Karenia brevis causes high mortality and impaired swimming behavior of Florida stone crab larvae.

The dinoflagellate Karenia brevis causes harmful algal blooms commonly referred to as red tides that are prevalent along Florida's gulf coast. Severe blooms often cause fish kills, turbid water, and hypoxic events all of which can negatively impact local fisheries. The stone crab, Menippe mercenaria, is a ˜$25 million per year fishery that occurs primarily along Florida's gulf coast. On the west Florida shelf, red tides occur from fall through spring, although severe blooms can occur during the summer. During the summer, stone crabs are reproductive and release larvae that are transported offshore where K. brevis blooms originate. This study determined the effects of K. brevis exposure on the survivorship, vertical swimming behavior, and oxygen consumption of stage-1 larval stone crabs. Survivorship was determined by exposing larvae to high (> 1 × 106 cells L-1) and medium (˜1 × 105 cells L-1) K. brevis concentrations for 96-hrs and were compared to controls that had no algae present. Larval swimming behavior (i.e., geotaxis) and oxygen consumption were monitored after 6-hr exposure to K. brevis. After 96-hrs of exposure, mortality was 100% and 30% for larvae in the high and medium concentrations of K. brevis, respectively, relative to the control. Larval swimming behavior was reversed in the K. brevis treatment; however oxygen consumption rates did not differ among treatments. These results suggest that severe blooms during the summer may reduce larval supply and serve as a potential bottleneck for new individuals recruiting into the fishery in years following a K. brevis bloom.

The effects of red tide (Karenia brevis) on reflex impairment and mortality of sublegal Florida stone crabs, Menippe mercenaria.

The Florida stone crab, Menippe mercenaria, is a major commercial fishery that occurs primarily along Florida's west coast, where harmful algal blooms of Karenia brevis frequently develop. To determine sublethal and lethal effects of K. brevis on M. mercenaria, we exposed sublegal stone crabs to three seawater treatments in laboratory conditions: no K. brevis (control), a low-toxin K. brevis strain (Wilson LT), and a toxic K. brevis (New Pass strain). Total food consumed, reflex impairment and survivorship of each crab was monitored throughout the nine-day experiment. Crabs in the toxic treatment consumed 67% less food. The probability of an individual losing a reflex significantly increased with time (days), and there was a 42% decrease in survivorship in the toxic treatment. This is the first study to demonstrate negative effects of K. brevis on the stone crab, presenting the critical need of further investigation to fully understand how red tide may impact sustainability of the fishery.

Effects of field and laboratory exposure to the toxic dinoflagellate Karenia brevis on the reproduction of the eastern oyster, Crassostrea virginica, and subsequent development of offspring.

Blooms of the brevetoxin-producing dinoflagellate, Karenia brevis, are a recurrent and sometimes devastating phenomenon in the Gulf of Mexico. The eastern oyster, Crassostrea virginica, is exposed regularly to these blooms, yet little is known about the impacts of K. brevis upon this important species. The present study considered the effects of exposure to both a natural bloom and cultured K. brevis on the reproductive development of C. virginica. Oysters had been exposed to a bloom of K. brevis that occurred in Lee County, Florida, from September 2012 through May 2013, during a period of gametogenesis and gamete ripening. Ripe adult oysters were collected from this bloom-exposed site and from a site 200 miles north which was not exposed to any bloom. In addition, responses to two 10-day laboratory exposures of either unripe or ripe adult oysters to whole cells of K. brevis at high bloom concentrations (1000 and 5000cellsmL-1) were determined. Both field- and laboratory-exposed adult oysters accumulated PbTx (attaining ∼22×103ngg-1 and 922ngg-1 PbTx-3 equivalents in the laboratory and the field, respectively), and significant mucal, edematous, and inflammatory features, indicative of a defense response, were recorded in adult tissues in direct contact with K. brevis cells. Laboratory-exposed oysters also showed an increase in the total number of circulating hemocytes suggesting that: (1) new hemocytes may be moving to sites of tissue inflammation, or, (2) hemocytes are released into the circulatory system from inflamed tissues where they may be produced. The area of oyster tissue occupied by gonad (representative of reproductive effort) and reactive oxygen species production in the spermatozoa of oysters exposed to the natural bloom of K. brevis were significantly lower compared to oysters that were not exposed to K. brevis. Additionally, following 10-day exposure of ripe oysters, a significant, 46% reduction in the prevalence of individuals with ripe gametes was obtained in the 5000cellsmL-1K. brevis treatment. Brevetoxin (PbTx) was recorded within the spermatozoa and oocytes of naturally exposed oysters and was estimated to be 18 and 26% of the adult PbTx load, respectively. Larvae derived from gametes containing PbTx showed significantly higher mortalities and attained a smaller larval size for the first 6 days post-fertilization. These negative effects on larval development may be due to the presence of PbTx in the lipid droplets of the oocytes, which is mobilized by the larvae during embryonic and lecithotrophic larval development. Provision of a non-contaminated food source to larvae however, appeared to mitigate the early negative effects of this neonatal PbTx exposure. Results herein show that adult eastern oysters and their offspring are susceptible to exposure to K. brevis. Caution should therefore be exercised when identifying oyster reef restoration areas and in efforts to establish aquaculture in areas prone to red tides.

Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis.

Biomarkers of brevetoxin exposure and composite toxin levels in hard clam (Mercenaria sp.) exposed to Karenia brevis blooms.

Brevetoxins in clams (Mercenaria sp.) exposed to recurring blooms of Karenia brevis in Sarasota Bay, FL, were studied over a three-year period. Brevetoxin profiles in toxic clams were generated by ELISA and LC-MS. Several brevetoxin metabolites, as identified by LC-MS, were major contributors to the composite brevetoxin response of ELISA. These were S-desoxyBTX-B2 (m/z 1018), BTX-B2 (m/z 1034), BTX-B5 (m/z 911), open A-ring BTX-B5 (m/z 929), and BTX-B1 (m/z 1018). Summed values of these metabolites were highly correlated (R(2) = 0.9) with composite B-type brevetoxin measurements by ELISA. S-desoxyBTX-B2, BTX-B2, and BTX-B1 were the most persistent and detectable in shellfish for several months after dissipation of blooms. These metabolites were selected as LC-MS biomarkers of brevetoxin exposure and reflective of composite B-type brevetoxins in hard clam. ELISA and LC-MS values were moderately correlated with toxicity of the shellfish by mouse bioassay. ELISA and LC-MS methods offer rapid screening and confirmatory determination of brevetoxins, respectively, as well as toxicity assessment in clams exposed to K. brevis blooms.

Compositional changes in neurotoxins and their oxidative derivatives from the dinoflagellate, Karenia brevis, in seawater and marine aerosol.

The harmful alga, Karenia brevis, produces a suite of polyether neurotoxins, brevetoxins or PbTx, that cause marine animal mortality and neurotoxic shellfish poisoning (NSP). A characteristic of K. brevis blooms is associated airborne toxins that result in severe respiratory problems. This study was undertaken to determine the composition of aerosolized brevetoxins and oxidative derivatives to which beachgoers are exposed during a K. brevis bloom. The suite of brevetoxins and derivatives in seawater is comprised of intra-cellular (IC) and extra-cellular (EC) compounds. We hypothesized that aerosolized compounds are generated primarily from EC, hydrophobic compounds in seawater by bubble-mediated transport. Thus the composition of aerosolized brevetoxins and derivatives, to which beachgoers are exposed, would reflect the EC composition of the source matrix (the local surf zone). Brevetoxins were extracted from water collected along the shore and from marine aerosols along Siesta Beach and Lido Beach in Sarasota, FL, USA, during K. brevis blooms. Water samples were further processed into IC and EC components. The primary brevetoxins observed in water and air included PbTx-1, -2, -3, -PbTx-2-carboxylic acid, and brevenal. Oxidation and/or hydrolysis products of PbTx-1, -2, -3 and -7 were also found in EC water and in aerosol, but not IC.

