Karenia Brevis Research Articles

Detection of dinoflagellates by the light scattering properties of the chiral structure of their chromosomes

One of the most prominent properties of dinoflagellates is their large sized and highly chromosome-laden nucleus, which contains dozens of cylindrically shaped chromosomes. With such high chromatic concentration, these chromosomes condense into ordered helical structures and were claimed to be responsible for the large circular polarization effects observed in the light scattering from dinoflagellates. In previous research, a thin helix model of a chromosome was used to compare the Discrete Dipole Approximation (DDA) and the analytical Born approximation calculations. However, for such a simplified model only modest qualitative agreements with experimental measurements were achieved. Moreover, only one chromosome in one nucleus was simulated, overlooking the effects of interactions between chromosomes. In this work, we adopt the helical plywood liquid crystal model with a capsule shape, in which parallel fibrils lie in plains perpendicular to the helix axis and the orientations of these fibrils twist at a constant angle between two neighboring layers. The ADDA code is applied to calculate the 16 Mueller matrix elements of light scattering from a single chromosome and from the nucleus, which is composed of a collection of randomly positioned and randomly orientated chromosomes. Special attention is paid to the S14 Mueller matrix element, which describes the ability of differentiating left and right circularly polarized light. Our results show that large S14 back scattering signals from the dinoflagellate nucleus results from the underlying helical structures of its chromosomes. These signals are sensitive to the light wavelength and pitch of the chromatic helix, the latter of which is species specific. Therefore, detecting back scattering S14 signal could be a promising method to monitor dinoflagellates such as Karenia brevis, the causal agent of the Florida red tide.

Bioaccumulation and depuration of brevetoxins in the eastern oyster (Crassostrea virginica) and the northern quahog (= hard clam, Mercenaria mercenaria)

The eastern oyster (Crassostrea virginica) and northern quahog (= hard clam, Mercenaria mercenaria) are two species of economic and ecological significance in east coast waters of the United States and the Gulf of Mexico. Commercial industries for these species, especially within the state of Florida, are significant. The current study was undertaken to build upon the already established body of knowledge surrounding effects of the toxic dinoflagellate Karenia brevis on shellfish, to provide an understanding of the kinetics of brevetoxins within shellfish tissues, and to provide an estimate of brevetoxin retention times in these shellfish after a bloom event.Individual clams and oysters were exposed to the toxic dinoflagellate, K. brevis at a bloom concentration of 5 × 105 cells·L−1 for eight days and then transferred to filtered water for depuration. Individuals were sampled periodically to determine depuration rates. Concentrations of brevetoxins (and/or their metabolites measured as PbTx-3 equivalent) in tissues were determined using an Enzyme Linked Immunosorbent Assay (ELISA). After five days of exposure, brevetoxin levels in tissues of both species reached concentrations well above the regulatory limit of 800 ng g−1 (Pb-TX3 equivalent). Averaged concentration of brevetoxins in clams was 1000 ng g−1, while the oysters averaged 1986 ng g−1. After two weeks of depuration, tissue concentrations in both species were below regulatory levels with clams averaging ∼204 ng g−1 and oysters averaging ∼437 ng g−1. Toxins (or their metabolities) remained detectable in both clams (139 days) and oysters (82 days) for the duration of the experiment.

Biofouling ascidians on aquaculture gear as potential vectors of harmful algal introductions

