Toshiya Katano

Pseudomonas fluorescens HYK0210‐SK09 offers species‐specific biological control of winter algal blooms caused by freshwater diatom Stephanodiscus hantzschii

Aims:  The study of an algicidal activity and mechanism of the isolated Pseudomonas fluorescens HYK0210‐SK09 (SK09) against a winter bloomed harmful diatom, Stephanodiscus hantzschii.

Monitoring of algicidal bacterium, Alteromonas sp. Strain A14 in its application to natural Cochlodinium polykrikoides blooming seawater using fluorescence in situ hybridization

The red tide of dinoflagellate, Cochlodinium polykrikoides has frequently occurred in coastal waters, causing severe damage to fisheries. In the present study, the algicidal bacterium Alteromonas sp. A14 isolated from the southern coast of Korea was applied to a red tide of C. polykrikoides in a laboratory experiment. In the experiment, the abundance of the strain A14 was monitored using fluorescence in situ hybridization. Inoculation of the A14 at a final cell density of 9.0×105 cells/ml caused a significant decrease in C. polykrikoides abundance from 1,830 to 700 cells/ml during 2 days, while abundances of harmless diatoms rapidly increased from 3 days. Abundances of both A14 and other bacteria increased to 1 day. After 1 day, with flagellate abundance increased, bacterial abundance decreased. Finally, algicidal bacterial abundance decreased to 3.5×104 cells/ml. In the biological control of harmful algal blooms, in addition to decrease in target algal abundance and not occurrence of other harmful blooms, decrease in abundance of utilized organism is also important. This study emphasizes the importance of monitoring the inoculated bacterium when applying bacterium to natural seawater.

Fixation of Chattonella antiqua and C. marina (Raphidophyceae) using Hepes-buffered paraformaldehyde and glutaraldehyde for flow cytometry and light microscopy

Katano T., Yoshida M., Lee J., Han M.-S. and Hayami Y. 2009. Fixation of Chattonella antiqua and C. marina (Raphidophyceae) using Hepes-buffered paraformaldehyde and glutaraldehyde for flow cytometry and light microscopy. Phycologia 48: 473–479. DOI: 10.2216/08-102.1. Chattonella antiqua and C. marina are harmful algal species that cause massive fish kills in coastal environments. Generally, Chattonella cells cannot be preserved well by fixation because of their fragile nature. In the present study, we developed a new fixative for Chattonella. Phosphate-buffered saline, which is generally used as a buffer for fixative, caused precipitation after fixation. In contrast, Hepes or sodium cacodylate prevented the precipitation. Moreover, these buffers worked well in the fixation to preserve cell morphology. Since Hepes is not as noxious as cacodylate, we selected Hepes as the buffer for the fixative. Cell counting revealed that the decrease in cell numbers by fixation was negligible and did not start until at least 8 days after fixation. We successfully analysed the DNA amount in fixed cells of Chattonella by flow cytometry. The present study demonstrated that Hepes-buffered paraformaldehyde and glutaraldehyde are superior to other fixatives for flow cytometry and light microscopy.

Comprehensive comparisons of three pennate diatoms, Diatoma tenuae, Fragilaria vaucheriae, and Navicula pelliculosa, isolated from summer Arctic reservoirs (Svalbard 79°N), by fine-scale morphology and nuclear 18S ribosomal DNA

Here we report morphological and molecular characteristics of dominant freshwater diatoms in summer Arctic reservoirs of Svalbard (Norway), using four culture isolates, when we collected the samples in the field on 15 August 2005. Analyses of morphology and BLAST searches with 18S rDNA sequences identified them to Diatoma tenue (HYNP006, HYNP013), Navicula pelliculosa (HYNP021), and Fragilaria vaucheriae (HYNP022), respectively. Comparative studies of morphology revealed that the body shapes of the three polar diatoms were nearly identical to the known morphology of each species; however, they were considerably shorter in body length than previously described identical species from other locations. The 18S rDNA sequences of the diatoms were nearly identical to the same species from temperate and other regions. Phylogenetic analysis showed that the polar diatoms each formed a clade with their identical species and genera according to their taxonomic positions. This suggests that the polar diatoms may possess little or no genetic or morphological variation compared to more temperate strains.

