Shigeru Itakura

Isolation and Characterization of a Novel Single-Stranded RNA Virus Infecting the Bloom-Forming Diatom Rhizosolenia setigera

ABSTRACT A novel single-stranded RNA (ssRNA) virus specifically infecting the bloom-forming diatom Rhizosolenia setigera (R. setigera RNA virus [RsRNAV]) was isolated from Ariake Sea, Japan. Viral replication occurred within the cytoplasm, and the virus particle was icosahedral, lacked a tail, and was 32 nm in diameter on average. The major nucleic acid extracted from the RsRNAV particles was an ssRNA molecule 11.2 kb in length, although smaller RNA molecules (0.6, 1.2, and 1.5 kb) were occasionally observed. The major structural proteins of RsRNAV were 41.5, 41.0, and 29.5 kDa. Inter- and intraspecies host specificity tests revealed that RsRNAV is not only species specific but also strain specific and that its intraspecies host specificity is diverse among virus clones. The latent period of RsRNAV was 2 days, and the burst sizes were 3,100 and 1,010 viruses per host cell when viruses were inoculated into the host culture at the exponential and stationary growth phases, respectively, at 15°C under a 12-h-12-h light-dark cycle of ca. 110 μmol of photons m−2 s−1 with cool white fluorescent illumination. To our knowledge, this is the first report describing the biological properties of a virus infecting a diatom. Further studies on RsRNAV will be helpful in understanding the ecological relationship between diatoms and viruses in nature.

MICROSATELLITE MARKERS REVEAL POPULATION GENETIC STRUCTURE OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN JAPANESE COASTAL WATERS1

This is the first report to explore the fine‐scale diversity, population genetic structure, and biogeography of a typical planktonic microbe in Japanese and Korean coastal waters and also to try to detect the impact of natural and human‐assisted dispersals on the genetic structure and gene flow in a toxic dinoflagellate species. Here we present the genetic analysis of Alexandrium tamarense (Lebour) Balech populations from 10 sites along the Japanese and Korean coasts. We used nine microsatellite loci, which varied widely in number of alleles and gene diversity across populations. The analysis revealed that Neis genetic distance correlated significantly with geographic distance in pair‐wise comparisons, and that there was genetic differentiation in about half of 45 pair‐wise populations. These results clearly indicate genetic isolation among populations according to geographic distance and restricted gene flow via natural dispersal through tidal currents among the populations. On the other hand, high P‐values in Fishers combined test were detected in five pair‐wise populations, suggesting similar genetic structure and a close genetic relationship between the populations. These findings suggest that the genetic structure of Japanese A. tamarense populations has been disturbed, possibly by human‐assisted dispersal, which has resulted in gene flow between geographically separated populations.

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.

Development of a quantification assay for the cysts of the toxic dinoflagellate Alexandrium tamarense using real-time polymerase chain reaction

The cysts of toxic dinoflagellate Alexandrium tamarense are the seed population for the bloom responsible for paralytic shellfish poisoning (PSP). However, it is impossible to identify the Alexandrium spp. cyst on the basis of morphological features. In this study, we prepared A. tamarense cysts by sexual conjugation in laboratory conditions and developed an efficient DNA extraction method for polymerase chain reaction (PCR) assay. Using the A. tamarense cysts, we established the identification and quantification method showing the species specificity and the high sensistivity for A. tamarense cysts using real-time PCR. This assay was also able to detect and quantify the A. tamarense cysts accurately when mixed with excess cysts of A. catenella (Whedon and Kofoid) Balech prepared by conjugation experiment.

Germination characteristics of naturally occurring cysts of Alexandrium tamarense (Dinophyceae) in Hiroshima Bay, Inland Sea of Japan

Abstract In order to examine temporal changes in the germination ability, time to germination and autofluorescence properties of the resting cysts of the toxic dinoflagellate Alexandrium tamarense, a long-term investigation was conducted in Hiroshima Bay. In Hiroshima Bay, a spring bloom (March to May) of A. tamarense has been observed almost every year since 1992. Approximately 50 resting cysts were isolated monthly from the bottom sediment between June 1994 and June 1997. The cysts were incubated from the day of sampling onwards under in situ bottom water temperature conditions, and germination success and the emission of autofluorescence were checked every day. High germination success rates (>50%) were observed between December and April each year (bottom water temperature = 10.0–16.5°C), with an average germination time of 10.2 days (n = 455). Resting cysts were found to start to emit red autofluorescence a few days before germination (average duration = 3.1 days, n = 449), and germination time was nearly constant within the temperature range 10–20°C. From June to November, germination success rates were considerably lower (0–40%, bottom water temperature = 14.6–23.9°C). No germination at all was observed in September (bottom temperature = 23.6–23.9°C). The relationship between the incubation temperature and the rate of germination success indicates that the resting cysts have a temperature ‘window’ (c. 10–15°C) for germination, which controls the seasonal change in germination ability. The present results indicate that the germination characteristics of A. tamarense resting cysts are well adapted to the ambient water temperature rhythm in temperate shallow coastal environments, allowing A. tamarense to seed vegetative cell populations for the spring bloom.