Biofouling ascidians are ubiquitous in coastal ecosystems and are among the main colonizers of aquaculture gear. Our study tested the hypothesis that the transport, removal, and transfer of fouling ascidian species by aquaculturists provide a mechanism for concentration and distribution of harmful-algal cells to new areas. Wild-caught specimens of common, biofouling ascidian species (Styela clava, Ciona intestinalis, Molgula manhattensis, Botrylloides violaceus, Didemnum vexillum, and Botryllus schlosseri) were exposed individually to cultured strains of co-occurring harmful algae (Prorocentrum minimum, Alexandrium fundyense, Alexandrium monilatum, Karenia brevis, Aureococcus anophagefferens, or Heterosigma akashiwo) at simulated bloom cell densities of each HAB species. After feeding, ascidians were transferred to ultrafiltered seawater. Immediately after exposure, and after 24 and 48h in ultrafiltered seawater, biodeposits were collected and observed microscopically for the presence of intact, potentially viable cells. Subsamples of biodeposits were transferred into culture tubes with ultrafiltered seawater and monitored for algal growth during 8 weeks. Cells of all HAB species were found to pass intact through the ascidian digestive system, remained viable, and in many cases were capable of re-establishing populations at least 48h post-ingestion. The results of our study will inform industry and managers of the potential threat and ecological impact of spreading biofouling ascidians, and practices to mitigate adverse impacts. Additionally, these management practices have been formally incorporated into a new cost-share program developed to help shellfish producers prevent the further spread of ascidians and associated HAB species.

Real-time isothermal RNA amplification of toxic marine microalgae using preserved reagents on an integrated microfluidic platform

Quantitation of specific RNA sequences is a useful technique in marine biology that can elucidate cell abundance, speciation and viability, especially for early detection of harmful algal blooms. We are thus developing an integrated microfluidic system for cell concentration and lysis, RNA extraction/purification and quantitative RNA detection for environmental applications. The portable system is based on a microfluidic cartridge, or "lab-card", using a low-cost injection moulded device, with a laminated lid. Here we present real-time isothermal RNA amplification using reagent master-mixes preserved on-chip in a gel at 4 °C for up to eight months. We demonstrate quantitation by reference to an internal control in a competitive assay with 500 cell equivalents of the toxic microalga Karenia brevis. Annealing of primers, amplification at 41 °C and real-time fluorescence detection of the internal control and target using sequence-specific molecular beacons were all performed on-chip.

Genetic diversity among clonal isolates of Karenia brevis as measured with microsatellite markers

Karenia brevis is the major harmful bloom-forming dinoflagellate in the Gulf of Mexico yet little is known about the intraspecific genetic diversity of this species. Here we describe nine new microsatellite markers and, combined with nine previously described microsatellites, use them to genotype 40 cultured isolates of K. brevis. Genetic diversity identified from cultured isolates was compared with the genetic diversity identified from two field samples to assess how well the current cultures represent the field population. Thirty-nine unique haplotypes were identified from 40 cultured isolates of K. brevis using 18 microsatellite markers. Genetic diversity was similar between cultured isolates and the two field samples. The success of 18 microsatellite markers to distinguish individual isolates supports the use of microsatellites as a genetic tool for diagnostic identification of cultured isolates of K. brevis.

Putative Monofunctional Type I Polyketide Synthase Units: A Dinoflagellate-Specific Feature?

Marine dinoflagellates (alveolata) are microalgae of which some cause harmful algal blooms and produce a broad variety of most likely polyketide synthesis derived phycotoxins. Recently, novel polyketide synthesase (PKS) transcripts have been described from the Florida red tide dinoflagellate Karenia brevis (gymnodiniales) which are evolutionarily related to Type I PKS but were apparently expressed as monofunctional proteins, a feature typical of Type II PKS. Here, we investigated expression units of PKS I-like sequences in Alexandrium ostenfeldii (gonyaulacales) and Heterocapsa triquetra (peridiniales) at the transcript and protein level. The five full length transcripts we obtained were all characterized by polyadenylation, a 3′ UTR and the dinoflagellate specific spliced leader sequence at the 5′end. Each of the five transcripts encoded a single ketoacylsynthase (KS) domain showing high similarity to K. brevis KS sequences. The monofunctional structure was also confirmed using dinoflagellate specific KS antibodies in Western Blots. In a maximum likelihood phylogenetic analysis of KS domains from diverse PKSs, dinoflagellate KSs formed a clade placed well within the protist Type I PKS clade between apicomplexa, haptophytes and chlorophytes. These findings indicate that the atypical PKS I structure, i.e., expression as putative monofunctional units, might be a dinoflagellate specific feature. In addition, the sequenced transcripts harbored a previously unknown, apparently dinoflagellate specific conserved N-terminal domain. We discuss the implications of this novel region with regard to the putative monofunctional organization of Type I PKS in dinoflagellates.

