Annual life cycle of Chattonella spp., causative flagellates of noxious red tides in the Inland Sea of Japan

Effects of storage temperature on the dormancy and maturation of dormant cells of Chattonella spp. were investigated by the extinction dilution method using sediment samples collected in 1984 after the blooming in Harima-Nada. Seasonality of germinability in fresh sediments was also studied in 1985. A storage period of more than 4 months with a low temperature (11°C) was essential for the maturation to induce the germination capacity of dormant cells. The optimal temperature for germination (22°C) had no effects on the maturation. Using fresh Suo-Nada sediments, a marked seasonality of germinability was confirmed. It was weak from autumn to early winter, then strengthened gradually up to a high level, which was maintained between spring and early summer, and again decreased rapidly during summer. The annual life cycle of Chattonella spp., including vetetative and dormant phases, was summarized as follows: (1) vegetative cells in early summer originate from germination of dormant cells in sediments; (2) they form overwintering dormant cells during the summer season; (3) dormant cells spend a period of spontaneous dormancy until next spring; (4) the duration of post dormancy, an enforced one due to low temperatures, follows until early summer. The life cycle of Chattonella spp. is therefore well adapted to the temperature regime in temperate seas such as the Seto Inland Sea of Japan.     

Cyst formation of the noxious red tide flagellate Chattonella marina (Raphidophyceae) in culture

Cyst formation of the noxious red tide flagellate Chattonella marina (Raphidophyceae), cultured from a cyst in sediments of Suo-nada, Japan in 1986, was investigated by culture experiments. Nitrogen limitation was effective in inducing cyst formation. After incubation in N-limited medium at 25°C with 10000 lux on a 14 h light, 10 h dark photo-cycle, cultures were subjected to six different light intensities (0 10000 lux) in order to determine effects of light intensity on cyst formation. Cysts were formed under light intensities of 1000 lux or below, but not at 10000 lux. Cysts formed in culture displayed morphological characteristics quite similar to those natural cysts observed in sediments collected from the Inland Sea of Japan. Observations on nucleus, using diaminidinophenylindole (DAPI)-staining and epifluorescence microscopy, indicated that the majority of cysts formed in culture were uninucleate, a few were binucleate. Germination of cysts was observed under adequate conditions for germination (22°C, 3500 lux with a 14 h light, 10 h dark photo-cycle) even after storage at 11°C in the dark for more than 4 mo.     

Importance of cysts in the population dynamics of the red tide flagellate Heterosigma akashiwo (Raphidophyceae)

To elucidate roles of cysts in occurrences of Heterosigma akashiwo blooms, cyst dynamics were studied in northern Hiroshima Bay, the Seto Inland Sea of Japan, where H. akashiwo regularly forms red tide in June. Monthly measurements of seasonal changes in the densities of vegetative cells of H. akashiwo and their germinable cysts in surface sediments (top 1-cm layer) were made for 2 years at three stations. Vegetative cells of H. akashiwo could be detected from April through December throughout the water column, and the existence of vegetative cells was confirmed in surface waters even in winter after incubation of sampled seawater in culture medium. Germinable cysts, enumerated by the extinction dilution method, existed in sediments in all seasons, even before and after the seasonal bloom. The effects of incubation temperature on the germination of natural cysts of H. akashiwo in sediments were examined. Germination was not observed at 5 °C, was low at 10 °C, while it increased at 15 °C, and maintained a high level to 25 °C. The bottom water temperature reached 15 °C (suitable for the germination of cysts) and the surface about 18 °C or more (suitable for the growth of vegetative cells) 2 to 3 weeks before the blooms. The dark survival of H. akashiwo cysts was tested, and it was found that the cysts were viable for at least 650 d at 11 °C, and for 165 d at 25 °C, indicating a significant role of cysts in the survival during winter and summer seasons. The cysts presumably also play an important role in seeding primary populations into water columns when the bottom water reaches a suitable temperature (around 15 °C); thereafter the populations develop with great annual regularity to bloom in June. These results suggest that initiation of H. akashiwo red tides in the Seto Inland Sea could be triggered by bottom water temperature. Received: 3 July 1998 / Accepted: 12 January 1999     

