| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2001: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
In the present research, elucidation of the speciation process of protozoan and nematode parasites of amphibians was attempted on genetic differences by analyzing DNA sequences. On trypanosome protozoans, DNA analysis was conducted using crude blood from Rana nigromaculata with Trypanonsoma sp. infection, and about 640 bases of 18SrDNA were successfully sequenced. It was demonstrated that under a suitable PCR condition, only 1 to 5μl of crude blood with a few trypanosomes was enough for analysis. This method is much more advantageous than the usual protocol in which mass culture system is required. It is also expected that DNA sequencing becomes possible using the blood smear slides by developing this technique. Among nematodes, CO1, ITS2, 28SrDNA, and 5SrDNA were analyzed on the genera Spiroxys, Rhabdias, Cosmocerca, Cosmocercoides and Amphibiocapillaria. Phylogenetic trees were made for gnathostomatids I.e., Spiroxys japonica from frogs, Spiroxys hanzaki from giant salamanders and Gn
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athostoma nipponicum from weasel, with a hedrurid, Hedruris . ijimai, from frogs as outgroup. The DNA base sequences of Rhabdias tokyoensis were almost identical between the hosts Cynops pyrrhogaster and Cynops ensicauda. No difference was found in the DNA sequences of Rhabdias japonica among examples from Rana nigromaculata, Rana porosa porosa and Rana rugosa. The DNA base sequences of Cosmocerca japonica were almost identical among the examples from Hyla japonica, Rana nigromaculata, Rana porosa porosa, Rana rugosa and Rhacophorus schlegelii, demonstrating that this nematode is shared by frogs of plural genera. About 30 bases were different in 28SrDNA sequence between genera Cosmocerca and Cosmocercoides, and about 10 bases differed between Cosmocercoides from Cynops ensicauda of Okinawa and Bufo japonicus of mainland Japan. The sequences of 18SrDNA of Amphibiocapillaria from Cynops ensicauda and Andriasjaponicus were highly identical. During the course of research, the life history of Spiroxys hanzaki was elucidated and it was demonstrated that gnathostomatids possess only 4 (3 larval and adult) stages. A new nematode genus Kamegainema was proposed for Filaria cingula of giant salamanders. A comparative study was also made on helminths from Japanese giant salamanders and hellbenders from U.S.A. Less
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