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Viruses are considered key players in phytoplankton population control in oceans. However, mechanisms that control viral gene expression in prominent microalgae such as diatoms remain largely unknown. In this study, potential promoter regions isolated from several marine diatom-infecting viruses (DIVs) were linked to the egfp reporter gene and transformed into the Pennales diatom Phaeodactylum tricornutum. We analysed their activity in cells grown under different conditions. Compared to diatom endogenous promoters, novel DIV promoter (ClP1) mediated a significantly higher degree of reporter transcription and translation. Stable expression levels were observed in transformants grown under both light and dark conditions, and high levels of expression were reported in cells in the stationary phase compared to the exponential phase of growth. Conserved motifs in the sequence of DIV promoters were also found. These results allow the identification of novel regulatory regions that drive DIV gene expression and further examinations of the mechanisms that control virus-mediated bloom control in diatoms. Moreover, the identified ClP1 promoter can serve as a novel tool for metabolic engineering of diatoms. This is the first report describing a promoter of DIVs that may be of use in basic and applied diatom research.

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.

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Benthic dinoflagellates of the genus Ostreopsis produce palytoxin (PTX)-like compounds. The worldwide distributed Ostreopsis ovata/O. cf. ovata is potentially responsible for outbreaks of human health problems around the coasts of tropical, subtropical, and temperate regions. The present study examined growth responses of an O. cf. ovata strain s0662 collected from coastal waters of Japan with 35 different combinations of temperature (15-35°C) and salinity (20-40) and discusses the bloom dynamics of the organism in Japanese coastal environments. The O. cf. ovata strain s0662 tolerated a wide range of temperature (17.5-35°C) and salinity (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Results of a two-way ANOVA showed significant effects of temperature-salinity interaction on growth rates and biomass yields of the O. cf. ovata strain (F(24,70) > 127, P < 0.001). The strain showed a maximal growth rate (1.03 divisions day -1 ) and biomass yield (240 relative fluorescence) at temperature 25°C and salinity 30. The high growth rates of over 1.0 division day -1 were obtained in conditions of temperature 25-30°C and salinity 30-35, which indicates that strain s0662 prefers high temperature and salinity conditions. The growth rates of O. cf. ovata under the optimal conditions were higher than those of other benthic toxic-dinoflagellates, Coolia monotis, Gambierdiscus toxicus, and Prorocentrum lima (Dinophyceae) previously reported. Taken together, we suggest that O. cf. ovata is able to grow faster than the other benthic dinoflagellates in waters of high temperature and salinity. The physiological feature probably confers an ecological advantage on O. cf. ovata in the bloom development during warmer seasons in Japan and may be responsible for outbreaks of PTX-like poisoning in the region especially during the warmer seasons.