Characterization of the complete mitochondrial genome of Acanthogyrus (Acanthosentis) bilaspurensis Chowhan, Gupta & Khera, 1987 (Eoacanthocephala: Quadrigyridae), the smallest mitochondrial genome in Acanthocephala, and its phylogenetic implications.

The phylum Acanthocephala is an important group of parasites with more than 1,300 species parasitizing intestine of all major vertebrate groups. However, our present knowledge of the mitochondrial genomes of Acanthocephala remains very limited. In the present study, we sequenced and annotated the complete mitochondrial genome of Acanthogyrus (Acanthosentis) bilaspurensis (Gyracanthocephala: Quadrigyridae) for the first time based on the specimens recovered from the intestine of common carp Cyprinus carpio Linnaeus (Cyprinidae) in Pakistan. The mitochondrial genome of A. bilaspurensis is 13,360 bp in size and contains 36 genes, representing the smallest mitogenome of acanthocephalans reported so far. The mitogenome of A. bilaspurensis also has the lowest level of overall A+T contents (59.3%) in the mitogenomes of Eoacanthocephala, and the non-coding region 3 (NCR3) lies between trnS2 and trnI, which is different from all of the other acanthocephalan species. Phylogenetic analyses based on concatenating the amino acid sequences of 12 protein-coding genes using maximum likelihood (ML) and Bayesian inference (BI) methods revealed that the family Pseudoacanthocephalidae is a sister to the Arhythmacanthidae rather than the Cavisomatidae, and the families Rhadinorhynchidae and Cavisomatidae showed sister relationships.

Circulatory microRNAs in helminthiases: Potent as diagnostics biomarker, its potential role and limitations.

Infections caused by helminths are responsible for severe public health problems and economic burden on continental scale. Well-timed and precise diagnosis of helminth infections is critical for taking by appropriate approaches for pathogen control. Circulating miRNAs are stable diagnostic tool for different diseases found in a variety of body fluid. As diagnostic biomarkers in infectious diseases, miRNAs detection in body fluids of helminth infected hosts is growing promptly. Uncovering miRNAs is a relatively new tool, used for early-stage detection of helminth infection from experimental or non-invasive clinical samples. miRNAs can be detected in body fluids such as serum, saliva, urine, and tissues of helminth infected host, mainly blood offering important benefits for diagnosis accurately. In this review, we discuss different characteristics of helminth parasite-derived circulating and EV miRNAs, supporting its potential uses in for helminth diagnosis and treatment efficiency.

Schistosomiasis related circulating cell-free DNA: A useful biomarker in diagnostics.

Schistosoma is a genus of trematodes causing schistosomiasis, a major neglected tropical disease infecting more than 240 million people and with 700 million people at the risk of infection in the tropical and subtropical regions of the world, especially low-income countries. For the elimination of the disease, accurate diagnostic tools are needed. Besides allowing early treatment, early detection prevents environmental contamination and in turn ensures safe water sources in the endemic areas. Cell-free DNA (cfDNA) biomarker detection is a relatively new tool, used for the diagnosis of schistosomiasis in the early stages of infection from non-invasive clinical or experimental samples. cfDNA can be detected in Schistosoma infected host body fluids such as urine, serum, saliva and tissues, mainly in blood offering significant benefits for accurate diagnosis. In the current review, we described different characteristics of cfDNA, evidencing and supporting its potential uses in Schistosoma diagnosis and the improvement of treatment effectiveness.

Morphology and molecular characterization of Acanthogyrus (Acanthosentis) bilaspurensis Chowhan, Gupta & Khera, 1987 (Acanthocephala: Gyracanthocephala: Quadrigyridae) from the common carp Cyprinus carpio Linnaeus (Cypriniformes: Cyprinidae) in Pakistan.

