The Mitochondrial Genome of Paramphistomum cervi (Digenea), the First Representative for the Family Paramphistomidae Hong-Bin Yan1., Xing-Ye Wang1,4., Zhong-Zi Lou1,LiLi1, David Blair2, Hong Yin1, Jin-Zhong Cai3, Xue-Ling Dai1, Meng-Tong Lei3, Xing-Quan Zhu1, Xue-Peng Cai1*, Wan-Zhong Jia1* 1 State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Key Laboratory of Veterinary Public Health of Agriculture Ministry, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, PR China, 2 School of Marine and Tropical Biology, James Cook University, Queensland, Australia, 3 Laboratory of Plateau Veterinary Parasitology, Veterinary Research Institute, Qinghai Academy of Animal Science and Veterinary Medicine, Xining, Qinghai Province, PR China, 4 College of Veterinary Medicine, Northwest A&F University, Yangling, Shanxi Province, PR China Abstract We determined the complete mitochondrial DNA (mtDNA) sequence of a fluke, Paramphistomum cervi (Digenea: Paramphistomidae). This genome (14,014 bp) is slightly larger than that of Clonorchis sinensis (13,875 bp), but smaller than those of other digenean species. The mt genome of P. cervi contains 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions (NCRs), a complement consistent with those of other digeneans. The arrangement of protein-coding and ribosomal RNA genes in the P. cervi mitochondrial genome is identical to that of other digeneans except for a group of Schistosoma species that exhibit a derived arrangement. The positions of some transfer RNA genes differ. Bayesian phylogenetic analyses, based on concatenated nucleotide sequences and amino-acid sequences of the 12 protein-coding genes, placed P. cervi within the Order Plagiorchiida, but relationships depicted within that order were not quite as expected from previous studies. The complete mtDNA sequence of P. cervi provides important genetic markers for diagnostics, ecological and evolutionary studies of digeneans. Citation: Yan H-B, Wang X-Y, Lou Z-Z, Li L, Blair D, et al. (2013) The Mitochondrial Genome of Paramphistomum cervi (Digenea), the First Representative for the Family Paramphistomidae. PLoS ONE 8(8): e71300. doi:10.1371/journal.pone.0071300 Editor: Kelly A. Brayton, Washington State University, United States of America Received March 25, 2013; Accepted June 26, 2013; Published August 22, 2013 Copyright: ß 2013 Yan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the ‘‘Special Fund for Agro-scientific Research in the Public Interest’’ (Grant No. 201303037), the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006), and NBCITS, MOA (CARS-38), the People’s Republic of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (WZJ); [email protected] (XPC) . These authors contributed equally to this work. Introduction misinterpretation or misdiagnosis because the presence of adult paramphistomes (hence their eggs) is not necessarily a cause of Paramphistomosis, due to paramphistomes (Trematoda: Dige- disease [7,13,14]. Early diagnosis of paramphistomosis is essential nea: Paramphistomidae), has recently emerged as a major cause of for prompt treatment before irreparable damage to the rumen and productivity loss in ruminants. Adult worms often inhabit the bile ducts occurs [9]. Immunological diagnosis may be a rumen and reticulum of cattle, water buffaloes, sheep and goats. dependable means for monitoring the infection, and be supple- Their presence in these sites may elicit few apparent signs or mented by the finding of eggs. In order to develop this method, symptoms. However, acute parasitic gastroenteritis causing high whole worm extract of adult P. cervi has been subjected to immuno- morbidity and mortality may occur as large numbers of immature blotting using sera from bovines infected with P. cervi. This paramphistomes migrate through the intestine towards the rumen method, however, has not been widely adopted [15]. and reticulum. Severity of disease is greatest in young animals [1– Rapid development of molecular biology techniques, especially 6]. As a consequence of frequent under-diagnosis, the significance the polymerase chain reaction (PCR), may provide valuable of subclinical infection in many animals remains unclear and supplementary tools for the differential identification of digenean economic losses may exceed those caused by many other helminth infection to overcome limitations of current morphological- parasites [5,7]. Paramphistomosis is widespread [6–10], with diagnostic methods. Due to their high