Ichiro Imai

Killing of marine phytoplankton by a gliding bacterium Cytophaga sp., isolated from the coastal sea of Japan

A marine gliding bacterium Cytophaga sp. (strain J18/M01) was isolated from Harima-Nada, eastern Seto Inland Sea, Japan in 1990. This bacterium preys upon various species of marine phytoplankton. All of the five raphidophycean flagellates, all of the four diatoms, and one of the two dinoflagellates examined were killed within a few days when cultured with the bacterium. The bacterium presumably achieves this by direct attack, because the culture filtrate in which host organisms were totally destroyed had no significant effects on the growth of the same host organism (Chattonella antiqua). If one or a few bacterial cells were inoculated into C. antiqua culture, all of the host organisms were killed. The bacterium proliferated in filter-sterilized seawater, suggesting its ubiquitous existence in the coastal sea. The killing of phytoplankton by bacteria such as Cytophaga sp. J18/M01 may be a significant factor influencing the population dynamics of phytoplankton in nature and may contribute to the sudden disappearance of red tides in the coastal sea. Bacterial destruction of phytoplankton may also be a factor that regulates primary productivity in marine ecosystems.

Virus-like particles in Heterosigma akashiwo (Raphidophyceae): a possible red tide disintegration mechanism

Electron microscopical observations on algal samples collected in 1992 in the middle or final stages of a red tide in Hiroshima Bay, Japan, revealed virus-like particles (VLPs) in the red tide alga Heterosigma akashiwo (Class Raphidophyceae). The host cells appeared moribund and the VLPs were located in and around the nuclear area. The VLPs were icosahedral, ca. 185 nm in diameter, and generated from the periphery of several viroplasms. VLPs were also observed in three other types of H. akashiwo-like cells, which were morphologically distinguishable from each other. The appearance of VLPs in the red tide alga could explain the dramatically rapid termination of this red tide.

Relationships between dynamics of red tide-causing raphidophycean flagellates and algicidal micro-organisms in the coastal sea of Japan

Temporal fluctuations of algicidal micro‐organisms against the red tide causing raphidophycean flagellates Chattonella antiqua (Hada) Ono and Heterosigma akashiwo (Hada) Hada ex Hara et Chihara were investigated using the microplate most probable number (MPN) method in northern Hiroshima Bay and Harima‐Nada, the Seto Inland Sea, in 1992 and 1993. In Har‐ima‐Nada, both flagellates appeared at low levels (< 1 cell mL−1), and killer micro‐organisms against the two flagellates (C‐killer for C. antiqua and H‐killer for H. akashiwo) also appeared at low densities (< 2 mL−1). In northern Hiroshima Bay, C. antiqua cells were scarce (< 1 cell mL−1), and C‐killers occurred at a low level (≤ 3.4 mL−1). Conversely, red tides of H. akashiwo occurred there in June of both years. The dynamics of H‐killers revealed a close relationship with that of H. akashiwo populations. H‐killers followed the increase of H. akashiwo cells, reached a maximum level after the beginning of decline of H. akashiwo, maintained a high level for at least 1 week after the crash of bloom, and then decreased. C‐killers consistently remained at low densities during the period of H. akashiwo red tides in both 1992 and 1993. Hence, algicidal micro‐organisms specifically associated with the occurrence and crash of H. akashiwo red tides, and presumably contributed to the rapid termination of the red tides in the coastal seas such as northern Hiroshima Bay.

Life cycle strategies of the red tide causing flagellates Chattonella (raphidophyceae) in the Seto Inland Sea

