Growth,death, and photobiology of dinoflagellates (Dinophyceae) under bacterial-algicide control

Naturally occurring allelopathic compounds, specific to some phytoplankton, may be a good source of bio-control agents against microalgae responsible for harmful algal blooms (HABs). Global expansion of HABs has invigorated research into different approaches to control these algae, including the search for naturally derived algicidal compounds. Here, we investigated the effects of a filtrate from the algicidal marine bacterium Shewanella sp. IRI-160 on photochemical function of four cultured dinoflagellates, Karlodinium veneficum, Gyrodinium instriatum, Prorocentrum minimum, and Alexandrium tamarense. The filtrate (designated IRI-160AA) contains bioactive compound(s), which were recently shown to inhibit growth of several dinoflagellate species. Results of this study show that all dinoflagellates but P. minimum exhibited photosystem II (PSII) inhibition, loss of photosynthetic electron transport, and varying degrees of cellular mortality. Exposure assays over 24 h showed that PSII inhibition and loss of cell membrane integrity occurred simultaneously in G. instriatum, but not in K. veneficum, where PSII activity declined prior to losing outer-membrane integrity. In addition, PSII inhibition and population growth inhibition were dose-dependent in K. veneficum, with an average EC-50 of 7.9 % (v/v) IRI-160AA. Application of IRI-160AA induced significantly higher PSII inhibition and cell mortality in K. veneficum subjected to continuous darkness as compared to cells maintained with 12:12 h light/dark cycles, while no such dark effect was noted for G. instriatum. The marked differences in the rate and impact of this algicide suggest that multiple cellular targets and different cascades of cellular dysfunction occur across these dinoflagellates.     

Roles of mixotrophy in blooms of different dinoflagellates: Implications from the growth experiment

Studies over the last two decades suggested that mixotrophy could be an important adaptive strategy for some bloom-forming dinoflagellates. In the coastal waters adjacent to the Changjiang River estuary in the East China Sea, recurrent blooms of dinoflagellates Prorocentrum donghaiense, Karenia mikimotoi and Alexandrium catenella started to appear from the beginning of the 21 century, but roles of mixotrophy in the formation of dinoflagellate blooms were not well understood. In the current study, mixotrophy-based growth of four selected bloom-causative dinoflagellate species, i.e. K. mikimotoi, A. catenella, P. donghaiense and Prorocentrum micans, were studied. Dinoflagellates were co-cultured with different prey organisms, including bacterium Marinobacter sp., microalgae Isochrysis galbana and Hemiselmis virescens, under a variant of nutrient conditions. It was found that growth of dinoflagellate K. mikimotoi was significantly promoted with the presence of prey organisms. Growth of P. donghaiense and P. micans was only slightly improved. For A. catenella, the addition of prey organisms has no effects on the growth, while both of the two prey microalgae I. galbana and H. virescens were killed, probably by allelochemicals released from A. catenella. There was no apparent relationship between nutrient conditions and the mixotrophy-based growth of the tested dinoflagellates. Based on the results of the growth experiment, it is implicated that mixotrophy may play different roles in the growth and bloom of the four dinoflagellate species. It can be an important competitive strategy for K. mikimotoi. For the two Prorocentrum species and A. catenella, however, the role of mixotrophy is much limited. They may depend more on other competitive strategies, such as phototrophy-based growth and allelopathic effect, to prevail in the phytoplankton community and form blooms.     

Investigation of the algicidal exudate produced by Shewanella sp. IRI-160 and its effect on dinoflagellates

The bacterium, Shewanella sp. IRI-160, was previously shown to have negative effects on the growth of dinoflagellates, while having no negative effects on other classes of phytoplankton tested (Hare et al., 2005). In this study, we investigated the mode of algicidal activity for Shewanella sp. IRI-160 and found that the bacterium secretes a bioactive compound. The optimum temperature for production of the algicidal compound by this bacterium was at 30 °C. Bacteria-free filtrate of medium containing the algicide (designated IRI-160AA) was stable at temperatures ranging from −80 °C to 121 °C, and could be stored at room temperature for at least three weeks with no loss in activity. Algicidal activity was eluted in the aqueous portion after C18 extraction, suggesting that the active compound is likely polar and water-soluble. The activity of IRI-160AA was examined on a broad range of dinoflagellates (Karlodinium veneficum, Karenia brevis, Gyrodinium instriatum, Cochlodinium polykrikoides, Heterocapsa triquetra, Prorocentrum minimum, Alexandrium tamarense and Oxyrrhis marina) and three species from other classes of algae as controls (Dunaliella tertiolecta, Rhodomonas sp. and Thalassiosira pseudonana). Algicidal activity was observed for each dinoflagellate and little to no negative effect was observed on chlorophyte and cryptophyte cultures, while a slight (non-significant) stimulatory effect was observed on the diatom culture exposed to the algicide. Finally, the effect of the algicide at different growth stages was investigated for K. veneficum and G. instriatum. IRI-160AA exhibited a significantly greater effect during logarithmic growth compared to stationary phase, suggesting a potential application of the algicide for prevention and control of harmful dinoflagellate blooms in the future.     