Acanthogyrus (Acanthosentis) bilaspurensis Chowhan, Gupta & Khera, 1987 is a poorly known acanthocephalan species reported from the reba carp Cirrhinus reba (Hamilton) (Cypriniformes: Cyprinidae) in India. In the present study, the detailed morphology of A. (Acanthosentis) bilaspurensis were studied using light microscopy and, for the first time, scanning electron microscopy (SEM), based on newly collected specimens from the common carp Cyprinus carpio Linnaeus (Cypriniformes: Cyprinidae) in Pakistan. The SEM observations revealed the outer shell of eggs with numerous remarkable protuberances and the gonopore of female located at a discoid protrusion surrounded by some very small spines. This is the first time that A. (Acanthosentis) bilaspurensis was reported in Pakistan, and the common carp represents a new host for this species. In addition, the molecular characterization of the 18S and 28S rDNA of A. (Acanthosentis) bilaspurensis is provided for the first time. There was no intraspecific genetic variation detected in the 18S and 28S regions between different individuals of A. (Acanthosentis) bilaspurensis, but high level of interspecific nucleotide divergence was found in the 18S (4.15-16.7%) and 28S regions (3.60-15.4%). Phylogenetic results based on the 18S, 28S and 18S + 28S sequence data, respectively, all revealed the Quadrigyridae, Pallisentinae and the genus Neoechinorhynchus are not monophyletic groups, and the genera Acanthogyrus and Pallisentis have far relationship. The systematic status of the genus Acanthogyrus and some species of Neoechinorhynchus need further clarify based on different mtDNA and nuclear DNA data including broader representatives of the class Eoacanthocephala.

Mitochondrial genomes of two eucotylids as the first representatives from the superfamily Microphalloidea (Trematoda) and phylogenetic implications.

BACKGROUND: The Eucotylidae Cohn, 1904 (Superfamily: Microphalloidea), is a family of digeneans parasitic in kidneys of birds as adults. The group is characterized by the high level of morphological similarities among genera and unclear systematic value of morphological characters traditionally used for their differentiation. In the present study, we sequenced the complete or nearly complete mitogenomes (mt genome) of two eucotylids representing the genera Tamerlania (T. zarudnyi) and Tanaisia (Tanaisia sp.). They represent the first sequenced mt genomes of any member of the superfamily Microphalloidea. METHODS: A comparative mitogenomic analysis of the two newly sequenced eucotylids was conducted for the investigation of mitochondrial gene arrangement, contents and genetic distance. Phylogenetic position of the family Eucotylidae within the order Plagiorchiida was examined using nucleotide sequences of mitochondrial protein-coding genes (PCGs) plus RNAs using maximum likelihood (ML) and Bayesian inference (BI) methods. BI phylogeny based on concatenated amino acids sequences of PCGs was also conducted to determine possible effects of silent mutations. RESULTS: The complete mt genome of T. zarudnyi was 16,188 bp and the nearly complete mt genome of Tanaisia sp. was 13,953 bp in length. A long string of additional amino acids (about 123 aa) at the 5' end of the cox1 gene in both studied eucotylid mt genomes has resulted in the cox1 gene of eucotylids being longer than in all previously sequenced digeneans. The rrnL gene was also longer than previously reported in any digenean mitogenome sequenced so far. The TΨC and DHU loops of the tRNAs varied greatly between the two eucotylids while the anticodon loop was highly conserved. Phylogenetic analyses based on mtDNA nucleotide and amino acids sequences (as a separate set) positioned eucotylids as a sister group to all remaining members of the order Plagiorchiida. Both ML and BI phylogenies revealed the paraphyletic nature of the superfamily Gorgoderoidea and the suborder Xiphidiata. CONCLUSIONS: The average sequence identity, combined nucleotide diversity and Kimura-2 parameter distances between the two eucotylid mitogenomes demonstrated that atp6, nad5, nad4L and nad6 genes are better markers than the traditionally used cox1 or nad1 for the species differentiation and population-level studies of eucotylids because of their higher variability. The position of the Dicrocoeliidae and Eucotylidae outside the clade uniting other xiphidiatan trematodes strengthened the argument for the need for re-evaluation of the taxonomic content of the Xiphidiata.

