Specific toxic effect of dinoflagellate Heterocapsa circularisquama on the rotifer Brachionus plicatilis

Heterocapsa circularisquama (Dinophyceae), a noxious red tide dinoflagellate, is known to have a specifically lethal effect on shellfish, especially bivalves such as pearl oyster (Pinctada fucata), but no detrimental effects of this alga on fishes have not been observed so far. In this study, we found that H. circularisquama was toxic to a microzooplankton, a rotifer (Brachionus plicatilis) in a cell concentration-dependent manner, while the cultured supernatant or ultrasonic ruptured H. circularisquama had no significant toxic effect on the rotifer. Since no such toxic effects on the rotifer were observed in Chattonella marina, Heterosigma akashiwo, or Cochlodinium polykrikoides, other species of harmful red tide plankton, H. circularisquama may have a strictly specific toxic mechanism against the rotifer as well as bivalves.     

Growth characteristics and intraspecies host specificity of a large virus infecting the dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30 degrees C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20 degrees C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

Diverse responses of the bivalve-killing dinoflagellate Heterocapsa circularisquama to infection by a single-stranded RNA virus

Viruses are believed to be significant pathogens for phytoplankton. Usually, they infect a single algal species, and often their infection is highly strain specific. However, the detailed molecular background of the strain specificity and its ecological significance have not been sufficiently understood. Here, we investigated the temporal changes in viral RNA accumulation and virus-induced cell lysis using a bloom-forming dinoflagellate Heterocapsa circularisquama and its single-stranded RNA virus, HcRNAV. We observed at least three host response patterns to virus inoculation: sensitive, resistant, and delayed lysis. In the sensitive response, the host cell culture was permissive for viral RNA replication and apparent cell lysis was observed; in contrast, resistant cell culture was nonpermissive for viral RNA replication and not lysed. In the delayed-lysis response, although viral RNA replication occurred, virus-induced cell lysis was faint and remarkably delayed. In addition, the number of infectious virus particles released to the culture supernatant at 12 days postinoculation was comparable to that of the sensitive strain. By further analysis, a few strains were characterized as variants of the delayed-lysis strain. These observations indicate that the response of H. circularisquama to HcRNAV infection is highly diverse.     

Virus resistance in the toxic bloom-forming dinoflagellate Heterocapsa circularisquama to single-stranded RNA virus infection

HcRNAV is the only known cultured dinoflagellate-infecting RNA virus. Lysis of its host dinoflagellate Heterocapsa circularisquama caused by HcRNAV is followed by apparent cell regrowth. Here we investigate the mechanism supporting the survival phenomenon. The proportion of normal cells with intact nucleus decreased to ∼8% by 3 days post infection, and then, increased to > 90% at 15 days post infection. There were abnormal cells lacking an intact nucleus, and this was followed by propagation of virus-resistant survivor cells. The proportion of HcRNAV-resistant cells in three different subcultures and temporal fluctuations were compared: a clonal H. circularisquama culture without virus inoculation (virus-sensitive, VS), a surviving isolate from the HcRNAV-inoculated Culture-VS incubated in autoclaved medium (virus-resistant, VR) and a portion of Culture-VR incubated with HcRNAV (VR incubated with virus, VR + V). The proportion of HcRNAV-resistant cells in Culture-VS was 0% and in Culture-VR + V was > 94% during the experiment; and Culture-VR fluctuated from 4% to 71%. Hence, the virus resistance was assumed to be reversible. Using Northern hybridization, viral genome accumulation was not detected in Culture-VR + V cells either inoculated with HcRNAV or transfected with HcRNAV-genome; thus, intracellular viral RNA replication was assumed to be interrupted in the virus-resistant cells.     

