Isolation,identification, and algicidal activity of marine bacteria against <Emphasis Type="Italic">Cochlodinium polykrikoides</Emphasis>

The aim of this study was to isolate and identify algicidal bacteria against the dinoflagellate Cochlodinium polykrikoides, and to determine the algicidal activity and algicidal range. During the declining period of C. polykrikoides blooms, seven algicidal bacteria were isolated. The algicidal bacteria against C. polykrikoides were enumerated using the most probable number (MPN) method. The number of algicidal bacteria was high (3.7 × 10³ mL⁻¹). Algicidal bacteria were identified on the basis of biochemical and chemotaxonomic characteristics, and analysis of 16S rDNA sequences. Seven algicidal bacteria isolated in this study belonged to the genera Bacillus, Dietzia, Janibacter, and Micrococcus. The most algicidal bacterium, designated Micrococcus luteus SY-13, is assumed to produce secondary metabolites. When 5% culture filtrate of this strain was applied to C. polykrikoides cultures, over 90% of C. polykrikoides cells were destroyed within 6 h. M. luteus SY-13 showed significant algicidal activities against C. polykrikoides and a wide algicidal range against various harmful algal bloom (HAB) species. Taken together, our results suggest that M. luteus SY-13 could be a candidate for controlling HABs.     

Preliminary observations on the concentration of marine bacteriophages in the water around Helgoland

In a preliminary survey, conducted between August 28 and October 9, 1990, the concentration of bacteriophages in seawater sampled at intervals of 1 to 4 days near Helgoland (station Kabeltonne) was determined by using indicator bacteria which had been isolated from seawater sampled only some weeks before. With a number of bacterial strains, phage concentrations ranging between 2 and 7×10²ml⁻¹ were found. However, during the course of this investigation maximal concentrations lasted for a few days only. With most indicator bacteria employed, the concentration of plaque-forming units (PFU) varied in the range of <1 and 20–30 PFU ml⁻¹.     

Further investigations on the concentration of marine bacteriophages in the water around Helgoland,with reference to the phage-host systems encountered

Between April 3 and September 24, 1991, the concentrations of bacteriophages infecting bacterial strains, isolated in 1990 and during this investigations, were determined in 35 samples of seawater taken at station ‘Kabeltonne’ adjacent to Helgoland. Similar to the findings of 1990, phage concentrations of several hundred plaque forming units (PFU) ml⁻¹ were observed with a number of indicator strains, the maximum concentration being at least 1.5×10³ PFU ml⁻¹. These high concentrations lasted for only a few days, generally decreasing at rates between 0.6 and 0.9 day⁻¹. Phage concentrations of 0 to 2 PFU ml⁻¹ were found to be predominant until the end of June, occasionally attaining 5 PFU ml⁻¹. From July through September, when high phage concentrations were observed with some indicator strains, between 0 and 10 PFU ml⁻¹ were found in the majority of tests. As revealed by a final phage-host cross-reaction test, the greater part of 138 indicator bacteria is genetically related, and almost half of the 200 phage strains tested are propagated only by their original indicator bacterium. The possible importance of mutational events for the maintenance of phage-host systems in nature is discussed.     

The effect of <Emphasis Type="Italic">Poterioochromonas</Emphasis> abundance on production of intra- and extracellular microcystin-LR concentration