Comparative analysis of three brevetoxin-associated bottlenose dolphin (Tursiops truncatus) mortality events in the Florida Panhandle region (USA).

In the Florida Panhandle region, bottlenose dolphins (Tursiops truncatus) have been highly susceptible to large-scale unusual mortality events (UMEs) that may have been the result of exposure to blooms of the dinoflagellate Karenia brevis and its neurotoxin, brevetoxin (PbTx). Between 1999 and 2006, three bottlenose dolphin UMEs occurred in the Florida Panhandle region. The primary objective of this study was to determine if these mortality events were due to brevetoxicosis. Analysis of over 850 samples from 105 bottlenose dolphins and associated prey items were analyzed for algal toxins and have provided details on tissue distribution, pathways of trophic transfer, and spatial-temporal trends for each mortality event. In 1999/2000, 152 dolphins died following extensive K. brevis blooms and brevetoxin was detected in 52% of animals tested at concentrations up to 500 ng/g. In 2004, 105 bottlenose dolphins died in the absence of an identifiable K. brevis bloom; however, 100% of the tested animals were positive for brevetoxin at concentrations up to 29,126 ng/mL. Dolphin stomach contents frequently consisted of brevetoxin-contaminated menhaden. In addition, another potentially toxigenic algal species, Pseudo-nitzschia, was present and low levels of the neurotoxin domoic acid (DA) were detected in nearly all tested animals (89%). In 2005/2006, 90 bottlenose dolphins died that were initially coincident with high densities of K. brevis. Most (93%) of the tested animals were positive for brevetoxin at concentrations up to 2,724 ng/mL. No DA was detected in these animals despite the presence of an intense DA-producing Pseudo-nitzschia bloom. In contrast to the absence or very low levels of brevetoxins measured in live dolphins, and those stranding in the absence of a K. brevis bloom, these data, taken together with the absence of any other obvious pathology, provide strong evidence that brevetoxin was the causative agent involved in these bottlenose dolphin mortality events.

Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins

Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY®-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY®–PbTx-2 conjugate was found to displace [3H]–PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [3H]–PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

The toxic effects of three dinoflagellate species from the genus Karenia on invertebrate larvae and finfish

Three species of Kareniaceae, Karenia brevis, Karenia brevisulcata and Karenia mikimotoi were assessed for toxicity to juvenile Chinook salmon (Oncorhynchus tshawytscha), snapper (Pagrus auratus) and six species of marine invertebrate larvae (GreenshellTM mussel, pacific oyster, paua [New Zealand abalone], sea slug, sea urchin and brine shrimp). Karenia brevis was ichthyotoxic to juvenile salmon with a LT50 of 0.75 h at 0.18 × 106 cells L–1 but juvenile snapper were less sensitive (LT50 of 4.5 h at 5.9 × 106 cells L–1). Karenia brevisulcata also showed differential toxicity to juvenile salmon and snapper with LT50 of 0.48 h at 11 × 106 cells L–1 and 7.5 h at 16 × 106 cells L–1 respectively. However, no acute symptoms were observed in the fish following 48 h exposure to K. mikimotoi at 3.4 × 106 cells L–1. Karenia brevis at the highest level tested (8.7 × 106 cells L–1) did not cause observable symptoms in any of the invertebrate larvae over 24 h. Karenia mikimotoi only affected paua larvae (LT50 10 h at 12 × 106 cells L–1). Karenia brevisulcata was highly toxic to mussel, sea urchin and paua larvae (LT50 1–6.1 h at 7.5–9.6 × 106 cells L–1), toxic to sea slug and oyster larvae (LT50 6 h at 80 × 106 cells L–1 and 9.2 h at 34 × 106 cells L–1 respectively) but did not affect brine shrimp. Analysis of test cultures by liquid chromatography-mass spectrometry confirmed that K. brevis produced brevetoxins (52 pg cell–1; mainly brevetoxin-2). Karenia brevisulcata produced brevisulcatic acids (BSXs) and the more toxic Karenia brevisulcata toxins (KBTs). Karenia mikimotoi did not produce brevetoxin, BSXs or KBTs. The present study confirms the broad threats that blooms of K. brevisulcata pose to marine ecosystems and aquaculture, and the hazards of K. brevis to finfish and K. mikimotoi to abalone respectively.

