Investigation And Species Identification Of Acanthus And Nematode Infection In Two Frogs

The amphibian, a large and ancient family,was easily ignored by people, in particular the frog, when they pay more and more attention to the protection of mammals and birds. Frogs are rich and there are more than 340 species of frogs in China according to statistics. The frog can adapt to different living conditions of land and water, to changing living environment, so that the possibility of parasit ic infection is higher accordingly, which will not only affect their own frogs, and would pose a threat to human health.1. Species identification of frogs and toadsIn order to determine the species of 98 frogs and 84 toads, mitochondrial 12 S sequence wasuesd. DNA was extractedfrom liver, intestine and blood of frogs and toads in batches and amplified by PCR, then sequenced. After sequenceswere BLAST, the phylogenetic tree was constructed using the maximum likelihood method. The results showed that there are 90 Rana nigromaculata, 8 Rana rugulosa, and 84 Bufo gargarizans, which is consistent with that of traditional morphological observation.2. Preliminary investigation of acanthocephalan and nematode infection in frogs and toadsTo understand the infection of parasitic worms in the frog from Shanghai, 90 Rana nigromaculataand 84 Bufo gargarizans collected between 2013 and 2014 were numbered and necropsied under the help of the Shanghai Wildlife Protection Station. Helminthswere collected from different organs, moreover, the parasitic locations, prevalence and intensity of acanthocephalanand nematode were recorded. The results showed that 550 parasites(314 acanthocephalans and 236 nematodes) were collected from 90 Rana nigromaculata. The overall infection rate was 65.56%(41.11% for acanthocephalans and 50% for nematodes) andmean intensity was 9.32. But the prevalence of acanthocephala and nematode was highest in intestine of Rana nigromaculata, followed by in stomach and lung. However, there were 1422 worms(207 acanthocephalans and 1215 nematodes) in 84 Bufo gargarizans. The overall infection rate was 86.90%(28.57% for acanthocephalans and 84.52% for nematodes) andmean intensity was 19.48. What is more, in different organs of Bufo gargarizans, the lung had the highest infection rate, followed by the intestine and reproductive system. According to preliminary morphological observations, there were 2 genera and 1 family in 521 acanthocephalans and 5 species in 1451 nematodes.But it needs further study to identify the specific species of acanthocephalans and nematodes.The findingsprovide a basic basis for graspingthe prevalence of acanthocephalans and nematodesin Rana nigromaculata and Bufo gargarizans from Shanghai and preventing and curing parasitic diseases better. 3. Identification of acanthocephalan from frogs anf toadsIn 521 acanthocephalans collected from Rana nigromaculata and Bufo gargarizans, 65 mounted slides were made according to at least one slide in each organ method. In morphology, the numbers and arrangement of hooks and cement glands, spines, position and arrangement of tests, and shapesof eggsof acanthocephalanwere measured carefully, and then compared with known species. We found 2 genera 7 species of acanthocephalan from frogs and toads, such as Pseudoacanthocehpalus bufonis, P. nguyenthileae, P. reesei, P. lutzi, P. smalesi, Acanthocephalus ranae and A. lucidus. 4. Preliminary identification of nematode from frogs and toadsSince the defects of morphological identification of species, we used molecular biological methods firstly to determine the genus and family of 1451 nematodes collected from from 90 Rana nigromaculata and 84 Bufo gargarizans, then identify the species of nematodes by morphological observation. In short, DNA was extracted from different organs, for example, intestine, lung, body cavity, stomach, lung, reproductive system, amplified using cox1 primers by PC R and aligned according to BLAST, and then ML tree was built.The results showed that there were 5 species, 2 genera and 2 families in 1451 nematodes, including Rhabdias sp.and Contracaecum sp. But the identification of specific species needs further morphological observation. This study revealed that the combination of morphological and molecular methods among identification of worm species not only can make up each other’s deficiencies, but also determine the parasite species more scientifically. 5. The species and geographic distribution of acanthocephala nand ne matode from frogs in ChinaIn order to understand the species and geographic distributions of acanthocephalan and nematode from frogs in China, we have collected relevant literatures that reported acanthocephalan and nematode in frogs of China and organized into the list according to newer classification system about acanthocephalan and nematode. In summary, 8 species of acanthocephalans and 48 species of nematodes have been recorded in frogs of C hina. Among them, the species of acanthocephalans belong to 3 genera, 2 families and the species of nematodes belong to 23 genera, 17 families. We have listed the Chinese and Latin names, the namer and naming years, the parasitic hosts a nd sites, the geographical distributions and the sources of literatures of these acanthocephalans and nematodes in this paper, which provides the basic materials for a more comprehensive grasp about species of acanthocephalans and nematodes from frogs in China.