Analysis of the complete Fischoederius elongatus (Paramphistomidae, Trematoda) mitochondrial genome
- Xin Yang†1,
- Yunyang Zhao†1,
- Lixia Wang2,
- Hanli Feng3,
- Li Tan1,
- Weiqiang Lei1,
- Pengfei Zhao1,
- Min Hu1 and
- Rui Fang1Email author
© Yang et al.; licensee BioMed Central. 2015
Received: 17 March 2015
Accepted: 9 May 2015
Published: 20 May 2015
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.
Paramphistomes are distributed worldwide and have been reported in many countries, such as Bulgaria, France, Poland, Hungary, Italy, India, Russia, Sardinia and Yugoslavia . 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 . In Arumeru District, the prevalence rate of paramphistomes is as high as 56.7 % in cattle .
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 . 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 , 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 [6–10]. 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.
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 . Subsequently, one worm was stained for identification , and the rest were fixed in 75 % alcohol (V/V) and stored at −20 °C until use . Total genomic DNA was isolated from one worm . The ITS-2 region of F. elongates was amplified and sequenced as reported previously , it was 100 % similar to that of F. elongates (GenBank accession no. JQ688410.1).
Amplification and sequencing of F. elongates mt genome
Primers used in the present study
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. PCRs (50 μl) were performed in reactions containing 0.4 mM each of dNTPs, 5 μl 10× LA Taq buffer II(Mg2+ Plus), 2.5 μM of each primer, 2.5 U LA Taq polymerase (Takara) and 2.5 μl genomic DNA. And the reactions were run under the following program: 94 °C for 5 min, followed by 35 cycles of 94 °C/30 s, 50 °C/30 s and 72 °C/1-5 min (depending on the size of F. hepatica). Amplicons were cloned into pGEM-T-Easy vector (Promega, USA) and then sequenced using a primer-walking strategy .
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 . 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 . 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 , C. sinensis  and P. cervi . Amino acid sequences of 12 protein-coding 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 .
Nucleotide variation analysis
The nucleotide variation between F. elongates and P. cervi was analysed by sliding window analysis as reported .
Amino acid sequences translated from individual genes of the mt genome of F. elongates were aligned with those predicted from mt genomes of selected trematodes, including C. sinensis (NC_012147) , Dicrocoelium dendriticum (NC_025280.1) , F. hepatica (NC_002546) , Haplorchis taichui (NC_022433.1) , Metagonimus yokogawai (KC330755.1), Opisthorchis viverrini (JF739555.1) , P. cervi (NC_023095.1) , Schistosoma haematobium (NC_008074) , Schistosoma japonicum (AF215860) , Schistosoma mekongi (NC_002529) , Schistosoma spindale (NC_008067) , and the cestode Taenia solium (outgroup) (NC_004022.1) . The amino acid sequences of selected trematodes were aligned using MEGA 5.0 , and phylogenetic analysis of the aligned amino acid sequences was conducted in MEGA 5.0 using the Maximum Likelihood (ML) method.
Results and discussion
Features of the mt genome of F. elongates
The complete mitochondrial genome of F. elongates (GenBank accession no. KM_397348) is 14,120 bp in length. The length of the F. elongates mt genome is larger than the mtDNA genomes of C. sinensis (13,875 bp) and S. japonicum (14,085 bp), but smaller than D. dendriticum (14,884 bp), F. hepatica (14,462 bp), H. taichui (15,130 bp), M. yokogawai (15,258 bp), S. haematobium (15,003 bp), S. mekongi (14,072 bp) and S. spindale (16,901 bp).
The organization of the mitochondrial genome of Fischoederius elongatus
Number of aa1
Nucleotide contents of genes and the non-coding region within the mitochondrial genome of Fischoederius elongatus
A + T(%)
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.
Codon usage for 12 protein-coding genes in the mitochondrial genome of Fischoederius elongatus
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 , C. sinensis  and P. cervi .
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 .
Nucleotide variability between F. elongates and P. cervi
Sincere thanks to Professor Bang Shen for comments on the manuscript. This work was supported in part by the “National Key Basic Research Program (973 Program) of China” (Grant No. 2015CB150300), the “Special Fund for Agro-scientific Research in the Public Interest” (Grant No. 201303037) and “Huazhong Agricultural University Students Research Fund” (Grant No. 2015054).
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