Bacteriocidal effects of brevetoxin on natural microbial communities

The sensitivity of bacteria to the marine neurotoxins, brevetoxins, produced by the dinoflagellate Karenia brevis and raphidophytes Chattonella spp. remains an open question. We investigated the bacteriocidal effects of brevetoxin (PbTx-2) on the abundance and community composition of natural microbial communities by adding it to microbes from three coastal marine locations that have varying degrees of historical brevetoxin exposure: (1) Great Bay, New Jersey, (2) Rehoboth Bay, Delaware and (3) Sarasota Bay, Florida. The populations with limited or no documented exposure were more susceptible to the effects of PbTx-2 than the Gulf of Mexico populations which are frequently exposed to brevetoxins. The community with no prior documented exposure to brevetoxins showed significant (p = 0.03) changes in bacterial abundance occurring with additions greater than 2.5 μg PbTx-2 L⁻¹. Brevetoxin concentrations during K. brevis blooms range from ∼2.5 to nearly 100 μg L⁻¹ with typical concentrations of ∼10–30 μg L⁻¹. In contrast to the unexposed populations, there was no significant decrease in bacterial cell number for the microbial community that was frequently exposed to brevetoxins, which implies variable sensitivity in natural communities. The diversity in the bacterial communities that were sensitive to PbTx-2 declined upon exposure. This suggests that the PbTx-2 was selecting for or against specific species. Mortality was much higher in the 200 μg PbTx-2 L⁻¹ treatment after 48 h and >37% of the species disappeared in the bacterial communities with no documented exposure. These results suggest that toxic red tides may play a role in structuring bacterial communities.     

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.     

Loss of waterborne brevetoxins from exposure to phytoplankton competitors

We tested whether interactions among phytoplankton competitors affect toxin dynamics involving the red tide dinoflagellate Karenia brevis, whose brevetoxins incapacitate and kill coastal wildlife. The addition of a live diatom, Skeletonema costatum, led to decreased concentrations of brevetoxin B (PbTx-2) associated with K. brevis cells in co-culturing experiments and with two of three natural bloom samples containing K. brevis. Similar decreases in PbTx-2 concentration, but not PbTx-3 concentration, occurred when a mixture of brevetoxins (without live K. brevis cells) was exposed to S. costatum, indicating that S. costatum metabolizes waterborne PbTx-2. Liquid chromatography–mass spectrometry (LC–MS) and ELISA analyses indicated that PbTx-2 is probably not transformed into other brevetoxins or into known brevetoxin metabolites, and instead is biotransformed by a previously unrecognized mechanism. Four different S. costatum strains from around the world caused similar loss of PbTx-2, suggesting that evolutionary experience with K. brevis is not a pre-requisite for the ability to metabolize PbTx-2. Additionally, phytoplankton-associated bacteria were found to play no role in the loss of PbTx-2, as bacteria-free S. costatum strains metabolized PbTx-2. Finally, loss of waterborne PbTx-2 caused by exposure to a dinoflagellate, a cryptophyte, and two additional diatom species indicates that this phenomenon is widespread among phytoplankton. Our results unexpectedly suggest that competing phytoplankton species present during K. brevis blooms, and possibly other red tides, could mediate bloom toxicity and therefore ecosystem-level consequences of red tides.     

Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis

Karenia brevis is a toxic marine dinoflagellate endemic to the Gulf of Mexico. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Previously, PKS encoding genes were amplified from K. brevis culture and their similarity to a PKS gene from the closely related protist, Cryptosporidium parvum, suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. Herein we report the localization of PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate.     