Synchronous recruitment and growth pattern of planktonic larvae of the amphioxus Branchiostoma belcheri in the Seto Inland Sea, Japan

Planktonic larvae of the amphioxus Branchiostoma belcheri were collected in the western part of the Seto Inland Sea, Japan, in order to document their seasonal occurrence, recruitment, and growth patterns. The larvae appeared from mid-July to early October and their size ranged from 0.6 to 5.5 mm. Three distinct cohorts were observed during the summers, indicating multiple, synchronous spawning within the population. The length increment pattern of a cohort demonstrates slow growth for at least a week during the early larval stage. July 10, 20, and 30 are the inferred dates when spawnings started to produce the three cohorts in 2001. Favorable temperature range for spawning was 21–23°C. Bias in population structure due to advection can be assumed to be negligible; therefore the length increment of the cohort, about 0.05 mm day⁻¹, is regarded as the larval growth rate after the initial slow-growth stage. The planktonic stage of the first cohort in 2001 is estimated to have lasted around 55 days. The relative proportions of the second and third cohorts in samples from surface and mid-depth waters suggest downward migration of the planktonic larvae, as they age.     

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.     

Interlinked temporal changes in environmental conditions, chemical characteristics of sediments and macrofaunal assemblages in an estuarine intertidal sandflat (Seto Inland Sea, Japan)

Five field surveys were conducted in an estuarine intertidal sandflat of the Seto Inland Sea (Japan) between April 1994 and April 1995. Chlorophyll a, pheopigments, total organic carbon and acid-volatile sulphides (AVS) of surface and subsurface sediments, and macrofaunal assemblages were investigated in parallel at 15 stations. Monthly hydrological data of low-tide creek water adjacent to the flat were used as a complementary environmental characterisation of the study area. Strong temporal changes were found among sampling dates, most remarkably in autumn with a major increase of algal detritus and AVS, a sharp reduction in macrofaunal abundances and species richness, and a massive mortality of the clam Ruditapes philippinarum. This dystrophic event was preceded by a photoautotrophic and hypertrophic spring–summer characterized by abundant fresh (i.e., living) algal material, including microphytobenthos and macroalgae (Ulva sp.). In summer, abundant macrofaunal assemblages reached the highest biomass values (455 g wet weight m⁻² or 60.6 g ash free dry weight m⁻²), with a major contribution of filter-feeding bivalves Musculista senhousia and R. philippinarum. These are among the highest values reported in the literature for sedimentary shores. From autumn, there was a progressive recolonisation of macrofauna, initiated by few opportunistic polychaetes (e.g., Cirriformia tentaculata and Polydora sp.), apparently promoting a fast sediment recovery in winter, and followed by new bivalve recruits in the next spring. This study provides the first evidence of significant and interlinked within-year changes in chemical characteristics of sediments and macrofaunal assemblages in an estuarine intertidal flat at a small spatial scale (i.e., tens of meters). This demonstrates the high temporal variability of species–environment relations in these systems and a close relationship in seasonally driven trophodynamic processes among primary producers and benthic consumers. We conclude that a thorough parallel evaluation of the temporal changes in chemical characteristics of sediments should be taken into account in assessing the year-round distribution and changes of intertidal macrofauna, particularly in eutrophic, estuarine intertidal flats.An erratum to this article can be found at     

“Seed bank” of coastal planktonic diatoms in bottom sediments of Hiroshima Bay, Seto Inland Sea, Japan

Abundance and temporal distribution of viable (able to germinate) resting stage cells of planktonic diatoms in bottom sediments have been investigated almost monthly during 1989 to 1992 in Hiroshima Bay, western part of Seto Inland Sea, Japan. The abundance of viable resting stages in bottom sediments was enumerated with the extinction dilution method (most probable number method, MPN). In bottom sediments of Hiroshima Bay, dominantly distributed species and/ or genera of the diatom resting stages were Skeletonema costatum, Chaetoceros spp. and Thalassiosira spp. Viable resting stages of these diatoms were densely distributed on the orders of 10³ to 10⁶ (MPN g⁻¹ wet sediments), and persisted in bottom sediments throughout the investigation period. Conversely, vegetative cells of these diatoms fluctuated remarkably in the water column and disappeared sporadically. Survival of the resting stages in a collected sediment sample was also determined with the MPN method, at different storage temperatures (5, 10, 15, 20, 25 °C). The survival test demonstrated that the diatom resting stages could survive in the dark for several months or years in sediments. Resting stages survived longer at the lower storage temperature, and the order of longevity was consistent within three diatoms (Chaetoceros spp. > Thalassiosira spp. > S. costatum) at each storage temperature. The present study suggests that these diatom resting stages in the coastal bottom sediments could serve as a “seed bank”, analogous to those of terrestrial plants. The seed bank would ensure the survival of diatoms within highly fluctuating coastal environments, while it would also be the source of sporadic and autochthonous diatom blooms in coastal waters. Received: 29 November 1996 / Accepted: 16 December 1996     

