Virus-specific responses of Heterosigma akashiwo to infection

We used flow cytometry to examine the process of cell death in the bloom-forming alga Heterosigma akashiwo during infection by a double-stranded DNA virus (OIs1) and a single-stranded RNA virus (H. akashiwo RNA virus [HaRNAV]). These viruses were isolated from the same geographic area and infect the same strain of H. akashiwo. By use of the live/dead stains fluorescein diacetate and SYTOX green as indicators of cellular physiology, cells infected with OIs1 showed signs of infection earlier than HaRNAV-infected cultures (6 to 17 h versus 23 to 29 h). Intracellular esterase activity was lost prior to increased membrane permeability during infection with OIs1, while the opposite was seen with HaRNAV-infected cultures. In addition, OIs1-infected cells accumulated in the cultures while HaRNAV-infected cells rapidly disintegrated. Progeny OIs1 viruses consisted of large and small morphotypes with estimated latent periods of 11 and 17 h, respectively, and about 1,100 and 16,000 viruses produced per cell, respectively. In contrast, HaRNAV produced about 21,000 viruses per cell and had a latent period of 29 h. This study reveals that the characteristics of viral infection in algae are virus dependent and therefore are variable among viruses infecting the same species. This is an important consideration for ecosystem modeling exercises; calculations based on in situ measurements of algal physiology must be sensitive to the diverse responses of algae to viral infection.     

Virus-Specific Responses of Heterosigma akashiwo to Infection

We used flow cytometry to examine the process of cell death in the bloom-forming alga Heterosigma akashiwo during infection by a double-stranded DNA virus (OIs1) and a single-stranded RNA virus (H. akashiwo RNA virus [HaRNAV]). These viruses were isolated from the same geographic area and infect the same strain of H. akashiwo. By use of the live/dead stains fluorescein diacetate and SYTOX green as indicators of cellular physiology, cells infected with OIs1 showed signs of infection earlier than HaRNAV-infected cultures (6 to 17 h versus 23 to 29 h). Intracellular esterase activity was lost prior to increased membrane permeability during infection with OIs1, while the opposite was seen with HaRNAV-infected cultures. In addition, OIs1-infected cells accumulated in the cultures while HaRNAV-infected cells rapidly disintegrated. Progeny OIs1 viruses consisted of large and small morphotypes with estimated latent periods of 11 and 17 h, respectively, and about 1,100 and 16,000 viruses produced per cell, respectively. In contrast, HaRNAV produced about 21,000 viruses per cell and had a latent period of 29 h. This study reveals that the characteristics of viral infection in algae are virus dependent and therefore are variable among viruses infecting the same species. This is an important consideration for ecosystem modeling exercises; calculations based on in situ measurements of algal physiology must be sensitive to the diverse responses of algae to viral infection.     

A thylakoidal processing peptidase from the heterokont alga Heterosigma akashiwo

Heterokont algae such as diatoms, brown seaweeds and the raphidophyte Heterosigma akashiwo acquired their chloroplasts via a secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host, resulting in chloroplasts surrounded by four membranes rather than two. The precursor of a nuclear-encoded thylakoid lumen protein, PsbO, from Heterosigma has a presequence composed of a typical ER signal peptide followed by putative stromal and thylakoid targeting domains. A processing enzyme associated with Heterosigma thylakoids cleaved the presequence (with or without the ER signal sequence) in a single step, giving a product of the size of the mature protein. Its sensitivity to a penem inhibitor and insensitivity to other protease inhibitors suggest that it is a member of the Type I signal peptidase family. Furthermore the Heterosigma enzyme appeared to have similar substrate specificity to the pea thylakoidal processing peptidase.     

