First report of Taenia laticollis and a new species, “Taenia sp. Eurasian lynx” from the Eurasian lynx (Lynx lynx) in Northwestern China

Background The Eurasian lynx (Lynx lynx) is a medium-sized wild cat species distributed throughout Eurasia, from Europe to the Far East. There has been no report on Taenia species (Cestoda: Cyclophyllidea) infecting this felid in China. Methods c and phylogenetic of the that i) a single tapeworm from lynx #2 shared 100% identity with Taenia laticollis genotype C detected in in and the remaining 23 tapeworms, provisionally named here as “Taenia sp. Eurasian lynx”, had two nucleotide substitutions but phylogenetically clustered together. The latter showed only 93.25% sequence identity to T. hydatigena from sheep (Ovis aries) in Slovakia. The scolex morphology is characteristic enough to distinguish “Taenia sp. Eurasian lynx” from other species of its genus.


Background
The Eurasian lynx (Lynx lynx) is a very widespread species, with a distribution range covering much of Europe and Asia. It has ve subspecies (northern lynx, Balkan lynx, Carpathian lynx, Caucasian lynx, Siberian lynx and Central Asian lynx). The Central Asian lynx inhabits Afghanistan, Bhutan, China, India, Kazakhstan, Kyrgyzstan, Nepal, Pakistan, Tajikistan, Turkmenistan and Uzbekistan [1].
In China, the Central Asian lynx is mainly distributed in Xinjiang, Qinghai, Gansu, Inner Mongolia and Tibet Province. The aim of the present study was to identify tapeworms in the Eurasian lynx (Lynx lynx) from Xinjiang, northwestern China.

Sample collection
Two Eurasian lynxes were found during our eld investigation on ticks and eas in the West Junggar Mountains, Xinjiang, northwestern China. One (adult female, #1) was road-killed in 2018. Another (adult male, #2) died due to natural causes in 2019. During a routine necropsy of the small intestine, 9 and 15 tapeworms were collected from lynxes #1 and #2, respectively (Supplementary Fig. 1). All tapeworms were washed in physiological saline prior to DNA extraction or morphological identi cation.
DNA extraction and molecular-phylogenetic analyses DNA was extracted from a small part of the strobila using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China). Molecular identi cation was performed from all tapeworm specimens based on two genetic markers of their mitochondrial genome: a 450-bp-long fragment of the cytochrome c oxidase subunit I (cox1) gene and a 526-bp-long fragment of the 16S rDNA (16S rDNA) gene as reported previously [10,11]. Sequences from this study were compared to those in GenBank with the BLASTn program (https://blast.ncbi.nlm.nih.gov). New sequences were deposited in GenBank (cox1: MW846305, MW846313 and MW843568; 16S rRNA: MW854635, MW854636 and MW843496). A phylogenic tree was constructed using the Neighbor-Joining method in MEGA 7.0.

Morphological identi cation
The staining procedure was performed as reported [12]. Brie y, tapeworm specimens were sequentially xed with 30%, 50% and 70% ethanol, and stained with acetate carmine. The decolorization was done with hydrochloric acid in alcohol (2 ml hydrochloric acid and 100 ml 70% ethanol). For the dehydration, 80%, 95% and 100% alcohol solutions were used sequentially, and then transparency was ensured with xylene. Finally, the specimens were mounted in Canada balsam. The scolex, neck, strobila (mature proglottid, gravid proglottid) of tapeworms were observed by microscopy.

Ethical approval
This study was approved by the Animal Ethics Committee of Shihezi University (Approval No. A2018-143-01).

Molecular identi cation
Molecular and phylogenic analysis of cox1 sequences showed that i) a single tapeworm from lynx #2 shared 100% sequence identity with T. laticollis genotype C (JX860623) found in Eurasian lynx in Finland, ii) the remaining 23 tapeworms, provisionally named here as "Taenia sp. Eurasian lynx", had two nucleotide substitutions but clustered together in the phylogenetic tree. These showed only 93.25% (400/444 bp) sequence identity to T. hydatigena (MW336935) from sheep (Ovis aries) reported in Slovakia (Fig. 1). Analysis of the COX1 protein amino acid sequences showed that "Taenia sp. Eurasian lynx" differs from T. hydatigena, Taeniaregis and T. lynciscapreoli with 2-4 amino acids ( Supplementary  Fig. 2).

