Analysis of Internal Transcribed Spacer Regions II Gene and Morphology of Paragonimus From Yunnan Province, China

For a long time, there is no clear-cut to identify some species of paragonimus in Yunnan Province, China. This paper involved the distribution of Paragonimus in Jinping country and Baoshan city, Yunnan province. In this experiment, the metacercariae, excysted metacercariae, eggs, adult worms were obtained from different hosts were observed and measured. Cats have been described as the appropriate denitive host of paragonimus sp., which are closed to P. cheni according to morphology. Especially the ovaries of adult worms are few and has no third branches. With SEM observation, their spines in the surface are single, a sharp-pointed knife or half-moon in shape and the end of a few of spines are bifurcate. While the clustered sequences strains of this study in the ITS2 tree clustered of Yunan is out sider of P. skirjabini complex and have the genetically greater distances than other isolates of the P. skrjabini complex. Therefore, the Paragonimus sp. of this study from Jinping County and Baoshan city are the same subspecies of P. skrjabini complex.


Introduction
Paragonimus is the pathogen of paragonimiasis that is a severe zoonosis. Around 50 species have been described since this genus was erected by Braun in 1899 [1]. Among the Paragonimus most species are found in Asia. And 32 species have been reported in China. As the province that located in the southwestern border of China Yunnan's climate and geography are pro t to the life cycle of paragonimus. So It had been reported some species of paragonimus in Yunnan, like Paragonimus heterotremus [2], P. proliferus [3], P. microrchis [4], P. bangkokensis [5], P. cheni [6] and P. skrjabini [7].
Pagumogonimus skrjabini is reported in 1959 by Xintao Chen[8], regional distribution is very broad, mainly in Sichuan, Yunnan, Guizhou, Hubei, Fujian and other 14 provinces of China. P. skrjabini can cause cutaneous larva migrancy and visceral larva migrancy, the main performance is migratory subcutaneous nodules, but also can invade the liver, brain and other tissues, easy to cause clinical misdiagnosis. Although the sequences of P. skrjabini collected from Hubei province, Sichuan province, Fujian province, Guangdong province, Guangxi province and Yunnan Province had been done[8], the phylogenetic trees had been built. But only one samples from Yunnan and not described in detail about morphology, However, about species of P. skjarbini is lacking detailed research data collected from Yunnan. And the same condition happened in 2002, it was reported that there are P. skjarbini in Jinping county by researching little samples and Not described in detail.
Given the relative paucity of morphological characters available for distinguishing among Paragonimus species, the morphotypes were indistinguishable using light microscopy. Scanning electron microscopy has often been used to reveal details of subtle morphological features, such as tegumentary papillae, that might assist in taxonomy [9,10]. Given this background, it is important to discern whether different morphotypes of metacercariae represent different species, a task well suited to molecular studies. This approach has been used only rarely for metacercariae of Paragonimus species in Latin America.
Hernández and Cavaleiro [11,12] reported two different morphotypes of metacercariae in an ultrastructural study of the papillae on the ventral sucker.
To date, molecular studies have identi ed four complexes of Paragonimus species [13]. Among these are the P. westermani complex, including P. westermani and P. siamensis, while the P. ohirai complex, including numbers of nominal species such as P. ohirai and P. harinasutai [14]. Sequence data of the ribosomal RNA (rRNA) gene, in particular the two highly variable internal transcribed spacer regions (ITS1 and ITS2), have been successfully used to resolve taxonomic questions and to determine phylogenetic a nities among closely related paragonimus species and other parasites. At present, morphology is an important method to the taxonomy of the paragonimus. The size and layer of metacercaria, shape of ovary and spines of adult worms are still the main basis for the identi cation and classi cation of the species.
We then collected the crabs in distribution of Paragonimus in Jinping country and Baoshan cith, Yunnan province, China. Metacercariae, excysted metacercariae, eggs, adult worms were observed and measured. The surface structures of adult worms were observed with scanning electron microscope (SEM). The sympathism of two hosts was compared. From these we can understand the infection condition of Paragonimus' host (rats, cats and crabs). We sequence analyses of ITS2 genes of Paragonimus in Jinping county and Baoshan cith in Yunnan province. All these were done to de nite the genus relationship and genetic relationship with other Paragonimus.

