| Research Abstract |
An assessment of water quality on the basis of quick and correct identification of chironomid larvae was tried by seeking species-specific hemoglobin (Hb) -coding DNA sequences of genus Polypedilum as well as by clarifying occurrence spectrum of polypedilum species corresponding to eutrophication level by comparison of species composition of this genus in waters at different trophic levels.
As a result, at rapid of river, the following occurrence spectrum was estimated ; aviceps, pedestre, etc.*hiroshimaense, etc.*japonicum, surugense, etc.*tamaharaki, etc.*convictum*paraviceps, etc.*okigrandis. At pool of river, the following spectrum was estimated ; tamahosohige, etc.*asakawaense, etc.*togaexpansum, etc.*nubeculosum, etc. At littoral of lake, the following spectrum was estimated ; masudai, etc.*cultellatum, etc.*nubifer.
On the other hand, gene cloning of homologous Hbs were undertaken from P.cultellatum, P.japonicum, P.nubeculosum and P.nubifer, each belonging to different group of this genus. However, this was successful only for a monomeric Hb of P.nubifer (PNf-MV). Therefore, an estimated amino acid sequence of PNf-MV was compared with those of 12 Hbs of a European species, Chironomus thummi thummi. As a result, 2 relatively long conserved regions, each located at N-terminal and C-terminal region, were revealed. Thus, RT-PCR with degenerative primers corresponding these two regions, using larval total RNA as a template, amplified DNA fragments of about 260bp, probably corresponding to 86 residues. DNA sequence determination and search of species-specific sequences, which are possible to be used for species identification, are now in progress.
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