Complete mitochondrial genome of the giant liver fluke Fascioloides magna (Digenea: Fasciolidae) and its comparison with selected trematodes.

BACKGROUND: Representatives of the trematode family Fasciolidae are responsible for major socio-economic losses worldwide. Fascioloides magna is an important pathogenic liver fluke of wild and domestic ungulates. To date, only a limited number of studies concerning the molecular biology of F. magna exist. Therefore, the objective of the present study was to determine the complete mitochondrial (mt) genome sequence of F. magna, and assess the phylogenetic relationships of this fluke with other trematodes based on the mtDNA dataset.
FINDINGS: The complete F. magna mt genome sequence is 14,047 bp. The gene content and arrangement of the F. magna mt genome is similar to those of Fasciola spp., except that trnE is located between trnG and the only non-coding region in F. magna mt genome. Phylogenetic relationships of F. magna with selected trematodes using Bayesian inference (BI) was reconstructed based on the concatenated amino acid sequences for 12 protein-coding genes, which confirmed that the genus Fascioloides is closely related to the genus Fasciola; the intergeneric differences of amino acid composition between the genera Fascioloides and Fasciola ranged 17.97-18.24 %.
CONCLUSIONS: The determination of F. magna mt genome sequence provides a valuable resource for further investigations of the phylogeny of the family Fasciolidae and other trematodes, and represents a useful platform for designing appropriate molecular markers.

Background

Fascioloides magna (Bassi, 1875), the type- and only species of the genus Fascioloides Ward, 1917, was first described as Distomum magnum in 1875 [1]. Latter in 1917, Ward erected the genus Fascioloides for Fasciola magna (Bassi, 1875) [2]. Fascioloides magna, known as the large American liver fluke, giant liver fluke or deer fluke, is an important digenetic trematode of the family Fasciolidae [3, 4]. This species, which is of North America origin [5, 6] and invasive in European countries [7], has high potential to colonize new geographic territories (a variety of wild and domestic ungulates [3, 8–10]), and can establish expanding populations from a natural epidemic focus through translocated hosts [5, 6, 11]. Migration of F. magna immature flukes within the host body often leads to profound damage to the liver and other organ tissues [8, 12], causing economic losses worldwide [13].

The consequences of infection of various intermediate and definitive hosts by F. magna has been intensively studied [8, 12], but the relevant molecular research of this fluke has not received enough attention [4, 9]. To date, a sequence of nuclear ribosomal DNA (rDNA) of F. magna was obtained in 2008 [14], partial sequences of mitochondrial (mt) genes, such as cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) were characterized [3]. According to these data, F. magna was divided into two mt haplotype groups [5, 14, 15], the first haplotype representing isolates from western North America and Italy, and the second haplotype representing isolates from eastern North America and some European countries such as Czech Republic, Poland and Croatia [3, 5]. Recently, the F. magna transcriptome was reported, which provides a useful platform for further fundamental studies of this fluke [16], but complete mt genome of F. magna is still unavailable.

Molecular tools, using genetic markers in mitochondrial DNA (mtDNA) sequences, have been proven reliable in identification and differentiation of trematode species [17-20]. In the present study we determined the mitochondrial genome sequence of F. magna (Czech isolate) using PCR-coupled sequencing technique combined with bioinformatic analysis, and for the first time assessed its phylogenetic relationship with selected trematodes based on the nucleotide- and inferred amino acid sequences of the protein-coding genes.

Methods

Sampling and DNA extraction

Three adult F. magna worms were isolated from livers of naturally infected red deer (Cervus elaphus), hunted at Kokořínsko area, Czech Republic. Worms were washed in 0.1 M phosphate-buffered saline (PBS), pH 7.2, fixed in 70 % (v/v) ethanol and preserved at -20 °C, until further use. Total genomic DNA was extracted from individual F. magna specimens using sodium dodecyl sulfate (SDS)/proteinase K treatment [21] and column-purification (Wizard® SV Genomic DNA Purification System, Promega, Madison, USA), according to the manufacturer’s protocol.

