Detrimental impacts of the dinoflagellate Karenia mikimotoi in Fujian coastal waters on typical marine organisms

Blooms of the toxic dinoflagellate Karenia mikimotoi (K. mikimotoi) have occurred frequently in the East China Sea in recent decades and were responsible for massive mortalities of abalones in Fujian coastal areas in 2012, however, little is known about the effects of these blooms on other marine organisms. In this study, the toxic effects and the possible mechanisms of toxicity of K. mikimotoi from Fujian coastal waters on typical marine organisms at different trophic levels, including zooplankton (Brachionus plicatilis, Artemia salina, Calanus sinicus, and Neomysis awatschensis) and aquaculture species (Penaeus vannamei and Scophthalmus maximus) were investigated. At a bloom density of 3 × 10⁴ cells/mL, the Fujian strain of K. mikimotoi significantly affected the tested organisms, which had mortality rates at 96 h of 100, 23, 20, 97, 33, and 53%, respectively. Moreover, the intact cell suspension was toxic to all tested species, whereas cell-free culture and the ruptured cell suspension had no significant effects on the tested organisms. Possible mechanisms for this toxic effect, including reactive oxygen species (ROS) and hemolytic toxins, were evaluated. For K. mikimotoi, 0.014 ± 0.004 OD/(10⁴ cells) superoxide (O₂⁻) and 3.00 ± 0.00 nmol/(10⁴ cells) hydrogen peroxide (H₂O₂) were measured, but hydrogen peroxide did not affect rotifers at that concentration, and rotifers were not protected from the lethal effects of K. mikimotoi when the enzymes superoxide dismutase and catalase were added to counteract the ROS. The lipophilic extract of K. mikimotoi had a hemolytic effect on rabbit erythrocytes but exhibited no significant toxicity. These results suggest that this strain of K. mikimotoi can have detrimental effects on several typical marine organisms and that its toxicity may be associated with intact cells but is not related to ROS or hemolytic toxins.     

Novel toxins produced by the dinoflagellate Karenia brevisulcata

Karenia brevisulcata (Chang), a new toxic dinoflagellate of the genus Karenia was isolated from a harmful algal bloom that occurred in Wellington Harbour, New Zealand in 1998. The bloom severely affected most marine biota resulting in long-term ecological damage and causing respiratory distress in harbour bystanders. Cultures of K. brevisulcata produced a range of novel toxins including ten lipid-soluble K. brevisulcata toxins (KBTs) and six water-soluble brevisulcatic acids (BSXs). Brevetoxins were not detected. KBT-F, KBT-G, BSX-1 and BSX-2 were isolated from 1450 L of bulk cultures and purified in mg quantities. Preliminary chemical and toxicological investigations show that KBT-F (M 2054 C₁₀₇H₁₆₀O₃₈) and KBT-G (M 2084 C₁₀₈H₁₆₂O₃₉) are complex polycyclic ethers with UVmax at 227 nm. NMR data gave characteristics of ladder frame polyether structures and a 2-methylbut-2-enal side chain, similar to gymnocins. The mouse i.p. LD₅₀s for KBT-F and -G were 0.032 and 0.040 mg kg⁻¹, respectively. These KBTs were also highly cytotoxic and haemolytic. BSX-1 (M 916 C₄₉H₇₂O₁₆) and BSX-2 (M 872 C₄₇H₆₈O₁₅) are polycyclic ether dicarboxylates with UVmax 196 nm. BSX-4 and BSX-5, the lactone ring-closed analogues and the presumed primary toxins in the algal cells, were isolated in smaller quantities. Preliminary structural information from NMR and MS showed a carboxylated side chain and some similarities to brevetoxin-A. However, the structures have not yet been fully elucidated due to conformers confounding the NMR. The mouse i.p. LD₅₀ for BSX-1 was 3.9 mg kg⁻¹ while no deaths were seen in mice injected with BSX-2 at 6.6 mg kg⁻¹. The LD₅₀s for the lactones BSX-4 and -5 were 1.4 and 1.6 mg kg⁻¹ respectively. BSX-4 and -5 were agonists of voltage-gated sodium channels but only weakly haemolytic. Activities in the Neuro-2a cytotoxicity assay were ca 10% of dihydrobrevetoxin-2 and were fully antagonised by saxitoxin.     