Due to its capability for producing various microcystins, Microcystis aeruginosa is recognized as one of the most toxic, bloom-forming cyanobacteria. In this study, the fates of intra- and extracellular microcystin-LR (MC-LR) were investigated when the mixotrophic golden alga Poterioochromonas sp. (ZX1) was grazing on M. aeruginosa cells. In the control groups, the total MC-LR concentration increased with the growth of M. aeruginosa with an MC-LR content per cell of 0.5–1.5 × 10⁻⁸ μg cell⁻¹. In the treatment with ZX1, the total MC-LR decreased linearly throughout the incubation period. In particular, intracellular MC-LR disappeared with a loss of M. aeruginosa cells in the first few days. Part of the intracellular MC-LR was released to the medium under the grazing stress, resulting in an increase of extracellular MC-LR. The degradation rate of MC-LR was positively related to the initial abundance of ZX1 and negatively related to that of M. aeruginosa. The inhibition ratio of MC-LR production dropped sharply from 98 to 67% when the initial abundance of M. aeruginosa increased from 10⁶ to 10⁷ cells ml⁻¹. However, it increased from 84 to 99% when the initial ZX1 abundance increased from 10⁴ to 10⁵ cells ml⁻¹. The effective removal of both M. aeruginosa cells and MC-LR was observed under lower M. aeruginosa abundance (<10⁶ cells ml⁻¹) and higher ZX1 abundance (>1% of M. aeruginosa abundance). Light had little impact on MC-LR degradation, but MC-LR degradation decreased due to the loss of ZX1 after 10 days of darkness. This study showed that the interactions between M. aeruginosa and ZX1 were strongly influenced by their initial abundances.     

Occurrence of virus-like particles in the Dead Sea

Electron-microscopic examination of water samples from the hypersaline Dead Sea showed the presence of high numbers of virus-like particles. Between 0.9 and 7.3 × 10⁷ virus-like particles ml⁻¹ were enumerated in October 1994 in the upper 20 m of the water column during the decline of a bloom of halophilic Archaea. Virus-like particles outnumbered bacteria by a factor of 0.9–9.5 (average 4.4). A variety of viral morphologies were detected, the most often encountered being spindle-shaped, followed by polyhedral and tailed phages. In addition, other types of particles were frequently found, such as unidentified algal scales, and virus-sized star-shaped particles. Water samples collected during 1995 contained low numbers of both bacteria and virus-like particles (1.9–2.6 × 10⁶ and 0.8–4.6 × 10⁷ ml⁻¹ in April 1995), with viral numbers sharply declining afterwards (less than 10⁴ ml⁻¹ in November 1995–January 1996). It is suggested that viruses may play a major role in the decline of halophilic archaeal communities in the Dead Sea, an environment in which protozoa and other predators are absent. Received: February 5, 1997 / Accepted: May 24, 1997     

Effects of water velocity on picoplankton uptake by coral reef communities

In previous experiments, rates of picoplankton uptake into coral communities were controlled by sponge and ascidian biomass. Those experimental communities, however, had relatively few sponges and ascidians. In contrast, turbulent transport of particles into the momentum boundary layers can limit particle removal by layered, dense bivalve populations. In this study, the role of water velocity in controlling particulate nutrient-uptake by rubble communities was evaluated, in which the rubble was more completely covered by sponges and ascidians. Picoplankton uptake was proportional to concentration over a range of cell concentrations from 3.0 × 10⁵ to 9.5 × 10⁵ heterotrophic bacteria ml⁻¹, 4.1 × 10⁴ to 1.2 × 10⁵ Synechococcus sp. ml⁻¹ and 6.3 × 10³ to 1.8 × 10⁴ picoeukaryotes ml⁻¹. The first-order uptake rate constants, normalized to sponge and ascidian biomass, were similar to previous experimental communities. Picoplankton uptake increased 1.6-fold over a 7-fold change in water velocity, 0.05–0.35 m s⁻¹. This increase has been interpreted as a result of higher turbulent transport within the rough coral community (canopy), as indicated by a 1.6-fold increase in the bottom friction with increasing water velocity.     