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.

A 1-D simulation analysis of the development and maintenance of the 2001 red tide of the ichthyotoxic dinoflagellate Karenia brevis on the West Florida shelf

A one-dimensional (1-D) ecological model, HABSIM, examined the initiation and maintenance of the 2001 red tide on the West Florida shelf (WFS). Phytoplankton competition among toxic dinoflagellates (Karenia brevis), nitrogen fixing cyanophytes (Trichodesmium erythraeum), large siliceous phytoplankton (diatoms), and small non-siliceous phytoplankton (microflagellates) explored the sequence of events required to support the observed red tide from August to December 2001. The ecological model contained 24 state variables within five submodels: circulation, atmospheric (iron deposition), bio-optics, pelagic (phytoplankton, nutrients, bacteria, zooplankton, and fish), and benthic (nutrient regeneration). The 2001 model results reaffirmed that diazotrophs are the basis for initiation of red tides of K. brevis on the WFS. A combination of selective grazing pressure, iron fertilization, low molar nitrogen to phosphorus ratios, and eventual silica limitation of fast-growing diatoms set the stage for dominance of nitrogen fixers. “New” nitrogen was made available for subsequent blooms of K. brevis through the release of ammonium and urea during nitrogen fixation, as well as during cell lysis, by the Trichodesmium population. Once K. brevis biomass reached ichthyotoxic levels, rapid decay of subsequent fish kills supplied additional organic nutrients for utilization by these opportunistic toxic algae. Both nutrient vectors represented organic non-siliceous sources of nitrogen and phosphorus, further exacerbating silica limitation of the diatom population. The model reproduced this spring transition from a simple estuarine-driven, diatom-based food chain to a complex summer–fall system of Trichodesmium and toxic dinoflagellates. While the model was able to replicate the initiation and maintenance of the 2001 red tide, bloom termination was not captured by this 1-D form on the WFS. Here, horizontal advection and perhaps cell lysis loss terms might play a significant role, to be addressed in future three-dimensional simulations.

Biogeographic effects of the Gulf of Mexico red tide dinoflagellate Karenia brevis on Mediterranean copepods