Circulating cell-free mitochondrial DNA fragment: A possible marker for early detection of Schistosoma japonicum.

Schistosomiasis is a major public health problem that is included in the neglected tropical diseases. The early diagnosis and detection of the pathogen are of critical importance in the control of the disease. The diagnostic techniques in use include the detection of worm's eggs in fecal examination or detection of circulating antigens in immunological based assays. These traditional strategies lack sensitivity in earlier detection of the schistosomiasis. Cell-free DNA (cfDNA) that includes the fragments of parasitic DNA circulating in the body fluids of host offers an alternative mean for the rapid pathogen detection and thus is a useful diagnostic tool. In this study, we explored the usefulness of the mitochondrial cfDNA markers for the diagnosis of schistosomiasis from the experimentally infected hosts (rabbits and mice). In this study we found mitochondrial DNA fragment cytochrome B gene as persistent and useful cfDNA marker for the early detection of schistosomiasis. We evaluated the sensitivity of cfDNA marker with varying numbers of cercaria. Overall, our results suggest that cfDNA markers can be useful for developing a diagnostic tool for the detection of S. japonicum infection.

Preliminary information on the prevalence and molecular description of Taenia hydatigena isolates in Pakistan based on mitochondrial cox1 gene.

Taenia hydatigena is a cestode of veterinary importance. Infection with the metacestode larval stage results in cysticercosis, which poses a serious challenge to the livestock industry worldwide. Globally, there are numerous reports on cysticercosis caused by T. hydatigena in sheep and goat but a lack of data on the prevalence and genetic diversity exists for Pakistan. We designed this study to provide an insight into the disease status as well as investigate the genetic variation among the recovered isolates based on the mitochondrial cox1 gene. In this study, we examined small ruminants (sheep and goats) slaughtered in Faisalabad in eastern Punjab province of Pakistan for T. hydatigena metacestodes and described the population structure and genetic variation using the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene. Overall, a prevalence of 4.40% (goat =4.67% sheep = 4.07%) from a total of 2225 small ruminant carcasses (sheep = 983, goats = 1242) was observed. Based on the NCBI BLAST search and Bayesian phylogeny, the identity of all isolates was confirmed via their nucleotide sequences. The diversity indices indicated a high haplotype and a low nucleotide diversity with 43 haplotypes from 98 isolates. The results also show the existence of unique haplotypes of T. hydatigena in Pakistan as demonstrated by the significant negative values of Tajima's D and Fu's Fs neutrality test suggesting a recent population expansion. The median-joining network of the partial cox1 sequence dataset showed the existence of two main haplotypes detected in both sheep and goat populations. This study shows that the prevalence of cycticercosis due to T. hydatigena is below 5% in sheep and goats in Faisalabad, Punjab, Pakistan. The molecular analysis of the partial cox1 gene also indicates a high degree of genetic variation with the existence of rare haplotypes. These findings represent a preliminary report on the prevalence and genetic variation of T. hydatigena in Pakistan and serve as baseline information for future studies on the prevalence and population structure of T. hydatigena in the country.

Morphological and genetic characterisation of Centrorhynchus clitorideus (Meyer, 1931) (Acanthocephala: Centrorhynchidae) from the little owl Athene noctua (Scopoli) (Strigiformes: Strigidae) in Pakistan.

Centrorhynchus Lühe, 1911 is a large genus of acanthocephalans mainly parasitic in various strigiform and falconiform birds. Some species of Centrorhynchus have not been adequately described. Here, the detailed morphology of C. clitorideus (Meyer, 1931) was studied using light and, for the first time, scanning electron microscopy, based on newly collected specimens from the little owl Athene noctua (Scopoli) (Strigiformes: Strigidae) in Pakistan. Partial sequences of the 18S and 28S nuclear ribosomal RNA genes and the mitochondrial cytochrome c oxidase subunit 1 (cox1) of C. clitorideus were generated for the first time. No nucleotide variation was detected for the partial 18S and 28S regions, but 3.30% of intraspecific nucleotide divergence was found for the cox1 gene. Phylogenetic analyses based on 28S and 18S sequence data showed that C. clitorideus formed a sister relationship with Centrorhynchus sp. MGV-2005 or Centrorhynchus sp. MGV-2005 + C. microcephalus (Bravo-Hollis, 1947), respectively.