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.     

Occurrence of tetrodotoxin in the starfishes Astropecten polyacanthus and A. scoparius in the Seto Inland Sea

Two starfishes, togemomijigai Astropecten polyacanthus and momijigai A. scoparius were collected from the Seto Inland Sea in October 1983 through November 1984, and assayed for toxicity by the standard method for tetrodotoxin (TTX). Most of the 54 A. polyacanthus specimens assayed were toxic, with the highest toxicity score being 520 mouse units (MU) g^-1. All seven A. scoparius were toxic, with the highest score being 46 MU g^-1. The toxin from A. polyacanthus was purified by a method which consisted mainly of activated charcoal treatment, and chromatography on CM-Sephadex C-25 and Bio-Rex 70 columns. The purified starfish toxin showed a specific toxicity of 4 700 MU mg^-1, a value which was almost comparable to that of authentic TTX. From the thin-layer chromatographic and electrophoretic behavior, along with ¹H-NMR spectrum, A. polyacanthus toxin was identified as TTX.     

The impact of grazing by microzooplankton in northern Hiroshima Bay,the Seto Inland Seam,Japan

The abundance of microzooplankton and their grazing impact on phytoplankton were studied using the dilution technique from May 1990 to November 1991 in northern Hiroshima Bay, a typical eutrophic area in the Seto Inland Sea. Microzooplankton, dominated in number by tintinnid ciliates, were abundant from June to September when chlorophyll-a concentrations were high. Maximum density of microzooplankton ranged from 3.8×10³ to 25.4×10³ ind l^-1. During the period of investigation, mean microzooplankton density and mean chlorophyll-a concentration of the <20-m fraction increased toward the inner region of the bay. The microzooplankton grazing on phytoplankton increased from summer to early autumn, and decreased from late autumn to winter. At an offshore station, the annual means of the daily grazing loss for total chlorophyll-a and the chlorophyll-a of the <20-m fraction were 12 and 15% of the initial standing stock, respectively. At an estuarine station, the microzooplankton grazed 19 and 29% of the total and <20-m initial standing stock, respectively. The quantity of grazed chlorophyll-a correlated positively and linearly with the potential production of chlorophyll-a at both stations. The quantity of chlorophyll-a grazed by microzooplankton and the potential production of chlorophyll-a were nearly equivalent in the <20-m fraction at the estuarine station. This suggests that the microzooplankton assemblage was able to consume almost all the nanoplankton newly produced in the eutrophic estuary.     

Population dynamics and production of the planktonic copepods in a eutrophic inlet of the Inland Sea of Japan. III.Paracalanus sp.

Population dynamics and production of the calanoid copepodParacalanus sp. were studied from November 1986 to November 1987 in Fukuyama Harbor, a eutrophic inlet of the Inland Sea of Japan. This species was perennial, with a large abundance peak in June/July and small peaks in September/October and November/December. During a year of investigation, 15 generations Gould be detected. For each generation, the mean population egg production rate and the mean daily midstage abundance front NIII to CV were determined to obtain a survival curve from egg to CV. The mortality was extremely high during the early life stages: on average only 7.1% of the eggs produced might survive into NIII. This high mortality might be caused by predation by sympatric omnivorous copepods, in addition to sinking loss of eggs from the waten column. The biomass ofParacalanus sp. showed marked seasonal variations largely in parallel with numerical abundance. The instantaneous growth rate of each developmental stage increased exponentially with temperature up to 20 °C, above which the rate was constant. The annual integrated production rate was 734 mg C m^–3 yr^–1 or 5.5 g C m^–2 yr^–1.     