There may be biogeographic aspects of interactions between toxic phytoplankton and their zooplankton grazers. Grazers that naturally co-occur with a given species of toxic phytoplankton may have evolved mechanisms to counteract adverse effects of phytoplankton toxins. Conversely, grazers that have no co-evolutionary experience with a toxic phytoplankter may be more susceptible to its effects. This may also be true for toxic metabolites other than neurotoxins that adversely affect copepod reproduction and development such as the well-described diatom oxylipins. In order to test this hypothesis, we investigated interactions in terms of feeding, reproductive success and survival of a copepod feeding on a potentially toxic dinoflagellate species with which the copepod does not naturally co-occur: the red tide dinoflagellate Karenia brevis from the Gulf of Mexico and the copepod Calanus helgolandicus from the Mediterranean. We additionally compared interactions of these two allopatric plankters with various co-occurring sympatric ones, including the copepod Temora stylifera and a non-neurotoxic strain of the dinoflagellate Alexandrium tamarense, both from the Mediterranean, and as a control, the non-toxic dinoflagellate Prorocentrum minimum. Both Mediterranean copepods fed, reproduced and survived on diets of K. brevis from the Gulf of Mexico, but neither copepod did as well on this diet as on diets of A. tamarense from the Mediterranean or the control diet of P. minimum. Of the two copepods, T. stylifera fared worse on a diet of K. brevis than did C. helgolandicus. Egg hatching success for both copepods feeding on K. brevis was sharply diminished over the seven days of the experiments. This was surprising, since we subsequently discovered that our culture of K. brevis contained no brevetoxins. However, our culture appeared to contain another as yet unidentified compound. Similarly, we have previously demonstrated that our culture of A. tamarense, which contained no saxitoxins or aldehydes, did contain another unidentified compound that reduced egg hatching success by inhibiting sperm from fertilizing eggs. Thus, although diets of the dinoflagellate from the Gulf of Mexico (K. brevis) caused adverse effects on both of the Mediterranean copepods, T. stylifera was also adversely affected by the Mediterranean dinoflagellate (A. tamarense). The copepod T. stylifera which co-occurs in the Gulf of Mexico with the dinoflagellate K. brevis was far more adversely affected by feeding on a sole diet of this dinoflagellate than was the Mediterranean copepod (C. helgolandicus) which never naturally co-occurs with this dinoflagellate. Thus, any putative biogeographic effects of copepods feeding on allopatric versus sympatric dinoflagellates did not clearly emerge from our data. However, since both species of dinoflagellates appear to contain additional undescribed chemicals other than the neurotoxins for which they are best known, and these compounds are potentially related to the adverse effects we recorded, it appears that understanding of the biogeography must await further defining the biochemistry of some harmful algae:grazer interactions.

Binational collaboration to study Gulf of Mexico's harmful algae

Blooms of the toxic marine dinoflagellate Karenia brevis cause massive fish kills and other public health and economic problems in coastal waters throughout the Gulf of Mexico [Steidinger, 2009]. These harmful algal blooms (HABs) are a gulf‐wide problem that require a synoptic observing system for better serving decision‐making needs. The major nutrient sources that initiate and maintain these HABs and the possible connectivity of blooms in different locations are important questions being addressed through new collaborations between Mexican and U.S. researchers and government institutions. These efforts were originally organized under the U.S./Mexico binational partnership for the HABs Observing System (HABSOS), led by the U.S. Environmental Protection Agency's Gulf of Mexico Program (EPAGMP) and several agencies in Veracruz, Mexico, since 2006. In 2010 these efforts were expanded to include other Mexican states and institutions with the integrated assessment and management of the Gulf of Mexico Large Marine Ecosystem (GoMLME) program sponsored by the Global Environment Facility (GEF), the United Nations Industrial Development Organization (UNIDO), the Secretaría de Medio Ambiente y Recursos Naturales (SEMARNAT), and the National Oceanic and Atmospheric Administration (NOAA).

Trophic transfer of brevetoxins to the benthic macrofaunal community during a bloom of the harmful dinoflagellate Karenia brevis in Sarasota Bay, Florida