Morphological description
The tapeworm, identi ed here as T. laticollis, measured 8 cm in length and 0.25 cm in width, similarly to T. laticollis from lynx reported in Alaska and Canada [13]. Out of the remaining 23 tapeworms, tentatively referred to as "Taenia sp. Eurasian lynx", three specimens were measured and further characterized (Fig.   2). Their lengths were 60.8 cm, 67.9 cm and 62.8 cm, whereas their widths were 0.51 cm, 0.60 cm and 0.64 cm. The diameter of their scolex, rostellum and sucker were 1474-1730 mm, 641-657 mm and 384-448 mm, respectively. Further measurements (ratio of total length to total width, posterior length, anterior length and guard length about large and small rostellar hooks) are shown in Supplementary Table 1.

Discussion
Here we report a novel Taenia species, provisionally named as "Taenia sp. Eurasian lynx", from the Eurasian lynx. Some common herbivorous prey items of this wild felid, such as the common hare (Lepus capensis) and the ibex (Capra ibex) are the most likely candidates to act as intermediate hosts of this novel species, which should be investigated further. In addition, another tapeworm species, T. laticollis was found in Eurasian lynx for the rst time in China.
"Taenia sp. Eurasian lynx", is phylogenetically closely related to T. hydatigena, and together these form a sister clade to T. regis reported from lion (Panthera leo) in Kenya and T. lynciscapreoli from the grey wolf, Eurasian lynx in Russia, Finland and Poland [4,5,[13][14][15]. Analysis of the COX1 protein amino acid sequences showed that in comparison with T. hydatigena, T. regis and T. lynciscapreoli, "Taenia sp. Eurasian lynx" has 2-4 amino acids substitutions (Supplementary Fig. 2). These ndings con rm "Taenia sp. Eurasian lynx" as a novel Taenia species, which is also supported by the morphological characteristics of the scolex and rostellar hooks showing differences from those of other Taenia spp. that are known to infect lynxes [4,15].
As reported previously, the de nitive hosts of T. laticollis include the Eurasian lynx, the Canada lynx (Lynx canadensis), the timber wolf (Canis lupus lycaon) and the coyote (Canis latrans) [16 -19]. Here T. laticollis haplotype C was also found in Eurasian lynx in China. This nding suggests that the host speci city of T. laticollis in lynxes and wolves should be evaluated further according to haplotypes.
The de nitive hosts of T. hydatigena, the species closest related to "Taenia sp. Eurasian lynx", include the coyote (Canis latrans), the timber wolf (Canis lupus lycaon), the black-backed jackal (Lupulellamesomelas), the Eurasian lynx (Lynx lynx), the Canada lynx (Lynx canadensis), the brown bear (Ursus arctos), whereas its intermediate hosts are the black-tailed jack rabbit (Lepus californicus), the European ground squirrel (Spermophilus citellus), domesticated ruminants, the springbok (Antidorcas marsupialis), the black wildebeest (Connochaetus gnou), the impala (Aepyceros melampus), the hartebeest (Alcelaphus buselaphus), the blue wildebeest (Connochaetus taurinus), the blesbuck (Damaliscus pygargus phillipsi) and the tsessebe (Damaliscus lunatus) [5,7,13,17,18,[20][21][22]. The West Junggar Mountains, between the Tianshan and Altai mountain belts, are located on the western rim of the Junggar Basin in northwestern China. Their landscape can be characterized as discontinuous treeless hills, with an altitude range from 2000-3000 m above sea level. In this region, the wildlife fauna relevant to the life cycles of cyclophyllidean cestodes of the family Taeniidae include the Pallas's cat (Otocolobus manul), the Eurasian lynx (Lynx lynx), the snow leopard (Panthera uncia), the grey wolf (Canis lupus), the red fox (Vulpes vulpes), the corsac fox (Vulpes corsac) and the Cape hare (Lepus capensis), all of which are sporadically distributed [23]. Since the number of Eurasian lynxes is limited in the West Junggar Mountains, other felids, as well as canids and lagomorphs should be investigated to systematically explore the de nitive and intermediate hosts of T. laticollis and "Taenia sp. Eurasian lynx" in the near future.

Declarations
Ethical approval and consent to participate This study was approved by the Animal Ethics Committee of Shihezi University (Approval No. A2018-143-01).

Consent for publication
Not applicable.

Availability of data and materials
The sequences obtained and analyzed during the present study are deposited in the GenBank database, under the following accession numbers: MW846305, MW846313 and MW843568 (cox1); MW854635, MW854636 and MW843496 (16S rRNA). Phylogenetic relationships of Taenia species from two Eurasian lynxes (marked with black circle and triangle) based on cox1 sequences.