Acquisition of ITS rDNA and sample identification

The internal transcribed spacer (ITS) rDNA region of each of the three F. magna specimens, spanning partial 18S rDNA, the complete ITS-1, 5.8S rDNA, ITS-2, and partial 28S rDNA, was amplified using primers BD1 (forward; 5’-GTC GTA ACA AGG TTT CCG TA-3’ and BD2 (reverse; 5’-ATG CTT AAA TTC AGC GGG T-3’) [22] and sequenced using the same primers. These F. magna samples had ITS-1 and ITS-2 sequences identical to the corresponding sequences available on GenBank (EF051080).

Long-range PCR-based sequencing of mt genome

The primers were designed based on relatively conserved regions of mtDNA sequences from Fasciola hepatica and Fasciola gigantica. The entire mt genome from a single specimen of F. magna was amplified in 5 overlapping fragments, using the primers shown in Additional file 1: Table S1.

PCR reactions were conducted in a total volume of 50 μl, using 25 μl PrimeStar Max DNA polymerase premix (Takara, Dalian, China), 25 pmol of each primer (synthesized in Genewiz, Suzhou, China), 0.5 μl DNA templates, and H2O, in a thermocycler (Biometra, Göttingen, Germany). PCR cycling conditions started with an initial denaturation at 98 °C for 2 min, followed by 22 cycles of denaturation at 92 °C for 18 s, annealing at 52–65 °C for 12 s and extension at 60 °C for 1–5 min, followed by 92 °C denaturation for 2 min, plus 25 cycles of 92 °C for 18 s (denaturation), 50–67 °C for 12 s (annealing) and 66 °C for 3–6 min, with a final extension step for 10 min at 66 °C. A negative control (no DNA) was included in each amplification run. Amplicons (2.5 μl) were electrophoresed in a 2 % agarose gel, stained with Gold View I (Solarbio, Beijing, China) and photographed by GelDoc - It TS™ Imaging System (UVP, USA).

Assembly, annotation and bioinformatics analysis

Sequences were assembled manually and aligned against the entire mt genome sequences of Fa. hepatica (GenBank accession No. NC_002546) and Fa. gigantica (NC_024025) using MAFFT 7.122 to infer boundaries for each gene. Amino acid sequences of 12 protein-coding genes were translated using MEGA v.6.06 and NCBI translation Table 21 (Trematode Mitochondrial Code). The tRNA genes were affirmed using the programs tRNAscan-SE [23] and ARWEN (http://130.235.46.10/ARWEN/) or by comparison with those from the Fa. hepatica and Fa. gigantica mt genomes. The two rRNA genes were identified by comparison with those of Fa. hepatica and Fa. gigantica.

A comparative analysis of the nucleotide sequences of each protein-coding gene, the amino acid sequences, two ribosomal RNA genes, 22 tRNA genes as well as non-coding regions (NCRs) among F. magna, Fa. hepatica and Fa. gigantica was conducted.

Phylogenetic analysis

The concatenated amino acid sequences of F. magna mt genome, conceptually translated from individual genes of each mt genome, were aligned with those of published mt genomes from selected trematodes, including Opisthorchis felineus (GenBank acession No. EU_921260) and Clonorchis sinensis (FJ_381664) (Opisthorchiidae); Metagonimus yokogawai (KC_330755) and Haplorchis taichui (KF_214770) (Heterophyidae); Paragonimus westermani Japanese isolate (AF219379) and Paragonimus westermani Indian isolate (NC_027673) (Paragonimidae); Fa. hepatica, Fasciola sp. (KF_543343) and Fa. gigantica (Fasciolidae); Hypoderaeum sp. (KM111525) (Echinostomatidae); Paramphistomum leydeni (KP341657) and Fischoederius elongatus (KM397348) (Paramphistomatidae); Diplostomum spathaceum (KR269763) and Diplostomum pseudospathaceum (KR269764) (Diplostomidae); Ogmocotyle sikae (KR006934) (Notocotylidae); Eurytrema pancreaticum (KP241855) (Dicrocoeliidae); Schistosoma turkestanicum (HQ_283100) and Schistosoma japonicum (HM_120842) (Schistosomatidae). The sequence for the monogenean Gyrodactylus derjavinoides (NC_010976) (Gyrodactylidae), was included as the outgroup.