Temporal variability of algicidal and growth-inhibiting bacteria at an eelgrass bed in the Ariake Sea,Japan

Abstract

Temporal changes of algicidal and growth-inhibiting bacteria on the fish-killing raphidophyte flagellate, Chattonella antiqua, at an eelgrass (Zostera marina) bed in southern Ariake Sea, Japan in 2011 was investigated. The maximum value (5.1?×?10⁷ CFU g^?1 wet leaf) of algicidal bacteria (AB) was detected from a biofilm formed on Z. marina on August 1 when AB in the adjacent seawater had also peaked (1.2?×?10⁴ CFU mL^?1). Two causative bacteria isolated from the biofilm and seawater on August 1 were both identified to be of the genus Alteromonas (γ-proteobacteria). AB and growth-inhibiting bacteria (GIB) were present from the beginning of sampling (May 20) to August 26, fluctuating between 8.6?×?10² and 1.2?×?10⁴, 1.2?×?10³ and 9.3?×?10³ CFU mL^?1, respectively. The highest phytoplankton density observed was 6423 cells mL^?1 on September 29 and was comprised of centric diatoms such as Chaetoceros, Skeletonema, and Thalassiosira and coincided with the absence of AB and GIB where the decline of Z. marina was also observed. These findings provide a new ecological insight on AB and GIB associated with Z. marina beds, indicating eelgrass beds have the important role as the nursery of those bacteria that can be utilized as mitigation measures of harmful algal blooms (HABs) in the future.     

Isolation of algicidal compounds from the red alga <Emphasis Type="Italic">Corallina pilulifera</Emphasis> against red tide microalgae

We determined the structure of two compounds, namely, 5,8,11,14,17-eicosapentaenoic acid (EPA) and di-n-octylphthalate (DnOP), which have algicidal activity against the toxic dinoflagellate, Cochlodinium polykrikoides. The polyunsaturated fatty acid EPA and the anthropogenic DnOP were isolated from the MeOH extract of the red alga Corallina pilulifera. We also found that a commercial EPA has algicidal activity identical to that of the EPA purified from C. pilulifera. At low inoculum (5.0 × 10² cells mL⁻¹), the highest algicidal activity of a commercial EPA exhibited approximately 92.6% algicidal activity after 1 h and 96.8% after 6 h treatment at 6 μg mL⁻¹, respectively. At high inoculum (1.0 × 10⁴ cells mL⁻¹), the strongest algicidal activity of EPA showed 69.5% after 1 h and 75.5% algicidal activity after 6 h treatment at 6 μg mL⁻¹, respectively. However, EPA did not show algicidal activity against several microalgae used in aquaculture such as Pavlova lutheri, Tetraselmis suecica, Isochrysis galbana, and Nannochloris oculata for 6 h treatment at 6 μg mL⁻¹. The algicidal activity of the five red tide strains to EPA (3 μg mL⁻¹) showed about 86.6%, 86.6%, and 67.3% algicidal activity against Skeletonema costatum, Chaetoceros curvisetus, and C. polykrikoides after 1 h treatment at low inoculum (5.0 × 10² cells mL⁻¹), respectively, but not against Prorocentrum minimum and Scrippsiella trochoidea. We concluded that EPA might be useful as a controlling agent of harmful algal blooms.     

Effect of two microalgae concentrations on body size and egg size of the rotifer <Emphasis Type="Italic">Brachionus calyciflorus</Emphasis>

The rotifer, Brachionus calyciflorus, was grown with two algae species (Chlorella sp. and Scenedesmus obliquus) at different concentrations (0.1, 1 and 10 × 10⁶ cells ml⁻¹). The body size (lorica biovolume) of individual rotifer and their egg size were measured when the populations were roughly in the exponential phase of population growth. The body size of the rotifers differed significantly (P < 0.05) among the two algae species used, however this effect was not observed for egg size. The body size of rotifers fed on higher densities of Chlorella sp. (10 × 10⁶ cells ml⁻¹) was significantly larger than for those fed on lower and medium densities (0.1 and 1 × 10⁶ cells ml⁻¹). Body size and egg size of rotifers fed with different amounts of Scenedesmus did not differ significantly. The egg size was significantly larger at higher food level of Chlorella. A significantly positive correlation was observed between the adult rotifer body size and their egg size.     