Mitochondrial DNA dataset suggest that the genus Sphaerirostris Golvan, 1956 is a synonym of the genus Centrorhynchus Lühe, 1911.

Our present genetic data of Acanthocephala, especially the mitochondrial (mt) genomes, remains very limited. In the present study, the nearly complete mt genome sequences of Sphaerirostris lanceoides (Petrochenko, 1949) was sequenced and determined for the first time based on specimens collected from the Indian pond heron Ardeola grayii (Sykes) (Ciconiiformes: Ardeidae) in Pakistan. The mt genome of S. lanceoides is 13 478 bp in size and contains 36 genes, including 12 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and two ribosomal RNA genes (rRNAs). Moreover, in order to clarify the phylogenetic relationship of the genera Centrorhynchus and Sphaerirostris, and to test the systematic position of S. lanceoides in the Centrorhynchidae, the phylogenetic analyses were performed using Bayesian inference and maximum likelihood methods, based on concatenated nucleotide sequences of 12 PCGs, rRNAs and tRNAs. The phylogenetic results further confirmed the monophyly of the order Polymorphida and the paraphyly of the order Echinorhynchida in the class Palaeacanthocephala. Our results also challenged the validity of the genus Sphaerirostris (Polymorphida: Centrorhynchidae) and showed a sister relationship between S. lanceoides and S. picae (Rudolphi, 1819).

Characterization of the complete mitogenome of Centrorhynchus clitorideus (Meyer, 1931) (Palaeacanthocephala: Centrorhynchidae), the largest mitochondrial genome in Acanthocephala, and its phylogenetic implications.

Species of Centrorhynchus (Polymorphida: Centrorhynchidae) commonly parasitize various falconiform and strigiform birds worldwide. In the present study, the complete mitochondrial (mt) genome sequences of Centrorhynchus clitorideus was sequenced and annotated for the first time based on specimens collected from the little owl Athene noctua (Scopoli) (Strigiformes: Strigidae) in Pakistan. The complete mt genome sequences of C. clitorideus is 15,884 bp in length, and contained 36 genes [two rRNA genes (rrnL and rrnS), 22 tRNA genes and 12 protein-coding genes (PCGs) (lacking atp8)] and two non-coding regions (NCR1 and NCR2), which represents the largest mt genome of acanthocephalan reported so far. In order to assess the systematic position of C. clitorideus and the interrelationship of the family Centrorhynchidae and the other families in order Polymorphida, the phylogenetic tree was constructed using Bayesian inference (BI) based on amino acid sequences of 12 PCGs. Phylogenetic results supported C. clitorideus formed a sister relationship to C. milvus in Centrorhynchidae, which has a sister relationship to the representatives of Polymorphidae + Plagiorhynchidae. Our results revealed the monophyly of Polymorphida and paraphyly of Echinorhynchida in the class Palaeacanthocephala. The validity of the genus Sphaerirostris (Polymorphida: Centrorhynchidae) was also challenged by our phylogenetic results, which seems to be a synonym of Centrorhynchus. Moreover, the present phylogenetic analysis indicated that the family Quadrigyridae and subfamily Pallisentinae (A. cheni and P. celatus) are polyphyletic.

Molecular phylogenetics and mitogenomics of three avian dicrocoeliids (Digenea: Dicrocoeliidae) and comparison with mammalian dicrocoeliids.