All inferred amino acid sequences were aligned using MAFFT 7.122. Poorly aligned sites and divergent regions of the alignment were eliminated using Gblocks Server v. 0.91b (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) using default settings, selecting the option of less strict conservation of flanking positions. The alignment was then converted into nexus format using Clustal X1.83 and subjected to phylogenetic analysis using Bayesian inference (BI). A mixed model was used in BI analysis using MrBayes 3.1.1 [24], because the most suitable amino acid evolution model JTT + G + F, selected by ProTest 3.4 based on the Akaike information criterion (AIC) [25], was not available in the current MrBayes version. Four independent Markov chain were run for 10,000,000 metropolis-coupled MCMC generations, sampling trees every 1,000 generations. The first 2,500 trees (25 %) were discarded as ‘burn-in’, and the remaining trees were used for calculating Bayesian posterior probabilities. The analysis was regarded as completed when the potential scale reduction factor was close to 1, and the average standard deviation of split frequencies was below 0.01. Phylograms were prepared using FigTree v. 1.42 [26].

Findings

Genome content and organization

The complete mt genome sequence of F. magna (GenBank accession no. KR006934) is 14,047 bp in length (Fig. 1) and contains 36 genes that are transcribed in the same direction, including 12 protein-coding genes (nad1-6, nad4L, cox1-3, atp6 and cytb), 22 tRNA genes and two rRNA genes (rrnL and rrnS), lacking the atp8 gene (Table 1), consistent with those of selected trematode species available on GenBank [17–19, 27, 28]. There is only one NCR in F. magna mt genome, whereas the mt genomes of Fasciola flukes have two non-coding regions [17, 27].

Fig. 1

Organization of the mitochondrial genome of Fascioloides magna. The scales are approximate. All genes are transcribed in the clockwise direction, using standard nomenclature. “NCR” refers to the only non-coding region in F. magna data. The A + T content is shown for each gene or region of the mt genome and represented by colour

Table 1

The features of the mitochondrial genomes of Fascioloides magna

Gene	Coding position (5’–3’)	Length (bp)	Start/Stop codons	No. of amino acids	Intergenic nucleotides
cox3	1–645	645	ATG/TAA	215	4
trnH	650–713	64			1
cytb	715–1827	1113	ATG/TAG	371	7
nad4L	1835–2107	273	GTG/TAG	91	-40
nad4	2068–3348	1281	GTG/TAG	427	1
trnQ	3350–3412	63			11
trnF	3424–3486	63			14
trnM	3501–3566	66			0
atp6	3567–4085	516	ATG/TAA	172	4
nad2	4090–4959	870	ATG/TAG	290	2
trnV	4962–5023	62			7
trnA	5031–5092	62			6
trnD	5099–5160	62			1
nad1	5162–6064	903	GTG/TAG	301	7
trnN	6072–6137	66			4
trnP	6142–6210	69			0
trnI	6211–6273	63			5
trnK	6279–6343	65			0
nad3	6344–6700	357	ATG/TAA	119	4
trnS1	6705–6763	59			8
trnW	6772–6836	65			3
cox1	6840–8384	1545	GTG/TAG	515	23
trn T	8408–8469	62			0
rrnL	8470–9453	984			2
trnC	9456–9518	63			-2
rrnS	9517–10281	765			2
cox2	10284–10886	603	ATG/TAG	201	32
nad6	10919–11371	453	GTG/TAG	151	0
trnY	11372–11428	57			12
trnL1	11441–11504	64			2
trnS2	11506–11566	60			10
trnL2	11577–11642	66			-3
trnR	11640–11705	66			-2
nad5	11704–13272	1569	GTG/TAG	523	10
trnG	13283–13348	66			6
trnE	13355–13422	68			0
NCR	13423–14047	520			0

Abbreviation: NCR Non-coding region

The arrangement of genes in the F. magna mt genome is similar to that of Fasciola spp. [17], except that only one non-coding region (NCR) in F. magna mt genome is located between trnE (13,355–13,422) and cox3 (1–645) (Table 1). The gene order of F. magna mt genes is similar to that in species of the Paramphistomatidae, Notocotylidae, Echinostomatidae, Heterophyidae and Opisthorchiidae, but is distinct from some flukes of the Schistosomatidae (S. mansoni, S. spindale and S. haematobium) [29].

