. This study aimed to further resolve the genetic relationship of Echinococcus shiquicus found present as cysts in various organs of trapped pikas from Darlag County, Qinghai province, People’s Republic (PR) of China using mitochondrial gene sequencing. A further aim was to develop a useful tool based on a new restriction 4

ACCEPTED MANUSCRIPT endonuclease analysis method involving a fragment of the nad1 gene to differentiate E. shiquicus and E. multilocularis for epidemiological investigations where the two

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species overlap.

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2. Materials and methods 2.1. Ethical statement

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The collection and autopsy of the trapped pikas was conducted under strict Chinese experimental animal clearances in accord with animal ethics procedures and guidelines for animal husbandry and wildlife protection. The study was approved by

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the Institutional Ethics Committee of Lanzhou Veterinary Research Institute, Chinese

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Academy of Agricultural Sciences (Approval No. LVRIAEC2010-005).

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2.2. Collection of samples

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Eighty plateau pikas were trapped in June, 2013, in Darlag county (located at 99°38′E, 33°43′N; altitude: 4070 m; Fig. 1), Qinghai province, PR China. If the animals trapped were not found dead, they were euthanized using anaesthesia with diethyl ether before dissection. All organs in the abdominal and chest cavities were examined. The organ location of any cystic lesion identified was recorded and the isolate examined macroscopically at the survey site, then microscopically in the laboratory at Lanzhou Veterinary Research Institute, Gansu province. All lesions were detached from infected organs, placed in 50% (v/v) ethanol and then frozen at -20℃ until used for DNA extraction. 5

ACCEPTED MANUSCRIPT E. multilocularis reference DNA was obtained from cystic lesions, excised from 30 Qinghai vole livers, provided by colleagues at the Center of Disease Control (CDC),

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Darlag County, Qinghai Province, which neighbors our sampling area. The isolates were preserved in 50% (v/v) ethanol, their DNA was extracted and their identity

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confirmed as E. multilocularis by PCR and sequencing of the nad1 and nad5 genes

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(Jia et al., 2010).

2.3. DNA extraction

Individual intact cysts (either fertile - protoscoleces present - or infertile) from

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infected O. curzoniae were ground in a pestle and mortar with repeat freeze-thawing

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in liquid nitrogen before being digested with proteinase K. Total genomic DNA was extracted using a spin column kit according to the manufacturer′s instructions

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(QIAamp DNA FFPE Tissue kit, Qiagen, Hilden, Germany). The extracted DNA was

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stored at -20℃ until use.

2.4. PCR-restriction endonuclease analysis One pair of universal primers (Es∕Em-F: 5′-TAAGWTRAGTGTGTGTGTTGGT-3′ and Es∕Em-R: 5′-TAARCAAACCTCTCAACGAGAC-3′) (W=A/T; R=A/G) was designed for the PCR amplification of the target nad1 locus (approximately 1400 bp length) in the mt genomes of E. shiquicus (NCBI accession no. AB208064) (Nakao et al., 2007) and E. multilocularis (NCBI accession no. AB018440) (Yang et al., 2005), which contains two optimal restriction sites of the endonucleases EcoRI (recognizing 6

ACCEPTED MANUSCRIPT GAATTC) and SspI (recognizing AATATT). The 50 μl PCR mixture was composed of 5×PrimeSTARTM Buffer (Mg2+ plus) 10

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μl, 2.5 mM of each dNTP 4 μl, primer F (10 μM) 1 μl, primer R (10 μM) 1 μl, template DNA (50-200 ng) and Prime STARTM HS DNA Polymerase with fortissimo

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3′-5′ exonuclease proofreading activity (2.5 U/μl) 0.5 μl (Takara Biomedicals, Shiga, Japan). The PCR reaction was performed under the following conditions: 94℃, 4 min

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denaturation; then 35 cycles for 94℃, 40 s denaturation; 55℃, 30 s annealing; 72℃, 90 s extension; followed by a final step at 72℃ for 10 min. PCR products were purified using the following steps: 2.5 vol of anhydrous ethanol

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were added to the tubes containing the PCR products and the tube contents were

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mixed thoroughly. The mixture was kept at -20℃ for 1 h, and then centrifuged at 20000g for at least 10 min at 4℃. The pellet was rewashed with 75% (v/v) ethanol

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solution. Finally, after drying in a biosafety cabinet, the sediments were used in

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restriction enzyme digestion reactions. The digestion reaction (20 μl), containing 1 μl EcoRI (15 U/μl) or SspI (10 U/μl), 2 μl 10×H Buffer and DNA of PCR products (0.5-1 µg), was maintained at 37℃ in a water bath for 1 h. The digestion products were mixed with 10×Loading Buffer and examined in 1.0% (w/v) agarose gels with ethidium bromide under UV light.