Personal exposure to aerosolized red tide toxins (brevetoxins).

Florida red tides occur annually in the Gulf of Mexico from blooms of the marine dinoflagellate, Karenia brevis, which produces highly potent natural polyether toxins, brevetoxins. Several epidemiologic studies have demonstrated that human exposure to red tide aerosol could result in increased respiratory symptoms. Environmental monitoring of aerosolized brevetoxins was performed using a high-volume sampler taken hourly at fixed locations on Siesta Beach, Florida. Personal exposure was monitored using personal air samplers and taking nasal swab samples from the subjects who were instructed to spend 1 hr on Sarasota Beach during two sampling periods of an active Florida red tide event in March 2005, and in May 2008 when there was no red tide. Results showed that the aerosolized brevetoxins from the personal sampler were in modest agreement with the environmental concentration taken from a high-volume sampler. Analysis of nasal swab samples for brevetoxins demonstrated 68% positive samples in the March 2005 sampling period when air concentrations of brevetoxins were between 50 to 120 ng/m(3) measured with the high-volume sampler. No swab samples showed detectable levels of brevetoxins in the May 2008 study, when all personal samples were below the limit of detection. However, there were no statistical correlations between the amounts of brevetoxins detected in the swab samples with either the environmental or personal concentration. Results showed that the personal sample might provide an estimate of individual exposure level. Nasal swab samples showed that brevetoxins indeed were inhaled and deposited in the nasal passage during the March 2005 red tide event.

Characterization of Florida red tide aerosol and the temporal profile of aerosol concentration.

Red tide aerosols containing aerosolized brevetoxins are produced during the red tide bloom and transported by wind to coastal areas of Florida. This study reports the characterization of Florida red tide aerosols in human volunteer studies, in which an asthma cohort spent 1h on Siesta Beach (Sarasota, Florida) during aerosolized red tide events and non-exposure periods. Aerosol concentrations, brevetoxin levels, and particle size distribution were measured. Hourly filter samples were taken and analyzed for brevetoxin and NaCl concentrations. In addition, the aerosol mass concentration was monitored in real time. The results indicated that during a non-exposure period in October 2004, no brevetoxin was detected in the water, resulting in non-detectable levels of brevetoxin in the aerosol. In March 2005, the time-averaged concentrations of brevetoxins in water samples were moderate, in the range of 5-10 microg/L, and the corresponding brevetoxin level of Florida red tide aerosol ranged between 21 and 39 ng/m(3). The temporal profiles of red tide aerosol concentration in terms of mass, NaCl, and brevetoxin were in good agreement, indicating that NaCl and brevetoxins are components of the red tide aerosol. By continuously monitoring the marine aerosol and wind direction at Siesta Beach, we observed that the marine aerosol concentration varied as the wind direction changed. The temporal profile of the Florida red tide aerosol during a sampling period could be explained generally with the variation of wind direction.

Cellular metabolism of brevetoxin (PbTx-2) by a monocyte cell line (U-937).

Blooms of Karenia brevis produce brevetoxins which cause neurotoxic shellfish poisoning and respiratory symptoms in humans as well as harmful effects on sea life. To investigate potential effects of brevetoxins on immune system components, a monocyte cell line (U-937) was exposed in vitro to PbTx-2. U-937 cells metabolized PbTx-2 through cellular detoxification mechanisms, as evidenced by depletion of intracellular glutathione and formation of glutathione and cysteine conjugates. Total intracellular glutathione was significantly decreased in toxin-treated cells compared to control cells, as measured using an enzymatic recycling method. LC/MS was used to detect the following brevetoxin metabolites: a cysteine-PbTx-2 conjugate (m/z 1018) and two putative glutathione-PbTx-2 conjugates (m/z 1204 and 1222). During 3h incubation, glutathione conjugates were detectable as early as 1h and increased in concentration after 2 and 3h. A cysteine-PbTx-2 conjugate appeared after 2h and increased in concentration after 3h. Detectable levels of brevetoxin conjugates were present in response to toxin concentrations of 1muM. Depletion of intracellular glutathione and formation of brevetoxin metabolites, with changes in concentrations over time, suggest immune cells (U-937) have important cellular detoxification pathways for PbTx-2.