Effects of a bacterial algicide,IRI-160AA,on dinoflagellates and the microbial community in microcosm experiments

Filtrates from the bacterium Shewanella sp. IRI-160 (termed IRI-160AA) have been shown to inhibit population growth and kill a variety of dinoflagellates grown in culture. Here we test the immediate efficacy of IRI-160AA in laboratory microcosms initiated from three natural dinoflagellate blooms (Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum). We measured target dinoflagellate abundance, total chlorophyll-a, photosystem II (PSII) photochemistry, and changes to the prokaryotic and eukaryotic community composition over 2–3 days of IRI-160AA incubation. Naked dinoflagellates were impacted more, while abundance of the thecate P. minimum was not affected. However, dinoflagellate growth inhibition was generally lower than that observed in uni-algal cultures, and took longer to occur. Eukaryotic community composition in IRI-160AA treated microcosms was significantly different from control incubations, and was driven predominantly by increases in heterotrophic protists (e.g. Euplotes sp. and Paraphysomonas sp.). Similarly, significant changes to the prokaryotic community structure were evident. Microcosms of G. instriatum with higher algicide concentrations indicated that algicidal activity was enhanced in a dose dependent manner. Furthermore, total ciliate abundance as well as a bactivorous chyrsophyte (Paraphysomonas sp.) increased in a dose dependent manner. Total diatom abundance increased at lower IRI-160AA concentrations, but increased less with increasing dose. Overall, the bio-activity of IRI-160AA on naturally occurring dinoflagellates in mixed natural microbial communities is encouraging from the applied perspective of using the active compound(s) in IRI-160AA as natural agent(s) to manage harmful dinoflagellate blooms.     

Biochemical compositions of two dominant bloom- forming species isolated from the Yangtze River Estuary in response to different nutrient conditions

Variations of cellular total lipid, total carbohydrate and total protein content of two dominant bloom-forming species (Skeletonema costatum and Prorocentrum donghaiense) isolated from the Yangtze River Estuary were examined under six different nutrient conditions in batch cultures. Daily samples were collected to estimate the cell growth, nutrient concentration and three biochemical compositions content during 7 days for S. costatum and the same sampling procedure was done every other day during 10 days for P. donghaiense. Results showed that for S. costatum, cellular total lipid content increased under phosphorus (P) limitation, but not for nitrogen (N) limitation; cellular carbohydrate were accumulated under both N and P limitation; cellular total protein content of low nutrient concentration treatments were significantly lower than that of high nutrient concentration treatments. For P. donghaiense, both cellular total lipid content and total carbohydrate content were greatly elevated as a result of N and P exhaustion, but cellular total protein content had no significant changes under nutrient limitation. In addition, the capability of accumulation of three biochemical constituents of P. donghaiense was much stronger than that of S. costatum. Pearson correlation showed that for both species, the biochemical composition of three constituents (lipid, carbohydrate and protein) had no significant relationship with extracellular N concentration, but had positive correlation with extracellular and intracellular P concentration. The capability of two species to accumulate cellular total lipid and carbohydrate under nutrient limitation may help them accommodate the fluctuating nutrient condition of the Yangtze River Estuary. The different responses of two species of cellular biochemical compositions content under different nutrient conditions may provide some evidence to explain the temporal characteristic of blooms caused by two species in the Yangtze River Estuary.     