Viral Impacts on Total Abundance and Clonal Composition of the Harmful Bloom-Forming Phytoplankton Heterosigma akashiwo

Recent observations that viruses are very abundant and biologically active components in marine ecosystems suggest that they probably influence various biogeochemical and ecological processes. In this study, the population dynamics of the harmful bloom-forming phytoplankton Heterosigma akashiwo (Raphidophyceae) and the infectious H. akashiwo viruses (HaV) were monitored in Hiroshima Bay, Japan, from May to July 1998. Concurrently, a number of H. akashiwo and HaV clones were isolated, and their virus susceptibilities and host ranges were determined through laboratory cross-reactivity tests. A sudden decrease in cell density of H. akashiwo was accompanied by a drastic increase in the abundance of HaV, suggesting that viruses contributed greatly to the disintegration of the H. akashiwo bloom as mortality agents. Despite the large quantity of infectious HaV, however, a significant proportion of H. akashiwo cells survived after the bloom disintegration. The viral susceptibility of H. akashiwo isolates demonstrated that the majority of these surviving cells were resistant to most of the HaV clones, whereas resistant cells were a minor component during the bloom period. Moreover, these resistant cells were displaced by susceptible cells, presumably due to viral infection. These results demonstrated that the properties of dominant cells within the H. akashiwo population change during the period when a bloom is terminated by viral infection, suggesting that viruses also play an important role in determining the clonal composition and maintaining the clonal diversity of H. akashiwo populations. Therefore, our data indicate that viral infection influences the total abundance and the clonal composition of one host algal species, suggesting that viruses are an important component in quantitatively and qualitatively controlling phytoplankton populations in natural marine environments.     

Viral impacts on total abundance and clonal composition of the harmful bloom-forming phytoplankton Heterosigma akashiwo

Recent observations that viruses are very abundant and biologically active components in marine ecosystems suggest that they probably influence various biogeochemical and ecological processes. In this study, the population dynamics of the harmful bloom-forming phytoplankton Heterosigma akashiwo (Raphidophyceae) and the infectious H. akashiwo viruses (HaV) were monitored in Hiroshima Bay, Japan, from May to July 1998. Concurrently, a number of H. akashiwo and HaV clones were isolated, and their virus susceptibilities and host ranges were determined through laboratory cross-reactivity tests. A sudden decrease in cell density of H. akashiwo was accompanied by a drastic increase in the abundance of HaV, suggesting that viruses contributed greatly to the disintegration of the H. akashiwo bloom as mortality agents. Despite the large quantity of infectious HaV, however, a significant proportion of H. akashiwo cells survived after the bloom disintegration. The viral susceptibility of H. akashiwo isolates demonstrated that the majority of these surviving cells were resistant to most of the HaV clones, whereas resistant cells were a minor component during the bloom period. Moreover, these resistant cells were displaced by susceptible cells, presumably due to viral infection. These results demonstrated that the properties of dominant cells within the H. akashiwo population change during the period when a bloom is terminated by viral infection, suggesting that viruses also play an important role in determining the clonal composition and maintaining the clonal diversity of H. akashiwo populations. Therefore, our data indicate that viral infection influences the total abundance and the clonal composition of one host algal species, suggesting that viruses are an important component in quantitatively and qualitatively controlling phytoplankton populations in natural marine environments.     

A Sodium Pump in the Plasma Membrane of the Marine Alga Heterosigma akashiwo

ATP-dependent transport of ²²Na⁺ into liposomes reconstitutedfrom plasma membrane proteins of Heterosigma akashiwo was examined.The apparent K^m values for transport of Na⁺ were 400 µMfor ATP and 7 mM for Na⁺. ATP-dependent transport of ²²Na⁺ wasnot inhibited by a protonophore or a membrane-permeable cationbut was inhibited by an inhibitor of P-type ATPases. (Received October 2, 1995; Accepted February 1, 1996)     

Photoperiodic Regulation of Cell Division and Chloroplast Replication in Heterosigma akashiwo