2.5. Sequencing of the complete nad1 and cox1 genes Two pairs of primers were designed to amplify the entire nad1 and cox1 genes of E. shiquicus based on the available mt genome sequence (GenBank accession no. 7

ACCEPTED MANUSCRIPT AB208064 or NC_009460): nad1-F: 5′-ATTGTTGAGTTGAGTAAAGC-3′ and 5′-TACAGACACAAAAAAGACTC-3′; and

cox1-F: cox1-R:

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5′-TTGACTTTCTCTTGGTGGGT-3′

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nad1-R:

5′-TAAACCCAAACAATCAATCCA-3′. The PCR products were purified using an

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Axy PrepTM DNA Gel Extraction kit (Axygen Biosciences, Union City, USA) and were then sent to commercial companies (Sangon Biotech, Shanghai, China and

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Genewiz Biotech, Beijing, China) for independent sequencing.

2.6. Genetic diversity and nucleotide sequence analyses

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Nucleotide sequences of the complete nad1 and cox1 genes obtained for each PCR

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sample were edited and aligned using Clustal Omega (online software, http://www.ebi.ac.uk/Tools/msa/clustalo/, serviced by EBI, the European Bioformatics

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Institute). Based on previous reports of Echinococus spp. on the Qinghai-Tibet

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Plateau, the complete nad1 and cox1 gene sequences (selected from the mt genome, GenBank accession no. AB208064 or NC_009460) of E. shiquicus (Nakao et al., 2007) were chosen as reference sequences designated as the H1 haplotype, whereas those of E. multilocularis (AB018440 or NC_000928) were used as an out group for phylogenetic tree construction (Jia et al., 2010; Kimura, 1980; Nakao et al., 2013; Nakao et al., 2010; Nakao et al., 2007; Nakao et al., 2002; Umhang et al., 2013; Yang et al., 2005). Both gene sequences of all larval Echinococcus isolates obtained, plus the reference sequences, were used to measure levels of genetic diversity using the Kimura 2-parameter model (Saitou and Nei, 1987). Based on this analysis, one 8

ACCEPTED MANUSCRIPT sequence from each genotypic group (having sequences with 100% homology) was selected as a representative sequence. Phylogenetic trees were generated using a

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concatenated dataset of the nad1 and cox1 genes with the neighbour-joining (NJ) method and MEGA software version 5.2.1 (Jia et al., 2010; Li et al., 2008; Tamura et

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al., 2011). Confidence limits for each branch of the trees were determined by 1000 bootstrap replications. Genetic diversities within E. shiquicus (H1 to H7) were also

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analyzed by TCS 1.21 software (Clement et al., 2000) of the haplotypes network. The network estimation was run at 95% connection limit.

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3.1. E. shiquicus cysts

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3. Results

Of 80 pikas examined, 19 animals (23.75%) had E. shiquicus cyst(s) (71 cysts in

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total). All cysts in one animal were confirmed to have the same haplotype as

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determined by nad1 and cox1 gene sequencing. Of these, 17 animals had multiple lesions located only in the lungs, one individual (pika 19) had cysts in the lung and spleen, and the remaining pika (18) had a single cyst in the kidney (Table 1; Fig. 2). It is noteworthy that none of the infected animals had cysts in the liver.

3.2. Restriction enzyme analysis of DNA The nad1 PCR products were 1426 bp and 1417 bp in size, using E. shiquicus DNA and E. multilocularis DNA as templates, respectively. After digestion with EcoRI, the E. shiquicus target fragment containing the nad1 locus was cut into two fragments of 9

ACCEPTED MANUSCRIPT 252 and 1174 bp in size, but with E. multilocularis the target fragment remained intact as there is no EcoRI cut site present in the gene. The E. shiquicus target fragment was

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digested into two fragments of 253 and 1173 bp using SspI, whereas with E. multilocularis, the target fragment was cut into three fragments of 260, 496 and 661

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bp in size (Fig. 3). None of the examined pikas harbored a co-infection based on the restriction enzyme patterns obtained with the cystic samples, all of which were typed

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as E. shiquicus.

