The application of species specific DNA-targeted probes and fluorescently tagged lectin to differentiate several species of Pseudo-nitzschia (Bacillariophyceae) in Chinhae Bay, Korea

Abstract

We applied molecular methods using a DNA probe and fluorescently tagged lectins to discrimenate toxic Pseudo-nitzschia multiseries from the Chinhae Bay for a Korean harmfulalgae monitoring program. From the binding activity of lectins, P. multistriata, P. subfraudulenta, P. pungens, P. multiseries and P. cuspidata bound ConA. whereas P. subpacifica and P. delicatissima did not. Ribosomal RNA-targeted oligonucleotide probes (muD1, pUD1 and deD1) specifically reacted to P. multiseries, P. pungens and P. delicatissima, respectively, whereas auD1, frD1 and amD1 probes did not bind to P. multiseries, P. pungens, P. cuspidata, P. multistriata, P. subpacifica, P. subfraudulenta and P. delicatissima. In particular, flourescent FITC-conjugated WGA specifically bound to P. multiseries but not to P. pungens, indicating that this is a desirable method for their rapid and easy discrimination. In addition, we tested a species-specific oligonucleotide DNA probe (muD1) using the whole cell hybridization filter tube system, and the WGA lectin to discriminate P. multiseries in the field. The oligonucleotide probe and fluorescent WGA bound specifically to P. multiseries and these labelled cells were correlated to label cell abundance. These results imply that DNA and lectin probes are appropriate tools for counting P. multiseries and distinguishing morphologically similar Pseudo-nitzschia species in natural samples, therefore, these methods are especially pertinent since rapid separation and quantitative estimation of cell abundance of P. multiseries are now important for a routine harmful algae monitoring program in Korean waters.