Molecular Biological Assessment of Soil Environment Using Earthworm (Annelida, Olagoch aeta)

蚯蚓对环境非常敏感，因此，蚯蚓可能是土壤环境的指示器。蚯蚓的实地调查在奥多摩林区和我校进行了2年。解剖分析发现，90%以上的蚯蚓具有不完整的雄性性腺，而所有蚯蚓都具有完整的雌性性腺。目前尚不清楚雄性性腺异常是环境污染所致，还是雌雄同体的物种特征所致。为了利用分子生物学技术对土壤环境进行评价，我们首先建立了世界范围内广泛分布的杀光剂爱森ia fetida的清洁栽培。在我们的实验室中，恶臭杆菌可以在有米糠的木屑培养基中生长，每月繁殖一次。雌蝶在含合成雌激素DES的生长培养基中表现出明显的生长迟缓，提示尽管没有明显的内分泌器官，但雌蝶接受类固醇性激素的共同机制。它们的遗传信息极为稀少，但对性腺分化的分子机制的研究是有价值的。我们正在对从线虫、果蝇和哺乳动物中分离的雄性性腺决定基因DMRT同源基因进行分子克隆。日本赤虾以独特的无性繁殖方式繁殖，成熟的日本赤虾分成约10个片段，每个片段在10天内长成成熟的蠕虫。然而，在饥饿和低密度培养下，性腺分化为雌雄同体并有性繁殖。17-b-雌二醇或DES既不能诱导性腺分化也不能诱导有性生殖。然而，17-b-雌二醇诱导了剂量依赖性的生长延迟，而DES表现出毒性作用。为了进一步研究这些繁殖和再生过程，采用抗磷酸酪氨酸和抗brdu抗体建立了免疫染色程序。

Earthworms are very sensitive to their circumstance, and thus, a possible indicator for soil environment. The field investigations of earthworm were carried out in Okutama forestry region and in our campus for 2 years. Anatomical analysis of Pheretimoid earthworms revealed more then 90% of them possess incomplete male gonads, whereas all possess complete female gonad. It is not yet clear whether the anomaly of male gonad come from environmental contaminations or the species characteristics of Pheretimoids.In order to assess soil environment in molecular biological technique, we first established the clean cultivation of Eisenia fetida, which is a world-widely distributed Lumricide. E. fetida can grow in a sawdust media with rice bran in our laboratory, and reproduce once a month. E. fetida shows the significant growth retardation in synthetic estrogen DES containing-growth media, suggesting the common mechanism for steroid sex hormones reception despite of no apparent endocrine organ. Their genetic information is extremely scarce, but it is worthy to investigate the molecular mechanism of gonad differentiation. We are carrying out the molecular cloning of homolog(s) of DMRT, which is isolated from nematode C. elegans, fruit fly D. melanogaster and also mammals as male gonad-determining gene.Enchytraeus japonensis reproduces in a unique asexual manner, in which the mature E. japonensis divided into 〜10 fragments and each fragment grow into mature worms within 10 days. However, under starved and low-density culture, gonad differentiation into hermaphrodites and sexual reproduction takes place. 17-b-estradiol or DES induces neither gonad differentiation nor sexual reproduction. However, 17-b-estradiol induces a dose-dependent delay of growth, while DES exhibits toxic effect. To further investigate these reproduction and regeneration process, immunological staining procedures were established using anti-phosphotyrosine and anti-BrdU antibodies.