Prorocentrum minimum blooms: potential impacts on dissolved oxygen and Chesapeake Bay oyster settlement and growth

The cosmopolitan dinoflagellate Prorocentrum minimum is a recurrent bloom forming species in the Chesapeake Bay and its tributaries, generally observed at its highest levels in late spring and summer. Laboratory studies were conducted to assess potential bloom impacts on diel oxygen concentrations in shallow littoral zones as well as settlement success and post-set growth of the eastern oyster Crassostrea virginica. Using light–dark and dark cultures and periodic diel sub-sampling, bloom levels of P. minimum produced supersaturated oxygen levels at the end of each day while darkened cultures were typified by rapid decreases in dissolved oxygen (DO) (1.1–1.3 mg L⁻¹ h⁻¹) to hypoxic and anoxic levels within 4 days. These data suggest shallow, poorly flushed systems and the biota in them will experience rapid and large diel variations in oxygen, implying recurrent P. minimum blooms need be considered as short-term oxygen stressors for Bay oyster spat and other living resources. Direct effects of P. minimum impacts on oysters were not as expected or previously reported. In one experiment, pre-bloom isolates of P. minimum were grown and then exposed to polyvinyl chloride (PVC) settlement plates to see whether dinoflagellate preconditioning of the hard substrate might affect oyster sets. No differences were noted between set on the PVC with P. minimum exposure to set recorded with filtered seawater, Instant Ocean^®, or Isochrysis. In the second oyster experiment, spat on PVC plates were exposed to field collected P. minimum blooms and a commercial mixture of several other food types including Isochrysis. Oyster growth was significantly higher in P. minimum exposures than noted in the commercial mix. These results, compared to results with other isolates from the same region, indicate substantial positive impact from some of the P. minimum blooms of the area while others separated in space, time, or nutrient status could severely curtail oyster success through toxin production induced by nutrient limitation.     

Inducible Mixotrophy in the Dinoflagellate Prorocentrum minimum

Prorocentrum minimum is a neritic dinoflagellate that forms seasonal blooms and red tides in estuarine ecosystems. While known to be mixotrophic, previous attempts to document feeding on algal prey have yielded low grazing rates. In this study, growth and ingestion rates of P. minimum were measured as a function of nitrogen (‐N) and phosphorous (‐P) starvation. A P. minimum isolate from Chesapeake Bay was found to ingest cryptophyte prey when in stationary phase and when starved of N or P. Prorocentrum minimum ingested two strains of Teleaulax amphioxeia at higher rates than six other cryptophyte species. In all cases ‐P treatments resulted in the highest grazing. Ingestion rates of ‐P cells on T. amphioxeia saturated at ~5 prey per predator per day, while ingestion by ‐N cells saturated at 1 prey per predator per day. In the presence of prey, ‐P treated cells reached a maximum mixotrophic growth rate (μ_max) of 0.5 d^?1, while ‐N cells had a μ_max of 0.18 d^?1. Calculations of ingested C, N, and P due to feeding on T. amphioxeia revealed that phagotrophy can be an important source of all three elements. While P. minimum is a proficient phototroph, inducible phagotrophy is an important nutritional source for this dinoflagellate.     

Effects of the bacterial algicide IRI-160AA on cellular morphology of harmful dinoflagellates

The algicide, IRI-160AA, induces mortality in dinoflagellates but not other species of algae, suggesting that a shared characteristic or feature renders this class of phytoplankton vulnerable to the algicide. In contrast to other eukaryotic species, the genome of dinoflagellates is stabilized by high concentrations of divalent cations and transition metals and contains large amounts of DNA with unusual base modifications. These distinctions set dinoflagellates apart from other phytoplankton and suggest that the nucleus may be a dinoflagellate-specific target for IRI-160AA. In this study, morphological and ultrastructural changes in three dinoflagellate species, Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum, were evaluated after short-term exposure to IRI-160AA using super resolution structured illumination microscopy (SR-SIM) and transmission electron microscopy (TEM). Exposure to the algicide resulted in cytoplasmic membrane blebbing, differing chloroplast morphologies, nuclear expansion, and chromosome expulsion and/or destabilization. TEM analysis showed that chromosomes of algicide-treated K. veneficum appeared electron dense with fibrous protrusions. In algicide-treated P. minimum and G. instriatum, chromosome decompaction occurred, while for P. minimum, nuclear expulsion was also observed for several cells. Results of this investigation demonstrate that exposure to the algicide destabilizes dinoflagellate chromosomes, although it was not clear if the nucleus was the primary target of the algicide or if the observed effects on chromosomal structure were due to downstream impacts. In all cases, changes in cellular morphology and ultrastructure were observed within two hours, suggesting that the algicide may be an effective and rapid approach to mitigate dinoflagellate blooms.     