Molecular ecological responses of the dinoflagellate Karenia mikimotoi to phosphate stress

Karenia mikimotoi is a toxic, widespread dinoflagellate which could produce hemolytic toxins and ichthyotoxins affecting fisheries within the area of its bloom. Previous ecophysiological studies indicated that the enhance of environmental phosphate concentration could promote the growth of K. mikimotoi. Intrinsic mechanisms regarding the effects of external phosphate on its photosynthesis, cell cycle succession and differential proteins’ expressions are still unknown. K. mikimotoi was cultured in phosphate-deprived medium, while the culture in f/2 medium (Guillard, 1975) was introduced as phosphate-sufficient control experiment. Cell counts and phosphate concentration detection were performed every other day. Flowcytometry was applied to measure cell cycle succession and chlorophyll fluorescence intensity fluctuation. Differential proteomics expression was examined by SDS-PAGE tandem LTQ Orbitrap MS/MS spectrometry. Functions of each differential protein were searched within NCBInr protein database and Swissprot database. Our study demonstrated that phosphate stress inhibited growth and cell cycle succession of K. mikimotoi remarkably (p < 0.01). Algal chlorophyll fluorescence intensity was significantly affected by phosphate deprivation (p < 0.05). 11 species of differential proteins were detected only in phosphate-limited culture sample which related to stress signal transduction, vacuolar phosphate release, phospholipid degradation, organic acid synthesis and phagotrophy. 4 kinds of differential proteins were identified only in f/2 medium culture sample which referred to cell proliferation, glycolysis, SAM cycle and polyamine production. Based on analysis of differential proteomic functional annotation, we hypothesized proteomic response mechanism of K. mikimotoi to phosphate stress. Molecular biological responses of dinoflagellate K. mikimotoi to phosphate stress was explored.     

Potential impact of an exceptional bloom of Karenia mikimotoi on dissolved oxygen levels in waters off western Ireland

In the summer of 2005 an exceptional bloom of the dinoflagellate Karenia mikimotoi occurred along Ireland's Atlantic seaboard and was associated with the mass mortality of both benthic and pelagic marine life. Oxygen depletion, cellular toxicity and physical smothering, are considered to be the main factors involved in mortality. In this paper we use a theoretical approach based on stoichiometry (the Anderson ratio) and an average K. mikimotoi cellular carbon content of 329 pg C cell⁻¹ (n = 20) to calculate the carbonaceous and nitrogenous oxygen demand following bloom collapse. The method was validated against measurements of biochemical oxygen demand and K. mikimotoi cell concentration. The estimated potential oxygen utilisation (POU) was in good agreement with field observations across a range of cell concentrations. The magnitude of POU following bloom collapse, with the exception of three coastal areas, was considered insufficient to cause harm to most marine organisms. This indicates that the widespread occurrence of mortality was primarily due to other factors such as cellular toxicity and/or mucilage production, and not oxygen depletion or related phenomena. In Donegal Bay, Kilkieran Bay and inner Dingle Bay, where cell densities were in the order of 10⁶ cells L⁻¹, estimated POU was sufficient to cause hypoxia. Of the three areas, Donegal Bay is considered to be the most vulnerable due to its hydrographic characteristics (seasonally stratified, weak residual flow) and hypoxic conditions (2.2 mg L⁻¹ O₂) were directly observed in the Bay post bloom collapse. Here, depending on the time of bloom collapse, depressed DO levels could persist for weeks and continue to have a potentially chronic impact on the Bay.     