BACKGROUND: The Dicrocoeliidae are digenetic trematodes mostly parasitic in the bile ducts and gall bladder of various avian and mammalian hosts. Until recently their systematics was based on morphological data only. Due to the high morphological uniformity across multiple dicrocoeliid taxa and insufficient knowledge of relative systematic value of traditionally used morphological characters, their taxonomy has always been unstable. Therefore, DNA sequence data provide a critical independent source of characters for phylogenetic inference and improvement of the system. METHODS: We examined the phylogenetic affinities of three avian dicrocoeliids representing the genera Brachylecithum, Brachydistomum and Lyperosomum, using partial sequences of the nuclear large ribosomal subunit (28S) RNA gene. We also sequenced the complete or nearly complete mitogenomes of these three isolates and conducted a comparative mitogenomic analysis with the previously available mitogenomes from three mammalian dicrocoeliids (from 2 different genera) and examined the phylogenetic position of the family Dicrocoeliidae within the order Plagiorchiida based on concatenated nucleotide sequences of all mitochondrial genes (except trnG and trnE). RESULTS: Combined nucleotide diversity, Kimura-2-parameter distance, non-synonymous/synonymous substitutions ratio and average sequence identity analyses consistently demonstrated that cox1, cytb, nad1 and two rRNAs were the most conserved and atp6, nad5, nad3 and nad2 were the most variable genes across dicrocoeliid mitogenomes. Phylogenetic analyses based on mtDNA sequences did not support the close relatedness of the Paragonimidae and Dicrocoeliidae and suggested non-monophyly of the Gorgoderoidea as currently recognized. CONCLUSIONS: Our results show that fast-evolving mitochondrial genes atp6, nad5 and nad3 would be better markers than slow-evolving genes cox1 and nad1 for species discrimination and population level studies in the Dicrocoeliidae. Furthermore, the Dicrocoeliidae being outside of the clade containing other xiphidiatan trematodes suggests a need for the re-evaluation of the taxonomic content of the Xiphidiata.

Characterization of the complete mitochondrial genome of Cavisoma magnum () (Acanthocephala: Palaeacanthocephala), first representative of the family Cavisomidae, and its phylogenetic implications.

The phylum Acanthocephala is a small group of endoparasites occurring in the alimentary canal of all major lineages of vertebrates worldwide. In the present study, the complete mitochondrial (mt) genome of Cavisoma magnum (Southwell, 1927) (Palaeacanthocephala: Echinorhynchida) was determined and annotated, the representative of the family Cavisomidae with the characterization of the complete mt genome firstly decoded. The mt genome of this acanthocephalan is 13,594 bp in length, containing 36 genes plus two non-coding regions. The positions of trnV and SNCR (short non-coding region) in the mt genome of C. magnum are different comparing to those of the other acanthocephalan species available in GenBank. Phylogenetic analysis based on amino acid sequences of 12 protein-coding genes using Bayesian inference (BI) supported the class Palaeacanthocephala and its included order Polymorphida to be monophyletic, but rejected monophyly of the order Echinorhynchida. Our phylogenetic results also challenged the validity of the genus Sphaerirostris (Polymorphida: Centrorhynchidae). The novel mt genomic data of C. magnum are very useful for understanding the evolutionary history of this group of parasites and establishing a natural classification of Acanthocephala.

Characterization of the complete mitochondrial genome of Centrorhynchus milvus (Acanthocephala: Polymorphida) and its phylogenetic implications.

The phylum Acanthocephala is a small group of obligate parasites of animals. However, the current classifications of Acanthocephala are still under debate. Moreover, our present knowledge of the complete mitochondrial genome of this parasite group remains limited. To fill this knowledge gap, the complete mitochondrial (mt) genome of Centrorhynchus milvusWard, 1956 (Palaeacanthocephala: Polymorphida) was firstly sequenced and determined based on specimens collected from the red kite (Milvus milvus) in Pakistan. The mitochondrial genome of C. milvus is 14,314 bp in length and contains 36 genes, including 12 protein-coding (PCGs) genes, 22 transfer RNA (tRNA) genes and ribosomal RNA (rRNA) genes (rrnL and rrnS). To elucidate the phylogenetic relationships of the four classes of Acanthocephala and the systematic position of C. milvus, phylogenetic analysis based on concatenated amino acid sequences of 12 PCGs was performed using Bayesian inference (BI). The results supported the monophyly of Archiacanthocephala and Palaeacanthocephala with strong support (BPP = 1) and also indicated that Archiacanthocephala is the sister clade to the remaining classes of Acanthocephala (Palaeacanthocephala, Eoacanthocephala and Polyacanthocephala). However, Polyacanthocephala with only one representative species (P. caballeroi) is nested within Eoacanthocephala. Our phylogenetic analysis also confirmed C. milvus as the member of the family Centrorhynchidae with a sister relationship to C. aluconis. Our present mt genomic data are very useful for studying the molecular epidemiology, population genetics and systematics of acanthocephalans.