Effects of macroalgae Ulva pertusa (Chlorophyta) and Gracilaria lemaneiformis (Rhodophyta) on growth of four species of bloom-forming dinoflagellates

The effects of fresh thalli and culture medium filtrates from two species of marine macroalgae, Ulva pertusa Kjellm (Chlorophyta) and Gracilaria lemaneiformis (Bory) Dawson (Rhodophyta), on growth of marine microalgae were investigated in co-culture under controlled laboratory conditions. A selection of microalgal species were used, all being identified as bloom-forming dinoflagellates: Prorocentrum donghaiense Lu sp., Alexandrium tamarense (Lebour) Balech, Amphidinium carterae Hulburt and Scrippsiella trochoide (Stein) Loeblich III. Results showed that the fresh thalli of either U. pertusa or G. lemaneiformis significantly inhibited the microalgal growth, or caused mortality at the end of the experiment. However, the overall effects of the macroalgal culture filtrates on the growth of the dinoflagellates were species-specific (inhibitory, stimulatory or none) for different microalgal species. Results indicated an allelopathic effect of macroalga on the co-cultured dinoflagellate. We then took P. donghaiense as an example to further assess this hypothesis. The present study was carried out under controlled conditions, thereby excluded the fluctuation in light and temperature. Nutrient assays showed that nitrate and phosphate were almost exhausted in G. lemaneiformis co-culture, but remained at enough high levels in U. pertusa co-culture, which were well above the nutrient limitation for the microalgal growth, when all cells of P. donghaiense were killed in the co-culture. Daily f/2 medium enrichment greatly alleviated the growth inhibition on P. donghaiense in G. lemaneiformis co-culture, but could not eliminate it. Other environmental factors, such as carbonate limitation, bacterial presence and the change of pH were also not necessary for the results. We thus concluded that allelopathy was the most possible reason leading to the negative effect of U. pertusa on P. donghaiense, and the combined roles of allelopathy and nutrient competition were essential for the effect of G. lemaneiformis on P. donghaiense.     

Newly discovered role of the heterotrophic nanoflagellate Katablepharis japonica,a predator of toxic or harmful dinoflagellates and raphidophytes

Heterotrophic nanoflagellates are ubiquitous and known to be major predators of bacteria. The feeding of free-living heterotrophic nanoflagellates on phytoplankton is poorly understood, although these two components usually co-exist. To investigate the feeding and ecological roles of major heterotrophic nanoflagellates Katablepharis spp., the feeding ability of Katablepharis japonica on bacteria and phytoplankton species and the type of the prey that K. japonica can feed on were explored. Furthermore, the growth and ingestion rates of K. japonica on the dinoflagellate Akashiwo sanguinea—a suitable algal prey item—heterotrophic bacteria, and the cyanobacteria Synechococcus sp., as a function of prey concentration were determined. Among the prey tested, K. japonica ingested heterotrophic bacteria, Synechococcus sp., the prasinophyte Pyramimonas sp., the cryptophytes Rhodomonas salina and Teleaulax sp., the raphidophytes Heterosigma akashiwo and Chattonella ovata, the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum donghaiense, Alexandrium minutum, Cochlodinium polykrikoides, Gymnodinium catenatum, A. sanguinea, Coolia malayensis, and the ciliate Mesodinium rubrum, however, it did not feed on the dinoflagellates Alexandrium catenella, Gambierdiscus caribaeus, Heterocapsa triquetra, Lingulodinium polyedra, Prorocentrum cordatum, P. micans, and Scrippsiella acuminata and the diatom Skeletonema costatum. Many K. japonica cells attacked and ingested a prey cell together after pecking and rupturing the surface of the prey cell and then uptaking the materials that emerged from the ruptured cell surface. Cells of A. sanguinea supported positive growth of K. japonica, but neither heterotrophic bacteria nor Synechococcus sp. supported growth. The maximum specific growth rate of K. japonica on A. sanguinea was 1.01 d⁻¹. In addition, the maximum ingestion rate of K. japonica for A. sanguinea was 0.13 ng C predator⁻¹d⁻¹ (0.06 cells predator⁻¹d⁻¹). The maximum ingestion rate of K. japonica for heterotrophic bacteria was 0.019 ng C predator⁻¹d⁻¹ (266 bacteria predator⁻¹d⁻¹), and the highest ingestion rate of K. japonica for Synechococcus sp. at the given prey concentrations of up to ca. 10⁷ cells ml⁻¹ was 0.01 ng C predator⁻¹d⁻¹ (48 Synechococcus predator⁻¹d⁻¹). The maximum daily carbon acquisition from A. sanguinea, heterotrophic bacteria, and Synechococcus sp. were 307, 43, and 22%, respectively, of the body carbon of the predator. Thus, low ingestion rates of K. japonica on heterotrophic bacteria and Synechococcus sp. may be responsible for the lack of growth. The results of the present study clearly show that K. japonica is a predator of diverse phytoplankton, including toxic or harmful algae, and may also affect the dynamics of red tides caused by these prey species.     

