Infection Investigation And Species Identification Of Two Species

Frogs and toads are common amphibian, and they are one of the most important components in the biological community. Trematodes and cestodes are common amphibian parasites. The growth of the frog can be adversely affected by parasites,including helminths, leading to limb deformities and even death. Because of the increasing use of frogs, their contacting with humam are more frequently, it can increase the risk of pathogens infecting human. Therefore, the research of trematodes and cestodes can not only protect frogs, but also conducive to improving people’s awareness of zoonotic parasitic disease. This study intends to investigate the infection situation and species of trematodes and cestodes from two species of frogs, and provide the basic date for the prevention of zoonotic parasites and protection of frog resources.1. Infection investigation of trematodes and cestodesTo investigate the infective status of trematodes and cestodes, 182 frogs from 3species(90 Rana nigromaculata, 8 Rana rugulosa and 84 Bufo gargarizans) were examined from July 2013 to July 2014. The parasites were collected and the infection rate and infection intensity were calculated. Of these, 86.67% of Rana nigromaculata were infected with parasites, including 1625 trematodes and 47 cestodes. The infection rates and intensities were 78.89% and 22.89 for trematodes, 13.33% and3.92 for cestodes, respectively. 85.71% of Bufo gargarizans were infected with parasites, including 1628 trematodes and 2 cestodes. The infection rates and intensities were 85.71% and 22.61 for trematodes, 2.38% and 1 for cestodes,respectively. No parasites were collected from Rana rugulosa. Our results indicated that, the infection rate and intensity of trematodes were higher than reported in the literature for other areas in China. Whereas the infection status of cestodes was lower than most reports. In this stady, all the cestodes were spirometra, although the number was very small, they could have a potential impact on human health.2. Species identification of monogeneansIn order to identify species of monogeneans, which were collected in the urinary bladde from Rana nigromaculata, we selected 30 parasites for morphological identification and 4 parasites for molecular identification. The molecular biology results showed that the 18 S r RNA gene sequences from 4 parasites displayed 99%homology and in the 18 S r RNA phylogenetic tree, all the parasites were in the same branch of Diplorchis. Finally we determined this species of parasite was Diplochis nigromaculatus combined with morphological identification results. Diplochis nigromaculatus was only recorded in Beijing ang Jiangsu province in China. This is the first report of Diplochis nigromaculatus in Shanghai.3. Species identification of digeneansWe used the morphological method to further determine the species of 2696 trematodes, through making part of mounting specimens, observating and measurementing the mounting and comparation to the reports in China. The results showed that the 2696 trematodes belonged to 6 species in 4 families and 4 orders. The Diplodiscus mehrai, Diplodiscus nigromaculati and Rauschiella ranae have been identified to species. Diplodiscus sp., Pleurogenoides sp., Haematoloechus sp.have been identified to genera. The identification results provide the basic data for the study of parasites in frogs from Shanghai.4. Species identification of cestodesIn order to identify species of cestodes, 8 parasites were randomly selected for molecular identification. We used the DNA barcoding to amply the ITS gene,compared the gene with all kinds of cestodes by Blast and analysied the reliability of the results by phylogenetic tree test. Results showed that the ITS gene sequences from8 parasites displayed the highest homology with Spirometra erinacei. Consequently the species of cestodes were Spirometra erinacei. Phylogenetic tree showed all the Spirometra and snake source sparganum were in the same branch, and mammal source sparganum were in the other branch. This proves that the snake source and frog source Spirometra erinacei has closer genetic relationship.5. Species and geographic distributions of trematodes from frogs in chinaIn order to understand the species and geographic distributions of trematodes from frogs in China, published reports relevant to trematodes in frogs of China were reviewed to organize a classification system of trematodes – order, family, genus and species in alphabetical order. In summary, 88 species of trematodes belonging to 29 genera, 13 families and 4 orders have been recorded in China. For example, the order Polystomatidea has 13 species in 2 genera and 1 family. In the order Echinostomida12 species in 2 genera and 1 family are present. The order Opisthorchiida consists of 3species in 1 genus and 1 family. The order Plagiorchiida includes 60 species in 24 genera and 10 families. The review also lists Chinese and Latin names, namers andnaming years, parasitic hosts and sites, geographical distributions and sources of literatures of all trematodes mentioned here. The overview provides basic informationon species and geographic distributions of trematode from frogs in China.