Dipetalonema gracile is a common parasite in squirrel monkeys (Saimiri sciureus), which can cause malnutrition and progressive wasting of the host, and lead to death in the case of massive infection. This study aimed to identify a suspected D. gracile worm from a dead squirrel monkey by means of molecular biology, and to amplify its complete mitochondrial genome by polymerase chain reaction (PCR) and sequence analysis. The results identified the worm as D. gracile, and the full length of its complete mitochondrial genome was 13,584 bp, which contained 22 tRNA genes, 12 protein-coding genes, two rRNA genes, one AT-rich region and one small non-coding region. The nucleotide composition included A (16.89%), G (20.19%), T (56.22%) and C (6.70%), among which A + T = 73.11%. The 12 protein-coding genes used TTG and ATT as start codons, and TAG and TAA as stop codons. Among the 22 tRNA genes, only trnS1AGN and trnS2UCN exhibited the TΨC-loop structure, while the other 20 tRNAs showed the TV-loop structure. The rrnL (986 bp) and rrnS (685 bp) genes were single-stranded and conserved in secondary structure. This study has enriched the mitochondrial gene database of Dipetalonema and laid a scientific basis for further study on classification, and genetic and evolutionary relationships of Dipetalonema nematodes.

We report the nearly complete mitochondrial genome of Rhabdosynochus viridisi – the first for this genus – achieved by combining shotgun sequencing of genomic and cDNA libraries prepared using low-input protocols. This integration of genomic information leads us to correct the annotation of the gene features. The mitochondrial genome consists of 13,863 bp. Annotation resulted in the identification of 12 protein-encoding genes, 22 tRNA genes and two rRNA genes. Three non-coding regions, delimited by three tRNAs, were found between the genes nad5 and cox3. A phylogenetic analysis grouped R. viridisi with three other species of diplectanid monogeneans for which mitochondrial genomes are available.

Ancylostoma tubaeforme may infect canids, felids and humans, and pose a potential risk to public health. Polymerase chain reaction (PCR) techniques were used to amplify the complete mitochondrial (mt) genome sequence of A. tubaeforme from cats and to analyse its sequence characteristics after molecular identification based on the internal transcribed spacer ITS1+ sequence. The results show that the complete mt genome sequence (GenBank accession number KY070315) of A. tubaeforme from cats was 13,730 bp in length, including 12 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, two non-coding regions and an AT-rich region. The nucleotide content of A and T was 77.93%, biased toward A and T. Twelve protein-coding genes used ATT, TTG and GTG as initiation codons, and TAA, TAG, TA and T as termination codons. The length of the 22 tRNA genes ranged from 52 to 62 bp, their predicted secondary structures were D loops and V loops. The lengths of the two rRNAs were 958 and 697 bp. Phylogenetic analyses showed that A. tubaeforme from cats was in the lineage of Ancylostoma, having a close phylogenetic relationship with A. caninum. This study reports for the first time the mt genome of A. tubaeforme from cats in China, which could enhance the mt genome database of Ancylostomatidae nematodes, and it offers the scientific basis for further studies in the genetic diversity of hookworms among different hosts.

The order Onchoproteocephalidea (Eucestoda) was recently erected to accommodate the hook-bearing tetraphyllideans and the proteocephalideans, which are characterized by internal proglottization and a tetra-acetabulate scolex. The recognized subfamilies in the Proteocephalidae appeared to be non-monophyletic based on 28S recombinant DNA (rDNA) sequence data. Other molecular markers with higher phylogenetic resolution, such as large mitochondrial DNA fragments and multiple genes, are obviously needed. Thus the mitochondrial genome of Gangesia oligonchis, belonging to the putative earliest diverging group of the Proteocephalidae, was sequenced. The circular mitogenome of G. oligonchis was 13,958 bp in size, and contained the standard 36 genes: 22 transfer RNA genes, two rRNA genes and 12 protein-coding genes, as well as two major non-coding regions. A short NCR and a large NCR (lNCR) region were 216 bp and 419 bp in size, respectively. Highly repetitive regions in the lNCR region were detected with that of 11 repeat units. The mitogenome of G. oligonchis shared 71.1% nucleotide identity with Testudotaenia sp. WL-2016. Phylogenetic analyses of the complete mitochondrial genomes with Bayesian inference and maximum likelihood methods indicated that G. oligonchis formed a sister clade with Testudotaenia sp. WL-2016 with maximum support. The ordinal topology is (Caryophyllidea, (Diphyllobothriidea, (Bothriocephalidea, (Onchoproteocephalidea, Cyclophyllidea)))). The mitogenomic gene arrangement of G. oligonchis was identical to that of Testudotaenia sp. WL-2016. Both mitogenomic and nuclear sequence data for many more taxa are required to effectively explore the inter-relationships among the Onchoproteocephalidea.

Tracheophilus cymbius (Trematoda: Cyclocoelidae) is a common tracheal fluke of waterfowl, causing serious loss in the poultry industry. However, taxonomic identification of T. cymbius remains controversial and confused. Mitochondrial (mt) genomes can provide genetic markers for the identification of closely related species. We determined the mt genome of T. cymbius and reconstructed phylogenies with other trematodes. The T. cymbius mt genome is 13,760 bp in size, and contains 12 protein-coding genes (cox 1–3, nad 1–6, nad 4L, cyt b and atp 6), 22 transfer RNA (tRNA) genes, two ribosomal RNA genes and one non-coding region. All are transcribed in the same direction. The A + T content is 62.82%. ATG and TAG are the most common initiation and termination codons, respectively. Phylogenetic analyses of concatenated nucleotide sequences show T. cymbius grouping in suborder Echinostomata, and clustering together, with high statistical support, as a sister taxon with Echinochasmus japonicus (Echinochasmidae), the two forming a distinct branch rooted to the ancestor of all Echinostomatidae and Fasciolidae species. This is the first report of the T. cymbius mt genome, and the first reported mt genome within the family Cyclocoelidae. These data will provide a significant resource of molecular markers for studying the taxonomy, population genetics and systematics of trematodes.

