Detection and enumeration of Heterosigma akashiwo and Fibrocapsa japonica (Raphidophyceae) using rRNA-targeted oligonucleotide probes

Abstract

Effective management of fish aquaculture stocks to mitigate the impact of fish-killing phytoplankton requires intensive spatial and temporal sampling to identify and quantify potentially harmful species. so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of large-subunit rRNA (LSU rRNA)–targeted oligonucleotide probes as tools to aid in the detection and enumeration of the fragile, fish-killing species Heterosigma akashiwo and Fibrocapsa japonica (Raphidophyceae). Oligonucleotides directed toward H. akashiwo and F. japonica were evaluated using fluorescent in situ hybridization (FISH). Probes that labelled those species well in the FISH format were then incorporated into a sandwich hybridization assay (SHA). SHAs were successfully developed for both H. akashiwo and F. japonica. Batch culture experiments showed that the response of the SHA using a constant number of H. akashiwo and F. japonica cells harvested in exponential vs stationary phase of growth varied by a factor of approximately two. Preliminary field trials indicate that the SHA appears to be a faster, more cost-effective, and easier-to-use method for detecting and estimating the abundance of H. akashiwo and F. japonica than is FISH or conventional light microscopy. This is particularly true when large numbers of samples need to be processed routinely and rapidly, and when the organism in question is fragile and subject to lysis or morphological distortion if chemically preserved prior to microscopical observation.