Flow cytometric detection and enumeration of DNA and RNA viruses infecting marine eukaryotic microalgae

Sample preparation protocols for flow cytometry (FCM) analysis with five algal viruses were optimized: Heterocapsa circularisquama virus (HcV), Heterosigma akashiwo virus (HaV), Chaetoceros salsugineum nuclear inclusion virus (CsNIV), Rhizosolenia setigera RNA virus (RsRNAV) and H. circularisquama RNA virus (HcRNAV). The optimum staining protocols differed significantly among the viruses tested. FCM counts for the large DNA algal viruses HaV and HcV (similar to 0.2 mu m in diameter) were similar to numbers determined by epifluorescence microscopy (EFM). In contrast, FCM counts of viruses smaller than 40 nm that harbor DNA (CsNIV) or RNA genomes (RsRNAV, HcRNAV) were comparable to or lower than most probable number (MPN) values, which indicate only infectious virus number, suggesting that the FCM counts were underestimates. This is presumably because their single particle fluorescence signals were below the detection limit for the flow cytometer. These results indicate that a large portion of the smaller viruses in the aquatic plankton virus community may be overlooked by FCNI.