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).
Phylogenetic relationships
The heuristic search of the ITS2 matrix resulted in 10,000 equally parsimonious trees of 146 steps, with high values of CI (0.8220) and RI (0.8779). In the strict consensus phylogram (Fig. 5), 14 isolates in China formed a branch (H2 and H3 BP=64%,) Paragonimus sp. (13 isolates from China shared a haploid H2 with 2 isolates from Xishuangbanna, Yunnan and 9 isolates from Vietnam) that was sister to the remaining members of subspecies P. skrjabini India (BP=52%). The haplotype H6 of proliferus was clustered with six haploids (H1, H5, H7-10) of P. skrjabini (P. miyazakii) (BP = 69%), and then, forming a cluster with Paragonimus sp., that formed a clade with P. vietnamensis (BP = 52%), juxtaposition with P. heterotremus (P. pseudoheterotremus). This branch went on with forming one clade with P. ohirai, P. bangkokensis and P. harinasutai. P. westermani is the base of Paragonimus.
For the BI analyses, the likelihood value of the 50% majority consensus tree (Fig. 6) was ln L=−1377.24. The average PSRF was 1.001. Overall, as well with maximum parsimony analyses, Paragonimus sp. which is composed of 14 geographical strains from Jinping and Baoshan in Yunnan, China, 2 strains from Xishuangbanna in Yunnan and 9 strains from Vietnam forms an evolutionary clade with a high posterior probability of 0.99, was sister to the P. skrjabini India strain from India (PP = 0.80). Similarly, the subspecies of P. skrjabini complex include H6 (P. proliferus), one of the six haploids (H1, H5, h7-10) of P. skrjabini (P. miyazakii) cluster (PP = 0.79) is juxtaposed with Paragonimus sp. The relationship of the remaining Paragonimus species is similar to that of Fig. 5.
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 intraspecific level, the haplotype network could intuitively reflect 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 five 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 filled patterns represent the corresponding geographical origin from which the haplotype was sample