Isolation, identification and characterization of algicidal bacteria against Stephanodiscus hantzschii and Peridinium bipes for the control of freshwater winter algal blooms

Five strains (HYY0510-SK04, HYY0511-SK09, HYK0512-SK12, HYK0512-PK04 and HYY0512-PK05) of algicidal bacteria against the harmful bloom forming diatom Stephanodiscus hantzschii and dinoflagellate Peridinium bipes, were isolated. Among these strains, HYY0510-SK04, HYY0511-SK09 and HYK0512-SK12 have an effective algicidal activity for S. hantzschii, while HYK0512-PK04 and HYY0512-PK05 have an algicidal effect against P. bipes. Sequence analysis of 16S rDNA showed that HYY0510-SK04 and HYY0511-SK09 were closely related to Acidovorax delafieldii ATCC 17505^T. HYK0512-SK12, HYK0512-PK04 and HYY0512-PK05 showed high homology with Variovorax paradoxus IAM 12373^T (98.9%), Hydrogenophaga palleronii ATCC 49743^T (98.8%) and Pseudomonas plecoglossicida ATCC 700383^T (98.3%), respectively. HYY0510-SK04, HYY0511-SK09 and HYK0512-SK12 degraded S. hantzschii cells within two weeks when those bacteria were inoculated at densities of ≥10⁷cells mL⁻¹ to the lag or logarithmic growth phase of the algal culture. HYK0512-PK04 and HYY0512-PK05 degraded more than 90% of P. bipes cells within 14 and 8 days, respectively, when these bacteria were inoculated at densities of ≥10⁷cells mL⁻¹. Among the five bacterial strains, HYK0512-SK12 and HYY0512-PK05 showed the most effective growth inhibition of all the algae and cyanobacteria tested. Biochemical assays revealed that the main algicidal substance from all isolates were likely to be extracellular substances. These results indicate that the bacterial strains isolated for this study are potential agents for the control of harmful algal blooms in eutrophic reservoirs.     

Use of clove oil to anaesthetize freshwater amphipods

The use of clove oil as a potential anaesthetic for freshwater amphipods was examined at 20 °C. Individuals of Gammarus minus, a common species in southern Illinois, USA, spanning the entire body size range (4.3–14.3 mm), were used to test four anaesthetic concentrations varying from 1.48 × 10⁻⁴ ml ml⁻¹ to 5.9 × 10⁻⁴ ml ml⁻¹. Small-bodied individuals (mean size = 5.4 mm ± 0.27SE) were used to test additional concentrations, up to 14.7 × 10⁻⁴ ml ml⁻¹, a 10-fold span, to identify potential lethal concentrations. At the lowest concentration, time to anaesthesia and recovery was constant at all body sizes. For the three next higher concentrations, time to anaesthesia decreased with increasing concentration while recovery time increased. Activity of amphipods was not affected by the ethanol carrier. In addition, activity did not differ between amphipods that had recovered from anaesthesia and unexposed amphipods. At clove oil concentrations of 8.84 × 10⁻⁴ ml ml⁻¹ and 14.7 × 10⁻⁴ ml ml⁻¹, mortality was 7 and 40%, respectively, indicating, that 5.9 × 10⁻⁴ ml ml⁻¹ was a safe working concentration. No mortality was observed with Gammarus acherondytes, a federally endangered cave amphipod on which the protocol with 80 μl of stock was used in the field. The method enabled us to obtain information on the endangered amphipod which normally would have required the sacrifice of individuals. Thus, research can continue on species for which population numbers are low and for which basic information is needed to formulate meaningful recovery plans.     

