Characterization of the intergenic spacer rDNAs of two pig nodule worms, Oesophagostomum dentatum and O. quadrispinulatum.
The ribosome has been identified as a central hub for sensing the nature of a nascent protein chain, recruiting protein folding and translocation components, and integrating mRNA and nascent chain quality control . Ribosomal RNA (rRNA) typically accounts for approximately 40% of all transcription within a cell, and ribosomal RNA comprises as much as 80% of the cellular RNA . The rRNA transcripts, including 18S, 28S, and 5.8S rRNA, mature through the excision of their tandem spacer regions, for example, internal transcribed spacers (ITSs) and intergenic spacers (IGSs) . The IGSs are biologically significant. Furthermore, the cell is capable of both regulating rRNA synthesis and sequestering large numbers of proteins to modulate essential molecular networks through the timely induction of various ribosomal IGS noncoding RNA (IGS RNA) transcripts .
IGS regions are composed of an extraordinary variety of repeats and RNA polymerase promoters and enhancers, which cause considerable inter- and intraspecific variations in parasites . These variations make IGS rDNA a suitable marker for inferring evolutionary relationships among more closely related species and among strains within the same species  as well as for developing molecular detection approaches for infectious diseases [7-12]. The IGS rDNA regions of Trypanosoma , Giardia , Leishmania , Toxoplasma gondii and Neospora caninum , Schistosoma haematobium, S. intercalatum, and S. mansoni , and S. japonicum  have been studied and showed some organizational features common to the majority of eukaryotes.
Oesophagostomiasis, which is caused by nodular worms (Oesophagostomum spp.) and is commonly observed in pigs, ruminants, and primates (including humans), is often neglected by researchers and practitioners due to its mild symptoms . However, severe infections can lead to significant socioeconomic problems and serious public health concerns [17-19]. Of the Oesophagostomum spp., O. dentatum and O. quadrispinulatum have been identified as the two main causative agents of oesophagostomiasis in pigs [20, 21]. Moreover, O. dentatum was proposed as a potential model for genomic studies of strongylid nematodes . The objective of this study was to determine the characteristics of the IGS rDNA regions of O. dentatum and O. quadrispinulatum collected from pigs at different geographical locations in Mainland China.
2. Materials and Methods
2.1. Parasite Samples. Isolates of O. dentatum (14 isolates) and O. quadrispinulatum (12 isolates) were collected from pigs at six geographical origins in Mainland China. Their codes, geographical origins, and accession numbers are listed in Table 1. Each adult parasite was washed extensively in physiological saline and was preliminarily identified at the species level based on its morphological characteristics .
2.2. DNA Isolation and PCR Amplification. Genomic DNA (gDNA) was extracted from individual adult worms through sodium dodecyl-sulfate/proteinase K treatment, column-purified using the Wizard SV Genomic DNA Purification System (Promega) and eluted with 40 [micro]L of [H.sub.2]O according to the manufacturer's recommendations. The DNA samples were then identified at the species level based on their ITS rDNA sequences  and stored at -20[degrees]C until further analysis.
The 28S-18S IGS rDNA sequences of O. dentatum and O. quadrispinulatum were amplified using PCR with the O28 (5'-ACGACAT GTATACT GGT CAAGG-3', forward) and O18 (5'-GCTTTGGTGCATGTATTAGCTC-3', reverse) primers. The PCR reactions included 3mM Mg[Cl.sub.2], 0.5 [micro]M of each primer, 2.5 [micro]L of Ex Taq buffer, 0.2 mM of each deoxyribonucleotide, 0.5 U of Ex Taq DNA polymerase (TAKARA), 1 [micro]L of DNA sample, and double-distilled water to a total volume of 25 [micro]L. The procedures were performed in a thermocycler (Biometra) under the following conditions: 94[degrees]C for 5 min (initial denaturation), followed by 35 cycles of 94[degrees]C for 30 s (denaturation), 55[degrees]C for 1 min (annealing), 72[degrees]C for 45 s (extension), and a final extension at 72[degrees]C for 5 min. An aliquot (5 [micro]L) of each amplicon was examined on 1.0% agarose-TBE gels, stained with ethidium bromide (EB) and photographed upon transillumination. The DL2000 marker (TAkArA) was used to estimate the sizes of the IGS+ rDNA amplicons.
2.3. Purification, Cloning, and Sequencing of the IGS PCR Products. Representative PCR products were purified using spin columns (Wizard PCR-Prep DNA Purification System, Promega), and the purified PCR products were ligated into the pGEM-T easy plasmid vector (Promega) according to the manufacturer's recommendations. The recombinant plasmid was then transformed into Escherichia coli JM109 competent cells (Promega), and positive transformants containing recombinant plasmids were selected by PCR amplification. Cell cultures with confirmed recombinant plasmids were sent to Shanghai sangon Biological Engineering Biotechnology Company for sequencing using an ABI 377 automated DNA sequencer (BigDye Terminator Chemistry).