Cell division and chloroplast replication in Heterosigma akashiwo(Hada) Hada occurred as separate synchronous events during thecell cycle when cells were subjected to light-dark regimes.Under three different photoperiodic cycles of 10L/14D (10 hlight/14 h dark), 12L/12D or 16L/8D, cell division began athour 19–20 and finished at hour 23–26 after theonset of the light period, while chloroplast replication beganat hour 20–22 after the onset of the dark period. Almostall the cells divided only once in the 12L/12D cycle. The rateof increase in chloroplast number during one light-anddark cyclewas always equal to that in cell number in every photoperiodexamined. Light was essential for both cell division and chloroplast replication,but the minimum light period necessary for each event differed.When the light period was shorter than 6 h, no cell divisionoccurred; when it was shorter than 3 h, no chloroplast replicationoccurred. (Received February 26, 1987; Accepted June 17, 1987)     

Cyst formation in the red tide flagellate Heterosigma akashiwo (Raphidophyceae)

A natural population of Heterosigma akashiwo was collected andincubated in the dark with autoclaved marine sediment. A portionof the cells in the population changed into cysts, which wereidentical to H.akashiwo cysts found in the field. The cystsrequired at least 2 weeks of mandatory dormancy before germination.     

Molecular cloning of P-type ATPases on intracellular membranes of the marine alga Heterosigma akashiwo

Two cDNA clones (HAA13 and HAA1) which include conserved regions of genes of P-type ATPases were isolated from the marine alga Heterosigma akashiwo by a method that included the polymerase chain reaction. The longer cDNA (3286 bp), HAA13, consisted of an open reading frame that encoded a 106 kDa polypeptide of 977 amino acids with several possible transmembrane domains and conserved regions of eukaryotic P-type ATPases. One transmembrane domain had a leucine zipper structure. HAA1 was not a full-length gene (2054 bp) and lacked the 5 region, but it also included the conserved regions and putative transmembrane domains. Antibodies against the polypeptides encoded by HAA13 and HAA1 that have been expressed in Escherichia coli reacted with 100 kDa and 95 kDa polypeptides, respectively, on intracellular membranes of H. akashiwo cells. Immunostaining of H. akashiwo cells revealed that the HAA13 antigen was distributed on membranes around chloroplasts and the HAA1 antigen was located on small vesicles.     

The role of interactions between Prorocentrum minimum and Heterosigma akashiwo in bloom formation

We examined the growth and interactions between the bloom-forming flagellates Prorocentrum minimum and Heterosigma akashiwo using bi-algal culture experiments. When both species were inoculated at high cell densities, growth of H. akashiwo was inhibited by P. minimum. In other combinations of inoculation densities, the species first reaching the stationary phase substantially suppressed maximum cell densities of the other species, but the growth inhibition effect of P. minimum was stronger than that of H. akashiwo. We used a mathematical model to simulate growth and interactions of P. minimum and H. akashiwo in bi-algal cultures. The model indicated that P. minimum always out-competed H. akashiwo over time. Additional experiments showed that crude extracts from P. minimum and H. akashiwo cultures did not affect the growth of either species, but both strongly inhibited the growth of the bloom-forming diatom Skeletonema costatum. Further experiments showed that it was unlikely that reactive oxygen species produced by H. akashiwo were responsible for the inhibition of P. minimum growth.     

The effects of Heterosigma akashiwo on juvenile Oncorhynchus tshawytscha and its implications for fish culture

The effects of natural blooms of Heterosigma akashiwo on freshwater-and saltwater-acclimated juvenile chinook salmon were assessed. Rates of fish mortality in the blooms were independent of acclimation of fish to seawater and the ambient oxygen levels, but were dependent on concentration of algae and ambient water temperatures. No pathological abnormality to gills or other internal organs in the fish were evident. Aeration or oxygenation of fish cages did not enhance or inhibit fish survival in a H. akashiwo bloom. Cause of death was considered to be due to a labile ichthyotoxic agent.     