The nucleotide composition of F. magna mt genome is obviously biased towards A and T. The value of total A + T content for F. magna mtDNA is 61.42 %, within the range recognized in other trematode mt genomes (54.38 % in Paragonimus westermani Indian isolates [30], 72.71 % in Schistosoma spindale [29]). The content of C is low (10.3 %) and that of T is high (44.0 %). The A + T content for each gene or region of F. magna mt genome ranged from 48.48 % (trnL2) to 68.18 % (trnG) (nad3, 64.43 %; cox2, 59.7 %). All 12 protein-coding genes of F. magna mtDNA possess a lower A + T percentage than those of Fa. hepatica and Fa. gigantica [17, 27], except for nad5 (Additional file 2: Table S2).

Annotation of F. magna mt genome

In the mt genome of F. magna, the protein-coding genes had ATG or GTG as start codons and TAG or TAA as stop codons (Table 1). Half of the protein-coding genes of F. magna were initiated with GTG (nad4L, nad4, nad1, cox1, nad6 and nad5). Incomplete codons were not detected in the mt genome of F. magna.

The 22 tRNA genes of F. magna mt genome ranged from 57 to 69 bp in length. The structure of all tRNA sequences is similar to those of Fa. hepatica and Fa. gigantica [17, 27]. The large ribosomal RNA gene (rrnL) and the adjacent small ribosomal RNA gene (rrnS) are located between trnT and cox2, and separated by trnC (9,456–9,518) (Table 1). The length of the rrnL and rrnS RNA genes is 984 bp and 765 bp, respectively. The only NCR of F. magna mt genome is of 520 bp in length, and is located between trnE and cox3. It contains two complete direct repeats: six copies of a 23 nt - repeat A (AGA TAG GAT AGG CAT CTG GTA TA) and five copies of a 37 nt - repeat B (GGT GCC CCC GGT GAA GGG GGA AAA GGA AGG TTG TAA G). There are five AB repeats, with one A at the end (located at positions 13,620–13,642).

Comparative analysis among mt genomes of F. magna, Fa. hepatica and Fa. gigantica

The difference between complete mt genomes of F. magna and Fa. hepatica was 22.66 % (3,290 nt), which is close to that between F. magna and Fa. gigantica (22.65 %, 3,297 nt) (Table 2). Considering the 12 protein-coding genes, different nucleotides were present at 18.80 % of positions (1,897 nt) between F. magna and Fa. hepatica, and at 18.62 % of positions (1,879 nt) between F. magna and Fa. gigantica. At the inferred amino acid level, there were 605 substitutions (17.97 %) of amino acids between F. magna and Fa. hepatica, and 614 substitutions (18.24 %) between F. magna and Fa. gigantica (Table 2).

Table 2

Comparison of nucleotides and predicted amino acids sequences among Fascioloides magna (Fm), Fasciola hepatica (Fh) and Fasciola gigantica (Fg)