Characterization of an algicidal bacterium Brevundimonas J4 and chemical defense of Synechococcus sp. BN60 against bacterium J4

As part of efforts to enhance the strategies explored to eliminate the adverse impacts of cyanobacterial blooms, we isolated an algicidal bacterium, J4, from Lake Taihu. Analysis of 16S rDNA sequence revealed that strain J4 belonged to the genus Brevundimonas. Bacterium J4 exhibited algicidal activity mainly through excretion of extracellular algicidal compounds that were further extracted with methanol and purified by silica gel chromatography and high performance liquid chromatography (HPLC). The compounds showed thermal stability, strong polarity and water solubility in J4 cultures. Study on the algicidal activity of J4 against two dominant cyanobacterial bloom-forming species in Lake Taihu showed that J4 exhibited lower algicidal rate against Synechococcus sp. BN60 (48.6%, t = 6 days) than against Microcystis aeruginosa 9110 (91.8%, t = 6 days). Additionally, rapid reduction in cell density of J4 was observed in co-cultures of Synechococcus sp. BN60 and bacterium J4 but not observed in co-cultures of M. aeruginosa 9110 and bacterium J4 during algicidal process, which was the main reason why the algicidal rate of J4 against BN60 was lower than against 9110. The reduction in cell density of J4 resulted from inducible production of antimicrobial-like compound secreted by Synechococcus sp. BN60 in co-cultures of Synechococcus sp. BN60 and bacterium J4, which reflected a kind of chemical defense from cyanobacteria (BN60) against algicidal bacteria (J4). However, M. aeruginosa 9110 had no chemical defense against J4, suggesting that whether cyanobacterial chemical defense occurs or not between cyanobacteria and algicidal bacteria depends on specific cyanobacteria–algicidal bacteria pairs. These results show that not only one-sided algicidal effect but also two-sided reciprocal inhibition interactions exist between algicidal bacteria and cyanobacteria, indicating the complexity of cyanobacteria–algicidal bacteria interactions in Lake Taihu and the need to take the cyanobacterial defensive responses into consideration when assessing potential use of algicidal bacteria.     

Production of recombinant herpes simplex virus protease in 10-L stirred vessels using a baculovirus–insect cell expression system

A gene expression system using recombinant Autographa californica nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external addition of air/O₂ mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks, Sf-9 cells were adapted to grow to high cell densities (6–10 × 10⁶ cells ml⁻¹) in shake flasks in serum-free media. With these procedures, cell densities of 5 × 10⁶ cells ml⁻¹ were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the 247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell⁻¹, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography with reproducible yields of 11–38 mg L⁻¹ of recombinant protein and ≥ 90% purity. Maximum production of this protein was observed at a cell density of 5.0 × 10⁶ cells ml⁻¹. Received 09 December 1996/ Accepted in revised form 13 April 1997     

Effects of Acetic Acid on the Regrowth of Heterotrophic Bacteria in the Drinking Water Distribution System

Summary Three laboratory-scale water pipe systems were set up to study the effects of adding two levels of acetic acid (10 and 50 μg acetate eq-C l⁻¹) on the bacterial regrowth in water pipes. The results of the water pipe test showed that nearly all carbon in the acetic acid could be readily utilized by bacteria and resulted in an increase in biomass concentration. The maximum heterotrophic plate counts in biofilm were equal to 3.5 × 10⁴, 8.9 × 10⁵ and 2.9 × 10⁷ c.f.u. cm⁻² while the maximum heterotrophic plate counts of free bacteria were equal to 1.2 × 10³, 5.0 × 10³ and 6.8 × 10⁴ c.f.u. ml⁻¹ for the blank and with addition of 10 and 50 μg acetate eq-C l⁻¹. These results showed that addition of acetic acid to drinking water has a positive effect on the assimilable organic carbon content of drinking water and bacterial regrowth in the distribution system. This effect is enhanced with addition of high-level acetic acid. Batch tests were also conducted using water samples collected from a Taiwanese drinking water distribution system. The bacterial regrowth potentials of the blank were equal to 4.3 × 10³, 1.5 × 10⁴, 4.9 × 10⁴ and 7.5 × 10⁴ c.f.u. ml⁻¹ for water samples collected from treatment plant effluent, commercial area, mixed area, and residential area, respectively. These results showed that the biological stability of drinking water is the highest in treatment plant effluent, followed by distributed water of the commercial area, distributed water of the mixed area, and then the distributed water of residential area.     