Effects of in vitro brevetoxin exposure on apoptosis and cellular metabolism in a leukemic T cell line (Jurkat).

Harmful algal blooms (HABs) of the toxic dinoflagellate, Karenia brevis, produce red tide toxins, or brevetoxins. Significant health effects associated with red tide toxin exposure have been reported in sea life and in humans, with brevetoxins documented within immune cells from many species. The objective of this research was to investigate potential immunotoxic effects of brevetoxins using a leukemic T cell line (Jurkat) as an in vitro model system. Viability, cell proliferation, and apoptosis assays were conducted using brevetoxin congeners PbTx-2, PbTx-3, and PbTx-6. The effects of in vitro brevetoxin exposure on cell viability and cellular metabolism or proliferation were determined using trypan blue and MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), respectively. Using MTT, cellular metabolic activity was decreased in Jurkat cells exposed to 5 - 10 microg/ml PbTx-2 or PbTx-6. After 3 h, no significant effects on cell viability were observed with any toxin congener in concentrations up to 10 microg/ml. Viability decreased dramatically after 24 h in cells treated with PbTx-2 or -6. Apoptosis, as measured by caspase-3 activity, was significantly increased in cells exposed to PbTx-2 or PbTx-6. In summary, brevetoxin congeners varied in effects on Jurkat cells, with PbTx-2 and PbTx-6 eliciting greater cellular effects compared to PbTx-3.

Extraction and analysis of lipophilic brevetoxins from the red tide dinoflagellate Karenia brevis.

Efficient extraction and accurate analysis of lipophilic brevetoxins (PbTxs), produced by the harmful algal bloom (HAB) species Karenia brevis, are essential when assessing the toxicological potential of this dinoflagellate. One of the most commonly used brevetoxin extraction methodologies employs C18 solid-phase extraction (SPE). In this study, C18 SPEC discs were tested for extraction of spiked PbTx-3 in seawater and naturally produced brevetoxins from K. brevis. Quantification of brevetoxin in the extracts was determined using four independent methods: receptor binding assay (RBA), radioimmunoassay (RIA), neuroblastoma (N2A) cytotoxicity assay, and liquid chromatography/mass spectrometry (LC/MS). In addition to quantification of the brevetoxin concentration, LC/MS analysis provided identification of individual congeners and each of their hydrolyzed products. SPEC disc extractions prepared from sonicated cultures of non-brevetoxin-producing Karenia mikimotoi cultures spiked with PbTx-3 yielded extraction efficiencies of 108, 99, and 125% as determined by the RBA, RIA, and N2A cytotoxicity assay, respectively. In SPEC disc extracts of brevetoxin-producing K. brevis (isolate SP3) cultures, LC/MS analysis yielded the highest total concentrations, possibly due to the concurrent detection of hydrolytic brevetoxin congeners that accounted for up to 20% of the congener profile. Relative to the brevetoxin concentration as determined by LC/MS, the RBA, RIA, and N2A cytotoxicity assay detected 73, 83, and 51% of the total brevetoxin concentration. Stability experiments demonstrated that brevetoxins remain stable on the SPEC discs for at least 30 days, making this extraction method suitable for shipboard collections.

Characterization of polar brevetoxin derivatives isolated from Karenia brevis cultures and natural blooms.

