Analysis of the complete Fischoederius elongatus (Paramphistomidae, Trematoda) mitochondrial genome

Fischoederius elongates is an important trematode of Paramphistomes in ruminants. Animals infected with F. elongates often don’t show obvious symptoms, so it is easy to be ignored. However it can cause severe economic losses to the breeding industry. Knowledge of the mitochondrial genome of F. elongates can be used for phylogenetic and epidemiological studies. The complete mt genome sequence of F. elongates is 14,120 bp in length and contains 12 protein-coding genes, 22 tRNA genes, two rRNA genes and two non-coding regions (LNR and SNR). The gene arrangement of F. elongates is the same as other trematodes, such as Fasciola hepatica and Paramphistomum cervi. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes by Maximum-likelihood and Neighbor-joining analysis method showed that F. elongates was closely related to P. cervi. The complete mt genome sequence of F. elongates should provide information for phylogenetic and epidemiological studies for F. elongates and the family Paramphistomidae.


Findings
Background Paramphistomes are distributed worldwide and have been reported in many countries, such as Bulgaria, France, Poland, Hungary, Italy, India, Russia, Sardinia and Yugoslavia [1]. The paramphistome can infect fishes, reptiles, birds and mammals, some of which can lead to huge economic losses related to seriously gastrointestinal diseases, low producitivity or death in ruminants [2]. In Arumeru District, the prevalence rate of paramphistomes is as high as 56.7 % in cattle [3].
Fischoederius elongates is an important member of paramphistomes, the parasite usually inhabits the rumen of cattle, buffaloes, sheep and goats. Ruminants are usually infected by ingesting snails, such as Lymnaea acuminata, Lymnaea succinea or Gyraulus euphraticus [4]. Ruminants infected with F. elongates show weakness, mental fatigue and eventually death. More seriously, F. elongates maybe a zoonotic trematode, a Chinese woman from Guangdong Province was reported to be the first human infection case [5], but it is still unknown how she was infected.
Untill now, the most common diagnostic method for F. elongates is the microscopical examination, but it's time-consuming, and hard to distinguish with other paramphistomes. As a useful marker, mt genome has been widely used for species identification [6][7][8][9][10]. The complete mt genome of F. elongates can provide alternative molecular markers for the species identification, epidemiology and genetic diversity of paramphistomes.
In the present study, we got the full sequence and gene arrangement of mt genome of F. elongates and compared it with selected trematodes. We found that F. elongates had the closest relationship with P. cervi.

Ethical approval
The study was performed under the instructions and approval of Laboratory Animals Research Centre of Hubei province in P. R. China and the ethics committee of Huazhong Agricultural University (Permit number: 4200695757).
Parasite collection and DNA isolation F. elongates adults were collected from the rumen and reticulum of naturally infected cattle in Zhanggang, Tianmen, Hubei province, PR China, according to the Animal Ethics Guidelines of Huazhong Agricultural University. Then, the adult worms were washed extensively in 0.9 % sodium chloride solution, and identified through morphological examinations [2]. Subsequently, one worm was stained for identification [11], and the rest were fixed in 75 % alcohol (V/V) and stored at −20°C until use [12]. Total genomic DNA was isolated from one worm [13]. The ITS-2 region of F. elongates was amplified and sequenced as reported previously [14], it was 100 % similar to that of F. elongates (GenBank accession no. JQ688410.1).
Then, 12 additional primers (Table 1) were designed based on the obtained sequencing results to amplify six regions from genomic DNA (~40-80 ng) by long-PCR. . Amplicons were cloned into pGEM-T-Easy vector (Promega, USA) and then sequenced using a primer-walking strategy [18].

Sequence analyses
F. elongates mt genome sequences were assembled manually and then aligned with the mt genome sequences of F. hepatica, C. sinensis and P. cervi using the program Clustal X 1.83 [19]. Open reading frames were identified by ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) using the echinoderm and flatworm mitochondrial code. Initiation and termination codons of the 12 protein-coding genes were identified as reported [15]. The 22 tRNA genes were predicted using tRNAscan-SE or manual adjustments [20,21]. The two rRNA genes were predicted by comparison with those of F. hepatica [15], C. sinensis [16] and P. cervi [17]. Amino acid sequences of 12 proteincoding genes were inferred using ExPASy Translate tool (http://web.expasy.org/translate/) using the echinoderm and flatworm mitochondrial codes, and aligned using MEGA 5.0 with default settings [22].