Using quantitative real-time PCR to study competition and community dynamics among Delaware Inland Bays harmful algae in field and laboratory studies

The Delaware Inland Bays (DIB) have experienced harmful algal blooms of dinoflagellates and raphidophytes in recent years. We used quantitative polymerase chain reaction (QPCR) techniques to investigate the community dynamics of three DIB dinoflagellates (Karlodinium veneficum, Gyrodinium instriatum, and Prorocentrum minimum) and one raphidophyte (Heterosigma akashiwo) at a single site in the DIB (IR-32) in summer 2006 relative to salinity, temperature and nutrient concentrations. We also carried out complementary laboratory culture studies. New primers and probes were developed and validated for the 18S rRNA genes in the three dinoflagellates. K. veneficum, H. akashiwo, and G. instriatum were present in almost all samples throughout the summer of 2006. In contrast, P. minimum was undetectable in late June through September, when temperatures ranged from 20 to 30 °C (average 25.7 °C). Dissolved nutrients ranged from 0.1 to 2.8 μM PO₄³⁻ (median = 0.3 μM), 0.7–30.2 μM NO_x (median = 12.9 μM), and 0–19.4 μM NH₄⁺ (median = 0.7 μM). Dissolved N:P ratios covered a wide range from 2.6 to 177, with a median of 40. There was considerable variability in occurrence of the four species versus nutrients, but in general P. minimum and H. akashiwo were most abundant at higher (>40) N:P ratios and dissolved nitrogen concentrations, while K. veneficum and G. instriatum were most abundant at low dissolved N:P ratios (<20) and dissolved nitrogen concentrations < 10 μM. The semi-continuous laboratory competition experiment used mixed cultures of K. veneficum, P. minimum, and H. akashiwo grown at dissolved N:P ratios of 5, 16, and 25. At an N:P of 16 and 25 P. minimum was the dominant alga at the end of the experiment, even at a temperature that was much higher than that at which this alga was found to bloom in the field (27 °C). P. minimum and H. akashiwo had highest densities in the N:P of 25. K. veneficum grew equally well at all three N:P ratios, and was co-dominant at times at an N:P of 5. H. akashiwo had the lowest densities of the three algae in the laboratory experiment. Laboratory and field results showed both interesting similarities and significant differences in the influences of important environmental factors on competition between these harmful algal species, suggesting the need for more work to fully understand HAB dynamics in the DIB.     

Pathology and immune response of the blue mussel (Mytilus edulis L.) after an exposure to the harmful dinoflagellate Prorocentrum minimum

The harmful dinoflagellate Prorocentrum minimum has different effects upon various species of grazing bivalves, and these effects also vary with life-history stage. Possible effects of this dinoflagellate upon mussels have not been reported; therefore, experiments exposing adult blue mussels, Mytilus edulis, to P. minimum were conducted. Mussels were exposed to cultures of toxic P. minimum or benign Rhodomonas sp. in glass aquaria. After a short period of acclimation, samples were collected on day 0 (before the exposure) and after 3, 6, and 9 days of continuous-exposure experiment. Hemolymph was extracted for flow-cytometric analyses of hemocyte, immune-response functions, and soft tissues were excised for histopathology. Mussels responded to P. minimum exposure with diapedesis of hemocytes into the intestine, presumably to isolate P. minimum cells within the gut, thereby minimizing damage to other tissues. This immune response appeared to have been sustained throughout the 9-day exposure period, as circulating hemocytes retained hematological and functional properties. Bacteria proliferated in the intestines of the P. minimum-exposed mussels. Hemocytes within the intestine appeared to be either overwhelmed by the large number of bacteria or fully occupied in the encapsulating response to P. minimum cells; when hemocytes reached the intestine lumina, they underwent apoptosis and bacterial degradation. This experiment demonstrated that M. edulis is affected by ingestion of toxic P. minimum; however, the specific responses observed in the blue mussel differed from those reported for other bivalve species. This finding highlights the need to study effects of HABs on different bivalve species, rather than inferring that results from one species reflect the exposure responses of all bivalves.     