A Nitrate-Inducible Plasma Membrane Protein of a Marine Alga, Heterosigma akashiwo

The rate of nitrate uptake by Heterosigma akashiwo cells thathad been cultured in medium with nitrate or ammonium ions asthe source of nitrogen was measured using¹⁵NO_3– The ratioof ¹⁵N/¹⁴N increased dramatically in nitrate-grown cells. Inammonium-grown cells, the ratio of ¹⁵N/¹⁴N did not increasefor 3 h but then it began to increase. Even when nitrate reductaseactivity was inhibited by tungstate, nitrate-grown cells couldtake up nitrate. Plasma membranes from nitrate-grown and ammonium-grown cellswere purified by the silica-microbead method, and polypeptidesassociated with the membranes were analyzed by SDS-PAGE andimmunostaining. A major polypeptide with a molecular mass of26 kDa appeared 3 h after the transfer of ammonium-grown cellsto nitrate-containing medium, and it disappeared 2 d after thetransfer of nitrate-grown cells to ammonium-containing medium.The 26 kDa polypeptide also appeared when cell growth shiftedfrom the logarithmic phase to the stationary phase and the ammoniumcontent of the medium decreased, even when the cells were culturedin ammonium-containing medium. (Received April 10, 1992; Accepted July 30, 1992)     

Possible physiological mechanisms for production of hydrogen peroxide by the ichthyotoxic flagellate Heterosigma akashiwo

Blooms of the toxic red tide phytoplankton Heterosigma akashiwo(Raphidophyceae) are responsible for substantial losses withinthe aquaculture industry. The toxicological mechanisms of H.akashiwoblooms are complex and to date, heavily debated. One putativetype of ichthyotoxin includes the production of reactive oxygenspecies (ROS) that could alter gill structure and function,resulting in asphyxiation. In this study, we investigated thepotential of H.akashiwo to produce extracellular hydrogen peroxide,and have investigated which cellular processes are responsiblefor this production. Within all experiments, H.akashiwo producedsubstantial amounts of hydrogen peroxide (up to 7.6 pmol min^–110⁴ cells^–1), resulting in extracellular concentrationsof ~0.5 µmol l^–1 H₂O₂. Measured rates of hydrogenperoxide production were directly proportional to cell density,but at higher cell densities, accuracy of H₂O₂ detection wasreduced. Whereas light intensity did not alter H₂O₂ production,rates of production were stimulated when temperature was elevated.Hydrogen peroxide production was not only dependent on growthphase, but also was regulated by the availability of iron inthe medium. Reduction of total iron to 1 nmol l^–1 enhancedthe production of H₂O₂ relative to iron replete conditions (10µmol l^–1 iron). From this, we collectively concludethat production of extracellular H₂O₂ by H.akashiwo occurs througha metabolic pathway that is not directly linked to photosynthesis.     

Competitive interactions between two allelopathic algal species: Heterosigma akashiwo and Phaeodactylum tricornutum

ABSTRACT

Both Heterosigma akashiwo and Phaeodactylum tricornutum have been reported to produce allelochemicals capable of inhibiting the growth of co-occurring microalgae. Here, potential allelopathy between H. akashiwo and P. tricornutum was evaluated using bi-algal culture, cell-free culture filtrate and a no-contact co-culturing system in nutrient-replete media. Experiments were also conducted in the no-contact co-culturing system under nutrient-limited conditions. In nutrient-replete bi-algal culture, the growth of P. tricornutum and H. akashiwo each tended to be strongly suppressed when the other species was inoculated at high cell densities. A mathematical model was used to simulate the growth interactions of the two species in bi-algal culture and showed that P. tricornutum outcompeted H. akashiwo over time with different initial cell densities under nutrient-sufficient conditions, indicating that P. tricornutum was more potent in allelopathy. Heterosigma akashiwo growth was inhibited both in the P. tricornutum culture filtrate and no-contact co-culturing system. This confirmed that the extracellular allelopathic compounds released by P. tricornutum were one of the sources of the H. akashiwo growth inhibition. Nutrient deficiency did not increase the extent of allelopathic activity of allelochemicals.     

