Application of tyramide signal amplification-fluorescence in situ hybridisation and flow cytometry to detection of Heterosigma akashiwo (Raphidophyceae) in natural waters

Figures & data

Table 1  Strains used in the present study and their probe reactivities for the HSIG 1451 probe.

Strain	Source	Locality	Probe reactivity
Akashiwo sanguinea GnSg-K01	KMMCC^1	Geoje Island, Korea
Chattonella sp.	SNU^2	Kunsan Bay, Korea	−
Heterosigma akashiwo CCMP1912	CCMP^3		+
H. akashiwo HYM06HA	HYU^4	Masan Bay, Korea	+
H. akashiwo NFHTS-AK-1	KMMCC^1	Masan Bay, Korea	+
Gymnodinium impudicum D80	KMMCC^1	Yosu Bay, Korea	−
Prorocentrum micans D77	KMMCC^1	Masan Bay, Korea	−
^1Korea Marine Microalgae Culture Center, Korea; ^2Seoul National University, Korea; ^3Center for Culture of Marine Phytoplankton, USA; ^4Hanyang University, Korea.

Figure 1  Light and fluorescence micrographs of Heterosigma akashiwo strain HYM06HA. A, Live cell. B, Chlorophyll in the live cell. C, Cell fixed with paraformaldehyde (PFA). D, Chlorophyll in the fixed cell. E, Cell following chlorophyll extraction with methanol after PFA fixation. F, Nucleus stained with propidium iodide (PI) after methanol treatment. G, Cell following chlorophyll extraction with ethanol after PFA fixation. H, Nucleus stained with PI after ethanol treatment.

Table 2  Oligonucleotide probes used in the present study.

Probe	Target	Sequence (5′–3′)	Target site^1	Tm ^2 (°C)	Reference
EUK1209	Eukaryote	GGG CAT CAC AGA CCT G	1195–	58.4	Giovannoni et al. (1988)
NON338	Antisense	ACT CCT ACG GGA GGC AGC	–	64.3	Wallner et al. (1993)
HSIG1451	Heterosigma	CCC TCG GCA AGT CAC AAT	1451–	63.7	Present study
^118S rRNA position of E. coli numbering, ARB database; ^2melting temperature value calculated using the nearest neighbour method according to Hugenholtz et al. (2001).

Table 3  Specificities of the HSIG1451 probe.

Organism	Target sequence
Heterosigma akashiwo	5′-AUUGUGACUUGCCGAGGG-3′
Chattonella sp.	5′-AUUGUGACUUGCCGUUAG-3′

Figure 2  Effect of probe concentration on fluorescent intensity (arbitrary units, AU) in Heterosigma akashiwo cells.

Figure 3  Effect of formamide concentration on fluorescent intensity (arbitrary units, AU) in Heterosigma akashiwo cells.

Figure 4  Epifluorescence micrographs. A, Algal cells after fluorescence in situ hybridisation using various probes. Panels a, f, and k: Heterosigma akashiwo, panels b, g and l: Chattonella sp., panels c and h: Gymnodinium impudicum, panels d and i: Akashiwo sanguine, panels e and j: Prorocentrum micans; upper panels (a, b, c, d, and e): cells hybridised with the EUK1209 probe, middle panels (f, g, h, I, and j): cells hybridised with the HSIG1451 probe, lower panels (k and l): cells hybridised with the NON338 probe. B, Natural seawater sample collected from Geoje Island. Panel a: cells hybridised with HSIG1451 probe, panel b: cells hybridised with EUK1209 probe.

Figure 5  Flow cytometric signatures of Heterosigma akashiwo. A–C, Signatures of H. akashiwo strain after fluorescence in situ hybridization (FISH). D–I, Signatures of cells collected from Korean coastal waters after FISH. A, Detection of H. akashiwo cells after FISH; red dots indicate 6 m beads; green dots indicate H. akashiwo cells detected with green fluorescence after FISH. B, Discrimination of doublet and singlet cells of H. akashiwo; pink dots indicate singlet cells discriminated by peak area and peak width (length) of red fluorescence of detected cells (green dots) in panel A. C, DNA histograms of H. akashiwo gated in panel B (composed of singlet cells discriminated in panel B). D–F, Samples hybridised with HSIG1284 probe. G–I, Samples hybridised with NON338 probe.

Figure 6  Diel cell cycle analysis of Heterosigma akashiwo from Shiwha Lake after fluorescence in situ hybridisation.