PMC:3091644 / 33659-43327 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3091644","sourcedb":"PMC","sourceid":"3091644","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3091644","text":"Methods\n\nParasites and DNA extraction\nSingle tapeworms each of T. multiceps and T. pisiformis tapeworm were collected for DNA extraction and sequencing. T. multiceps was collected from a dog infected experimentally with Coenurus cerebralis from naturally infected sheep (Gansu Provincial Huangcheng Wool Sheep Breeding Farm). A single cysticercus of T. pisiformis was isolated from a naturally infected rabbit (at a slaughterhouse in Shandong Province) in our laboratory, and a cyst of the same species was collected from a rabbit in Henan Province. One T. hydatigena cyst was collected from the abdominal cavity of a sheep at a slaughterhouse in Qinghai Province. Other adult worms, T. asiatica, T. saginata and T. solium from patients were also used for genomic DNA extraction. Fragments from the tapeworms and a protoscolex from the cyst were washed with cold phosphate-buffered saline and frozen in liquid nitrogen. Genomic DNA was isolated using Genomic DNA Purification Kit (Puregene® DNA Purification System, Gentra Systems, Minneapolis, Minnesota, USA) according to the manufacturer's instructions.\n\nAmplification of mtDNA fragments\nThe total length of the mt genome was amplified in 9 overlapping fragments using EX TaqTM polymerases with 3'-5' exonuclease proofreading activity (Takara Biotechnology Co. Ltd, Dalian, China) using total genomic DNA purified from a single cyst or worm as the template. The overlapping fragments of T. multiceps, T. hydatigena and T. pisiformis mtDNAs were amplified using nine pairs of oligonucleotide primers (Additional file 5), designed according to the conserved regions from published complete mtDNA sequences of taeniid cestodes. All PCR reactions comprised ~20-40 ng of the genomic DNA in a 50 μl reaction containing 1.5 U Taq polymerase, 10 mM Tris-HCl pH9, 50 mM KCl, 2 mM MgCl2, 200 μM of each dNTP. PCR amplifications each proceeded with 35 cycles of 94°C for 1 min, 52°C for 45 s, 72°C for 2 to 4 min depending on product length. The amplicons were then cloned into the pGEM-T Easy vector (Promega Co., Winsconsin, USA). At least 3 clones from each amplicon were double-stranded sequenced.\n\nSequencing and assembling of DNA fragments\nAll sequencing was performed using terminator-based cycle sequencing with BigDye chemistry (Applied Biosystems, Foster City, CA, USA) on an ABI 3730 or 373 DNA sequencer (Applied Biosystems) at Shanghai Sangon or Takara Biotechnology Co. Amplicons were sequenced to completion by primer walking. Chromatograms were visualized using reports were analyzed using Chromas 2.33 software http://www.technelysium.com.au, and sequences were assembled using CUGI's New CAP3 Server online (The Clemson University Genomics Institute, from http://www.genome.clemson.edu/) [68]. Sequence data were analyzed with the SeqMan and MegAlign programs, and the consensus sequence of each amplicon was used as the final sequence (DNASTAR Inc., Madison, WI, USA).\nNucleotide sequences identified in this study have been submitted to GenBank, and the accession numbers for T. multiceps, T. hytigena and T. pisiformis mtDNAs are GQ228818, GQ228819 and GU569096, respectively. The published mtDNA sequences for other Cestoda used in this study include: T. solium (NC_004022), T. saginata (NC_009938), T. asiatica (NC_004826), T. crassiceps (NC_002547), Echinococcus multilocularis (NC_000928), E. oligarthrus (NC_000928) and Hymenolepis diminuta (NC_002767).\n\nPrediction of protein-coding genes\nThe protein-coding regions were identified using BLAST searches, ORF finder of DNAStar and comparisons with other sequences of Platyhelminthes available in the GenBank database http://www.ncbi.nlm.nih.gov/BLAST/. Genetic codes were based on translation table nine and those in cestodes [49,52].\n\nPrediction of tRNAs and genes for rrnL and rrnS\nPutative tRNA genes were identified using the software ARWEN http://130.235.46.10/ARWEN/[55], combined with visual inspection of aligned mtDNAs and tRNA genes. Genes for rrnL and rrnS were identified from sequence similarities to the published cestode mitochondrial rRNA genes [43]. Putative stem-loop structures of non-coding mitochondrial regions (LNR and SNR) were inferred using the program RNAstructure v. 4.6) [69,70].\n\nMitochondrial gene arrangement\nMitochondrial gene arrangements were compared by eye for gene adjacencies in all pairwise combinations for T. multiceps, T. hydatigena and T. pisiformis according to T. solium, T. saginata, T. asiatica, T. crassiceps and E. multilocularis.