Molecular mechanism of glucose-6-phosphate utilization in the dinoflagellate Karenia mikimotoi

Phosphorus (P) is an essential nutrient for marine phytoplankton as for other living organisms, and the preferred form, dissolved inorganic phosphate (DIP), is often quickly depleted in the sunlit layer of the ocean. Phytoplankton have developed mechanisms to utilize organic forms of P (DOP). Hydrolysis of DOP to release DIP by alkaline phosphatase is believed to be the most common mechanism of DOP utilization. Little effort has been made, however, to understand other potential molecular mechanisms of utilizing different types of DOP. This study investigated the bioavailability of glucose-6-phosphate (G6P) and its underlying molecular mechanism in the dinoflagellate Karenia mikimotoi. Suppression Subtraction Hybridization (SSH) was used to identify genes up- and down-regulated during G6P utilization compared to DIP condition. The results showed that G6P supported the growth and yield of K. mikimotoi as efficiently as DIP. Neither DIP release nor AP activity was detected in the cultures grown in G6P medium, however, suggesting direct uptake of G6P. SSH analysis and RT-qPCR results showed evidence of metabolic modifications, particularly that mitochondrial ATP synthase f1 gamma subunit and thioredoxin reductase were up-regulated while diphosphatase and pyrophosphatase were down-regulated in the G6P cultures. All the results indicate that K. mikimotoi has developed a mechanism other than alkaline phosphatase to utilize G6P.     

Allelopathic interactions between the macroalga Hizikia fusiformis (Harvey) and the harmful blooms-forming dinoflagellate Karenia mikimotoi

The effects of algal blooms on seaweeds have been rarely studied, although harmful algal blooms (HABs) are now normally regarded as worldwide incidents. In the present study, the effects of dense Karenia mikimotoi cells on the growth and photosynthesis of Hizikia fusiformis, a common and commercially cultivated macroalga in coastal waters of the East China Sea (ECS), were studied to understand the possible consequences when the mariculture encountered a dense harmful algal bloom. Furthermore, the counteraction of the latter on the growth and photosynthetic activities of K. mikimotoi was determined to evaluate the contribution of H. fusiformis commercial cultivation to environmental improvements. The results showed that the chlorophyll a (Chl a) contents, maximal photochemical efficiency (F_v/F_m) and relative electron transfer rate (rETR) of gas vesicles (specialized leaves), adult and young receptacles of H. fusiformis were all significantly (P < 0.05) inhibited compared with the mono-cultured ones. When compared with mono-cultured H. fusiformis (without K. mikimotoi), the Chl a contents in gas vesicles, adult and young receptacles decreased by 20.6%, 17.6% and 33.2% within 2 weeks. Correspondingly, the F_v/F_m decreased by 7.9%, 37.4% and 43.7%; the apparent photosynthetic efficiency (α) decreased by 9.4%, 47.1% and 48.3%; and rETR decreased by 19.5%, 52.6% and 68.2%, respectively. The Chl a concentration of the mono-cultured K. mikimotoi (without H. fusiformis) increased to 2247.97 μg l⁻¹ from 958.11 μg l⁻¹ within 14 d. Those of the co-cultivated ones (with H. fusiformis), however, increased to 1591.31 μg l⁻¹ on the 8th day and then decreased rapidly to 254.99 (±37.73) μg l⁻¹ after the next 6 days. Furthermore, compared with the mono-cultured K. mikimotoi cells, the F_v/F_m, α and rETR_max of co-cultivated ones decreased by 9.4%, 36.3% and 30.6%, respectively. The results indicated that the mature sporophytes of H. fusiformis were resistant to dense K. mikimotoi blooms and this resistance was organ-dependent as: gas vesicle > adult receptacles > young receptacles. On the other hand, commercial mariculture of H. fusiformis demonstrated the potential of preventing the occurrence of algal blooms.     

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.     