3.3. Phylogenetic and haplotype network analyses

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Analysis by BLAST indicated the DNA sequences of all cystic lesions examined

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aligned with the nad1 (897 bp) and cox1 (1608 bp) genes of E. shiquicus available in GenBank, thereby confirming the identity of the isolates. Phylogenetic analysis

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revealed that the genetic diversity of the nad1 and cox1 genes varied in 0.1-1.2% and

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0.1-1.0%, respectively. Using Clustal W, the E. shiquicus isolates were distinguishable into 6 different haplotypes (H2-H7) based on the combined nad1 and cox1 gene sequences. The relationship between the haplotypes and the cyst samples are shown in Table 1, where the reference sequence for E. shiquicus (GenBank accession no. AB208064 or NC_009460) is designated as H1. Haplotype network analysis on the sequences of the nad1 and cox1 genes provided the different haplotype percentages that ranged from 4.2% to 29.6% (Fig. 5), demonstrating the high haplotype diversities of E. shiquicus occurring in Darlag County. Both analyses ( Fig. 4 and Fig. 5), based on the concatenated nad1 and cox1 nucleotide sequences, further confirmed that all 10

ACCEPTED MANUSCRIPT the isolates obtained from the plateau pikas clustered as E. shiquicus. When compared with the reference sequences (AB208064 or NC_009460) for the

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nad1 and cox1 genes, the genetic distances for the sequences of the H2 haplotypes (cysts 1 to 3) were 100% and 99.9%; for H3 (cysts 4 and 5) were both 99.8%; for H4

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(cysts 6 and 7) were 99.9% and 99.4%, respectively; for H5 (cyst 8) were 99.7% and 99.6%, respectively; for H6 (cysts 9 to 16) were 99.4% and 99.1%, respectively; and

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for H7 (cysts 17 to 20) were both 99.1% (Table 1).

The H2, H3, H4, H5 and H6 haplotype sequences were found in lung cysts (varying in number/individual from 1 to 13) from 16 pikas (Table 1). H7 sequences originated

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from four lesions located in the lungs and one in spleen of one animal and in the

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kidney of another pika (cyst 18) ( Table 1). The genetic distance of haplotype H7 (cysts 17 to 20) was 1.0% when compared

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with haplotypes H3 (cysts 4 and 5) and H6 (cysts 9 to 16), and 0.9% when compared

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with the sequences of haplotypes H2 (cysts 1 to 3), H4 (cysts 6 and 7) and H5 (cyst 8). Apart from these, all other comparisons between sequences from the different isolates had genetic distances which were ≤ 0.6. Therefore, the H7 haplotype appears to have diverged the most compared with the other haplotypes during the course of intra-specific evolution in E. shiquicus ( Fig. 4). Variant hotspots within the E. shiquicus haplotypes are shown in Tables 2 and 3. For the complete nad1 gene (Table 2), there were 16 mutational sites among the different haplotypes with both transitions and transversions occurring within the different haplotypes. For the cox1 gene there were 26 mutational hotspots among the 11

ACCEPTED MANUSCRIPT different haplotypes with only transitions evident (Table 3).

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4. Discussion

The findings presented here are the first evidence of extra-hepatic larval E.

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shiquicus in the Tibetan pika. E. shiquicus is a recently described new species of Echinococcus found in wildlife hosts from the eastern Tibetan plateau of China (Xiao

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et al., 2005); its potential in zoonotic transmission is unknown (Li et al., 2008; McManus, 2013). The Tibetan fox is the major definitive host for E. shiquicus although there are reports that the domestic dog may also act as a cryptic definitive

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host. However, the plateau pika (O. curzoniae) is the only species of intermediate host

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so far implicated in the life cycle (Boufana et al., 2013; Ma et al., 2012; Silvestri, 1964). Previous reports have shown that the larvae of E. shiquicus, develop into

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unilocular cysts mainly in the liver of O. curzoniae (McManus, 2013; Xiao et al.,

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2006), whereas cysts reported in the current study occurred mainly in the lungs, with none recorded in the liver. The spleen (fertile cyst) and kidney (infertile cyst) were also found to be infected organs, albeit at a low rate, for the first time in this study. It appears that E. shiquicus may have similar site preferences to E. granulosus (Moro and Schantz, 2009), with cystic lesions found in all organs with different frequencies. The prevalence (25%) we report for E. shiquicus in the lungs of O. curzoniae is higher than previously recorded from the Darlag area (Han et al., 2009). The sampling area for the current study (Fig. 1) is located at high elevation (4070 m above sea level), where the reduced environmental oxygen level would result in less oxygen available 12