nucleotide substitution different species predominating in different places. However, rates, partial or complete mitochondrial (mt) genomes of parasitic Paramphistomum cervi is perhaps the most widespread species, being flatworms have become very popular markers for detecting their reported from many parts of Eurasia [9,11] and North America presence in animals and for investigating their phylogenetic [12]. Conventional diagnosis of paramphistomosis is based on the relationships at different levels [16–29]. history and clinical signs of the disease. Further confirmation can The lack of knowledge of mt genomics for P. cervi is a major be obtained by collection of fecal samples from the host and limitation for the development of molecular diagnostic techniques, examination for parasite eggs. However, this can lead to PLOS ONE | www.plosone.org 1 August 2013 | Volume 8 | Issue 8 | e71300 The Mitochondrial Genome of Paramphistomum cervi Table 1. Primers for amplification of mt DNA genome of P. Seven pairs of oligonucleotide primers were designed based on the cervi. conserved regions from published complete mtDNA sequences of Fasciola hepatica [27,28], Clonorchis sinensis [30,31], Opisthorchis felineus [30] and Paragonimus westermani (GenBank Accession No. Primer name (positions) Sequence of primer (59R39) AF219379) (Table 1). These sets of primers amplified overlapping fragments to facilitate eventual assembly using Taq polymerase – PC1-F_nad5 (12177–12198) TTDCKTCTCGNTTBGGKGATGT KOD FX Neo (TOYOBO, Japan). The cycling conditions used PC1-R_cytb (1497–1519) ARAAARTAYCACTCNGGCTTWAT were 94uC for 5 min (initial denaturation); then 94uC for 1 min PC2-F_cytb (1110–1132) TATTGRGCTGCTACDGTTTTGAC (denaturation), 50uC for 35 s (annealing), 72uC for 1–3 min PC2-R_nad2 (4623–4648) CATCATATGACACCAACAATAATACC (extension) for 30 cycles and a final extension at 72uC for 10 min. Each PCR reaction yielded a single band detected in a 1.0% (w/v) PC3-F_nad2 (4054–4076) TTTKTTTATGAGRTYTTTGTBGC agarose gel stained with ethidium-bromide [24]. PCR products PC3-R_nad1 (5839–5963) AYTCDCTYTCVGMCTCMSCRTAATC were directly sequenced on an ABI 3370 DNA sequencer at PC4-F_nad1 (5350–5372) CGTAAGGGKCCDAAHAAGGTTGG Sangon Company (Shanghai, China) using a primer walking PC4-R_cox1 (7603–7628) CCAAARAAYCAAAAYAWATGYTGAAA strategy. The complete mtDNA sequence of P. cervi was assembled PC5-F_cox1 (7285–7307) GTTGGKTGRACTTTTTATCCWCC using DNAStar software as a sequence editor [32]. PC5-R_s-rRNA (9455–9474) AGATAAGAACCGACCTGGCT PC6-F_s-rRNA (9296–9318) TTACCTYGGGGATAACTRRGTAA Prediction of protein-coding genes, tRNAs and genes for rrnL and rrnS PC6-R_nad6 (10851–10876) GCACCACAHAAYTCMSTACARTAHCC The ORF finder tool at NCBI (http://www.ncbi.nlm.nih.gov/ PC7-F_cox2 (10265–10292) TAGCTCTGATAAGTCGTAACATGGTAAT gorf/gorf.html) was used to find protein-coding gene sequences, PC7-R_nad5 (12326–12343) HGGAGCVCGCATHGCYTC which were subsequently used to search for homologous digenean TM Notes: D = A/T/G; H = A/C/T; K = G/T; M = A/C; N = A/C/G/T; R = A/G; V = A/C/G; sequences deposited in the GenBank by using tBLASTn. The W = A/T; Y = T/C. The positions of primers in the study are based on the mt rhabditophoran platyhelminth genetic code [33] was specified. genome sequence of P. cervi. Gene boundaries were confirmed based on comparison and doi:10.1371/journal.pone.0071300.t001 alignment with other published mt genomes of species in Fasciolidae, Opisthorchiidae and Paragonimidae [27,28,30,31]. for analyses of population and genetic variation within this species, Putative tRNA genes were identified using the program and for phylogenetic studies of the Digenea in general. tRNAscan-SE [34] and the online tool ARWEN [35] combined In our present study, we determined the complete mt with observations and alignments by eye. Genes for large (rrnL) nucleotide sequence of P. cervi, which was collected from Qinghai and small (rrnS) subunit ribosomal RNA genes were identified by Province, China. Phylogenetic analyses were performed using comparison with the mt rRNA genes of F. hepatica, C. sinensis, O. concatenated mt sequences of 12 protein-coding genes of felineus, P. westermani and other flatworms [27,28,31]. digenean species available in GenBank to date. The new mt Phylogenetic analyses. DNA sequences of the 12 protein- genome sequence may provide useful information on both coding genes were concatenated and imported into BioEdit [36]. genomics and the evolution of Paramphistomidae,