Vertical migration of Karenia brevis in the northeastern Gulf of Mexico observed from glider measurements

The toxic marine dinoflagellate, Karenia brevis (the species responsible for most of red tides or harmful algal blooms in the Gulf of Mexico), is known to be able to swim vertically to adapt to the light and nutrient environments, nearly all such observations have been made through controlled experiments using cultures. Here, using continuous 3-dimensional measurements by an ocean glider across a K. brevis bloom in the northeastern Gulf of Mexico between 1 and 8 August 2014, we show the vertical migration behavior of K. brevis. Within the bloom where K. brevis concentration is between 100,000 and 1,000,000 cells L⁻¹, the stratified water shows a two-layer system with the depth of pycnocline ranging between 14–20 m and salinity and temperature in the surface layer being <34.8 and >28 °C, respectively. The bottom layer shows the salinity of >36 and temperature of <26 °C. The low salinity is apparently due to coastal runoff, as the top layer also shows high amount of colored dissolved organic matter (CDOM). Within the top layer, chlorophyll-a fluorescence shows clear diel changes in the vertical structure, an indication of K. brevis vertical migration at a mean speed of 0.5–1 m h⁻¹. The upward migration appears to start at sunrise at a depth of 8–10 m, while the downward migration appears to start at sunset (or when surface light approaches 0) at a depth of ∼2 m. These vertical migrations are believed to be a result of the need of K. brevis cells for light and nutrients in a stable, stratified, and CDOM-rich environment.     

Detection of Karenia brevis blooms on the west Florida shelf using in situ backscattering and fluorescence data

Using shipboard data collected from the central west Florida shelf (WFS) between 2000 and 2001, an optical classification algorithm was developed to differentiate toxic Karenia brevis blooms (>10⁴ cells l⁻¹) from other waters (including non-blooms and blooms of other phytoplankton species). The identification of K. brevis blooms is based on two criteria: (1) chlorophyll a concentration ≥1.5 mg m⁻³ and (2) chlorophyll-specific particulate backscattering at 550 nm ≤ 0.0045 m² mg⁻¹. The classification criteria yielded an overall accuracy of 99% in identifying both K. brevis blooms and other waters from 194 cruise stations. The algorithm was validated using an independent dataset collected from both the central and south WFS between 2005 and 2006. After excluding data from estuarine and post-hurricane turbid waters, an overall accuracy of 94% was achieved with 86% of all K. brevis bloom data points identified successfully. Satisfactory algorithm performance (88% overall accuracy) was also achieved when using underway chlorophyll fluorescence and backscattering data collected during a repeated alongshore transect between Tampa Bay and Florida Bay in 2005 and 2006. These results suggest that it may be possible to use presently available, commercial optical backscattering instrumentation on autonomous platforms (e.g. moorings, gliders, and AUVs) for rapid and timely detection and monitoring of K. brevis blooms on the WFS.     

Brevenal,a brevetoxin antagonist from Karenia brevis,binds to a previously unreported site on mammalian sodium channels

Brevetoxins are a family of ladder-frame polyether toxins produced by the marine dinoflagellate Karenia brevis. During blooms of K. brevis, inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in persons at the beach. Consumption of either shellfish or finfish contaminated by K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to binding at a defined site on, and subsequent activation of, voltage-sensitive sodium channels (VSSCs) in cell membranes (site 5). In addition to brevetoxins, K. brevis produces several other ladder-frame compounds. One of these compounds, brevenal, has been shown to antagonize the effects of brevetoxin. In an effort to further characterize the effects of brevenal, a radioactive analog ([³H]-brevenol) was produced by reducing the terminal aldehyde moiety of brevenal to an alcohol using tritiated sodium borohydride. A K_D of 67 nM and B_max of 7.1 pmol/mg protein were obtained for [³H]-brevenol in rat brain synaptosomes, suggesting a 1:1 matching with VSSCs. Brevenal and brevenol competed for [³H]-brevenol binding with K_i values of 75 nM and 56 nM, respectively. However, although both brevenal and brevenol inhibited brevetoxin binding, brevetoxin was completely ineffective at competition for [³H]-brevenol binding. After examining other site-specific compounds, it was determined that [³H]-brevenol binds to a site that is distinct from the other known sites on the sodium channel, including the brevetoxin site, (site 5) although some interaction with site 5 is apparent.     

