Genetic Characterization and Phylogenetic Analysis of Fasciola Species Isolated From Yaks on Qinghai-Tibet Plateau, China

The present study determined the complete mitochondrial DNA (mt DNA) sequence of Fasciola intermediate (isolated from yaks) based on gene content and genome organization. According to our findings, the genome of Fasciola intermediate was 13,960 bp in length, containing 2 ribosomal RNA (rRNA) genes, 12 protein-coding genes (PCGs), and 22 transfer RNA (tRNA) genes. The A+T content of genomes was 63.19%, with A (15.17%), C (9.31%), G (27.51%), and T as the nucleotide composition (48.02%). Meanwhile, the results showed negative AT-skew (-0.52) and positive GC-skew (0.494). The AT bias significantly affected both the codon usage pattern and amino acid composition of proteins. There were 2715 codons in all 12 protein-coding genes, excluding termination codons. Leu (16.72%) was the most often used amino acid, followed by Val (12.74%), Phe (10.90%), Ser (10.09%), and Gly (8.39%). A phylogenetic tree was built using Maximum-Likelihood (ML) through MEGA 11.0 software. The entire mt DNA sequence of Fasciola intermediate gave more genetic markers for investigating Trematoda population genetics, systematics, and phylogeography. Hence, for the first time, our study confirmed that yaks on the Qinghai-Tibet plateau have the infestation of Fasciola intermediate parasite.


INTRODUCTION
Yak is a unique bovine specie on the Qinghai-Tibet plateau, China. The Qinghai-Tibet plateau is home to around 14 million yaks (a small amount of distribution in India, Bhutan, Sikkim, Afghanistan and Pakistan) (1). The yaks are necessary for herders in this area because of their milk, wool, and meat (2). Fasciolosis is one of the most important parasitic zoonotic issue, mainly caused by Fasciola hepatica (F. hepatica), Fasciola gigantica (F. gigantica) (3,4). The infection occurs mostly through the oral route (5), causing emaciation, fever, hepatomegaly, cholangitis, and jaundice, and even death (5). Approximately 2.4 million humans and more than 600 million animals are infected per year, causing serious public health threats and considerable economic loss to the livestock industry (6,7).
Previous research has identified the species of Fasciola in buffaloes and sheep, but not in yaks of the Qinghai-Tibet plateau. Therefore, in this study, the complete mitogenome of F. intermediate from yaks was initially sequenced and compared with other Trematoda mitogenomes (4

Gene Annotation and Sequence Analysis
DNA samples were randomly interrupted, the required length of DNA fragments were collected, the base "A" to 3 ′ -end was added, DNA fragments and 3 ′ -end were connected with "T" base special joint, and finally used for cluster preparation and sequencing. The mitogenome of F. intermediate was sequenced by nextgeneration sequencing (NGS). Two lanes for F. intermediate were sequenced as 400 bp reads using Illumina MiSeq (1 GB raw). The raw data was saved by Paired-End FASTQ and generated high-quality sequences by using AdapterRemoval (version 2) and SOAPec (version 2.01) based on K-mer distribution (13).
A5-miseqv20150522 and SPAdesv3.9.0 were used to assemble high-quality second-generation sequencing data to construct contig and scaffold sequences (14,15). Mitochondrial sequences of each splicing result were selected by Blastn (BLAST v2.2.31+), compared between the sequences with high sequencing depth and the NT library on NCBI. By mummerv3.1 software. The results of mitochondrial splicing were used for collinearity analysis. The location relationship was determined, and then gap between contig was filled (16).
The complete mitochondrial genome sequences of splicing were uploaded to MITOS web server (http://mitos.bioinf.unileipzig.de/) for functional annotation (17). The Genetic Code was set to 05-invertebrate; the rest was set as the default parameter by MITOS. The secondary structure of each predicted tRNA can be obtained in the MITOS web server. The complete genome circle map of mitochondria was drawn using CGview visualization software (18).

Phylogenetic Analysis
The taxonomic status of F. intermediate with available Trematoda was estimated by reconstructing phylogenetic trees. The complete nucleotide sequences of 36 Trematoda and one outgroup are available at GenBank (https://www.ncbi.nlm.nih. gov/genbank/).
Nucleotide sequences of each gene and their deduced amino acid sequences were aligned separately by using https://www. novoprolabs.com/ and MEGA 11.0 (19). The phylogenetic tree was built using Maximum-Likelihood (ML) through MEGA 11.0 software. The genome information used in this study is shown in Table 1.

A+T Skewness and Transfer RNAs
In the mitogenome of F. intermediate, the skew of AT was negative and the skew of GC was positive, indicating an obvious bias toward the use of T and A ( Table 3). Like most mt DNA, the F. intermediate mitogenome contained a set of 22 tRNAs genes. The tRNAs ranged in size from 57 to 71 bp. All the tRNA genes were present on the N strand and all the tRNA genes had the typical cloverleaf structure (Figure 3).

