| Objective By investigating the species and the distribution of Paragonimus in Dali prefecture in Yunnan Province, and finding out the natural infection situation of the second intermediate host crabs, based on morphological classification and combined with DNA sequence analysis, this paper aimed to identify the independence of Pagumogonimus cheni, and provide a scientific basis for studying the taxonomy of the genus Paragonimus, the epidemic district distribution of paragonimiasis and its prevention and treatment in Yunnan Province.Methods From the mountain stream in Binchuan County, which has good cover degree of forest in Dali prefecture, fresh crabs were captured and identified the species. Metacercariae were separated, counted, measured, classified, and then fed to experimental cats, whose fresh dejecta was collected and eggs were fixed in10%formalin. Two cats dissected, adult worms and eggs were collected and made into stained specimens, which were observed and measured with light microscope. By using reagent kit DNA was respectively extracted from metacercariae, excysted metacercariae and adult worms. With the corresponding primers, the ITS2and CO I gene fragments were respectively amplified in vitro, and then the DNA sequencings were obtain. Retrieving ITS2and CO I gene sequences from the GenBank, these sequences obtained were analysed with SeqMan program of DNAStar software, and phylogenetic trees were constructed with MEGA4.0software. Results1. The fresh crabs from Binchuan County are all small and dark brown. The crabs were preliminarily identified as a new species, and named for Potamon lawuense sp., which belongs to potamodiea, potamidea and potamon. The crab natural infection rate from Binchuan County was13.24%, the average infection degree of the crabs is3.67, and the highest infection degree of single crab is24. The adult worms recovery rate from two cats infected artificially Paragonimus metacercariae are respectively77.27%and27.27%.2.66metacercariae examined from Binchuan County are mostly quasi-circular, and all of them are single cystwall. The averaged size of living metacercariae of Pagumogonimus cheni is (435.28±25.84) μm×(424.25±26.92) μm, the averaged thickness of the cystwall is (13.65±1.80) μm. Excysted metacercaria nearly fills to the full cystic space, and there is no obvious lacuna, the black excretory vesicle and curved intestinal canal of which are clearly visible.Living adult worm of Pagumogonimus cheni is flesh red, big and fleshy, and its movement is leech-like. After pressing and fixing lightly, it looks like gray in colour and shows narrow and elongated in shape.21specimens were measured. The average size of adult worms is (10.529±2.081) mm×(3.648±0.827) mm. The width to length ratio of adult worms is1:2.92. The ratio of transverse diameter of oral sucker to ventral sucker is0.743:1, and so the size of ventral sucker is apparently bigger than oral sucker. Ovary mostly has obvious centrebody and3-6lobes, which shows fingerlike with lobular shape. Utero full of eggs convolutes into a group, the shape of which is like a tube, and parallels arrangement with ovary. Two testes show long with lobular shape, and their centrebody is small. The testis have about3-5branches each, the bottom of which swells apparently. Eggs of Pagumogonimus cheni are always oval in shape, and golden yellow in color.50specimens were measured. The average size of eggs is (73.01±6.93) μm×(46.80±3.33) μm. The widest point is located at the first1/3of the egg. The thickness of egg shows uneven, and the end edge of eggs looks like thicker apparently. The egg cover always locates to the relative width end with a clear acromion. Approximately88%of the end edge of eggs have visible verrucous swelled. According to the morphological characteristics of metacercariae, excysted metacercariae and adult worms, the species of Paragonimus examined from Binchuan County is preliminarily identified as Pagumogonimus cheni.3. DNA sequence analysis of Pagumogonimus cheni from Binchuan County in Dali prefecture in Yunnan Province suggested that the obtained target fragments length by PCR amplification of ITS2å'ŒCO I gene was around500bp. Binchuan strains, Baoshan strains and Jinping strains, which have similar genetic distance and high homology by multiple sequences alignment, are all Pagumogonimus cheni. The phylogenetic trees of ITS2and CO I gene sequences demonstrate Pagumogonimus cheni from Binchuan County and Pagumogonimus skrjabini from Mengla County of Xishuang banna state of Yunnan Province and Thanh Hoa of Vietnam cluster together one branch.(The confidences of ITS2and CO I gene are respectively90%and97%.)Conclusion1. It is first reported that Binchuan County in Dali prefecture is the distribution district of Pagumogonimus cheni.2. Potamon lawuense sp. from Yunnan is the second intermediate host of Pagumogonimus cheni. Cat is susceptible to Pagumogonimus cheni, and is its appropriate experimental definitive host and ideal animal model.3. Based on morphological classification and combined with molecular biology method, this paper further identified the independence and taxonomic status of Pagumogonimus cheni from macroscopic and microscopic aspects.4. Pagumogonimus cheni from different areas in Yunnan has similar genetic distance and highly identity in DNA sequence analysis, and clusters to one branch and separates apart from other species in phylogenetic trees. As far as the sequence analysis of ITS2and CO I gene to be concerned, Pagumogonimus skrjabini from Mengla County of Xishuang banna state of Yunnan Province and Thanh Hoa of Vietnam are the same as Pagumogonimus cheni from Binchuan County, so they all belong to the same species of Pagumogonimus cheni.5. This research proves DNA extraction of Paragonimus from the specimens of different stages such as adult worm, metacercaria and excysted metacercaria, has no effect on the taxonomic study of the Paragonimus. |
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