Molecular analysis
The metacercariae of Paragonimus were isolated from individual crabs which were collected from Jinping county and Baoshan city, Yunnan province, China. The metacercariae which are all taken from above places and the adult and the egg obtain from the cat that was infected by the metacercariae collected from Jinping county and Baoshan city as the molecular samples. These samples were immediately xed in 100% ethanol and stored at 4°C before extracting the genomic DNA.
To amplify the nuclear ribosomal second internal transcribed spacer region 2 (ITS2), the following primers were used 3S (5′-GGT ACC GGT GGA TCA CTC GGC TCG TG-3′) and BD2 (5′-TAT GCT TAA ATT CAG CGG GT-3′) also previously described by Bowles and Bowles et al. (1993). The initial denaturazed step of ITS2 (95℃for 1 min) was followed by 35 cycles at 95℃(50 s), 68℃(2 min) and extension of 72℃(10 min). The quality of PCR products was indicating by electrophoresis in a 1% agarose gel. The ampli ed DNA products were separated by electrophoresis in a 1% low melting point agarose gel in TAE buffer. The polymerase chain reaction (PCR) products were puri ed using PCR puri cation kit, primed using Big-Dye terminator cycle sequencing kit v3.1 (ABI), and both strands directly sequenced by Sangon Biotech (Shanghai) Co..

Phylogenetic analyses
For one thing the rst or last 24-29 base pairs (bp) of each sequence were uniformly excluded because in our sequences the rst 24-29 base pairs beyond each primer were often rich in ambiguities. We got the ITS2 sequence which had 463 bp. All sequences are available from GenBank (Accession # EU769097-EU769103). Then all Aligned ITS2 sequences obtained in this study were compared with 39 sequences of each ITS2 genes of several Paragonimus species, respectively, obtained from previous studies species were obtained from previous study. Phylogenetic tree was constructed based on sequences. Phylogenetic tree was reconstructed based on sequence data sets ITS2. They were aligned using Clustal-X v1.83 [15]with default options. The aligned matrix from this procedure was veri ed to have the same length, and minor adjustments were then made manually using SeaView v.4.2.5 [16]. The data matrices are available from the corresponding author. The haplotype analyses were performed to 39sequences using DAMBE software for 50 sequences [17]. Distances from the predicted amino acid sequences were determined with the p-distance models which were computed by MEGA v. 4.1 [18].
Phylogenetic hypotheses of Leishmania were generated with ITS2 rRNA segments using two types of commonly applied phylogenetic method: heuristic searches using equally weighted maximum parsimony (MP) analyses performed with the program PAUP* [19] and Bayesian inference (BI) with the program MrBayes v.3.2 [20]. For heuristic searches under parsimony, invariant characters were removed from the dataset, and all remaining characters were treated as equally weighted. Each search involved ten random addition replicates, one tree held at each step, TBR branch swapping, steepest descent on, and a maximum of 10,000 saved trees; all other search settings were left at default values. Non-parametric bootstrapping was used to generate phylogeny con dence values [21], with 1,000 pseudoreplicates using a heuristic tree search for each pseudoreplicate. Euparagonimus cenocopious (AF159601). Because intraspeci c gene evolution cannot always be represented Phylogenetic hypotheses of paragonimus were generated with ITS2 segments using Bayesian inference (BI) using the MrBayes v.3.2 program. In BI analyses, gaps were treated as missing data. Euparagonimus cenocopious (AF159601) was used to root the trees. Prior to Bayesian analyses, the best-t model of evolution, HKY+G, was selected using Modeltest 3.7 under the Bayesian information criterion, following recent recommendations. We estimated the posterior probability distributions by allowing four incrementally heated Markov chains (default heating values) to proceed to four million generations, and with samples were taken every 200 generations. Analyses were repeated beginning with different starting trees to ensure that the analyses were not restricted from the global optimum. Convergence was rst tested by examining the average deviation of the split frequencies of the two runs, in order to determine whether the two runs had converged. MCMC convergence was also explored by examining the potential scale reduction factor (PSRF) convergence diagnostics for all model parameters (provided by the sump and sumt commands). The rst one million generations before this chain became stationary were discarded, and the remaining samples from the independent runs were pooled to obtain the nal approximation of the posterior tree distribution.
Because intraspeci c gene evolution cannot always be represented by a bifurcating tree, haplotype networks may more effectively portray the relationships among haplotypes within species [22]. Therefore, we constructed unrooted parsimony networks of haplotypes for P. skrjabini complex and Paragonimus sp. (see below) using TCS v.1.21 [23].