Distribution and abundance of finless porpoise in the Inland Sea of Japan

Aerial sighting surveys were conducted in 2000 to evaluate the distribution and abundance of finless porpoise Neophocaena phocaenoides in the Inland Sea of Japan. We flew 60 north–south transects (2,218 km in total) at intervals of 6.43 km over the study area between 131°3′ and 134°59′ E. In total, 148 groups were detected by two observers. The average group size was 1.56 individuals. The effective strip half-width of each observer was estimated to be 107 m (coefficient of variation [CV] = 8.26%). Porpoise abundance was estimated at 7,572 individuals and the density was as low as 0.506 individuals/km² (CV = 17.3%). The sea is among the Japanese waters with the lowest density in spite of its favorable topographical conditions. In the western stratum of the study area, where the estimated density was 1.31 individuals/km², we observed a relatively regular distribution. In the central-eastern stratum, which had an estimated density of 0.208 individuals/km², we confirmed a clumped distribution that was restricted to inshore waters or near islands. No individuals were observed in waters between 132°51′ and 133°11′E, and between 133°43′ and 133°59′E, where sand dredging and other human activities have been active, suggesting that habitat fragmentation has occurred.     

Ultrastructural alterations on chloride cells of the yellowtail Seriola quinqueradiata,following exposure to the red tide species Chattonella antiqua

By means of scanning and transmission electron microscopy, the influence of the red tide species Chattonella antiqua was examined with respect to the surface ultrastructures of chloride cells of the yellowtail Seriola quinqueradiata. Conspicuous ultrastructural alterations occurred on the apical surface of these cells. The majority of chloride cells in the control gills showed an apical surface with numerous cellular extensions, while more than half the chloride cells affected by red tide organisms exhibited an apical surface with fewer and smaller extensions, a wrinkled apical surface, or a protruded apical surface. These ultrastructural alterations of chloride cell surface may be due to the partial disturbance of salinity by C. antiqua, and reflect the changes of the ion-transport function in yellowtail gills exposed to red tide water.     

Feeding habits of marine cladocerans in the Inland Sea of Japan

Natural food items of five species of marine cladocerans, Evadne nordmanni, E. tergestina, Penilia avirostris, Podon leuckarti and P. polyphemoides, were investigated in the Inland Sea of Japan between April 1986 and May 1987. Gut content examination with SEM (scanning electron microscopy) revealed that feeding was largely limited to centric diatoms and a few exceptions of pennate diatoms and dinoflagellates. No animal remains were detected, and some unidentified materials were also found. Phytoplankton smaller than 35 m in size (cell diameter in centric diatoms and longest dimension in others) was found most frequently in the gut of cladocerans. The role of grazing of marine cladocerans in trophodynamic pathways of the pelagic realm is discussed.     

Possible involvement of the glycocalyx in the ichthyotoxicity of Chattonella marina (Raphidophyceae): immunological approach using antiserum against cell surface structures of the flagellate

Although the ichthyotoxic mechanism of Chattonella marina is still unknown, several lines of evidence suggest that the reactive oxygen species (ROS), such as superoxide anion (O₂^-), hydrogen peroxide (H₂O₂)_, and hydroxyl radical (·OH), produced by C. marina are involved in the mortality of fish exposed to this flagellate. Recently, we found that the cell-free supernatant prepared from C. marina, which is considered to contain the glycocalyx, showed NADPH-dependent O₂^- generation. In this study, we prepared antiserum against the crude glycocalyx of C. marina. Using indirect immunofluorescence, it was confirmed that the antiserum specifically reacted with C. marina cells. In addition to C. marina, the antiserum also reacted with other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica, whereas no reactivity was observed against six other flagellate species tested. These results suggest that raphidophycean flagellates have common epitopes recognized by the antiserum. Interestingly, immunohistochemical analysis of paraformaldehyde-fixed gill lamellae from yellowtail exposed to C. marina revealed that the antiserum stained the surface of gill lamellae, while no such staining pattern was observed in control gill lamellae. These results suggest that the glycocalyx may be discharged when C. marina cells are inhaled into the fishes' mouths and then come into contact with the gill surface. Based on the present results, together with our previous findings, we propose that continuous accumulation of the discharged glycocalyx on the gill surface occurs during C. marina exposure, which may be responsible for the ROS-mediated severe gill tissue damage leading to fish death.     

