The effects of light intensity on the growth of Japanese Gambierdiscus spp. (Dinophyceae).

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a form of seafood poisoning that is widespread in tropical, subtropical and temperate regions worldwide. The distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3 have been reported for the waters surrounding the main island of Japan. To explore the bloom dynamics and the vertical distribution of these Japanese species and phylotypes of Gambierdiscus, the effects of light intensity on their growth were tested, using a photoirradiation-culture system. The relationship between the observed growth rates and light intensity conditions for the four species/phylotypes were formulated at R>0.92 (p<0.01) using regression analysis and photosynthesis-light intensity (P-L) model. Based on this equation, the optimum light intensity (Lmax) and the semi-optimum light intensity range (Ls-opt) that resulted in the maximum growth rate (µmax) and ≥80% µmax values of the four species/phylotypes, respectively, were as follows: (1) the Lmax and Ls-opt of G. australes were 208µmol photons m-2s-1 and 91-422µmol photons m-2s-1, respectively; (2) those of G. scabrosus were 252 and 120-421µmol photons m-2s-1, respectively; (3) those of Gambierdiscus sp. type 2 were 192 and 75-430µmol photons m-2s-1, respectively; and (4) those of Gambierdiscus sp. type 3 were ≥427 and 73-427µmol photons m-2s-1, respectively. All four Gambierdiscus species/phylotypes required approximately 10µmol photons m-2s-1 to maintain growth. The light intensities in coastal waters at a site in Tosa Bay were measured vertically at 1m intervals once per season. The relationships between the observed light intensity and depth were formulated using Beer's Law. Based on these equations, the range of the attenuation coefficients at Tosa Bay site was determined to be 0.058-0.119m-1. The values 1700µmol photons m-2s-1, 500µmol photons m-2s-1, and 200µmol photons m-2s-1 were substituted into the equations to estimate the vertical profiles of light intensity at sunny midday, cloudy midday and rainy midday, respectively. Based on the regression equations coupled with the empirically determined attenuation coefficients for each of the four seasons, the ranges of the projected depths of Lmax and Ls-opt for the four Gambierdiscus species/phylotypes under sunny midday conditions, cloudy midday conditions, and rainy midday conditions were 12-38m and 12-54m, 1-16m and 1-33m, and 0m and 0-16m, respectively. These results suggest that light intensity plays an important role in the bloom dynamics and vertical distribution of Gambierdiscus species/phylotypes in Japanese coastal waters.

Occurrence of Karenia papilionacea (Dinophyceae) and its novel sister phylotype in Japanese coastal waters.

Several species of the genus Karenia (Dinophyceae) form blooms and often cause the mortality of cultured and wild fish. In Japan, blooms caused by two species - namely Karenia mikimotoi and Karenia brevis - have been reported so far. On the basis of morphological and molecular-phylogenic examinations, the present investigation found Karenia papilionacea and its novel sister phylotype for the first time in the coastal waters of the various regions of Japan. Of 34 strains isolated from the coastal waters, 27 strains displayed the typical morphological characteristics of K. papilionacea and further showed consensus DNA sequences corresponding to those of the originally described K. papilionacea. The other 7 strains displayed the same morphological characteristics of K. papilionacea, but showed divergent DNA sequences, at a genetic distance of over 0.04 (Internal Transcribed Spacer regions) from those of the original phylotype of K. papilionacea. These divergent strains were characterized as a novel sister phylotype (phylotype-I) of K. papilionacea. In the coastal waters of Japan, K. papilionacea-like (K. papilionacea and/or its phylotype-I) formed blooms at 20.3-30.4°C and salinity 30.1-33.9. No K. brevis was identified in Japanese coastal waters during this study. These findings demonstrated that K. papilionacea occurs along the coasts of western Japan and possibly shares several coastal regions with K. mikimotoi and K. papilionacea phylotype-I. In order to assess the risks of Karenia blooms to aquaculture, it is essential that the growth physiology and ichthyotoxicity of K. papilionacea and its novel phylotype should be characterized.

Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum.

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.

Quantitative PCR method for enumeration of cells of cryptic species of the toxic marine dinoflagellate Ostreopsis spp. in coastal waters of Japan.

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.