# Characterization of the Complete Mitochondrial Genome ofFischoederius elongatus Derived from Cows in Shanghai, China

Authors: Zhaoqing Han; Kun Li; Houqiang Luo; Muhammad Shahzad; Khalid Mehmood
Journal: BioMed Research International
 (2020)

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## Abstract

A study was conducted to reveal the characterization of the complete mitochondrial genome ofFischoederius elongatus derived from cows in Shanghai, China. Results indicated that the complete mt genome of F. elongatus was 14,288 bp and contained 12 protein-coding genes (cox1-3, nad1-6, nad4L, atp6, and cytb), 22 transfer RNA genes, and two ribosomal RNA genes (l-rRNA and s-rRNA). The overall A + T content of the mt genome was 63.83%, and the nucleotide composition was A (19.83%), C (9.75%), G (26.43%), and T (44.00%). A total of 3284 amino acids were encoded by current F. elongatus isolate mt genome, TTT (Phe) (9.84%) and TTG (Leu) (7.73%) codon were the most frequent amino acids, whereas the ACC (Thr) (0.06%), GCC (Ala) (0.09%), CTC (Leu) (0.09%), and AAC (Asn) (0.09%) codon were the least frequent ones. At the third codon position of F. elongatus mt protein genes, T (50.82%) was observed most frequently and C (5.85%) was the least one. The current results can contribute to epidemiology diagnosis, molecular identification, taxonomy, genetic, and drug development researches about this parasite species in cattle.


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## Body

## 1. Introduction

Fischoederius elongatus is a representative of Paragonimus genus, which was frequently discovered in ruminants in tropical and subtropical regions [1]. Weight loss and decease of milk production were the common effects of infections caused by F. elongatus [2]. Due to the serious economic losses caused by this trematode, a growing trend of attentions was paid towards the F. elongatus [3]. According to National Bureau of Statistics of China, population of 89.15 and 297.13 million heads of cattle and sheep, respectively, was estimated in 2018 (http://data.stats.gov.cn/easyquery.htm?cn=C01). In a study, the prevalence of F. elongatus infection in cattle and sheep was found to be 50% and 10%, respectively, in Jiangjin, China [4]. However, little knowledge is known about the genetic information of F. elongatus in cows in China.As a maternal inheritance circular genome, mitochondrial genome is popularly utilized in studies for taxonomy and phylogenetic analysis [5, 6]. A higher mutational rate has been observed in mt DNA than that of nuclear DNA [7], and due to the alterations in contents of mt DNA, it has been reported to be highly related to various diseases [8]. Hundreds of parasitic mt genomes are available at NCBI (https://www.ncbi.nlm.nih.gov/nucleotide/); however, there is relatively a lack of data about the mt genomes of trematodes. Until now, to the best of our knowledge, only one report is available about the mt genome of F. elongatus isolated from cattle [3]. The present research herein was aimed to reveal the characterization of the complete mt genome of F. elongatus derived from cows in Shanghai, China.


## 2. Materials and Methods

### 2.1. Ethical Statement

All procedures adopted in the current research were followed according to the laws, regulations, and guidelines of the Laboratory Animals Research Centre of Hubei province, P. R. China, and the Ethics Committee of Huazhong Agricultural University.


### 2.2. Parasite Collection

Adult trematodes were collected from the cows in Shanghai in 2019. Morphological examination was conducted after extensive washing in 0.9% sodium chloride solution [9]. All the samples were fixed in 75% alcohol (V/V) and kept at −2°C for further utilization as narrated in a previous research [5].


### 2.3. Mitochondrial DNA Sequencing

The extraction ofmt DNA of F. elongatus was performed by employing a commercial Mitochondria Isolation Kit (Sigma-Aldrich, China). The agarose gel electrophoresis method and nanodrop detection were used for the integrity and purity of DNA. All the DNA samples were quantified via a QubitFluorometer (3.0). DNA samples were disrupted into fragments randomly via the ultrasonic method. End repair, A-tailing, index adapter adding, amplification, and purification were performed for library constriction according to the manufacturer’s instructions (Illumina). These libraries were sent to commercial sequencing via an Illumina HiSeq X sequencing system at Personalbio in Shanghai, China.


### 2.4. Sequencing Analysis and Genome Annotation

To obtain high accurate sequencing clean data, all the obtained raw reads were filtered with quality score (Q < 10) (90%), uncalled bases (“N” characters) (>10%), and duplicated sequences. The mt genome of F. elongatus was assembled via SPAdes v3.11.1 (http://cab.spbu.ru/software/spades/). The mt genome assembling and annotation of F. elongatus were performed online using the DOGMA tool and MITOS [10, 11]. The circular mt genome of the F. elongatus genomic map was drawn via OGDraw v1.2 [12].