Contribution of diazotrophy to nitrogen inputs supporting Karenia brevis blooms in the Gulf of Mexico

Blooms of Karenia brevis plague the West Florida Shelf (WFS) region in the Gulf of Mexico (GOM) where they exert harmful effects on aquatic biota and humans. Because productivity on the WFS is N limited, new N inputs into the region are thought to trigger blooms of K. brevis. Here we examine the potential for new N inputs via N₂ fixation by Trichodesmium and other diazotrophic plankton to contribute to the N demand of K. brevis. Because of possible methodological biases, we also compared N₂ fixation rates by cultured Trichodesmium using the ¹⁵N₂ bubble addition method and the ¹⁵N₂ saturated seawater. Both methods yielded identical results in 12 and 24 h incubations; however, there was more variability in rate estimates made using the bubble addition method. Pelagic N₂ fixation rates by other planktonic diazotrophs ranged from 0 to 13.6 nmol N L⁻¹ d⁻¹, comparable to or higher than rates observed in oligotrophic gyres. These rates should be considered conservative estimates because they were made using the bubble addition method. Integrating over our study area, we estimate that new inputs of N to the WFS via N₂ fixation are on the order of 0.011 Tmol N annually. Further, we measured directly the trophic transfer of recently fixed N₂ to co-occurring plankton that included K. brevis and found that up to 47% of N₂ fixed was transferred to non-diazotrophic plankton even in short (<6 h) incubations where N₂ fixation was likely underestimated.     

Comparative diel oxygen cycles preceding and during a Karenia bloom in Sarasota Bay,Florida, USA

The diel change in dissolved oxygen concentrations were recorded with an automated incubator containing a pulsed oxygen sensor in Sarasota Bay, Florida. The deployments occurred during a ‘pre-bloom’ period in May to June 2006, and during a harmful algal bloom dominated by Karenia brevis in September 2006. The diurnal (daylight) increase in dissolved oxygen concentrations varied from 16 to 104 μmol O₂ l⁻¹ with the corresponding nocturnal decrease in oxygen varying from 16 to 77 μmol O₂ l⁻¹. Nocturnal respiration consumed 42–113% of the diurnal net oxygen production with the minimum and maximum during the pre-bloom period. Hourly production rates closely followed fluctuations in irradiance with maximum rates in the late morning. Hourly oxygen utilization rates (community respiration) at night were highest during the first few hours after sunset.     

Three-dimensional structure of a Karenia brevis bloom: Observations from gliders,satellites, and field measurements

Autonomous underwater gliders with customized sensors were deployed in October 2011 on the central West Florida Shelf to measure a Karenia brevis bloom, which was captured in satellite imagery since late September 2011. Combined with in situ taxonomy data, satellite measurements, and numerical circulation models, the glider measurements provided information on the three-dimensional structure of the bloom. Temperature, salinity, fluorescence of colored dissolved organic matter (CDOM) and chlorophyll-a, particulate backscattering coefficient, and K. brevis-specific chlorophyll-a concentrations were measured by the gliders over >250 km from the surface to about 30-m water depth on the shallow shelf. At the time of sampling the bloom was characterized by uniform vertical structures, with relatively high chlorophyll-a and CDOM fluorescence, low temperature, and high salinity. Satellite data extracted along the glider tracks demonstrated coherent spatial variations as observed by the gliders. Further, the synoptic satellite observations revealed the bloom evolution during the 7 months between late September 2011 and mid April 2012, and showed the maximum bloom size of ∼3000 km² around 23 November. The combined satellite and in situ data also confirmed that the ratio of satellite-derived fluorescence line height (FLH) to particulate backscattering coefficient at 547 nm (b_bp(547)) could be used as a better index than FLH alone to detect K. brevis blooms. Numerical circulation models further suggested that the bloom could have been initiated offshore and advected onshore via the bottom Ekman layer. The case study here demonstrates the unique value of an integrated coastal ocean observing system in studying harmful algal blooms (HABs).     