Identification of cultured and uncultured picocyanobacteria from a mesotrophic freshwater lake based on the partial sequences of 16S rDNA

Abstract Eight cultured strains (OK01, OK02, OK03, OK05, OK07, OK08, OK09, and OK10) of picocyanobacteria were isolated from Lake Okutama. Five cyanobacterial DNA fragments (DGGE bands; B4, B5, B6, B7, and B8) were obtained from the lake water samples by denaturing gradient gel electrophoresis (DGGE) after polymerase chain reaction (PCR) amplification of 16S ribosomal genes. To classify the picocyanobacterial strains and the DGGE bands, a partial sequence of 16S rDNA was used. Among seven strains, OK01, OK07, and OK09 were identified as the genus Synechococcus and OK02 and OK05 as the genus Phormidium. OK03 was identified as the genus Oscillatoria and was closely related to B4 (100% homology). B5, B6, B7, and B8 were related to the genus Synechococcus. These results revealed that the picocyanobacteria in the lake are phylogenetically diverse. PCR-DGGE analysis is a useful tool to determine picocyanobacterial community structure in freshwater environments.

Nutrient limitation of the primary production of phytoplankton in Lake Baikal

Nutrient limitation of the primary production of phytoplankton at some stations in southern and central Lake Baikal was studied by nutrient enrichment experiments in August 2002. Chlorophyll (Chl.) a concentrations ranged from 0.7 to 5.8μgl−1. Inorganic nutrient concentrations were low: soluble reactive phosphorus ranged from 0.05 to 0.20μmoll−1, ammonia from 0.21 to 0.41μmoll−1, and nitrite plus nitrate from 0.33 to 0.37μmoll−1. In the five enrichment experiments, phosphate spikes and phosphate plus nitrate spikes always stimulated primary production. Nitrate spikes also stimulated primary production in four of the experiments. Significant differences were detected between the controls and phosphate spikes and between the controls and phosphate plus nitrate spikes. Thus, the first limiting nutrient is thought to be phosphorus, but once phosphorus is supplied to the surface water, the limiting nutrient will quickly shift from phosphorus to nitrogen.

Dense winter bloom of the dinoflagellate Heterocapsa triquetra below the thick surface ice of brackish Lake Shihwa, Korea

We investigated the seasonal abundance of the dinoflagellate Heterocapsa triquetra (Ehrenberg) F. Stein, as well as the relevant in situ environmental factors, in brackish Lake Shihwa, Korea. We also examined the growth rates and morphological characteristics of the species in laboratory cultures. In the field, the population densities of H. triquetra remained at low levels from late spring to early summer, and then completely disappeared from August to November 2007. Interestingly, a dense bloom of H. triquetra appeared below the ice surface on 17 January 2008; identities of the cells were confirmed by rDNA sequence comparisons. The second peak reached a density of 672 ×  103 cells L−1 on 28 March 2008, at a water temperature of 9.1°C. Laboratory experiments showed that growth rates of H. triquetra increased with incremental temperature increases within the range of 10 and 20°C. The highest growth rate reached by H. triquetra was 0.62 d−1 at 20°C with a salinity of 30. Above 25°C, the dinoflagellate was unable to grow between salinities of 10 and 15, and reached only relatively low growth rates (<0.12 d−1) under other salinity conditions. However, under continuous cultures at 5°C and 8°C, H. triquetra cells retained its growth capability for more than 12 days, implying that H. triquetra can survive and grows even at very low temperatures. The equivalent spherical diameter (ESD) of H. triquetra did not change markedly between 10 and 25°C, but the equivalent spherical diameter was significantly different at 5°C. The cell volume buildup of H. triquetra at low temperatures is one of the important survival strategies to overcome the harsh environmental conditions. These characteristics make H. triquetra a consistently dominant dinoflagellate in Lake Shihwa during the cold winter season.