Harmful algal blooms can cause mass mortalities of top predators such as fish, marine mammals and seabirds but the food web transfer from toxic phytoplankton to these organisms has not been fully elucidated. Macrobenthic invertebrates in coastal waters, including bivalve suspension- and deposit-feeders, carnivorous gastropods, deposit-feeding amphipods and polychaetes, are a major food source for a wide variety of predators and can thus play a critical role in the trophic transfer of algal toxins to higher trophic levels. The objective of this study was to investigate toxin accumulation in transplanted juvenile hard clams, Mercenaria mercenaria, a species naturally occurring in the region, and in various macrobenthic functional groups from Florida coastal waters during a natural bloom of the dinoflagellate, Karenia brevis, a producer of brevetoxins. Bloom concentrations in the water column ranged from 100 to 1200cellsml−1 over the course of the experiment. This study revealed that these lipophilic toxins can be rapidly accumulated by both suspension- and deposit-feeding benthos, especially bivalve molluscs [1.9–2.8μg PbTx-3 eq (g wet weight)−1]. Transplanted M. mercenaria rapidly accumulated toxins from the water column attaining ∼0.5μg PbTx-3 eq (g wet tissue)−1 after only 4h-exposure to the K. brevis bloom and a maximum value of 1.5±0.2μg PbTx-3 eq (g wet tissue)−1 after 72h. Relatively high brevetoxin concentrations were also measured in co-occurring benthic carnivorous gastropods [1–2.6μg PbTx-3 eq (g wet weight, WW)−1]. Mean toxin concentrations in polychaetes and crustaceans varied in the range ∼0.04–0.2μg PbTx-3 eq (g WW)−1 over the study period, and thus were typically lower than in molluscs. This study demonstrated in situ toxin accumulation by benthic primary and secondary consumers during a natural Florida red tide. Accumulation by primary consumers may be highly variable in space and time (as shown in bivalves from the natural benthic community) and among taxonomic groups. Toxin transfer further up the food web will thus depend on the toxin level accumulated in prey, the number of pathways from which the predator may accumulate toxins and on possible biological magnification of lipophilic toxins. Overall, this study revealed qualitatively and quantitatively that benthic consumers of a number of taxa can serve as vectors for transporting brevetoxins within the food web.

Toxicity and nutritional inadequacy of Karenia brevis: synergistic mechanisms disrupt top-down grazer control

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 444:15-30 (2012) - DOI: https://doi.org/10.3354/meps09401 Toxicity and nutritional inadequacy of Karenia ­brevis: synergistic mechanisms disrupt top-down grazer control Rebecca J. Waggett1,2,*, D. Ransom Hardison1, Patricia A. Tester1 1National Ocean Service, National Oceanic and Atmospheric Administration, 101 Pivers Island Road, Beaufort, North Carolina 28516-9722, USA 2Present address: The University of Tampa, 401 W. Kennedy Blvd., Tampa, Florida 33606, USA *Email: rwaggett@ut.edu ABSTRACT: Zooplankton grazers are capable of influencing food-web dynamics by exerting top-down control over phytoplankton prey populations. Certain toxic or unpalatable algal species have evolved mechanisms to disrupt grazer control, thereby facilitating the formation of massive, monospecific blooms. The harmful algal bloom (HAB)-forming dinoflagellate Karenia brevis has been associated with lethal and sublethal effects on zooplankton that may offer both direct and indirect support of bloom formation and maintenance. Reductions in copepod grazing on K. brevis have been attributed to acute physiological incapacitation and nutritional inadequacy. To evaluate the potential toxicity or nutritional inadequacy of K. brevis, food removal and egg production experiments were conducted using the copepod Acartia tonsa and K. brevis strains CCMP 2228, Wilson, and SP-1, characterized using liquid chromatography-mass spectrometry (LC-MS) as having high, low, and no brevetoxin levels, respectively. Variable grazing rates were found in experiments involving mixtures of toxic CCMP 2228 and Wilson strains. However, in experiments with toxic CCMP 2228 and non-toxic SP-1 strains, A. tonsa grazed SP-1 at significantly higher rates than the toxic alternative. Additionally, A. tonsa experienced significantly greater mortality when exposed to toxic K. brevis strains, particularly after prolonged exposure. Egg production rates of copepods fed toxic K. brevis strains were similar to those of starved copepods, while those of copepods fed non-toxic SP-1 and the nutritious Rhodomonas salina were significantly higher. Analysis indicates that K. brevis impacts grazer populations via multiple synergistic mechanisms: (1) decreased ingestion rates, (2) decreased egg production, and (3) increased mortality of copepods through a combination of toxicity and nutritional inadequacy. KEY WORDS: Copepod grazing · Harmful algae · Toxic dinoflagellate · Chemical deterrent · Karenia brevis · Brevetoxin Full text in pdf format PreviousNextCite this article as: Waggett RJ, Hardison DR, Tester PA (2012) Toxicity and nutritional inadequacy of Karenia ­brevis: synergistic mechanisms disrupt top-down grazer control. Mar Ecol Prog Ser 444:15-30. https://doi.org/10.3354/meps09401 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 444. Online publication date: January 10, 2012 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2012 Inter-Research.

