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

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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.