\n\nAlignment and phylogenetic analysis\nTwo other taeniids, early divergent members of Echinococcus, E. multilocularis and E. oligarthus [48], and Hymenolepis diminuta were selected as suitable outgroups. Nucleotides of all Taenia mtDNAs and outgroups were aligned initially by eye, in frame where appropriate for protein-coding genes, using MacClade [71]. Amino acids translations were inferred using the flatworm mitochondrial code (Table nine, GenBank http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c#SG9) [52], exported and handled in a separate MacClade file. Nucleotide and amino acid alignments consisted of 11,983 and 3404 positions respectively, and each were exported to the Gblocks server http://molevol.cmima.csic.es/castresana/Gblocks_server.html) running Gblocks 0.91 b [72] under default parameters, to remove poorly aligned positions. Gblocks removed 4.9% (n = 590 nucleotides) and 2.3% (n = 80 amino acids) of the nucleotide and amino acid alignments respectively. For the nucleotide data set, individual genes were first scrutinized to see whether they were overly saturated. Using Xia et al.'s test [73], as implemented in Dambe[74], individual genes were tested using all sites, and for each protein-coding gene 1st, 2nd and 3rd positions were checked individually. The following 8 gene partitions passed the test and were included in the phylogenetic analysis (1,2 indicates sites 1 + 2 included only, otherwise all sites included): cox3, cytb, nad41,2, nad2, nad11,2, cox1, cox21,2 and nad5. Both ribosomal RNA genes, atp6, nad3, nad4L and nad6 were significantly saturated and were excluded. The 8 unstaurated gene partitions provided a total of 7,655 nucleotides available for phylogenetic analysis.\nBayesian analyses of the nucleotide and amino acid analyses were each carried out using MrBayes, v.3.1.2 [75]. Following the recommendations of Rota-Stabelli et al. [76], for the amino acid data set the mtZoa model was applied (settings were rates = gamma, ngammacat = 5, aamodel = fixed (rtrev) ); two chains (temp = 0.2) were run for 5,000,000 generations and sampled every 1,000 generations. Using the unsaturated gene partitions only, Modeltest 3.7maxX [77] was used to estimate a suitable model for nucleotide substitution; this was equivalent to GTR+I+G and settings were nst = 6, rates = invgamma, ngammacat = 4. Four chains (temp = 0.2) were run for 5,000,000 generations and sampled every 1,000 generations. For each analysis convergence was assessed using Tracer v 1.4 [78], with a discarded burn-in period of 5,000 trees. Posterior probabilities provided evidence of nodal support. All trees were rooted against Hymenolepis diminuta.\nThe morphological data set of Hoberg et al. [6] was incorporated into a MacClade [71] file for each of the 7 Taenia species studied here, to enable character mapping onto alternative tree topologies offered by alternative data sets. Only unambiguous changes were traced to determine whether novel tree topologies from molecular data were supported by morphological characters.\n\nSliding window analysis of nucleotide variation\nThe complete alignment of nucleotides of the 7 Taenia mtDNAs was used to effect sliding window analyses using DnaSP v.5 [64]. A sliding window of 300 bp and steps of 10 bp was used to estimate nucleotide diversity (π) for the entire alignment. Nucleotide diversity for the entire alignments was plotted against midpoint positions of each window, and gene boundaries indicated. PAUP* v. 4.0b10 [79] was used to determine the position of phylogenetically informative positions under the principle of parsimony, and the sum of these sites was calculated using a sliding window approach with the same parameters (window size = 300 bp; step size = 10 bp) and plotted on the same graph.\n\nDesign of novel mitochondrial markers for Taenia and their applications in PCR\nUsing the same complete nucleotide alignment of mtDNAs, but with outgroups and deletions common to all taxa removed, novel PCR primers were sought using PriFi [67]. This software looks for primer pairs that fit given criteria, with the added benefit of being designed on the basis of all taxa in the alignment. PriFi only accepts relatively short alignments and so 4,000 bp segments of the complete alignment were subjected to analysis; each segment overlapped the next by 1,000 bp and a segment including both ends of the linear alignment (to complete the mtDNA circle) was also analyzed. Settings differed from default parameters as follows: minimum melting temperature = 45.0˙C, critical melting temperature = 55.0˙C, minimum number of 3'-end matches = 3, optimal primer length interval = (22, 35 bp), optimal PCR product length interval = (400, 500, 800, 1,000 bp), minimum product length = 350 bp, conservation window length = 50 bp.\nAll PCR reactions comprised ~20-40 ng of the genomic DNA in a 50 μl reaction containing 1.25 U Taq polymerase, 10 mM Tris-HCl pH9, 50 mM KCl, 2 mM MgCl2, 200 μM of each dNTP, and 0.2 μM each primer. PCR amplifications each proceeded with 35 cycles of 94°C for 1 min 30 s, 52~57°C for 30 s, 72°C for 1 min. 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