### 2.5. Nucleotide Variation Analysis

The nucleotide variation ofF. elongatus between the Tianmen isolate (KM397348.1) and the current isolate was analyzed by employing DnaSp 5.0.


### 2.6. Phylogenetic Analysis

The phylogenetic relationships ofF. elongatus and other available trematodes were based on mt genome using the neighbor-joining method with Kimura two-parameter analysis and bootstrap analysis of 1000 replicates (MEGA 6.0). The available trematodes were F. elongatus (KM397348.1), F. cobboldi (KX169164.1), Gastrothylax crumenifer (KM400624.1), Paramphistomum cervi (KT198987.1, KF475773.1), Calicophoron microbothrioides (KR337555.1), Orthocoelium streptocoelium (KM659177.1), Explanatum explanatum (KT198989.1), Homalogaster paloniae (KT266674.1, KX169165.1), and Ogmocotyle sp. (KR006935.1). The numbers on the branches indicate the percentage of replicates that reproduced the topology for each clad.


## 2.1. Ethical Statement

All procedures adopted in the current research were followed according to the laws, regulations, and guidelines of the Laboratory Animals Research Centre of Hubei province, P. R. China, and the Ethics Committee of Huazhong Agricultural University.


## 2.2. Parasite Collection

Adult trematodes were collected from the cows in Shanghai in 2019. Morphological examination was conducted after extensive washing in 0.9% sodium chloride solution [9]. All the samples were fixed in 75% alcohol (V/V) and kept at −2°C for further utilization as narrated in a previous research [5].


## 2.3. Mitochondrial DNA Sequencing

The extraction ofmt DNA of F. elongatus was performed by employing a commercial Mitochondria Isolation Kit (Sigma-Aldrich, China). The agarose gel electrophoresis method and nanodrop detection were used for the integrity and purity of DNA. All the DNA samples were quantified via a QubitFluorometer (3.0). DNA samples were disrupted into fragments randomly via the ultrasonic method. End repair, A-tailing, index adapter adding, amplification, and purification were performed for library constriction according to the manufacturer’s instructions (Illumina). These libraries were sent to commercial sequencing via an Illumina HiSeq X sequencing system at Personalbio in Shanghai, China.


## 2.4. Sequencing Analysis and Genome Annotation

To obtain high accurate sequencing clean data, all the obtained raw reads were filtered with quality score (Q < 10) (90%), uncalled bases (“N” characters) (>10%), and duplicated sequences. The mt genome of F. elongatus was assembled via SPAdes v3.11.1 (http://cab.spbu.ru/software/spades/). The mt genome assembling and annotation of F. elongatus were performed online using the DOGMA tool and MITOS [10, 11]. The circular mt genome of the F. elongatus genomic map was drawn via OGDraw v1.2 [12].


## 2.5. Nucleotide Variation Analysis

The nucleotide variation ofF. elongatus between the Tianmen isolate (KM397348.1) and the current isolate was analyzed by employing DnaSp 5.0.


## 2.6. Phylogenetic Analysis

The phylogenetic relationships ofF. elongatus and other available trematodes were based on mt genome using the neighbor-joining method with Kimura two-parameter analysis and bootstrap analysis of 1000 replicates (MEGA 6.0). The available trematodes were F. elongatus (KM397348.1), F. cobboldi (KX169164.1), Gastrothylax crumenifer (KM400624.1), Paramphistomum cervi (KT198987.1, KF475773.1), Calicophoron microbothrioides (KR337555.1), Orthocoelium streptocoelium (KM659177.1), Explanatum explanatum (KT198989.1), Homalogaster paloniae (KT266674.1, KX169165.1), and Ogmocotyle sp. (KR006935.1). The numbers on the branches indicate the percentage of replicates that reproduced the topology for each clad.