On the Mechanisms of Diurnal Vertical Migration Behavior of Heterosigma akashiwo (Raphidophyceae)

The marine raphidophycean biflagellate, Heterosigma akashiwo,clearly showed diurnal vertical migration under a 12 h light-12h dark photoperiod appearing at the surface of the culture mediumduring the light period and at the bottom during the dark period.The upward migration commenced a few hours before the lightwas turned on and the downward migration a few hours beforeit was turned off. The diuranal vertical migration behaviorwas closely correlated with diurnal changes in the specificgravity of the cells, those near the surface of the culturemedium had a smaller specific gravity than those at the bottom.The migration behavior was also correlated with the directionof cell swimming. More cells had flagella furrow facing upwardthan downward in the light phase, and vice versa in the darkphase. Phototaxis was not the main factor inducing the verticalmigration, though the cells did show a tactic respose to light.Chemotactic responses to O₂, N₂ or CO₂ gas did not occur. (Received August 9, 1984; Accepted January 9, 1985)     

Viral mortality in the final stage of Heterosigma akashiwo (Raphidophyceae) red tide

During a bloom of Heterosigma akashiwo (Raphidophyceae) in northernHiroshima Bay, Japan, in June 1993, the proportion of H.akashiwocells containing virus-like particles (VLPs) was monitored bytransmission electron microscopy. Until 3 days before the terminationof the red tide, no VLP-containing cells were detected, andthe proportion of VLP-containing cells was <1% on the last2 days of the red tide. However, the sample collected on thelast day, continuously incubated for 26 h at 22°C, revealeda high frequency (11.5%) of VLP-containing cells. These findingssuggest that viral mortality occurs in quite a short time andmay play an important role in regulating the disintegrationof H.akashiwo red tide.     

Concanavalin A-induced discharge of glycocalyx of raphidophycean flagellates, Chattonella marina and Heterosigma akashiwo

Chattonella marina and Heterosigma akashiwo, known as red tide phytoplankton, are naturally wall-less and have quite fragile cell structures. In this study, we found that an equilibrium dialysis technique allowed the study of lectin binding to these flagellates. The results suggested that concanavalin A (Con A) binds to these flagellate cells through the specific carbohydrate moieties on the cell surface. Interestingly, the binding of an excess of Con A on the cell surface caused morphological changes concomitant with discharge of glycocalyx, a polysaccharide-containing common structure on the external cell surface of these flagellates. Fluorescent microscopic observation using FITC-labeled Con A (F-Con A) confirmed that F-Con A molecules are localized on the discharged glycocalyx.     

Galacturonic-acid-induced increase of superoxide production in red tide phytoplankton Chattonella marina and Heterosigma akashiwo

Red tide phytoplankton, Chattonella marina and Heterosigma akashiwo, are known to generate superoxide anion (O2-). We found that galacturonic acid (GaLUA) stimulated C. marina and H. akashiwo to generate increased amounts of O2-. Since such effect was not observed in any other monosaccharides tested, our results suggest that the binding of GalUA to specific sites on the flagellate cell surface may induce the increase of 02- production.     

Sinking of Heterosigma akashiwo results in increased toxicity of this harmful algal bloom species

Notable physiological responses such as toxicity and sinking rates of the red tide forming raphidophyte Heterosigma akashiwo are correlated with high levels of macronutrient stress. Individual cells of this species are also capable of forming benthic vegetative cysts that overwinter in marine sediment and contribute to bloom propagation in subsequent seasons. It was hypothesized that there is variability in the rates of sinking within cell cultures and that sinking cells are more toxic than the neutrally buoyant or floating cells. Using laboratory-based settling columns, various isolates of H. akashiwo were allowed to separate, and the toxicities of sinking and floating populations were analyzed. Sinking and floating rates were significantly higher during the late stationary growth phase for all isolates. For two H. akashiwo isolates, sinking populations were significantly more toxic than those that were positively buoyant. A similar trend was observed in a third strain, however the relationship was not significant. Differences in adaptive ecophysiology among the different strain likely caused the variation. It is suggested that the most toxic cells within a bloom are those found at the lower depths, potentially interacting with the benthic community or ensuring that subsequent bloom propagation contains cells with the potential for toxicity.