Rapid Detection of Karenia brevis (Dinophyceae) by Loop-mediated Isothermal Amplification (LAMP)

Karenia brevis(Gymnodinium breve) is an unarmored,non-peridinin-containing dinoflagellate.It produces lipophilic brevetoxin that can harm many marine animals,eg.fish,birds and shellfish.As for human,brevetoxin can cause respiratory distress by inhalation and food poisoning by consumption of contaminated shellfish.Previous methods for the detection of K.brevis depend on microscopy analysis,which is time-consuming and requires a considerable amount of expertise and skill.Molecular methods have also been applied to detect K.brevis,e.g.real-time PCR and DNA hybridization assay.However,there are some inevitable disadvantages for the two methods,such as expensive reagents and equipment,or fussy approaches.Loop-mediated isothermal amplification(LAMP) is a specific nucleic acid amplification method that is easy to perform.The LAMP method can amplify nucleic acids under isothermal conditions at approximately 65 ℃.As a result,it allows for the use of simple and inexpensive reaction equipment.The identification system of microalgae K.brevis was successfully established in this study using LAMP method.The specificity and sensitivity were validated based on this detection system.Nine different algae were investigated to confirm the specificity of the LAMP for detecting K.brevis.The results showed that only K.brevis and the samples containing K.brevis were amplified;no amplification occurred in all the other samples including K.mikimotoi.Moreover,LAMP was able to detect template at a 100 to 10-3 dilution,whereas regular PCR could amplify the sample at only a 100 to 10-2 dilution.Therefore,the detection limit of the LAMP system was approximately 5.0×10-2 ng(50 pg) DNA,which was 10 times more sensitive than conventional PCR.LAMP products could be identified by adding SYBR Green I dyes or by the production of a white precipitate.This visual inspection has predominance because there is no need for gel electrophoresis and staining with ethidium bromide.So this method illustrates a novel application prospect for forecasting red-tides caused by K.brevis because of its high sensitivity,rapid reaction and the need for only simple equipment.

Exocytic Mechanisms of Storage and Release of Brevetoxin in the Dinoflagellate Karenia Brevis

Karenia brevis is a broadly distributed toxic dinoflagellate responsible for Red Tide outbreaks throughout the world. Deleterious effects of these blooms are caused by brevetoxin, a potent neurotoxin that binds to sodium channels in nerve and muscle cell leading to substantial marine life mortality and human morbidity. Little is known about the mechanisms of storage and release of toxins in algal species responsible for harmful algal blooms. Toxins have been thought to be exported from these unicellulars by an ill-defined “exudation” mechanism. Although secretion is a standard strategy of material export in plants and animal cells, regulated exocytosis has only recently begun to be explored in dinoflagellates (FEBS Letters 2006, 580:2201-2206). Results presented here using fluorescently-labeled antibrevetoxin antibodies show that brevetoxin is present in Karenia's secretory vesicles and is released following blue light-stimulated exocytosis. The matrix of secretory granules functions as a caging polymer network that holds immobilized high payloads of active molecules including hormones, antibacterial peptides, or in this case toxins (Ann. Rev. Physiol 1990.52: 157-176). Upon release, Karenia's vesicle matrix undergoes typical phase transition from condensed to solvated phase, with characteristic first order kinetics swelling, and release of its brevetoxin payload. These observations support the notion that Karenia b functions as a typical secretory cell, opening the way for a better understanding of Red Tide blooms dynamics.

Karenia brevis red tides and brevetoxin-contaminated fish: a high risk factor for Florida’s scavenging shorebirds?