ACCEPTED MANUSCRIPT in the lungs of O. curzoniae with weaker local immunological capability resulting. The Darlag study areas reported on by others could have been at a lower altitude from

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our sampling zone, though this presumption cannot be confirmed due to the lack of geographic information provided in these previous reports (Han et al., 2009; Xiao et

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al., 2005, 2006). Also we found multiple E. shiquicus cysts were commonly found in

sources were at high density.

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a single individual of O. curzoniae (Table 1; Fig. 2), suggesting the transmission

The genetic diversity analysis undertaken revealed that the maximum variation values were 1.2% and 1.0% in the nad1 and cox1 genes for the E. shiquicus isolates

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examined, respectively, indicating that the nad1 gene had more diversity than the cox1

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gene, and that the mitochondrial locus in Echinococcus evolved without bottleneck effects supporting the hypotheses previously suggested by Nakao and co-workers

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(Nakao et al., 2010). Transversion mutations are generally less common than

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transition mutations whereas it was a feature of the nad1 gene among the E. shiquicus isolates analyzed in this study, suggesting that the nad1 gene is more susceptible to mutation than cox1, a feature different from that previously reported for the genus Echinococcus (Nakao et al., 2007). The gene sequences (H2 to H7 from the total of 71 cysts) determined in this study showed none had the same sequences as the reference haplotype (H1) of E. shiquicus samples which found only in the liver as reported by others (Xiao et al., 2005, 2006). The genetic differences between ours and these previous reports may reflect adaptive mutations for parasitism under environmentally selective pressure. Furthermore, the 13

ACCEPTED MANUSCRIPT two distinct clusters (Ⅰ and Ⅱ) we describe were clearly distinguishable, based on the constructed phylogenetic tree and the genetic divergence of the full-length cox1

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and nad1 genes for the collected E. shiquicus isolates. Moreover, with the exception of two isolates (Cysts 17 and 19), the majority of lung cysts were grouped into Cluster

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Ⅱ. Thus, we infer that the isolates in Cluster Ⅱ may represent a discrete strain or genotype of E. shiquicus considering their anatomical location and their relatively

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high nucleotide diversities in the constructed phylogeny, an issue worthy of further study.

Notably, none of the trapped O. curzoniae were infected with E. multilocularis in

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our investigation although a previous study in Darlag County (Fig. 1) indicated dogs

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had a high prevalence (11.8%) in this locality (Han et al., 2009). A new PCR-restriction endonuclease-based method was also developed in the

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current study. The procedure can be used to distinguish E. shiquicus from E.

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multilocularis, which will be of value with both species reported to infect O. curzoniae on the Tibetan plateau (Han et al., 2009; Xiao et al., 2006). The method has two basic advantages in that it can be completed in a relatively short time period (4-8 hours), and is less expensive than sequencing, due in part to the low cost of the EcoRI and SspI enzymes employed (Boufana et al., 2013; Han et al., 2009; Li et al., 2013; Xiao et al., 2006).

Acknowledgments This study was financially supported by Projects provided by the Science Fund for 14

ACCEPTED MANUSCRIPT Gansu Provincial Key Science and Technology Projects (1203NKDA039); Creative Research Groups of Gansu Province (1210RJIA006); Special Fund for Agro-scientific

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Research in the Public Interest (201303037; 200903036-07), the People’s Republic of China and National Health and Medical Research Council (NHMRC) Project

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(APP-1009539), Australia. We thank the veterinarians and all other colleagues at the Center for Animal Disease Prevention and Control of Darlag County, Qinghai

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Province. We are also particularly grateful to Rongchuan Xiong from Liupanshui Normal College for his advice on the haplotype network analysis. DPM is a NHMRC Senior Research Fellow and acknowledges financial support from NHMRC for his

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studies on echinococcosis.

Author’s contributions

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Conceived and designed the experiments: YLF ZZL DMP YRY WZJ. Performed

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the experiments: YLF ZZL LL QYL FZ JQL CNL JZC MTL WGS WZJ. Analyzed the data: YLF DPM LL WZJ. Contributed reagents/ materials/ analysis tools: YLF HBY LL ZZL MTL JZC YRY WZJ. Wrote the paper: YLF YRY DPM WZJ.

