Mitochondrial and Nuclear Ribosomal DNA Evidence Supports the Existence of a New Trichuris Species in the Endangered François’ Leaf-Monkey

The whipworm of humans, Trichuris trichiura, is responsible for a neglected tropical disease (NTD) of major importance in tropical and subtropical countries of the world. Whipworms also infect animal hosts, including pigs, dogs and non-human primates, cause clinical disease (trichuriasis) similar to that of humans. Although Trichuris species are usually considered to be host specific, it is not clear whether non-human primates are infected with T. trichiura or other species. In the present study, we sequenced the complete mitochondrial (mt) genome as well as the first and second internal transcribed spacers (ITS-1 and ITS-2) of Trichuris from the François’ leaf-monkey (langur), and compared them with homologous sequences from human- and pig-derived Trichuris. In addition, sequence comparison of a conserved mt ribosomal gene among multiple individual whipworms revealed substantial nucleotide differences among these three host species but limited sequence variation within each of them. The molecular data indicate that the monkey-derived whipworm is a separate species from that of humans. Future work should focus on detailed population genetic and morphological studies (by electron microscopy) of whipworms from various non-humans primates and humans.


Introduction
Neglected tropical diseases (NTD S ) have a devastating effect on animal and human health and food production globally. For instance, it is estimated that more than two billion people are infected with geohelminths, including the Ascaris (common roundworm), Necator, Ancylostoma (hookworms) and Trichuris (whipworm), mainly in underprivileged areas of the world [1]. Trichuris trichiura is a very common parasite of humans in developing countries, and causes trichuriasis in , 600 million people worldwide, mainly in children aged between 5 and 15 years [2]. Trichuriasis can be associated with intestinal symptoms, such as abdominal pain, dysentery, nausea, vomiting, anorexia, constipation and chronic appendiceal syndrome [2]. Whipworms also infect a broad range of other hosts, including pigs (T. suis), dogs (T. vulpis), sheep (T. ovis), goats (T. skrjabini), rats (T. muris) and nonhuman primates, and can cause clinical disease similar to trichuriasis of humans [3][4][5][6][7].
Trichuris infects non-human primates in many countries, including Belgium [7], China [8], Ethiopia [9], Kenya [10,11], Peru [12], South Africa [13]. In spite of the high prevalence of Trichuris sometimes reported in non-human primates [13], it is not clear whether the non-human primates harbour T. trichiura or other congeners. Based on morphological features of adult worms, Trichuris of non-human primates (including Trichuris cynocephalus and T. rhinopithecus) have been regarded as T. trichiura [14,15]. However, the identification of Trichuris to species using morphological criteria alone is not reliable. Moreover, neither larval or egg stages of Trichuris from humans, pigs and non-human primates can be identified or differentiated unequivocally to species using classical diagnostic approaches [14,16]. Therefore, there is a need for suitable molecular approaches to accurately identify and distinguish closely-related Trichuris species from different hosts.
Molecular tools, using genetic markers in mitochondrial (mt) DNA and in the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA), have been used effectively to identify nematode species [17][18][19][20][21]. For whipworms, mtDNA has been used in China to show clear genetic distinctiveness between human-and pig-derived Trichuris [22], and between T. ovis and T. discolor from ruminant hosts [23]. Using ITS rDNA, recent studies of Trichuris specimens obtained from humans and pigs [24,25] also indicate that T. trichiura and T. suis are separate species. Cutillas et al. [26] used the ITS rDNA to infer the existence of two separate Trichuris species in murid and arvicolid rodents. In other studies from Spain, ITS rDNA has also been employed to distinguish among T. suis from swine, T. vulpis from dogs [27] and T. trichiura from the non-human primates (i.e. Pan troglodytes, Colobus guereza kikuyensis and Nomascus gabriellae) [15]. Although a recent investigation has shown two distinct Trichuris genotypes infecting both humans and non-human primates [28], there is still a paucity of information on Trichuris from different species of primates and countries around the world. Therefore, in the present study, we characterized the mt genomic and ITS rDNA sequences of Trichuris from the endangered François' leaf-monkey (Trachypithecus (Presbytis) françoisi), which usually lives in close proximity to human populations in southern China [29], and we compared them with homologous sequences of human-and pig-derived Trichuris, and then tested the hypothesis that this monkey-derived Trichuris is a separate species.

Ethics Statement
This study did not require approval by an ethics committee. Two François' leaf-monkeys, from which Trichuris specimens were collected from their caeca post-mortem, were handled and housed in a zoo in strict accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People's Republic of China. The monkeys were caged, and there were two rooms in a cage; one was indoor and the other was outdoor. They were fed fruits and vegetables. The monkeys were under the care and treatment of a licensed veterinarian at the zoo, and were euthanized due to acute gastric dilation.

Parasites and Isolation of Total Genomic DNA
Two adult specimens of Trichuris (designated ''monkey-Trichuris'') were collected from each of the two François' leaf-monkeys, and were washed in physiological saline, identified morphologically [14], fixed in 70% (v/v) ethanol and stored at -20uC until use. Total genomic DNA was isolated separately from four individual worms (coded TH1-TH4) using an established method [30].