## 3. Results and Discussion

In our study, the completemt genome of the F. elongatus isolate was 14,288 bp long (Figure 1), which is longer (by 168 bp) than that of the F. elongatus isolated from Tianmen, China (14,120 bp) [3]. The difference may be because of employing different techniques and possibly the genetic prediction error; however, the gene and length are in line with each other. The present sequence of the mt genome has been submitted to the GenBank with the Accession number: MN537973. The circular mt genome of F. elongatus contains 12 protein-coding genes (cox1-3, nad1-6, nad4L, atp6, and cytb), 22 transfer RNA genes, and two ribosomal RNA genes (l-rRNA and s-rRNA) (Figure 1, Table 1); however, it lacks atp8, which is in line with F. elongatus of the Tianmen isolate and mt genomes of other trematodes, such as Gastrothylax crumenifer and Paramphistomum cervi [3, 13, 14]. The protein-coding genes of current F. elongatus isolates were transcribed in the same direction, and those genes were assembled in line of cox3, cytb, nd4L, nd4, atp6, nd2, nd1, nd3, cox1, l-rRNA, s-rRNA, cox2, nad6, and nad5 which was in accordance with previously reported results [3, 13, 14] (Figure 1; Table 1).Figure 1
Arrangement of themt genome of F. elongatus.Table 1
Themt genome of F. elongatus isolated from cows using MITOS.
Gene
Position
Length (bp)
Start/stop codon of PCGs
Anticodons
COX3
1–645
645
ATG/TAG
tRNA-His
646–713
68
GTC
CYTB
714–1829
1116
ATT/TAA
AT-loop
1830–1892
63
ND4L
1893–2156
264
ATG/TAG
ND4
2117–3397
1281
GTG/TAA
tRNA-Gln
3409–3471
63
GTT
tRNA-Phe
3485–3549
65
TTC
tRNA-Met
3549–3612
64
AGA
ATP6
3613–4128
516
ATG/TAG
ND2
4133–5008
876
GTG/TAG
tRNA-Val
5041–5101
61
AAG
tRNA-Ala
5109–5179
71
AAC
tRNA-Asp
5431–5500
70
CAT
ND1
5530–6399
870
TTG/TAG
tRNA-Asn
6419–6484
66
TGG
tRNA-Pro
6489–6552
64
CAG
tRNA-Ile
6554–6616
63
CCG
tRNA-Lys
6623–6687
65
CTG
ND3
6701–7048
348
GTG/TAG
tRNA-Ser
7060–7119
60
GAG
tRNA-Trp
7132–7196
65
AGT
COX1
7200–8741
1542
GTG/TAA
l-rRNA
8575–9865
1291
tRNA-Thr
8751–8814
64
GAA
s-rRNA
9815–10,603
789
COX2
10,624–11,205
582
ATG/TAG
ND6
11244–11651
408
ATG/TAG
tRNA-Tyr
11,673–11,737
65
CAG
tRNA-Leu
11,757–11,820
64
TAA
tRNA-Ser
11,822–11,890
69
GTG
tRNA-Leu
11,897–11,961
65
ATA
tRNA-Arg
11,965–12,030
66
TGA
ND5
12,031–13,611
1581
GTG/TAG
tRNA-Gly
13,615–13,679
65
ACG
tRNA-Glu
13,692–13,755
64
TTG
AT-loop
13,756–14,228
473The overall A + T content of themt genome of the current F. elongatus isolate was found to be 63.83%, and the nucleotide composition was A (19.83%), C (9.75%), G (26.43%), and T (44.00%). Moreover, T was the most favored nucleotide, while C was the least common one. These findings are also in accordance with the isolate results of Tianmen [3].Among the 12 protein genes of the presentF. elongatus isolate, ATG (5/12) and GTG (5/12) were the most common start codons and TAA (9/12) was the predominant stop codon (Table 1). In current results herein, the 3′-end of genes of nd1, nd3, nd4, nd5, dn6, atp6, cox3, and cox1 was found immediately adjacent to a downstream tRNA gene (Table 1), which was in parallel arrangement with F. elongatus Tianmen isolates of Gastrothylax crumenifer and Paramphistomum cervi [3, 13, 14].In our study, a total of 3284 amino acids were encoded from theF. elongatus isolate mt genome excluding the termination codons. All of the 63 possible codons except CGC were found in the F. elongatus isolate. TTT (Phe) (9.84%) and TTG (Leu) (7.73%) codon were the most frequent amino acids found, whereas the ACC (Thr) (0.06%), GCC (Ala) (0.09%), CTC (Leu) (0.09%), and AAC (Asn) (0.09%) codon were the least frequent ones uncovered in F. elongatus (Table 2). Preferable codons were commonly uncovered with important functional gene regions, as those bias codons with silent sites were found to be related to maximize