Examination of six commonly used laboratory fixatives in HAB monitoring programs for their use in quantitative PCR based on Taqman probe technology

Due to the need for more rapid and reliable detection, quantification and enumeration of harmful algal species the use of molecular methods are increasingly being used in monitoring and field studies. However, many studies often require sample fixation to allow for transportation before analyses are conducted. Here, we describe the effects of six fixatives (acidified Lugol's iodine with or without sodium thiosulphate, glutaraldehyde, paraformaldehyde (PFA), formalin and ethanol) on quantitative real-time polymerase chain reaction (qPCR) amplification with Taqman probes. We applied extracted total genomic DNA from four harmful algal species from Danish waters, representing three dinoflagellates (Alexandrium tamarense, Karenia mikimotoi, Karlodinium veneficum and a haptophyte (Prymnesium parvum). The C_q values generated on the qPCR amplification plot were compared to those of an unfixed sample that acted as a control. For all species positive amplifications were achieved from DNA templates from all preserved samples. However, amplification efficiencies between fixatives and species varied. Yet it was found that Lugol's iodine was the most ideal short-term fixative for enumeration of cells by qPCR as well as being the safest to handle. The effect of age on Lugol's iodine fixed samples was also addressed. Samples were fixed and stored at 5 °C in the dark and total genomic DNA extracted after 24 h, 72 h, 1 week, 2 weeks, 1 month and 2 months. Samples remained stable for 1 month for A. tamarense and K. veneficum and 2 months for K. mikimotoi and P. parvum.     

Nitrogen uptake kinetics in field populations and cultured strains of Karenia brevis

This study represents the most comprehensive assessment of kinetic parameters for Karenia brevis to date as it encompasses natural populations sampled during three different bloom years in addition to cultured strains under controlled conditions. Nitrogen (N) uptake kinetics for ammonium (NH₄⁺), nitrate (NO₃⁻), urea, an amino acid mixture, individual amino acids (glutamate and alanine), and humic substrates were examined for the toxic red tide dinoflagellate, K. brevis, during short term incubations (0.5–1 h) using ¹⁵N tracer techniques. Experiments were conducted using natural populations collected during extensive blooms along the West Florida Shelf in October 2001, 2002, and 2007, and in cultured strains (CCFWC 251 and CCFWC 267) obtained from the Florida Fish and Wildlife Institute culture collection. Kinetic parameters for the maximum uptake velocity (V_max), half-saturation concentration (K_s), and the affinity constant (α) were determined. The affinity constant is considered a more accurate indicator of substrate affinity at low concentrations. K. brevis took up all organic substrates tested, including N derived from humic substances. Uptake rates of the amino acid mixture and some NO₃⁻ incubations did not saturate even at the highest substrate additions (50–200 μmol N L⁻¹). Based upon the calculated α values, the greatest substrate preference was for NH₄⁺ followed by NO₃⁻ ≥ urea, humic compounds and amino acids. The ability of K. brevis to utilize a variety of inorganic and organic substrates likely helps it flourish under a wide range of nutrient conditions from bloom initiation in oligotrophic waters offshore to bloom maintenance near shore where ambient nutrient concentrations may be orders of magnitude greater.     

Effects of nutrient-limiting supply ratios on toxin content of Karenia brevis grown in continuous culture

Toxins produced as secondary metabolites can play important roles in phytoplankton communities and contribute to the ecological success of harmful algal bloom (HAB) taxa. Toxin composition and content in phytoplankton are affected by a suite of environmental factors, including nutrient availability. Changes in nutrient availability can increase or decrease toxin content and alter toxin composition, depending on toxin stoichiometry and the mechanisms by which nutrient limitation affects toxin production. The studies that have assessed the effects of nutrient availability on brevetoxin content of the HAB species Karenia brevis have reported contradictory results, although there is growing support that nutrient limitation increases brevetoxin content. In this study, we assessed the effects of decreased nitrogen (N) and phosphorus (P) availability on brevetoxin content and composition of K. brevis grown in chemostats at steady state by altering the nutrient supply ratios of incoming media from the Redfield Ratio. Overall, brevetoxin content was greatest in cultures grown at the lowest rate, regardless of the nutrient supply ratio (i.e., under both Redfield and N-limiting supply ratios). Compared to cultures grown at 0.2 d⁻¹, cultures grown at 0.1 d⁻¹ exhibited 5-fold increases in intracellular toxin content. In contrast, at constant growth rates, N-limiting supply ratios decreased intracellular brevetoxin content by approximately one-third, although this result was significant only in cultures growing at the fastest rate of 0.23 d⁻¹. P-limiting supply ratios had no effect on brevetoxin content or composition. In addition, when cultures grown at rates of 0.2 d⁻¹ were supplied with balanced/Redfield N:P supply ratios, but different absolute nutrient concentrations, toxin content was greater under greater nutrient concentrations. These findings suggest that when growth rate is not nutrient limited, there is a positive relationship between nutrient availability and brevetoxin content. This work contributes to previous studies by demonstrating strong growth rates effects on brevetoxin content and that growth rate and nutrient availability can independently or together affect toxin content of K. brevis. Moreover, our work underscores the value of the chemostat as a tool to elucidate the mechanisms by which nutrient availability and growth rate affect toxin production and content of HAB species.     