Abstract Chattonella antiqua (Hada) Ono and Chattonella marina (Subrahmanyan) Hara et Chihara are the most noxious red tide flagellates which cause serious damage to fish farming, especially to yellowtail culture, in Japanese coastal waters such as the Seto Inland Sea during summer. Cysts of Chattonella were identified from sediments of the Seto Inland Sea. These cysts overwinter in sea bottom and play an important role in initiating the summer red tides. Most of the cysts adhere to solid surfaces such as diatom frustules and sand grains, which may aid in keeping the cyst populations within seed beds for the red tides. Temperature is a principal factor affecting the physiology of cysts of Chattonella . No cysts germinate at 10 ° C. Optimum temperature range is between 20 ° and 25 ° C for germination. For maturation (acquisition of germinability) of the cysts, low storage temperature of 11 ° C or below for more than four months is essential, whereas no significant maturation is observed at 20 ° C or more. In freshly collected sediments, marked seasonality of germinability was confirmed in Suo-Nada. The cysts have germinability between spring and early summer. The cysts spend a period of spontaneous dormancy between autumn and the next spring, and they mature during the winter season. The life cycle of Chattonella is therefore well adpted to the seasonal temperature fluctuation in the Seto Inland Sea. And further, alternation between benthic and planktonic stage is presumably unconstrained by virtue of shallowness of the Seto Inland Sea. The life cycle of Chattonella can be regarded as superior strategies in conclusion for the occurrences of the red tides in temperate waters such as the Seto Inland Sea.

Growth and uptake kinetics of a facultatively oligotrophic bacterium at low nutrient concentrations.

In oligotrophic waters, not only community structure but also physiological properties of heterotrophic bacteria are influenced by the concentration of organic matter.The relationship between growth rate of two facultatively oligotrophic strains ofAeromonas sp. No. 6 andFlavobacterium sp. M1 was studied in comparison with that of two eutrophic strains ofEscherichia coli 7020 andFlavobacterium sp. M2. These strains had two or three different substrate constants (Ks values) depending on substrate concentrations: Ks values for the two former were remarkably lower than those for the two latter. For instance, Ks value forAeromonas sp. No. 6 was about 8.9μM when substrate concentration was greater than 53μM and about 1.1μM when substrate concentration was less man 53μM. InE. coli the Ks value was about 260μM at greater than 5600μM and about 47μM at less than 5600μM substrate concentration.Uptake kinetics ofAeromonas sp. grown in a medium containing 2.7 mM glutamate (H-cell) and 0.11μM glutamate (L-cell) have been determined for the intact cells. H-cell had two distinct values of Km for glutamate assimilation and respiration, and L-cell had three distinct values of Km for glutamate assimilation and respiration: In H-cell Km of assimilation was 2.8×10−7 M and 1.5×10−4 M, and Km of respiration was 2.3×10−7 M and 1.7×10−4 M; in L-cell Km of assimilation was 7.4×10−8 M, 8.3×10−6 M, and 1.3×10−4 M, and Km of respiration was 2.5×10−7, 8.9×10−6M, and 1.7×10−4 M. More than 60% of glutamate taken up by the H- and L-cells was respired when the substrate concentration was less than 10−6 M, although at greater than 10−6 M, 50% and 30% of glutamate was respired by H-cells and L-cells, respectively. These results suggest that the facultatively oligotrophic bacteria grow with high efficiency in environments with extremely low nutrient concentration, such as oligotrophic waters of lakes and ocean, as compared with in their growth in conditions of high nutrient concentraton, such as nutrient broth.

Induction of apoptosis in DLD-1 human colon cancer cells by peridinin isolated from the dinoflagellate, Heterocapsa triquetra

Peridinin, which is uniquely present in dinoflagellates, is one of the most abundant carotenoids found in nature. We evaluated the apoptotic effect of peridinin on DLD-1 human colorectal cancer cells. Peridinin significantly reduced the cell viability in a dose-dependent manner (0–20 μM) and induced apoptosis by activating both caspase-8 and caspase-9. Our findings could be important for the high-performance utilization of marine bioproducts.

