| [Objective] Applicating morphological classification and DNA sequence analysis method to deeply study Paragonimus microrchis Hsia Zhou & Chang, 1978 of the independence of the species and taxonomic status in Paragonimus, as well as the relations with other Paragonimus species , clarificating gradually the controversial problem of Paragonimus species, explore more objective methods of taxonomy and easy identification ,for further study the biologic characteristics of species, geographical distribution, and pathogenicity for human , to lay an important foundation for prevention and treatment.[Methods] Collect the second intermediate host of Paragonimus microrchis in the endemic area, separation metacercaria, encysted metacercaria from collect the crabs. Infecte the experimental animals with metacercaria, encysted metacercaria and get adults and eggs. Observate and measure the adults, the metacercaria, the encysted metacercaria and eggs with stereomicroscope or microscope. Two adults for scanning electron microscopy observating its spines morphology. In addition, combinate DNA sequence analysis, Blast homology comparison and MEGA construct phylogenetic trees.[Results] The adult of Paragonimus microrchis ,the live is pink and the fixed is gray. Its size is 8.5-12.9×3.6-5.0mm, average 10.47±1.94×4.35±0.74mm. Width length ratio is 1: 2.1-2.8, average 1: 2.41. Oral sucker diameter 0.511-0.778mm, average 0.657±0.103mm. Ventral sucker is located in front 1/3 and 1/3 later or edge line. The size is 0.648-0.821×0.648-0.828mm, average 0.748±0.107×0.745±0.102mm. Two sucker diameter ratio is 1: 1.13. Ovary is located at the left or the lower right of ventral sucker ,even partially overlap. The size is 0.720-1.598× 0.662-1.397mm, average 1.238±0.475×1.032±0.376mm, The centrosome obvious, since the center body to issue 6 to 8 leaves, each leaf and may be issued 3 to 4 lobules. Testis at body 1/3 and 1/3 line or the front, the center block, and no branches or buds 1 to 5 like protrusions, the size of the left testis 0.662-1.390×0.504-0.979mm , average 0.974±0.334×0.732±0.250mm; right testis for 0.526-1.570×0.504-1.210mm, average 0.989±0.437×0.701±0.344mm, obviously smaller than the ovary. Adult body wall besides the oral suker and ventrall sucker almost have the single spines. Their shape are like triangle or dagger. The anterior body of spines are intensive and regular. The posterior body are sparse and irregular. All of them tilt backward. The oral sucker is vertical plica, and the ventral sucker is ring plica.The encysted metacercaria is oblong oval.The live is vivo white and the fixed is off-white. Its excretory bladder is huge, between the two intestinal branch. Post-fixed encysted metacercaria measured the size is 0.878-1.210×0.432-0.634mm, averagel.034±0.167×0.509±0.108mm. Oral sucker is 0.115×0.180mm, average 0.140±0.025mm. Ventral sucker is 0.130-0.202×0.130-0.209mm, average 0.170±0.027×0.173+0.031mm. Oral sucker and ventral sucker ratio is 1:1.236.The metacercaria is spherical, individual large, with double-wall, outer wall thin and transparent, easy to rupture, but the inter wall is thick. Sporocyst body is full of the inter capsular space. There is the small gap between the inner wall and sporocyst body. Twisting and refractive index of the intestinal branch can be seen. Excretory bladder like pocket located between the two intestinal branch, and sometimes we can see its oral sucker or ventral sucker. Measure its size without coverslip (including the outer wall) 0.760-1.406×0.689-1.399mm, average 1.006×0.951mm, outer wall thickness for 0.003-0.014mm, average 0.008mm. Measure with the same method (including inner wall) 0.632-0.852×0.596-0.831mm, average 0.728×0.703mm, inter wall thickness for 0.007-0.031mm, average 0.011 mm.The eggs were generally wide oval, golden yellow. The majority of eggs have covers, egg shell is uneven, with the thicken end. Measure with coverslip its size 73.1-95.9×42.3-55.3μm, average 81.37±9.90×47.67±5.58μm.Blast homology analysis results Paragonimus microrchis of ITS2 and CO1 are100% and 99% with Paragonimus harinasutai (Laos) hihgly homology in Genebank; with Paragonimus bangkokensis (Thailand) homology are 98% and 96%, with Paragonimus harinasutai (China Zhejiang )homology are 96% and 88%. ITS2 and CO1 phylogenetic trees showed that Paragonimus microrchis with Paragonimus harinasutai (Laos) clustered into the same clade. The one with paragonimus bangkokensis (Thailand) clustered into the another one, but Paragonimus harinasutai (China's Zhejiang) are sister to one.[Conclusion] Paragonimus microrchis adult, metacercaria, encysted metacercaria and eggs are unanimously with Professor Xia Daiguang (1978)and Professor Zhou Benjiang (1988)reported, in accordance with Paragonimus microrchis characteristics. Scanning electron microscopy firstly observated adult's spines are the single spines. Their shape are like triangle or dagger. The anterior body of spines are intensive and regular. The posterior body are sparse and irregular. The oral sucker is vertical plica, and the ventral sucker is ring plica. Its morphology is associated with cibation, coherence and movement. For sequnence, Paragonimus microrchis and Paragonimus harinasutai (Laos) are the same species. Paragonimus harinasutai (Laos) will be clarified in further. Paragonimus microchis with Paragonimus bangkokensis (Thailand) are high homology and close relationships. According to Blair.D's view, they are the same speices complex. |
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