Sequence Analyses
Sequences were assembled manually and aligned against the complete mt genome sequences of T. trichiura [22] using the computer program Clustal X 1.83 [33] to infer gene boundaries. The open reading frames (ORFs) were identified using ORFFinder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) employing the invertebrate mitochondrial code, and subsequently compared with that of T. trichiura [22]. Translation initiation and termination codons were identified based on comparison with those reported previously [22]. The secondary structures of 22 tRNA genes were predicted using tRNAscan-SE [34] and/or manual adjustment [35], and rRNA genes were identified by comparison with those known for Trichuris [22].

Phylogenetic Analyses
Amino acid sequences inferred from the 12 protein-coding genes (i.e. not atp-8) common among all of the nematodes included here were concatenated into a single alignment, and then aligned with those of four other enoplid nematodes (GenBank accession nos. GU385218, GU070737, JQ996232 and JQ996231 for T. trichiura, T. suis, T. ovis and T. discolor, respectively), using T. spiralis (accession no. NC_002681) [37] as an outgroup. Ambiguous sites and regions in the alignment were excluded using Gblocks (http:// molevol.cmima.csic.es/castresana/Gblocks_server.html) [38] using default parameters. The rrnL sequences determined here and those of human-and pig-derived Trichuris [22] were aligned and subjected to phylogenetic analysis using Trichinella spiralis (accession Table 1. Sequences of primers used to amplify mitochondrial DNA regions from monkey-Trichuris.

Results
Features of the Circular mt Genome of Trichuris from the François Leaf-monkey The complete mt genome sequence was 14,147 bp in length (GenBank accession no. KC461179). The mt genome contains 13 protein-coding genes (cox1-3, nad1-6, nad4L, cytb, atp6 and atp8), 22 transfer RNA genes and two ribosomal RNA genes (rrnS and rrnL) ( Table 2); the atp8 gene is encoded (Figure 1). The protein-coding genes are transcribed in different directions, as reported for T. trichiura and T. suis [22] (Table 2). Protein-coding genes were annotated by aligning sequences, and identifying translation initiation and termination codons by comparison with homologous sequences for other whipworms ( Table 2).
Twenty-two tRNA genes, which varied from 52 to 67 bp in length, were predicted from the mt genomes. The two ribosomal RNA genes (rrnL and rrnS) were inferred; rrnL is located between tRNA-Val and atp6, and rrnS is located between tRNA-Ser (AGN) and tRNA-Val. The lengths of rrnL and rrnS are 1,007 bp and 705 bp, respectively. The A+T contents of rrnL and rrnS are 69.02% and 70.21%, respectively.
Two AT-rich non-coding regions (NCRs) were inferred in the mt genome. For this genome, the long NCR (designated NCR-L; 124 bp in length) is located between the nad1 and tRNA-Lys (Figure 1), has an A+T content of 59.68%. The short NCR (NCR-S; 105 bp in length) is located between genes nad3 and tRNA-Ser (UCN) (Figure 1), with an A+T content of 79.25%.

Nuclear Ribosomal DNA Regions of Trichuris from the Monkey
The rDNA region including ITS-1, ITS-2 and intervening 5.8 rRNA gene sequenced from individual Trichuris samples (coded TH1-TH4) was 1,314 bp in length. Individual spacers were 570 bp (ITS-1) and 468 bp (ITS-2), and the 5.8S rRNA gene was 154 bp long.
Comparative Analyses Among Monkey-Trichuris, Human-Trichuris and Pig-Trichuris The mt genome sequence of monkey-Trichuris (accession no. KC461179) was 14,147 bp in length, 101 bp longer than that of human-Trichuris, and 289 bp shorter than that of pig-Trichuris. The arrangement of the mt genes (i.e., 13 protein genes, 2 rrn genes and 22 tRNA genes) and NCRs were the same. A pairwise comparison of the nucleotide sequences of each mt gene and the amino acid sequences conceptually translated from individual protein genes was made among the three taxa of Trichuris (from the three host species) ( Table 3). The sequence lengths of individual genes varied among these taxa, except for the nad1 gene, which was the same ( Table 3). The magnitude of sequence variation in each gene among the three taxa of Trichuris ranged from 24.2-50.9% for nucleotide sequences and 13.6-62.5% for amino acid sequences ( Table 3). The sequence difference across the entire mt genome between monkey-and human-Trichuris was 29.35% (a total of 4,152 nucleotide alterations). This difference across the entire mt genome between monkey-and pig-Trichuris was 33.49% (a total of 4835 nucleotide alterations). The greatest variation among the three taxa of Trichuris was in the atp8 gene (42.4-58.9%), whereas least differences (24.3%-31.5%) were detected in the rrnS and rrnL subunits, respectively (Table 3).
Amino acid sequences inferred from individual mt protein genes of monkey-Trichuris were compared with those of human-and pig-Trichuris. The difference across amino acid sequences of the 13 protein genes between the monkey-and human-Trichuris was 28.52% (a total of 1015 amino acid alterations) and 38.28% (a total of 1364 amino acid alterations) between the monkey-and pig-Trichuris, respectively. The amino acid sequence differences among three taxa of Trichuris ranged from 13.6-62.5%, with COX1 being the most conserved and ATP8 the least conserved protein. Phylogenetic analyses of concatenated amino acid sequence data sets, using T. spiralis as an outgroup, revealed that the monkey-Trichuris was more closely related to the human-Trichuris than to representative Trichuris species from porcine and ruminant hosts, with absolute support (pp = 1.00) (Figure 2).
Comparison of the mt genomes of monkey-Trichuris, human-Trichuris and pig-Trichuris showed that the rrnS and rrnL were the two most conserved genes (Table 3). Sequence variation in part of the rrnL gene was assessed among four individuals of Trichuris from monkeys. The rrnL sequences of the four monkey-Trichuris individuals (GenBank accession nos. KC481232-KC481235) were of the same length (616 bp). Nucleotide variation among the four monkey-Trichuris individuals was detected at 15 sites (15/616; 2.44%). The four monkey-Trichuris sequeces were aligned with 10 and six rrnL sequences (GenBank accession nos. AM993017-AM993032; [22]) reported previously for human-and pig-derived Trichuris, respectively. The alignment of the partial rrnL sequences revealed that all individuals of monkey-Trichuris differed at 140 nucleotide positions (140/430; 32.6%) when compared with human-and pig-Trichuris. Phylogenetic analysis of the rrnL Figure 1. Structure of the mitochondrial genome for Trichuris from the François' langur (Trichuris sp.). Genes are designated according to standard nomenclature, except for the 22 tRNA genes, which are designated using one-letter amino acid codes, with numerals differentiating each of the two leucine-and serine-specifying tRNAs (L1 and L2 for codon families CUN and UUR, respectively; S1 and S2 for codon families AGN and UCN, respectively). ''NCR-L'' refers to a large non-coding region; ''NCR-S'' refers to a small non-coding region. doi:10.1371/journal.pone.0066249.g001 sequence data from individual worms revealed strong support for the separation of monkey-Trichuris from human-Trichuris and pig-Trichuris (Figure 3).

