Application of fluorescence in situ hybridization (FISH) method to detect Heterosigma akashiwo

Species-specific probes against cytoplasmic rRNA and nuclear rDNA of Heterosigma akashiwo were designed on the basis of LSU and ITS sequences. FISH protocols for whole cell and nuclear were established to detect this alga. The results showed that the rRNA-targeted probe hybridized with cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively a fluorescent nucleus. The probes were specific, without cross-reacting to other test algae. The method was also applied successfully to distinguish H. akashiwo from mixed field samples. Consequently, the FISH method was proved promising for specific, rapid, precise and semi-quantitative detection for cultured algae as well as field samples. Introduction The golden-brown marine alga Heterosigma akashiwo (Hada) Hada (Chromophyta: Raphidophyceae) is well known as a eurythermal and euryhaline flagellate in temperate seas. This alga has been found to be responsible for mass mortality of cultured fish in Japan, China, Korea, Norway, Canada, Chile, New Zealand and USA, resulting in great loss to aquaculture (Yamochi 1987; Chang et al. 1990; Hard et al. 2000). It is crucial for the management of cultured fish in order to avoid stock loss to rapidly identify and quantify H. akashiwo, as well as other harmful algal species (Hallegraeff and Hara 1995). Detection of H. akashiwo prior to the onset of a bloom event is viewed as important for improving early warning capabilities. However, correct identification and enumeration of H. akashiwo is rather difficult and time-consuming. The cells are small, fragile and cell morphology often changes with different water conditions (Guo 2004). On the other hand, traditional monitoring methods for H. akashiwo rely on light microscopical examination of live or Lugol's iodine-stained cells. A long time is spent on shipping and counting samples, which greatly delays the warning of potential blooms. To solve these problems, much attention has focused on developing a simple, rapid, reliable, and effective identification and quantification method for this species. In the last decade, fluorescence in situ hybridization (FISH), as a promising cell detection technique, has been widely applied. Tyrrell et al. (2001) firstly explored the utility of rRNA-targeted oligonucleotide probes to detect H. akashiwo cells using FISH. Unfortunately, the authors argued that the application of FISH is problematic, and in their study, this detection method was overlooked and served as an intermediate step for the sandwich hybridization assay. Chen et al. (2008) established protocols of FISH for cultured H. akashiwo. However, applicability of this method to field samples has not been examined. Therefore, in this study, two specific probes (HA-lsu and HA-its) were designed based on the large-subunit (LSU) ribosomal RNA gene (28S rDNA regions D1-D2) and the internal transcribed spacers (ITS) containing 5.8S rDNA, respectively. These two probes were tested for cross-reactivity to other typical harmful microalgae and a FISH protocol was established for species-specific, quantitative, and rapid detection of H. akashiwo. Materials and methods Cultures. H. akashiwo, isolated from Jiaozhou Bay, Yellow Sea, was used as targeted species. Prorocentrum micans, Scrippsiella trochoidea, Amphidinium carterae, Prorocentrum lima and Amphidinium operculatum were used to confirm the species specificity of the designed probes and test for cross-reactivity. The cultures were kept in 250mL flasks containing 100mL f/2 medium at a salinity of 36 psu, at 20–22 °C and a 12:12-h light:dark cycle at an irradiance of 100 μmol photons·m −2 ·s −1 provided by cool white fluorescent tubes. The cultures were stirred