Phylogenetic Analysis
The phylogenetic relationship was analyzed based on the concatenated nucleotide sequences of 12 PCGs from 36 Trematoda and one outgroup. The result of analyses, generated a consistent tree topologies (Figure 4).
In this study, the ML analyses showed that each superfamily in the tree formed a monophyletic clade. Obviously, F. hepatica, F. intermediate, and F. gigantica clustered in one branch in the phylogenetic tree with high nodal support values (Figure 4), indicating that F. hepatica, F. intermediate, and F. gigantica have a sister group relationship. Additionally, the phylogenetic analyses revealed that F. hepatica, F. intermediate, and F. gigantica were grouped into one clade in fascioliases within Trematoda, which was consistent with a previous study (4).

Comparative mt Genomic Analyses With F. hepatica, F. intermediate, and F. gigantica
The complete mitogenome sequences in this study (F.

DISCUSSION
Fasciolosis is a zoonotic disease belonging to water-borne trematodes. The previous studies showed the prevalence of fasciolosis in Africa with the highest range in cattle (1.2-91.0%) and the lowest in sheep (0.19-73.7%). In America the highest range was in goats (24.5-100%) and the lowest in cattle (3.0-66.7%) in America. In Asia the highest range in cattle (0.71-69.2%) and lowest in goat (0.0-47.0%). In Australia, the highest range was in cattle (26.5-81.0%), and the lowest range was in sheep (5.5-52.2%). While in Europe, the highest range was in cattle (0.12-86.0%) and the lowest in goats (0.0-0.8%) (20). Annually 2000 million dollars are lost because of helminthic infection (21).
F. hepatica and F. gigantica are considered two effective species in the genus Fasciola. However, the researchers found that there was also an "intermediate type" of Fasciola (F. intermediate). This "intermediate type" of Fasciola was first discovered in Japan and was subsequently reported in China and South Korea (22). Many studies on Fascioliasis have limited information about the Fasciola types in yaks. Genome-based molecular identification has been more commonly employed     atp6  172  172  172  172  9  25  6   nad1  300  300  300  300  9  20  0   nad2  288  288  288  288  5  31  0   nad3  118  118  118  118  1  9  0   nad4  422  422  423  422  10  38  7   nad4L  90  90  90  90  0  4  1   nad5  521  521  521  521  1  58  5   nad6  150  150  150  150  0  9  11   cox1  513  513  513  513  4  24  3   cox2  200  200  200  200  0  10  1   cox3  213  213  213  213  1  25  5   cytb  370  370  370  370  2  17  5 to identify biological types in recent years due to advances in biotechnology. mt DNA is an ideal genetic marker due to the simple and stable structure, reflecting the maternal genetic background (23). Our study showed that F. intermediate from yak and F. intermediate from bovine belonged to the same branch and it was to be similar to F. gigantica compared with the F. hepatica through phylogenetic analysis. We compared the whole mitochondrial genome between Fi, Fin, Fg, and Fh. The results showed that Fi was no base site mutations in rrnL and rrnS compared with Fin, 10 base site mutations in rrnL and rrnS compared with Fg. Compared with Fh, Fi there were 99 base site mutations in rrnL and 92 base site mutations in rrnS, respectively. Hence, it would be the ideal genetic marker to distinguish the three species of Fasciola. Meanwhile, Fi had a deletion of nearly 500 bp in the AT-loop area, compared with Fin, Fg, and Fh, which may be due to the adaptability to plateau for Fi.

CONCLUSION
In this study, we sequenced the complete 13,960 bp mitogenome of F. intermediate from yaks, in which 36 genes (12 PCGs, 22tRNA genes and 2 rRNA genes) were located as a typical of Trematoda mitogenome. All PCGs were initiated by ATN codon, the cob genes had incomplete stop codons consisting of just T, and the other 11 PCGs stop with the canonical TAA or TAG. The AT-skew were negative, and the GC-skew were positive in the mitogenomes of F. intermediate, consistent with most sequenced Trematoda. The phylogenetic analyses support that F. intermediate from yaks was the same as the F. intermediate from bovine. This study would provide a basis for the accurate prevention, diagnosis, and treatment of Fasciolosis in yaks.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

ETHICS STATEMENT
The samples were collected under the permission of the relevant institutions. All procedures were approved and performed by Laboratory Animals Research Centre of Hubei province and Qinghai province in P.R. China, and the Ethics Committee of Huazhong Agricultural University, China (Permit number: 4200695757). All animal experiments and procedures were conducted under the relevant procedures of Proclamation of the Standing Committee of Hubei People's congress (PSCH No. 5), China.