Nutrition and growth kinetics in nitrogen- or phosphorus-limited cultures of the ‘novel red tide’ dinoflagellate Heterocapsa circularisquama (Dinophyceae)

Abstract Heterocapsa circularisquama is a dinoflagellate that has only recently been recorded as producing red tides; it causes severe damage to Japanese shellfish aquaculture by killing bivalves. In order to elucidate the mechanism of red tide outbreaks, the nutrition and growth kinetics of H. circularisquama were examined in nitrogen (N)– or phosphorus (P)–limited semicontinuous culture. Inorganic N compounds, such as nitrate, nitrite, and ammonium, were found to be good nitrogen sources for the growth, whereas organic nitrogen (urea and uric acid) was not utilized. Heterocapsa circuiarisquama could successfully use a wide variety of inorganic and organic phosphorus compounds of different molecular structures as the sole P source. Under N-limited steady-state conditions, the dilution rate (= growth rate) as a function of cell nitrogen quota followed the Droop equation. Under P-limited steady state culture too, the dilution rate as a function of cell P quota followed the Droop equation. Kinetic parameters Dm, (the maximum dilution rate) and Kq (minimum cell quota) were 0.9 d−1 and 1.1 pmol N cell−1 for N-limited cultures and 1.1 d−1 and 89.4 fmol P cell−1 for P-limited cultures. The nutrient availability and kinetic parameters of H. circularisquama are compared with other red-tide organisms and the ecological implications of these characteristics are discussed.

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.

Population Structure of Alexandrium (Dinophyceae) Cyst Formation-Promoting Bacteria in Hiroshima Bay, Japan

ABSTRACT A total of 31 bacterial isolates that have potential Alexandrium cyst formation-promoting activity (Alex-CFPB) were isolated from Hiroshima Bay (Japan), which is characterized by seasonal blooms of the toxic dinoflagellate Alexandrium tamarense. The population structure of Alex-CFPB was analyzed by means of restriction fragment length polymorphism analysis of the 16S rRNA genes (16S rDNA). Fourteen ribotypes, A to N, were observed among the 31 isolates of Alex-CFPB by using four restriction enzymes, MboI, HhaI, RsaI and BstUI. Among them, seven isolates, which were obtained from the seawater samples taken during the peak and termination periods of the A. tamarense bloom in 1998, belonged to ribotype A. This result suggests that bacterial strains of ribotype A may be dominant in the Alex-CFPB assemblages during these periods. The partial 16S rDNA-based phylogenetic tree of 10 ribotypes studied showed that nine of them fell into the Rhodobacter group of the α subclass of the Proteobacteria. Eight of nine ribotypes of the Rhodobacter group fell into the lineage of the Roseobacter subgroup, and one fell into the Rhodobacter subgroup. The non-Rhodobacter group type fell into the Marinobacterium-Neptunomonas-Pseudomonas group of the γ-Proteobacteria. Isolates of Alex-CFPB ribotypes A and C do not have clear growth-promoting activities but have strong cyst formation-promoting activities (CFPAs) under our laboratory conditions. These results show that the Alex-CFPB assemblage may consist of various bacteria that belong mainly to the Roseobacter group and have strong CFPAs. These results suggest that not only the Alexandrium cyst formation-inhibiting bacteria (Alex-CFIB) reported previously but also Alex-CFPB, especially bacteria of ribotype A, may play significant roles in the process of encystment and bloom dynamics of Alexandrium in the natural environment.

Study of DNA extraction methods for use in loop-mediated isothermal amplification detection of single resting cysts in the toxic dinoflagellates Alexandrium tamarense and A. catenella

In a previous study, we experienced instable amplification and a low amplification success in loop-mediated isothermal amplification (LAMP) reactions from naturally occurring vegetative cells or resting cysts of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella. In this study, we examined 4 methods for extracting DNA from single resting cysts of A. tamarense and A. catenella to obtain more stable and better amplification success and to facilitate unambiguous detection using the LAMP method. Apart from comparing the 4 different DNA extraction methods, namely, (1) boiling in Tris-EDTA (TE) buffer, (2) heating at 65 °C in hexadecyltrimethylammonium bromide buffer, (3) boiling in 0.5% Chelex buffer, and (4) boiling in 5% Chelex buffer, we also examined the need for homogenization to crush the resting cysts before DNA extraction in each method. Homogenization of resting cysts was found to be essential for DNA extraction in all 4 methods. The detection time was significantly shorter in 5% Chelex buffer than in the other buffers and the amplification success was 100% (65/65), indicating the importance of DNA extraction and the effectiveness of 5% Chelex buffer in the Alexandrium LAMP.