Several novel brevetoxin derivatives were isolated and identified in Karenia brevis cultures and natural blooms by using solid phase extraction (SPE) and LC/MS(MS) techniques. These analogs were more polar compared with previously described brevetoxins, and were poorly extractable by conventional non-polar solvent (chloroform) partitioning. Brevetoxin analogs were structurally confirmed as hydrolyzed (open A-ring) forms of brevetoxins PbTx-1, PbTx-7, PbTx-2, and PbTx-3, and of oxidized PbTx-1 and PbTx-2. Some of these open A-ring derivatives were in greater abundance than their non-hydrolyzed counterparts. All were in much greater abundance in bloom water filtrate compared with cell-rich fractions. Open A-ring compounds were cytotoxic in mouse neuroblastoma (N2a) cell assay. In the K. brevis bloom-exposed Eastern oyster, brevetoxin metabolites with opened A rings were identified (e.g., open-ring cysteine-PbTx conjugates), contributing to their overall toxin burden.

Effects of the dinoflagellate Karenia brevis on larval development in three species of bivalve mollusc from Florida.

The effects of Karenia brevis (Wilson clone) on larval survival and development of the northern quahog, Mercenaria mercenaria, eastern oyster, Crassostrea virginica and bay scallop, Argopecten irradians, were studied in the laboratory. Larvae were exposed to cultures of whole and lysed cells, with mean total brevetoxin concentrations of 53.8 and 68.9 microgL(-1), respectively. Survival of early (3-day-old) larvae was generally over 85% for all shellfish species at K. brevis densities of 100 cells ml(-1) or less, and not significantly different between whole and lysed culture. At 1000 cells ml(-1), survival was significantly less in lysed culture than whole culture for both M. mercenaria and C. virginica. Survival of late (7-day-old) larvae in all three species was not significantly affected by K. brevis densities of 1000 cells ml(-1) or less. At 5000 cells ml(-1), however, survival was reduced to 37%, 26% and 19% for A. irradians, M. mercenaria and C. virginica, respectively. Development of C. virginica and M. mercenaria larvae was protracted at K. brevis densities of 1000 cells ml(-1). These results suggest that blooms of K. brevis, and particularly their associated brevetoxins, may have detrimental consequences for Florida's shellfisheries by disrupting critical larval processes. Special attention should be paid to blooms of K. brevis where these shellfish occur naturally or where aquaculture and restoration activities are either ongoing or planned.

Brevetoxicosis: red tides and marine mammal mortalities.

Potent marine neurotoxins known as brevetoxins are produced by the 'red tide' dinoflagellate Karenia brevis. They kill large numbers of fish and cause illness in humans who ingest toxic filter-feeding shellfish or inhale toxic aerosols. The toxins are also suspected of having been involved in events in which many manatees and dolphins died, but this has usually not been verified owing to limited confirmation of toxin exposure, unexplained intoxication mechanisms and complicating pathologies. Here we show that fish and seagrass can accumulate high concentrations of brevetoxins and that these have acted as toxin vectors during recent deaths of dolphins and manatees, respectively. Our results challenge claims that the deleterious effects of a brevetoxin on fish (ichthyotoxicity) preclude its accumulation in live fish, and they reveal a new vector mechanism for brevetoxin spread through food webs that poses a threat to upper trophic levels.

Concentration and particle size of airborne toxic algae (brevetoxin) derived from ocean red tide events.

Red tides in the Gulf of Mexico are formed by blooms of the dinoflagellate Karenia brevis, which produces brevetoxins (PbTx). Brevetoxins can be transferred from water to air in the wind-powered whitecapped waves during red tide episodes. Inhalation exposure to marine aerosol containing PbTx causes respiratory problems. A liquid chromatograph/ tandem mass spectrometric method was developed for the detection and quantitation of several PbTxs in ambient samples collected during red tide events. This method was complemented by a previously developed antibody assay that analyzes the entire class of PbTx compounds. The method showed good linearity, accuracy, and reproducibility, allowing quantitation of PbTx compounds in the 10 pg/m3 range. Air concentrations of PbTxs and brevenal for individual samples ranged from 0.01 to 80 ng/m3. The particle size showed a single mode with a mass median diameter between 6 and 10 microm, which was consistent for all of the PbTx species that were measured. Our results imply that individual PbTxs were from the same marine aerosol or from marine aerosol that was produced from the same process. The particle size indicated the likelihood of high deposition efficiency in the respiratory tract with the majority of aerosol deposited in the upper airways and small but not insignificant deposition in the lower airways.