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ACCEPTED MANUSCRIPT Tables Table 1 Haplotypes* of E. shiquicus isolates, and their location in the organs of 19 trapped pikas. Cyst number/location

Parasite no.

Lungs

Spleen

Kidney

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Ref. isolate

Haplotype*

T

Pika no.

H1

Cyst 1

4

-

-

H2

Pika 2

Cyst 2

5

-

-

H2

Pika 3

Cyst 3

11

-

-

H2

Pika 4

Cyst 4

13

-

-

H3

Pika 5

Cyst 5

3

-

-

H3

Pika 6

Cyst 6

2

-

-

H4

Pika 7

Cyst 7

Pika 8

Cyst 8

Pika 9

Cyst 9

Pika 10

Cyst 10

Pika 11

Cyst 11

Pika 12

Cyst 12

Pika 13

Cyst 13

Pika 14

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Pika 1

-

-

H4

3

-

-

H5

2

-

-

H6

1

-

-

H6

4

-

-

H6

2

-

-

H6

1

-

-

H6

Cyst 14

6

-

-

H6

Pika 15

Cyst 15

2

-

-

H6

Pika 16

Cyst 16

3

-

-

H6

2

-

-

H7

Pika 18

Cyst 17 Cyst 18

-

-

1

H7

Cyst 19

4

1

-

H7

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Pika 19

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Pika 17

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1

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* Based on complete concatenated nad1 and cox1 gene sequences

Table 2 Mutation sites in the complete nad1 gene of different E. shiquicus haplotypes. Haplotype H1 (ref.) H2 H3 H4 H5 H6 H7

Mutation sites 30

39

136

246

280

330

397

405

483

486

528

534

606

696

745

894

G A

G A -

G A -

T C

G A A A A A

T C C -

T C

T G G G G G

A T

A T -

T A -

A G

T C C C C C

A C C C C C

A T

T C

-, Nucleotide is the same as the H1haplotype

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ACCEPTED MANUSCRIPT Table 3 Mutation sites in the complete cox1 gene of different E. shiquicus haplotypes. Mutation sites

Haplotype

16

27

225

240

327

384

459

474

582

747

777

819

H1 (ref.)

A

T

A

A

A

C

T

A

G

G

T

G

A

H2 H3 H4 H5 H6 H7

G

C -

G -

G G -

G

T

C

G G G G

A

849

873

882

888

1092

1104

1198

A G -

T C -

T C

G A -

G A A A A

G A A A A

C T

C -

A

G

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A -

1299

1300

1314

1462

1478

T C -

G A

C T T T T T T

C T

A G -

T C -

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H1 (ref.) H2 H3 H4 H5 H6 H7

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Figure Legends

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* -, Nucleotide is the same as the H1haplotype

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Fig. 1. Map showing the study sampled area of Darlag County, Qinghai Province,

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Fig. 2. Typical E. shiquicus cysts in a lung of the plateau pika (O. curzoniae). (Scale: 1 cm).

Fig. 3. Restriction endonuclease digestion of PCR-amplified E. shiquicus cyst mtDNA sequences. The restriction enzymes are marked at the top right hand corner. 1, E. multilocularis DNA fragment without enzyme digestion; 2, E. multilocularis DNA fragment with enzyme digestion; 3, E. shiquicus DNA fragment without enzyme 21

ACCEPTED MANUSCRIPT digestion; 4, E. shiquicus DNA fragment with enzyme digestion; 5-23, individual cyst

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sample DNA fragments (Cysts 1 to 19 except Cyst 16) with enzyme digestion.

Fig. 4. Neighbour-joining (NJ) tree constructed with the MEGA software version

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5.2.1 based on the complete nad1+cox1 gene sequences.

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Fig. 5. The frequency of each haplotype of complete nad1+cox1 genes of E. shiquicus in Darlag County, Qinghai, China calculated using the haplotype network software

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and the percentage of the different haplotypes is shown in blue.

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Highlights

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—– ‘Genetic diversity in Echinococcus shiquicus from the plateau pika (Ochotona curzoniae) in Darlag County, Qinghai, China’

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1. First report of extra-hepatic larval E. shiquicus in O. curzoniae 2. Unique finding showing E. shiquicus can be divided into two distinct genetic clusters. 3. Provision of a new DNA method to rapidly distinguish E. shiquicus from E. multilocularis.

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