Results
The morphology of paragonimus The shape of metacercaria from Jinping country and Baoshan city close to spherical. The capsule wall of metacercariae almost is monoptychial and the size of its from Jinping country are 0.401 mm×0.395mm, from Baoshan city are 0. 435mm×0. 433mm (Fig. 1a-b). Adult worms are all elliptic in shape and the average size from Jinping country and Baoshan city are 10. 107mm×4.064mmand 9.
59mm×43. 577mm respectively (Fig. 2). The rate of width to length of each adult worm from Jinping country and Baoshan city are 1:2.49 and 1:2.63 respectively. The size of their ventral sucker larger than oral sucker. The ovarys of these adults are all near the ventral sucker, the branches are simple. The testes' centrosomes are small, and the length are about 1/6 of the body. With SEM observation there are single spines in the surface of adult worms, the spines before the ventral sucker are triangle and a sharp-pointed knife in shape, after the ventral sucker are almost half-moon in shape and the end of a few of spines are bifurcate (Fig. 3-4). The morphology of metacercariaes, excysted metacercariae, eggs from Jinping country are nearly resemble but there are few different from the morphology of its from Baoshan city (Fig.   1c-f). The morphology of adult worms is identical.
The natural infection rate of the second intermediate host with Paragonimus from Jinping country and Baoshan city are 79.9% and 100%. The quantity of each crab infects metacercariae which were collected from Jinping country and Baoshan city are 1.8 and 13.3 respectively. The rate of worms recovered from experimentally infected rats and cats which infected metacercaria from Jinping country are 24% and 55%. The rate of worms recovered from rats and cats which infected metacercaria from Baoshan city are 5% and 67. 7%.

Molecular phylogenetic analyses
We obtained the complete 7 ITS2 of Paraginumus collected from Jinping county gene sequences in the GenBank nucleotide sequence databases with the accession numbers (EU769097-EU769103), from the metacercariae, adult and eggs samples. The ITS2 alignment was 463 bases in length separately. All the above sequences were completely identical with each other. These sequences and several related species within genus Paragonimus obtained from GenBank which had been aligned by us constructed the phylogenetic trees separately in the sequence data set of ITS2 (Table 1-2) genes. P distances among the Paragonimus species except Paragonimus sp. ranged from 0.004 (between P. proliferus and P. miyazakii) to 0.107 (between P. vietnamensis and P. ohirai). Most pairwise comparisons mentioned above had divergence values of less than 0.107, with 0.073 on average. Meanwhile, the divergence between Paragonimus sp. and other species ranged from 0.013 (Paragonimus sp. versus P. skrjabini India) to 0.019 (Paragonimus sp. vs. P. westermani), with an average of 0.017 (Table 3). P distances among subspecies of P. skrjbini complex (P. skrjabini, P. miyazakii and P. proliferus) ranged from 0.004 (between P. proliferus and P. miyazakii) to 0.009 (between P. skrjabini and P. proliferus), with an average of 0.006 (Table 3).
For the BI analyses, the likelihood value of the 50% majority consensus tree (Fig. 6)

Haplotype network
To get additional insight into the relationships among the P. skrjabini complex strains, we analyzed our data set, using the coalescent-based statistical parsimony network approach. The network of 10 haplotypes was shown as Fig. 7. H5 (strains from Fujian, China) and H1 seemed to be central haplotypes with 2 mutitianal steps. P. skrjbini (isolates from Sichuan and Hubei, China) and P. miyazakii (isolates from Japan and Hubei, China) share the one haplotype H1. P. skrjabini (H4, H5, H7, H10 and parts of H1) have a wider geographical distribution and revealed much more polymorphism than P. miyazakii (H8, H9 and other part of H1). The strains of this sutdy and other strain of Yunnan reported by Blair previously share one haplotype H2 with that from Vietnam. And H6, shared by P. proliferus from Yunnan, China and Vietnam. H1 (P. skrjbini and P. miyazakii) was most closely related to the H9 (P. skrjabini from Fujian, China), H7 (P. skrjabini from Guangxi, China) and H8 (P. miyazakii from Japan, with one mutational step. H10, shared by four strains from Guangdong, China, was also closely related to H9, with two mutational steps. Having an advantage over the bifurcating tree in detail at the intraspeci c level, the haplotype network could intuitively re ect the genetically greater distances between the singleton (H4 from India) and H2 (4 mutational steps, see Fig. 7). P. proliferus (H6) has 3 mutational steps genetically distances with H1 and one mutational step genetically distances with H5. Strains of this study (H2) have the genetically greater distances with the two-central haplotype (H1 with 7 mutational steps, H5 with ve mutational steps; see Fig. 7).
Numbers of haplotypes correspond to Table 1. In the network, solid circles indicate sampled haplotypes; small hollow circles indicate unsampled or extinct haplotypes. Each mutation step is shown as either a short or longer line connecting neighboring haplotypes (including observed and unobserved one). The size of the solid circles roughly represents the numbers of strains carrying the haplotype, with the scale given beside the network; different lled patterns represent the corresponding geographical origin from which the haplotype was sample