Algicidal activity of the thiazolidinedione derivative TD49 against the harmful dinoflagellate Heterocapsa circularisquama in a mesocosm enclosure

To assess the algicidal effects of the thiazolidinedione derivative TD49 on the unarmored dinoflagellate Heterocapsa circularisquama and to evaluate the response of the planktonic community and the environment to this chemical, we undertook mesocosm (1,300 L) and small-scale experiments. The reduction ratio for H. circularisquama in each experiment was dependent on the concentration of TD49. At a TD49 concentration >0.4 μM, the abundance of H. circularisquama decreased by 99 % in the small-scale experiment and by 84 % in the mesocosm during the initial 2 days. At 0.2 μM TD49, the abundance of H. circularisquama decreased by up to 85 % in the small-scale experiment, whereas the abundance in the mesocosm increased, implying the absence of an algicidal effect. The decrease in planktonic organisms, including H. circularisquama, following TD49 treatment was correlated with abrupt declines in culture pH and dissolved oxygen concentration. Following addition of TD49, there was a significant increase in the abundance of diatoms and cryptophyta species, and after 8 days, the dominant species in the TD49 treatments shifted to small pennate diatoms including Cylindrotheca and Entomoneis species. The growth of some species among the zooplankton community was promoted at low TD49 concentrations (≤0.4 μM), whereas high concentrations (≥1.0 μM) had a negative effect. This study demonstrates that TD49 is an effective agent for the control for H. circularisquama blooms and that large-scale mesocosms play a crucial role in assessing the application of algicides such as TD49 in natural environments.     

Cytotoxic action mode of a novel porphyrin derivative isolated from harmful red tide dinoflagellate Heterocapsa circularisquama

Heterocapsa circularisquama is known to cause lethal effect on bivalves, but toxic effect on fish has not been reported yet. Recently, we have found that H. circularisquama has potent light-dependent hemolytic toxins. Based on the chemical structural analysis, one of the hemolytic toxins named H2-a was found to be a novel porphyrin derivative with similar structure to pyropheophorbide a methyl ester (PME), a well-known photoactive hemolytic agent (Miyazaki et al., Aquatic Toxicol. 2005;73:382--393). To clarify the cytotoxic action mode of H2-a, we examined the effects of H2-a on HeLa cells in comparison with PME. The cytotoxicities of both reagents were strictly light dependent, and no significant cytotoxic effects including cellular morphological changes were induced without light illumination. The dose response curves revealed that H2-a showed stronger cytotoxicity to HeLa cells than PME. Fluorescence microscopic observation suggested that H2-a tends to accumulate in the plasma membrane, whereas PME seems to distribute entire cytoplasm. Although PME induced typical apoptotic nuclear morphological changes and DNA fragmentation in HeLa cells, no such apoptosis-inducing ability of H2-a was observed. Among the radical scavengers, histidine significantly inhibited the cytotoxic activity of H2-a, suggesting the involvement of singlet oxygen in the cytotoxicity. These results suggest that the cytotoxic mechanism of H2-a is necrotic rather than apoptosis differing from PME, even though these are structurally quite similar to each other. The relatively high affinity of H2-a to the plasma membrane might result in the potent and quick cytotoxicity without induction of apoptotic signal transduction.     

Ecological dynamics of the bivalve-killing dinoflagellate Heterocapsa circularisquama and its infectious viruses in different locations of western Japan

We studied the ecological relationships between the bloom-forming dinoflagellate Heterocapsa circularisquama and its infectious viruses in field surveys conducted in western Japan. The occurrence of H. circularisquama blooms in Imari Bay during 2002 and in Ago Bay during 2002 and 2004 was accompanied by specific increase in abundance of viruses lytic to H. circularisquama. Using northern dot-blot analysis, approximately 96% of the clonal virus isolates collected in the field surveys positively reacted with a molecular probe specific for HcRNAV (H. circularisquama RNA virus); hence, viral impacts on H. circularisquama population observed in these field surveys are considered largely due to HcRNAV and/or its closely related viruses. The dynamics of type UA viruses and type CY viruses having complementary host ranges to H. circularisquama clones were different in each survey and considered to reflect fluctuations in abundance of their suitable host cells in situ. The dynamics of H. circularisquama and its viruses in Ago Bay from 2002 to 2004 suggests the concentration of HcRNAV in the sediment prior to the host's blooming season is a significant factor in determining the size and length of the H. circularisquama blooms. These results support the hypothesis that HcRNAV infection is one of the significant factors affecting the population dynamics of H. circularisquama in both quantity (biomass) and quality (clonal composition).     