2.4. Sequence Analysis and Reconstruction of Phylogenetic Relationships. The characteristics of the 28S-18S IGS rDNA regions of O. dentatum and O. quadrispinulatum were determined by comparing these sequences with the previously published IGS rDNA sequences of Skrjabingylus chitwoodorum (AY295819), Cylicocyclus nassatus (AJ223348), and Cyathostomum catinatum (AJ223339); the 18S rDNA sequences of Chabertia ovina (AJ920341) and Labiostrongylus bipapillosus (AJ920337); and the 28S rDNA sequences of Chabertia ovina (AM039733) and Labiostrongylus bipapillosus (AJ512837).
The palindrome in EMBOSS 6.3.1  (http://mobyle .pasteur.fr/cgi-bin/portal.py?#forms::palindrome) was used to identify inverted repeats in the Oesophagostomum species. Direct repeats were identified using REPFIND  at http://cagt.bu.edu/page/REPFINDsubmit and Tandem Repeats Finder  at http://tandem.bu.edu/trf/trf.html. These repeats were identified with the criteria of nuclear match [greater than or equal to] 10 bp and mismatch [less than or equal to] 1.
The phylogenetic relationships of the O. dentatum and O. quadrispinulatum isolates from the different geographical origins were reconstructed based on their IGS rDNA sequences using the neighbor-joining (NJ) method within the Mega 4.0 software and the Kimura 2-parameter model . Phylograms were drawn using the TreeView program, version 1.65 .
3. Results and Discussion
The 28S-18S IGS rDNA sequences of O. dentatum and O. quadrispinulatum from Mainland China had dynamic and highly complex structures. The first indication of this finding became apparent upon amplification of the IGS rDNA, which presented variable lengths ranging from 1000 to 1400 bp (data not shown). After removal of the flanking 28S and 18S rDNA sequences, the lengths of the IGS rDNA sequences were 759-762 bp and 937-1128 bp for O. dentatum and O. quadrispinulatum, respectively The IGS of O. dentatum only contained 2 copies of one 11-nt direct repeat (A1 and A2) and one 10-nt inverted repeat (B and B rev comp). The IGS rDNA sequences of O. quadrispinulatum could be grouped into two types based on their lengths and characteristics. The longest, the IGS of OQHN1, exhibited the following features: (1) three complete 49-nt copies of direct repeat C beginning 409 nt downstream of the 5' end of the IGS rDNA; (2) two copies of direct repeat D beginning 89 nt downstream of the last copy of direct repeat C; (3) one complete inverted repeat I; and (4) six short, incomplete inverted repeats (inverted repeats F, G, H, J, K, and L). Compared with OQHN1, the shortest sequence, OQYC4, showed the following features: (1) a new, incomplete inverted repeat E and (2) no repeats including two copies of direct repeat C, repeat F, repeat G, or inverted repeat F (Figure 1).
Eukaryotic ribosomes are very important for protein synthesis, cellular growth, and organismal development . In most eukaryotes, rDNA is arranged in tandemly repeated units containing genes for the 18S, 5.8S, and 28S rRNAs, which are separated by spacers. The large intergenic spacer (IGS; formerly NTS) separating the 28S and 18S genes is internally repetitious: each repeat contains a tandem array of short subrepeat units [29, 30]. Alterations in these repeats mostly occur due to unequal crossing over during both sexual and asexual reproduction or in somatic cell lineages , and variations in the number of repeat units and, consequently, in the copy number of the regulatory elements can lead to the polymorphic lengths and structures observed in the IGS rDNA . In this study, the IGS rDNA sequences of O. dentatum and O. quadrispinulatum were found to contain many short, direct, and inverted repeats, and different types and numbers of repeats were found both between the two pig nodule species and within O. quadrispinulatum. These differences suggested that these species are independent and that genotypes or subspecies exist within the known species of pig nodule worms.
Homologous chromosome pairing precedes meiotic recombination and may initiate without strand breakage by way of "kissing" interactions between the loops of extruded stem-loop structures . Such recombination may be responsible for the existence of numerous repeats such as "cross-over hot-spot" (Chi) sequences . The Chi site, which contains sequences of 57-GCTGGTGG-37 in the template strand and 5,-CCACCAGC-37 in the complementary strand, has been found in the repetitive sequences within the first internal transcribed spacer of the rDNA of schistosomes . Although these two sequences were not found in the IGS rDNA sequences of the two pig nodule worms examined in this study, two similar sites, with sequences of 57GCTGGTGT-37 93 bp upstream of the 5' end of direct repeat B and of 5,-CCTGGCGG-37 9bp downstream of the 3' end of inverted repeat I, were found in the IGS rDNA sequences of O. dentatum and O. quadrispinulatum, respectively. These results indicated that the IGS rDNA might participate in homologous chromosome pairing.
