Tomoyuki Shikata

Extracellular polysaccharide-protein complexes of a harmful alga mediate the allelopathic control it exerts within the phytoplankton community.

The goal of this study was to examine the significance of allelopathy by the raphidophyte Heterosigma akashiwo in a multispecies phytoplankton community in the field. Towards this aim, we sought allelochemicals of H. akashiwo, which had allelopathic effect both in laboratory experiments and in the field. As an initial step, we showed that the allelopathic effects of H. akashiwo filtrate were both species-specific and dependent upon the cell density of the target species. Secondly, we found for the first time that extracellular, high-molecular weight allelochemicals [that is, polysaccharide-protein complexes (APPCs)] were produced by a marine phytoplankton species, H. akashiwo. Thirdly, we indicated that the purified APPCs selectively inhibited the growth of the diatom Skeletonema costatum that is a major competitor of H. akashiwo, and thereby tended to promote the formation of monospecific H. akashiwo blooms. Furthermore, we demonstrated that the inhibitory effect of APPCs on the growth of the diatoms was determined by binding to the cell surface of the target species. Finally, we succeeded in the detection of APPCs in the field samples at concentrations exceeding their experimentally determined action threshold during the H. akashiwo bloom. Strategies for ecosystem control, including mitigation of harmful algal blooms (HABs), should take into account that red-tide organisms like H. akashiwo are already part of complex webs involving inter-specific allelopathic inhibition and ecosystem control during their dense blooms.

Growth dynamics of Heterosigma akashiwo (Raphidophyceae) in Hakata Bay, Japan

In Hakata Bay, Japan, from April to June 2006, Heterosigma akashiwo dynamics were investigated by daily to weekly sampling in relation to environmental parameters. To test how well the water in the samples supported H. akashiwo growth, bioassays of growth of a cultured strain of H. akashiwo were also conducted on these samples. In Hakozaki, a semi-enclosed fishing port, H. akashiwo had bloomed at almost all stations, one week after DIN and DIP concentrations increased by the end of May. Thereafter, H. akashiwo declined and fluctuated at low densities. The H. akashiwo population almost completely disappeared associated with a rapid decrease in salinity at the end of June. In the bioassays, addition of phosphate and nitrate often promoted H. akashiwo growth during the investigation, but 1 week before the H. akashiwo bloom, the growth rates in seawater samples without addition of these nutrients became close to those with nutrients. Addition of vitamins and metals barely affected growth rates during the investigation period. Moreover, even when some phytoplankton species also bloomed densely, H. akashiwo grew in the seawater samples at rates similar to that when phytoplankton abundance was low, indicating absence of significant allelopathic control by other phytoplankton. The present study shows that H. akashiwo growth is limited by N- and P-sources in this bay.

The role of interactions between Prorocentrum minimum and Heterosigma akashiwo in bloom formation

We examined the growth and interactions between the bloom-forming flagellates Prorocentrum minimum and Heterosigma akashiwo using bi-algal culture experiments. When both species were inoculated at high cell densities, growth of H. akashiwo was inhibited by P. minimum. In other combinations of inoculation densities, the species first reaching the stationary phase substantially suppressed maximum cell densities of the other species, but the growth inhibition effect of P. minimum was stronger than that of H. akashiwo. We used a mathematical model to simulate growth and interactions of P. minimum and H. akashiwo in bi-algal cultures. The model indicated that P. minimum always out-competed H. akashiwo over time. Additional experiments showed that crude extracts from P. minimum and H. akashiwo cultures did not affect the growth of either species, but both strongly inhibited the growth of the bloom-forming diatom Skeletonema costatum. Further experiments showed that it was unlikely that reactive oxygen species produced by H. akashiwo were responsible for the inhibition of P. minimum growth.

Blue and red light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus.

Photophysiological and pharmacological approaches were used to examine light‐induced germination of resting spores in the red‐tide diatom Leptocylindrus danicus. The equal‐quantum action spectrum for photogermination had peaks at about 440 nm (blue light) and 680 nm (red light), which matched the absorption spectrum of the resting spore chloroplast, as well as photosynthetic action spectra reported for other diatoms. DCMU, an inhibitor of photosynthetic electron flow near photosystem II, completely blocked photogermination. These results suggest that the photosynthetic system is involved in the photoreception process of light‐induced germination. Results of pharmacological studies of the downstream signal transduction pathway suggested that Ca2+ influx is the closest downstream neighbor, followed by steps involving calmodulin, nitric oxide synthase, guanylyl cyclase, protein‐tyrosine‐phosphatase, protein kinase C and actin polymerization and translation.