Development of microsatellite markers in the noxious red tide‐causing dinoflagellate Heterocapsa circularisquama (Dinophyceae)

We isolated 15 polymorphic microsatellites from one of the most noxious red tide‐causing dinoflagellate species, Heterocapsa circularisquama. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from two to six, and the estimate of gene diversity from 0.205 to 0.684 across the 15 microsatellites. These loci have the potential to reveal genetic structure and gene flow among H. circularisquama populations.     

Uptake kinetics of nitrate,ammonia and phosphate by the dinoflagellate Heterocapsa circularisquama isolated from Hiroshima Bay,Japan

Heterocapsa circularisquama is a harmful dinoflagellate whose first bloom in Hiroshima Bay, Japan, appeared in 1992. As suggested by the authors’ group, in the Seto Inland Sea including Hiroshima Bay, oligotrophication particularly the reduction of phosphate starting 1980 is severe. The bloom caused serious damage to the bay's extensive oyster culture. In the present study, the uptake kinetics of nitrate, ammonia, and phosphate by this species were experimentally investigated. The maximum uptake rate (ρ_max) and the half‐saturation constant (Ks) were 0.41 pmol cell^?1 h^?1 and 4.45 μM, respectively, for nitrate, 2.02 pmol cell^?1 h^?1 and 11.1 μM for ammonium, and 0.079 pmol cell^?1 h^?1 and 1.79 μM for phosphate. The maximum specific uptake rates (V_max) for nitrate, ammonia, and phosphate were estimated to be 8.95, 44.1, and 21.3 day^?1, respectively. A comparison of V_max/Ks, which is also an index of affinity to nutrients, between this species and others suggested that H. circularisquama can utilize nitrate and ammonia efficiently, but not phosphate. Considering both reports describing that H. circularisquama has the ability to utilize dissolved organic phosphorus (DOP) and the DOP concentration is higher than phosphate in Hiroshima Bay, it was concluded that H. circularisquama became dominant due to the phosphate reduction measure.     

Physiological and Optical Responses of the Harmful Dinoflagellate Heterocapsa circularisquama to a Range of Salinity

The responses of division rate, cell volume, cellular carbon (C) and nitrogen (N), cellular pigments and optical characteristics to changes in salinity were examined in the dinoflagellate Heterocapsa circularisquama. The physiological and optical characteristics of H. circularisquama were significantly affected by changes in salinity. When cells were exposed to different salinities, they exhibited physiological acclimation by adjusting their cellular properties associated with growth. This could be a beneficial tactic for ensuring proliferation and limiting damages induced by adverse environmental factors. The results of this study could be essential for assisting in the development of growth models for H. circularisquama.     

Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

Heterocapsa circularisquama sp. nov. (Peridiniales, Dinophyceae): A new marine dinoflagellate causing mass mortality of bivalves in Japan

Heterocapsa circularisquama Horiguchi sp. nov. is described from Ago Bay, central Japan. The dinoflagellate produced large-scale red tides in the bays of central and western Japan and caused mass mortality of bivalves, notably the pearl oysters. The cell is small and is composed of a conical epitheca and a hemi-spheroidal hypothecs. The chloroplast is single and is connected to the single pyrenoid. The nucleus is elongated and is located in the left side of the cell. Thecal plate arrangement has been determined as: Po, cp, 5′, 3a, 7″, 6c, 5s, 5″′, 2″″. Heterocapsa circularisquama is morphologically very similar to Heterocapsa illdefina and it is almost impossible to distinguish these two species at light microscopical level. The characteristics which can be used to distinguish these two species are the morphology of body scales and the ultrastructure of the pyrenoid matrix. The body scales of H. circularisquama possess six radiating ridges on the circular basal plate; no such ridges can be observed on the roughly triangular basal plate of the scales of H. illdefina. Furthermore, the scales of the latter species possess substantially shorter spines compared to those of H. circularisquama. The pyrenoid matrix of H. circularisquama is hardly perforated by cytoplasmic tubules, while in H. tlldefina the pyrenoid matrix is always penetrated by many cytoplasmic tubules. Based on the arrangement of thecal plates, morphology of body scales, and ultra-structure of the pyrenoid, I am placing H. circularisquama sp nov. into the genus Heterocapsa.     