Shorebirds, including sanderlings ( Calidris alba ) and ruddy turnstones ( Arenaria interpres ), were observed scavenging beached fish, such as thread herring ( Opisthonema oglinum ), scaled sardine ( Harengula jaguana ) and mullet ( Mugil spp.) killed during a 2005 Karenia brevis red tide along the central west coast of Florida. Brevetoxin concentrations in dead fish tissues were analyzed to determine the potential exposure risk to scavenging shorebirds. This component of brevetoxin cycling in the food web has not previously been explored and the risks or benefits of this behavior for shorebirds are not currently understood. Toxin levels in freshly dead fish tissues ranged from 32 to 95,753 ng PbTx g -1 . Brevetoxins in shorebird livers were also confirmed (26–1313 ng PbTx g -1 ) in dead birds collected opportunistically from local beaches and rehabilitation centers during the red tide event, suggesting that brevetoxin exposure is a risk factor for mortality. These findings underscore the need to assess the ecological impacts of K. brevis blooms on Florida’s migratory and resident shorebird populations.

Resolving DOM fluorescence fractions during a Karenia brevis bloom patch on the Southwest Florida Shelf

Organic matter can be supplied naturally from land through rivers or produced in-situ in the marine environment. Current methods of examining natural bulk dissolved organic matter (DOM) are not able to discriminate multiple sources of DOM. A diagnostic tool to identify DOM sources is critical to determine possible sources of organic nutrients that influence K. brevis harmful algal bloom (HAB) development. This study applied multi-wavelength fluorescence coupled with a supervised pattern recognition technique (e.g., parallel factorial analysis (PARAFAC) using samples collected from river, estuary and shelf waters where the toxic dinoflagellate Karenia brevis off of Sanibel Island, Florida was observed. The PARAFAC model distinguished four different fractions of DOM components containing humic-like and protein-like components. The derived terrestrial humic-like material was indicative of land-based sources while the tryptophan-like component was likely produced from in-situ biological production. The study developed and tested the hypothesis that the direct relationship of the protein-like DOM fluorescence with K. brevis cell density indicated that the bloom patch was most likely supported by organic nutrients produced in-situ. The results demonstrated that multi-wavelength fluorescence analysis coupled with PARAFAC modeling technique simultaneously resolved DOM fluorescence fractions and their possible sources—information that are critical in explaining harmful algal bloom formation.

Growth and competition of several harmful dinoflagellates under different nutrient and light conditions

Three near-shore type harmful dinoflagellates, Prorocentrum minimum, Prorocentrum donghaiense and Karlodinium veneficum, and one off-shore dinoflagellate, Karenia brevis, were grown in laboratory monocultures and mixed batch cultures. The dinoflagellate cultures were grown on treatments of two ambient nitrogen (N):phosphorus (P) ratios; two N substrates (nitrate and urea) and two light intensities. The microalgae Rhodomonas and Synechococcus were also added in separate treatments to the mixed culture treatments as potential food sources. All tested species grew well on both N substrates. In mixed culture, P. minimum outgrew K. veneficum, and P. donghaiense outgrew K. brevis in most treatments reaching higher growth rates and higher biomass. However, when a third algae, Rhodomonas, was added, the growth of P. minimum was inhibited relative to that of K. veneficum. In contrast, when grown with K. brevis, the growth rate of P. donghaiense was not significantly affected by the addition of potential prey. K. brevis had a longer growth phase, and kept growing after P. donghaiense reached stationary phase, suggesting better adaptation of K. brevis to low inorganic nutrient conditions. The growth of K. brevis was also significantly limited in the low light treatment. K. veneficum overgrew P. minimum in the presence of Rhodomonas, a potential nutrient source. The growth rates of both K. brevis and P. donghaiense were reduced with the presence of Synechococcus. In addition to nutrient competition, mixotrophy and allelopathy were likely mechanisms in determining the dominant species.