the translation efficiency [15, 16]. At the third codon position of the current F. elongatus mt protein genes, T (50.82%) was the most frequently observed and C (5.85%) was used least frequently (Figure S1). In codons with ≥2 unique bias, the TT codons were noticed at the highest point while CC ones were least found (Figure S2). These interesting results may reveal that F. elongatus mt is biased toward utilizing T-rich amino acid codons which are suggestive of the nucleotide bias [17]. However, until now, it is still unclear whether this bias of codon usage contributes to parasite mt systems or not [18].Table 2
Codons usage ofF. elongatus mt DNA-encoded proteins.
Amino acid
Codon
Number
Frequency (%)
Amino acid
Codon
Number
Frequency (%)
Phe
TTT
323
9.84
Pro
CCT
34
0.80
Phe
TTC
27
0.82
Pro
CCC
4
0.12
Leu
TTA
163
4.96
Pro
CCA
11
0.26
Leu
TTG
254
7.73
Pro
CCG
8
0.19
Leu
CTT
41
1.25
Thr
ACT
52
1.58
Leu
CTC
3
0.09
Thr
ACC
2
0.06
Leu
CTA
17
0.52
Thr
ACA
18
0.55
Leu
CTG
25
0.76
Thr
ACG
16
0.49
IIe
ATT
128
3.90
Ala
GCT
98
2.98
IIe
ATC
5
0.15
Ala
GCC
3
0.09
IIe
ATA
73
2.22
Ala
GCA
14
0.43
Val
GTT
176
5.36
Ala
GCG
31
5.18
Val
GTC
13
0.40
Tyr
TAT
170
5.18
Val
GTA
56
1.71
Tyr
TAC
8
0.24
Val
GTG
167
5.09
His
CAT
42
1.28
Ser
TCT
116
3.53
His
CAC
6
0.18
Ser
TCC
8
0.24
Gln
CAA
13
0.40
Ser
TCA
23
0.70
Gln
CAG
25
0.76
Ser
TCG
32
0.97
Asn
AAT
55
1.67
Ser
AGT
80
2.44
Asn
AAC
3
0.09
Ser
AGC
13
0.40
Arg
CGT
45
1.37
Trp
TGG
74
2.25
Arg
CGC
0
0
Lys
AAA
20
0.61
Arg
CGA
6
0.18
Lys
AAG
52
1.58
Arg
CGG
11
0.33
Asp
GAT
61
1.86
Arg
AGA
30
0.91
Asp
GAC
4
0.12
Arg
AGG
36
1.10
Glu
GAA
20
0.61
Gly
GGT
160
4.87
Glu
GAG
64
1.85
Gly
GGC
20
0.61
Cys
TGT
116
3.53
Gly
GGA
24
0.73
Cys
TGC
7
0.21
Gly
GGG
50
1.52
Met
ATG
3.11
2.48A total of 21 tRNA gene sequences (61–71 bp) and 2 noncoding regions (NCR) (l-RNA and s-RNA) were found in the F. elongatus mt genome (Figure 1, Table 1) depicting one undetected tRNA gene in the current isolate, as other trematodes contain 22 tRNA gene sequences [3, 13, 14]. The L-RNA and s-RNA were found to be located between cox1 and cox2 and separated by tRNA-Thr. Though NCR have been commonly reported in trematodes, however, still scarce information were available regarding the function of these special sequences [19]. Two AT-loops (63 and 473 bp) were found in the F. elongatus mt genome located between Cytb and Nd4L and after tRNA-Glu, respectively. By comparing mt genome of F. elongatus isolated from the cows of Shanghai with 11 available trematodes and employing phylogenetic analysis; the current F. elongatus isolates was found highly homologous with the Tianmen isolate (KM397348). The identity between the present isolate and Tianmen isolate has been observed as 98.73% via Nucleotide Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Sliding window analysis of mt genome of these two F. elongatus isolates revealed the nucleotide diversity (Pi) of the 12 protein-coding genes (Figure 2). Nd2 and Nd6 were demonstrated to be the highest and at the lowest level of nucleotide variability, respectively (Table 3).Figure 2
A sliding window analysis of the completemt genome sequences of F. elongatus of the current isolate and Tianmen isolate (KM397348.1).Table 3
Multidomain analysis of the completemt genome sequences of F. elongatus of the current isolate and Tianmen isolate (KM397348.1).
Region
n
Sites
Net sites
S
Eta
Hap
Hd
VarHd
Pi
1–14,228
2
14,228
14,120
181
181
2
1.000
0.25000
0.01282In conclusion, completemt DNA sequences of F. elongatus isolated from cows can contribute to the epidemiological diagnosis, molecular identification, taxonomy, genetic and drug development researches about this parasite species [3], and to get benefits for control measures of Fischoederius sp. in cattle.



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*Source: 7975948-2020-01-13.xml*