Study on speciation of amphibian parasites by DNA analysis

DNA分析研究两栖类寄生虫的物种形成

• 批准号: 11640700
• 负责人: MIYATA Akira
• 金额: $ 1.98万
• 依托单位: OITA MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11640700/
• 关键词: Amphibia; Parasite; Molecular systematics; DNA; Speciation; Trypanosoma; Protozoa; Nematoda; 分子系統; Spiroxys; Gnathostoma; Hedriris; COI; ITS2; 遺伝子

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 … More 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

在本研究中，试图通过分析DNA序列来阐明两栖动物原生动物和线虫寄生虫的规范过程。在锥虫原生动物上，使用来自锥虫瘤的Rana nigromaculata的粗血进行了DNA分析。成功地测序了感染，约有640个基部的18srDNA。证明在适当的PCR条件下，只有1至5μl的粗血和几个锥虫足以分析。此方法比需要大众培养系统的通常协议更有优势。还可以预期，通过开发该技术，使用血液涂片幻灯片可能会进行DNA测序。在线虫，CO1，IS2，28SRDNA和5srDNA中，在螺旋属，rhabdias，Cosmocerca，Cosmocercoides和Amphibiocapillaria上进行了分析。制作了用于gnathostomatids的系统发育树，即来自青蛙的螺旋形japonica，来自巨型salamanders的Siroxys Hanzaki和GN…来自Weasel的Athostoma nipplem，来自Weasel，带有Hedrurid，Hedruris Hedruris。 ijimai，来自青蛙的群体。宿主cynops pyrrhogaster和cynops ensicauda之间的Rhabdias Tokyoensis的DNA碱基序列几乎相同。在Rana nigromaculata，Rana Porosa和Rana Rugosa的例子中，rhabdias japonica的DNA序列中没有发现差异。在Hyla Japonica，Rana Nigromaculata，Rana Porosa，Rana Porosa，Rana Rugosa和Rhacophorus schlegelii的示例中，Cosmocera的DNA碱基序列几乎是相同的，这表明这种线虫是由Plural Genera的Frogs共享的。在Cosmocerca和Cosmocercoides之间的28srDNA序列中，约有30个基础不同，而来自Oukinawa的Cynops Ensicauda和日本大陆的Bufo Japonicus的Cosmocercoides之间约有10个基部。来自cynops的二氧化二苯性下调的序列Ensicauda和Andriasjaponicus是高度同样的。在研究过程中，Hanzaki的螺旋氧基人生历史被阐明了，并且证明Gnathostomatids仅具有4个（3个幼虫和成人）阶段。提出了一种新的线虫属kamegainema属，用于巨型sal的丝状丝。还对日本巨人sal和美国地狱者的舵手进行了比较研究。

项目成果

Hasegawa,Hideo 他: "Kamegainema cingulum (Linstow,1902) n.gen.,n.sp.(Nematoda:Dracunculidae),a subcutaneous・・・"Journal of Parasitology. 86・3(印刷中). (2000)

Hasekawa, Hideo 等人：“Kamegainema cingulum (Linstow, 1902) n.gen., n.sp.（线虫纲：龙线虫科），皮下...”《寄生虫学杂志》86·3（印刷中）。 ）

Hasegawa, H., Doi, T., Araki, J., Miyata, A.: "Kamegainema cingulum (Linstow, 1902) n. gen., n. comb. (Nematoda : Dracunculidae), a subcutaneous parasite of cryptobranchids (Amphibia : Caudata)"Journal of Parasitology. 86・3. 583-587 (2000)

Hasekawa, H.、Doi, T.、Araki, J.、Miyata, A.：“Kamegainema cingulum (Linstow, 1902) n. gen., n. Com.（线虫纲：龙线虫科），一种隐鳃纲动物（两栖类）的皮下寄生虫：有尾目）“寄生虫学杂志.86・3.583-587（2000）

Imai, J., Hasegawa, H.: "Molting of Gnathostoma doloresi (Nematoda : Gnathostomatoidea) in the definitive host"Journal of Parasitology. 87・1. 14-18 (2001)

今井 J.、长谷川 H.：“最终宿主中颚口线虫 (Gnathostoma doloresi) 的蜕变”，寄生虫学杂志 87・1 (2001)。

Imai, J., Hasegawa, H.: "Molting of Gnathostoma doloresi (Nematoda : Gnathostomatoidea) in the definitive host"Journal of Parasitology. 87(1). 14-18 (2001)

Imai, J., Hasekawa, H.：“Gnathostoma doloresi（线虫纲：Gnathostomatoidea）在最终宿主中的蜕皮”寄生虫学杂志。

Hasegawa, H., Doi, T., Tochimoto, T., Miyata, A.: "Parasitic helminthes collected from the Japanese giant salamander, Andrias japonicus(Temminck,1837)(Amhibia : Crtobranchidae)in Japan."Comparative Parasitology. 69.1. 33-42 (2002)

Hasekawa, H.、Doi, T.、Tochimoto, T.、Miyata, A.：“从日本大鲵 Andrias japonicus（Temminck，1837）（Amhibia：Crtobranchidae）中收集的寄生蠕虫。”比较寄生虫学。