Gene	nt difference (%)			aa difference (%)
	Fm/Fh	Fm/Fg	Fh/Fg	Fm/Fh	Fm/Fg	Fh/Fg
cox3	19.8	18.7	13.4	20.1	22.4	13.1
cytb	16.1	16.4	8.3	11.6	13.2	6.5
nad4L	15.3	16.1	8.4	12.1	15.4	5.5
nad4	21.4	20.9	13.5	21.7	20.8	10.6
atp6	21.3	21.2	13.8	22.0	20.2	13.3
nad2	21.1	22.8	11.6	25.4	24.7	11.4
nad1	15.0	14.9	8.4	13.0	14.3	6.6
nad3	19.0	19.0	10.6	13.4	15.1	7.6
cox1	13.1	12.8	9.1	9.2	8.4	5.5
cox2	19.2	19.2	11.6	14.4	15.4	6.5
nad6	24.2	26.2	16.3	22.5	27.8	13.2
nad5	21.5	19.8	13.7	23.6	22.8	12.3
rrnL	18.3	16.6	10.6
rrnS	22.2	21.4	11.1
22 tRNAs	16.3	16.0	9.9
Overall	22.7	22.7	12.2	17.97	18.24	9.4

At the nucleotide level, sequence differences in protein-coding genes ranged from 13.1 to 24.2 % (between F. magna and Fa. hepatica) and from 12.8 to 26.2 % (between F. magna and Fa. gigantica), with cox1, nad1, nad4L and cytb being the most conserved genes, and nad6, nad5 and nad2 being the least conserved genes among those three species. At the amino acid level, sequence differences ranged from 9.2 to 25.4 % between F. magna and Fa. hepatica, and from 8.4 to 27.8 % between F. magna and Fa. gigantica: cox1, cytb, nad4L and nad1 were the most conserved protein-coding genes, while nad6, nad2 and nad5 were the least conserved.

Comparisons between the mt genomes of F. magna and Fasciola spp., at both nucleotide and amino acid levels, indicate that the most conserved and the least conserved gene in the Fasciolidae are cox1 and nad6, respectively. Besides, the nad5 is highly variable, and genes of nad4L and cytb are rather conserved. These characteristics are in accordance with flukes of the families Paramphistomatidae and Notocotylidae [18, 28].

Nucleotide differences were also found in ribosomal RNA genes: between F. magna and Fa. hepatica (rrnL, 18.3 %; rrnS, 22.2 %) and between F. magna and Fa. gigantica (rrnL, 16.6 %; rrnS, 21.4 %) as well as in tRNA genes (16.3 % between F. magna and Fa. hepatica and 16.0 % between F. magna and Fa. gigantica). Meaningful sequence comparisons of NCRs in mt genomes of the three fasciolid trematodes is not possible, because there is only one NCR present in F. magna mt genome, while in both Fa. hepatica and Fa. gigantica there are two NCRs.

In the phylogenic tree inferred from the concatenated amino acid sequence dataset of all 12 mt proteins (Fig. 2) F. magna clustered with three other Fasciola species with strong support (Bpp = 1). The closest family to the Fasciolidae is Echinostomatidae, represented by Hypoderaeum sp. The taxonomic relationships of the selected trematodes are in concordance with results of previous studies [17–19, 28]. Each node received the maximum possible nodal support (Bpp = 1).

Fig. 2

Phylogenetic relationships of Fascioloides magna and other trematodes. Tree inferred from the concatenated amino acid sequence dataset for 12 protein-coding genes from 19 trematodes was performed by Bayesian inference (BI). Gyrodactylus derjavinoides (NC_010976) was chosen as the outgroup

In several recent phylogenetic studies, the F. magna was characterized only based on partial 28S rDNA [31] and combined ITS1, ITS2 and nad1 sequences [32]. The relationship between the genera Fasciola and Fasciolopsis was considered as being very close and the genetic relationship between F. magna and Fasciola jacksoni (or Fascioloides jacksoni) is disputable [31-33]. Further studies are warranted to determine the mt genome of Fa. jacksoni and solve this controversy in the family Fasciolidae.

Conclusions

The present study determined the complete mt genome sequence of the pathogenic liver fluke F. magna and revealed its close relationship with the species of Fasciola. The complete mt genome data of F. magna provides a resource for further investigations of the phylogeny, epidemiology, biology and population genetics of the family Fasciolidae and other trematodes.

Abbreviations

mt, mitochondrial; mtDNA, mitochondrial DNA; rDNA, ribosomal DNA; BI, Bayesian inference; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulphate; ITS, internal transcribed spacer; NCR, non-coding region