Discussion
Up to now, it has 150 years in history since we study Paragonimus. The shape of metacercariae is important to the taxology of Paragonimus [36]. The capsule wall of metacercariae from Jinping and Baoshan are almost monoptychial. We changed the method that separate metacercariae under platoscope directly, which Zhou Benjiang has been used [37]. Then, the metacercariae. We had are all most monoptychial. Metacercariae with the single cyst wall are P.iloktsuenensis Chen, 1940, Paragonimus fukienensis Tang and Tang, 1962, P. cheni, P. proliferus, P.caliensis, P.uterobilateralis, but there are only rst 4 species distributing in China [38]. These four species are difference in the size. The size of metacercariae from Jinping and Baoshan are near to P. cheni.
The adult worms from Jinping and Baoshan are similar which gures are oval-shap and every rate of width to length are over 1:2.4. Paragonimus have been divided into 3 species according to gure and rate of width to length of adult worms [39]. Among those, which gures are fusiform shape and every rate of width to length are over 1:2.4, are Pagumogonimus skrjabini, P. cheni, P. macrorchis and P. proliferus and so on. But the size of metacercariae from Jinping and Baoshan are very difference from P. macrorchis and P. proliferus, which size are 0.280mm×0.259mm [36] and over 1mm respectively. The type of ovaries of adult worms from Jinping are similar with P. skrjabini and P. cheni [40] which branches are few and has no third branches. We observed 4 adult worms from Jinping and Baoshan respectively and the spines are resembled. There are single spines in the surface, and the spines before the ventral sucker are triangle and a sharp-pointed knife in shape, after the ventral sucker are almost half-moon in shape and the end of a few of spines are bifurcate. Different species of Paragonimus have different infection e ciency across various animal host. In this experiment, rats and cats have been infected, and cats have been described as the appropriate de nitive host of paragonimus from Jinping and Baoshan. Cats are suitable de nitive host of P. cheni [36].It indicates that the Paragonimus from Jinping and Baoshan are the same species according to that the morphology and the result of SEM. Although the morphology are both similar with P. skrjabini and P. cheni.
In the present study, we determined the DNA sequences of ITS2 genes of Paragonimus obtained from Jinping and Baoshan in Yunnan province where is in southern. Farther more our samples form one clade with P. skrjabini harvested from Xi shuangbanna Yunnan and P. skrjabini harvested from Vietnam [8,26]. In case of ITS2 gene, our samples of two places and the sequences of P. skrjabini yn are 100% identi ed with each other except the egg samples. So, the Paragonimus from three places of Yunnan are one species. In our previous study, P. hokuoensis [8] suggesting that P. hokuoensis is highly likely be a synonymy of P. proliferus [3]. And Blair et al. recognise two species within the P. skrjabini complex: P. hokuoensis in southern Yunnan and P. skrjabini in Guangdong, Sichuan, Hubei, Fujian and Japan (and other Chinese Province not sampled by them.) before this our research [8]. And in our study the clustered sequences of Yunnan include our samples and samples in Xishuangbannan of Yunan in the ITS2 tree clustered of Yunan is out sider of P. skirjabini complex. While haplotype networks show that Strains from this study, Xishuangbannan of Yunan and Vietnam (H2) have the genetically greater distances with the two-central haplotype (H1 with 7 mutational steps, H5 with ve mutational steps) than that H6, shared by P. proliferus from Yunnan, China and Vietnam. P. proliferus (H6) has 3 mutational steps genetically distances with H1 and one mutational step genetically distances with H5. Therefore, the clade of our sequences with the sequences of P. skrjabini yn [8] is a distinct subspecies in the P. skirjabini complex like P. proliferus.

Conclusions
The results of morphology indicate that the Paragonimus from Jinping and Baoshan are the same species and both similar with P. skrjabini and P. cheni. Meanwhile, the analysis of phylogenetic and network show that the clade of our sequences with the sequences of P. skrjabini yn is a distinct subspecies in the P. skirjabini complex like P. proliferus. Thus, according to our morphology and molecular result, the Paragonimus sp. of this study from Jinping County and Baoshan city and the strain of Xishuangbanna are the same species, which is a distinct subspecies in the P. skirjabini complex like P. proliferus and belong to the P. skirjabini complex. Figure 1 Metacercariaes Excyted metacercariaes and Eggs from Host a Metacercariae from Jinping single cyst wall ×100 ,b Metacercariae from Baoshan single cyst wall ×100 ;c Excyted metacercariae from Jinping ×100 ,d Excyted metacercariae from Baoshan ×100 ; e Egg from Jinping(×400, From cat's stool,80days), f Egg from Baoshan(×400, From cat's stool,80days).  Maximum parsimony consensus tree from 1,000 bootstrap replicates of ITS2 dataset by using PAUP*. Numbers above the branch represent percent recovery in bootstrap analysis (1,000 pseudoreplicates).

Declarations
Tree length=146, CI=0.8220, RI=0.8779 The 50% majority-rule consensus tree inferred from Bayesian inference of ITS2 dataset by using MrBayes v. 3.2. Numbers at nodes represent Bayesian posterior probabilities