Morphological and Molecular Phylogenetic Position of Prorocentrum micans sensu stricto and Description of Prorocentrum koreanum sp nov from Southern Coastal Waters in Korea and Japan

Prorocentrum micans is an extremely variable dinoflagellate species, with many different local forms reported worldwide. Because of this morphological diversity, it is important to establish whether these various forms belong to P. micans sensu stricto. For this study, P. micans-like specimens were isolated from several localities in the southern coastal waters of Korea and Japan. The morphological characteristics and the molecular signatures of P. micans were re-examined. Moreover, a new Prorocentrum species, Prorocentrum koreanum sp. nov. was established through detailed light microscopy and scanning electron microscopy observations. Examination of the periflagellar platelets revealed that P. koreanum sp. nov. differs from P. micans. Furthermore, P. koreanum and P. micans exhibited different distribution patterns of trichocyst pores. Through molecular phylogeny analysis of small subunit (SSU) rRNA, internal transcribed spacer (ITS), and large subunit (LSU) rRNA sequence, we found P. koreanum to be more closely related to P. mexicanum and P. rhathymum than to P. micans. Additionally, ITS2 compensatory base changes also provide strong evidence to support P. koreanum and P. micans being separate species.

The dynamics of microbial and herbivorous food webs in a coastal sea with special reference to intermittent nutrient supply from bottom intrusion

Seasonal changes in abundance of planktonic microorganisms, together with some physico-chemical variables, were monitored monthly from May 1999 to March 2002 in the surface water of a coastal bay where nutrients are mainly supplied by intermittent intrusions of deeper water (bottom intrusion). No significant bottom intrusion was detected in 1999 but large or frequent bottom intrusions were found from June to October in 2000, and again from mid-June only to late July in 2001. These results indicate that there is a different nutrient supply every year, and peaks in the abundance of dominant eukaryotic phytoplankton (diatoms and dinoflagellates) roughly corresponded to the occurrences of bottom intrusions. By contrast, there was a cyclic seasonal pattern of autotrophic picoplankton (APP) cell density, which reached maxima in August of every year at very similar levels (4.0-5.0 × 105 cells ml–1). Thus, the seasonal abundance of APP was apparently independent of the occurrence of bottom intrusions. Seasonal changes in cell densities of heterotrophic bacteria showed similar trends to the APP, and temperature-dependent growth of both was indicated. The present study suggests that the matter cycling in the bay varies as a result of shifts in the dominant food linkages, from a microbial food web to a herbivorous food web, due to intermittent nutrient supplies from bottom intrusions.

Effect of nutrient concentration and salinity on diel vertical migration of Chattonella marina (Raphidophyceae)

Abstract Vertical migration plays an important role in the development of Chattonella populations in the natural environment; however, this behaviour has remained largely undescribed. In the present study, the diel vertical migration of Chattonella marina in the Ariake Sea was examined along with nutrient concentrations and salinity at four sampling sites during the bloom in the summers of 2008, 2009 and 2010. Cells of Chattonella were abundant at the surface (0 m depth) in the daytime in most cases. However, when surface salinity was 9, which is the lower limit for growth, the population accumulated at a depth of 2 m, where salinity was 15, suggesting that Chattonella moves to avoid water with low salinity. Chattonella cells actively migrated, even under nutrient-replete conditions (mean DIN concentration, 12.73 µM). The present study demonstrates that Chattonella cell migration is independent of nutrient availability. In addition, Chattonella cells stop upward migration when the surface salinity is low (< 15). These insights are important for making predictions of Chattonella population dynamics after heavy rains.