Nucleotide variation analysis
The nucleotide variation between F. elongates and P. cervi was analysed by sliding window analysis as reported [17].
Overlapping nucleotides between mt genes of F. elongates ranged from 1 to 53 bp ( Table 2). The F. elongates mt genome has 26 intergenic spacers ranging from 1 bp to 148 bp in length ( Table 2). The nucleotide contents of A, C, T and G in the mt genome are 19.78 %, 9.62 %, 44.10 % and 26.50 %, respectively (Table 3), with T being the most favored nucleotide, followed by G, A and C, which is also the same as the mt genomes of F. hepatica   [15], C. sinensis [16] and P. cervi [17]. The A + T content of 12 protein coding genes and 22 rRNA genes of F. elongates ranged from 59.65 % (rrnS) to 66.97 % (cox3), and the overall A + T content of the mt genome is 63.88 %. The present F. elongates mt genome can provide useful information for the studies of epidemiology, species identification and genetic diversity of Fischoederius spp. At the same, it will also make contribution to the taxonomy study of Fischoederius spp. With the full mt genome of F. elongates, we can undertake a study within F. elongates from different regions or among Fischoederius spp. by combining the morphological features with genetic analyses (with molecular markers from mitochondria or ribosome, such as cox1, nad4, 18S, ITS-1 and ITS-2). Meanwhile, the mt genome of F. elongates may also provide information for the prevention and diagnosis of Fischoederius spp. and perhaps, this mt genome information may assist in the new drug, since mitochondria is the target of some drugs, such as decoquinate.

Transfer RNA and ribosomal RNA genes
The F. elongates mt genome encodes 22 tRNAs, and the length of 22 tRNA genes ranged from 60 bp to 71 bp ( Table 2). There are two non-coding regions in F. elongates mt genome, rrnS (751 bp) and rrnL (995 bp) ( Table 2). The location of rrnS is between tRNA-Cys and cox2 and the rrnL is between tRNA-Thr and tRNA-Cys, which is the same as other trematodes, such as F. hepatica [15], C. sinensis [16] and P. cervi [17].

Non-coding regions
Many flatworms have non-coding regions, it's common to find two non-coding regions in trematodes: one long  non-coding region (LNR) and one short non-coding region (SNR). In F. elongates, there is a short non-coding region (SNR: 62 nucleotides), which is located between cytb and nad4L. In addition, there is also a long non-coding region (LNR: 468 nucleotides) between tRNA-Phe and cox3 (Table 2), the LNR has two obvious features, one is microsatellite-like sequences, such as (TA)n (n <5); the other is homopolymer sequences, such as (T)n (n <7). People still don't understand clearly why the non-coding regions exist, and the function of them, people just knew the non-coding regions may participate in the replication of mitochondria [26].
Nucleotide variability between F. elongates and P. cervi A sliding window analysis of F. elongates and P. cervi using full mt genome sequences reflected the nucleotide diversity (π) for all the protein-coding genes (Fig. 2). The highest and lowest level of nucleotide variability was within nad6 and cox3, respectively. In our study, nad6 and cox2 are the most conserved genes, and cox3 and atp6 are the least conserved. With sliding window analysis, we could know the conserved regions of mt genome among species.

Genetic relationships
Concatenated amino acid sequence data representing 12 protein-coding genes of 11 digenean species (C. sinensis, D. dendriticum, F. hepatica, H. taichui, M. yokogawai, O. viverrini, P. cervi, S. haematobium, S. japonicum, S. mekongi and S. spindale) and one tapeworm (T. solium) were used for genetic relationship analysis (Fig. 3). In the tree, we can find two large clades with strong support (100 %): one clade consists of eight members representing five families (Heterophyidae, Opisthorchiidae, Fasciolidae, Paramphistomidae and Dicrocoeliidae); the other clade is Schistosomatidae. In the present analysis, F. elongates has the closest genetic relationship with P. cervi (100 %), followed by Fasciolidae, this is consistent with their relationship in the classification of biology. At the same time, we also used NJ method analysis (not shown), and there was no difference between these two methods.