Protozoan Bacterivory in the Ice and the Water Column of a Cold Temperate Lagoon

Abstract Bacterial abundance and bacterivorous protist abundance and activity were examined in ice-brine and water column communities of a cold temperate Japanese lagoon (Saroma-Ko Lagoon, Hokkaido, 44°N, 144°E), during the late winter phase of ice community development (February–March 1992). Bacterial abundance averaged 6 and 1 × 10⁵ cells ml⁻¹ in the ice-brine and plankton samples, respectively, and generally decreased during the sampling period. Bacterivorous protists, identified based on direct observation of short-term (<1 h) ingested fluorescently labeled bacteria (FLB) in their food vacuoles, were largely dominated by flagellates, mainly cryothecomonad-type and chrysomonad-like cells and small dinoflagellates of the genus Gymnodinium. Bacterivorous ciliates included mainly the prostomatid Urotricha sp., the scuticociliates Uronema and Cyclidium, the choreotrichs Lohmaniella oviformis and Strobilidium, and the hypotrich Euplotes sp. Protist abundance averaged 4 × 10³ and 8.1 cells ml⁻¹ in the ice-brine and 0.3 × 10³ and 1.2 cells ml⁻¹ in the plankton, for flagellates and ciliates, respectively. In contrast to bacteria, the abundance of protists generally increased throughout the sampling period, indicating predator–prey interactions. Protistan bacterivory, measured from the rate of FLB disappearance over 24 h, averaged 36% (ice) and 24% (plankton) of bacterial standing stock and exhibited the same seasonal pattern as for protist abundance. The calculated specific clearance (range, 2–67 nl protozoa⁻¹ h⁻¹) and ingestion (<1–26 particles protozoa⁻¹ h⁻¹) rates were likely to be minimal estimates and grazing impact may have been higher on occasion. Indications for the dependence of ``bacterivorous protists' on nonbacterial food items were also provided. Although alternative sources of bacterial loss are likely to be of importance, this study provides evidence for the potential of protozoan assemblages as bacterial grazers in both sea ice-brine biota and water column at the southern limit of sea ice in the northern hemisphere. Received: 30 July 1998; Accepted: 18 November 1998     

Transmission electron microscope analysis of virus-like particles in the freshwater lakes of Signy Island, Antarctica

Water samples from a range of fresh-water Antarctic lakes on Signy Island (South Orkney Islands: 60°45′S, 45 °38′W) were examined for the presence of virus-like particles (VLPs) during the 1998/1999 field season. It was discovered that VLPs were ubiquitous, morphologically diverse and abundant, with high concentrations ranging from 4.9 × 10⁶ ml⁻¹ to 3.1 × 10⁷ ml⁻¹. Likely hosts include bacteria, cyanobacteria and eukaryotic algae. In addition, an unusually large virus morphotype was observed with a head diameter 370 × 330 nm and a tail 1.3 μm long. Accepted: 15 May 2000     

Antarctic sea ice viral dynamics over an annual cycle

Viral abundance, burst sizes, lytic production and temperate phage were investigated in land-fast ice at two sites in Prydz Bay Antarctica (68°S, 77°E) between April and November 2008. Both ice cores and brine were collected. There was no seasonal pattern in viral or bacterial numbers. Across the two sites virus abundances ranged between 0.5 × 10⁵ and 5.1 × 10⁵ viruses ml⁻¹ in melted ice cores and 0.6 × 10⁵–3.5 × 10⁵ viruses ml⁻¹ in brine, and bacterial abundances between 2.7 × 10⁴ and 17.3 × 10⁴ cells ml⁻¹ in melted ice cores and 3.9 × 10⁴–32.5 × 10⁴ cells ml⁻¹ in brine. Virus to bacterium ratios (VBR) showed a clear seasonal pattern in ice cores with lowest values in winter (range 1.2–20.8), while VBRs in brine were lower (0.2–4.9). Lytic viral production range from undetectable to 2.0 × 10⁴ viruses ml⁻¹ h⁻¹ in ice cores with maximum rates in September and November. In brine maximum, lytic viral production occurred in November (1.18 × 10⁴ viruses ml⁻¹ h⁻¹). Low burst sizes were typical (3.94–4.03 viruses per bacterium in ice cores and 3.16–4.0 viruses per bacterium in brine) with unusually high levels of visibly infected cells—range 40–50%. This long-term investigation revealed that viral activity was apparent within the sea ice throughout its annual cycle. The findings are discussed within the context of limited data available on viruses in sea ice.     