Maternal and neonate diatom diets impair development and sex differentiation in the copepod Temora stylifera

Development and sex differentiation in the copepod Temora stylifera was studied in the presence of maternal and larval diets of the diatoms Thalassiosira rotula and Skeletonema marinoi, either provided alone or supplemented with the control dinoflagellate Prorocentrum minimum. Both diatoms had deleterious effects on growth compared to the control when used as pure diets, inducing very low or even zero survival from hatching to adulthood. This effect was deleted when the diet was supplemented with a good food (P. minimum) only in the case of T. rotula. By contrast, with a maternal or larval diet of S. marinoi, nauplii did not pass metamorphosis even when this alga was mixed with P. minimum. Arrested development was not due to lack of feeding since early and late nauplii (NII and NV) ingested all three algae at similar rates when used as single diets, and did not show any preference when the algae were offered as mixtures. Since mortality rates with a mixed diet of S. marinoi + P. minimum were similar or even higher than those obtained with a single diet of S. marinoi, we suggest that this diatom is more toxic than nutritionally deficient for T. stlyfera development. No males were produced in cohorts reared on pure diatom diets or with a mixture of S. marinoi + P. minimum, and intermediate male:female sex ratios were obtained with the mixed T. rotula + P. minimum diet. Possibly some diatoms produce compounds such as oxylipins or new molecules that alter sex differentiation in T. stylifera.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Quantification of toxic effects of the herbicide metolachlor on marine microalgae Ditylum brightwellii (Bacillariophyceae), Prorocentrum minimum (Dinophyceae), and Tetraselmis suecica (Chlorophyceae)

Toxic effects of the herbicide metolachlor (MC) were evaluated for three marine microalgae, Tetraselmis suecica (chlorophyte), Ditylum brightwellii (diatom), and Prorocentrum minimum (dinoflagellate). MC showed a significant reduction in cell counts and chlorophyll a levels. Median effective concentration (EC₅₀) was calculated based on chlorophyll a levels after a 72-h MC exposure. EC₅₀ values for T. suecica, D. brightwellii, and P. minimum were 21.3, 0.423, and 0.07 mg/L, respectively. These values showed that the dinoflagellate was most sensitive when exposed to the herbicide, at a concentration comparable to freshwater algae, suggesting its potential as an appropriate model organism for ecotoxicity assessments in marine environments.     

Development of an immunofluorescence technique for detecting <Emphasis Type="Italic">Prorocentrum donghaiense</Emphasis> Lu

Prorocentrum donghaiense Lu is a key harmful algal bloom (HAB) species which is widespread along the China Sea coast. In this study, P. donghaiense-specific antiserum was developed, and the detection method, based on immunofluorescence (IF), was optimized. The antiserum was raised using 0.5% paraformaldehyde-fixed whole cells (WC) as antigen. The titer and the specificity were examined using whole-cell IF. Results showed that ethanol was an effective decolorization reagent, and 80% ethanol was able to minimize autofluorescence of cells. Samples preserved by freezing at −20°C or −80°C remained above 85% detection efficiency after 1-month storage. The antiserum against WC had a high titer (1:10000), and exhibited high specificity at species level. The antiserum showed a weak cross reaction with the closely related species P. dentatum HK, P. dentatum CCMP1517 and P. minimum HK only at very low dilution (1:5). However, it did not cross-react with the species from the same genus or other phytoplankton species when the dilution reached or exceeded 1:100. Results from different P. donghaiense samples collected at different growth phases or grown under different nutrient conditions showed no significant difference in IF intensity. In addition, the antiserum exhibited high specific binding to P. donghaiense in both mixed phytoplankton samples and field samples. The results indicate that the technique is a potentially useful tool for the rapid identification of P. donghaiense and can facilitate the analysis of various natural samples.     

