報告書番号

V05007

タイトル（和文）

DNA解析によるアカフジツボ幼生の簡易検出法

タイトル（英文）

Identification of fouling species in barnacle larvae by DNA analysis

概要 （図表や脚注は「報告書全文」に掲載しております）

冷却水路系へのフジツボ付着は多くの発電所で深刻な問題となっているが、汚損性フジツボの種はアカフジツボなどに限られている。汚損性フジツボの付着時期を予測することはそれらの防除対策にとって重要であり、汚損性フジツボ幼生を正確かつ簡便に検出する技術が求められている。本研究では、日本沿岸に生息する主なフジツボの種判定をDNA解析により行うとともに、1個体の幼生からのDNA解析法を開発した。PCR法で12S rRNA遺伝子領域を増幅し、その塩基配列を解析することで、国内に棲息する15種のフジツボを正確に種判別することができた。また、微小なフジツボ幼生1個体をそのままPCRに供することで、ほぼ100％の幼生サンプルで種判定が可能となった。さらに制限酵素Spe Iを用いたPCR-RFLP法（制限酵素断片長多型解析）により代表的汚損種であるアカフジツボの幼生を簡便に検出する技術を開発した。

概要 （英文）

A large quantity of barnacles invade cooling water systems and cause serious problems at several electric power stations in Japan. The fouling barnacle species are limited but it is difficult to identify the species of larvae by their morphological features. In the present study, we examined inter-species genetic variation in several barnacles by DNA analysis in order to identify the species of larvae collected in the field and to forecast settlement period of the fouling barnacles. Fifteen common barnacle species were collected and applied to DNA analysis. Total DNA of each sample was extracted and applied to polymerase chain reaction (PCR) using specific primers for amplification of 12S rRNA and 16S rRNA genes encoded on mitochondrial DNA. Then the DNA sequences of the PCR products were compared. The results showed that the sequences of 12S rRNA gene are useful for identification of species. We also developed direct PCR method for individual barnacle larva using Advantage 2 Taq polymerase. Then, we established identification method of species in barnacle larvae by sequencing of 12S rRNA gene, which can be applied to samples collected from natural sea areas. The barnacle, Megabalanus rosa, is a major fouling species in Japan, and development of simple identification method for this species is thought to be useful. We found a specific sequence for the restriction enzyme Spe I in 12S rRNA gene of Megabalanus rosa, but the sequence was not found in the gene of the other 14 species. Then we performed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using Spe I, and succeeded in quick identification of M. rosa larvae in mixed larval samples. The present study showed that DNA analysis, particularly PCR-RFLP, is easy and precise technique for species identification of barnacle larvae and will be useful for forecast of settlement period in the fouling barnacle species.

報告者

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