THIS ARTICLE HAS BEEN RETRACTEDDevelopment of microsatellite markers in the noxious red tide‐causing dinoflagellate Heterocapsa circularisquama (Dinophyceae)

We isolated 15 polymorphic microsatellites from one of the most noxious red tide‐causing dinoflagellate species, Heterocapsa circularisquama. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from two to six, and the estimate of gene diversity was from 0.205 to 0.684 across the 15 microsatellites. These loci have the potential to reveal genetic structure and gene flow among H. circularisquama populations.     

Comparison of hemolytic activities among strains of Heterocapsa circularisquama isolated in various localities in Japan

Heterocapsa circularisquama (Dinophyceae), a red tide dinoflagellate, is toxic to bivalve molluscs such as the pearl oyster (Pinctada fucata), but no harmful effects of this alga on fish have been observed so far. We found that 7 strains of H. circularisquama showed hemolytic activities toward rabbit erythrocytes in a cell-density dependent manner, but to quite different extents. The strains which are known to be highly toxic to bivalves tend to show stronger hemolytic activities and vice versa, suggesting that the hemolytic activity is parallel with the shellfish toxicity of H. circularisquama. Since the hemolytic assay is simple, semiquantitative, and reproducible, this assay is useful not only to search for certain toxins responsible for the shellfish toxicity of H. circularisquama but also to estimate the potential toxicity of a newly appeared strain of H. circularisquama.     

Temporal changes in the ciliate assemblage and consecutive estimates of their grazing effect during the course of a Heterocapsa circularisquama bloom

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Temporal changes in the ciliate assemblage and consecutive estimates of their grazing effect during the course of a Heterocapsa circularisquama bloom

Temporal changes in ciliate assemblages during the course ofa bloom of the harmful microalga Heterocapsa circularisquama(Dinophyceae) were investigated and consecutive estimates ofspecies-specific maximum grazing losses were analyzed from Augustto September 1998 at a site in western Hiroshima Bay, the SetoInland Sea of Japan. Temporal increases of the H. circularisquamamean concentration in the water column were observed twice (25–29August and 7–10 September) with the maximum concentration(ca. 4000 cells mL^–1) being recorded on 25 August. Themain ciliate genera during the bloom were Favella, Tontonia,Eutintinnus, Tintinnopsis and Amphorellopsis. Increases of Favellaand Tontonia were observed when the concentration of H. circularisquamaranged from 260 to 1170 cells mL^–1. Total maximum grazingloss estimated from the abundance and ingestion rate of eachciliate species on H. circularisquama ranged from 1 to 75% standingstock removed d^–1 of the H. circularisquama concentration.High grazing losses mainly due to the genera Favella and Tontoniaoccurred during the period when the H. circularisquama concentrationwas decreasing. These results suggest that grazing by ciliateassemblages can influence the population dynamics of H. circularisquamadespite the potentially toxic nature of the phytoplankter.     

Plastid genes in a non-photosynthetic dinoflagellate

Dinoflagellates are a diverse group of protists, comprising photosynthetic and heterotrophic free-living species, as well as parasitic ones. About half of them are photosynthetic with peridinin-containing plastids being the most common. It is uncertain whether non-photosynthetic dinoflagellates are primitively so, or have lost photosynthesis. Studies of heterotrophic species from this lineage may increase our understanding of plastid evolution. We analyzed an EST project of the early-diverging heterotrophic dinoflagellate Crypthecodinium cohnii looking for evidence of past endosymbiosis. A large number of putative genes of cyanobacterial or algal origin were identified using BLAST, and later screened by metabolic function. Phylogenetic analyses suggest that several proteins could have been acquired from a photosynthetic endosymbiont, arguing for an earlier plastid acquisition in dinoflagellates. In addition, intact N-terminal plastid-targeting peptides were detected, indicating that C. cohnii may contain a reduced plastid and that some of these proteins are imported into this organelle. A number of metabolic pathways, such as heme and isoprenoid biosynthesis, seem to take place in the plastid. Overall, these data indicate that C. cohnii is derived from a photosynthetic ancestor and provide a model for loss of photosynthesis in dinoflagellates and their relatives. This represents the first extensive genomic analysis of a heterotrophic dinoflagellate.