Characterization of marine aerosol for assessment of human exposure to brevetoxins.

Red tides in the Gulf of Mexico are commonly formed by the fish-killing dinoflagellate Karenia brevis, which produces nine potent polyether brevetoxins (PbTxs). Brevetoxins can be transferred from water to air in wind-powered white-capped waves. Inhalation exposure to marine aerosol containing brevetoxins causes respiratory symptoms. We describe detailed characterization of aerosols during an epidemiologic study of occupational exposure to Florida red tide aerosol in terms of its concentration, toxin profile, and particle size distribution. This information is essential in understanding its source, assessing exposure to people, and estimating dose of inhaled aerosols. Environmental sampling confirmed the presence of brevetoxins in water and air during a red tide exposure period (September 2001) and lack of significant toxin levels in the water and air during an unexposed period (May 2002). Water samples collected during a red tide bloom in 2001 showed moderate-to-high concentrations of K. brevis cells and PbTxs. The daily mean PbTx concentration in water samples ranged from 8 to 28 microg/L from 7 to 11 September 2001; the daily mean PbTx concentration in air samples ranged from 1.3 to 27 ng/m(3). The daily aerosol concentration on the beach can be related to PbTx concentration in water, wind speed, and wind direction. Personal samples confirmed human exposure to red tide aerosols. The particle size distribution showed a mean aerodynamic diameter in the size range of 6-12 microm, with deposits mainly in the upper airways. The deposition pattern correlated with the observed increase of upper airway symptoms in healthy lifeguards during the exposure periods.

Charlotte Harbor initiative: assessing the ecological health of southwest Florida's Charlotte Harbor estuary.

Charlotte Harbor is the largest and one of the least impacted estuaries on the southwest Florida coast, encompassing about 270 square miles (700 km2) with a watershed of 4400 square miles (11,400 km2). The Harbor is distinguished by extensive phosphate mining in its watershed and declining freshwater inflows, more protected submerged and intertidal areas than most Gulf ecosystems, and is part of the National Estuary Program. A hypoxic event occurs annually in the Harbor for possibly natural rather than anthropogenic reasons providing an opportunity for the study of hypoxic effects on the ecology of a large subtropical ecosystem. A 5-year, multimillion dollar study was begun in 2001 to enable scientists of Mote Marine Laboratory (MML, Sarasota, Florida) to collaborate on ecological characterization of the estuary and provide data necessary for resource managers to predict consequences of future population growth in the region. Initial studies were organized around themes of preservation, conservation and restoration while subsequent years of work are organized around a core program of physical, chemical and biological studies that track the ecological consequences of freshwater inflow, hypoxia and anthropogenic-derived contaminants. Along with MML, scientists in federal and state agencies along with a number of colleges and universities are cooperating in the project.

Multi-Laboratory Study of Five Methods for the Determination of Brevetoxins in Shellfish Tissue Extracts.

A thirteen-laboratory comparative study tested the performance of four methods as alternatives to mouse bioassay for the determination of brevetoxins in shellfish. The methods were N2a neuroblastoma cell assay, two variations of the sodium channel receptor binding assay, competitive ELISA, and LC/MS. Three to five laboratories independently performed each method using centrally prepared spiked and naturally incurred test samples. Competitive ELISA and receptor binding (96-well format) compared most favorably with mouse bioassay. Between-laboratory relative standard deviations (RSDR) ranged from 10 to 20% for ELISA and 14 to 31% for receptor binding. Within-laboratory (RSDr) ranged from 6 to 15% for ELISA, and 5 to 31% for receptor binding. Cell assay was extremely sensitive but data variation rendered it unsuitable for statistical treatment. LC/MS performed as well as ELISA on spiked test samples but was inordinately affected by lack of toxin-metabolite standards, uniform instrumental parameters, or both, on incurred test samples. The ELISA and receptor binding assay are good alternatives to mouse bioassay for the determination of brevetoxins in shellfish.