Pairwise comparisons showed interspecific genetic variations of 54.6-56.8% between O. dentatum and O. quadrispinulatum isolates and intraspecific sequence differences of 01.3% and 0.2-15.1% for O. dentatum and O. quadrispinulatum, respectively. The phylogenetic relationships among O. dentatum, O. quadrispinulatum, and other known species were reconstructed using NJ analysis (Figure 2). From the NJ tree, two main clades were observed. All Oesophagostomum isolates were grouped in a sister clade, including Trichostrongylus colubriformis and Marshallagia marshalli, which suggested their close relationships with the two Oesophagostomum spp. Within the cluster of the two Oesophagostomum species, all O. dentatum isolates clustered together in one clade, whereas the O. quadrispinulatum isolates from different locations grouped into two distinct clusters. These results indicated the complicated genetic structure of O. quadrispinulatum.
The IGS is the most rapidly evolving region of rDNA, and the number and organization of the internal repeats are species-specific and often vary among populations, individuals, and even within a single cell . These variations in the structures of repetitive regions are common in many taxa and have been widely used in phylogenetic analysis and to quantify gene flow between populations [29, 30]. Analyses of inter- and intragenetic variations in the IGS rDNA sequences of two species of pig nodule worms showed that the sequence differences between O. quadrispinulatum isolates were larger than those between O. dentatum isolates, and phylogenetic analysis revealed two subclusters within a clade of O. quadrispinulatum. These results suggested that different genotypes or subspecies might exist in O. quadrispinulatum.
This study is the first to report the 28S-18S IGS rDNA sequences of O. dentatum and O. quadrispinulatum from different geographical locations in China. Genetic analysis revealed the sequence annotations and organizations of these sequences and demonstrated that these regions were polymorphic and contained direct and inverted repeats. Different types and numbers of repeats and IGS rDNA structures could serve as potential markers for differentiating these two species of pig nodule worms.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by the Open Funds of the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Grant no. SKLVEB2012KFKT003), and the Scientific and Technological Programs of Guangdong Province (Grant no. 2012A020100001).
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Rui-Qing Lin, (1) Li Shu, (1,2) Guang-Hui Zhao, (3) Tian Cheng, (1) Shang-Shu Zou, (1) Yuan Zhang, (1) and Ya-Biao Weng (1)
(1) Laboratory of Parasitology, College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong Province 510642, China
(2) Yong Nian Veterinary Station, Fushun, Sichuan 643200, China
(3) College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
Correspondence should be addressed to Ya-Biao Weng; email@example.com
Received 10 March 2014; Revised 16 July 2014; Accepted 17 July 2014; Published 13 August 2014
Academic Editor: Rajesh Jeewon
TABLE 1: Geographical origins, sample codes, and GenBank accession numbers of Oesophagostomum dentatum and O. quadrispinulatum samples used in the present study. Species Sample Geographical origin Accession code number O. dentatum ODHN1 Hunan (Huihua) KC991159 ODHN2 Hunan (Huihua) KC991160 ODYJ1 Guangdong (Yangjiang) KC991161 ODYJ2 Guangdong (Yangjiang) KC991162 ODYJ3 Guangdong (Yangjiang) KC991163 ODYJ4 Guangdong (Yangjiang) KC991164 ODYJ5 Guangdong (Yangjiang) KC991165 ODYC1 Chongqing (Yongzhou) KC991166 ODYC2 Chongqing (Yongzhou) KC991167 ODYC3 Chongqing (Yongzhou) KC991168 ODYC4 Chongqing (Yongzhou) KC991169 ODYC5 Chongqing (Yongzhou) KC991170 ODHLJ1 Heilongjiang (Jiaxing) KC991171 ODYB1 Chongqing (Yubei) KC991172 O. quadrispinulatum OQHN1 Hunan (Huihua) KC991173 OQRC1 Chongqing (Rongchang) KC991174 OQYC1 Chongqing (Yongchuan) KC991175 OQYC2 Chongqing (Yongchuan) KC991176 OQYC3 Chongqing (Yongchuan) KC991177 OQYC4 Chongqing (Yongchuan) KC991178 OQYC5 Chongqing (Yongchuan) KC991179 OQYC6 Chongqing (Yongchuan) KC991180 OQYC7 Chongqing (Yongchuan) KC991181 OQYC8 Chongqing (Yongchuan) KC991182 OQYC9 Chongqing (Yongchuan) KC991183 OQYC10 Chongqing (Yongchuan) KC991184 Trichostrongylus -- Iran HQ389237 colubriformis Marshallagia marshalli -- Iran HQ389236 Haemonchus contortus -- Iran HQ389234 Cylicostephanus minutus -- -- HM142941 Coronocyclus coronatus -- -- HM142939
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|Title Annotation:||Research Article|
|Author:||Lin, Rui-Qing; Shu, Li; Zhao, Guang-Hui; Cheng, Tian; Zou, Shang-Shu; Zhang, Yuan; Weng, Ya-Biao|
|Publication:||The Scientific World Journal|
|Date:||Jan 1, 2014|
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