Characterization of the complete mitochondrial genome of Sphaerirostris picae (Rudolphi, 1819) (Acanthocephala: Centrorhynchidae), representative of the genus Sphaerirostris.

The Centrorhynchidae (Acanthocephala: Palaeacanthocephala) is a cosmopolitan family commonly found in various avian and mammalian hosts. Within Centrorhynchidae, species of the genus Sphaerirostris Golvan, 1956 are usually parasitic in the digestive tract of various passerine birds. In the present study, adult specimens of Sphaerirostris picae (Rudolphi, 1819), the type species of this genus, were recovered from the small intestine of Acridotheres tristis (Sturnidae) and Dendrocitta vagabunda (Corvidae) in Pakistan. Molecular data from the nuclear or mitochondrial genome is either very limited or completely absent from this phylogenetically understudied group of acanthocephalans. To fill this knowledge gap, we sequenced and determined the internal transcribed spacers of ribosomal DNA (ITS rDNA) and the complete mitochondrial (mt) genome of S. picae. The ITS rDNA of S. picae was 95.2% similar to that of Sphaerirostris lanceoides which is the only member of the Centrorhynchidae whose ITS rDNA is available in GenBank. The phylogenetic tree based on the amino acid sequences of 12 mt protein-coding genes (PCGs) placed S. picae close to Centrorhynchus aluconis in a monophyletic clade of Polymorphida which also contain members of the families Polymorphidae and Plagiorhynchidae on separate branches. The mt gene arrangement, nucleotide composition and codon usage of 12 PCGs were discussed and compared with those of other acanthocephalan mt genomes. Within the Centrorhynchidae, S. picae and C. aluconis showed 67.7-86.8% similarity in the nucleotide sequences of 12 PCGs and 2 rRNAs, where nad4L is the most conserved gene while atp6 is the least conserved. The similarity in amino acid sequences ranged from 68.1 to 91.8%, where cox1 was recorded as the most conserved gene, while atp6 is highly variable among 12 PCGs. This novel mt genome of S. picae provides genetic resources for further studies of phylogenetics and molecular epidemiology of acanthocephalans.

Characterization of the complete mitochondrial genome of Uvitellina sp., representative of the family Cyclocoelidae and phylogenetic implications.

Mitochondrial (mt) DNA has been useful in revealing the phylogenetic relationship of eukaryotic organisms including flatworms. Therefore, the use of mitogenomic data for the comparative and phylogenetic purposes is needed for those families of digenetic trematodes for which the mitogenomic data are still missing. Molecular data with sufficiently rich informative characters that can better resolve species identification, discrimination, and membership in different genera is also required for members of some morphologically difficult families of trematodes bearing few autapomorphic characters among its members. Here, the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) and the complete mt genome of the trematode Uvitellina sp. (Cyclocoelidae: Haematotrephinae) was determined and annotated. The mt genome of this avian trematode is 14,217 bp in length, containing 36 genes plus a single non-coding region. The ITS rDNA sequences were used for the pairwise sequence comparison of Uvitellina sp. with European cyclocoelid species, and the mitochondrial 12 protein-coding genes (PCGs) and two ribosomal RNA genes were used to evaluate the position of the family within selected trematodes. The ITS rDNA analysis of Uvitellina sp. showed less nucleotide differences with Hyptiasmus oculeus (16.77%) than with other European cyclocoelids (18.63-23.58%). The Bayesian inference (BI) analysis using the 12 mt PCGs and two rRNA genes supported the placement of the family Cyclocoelidae within the superfamily Echinostomatoidea (Plagiorchiida: Echinostmata). The availability of the mt genome sequences of Uvitellina sp. provides a novel resource of molecular markers for phylogenetic studies of Cyclocoelidae and other trematodes.