A Mutant of Metarhizium anisopliae var. acridum with Enhanced Submerged Conidiation

Summary An insertional mutant of Metarhizium anisopliae is described with enhanced submerged conidiation. In a 500 ml submerged culture, this mutant produces a mean of 4.05 × 10⁸ propagules ml⁻¹ from an inoculum of 1 × 10⁶ conidia, where the parental strain accumulates only 3.75 × 10⁴ propagules ml⁻¹.     

Resistance of the fish-killing dinoflagellate Cochlodinium polykrikoides against algicidal bacteria isolated from the coastal sea of Japan

Noxious red tides of the dinoflagellate Cochlodinium polykrikoides tend to be long lasting and cause mass mortalities of cultured and natural fish and invertebrates along the western coast of Japan and the southern coast of Korea. In order to assess the tolerance of C. polykrikoides to attack by algicidal bacteria, the effects of algicidal bacteria strains on the growth of three C. polykrikoides strains were examined in laboratory culture experiments. Algicidal bacteria used were two strains of Cytophaga (J18/M01 and AA8-2, direct attack type and wide prey range), three strains of Alteromonas (S, K, D) and one strain of Pseudoalteromonas (R, indirect attack type), which were all isolated by using Chattonella antiqua as a prey organism. Neither Cytophaga strain showed any algicidal activity. In the cases of Alteromonas and Pseudoalteromonas, some cultures of C. polykrikoides were killed, but at least 10 days or more were required for the death of this dinoflagellate. C. polykrikoides survived in the presence of algicidal bacteria in concentrations up to 10⁶–10⁷ cells ml⁻¹, which is enough for other red tide microalgae to be killed. On the contrary, the algicidal effects of bacteria on C. antiqua were detected clearly within a few days. These results imply that C. polykrikoides is resistant to the six algicidal bacteria examined, which may reflect the capacity for mixotrophy. This resistance of C. polykrikoides to algicidal bacteria could provide a selective advantage for survival compared to other microalgae susceptible to attack by algicidal bacteria and hence prolong red tides caused by this harmful dinoflagellate.     

Resistance to glyphosate in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> as result of pre-selective mutations

Adaptation of Microcystis aeruginosa (Cyanobacteria) to resist the herbicide glyphosate was analysed by using an experimental model. Growth of wild-type, glyphosate-sensitive (G^s) cells was inhibited when they were cultured with 120 ppm glyphosate, but after further incubation for several weeks, occasionally the growth of rare cells resistant (G^r) to the herbicide was found. A fluctuation analysis was carried out to distinguish between resistant cells arising from rare spontaneous mutations and resistant cells arising from other mechanisms of adaptation. Resistant cells arose by rare spontaneous mutations prior to the addition of glyphosate, with a rate ranging from 3.1 × 10⁻⁷ to 3.6 × 10⁻⁷ mutants per cell per generation in two strains of M. aeruginosa; the frequency of the G^r allele ranged from 6.14 × 10⁻⁴ to 6.54 × 10⁻⁴. The G^r mutants are slightly elliptical in outline, whereas the G^s cells are spherical. Since G^r mutants have a diminished growth rate, they may be maintained in uncontaminated waters as the result of a balance between new resistants arising from spontaneous mutation and resistants eliminated by natural selection. Thus, rare spontaneous pre-selective mutations may allow the survival of M. aeruginosa in glyphosate-polluted waters via G^r clone selection.