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.     

Rapid detection and quantification of Prorocentrum minimum by loop-mediated isothermal amplification and real-time fluorescence quantitative PCR

The dinoflagellate Prorocentrum minimum was successfully detected using loop-mediated isothermal amplification (LAMP) and real-time fluorescence quantitative PCR (RTFQ-PCR). Both specificity and sensitivity testing in the two methods have been validated. In the LAMP assay, the specific ladder-like pattern of bands only appeared in those templates containing P. minimum. The sensitivity of LAMP was tenfold higher than conventional PCR. In RTFQ-PCR assay, only positive amplifications were detected from those samples containing P. minimum. RTFQ-PCR can detect 0.1 cells and 10 pg of DNA within 40 cycles, showing its high sensitivity. Cells could be quantified according to standard curves in agreement with the quantification by standard microscopy counting methods. The LAMP method therefore is appropriate for on-the-spot testing because of its rapidity and simplification, and the RTFQ-PCR is fit for laboratory testing owing to its accurate quantification. The two methods are of significance in forecasting red tides.     

A brief summary of the physiology and ecology of Karenia brevis Davis (G. Hansen and Moestrup comb. nov.) red tides on the West Florida Shelf and of hypotheses posed for their initiation,growth, maintenance,and termination

This contribution represents a review of the historical and recent literature describing the environmental factors that relate to the distribution, growth, primary production, nutrient requirements and utilization along with hypotheses that are extant for the initiation, growth, maintenance and termination of Karenia brevis blooms on the West Florida Shelf. Potential nutrient sources that support blooms and relate to recent questions on the duration, frequency, and intensity of WFS blooms are summarized and some thoughts are presented which relate to the question of why K. brevis, a slow growing dinoflagellate, becomes dominant in a nearshore shelf region that is typically dominated by diatoms.There is no single hypothesis that can account for blooms of K. brevis along the west coast of Florida. Of the approximately 24 thoughts and hypotheses described herein (including the 1880s speculation), seven are related to rainfall and/or riverine flux, six invoke the benthos or bottom flux in one form or another, seven involve water column hydrodynamics or are unrelated to the benthos or land sources, and four are primarily chemical/allelopathy based. Nutrient sources for growth and maintenance range from atmospheric deposition, N-fixation, riverine and benthic flux, and zooplankton excretion to decaying fish killed by the toxic dinoflagellate with no one source being conclusively identified as a primary contributor to prolonged bloom maintenance. Insufficient information is available to delimit specific mechanisms that may play a role in the termination of K. brevis blooms. However, general processes such as macro- and microzooplankton grazing, bacterial and viral cell lysis, and dispersal by physical advection and the break down of fronts, that originally may have acted as concentrating mechanisms, are reviewed.     

Use of dissolved inorganic and organic phosphorus by axenic and nonaxenic clones of Karenia brevis and Karenia mikimotoi

Nearly annual blooms of the marine dinoflagellate Karenia brevis, which initiate offshore on the West Florida Shelf in oligotrophic waters, cause widespread environmental and economic damage. The success of K. brevis as a bloom-former is partially attributed to its ability to use a diverse suite of nutrients from natural and anthropogenic sources, although relatively little is known about the ability of K. brevis and the closely related Karenia mikimotoi to use a variety of organic sources of phosphorus, including phosphomonoesters, phosphodiesters, and phosphonates. Through a series of bioassays, this study characterized the ability of axenic and nonaxenic K. brevis and K. mikimotoi clones isolated from Florida waters to use a variety of organic phosphorus compounds as the sole source of phosphorus for growth, comparing this utilization to that of inorganic sources of phosphate. Differing abilities of axenic and nonaxenic K. brevis and K. mikimotoi cultures to use phosphorus from the compounds evaluated were documented. Specifically, growth of axenic cultures was greatest on inorganic phosphorus and was not supported on the phosphomonoester phytate, or generally on phosphodiesters or phosphonates. The nonaxenic cultures were able to use organic compounds that the axenic cultures were not able to use, often after lags in growth, highlighting a potential role of co-associated bacterial communities to transform nutrients to bioavailable forms. Given the ability of K. brevis and K. mikimotoi to use a diverse suite of inorganic and organic phosphorus, bloom mitigation strategies should consider all nutrient forms.