Characterization of the complete mitochondrial genome of Plagiorchis maculosus (Digenea, Plagiorchiidae), Representative of a taxonomically complex digenean family.

Despite the highly divergent morphology, pathogenicity and worldwide distribution of digenean parasites belonging to one of the largest families, the Plagiorchiidae, there are no complete mitochondrial (mt) genomes published to date for plagiorchiids. In this study, we obtained nuclear ribosomal DNA (ITS region and 28S rDNA) sequences and the complete mt genome sequences of Plagiorchis maculosus (Rudolphi 1802) Braun, 1902, and assessed its phylogenetic relationship with other xiphidiates, based on the mtDNA sequences. The obtained ITS and 28S rDNA sequences were identical to the corresponding sequences of P. maculosus available in GenBank. The complete mitochondrial genome of P. maculosus (14,124 bp) contained 36 genes (atp8 is absent) and a long non-coding region (NCR) with two sets of repeated sequences of 283 nucleotides each. The phylogenetic tree resulting from Bayesian inference (BI) analyses based on concatenated nucleotide sequences of all 36 genes of P. maculosus and other xiphidiates mitochondrial genomes, indicated that P. maculosus (and the Plagiorchiidae) is phylogenetically closest to the Brachycladiidae and Paragonimidae. The present study describes the first mitochondrial genome from the type genus of the family Plagiorchiidae. The overall gene arrangement, nucleotide composition, A + T contents, AT and GC skew and codon usage with relative synonymous codon usage (RSCU) for 12 PCGs are described. Characterization of mitochondrial genomes from additional plagiorchiid taxa is necessary to make further progress in phylogenetic and epidemiological studies of these digeneans as well as accurate diagnostics of these parasites including those parasitic in humans.

Mitochondrial and nuclear ribosomal DNA dataset suggests that Hepatiarius sudarikovi Feizullaev, 1961 is a member of the genus Opisthorchis Blanchard, 1895 (Digenea: Opisthorchiidae).

The taxonomy and classification of the family Opisthorchiidae have been revised by several authors with the exclusion or synonymization of some genera. The genus Hepatiarius Feizullaev, 1961 accommodated two species: Hepatiarius sudarikovi Feizullaev, 1961 and H. longissimus Linstow, 1883. Recently, some experts have suppressed Hepatiarius as a junior synonym of Opisthorchis Blanchard, 1895 based on morphological features alone. Prior to the present study, no molecular data either from nuclear or from mitochondrial DNA was available for any species of this genus. In the present study, four specimens of H. sudarikovi Feizullaev, 1961 were recovered from the bile ducts of the little egret, Egretta garzetta. The complete sequences of the internal transcribed spacers (ITS-1 and ITS-2) of ribosomal DNA (rDNA) and the nearly complete mitochondrial genome sequences were determined and the phylogenetic relationship of H. sudarikovi with related taxa was assessed based on the mitochondrial (mt) DNA sequences. The sequence similarity in the ITS rDNA between H. sudarikovi and Opisthorchis felineus was higher (97.62% in ITS-1 and 96.22% in ITS-2) than with other opisthorchiids. Phylogenetic analysis using Bayesian inference (BI) based on the concatenated amino acid sequences of 12 protein-coding genes (PCGs) clustered H. sudarikovi into the clade of opisthorchiids, with O. felineus being the closest related species, which supports the affinity of H. sudarikovi with trematodes in the genus Opisthorchis. This is the first avian liver fluke whose nearly complete mitochondrial genome was sequenced. The mtDNA sequences of H. sudarikovi, in combination with its rDNA sequences, provide novel resources of genetic markers for the identification, species differentiation, and systematic studies of H. sudarikovi with other avian opisthorchiid flukes.