Chemical Response of the Toxic Dinoflagellate Karenia mikimotoi Against Grazing by Three Species of Zooplankton

We investigated the toxicity of Karenia mikimotoi toward three model grazers, the cladoceran Moina mongolica, the copepod Pseudodiaptomus annandalei, and the crustacean Artemia salina, and explored its chemical response upon zooplankton grazing. An induction experiment, where K. mikimotoi was exposed to grazers or waterborne cues from the mixed cultures revealed that K. mikimotoi might be toxic or nutritionally inadequate toward the three grazers. In general, direct exposure to the three grazers induced the production of hemolytic toxins and the synthesis of eicosapentaenoic acid (EPA). Both EPA and the hemolytic toxins from K. mikimotoi decreased the survival rate of the three grazers. In addition, the survival rates of M. mongolica, P. annandalei, and A. salina in the presence of induced K. mikimotoi that had previously been exposed to a certain grazer were lower than their counterparts caused by fresh K. mikimotoi, suggesting that exposure to some grazers might increase the toxicity of K. mikimotoi. The chemical response and associated increased resistance to further grazing suggested that K. mikimotoi could produce deterrents to protect against grazing by zooplankton and that the substances responsible might be hemolytic toxins and EPA.     

Ichthyotoxicity of four species of gymnodinioid dinoflagellates (Kareniaceae,Dinophyta) and purified karlotoxins to larval sheepshead minnow

Two species of Kareniaceae, Karlodinium veneficum (Swan and Huon River isolates) and Karlodinium conicum, and their respective purified karlotoxins (KmTx), were investigated for ichthyotoxicity on larval sheepshead minnow. Two non-karlotoxin producing species, Karenia mikimotoi and Karlodinium ballantinum were also tested. Algal treatments included live and lysed cells (homogenized and CuSO₄ treated) with fish mortalities observed from lysed Ka. veneficum and Ka. conicum but none observed from K. mikimotoi and Ka. ballantinum. The variance in ichthyotoxicity between live and lysed cells of Ka. veneficum (Swan and Huon River) and Ka. conicum (Southern Ocean) confirm that toxin is cell bound and ichthyotoxicity increases upon lysis. Ichthyotoxic blooms of Ka. veneficum in situ in the Swan River, Western Australia and Chesapeake Bay, Maryland, USA are unrelated to algal cell density as mortality was observed with low densities. In laboratory treatments, no fish mortalities were observed upon exposure to live intact cells of all four species at algal concentrations up to 2.5 × 10⁵ cells/mL in replete nutrient growth conditions. Lysed low density (3 × 10⁴ cells/mL) Ka. veneficum (Swan and Huon River) grown under P-limited nutrients caused quicker fish mortality than those cultured in replete nutrient conditions. Pure toxin isolated from Ka. veneficum (Swan and Huon River) and Ka. conicum (Southern Ocean) were toxic to sheepshead minnow larvae, with the lethal dose lowest for Km^HuonTx 2 (508.2 ng/mL), followed by Km^SwanTx 2-1 (563.2 ng/mL), and Km_conicumTx (762.4 ng/mL).