The abundance of potentially Microcystis aeruginosa-infectious cyanophages in freshwater was studied using g91 real-time PCR. A clear increase in cyanophage abundance was observed when M. aeruginosa numbers declined, showing that these factors were significantly negatively correlated. Furthermore, our data suggested that cyanophage dynamics may also affect shifts in microcystin-producing and non-microcystin-producing populations.The major bloom-forming cyanobacterial species Microcystis aeruginosa forms noxious blooms in many eutrophic freshwater lakes, ponds, and reservoirs. A limited population (limited number of strains) of M. aeruginosa in the environment produces potent hepatotoxins called microcystins (7). These potent toxins in the M. aeruginosa blooms have caused many cases of animal and human poisoning (3,8,16).Previously, most studies have focused on relationships among the cyanobacterial bloom dynamics and the changes in physicochemical factors (e.g., nutrient supply, light, and temperature) that influence cyanobacterial growth in the aquatic environment (28). Since the discovery that viruses are widespread in marine ecosystems (4), cyanophages that can infect cyanobacteria have been thought to be an alternative factor that may control the succession of cyanobacterial blooms (12,14,15,18,19). In addition, cyanophages can also influence the clonal composition of the host Synechococcus communities (14, 27) and could account for some of the cyanobacterial diversity observed in natural communities (22,25,30). Nevertheless, little is known about how freshwater cyanophages can affect the abundance and clonal composition of cyanobacterial blooms in lakes over time.Our aim is to determine if the cyanophages have potential quantitative and qualitative effects on the M. aeruginosa communities in Lake Mikata in Japan. We performed two independent real-time PCR assays to monitor the dynamics of M. aeruginosa and its cyanophage communities. To quantify M. aeruginosa, we used the phycocyanin intergenic spacer (PC-IGS) that was previously used to examine total M. aeruginosa numbers (9, 32). A second real-time PCR assay was used to quantitatively detect potentially M. aeruginosa-infectious cyanophages using the primers targeting the viral sheath proteinencoding gene (g91) previously identified by Takashima et al.(21). To determine the effect of the cyanophages on the internal dynamics of the total M. aeruginosa communities, we examined the fluctuation in the abundance of potentially microcystin-producing M. aeruginosa populations using the real-time PCR and microcystin synthetase gene (mcyA)-specific primers (32) and monitored the relative size of the microcystin-producing subpopulation compared to the total population in relation to the cyanophage numbers using a field survey of M. aeruginosa blooms in a Japanese lake.Water samples were collected from the surface layer (0.5 m) once per month from April to October in 2006 at a fixed point (35°33ЈN, 135°53ЈE) in Lake Mikata (Fig. 1). The cyanobacterial cells used for ...

The aim of this study was to develop a quantitative real-time reverse transcription-PCR (real-time RT-PCR) assay to detect and quantify mRNA of cyanophages within infected Microcystis aeruginosa cells in a freshwater pond. Laboratory-based data showed that the relative abundance of the cyanophage g91 mRNA within host cells increased before cyanophage numbers increased in culture. This transcriptional pattern indicated the kinetics of the viral infection suggesting the real-time RT-PCR method to be a potential tool for environmental monitoring of cyanophage infections. In this field survey, the numbers of infected M. aeruginosa cell populations estimated from cyanophage numbers were low at 0.01-2.9 cells mL −1 . The highest relative abundance of phage g91 RNA (10 −2 per rnpB transcript) was at about the same levels of expression as laboratory-based growth data for Ma-LMM01 (estimated density of infected host cells: 10 5 cells mL −1 ); and was observed when cyanophage numbers rapidly increased (as well as a decrease in host cell numbers). Quantification of cyanophage numbers is important to understand ecological relationships between the phage and its hosts. Our data suggest the quantification of phage gene transcripts within a natural host cell population to be a strong tool for investigating the quantitative effects of phage lysis during infection of the host population.Key words: cyanophage, Microcystis aeruginosa, real-time RT-PCR, succession, toxic cyanobacteria Microcystis aeruginosa is a well-known toxic cyanobacterial species that commonly develops blooms in eutrophic freshwater throughout the world. This species includes strains that can produce potent hepatotoxins called microcystins (2). There are several reports of deaths in wild and domestic animals as well as humans due to acute poisoning which causes massive hepatic hemorrhage (1,6,16).Cyanophages are considered to be a significant factor regulating the abundance, clonal diversity, and composition of their cyanobacterial host populations (9,10,12,14,15,19,20,25). The phages also play a major role in nutrient cycling and genetic transfer (21,22,26). In contrast to the vast majority of research having focused on marine cyanophages (12,14,15,19,20,25), there are few studies concerning freshwater cyanophages (9, 10). Reports suggest phages play an important role in regulating the bloom dynamics of M. aeruginosa blooms. Manage et al. (9, 10) observed an increase in cyanophage titers (the numbers of particles forming plaques on an M. aeruginosa lawn) accompanied by a large decrease in the abundance of M. aeruginosa in a natural freshwater environment. Recently, during a field survey of a Japanese freshwater lake, real-time monitoring of M. aeruginosa-cyanophage abundance with quantitative PCR assays showed the seasonal dynamics of the cyanophage community in freshwater that may affect shifts in the clonal composition of diverse M. aeruginosa populations (e.g., microcystin-producing and non-producing populations); rather than having a quantitative impact o...

We optimized conditions for cryopreservation of a cyanophage (Ma-LMM01) infecting the toxic cyanobacterium Microcystis aeruginosa. The quality of cryopreservation was estimated by comparing the phage titer before and after preservation at 4, −80, or −196°C and with or without a cryoprotectant: glycerol, dimethyl sulfoxide (DMSO), or Cellbanker. Storage at 4°C for 100 days resulted in ~90% loss of infectivity; whereas cryopreservation at −196°C resulted in stable preservation with or without cryoprotectant. Therefore, we established methods to stably preserve the phage, Ma-LMM01, that may be useful in further studies of cyanophages and may be used in isolating new phages.