RNA Sequencing Revealed Numerous Polyketide Synthase Genes in the Harmful Dinoflagellate Karenia mikimotoi

The dinoflagellate Karenia mikimotoi forms blooms in the coastal waters of temperate regions and occasionally causes massive fish and invertebrate mortality. This study aimed to elucidate the toxic effect of K. mikimotoi on marine organisms by using the genomics approach; RNA-sequence libraries were constructed, and data were analyzed to identify toxin-related genes. Next-generation sequencing produced 153,406 transcript contigs from the axenic culture of K. mikimotoi. BLASTX analysis against all assembled contigs revealed that 208 contigs were polyketide synthase (PKS) sequences. Thus, K. mikimotoi was thought to have several genes encoding PKS metabolites and to likely produce toxin-like polyketide molecules. Of all the sequences, approximately 30 encoded eight PKS genes, which were remarkably similar to those of Karenia brevis. Our phylogenetic analyses showed that these genes belonged to a new group of PKS type-I genes. Phylogenetic and active domain analyses showed that the amino acid sequence of four among eight Karenia PKS genes was not similar to any of the reported PKS genes. These PKS genes might possibly be associated with the synthesis of polyketide toxins produced by Karenia species. Further, a homology search revealed 10 contigs that were similar to a toxin gene responsible for the synthesis of saxitoxin (sxtA) in the toxic dinoflagellate Alexandrium fundyense. These contigs encoded A1–A3 domains of sxtA genes. Thus, this study identified some transcripts in K. mikimotoi that might be associated with several putative toxin-related genes. The findings of this study might help understand the mechanism of toxicity of K. mikimotoi and other dinoflagellates.

Light spectrum regulates cell accumulation during daytime in the raphidophyte Chattonella antiqua causing noxious red tides.

Most marine raphidophyte species cause noxious red tides in temperate coastal areas around the world. It is known that swimming abilities enable raphidophytes to accumulation of cells and to actively acquire light at surface layers and nutrients over a wide depth range. However, it remains unclear how the swimming behavior is affected by environmental conditions, especially light condition. In the present study, we observed the accumulation of the harmful red-tide raphidophyte Chattonella antiqua under various light conditions during the daytime in the laboratory. When exposed to ultraviolet-A/blue light (320-480nm) or red light (640-680nm) from above, cells moved downward. In the case of blue light (455nm), cells started to swim downward after 5-15min of irradiation at a photon flux density≥10μmolm(-2)s(-1). When exposed to monochromatic lights (400-680nm) from the side, cells moved away from the blue light source and then descended, but just moved downward under red light. However, mixing of green/orange light (520-630nm) diminished the effects of blue light. When exposed to a mixture of 30μmolm(-2)s(-1) of blue light (440nm) and ≥6μmolm(-2)s(-1) of yellow light (560nm) from above, cells did not move downward. These results indicate that blue light induces negative phototaxis and ultraviolet-A/blue and red lights induce descending, and green/orange light cancels out their effects in C. antiqua.

Adverse effects of strong light and nitrogen deficiency on cell viability, photosynthesis, and motility of the red-tide dinoflagellate Karenia mikimotoi

Abstract: The dinoflagellate Karenia mikimotoi is a red tide–forming alga that causes serious damage to aquaculture in coastal areas around the world. Its eco-physiological characteristics have been investigated extensively but conditions associated with declines of red tides of K. mikimotoi remain unknown. In the present study, we investigated the effects of light and nutrients on the viability, activity of photosystem II (PSII), and motility of K. mikimotoi. Cells were incubated under low (100 μmol photons m−2 s−1 on a 12:12 light:dark cycle of illumination) or high irradiance (1000 μmol photons m−2 s−1) for 4 h in the middle of the photoperiod, in different media. Under low light, most cells survived for about 1 month in media depleted of nitrogen (N) and phosphorus (P), similar to their survival pattern in complete medium. However, when cells that had been pre-incubated in N-depleted medium for 7 d were incubated under high light in an N-depleted medium, cell density and PSII activity declined within a few days. In P-depleted and complete media, there were no significant differences in cell density and PSII activity between low and high light, at least for 10 d. Furthermore, PSII activity was much more sensitive to photoinhibition in N-depleted than in complete medium. The rhythm of diurnal vertical migration of K. mikimotoi was observed in very low light (10 μmol photons m−2 s−1) in different media. The rhythm became obscure in N- and P-depleted media; in fact, most cells stayed in the surface layer all day in N-depleted medium. Moreover, cells accumulating in surface layers in N- and P-depleted media descended very little, even when irradiated with high light. These observations suggest that N-deficiency might trap cells in the surface layer and increase their susceptibility to photoinhibition of PSII, possibly leading to cell death. The combination of N-deficiency and high light thus appears to be an important factor that determines the decline of red tides of K. mikimotoi.