Ontogenetic diel vertical migration of the planktonic copepodCalanus sinicus in the Inland Sea of Japan

The ontogenetic diel vertical migration of the planktonic copepodCalanus sinicus was investigated in the Inland Sea of Japan in summer 1988, when the water was thermally stratified with a thermocline of ca 5 °C between 35 and 45 m. Stage-specific differences in the diel vertical migration behavior ofC. sinicus were found. Eggs were spawned primarily within the surface-waters between midnight and dawn by ascending females, and sank gradually to deeper waters until they hatched into nauplii. Non-feeding nauplius stages (NI and II) were distributed throughout the water column, but the first feeding stage (NIII) performed an ontogenetic upward migration. NIV to VI and copepodite (C) stages I to III continuously aggregated in the phytoplankton-rich euphotic layer. However, the depth of the median CI to III populations descended as stage progressed. The onset of prominent diel vertical migration took place in CIV, and the amplitude of vertical migration increased with age, being maximal in adult females (CVI). Adult males (CVI), however, remained in the layer below 20 m, and did not migrate dielly. The ecological significance of ontogenetic diel vertical migration is discussed.     

Distribution of the Lamellibrachia spp. (Siboglinidae,Annelida) and their trophosome endosymbiont phylotypes in the Mediterranean Sea

During the 2010/2011 Exploration vessel Nautilus expedition to the Mediterranean Sea, samples of Lamellibrachia (Siboglinidae, Annelida) were imaged in situ and collected from hydrothermal vent and methane “cold seeps.” An analysis of these Lamellibrachia and their endosymbiotic thioautotrophic gammaproteobacteria reveals two distinct endosymbiont phylotypes. Phylotype 1 was present in Lamellibrachia specimens from 947 m at the Eratosthenes seamount seep (a seep off Cyprus in the Eastern Mediterranean), and Phylotype 2 was found in siboglinids from 618 m at a hydrothermal vent within Palinuro volcanic complex in Tyrrhenian Sea. Both phylotypes coexist in siboglinids at 1,036 m from the Palmachim disturbance, a cold seep in the Eastern Mediterranean’s Levantine basin. Our results, combined with existing knowledge of siboglinid host and endosymbiotic bacteria biogeography, reveal that two major groups of endosymbionts coexist within lamellibranchids and escarpids. The phylogenetic clustering of these bacteria is primarily influenced by geographic location, rather than selection by the siboglinid host.     

Ontogenetic diel vertical migration of the planktonic copepod Calanus sinicus in the Inland Sea of Japan

The ontogenetic diel vertical migration of the planktonic copepod Calanus sinicus was investigated in the Inland Sea of Japan in June 1989, when the water column was thermally weakly stratified. Because of fewer eggs and less variation in their abundance, nocturnal spawning was not apparent. A pronounced upward migration occurred in NIII. NIII to CIII resided in the upper 20 m layer throughout the day, and from CIV on their median depths descended. CV and adult females underwent significant diel vertical migration, whereas adult males did not migrate. By integrating the results from the present study and those from our previous investigations (in August–September 1988, November 1988 and March 1989), we review seasonal variation in the ontogenetic diel vertical migration of C. sinicus. Spawning was largely nocturnal, reaching its maximum level around dawn, but spawning depth and fecundity changed seasonally. The distribution of pre-feeding stages, NI and NII, was similar to that of eggs. A pronounced upward migration always occurred in the first feeding stage, NIII, and late nauplii and early copepodites always resided in the food-rich upper layer, indicating that upward migration by NIII is feeding migration. As the stages progressed, they extended their vertical distribution range, and CV and adult females usually underwent diel vertical migration. However, the pattern and strength of this migration differed seasonally. Their day depths increased with the increase of relative biomass of planktivorous fish, indicating that predator avoidance induces their diurnal downward migration. High chlorophyll a concentrations in the upper layer (<15 m deep) relative to the lower layer (>20 m deep) amplified their diel vertical migrations. Diel vertical migration of C. sinicus is a phenotypic behavior.