Discussion
To date, more than 20 Trichuris species have been described from various mammalian hosts based on the microscopic features of the adult worms [42]. Some studies (e.g., [43,44]) have claimed that male spicule and body lengths are useful morphological parameters for the differentiation of Trichuris species. However, other studies have shown that these measurements are not necessarily reliable for specific identification [15]. For instance, Cutillas et al. (2009) [15] observed that the spicule lengths of T. trichiura and T. suis overlapped. While other workers considered that the presence of pericloacal papillae might be useful for species determination [45], also this criterion does not appear to allow accurate identification/delineation [15]. Clearly, these studies show that morphological characters or morphometrics should be interpreted with caution. For this reason, we employed here a molecular genetic approach, logically extending previous studies [22][23][24][25][26], so that comparative genetic analyses could be conducted.
The present investigation shows clear genetic distinctiveness between Trichuris from the François' langur and Trichuris from humans and livestock animals (i.e., T. suis, T. ovis and T. discolor) (Figure 2). Our and previous findings [22][23][24][25][26][27] support the contention that each Trichuris species has a very specific affiliation with a particular host species [16], although, to date, only small numbers of adult worms have been studied molecularly. Clearly, larger population genetic and molecular epidemiological studies should be conducted using the mt and nuclear markers defined in this and previous studies [22][23][24][25][26][27][28] to further test this hypothesis.

Gene/region
Nucleotide length (bp) Nucleotide difference (%) Number of aa aa difference (%)   MT  TT  TS  MT/TT  MT/TS  TT/TS  MT  TT  TS  MT/TT  MT/TS  TT/ [54,55]. Another likely threat to the François' langur, particularly in captive situations in conservation parks and zoos, is whipworm disease. This statement is supported by reports from China (e.g., [56][57][58][59]), indicating that Trichuris infection is common (14.3-100%) in this langur in zoos and conservation parks, as are clinical cases of trichuriasis. The direct life cycle of Trichuris, the accumulation of eggs in environments with relatively high population density of primates (animals) and the robustness and longevity of the infective stage (larvated eggs) in the environment [60] are all factors that contribute significantly to a gradual increase of trichuriasis in 'closed' environments, such as parks [61]. Although we expect the monkey-Trichuris studied herein to be specific to the François' langur, there is a possibility that this parasite is transmissible to other primates, including humans. However, this proposal needs to be assessed. In spite of molecular evidence for the existence of a unique Trichuris species in the endangered François' langur, the interpre-tations from the present study are guarded, at this stage, until detailed population genetic investigations have been conducted. Future studies should include (i) exploring, in detail, nucleotide variation in rDNA and mtDNA within and among Trichuris populations from a range of different primate species and countries, and to establish whether more than one Trichuris species infect non-human primates, (ii) establishing, using accurate molecular tools, whether cross-host species infection occurs or not, (iii) undertaking detailed morphological studies, by scanning electron microscopy and field emission scanning electron microscopy, of whipworms from various non-humans primates. This focus is important because, traditionally, the diagnosis of Trichuris infection in animals has relied mainly on the morphological identification of adult and egg stages.