Proposal of identification criteria for resting spores of Chaetoceros species (Bacillariophyceae) from a temperate coastal sea

Ishii K.-I., Iwataki M., Matsuoka K. and Imai I. 2011. Proposal of identification criteria for resting spores of Chaetoceros species (Bacillariophyceae) from a temperate coastal sea. Phycologia 50: 351–362. DOI: 10.2216/10-36.1 Chaetoceros is a large, ecologically important genus of marine planktonic diatoms. Many species of Chaetoceros form resting spores to survive environmental stress. Species identification based on morphological characteristics of resting spores is difficult. In the present study, we proposed a flow diagram for the identification of Chaetoceros species based on morphology. Resting spores of 18 Chaetoceros species were identified in water and sediment samples from southern Japan and the East China Sea based on morphological characteristics of resting spores and/or the vegetative cells containing them. A single ring of puncta was found to be the most important morphological feature of resting spores for species identification, by which the positions of primary and secondary valves were correctly determined. Other morphological characteristics such as features of the primary valve face, margins of the primary and secondary valve mantles, and secondary valve face are also useful for species identification. A combination of these morphological characteristics was used to construct a flow diagram for identification of the 18 Chaetoceros species found in our samples with light microscopy. This flow diagram is most applicable to Japanese waters, but the approach should be refined and improved to understand the biology and ecology of Chaetoceros species in any marine ecosystem.

Complexation of iron by microbial siderophores and effects of iron chelates on the growth of marine microalgae causing red tides

The growth rates of 13 species of abundant red tide algae in media with different iron species complexed with microbial siderophores (Ferrichrome and Ferrioxamine) and Fe‐Catechol were investigated. Our study demonstrated that the Fe‐chelates (at molar ratios = 1:1) were bioavailable to some red tide species. In Fe‐Catechol medium, growth was observed for the raphidophyte Heterosigma akashiwo, the dinoflagellates Heterocapsa circularisquama and Heterocapsa triquetra, the diatom Ditylum brightwellii, the cryptophyte Rhodomonas ovalis, the chlorophyte Oltmannsiellopsis viridis, and the haptophyte Cricosphaera roscoffensis. In Ferrioxamine medium, we found the growth of the dinoflagellate Karenia mikimotoi, the diatom Ditylum brightwellii, and the cryptophyte Rhodomonas ovalis. But, the existence of higher ligand concentrations (molar ratios ≥ 1:10) decreased the growth rates of most red tide species that were examined. Furthermore, all red tide species examined were not able to grow in Ferrichrome medium. In particular, the Chattonella species examined did not grow in the presence of Fe‐chelates. These results suggest that bioavailability of iron depends not only on ligand species, but also on the concentration of the ligands; moreover, microbial siderophores may play an important role in controlling the uptake of iron complexed with organic materials that exist in coastal water and the formation of red tides in coastal areas.

Esterification of xanthophylls by human intestinal Caco-2 cells

We recently found that peridinin, which is uniquely present in dinoflagellates, reduced cell viability by inducing apoptosis in human colon cancer cells. Peridinin is also found in edible clams and oysters because the major food sources of those shellfish are phytoplanktons such as dinoflagellates. Little is known, however, about the fate of dietary peridinin and its biological activities in mammals. The aim of the present study was to investigate the enzymatic esterification of xanthophylls, especially peridinin which is uniquely present in dinoflagellates, using differentiated cultures of Caco-2 human intestinal cells. We found that peridinin is converted to peridininol and its fatty acid esters in differentiated Caco-2 cells treated with 5mumol/L peridinin solubilized with mixed micelles. The cell homogenate was also able to deacetylate peridinin and to esterify peridininol. Other xanthophylls, such as fucoxanthin, astaxanthin and zeaxanthin, were also esterified, but at relatively lower rates than peridinin. In this study, we found the enzymatic esterification of xanthophylls in mammalian intestinal cells for the first time. Our results suggest that the esterification of xanthophylls in intestinal cells is dependent on their polarity.

Occurrence of the polyamines caldopentamine and homocaldopentamine in axenic cultures of the red tide flagellates Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae)

The polyamines caldopentamine and homocaldopentamine were detected in axenic strains of Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae), respectively, as well as spermidine, the most abundant polyamine in both phytoplankton species. Trace amounts of putrescine, diaminopropane and norspermine were also detected in both species. Spermine was detected only from C. antiqua. These long linear polyamines are characteristic components of thermophilic bacteria. The detection from two species of Raphidophyceae indicates that the occurrence of long linear polyamines is not restricted to thermophilic microorganisms.