Rapid monitoring for the potentially ichthyotoxic dinoflagellate Cochlodinium polykrikoides in Korean coastal waters using fluorescent probe tools

Abstract

Cochlodinium polykrikoides has occurred annually in Korea's coastal waters since 1982, where it has caused extensive fish mortality. Rapid and accurate monitoring is very important to reduce the economic damage caused by C. polykrikoides. However, before outbreaks of C. polykrikoides occur, single cells are often more abundant than chain-forming cells. During this period, it is very difficult to discriminate C. polykrikoides from closely related dinoflagellates and other phytoplankton species. The aim of the present study was to identify even single-celled C. polykrikoides and to assess counting methodologies using molecular probes. A new polyclonal antibody bound specifically to C. polykrikoides, and not to other phytoplankton, including the morphologically similar Gyrodinium impudicum and Gymnodinium catenatum. When cultured C. polykrikoides cells were fixed with 2.5% glutaraldehyde, they emitted a high-intensity fluorescent signal during the period of exponential growth. The correlation with cell density of a mixed culture including C. polykrikoides treated with an antibody and lectin probes (wheat germ agglutinin/Erythrina cristagalli agglutinin) was well matched (r2 = 0.83). The detection of C. polykrikoides was possible at concentrations as low as 5 cells ml-1. Reaction with C. polykrikoides antisera and antigen by western blotting analysis was at ≃30 kDa and gave no signal for G. impudicum, G. catenatum, Alexandrium tamarense or Prorocentrum micans. Consequently, the new antibody is of great importance for enumeration and identification of C. polykrikoides in Korea's waters.