Ortleppascaris sinensis (Nematoda: Ascaridida) is a dominant intestinal nematode of the captive Chinese alligator. However, the epidemiology, molecular ecology and population genetics of this parasite remain largely unexplored. In this study, the complete mitochondrial (mt) genome sequence of O. sinensis was first determined using a polymerase chain reaction (PCR)-based primer-walking strategy, and this is also the first sequencing of the complete mitochondrial genome of a member of the genus Ortleppascaris. The circular mitochondrial genome (13,828 bp) of O. sinensis contained 12 protein-coding, 22 transfer RNA and 2 ribosomal RNA genes, but lacked the ATP synthetase subunit 8 gene. Finally, phylogenetic analysis of mtDNAs indicated that the genus Ortleppascaris should be attributed to the family Heterocheilidae. It is necessary to sequence more mtNDAs of Ortleppascaris nematodes in the future to test and confirm our conclusion. The complete mitochondrial genome sequence of O. sinensis reported here should contribute to molecular diagnosis, epidemiological investigations and ecological studies of O. sinensis and other related Ascaridida nematodes.

Despite the worldwide distribution and pathogenicity of monogenean parasites belonging to the largest helminth genus, Dactylogyrus, there are no complete Dactylogyrinae (subfamily) mitogenomes published to date. In order to fill this knowledge gap, we have sequenced and characterized the complete mitogenome of Dactylogyrus lamellatus, a common parasite on the gills of grass carp (Ctenopharyngodon idella). The circular mitogenome is 15,187 bp in size, containing the standard 22 tRNA genes, 2 rRNA genes, 12 protein-encoding genes and a long non-coding region (NCR). There are two highly repetitive regions in the NCR. We have used concatenated nucleotide sequences of all 36 genes to perform the phylogenetic analysis using Bayesian inference and maximum likelihood approaches. As expected, the two dactylogyrids, D. lamellatus (Dactylogyrinae) and Tetrancistrum nebulosi (Ancyrocephalinae), were closely related to each other. These two formed a sister group with Capsalidae, and this cluster finally formed a further sister group with Gyrodactylidae. Phylogenetic affinity between Dactylogyrinae and Ancyrocephalinae was further confirmed by the similarity in their gene arrangement. The sequencing of the first Dactylogyrinae, along with a more suitable selection of outgroups, has enabled us to infer a much better phylogenetic resolution than recent mitogenomic studies. However, as many lineages of the class Monogenea remain underrepresented or not represented at all, a much larger number of mitogenome sequences will have to be available in order to infer the evolutionary relationships among the monogeneans fully, and with certainty.

Eurytrema pancreaticum is one of the most common trematodes of cattle and sheep, and also infects humans occasionally, causing great economic losses and medical costs. In this study, the sequences of the complete nuclear ribosomal DNA (rDNA) repeat units of five E. pancreaticum individuals were determined for the first time. They were 8306–8310 bp in length, including the small subunit (18S) rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), large subunit (28S) rDNA and intergenic spacer (IGS). There were no length variations in any of the investigated 18S (1996 bp), ITS1 (1103 bp), 5.8S (160 bp), ITS2 (231 bp) or 28S (3669 bp) rDNA sequences, whereas the IGS rDNA sequences of E. pancreaticum had a 4-bp length variation, ranging from 1147 to 1151 bp. The intraspecific variations within E. pancreaticum were 0–0.2% for 18S rDNA, 0–0.5% for ITS1, 0% for 5.8S rDNA and ITS2, 0–0.2% for 28S rDNA and 2.9–20.2% for IGS. There were nine types of repeat sequences in ITS1, two types in 28S rDNA, but none in IGS. A phylogenetic analysis based on the 18S rDNA sequences classified E. pancreaticum in the family Dicrocoeliidae of Plagiorchiata, closely related to the suborder Opisthorchiata. These results provide useful information for the further study of Dicrocoeliidae trematodes.

The parasite Cyathostomum pateratum, which occurs in the large intestine of equines, is a common species of the subfamily Cyathostominae. Cyathostominae nematodes are a complex nematode group for which only limited genetic information has been reported. To re-examine the phylogenetic relationships among Cyathostominae nematodes, we sequenced the complete mitochondrial (mt) genome of Cy. pateratum and compared it with the mt genome of the congeneric species Cyathostomum catinatum. The complete mtDNA sequence of Cy. pateratum was 13,822 bp in length, 16 bp shorter than that of Cy. catinatum. The mtDNA sequences of both species contained 12 protein-coding genes, two rRNA genes and 22 tRNA genes, and all 36 genes were transcribed in the same direction and in the same strand. Pairwise comparisons of the 12 predicted amino acid sequences between Cy. catinatum and Cy. pateratum revealed differences of 0.4–3.1%; the least conserved sequence was that of cytochrome c oxidase subunit 3 (cox3). Phylogenetic analysis of the concatenated amino acid sequences using Bayesian inference and maximum likelihood methods showed that Cy. catinatum and Cy. pateratum clustered together with very high nodal support, and Cylicostephanus goldi was closer to the Cyathostomum nematodes than to other Cyathostominae nematodes. The mtDNA sequence of Cy. pateratum is reported here for the first time. The study will shed some light on the genetic evolution among parasitic nematodes in Cyathostomum.