Toxicogenomic effects of marine brevetoxins in liver and brain of mouse

Although the polyether brevetoxins (PbTx's) produced by Karenia brevis (the organism responsible for blooms of the Florida red tide) are known to exert their acute toxic effects through ion-channel mediated pathways in neural tissue, prior studies have also demonstrated that at least one form of the toxin (PbTx-6) is bound avidly by the aryl hydrocarbon receptor (AhR). Since AhR binding of a prototypical ligand such as dioxin is the first step in a cascade pathway producing major changes in gene expression, we reasoned that PbTx-6 might produce similar genomic-wide changes in expression. Mice were injected i.p. with sub-lethal doses of PbTx's (either 1.5 or 3 mg/g body weight of PbTx-6; or 0.15 mg/g body weight of PbTx-2, a toxin not avidly bound by the AhR), and liver and brain tissues were sampled at 8, 24 and 72 h and RNA was isolated. Changes in gene-specific RNA levels were assessed using commercially available mouse cDNA arrays (Incyte) containing >9600 array elements, including many elements from AhR-mediated genes. Histopathology of the two organs was also assessed. We observed minor histopathological effects and a total of only 29 significant (>2.0-fold) changes in gene expression, most of which occurred in the liver, and most of which could be attributable to an 'acute phase' inflammatory response. These results argue against the hypothesis that PbTx-6 acts via a classic AhR-mediated mechanism to evoke gene expression changes. However, given the avidity with which PbTx-6 binds to the AhR, these findings have important implications for how PbTx's may act in concert with other toxicants that are sensed by the AhR.     

Impacts of Florida red tides on coastal communities

Over the last few decades, scientific research has helped to describe the disease neurotoxic shellfish poisoning (NSP) by identifying the causative organism, Karenia brevis, and by characterizing the disease-causing toxins, a suite of polyether toxins called brevetoxins. In addition to causing disease in exposed human populations, K. brevis blooms and associated management responses have been linked to other effects on coastal communities. Some of these effects are negative, such as the loss of tourism dollars and the increased burden on local health care services caused by increases in human disease incidence. However, some of the effects are positive, such as the significant improvement in detecting brevetoxins in environmental samples and clinical specimens. This review discusses the health and economic effects from K. brevis blooms on Florida coastal communities and the current efforts to identify the data needed to assess social and cultural effects.     

Competing phytoplankton undermines allelopathy of a bloom-forming dinoflagellate

Biotic interactions in the plankton can be both complex and dynamic. Competition among phytoplankton is often chemically mediated, but no studies have considered whether allelopathic compounds are modified by biotic interactions. Here, we show that compounds exuded during Karenia brevis blooms were allelopathic to the cosmopolitan diatom Skeletonema costatum, but that bloom allelopathy varied dramatically among collections and years. We investigated several possible causes of this variability and found that neither bloom density nor concentrations of water-borne brevetoxins correlated with allelopathic potency. However, when we directly tested whether the presence of competing phytoplankton influenced bloom allelopathy, we found that S. costatum reduced the growth-inhibiting effects of bloom exudates, suggesting that S. costatum has a mechanism for undermining K. brevis allelopathy. Additional laboratory experiments indicated that inducible changes to K. brevis allelopathy were restricted to two diatoms among five sensitive phytoplankton species, whereas five other species were constitutively resistant to K. brevis allelopathy. Our results suggest that competitors differ in their responses to phytoplankton allelopathy, with S. costatum exhibiting a previously undescribed method of resistance that may influence community structure and alter bloom dynamics.     

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.     

NOVEL STEROLS OF THE TOXIC DINOFLAGELLATE KARENIA BREVIS (DINOPHYCEAE): A DEFENSIVE FUNCTION FOR UNUSUAL MARINE STEROLS?1

The “red tide” organism Karenia brevis (Davis) Hansen & Moestrup (=Gymnodinium breve Davis) produces a mixture of brevetoxins, potent neurotoxins responsible for neurotoxic shellfish poisoning in humans and massive fish kills in the Gulf of Mexico and the southern Atlantic coast of the United States. The sterol composition of K. brevis was found to be a mixture of six novel and rare Δ⁸⁽¹⁴⁾ sterols. The two predominant sterols, (24R)‐4α‐methylergosta‐8(14), 22‐dienol and (24R)‐4α‐methyl‐27‐norergosta‐8(14), 22‐dienol, were named gymnodinosterol and brevesterol and represent potentially useful biomarkers for K. brevis. A possible function for such unusual marine sterols is proposed whereby structural modifications render the sterols non‐nutritious to marine invertebrates, reducing predation and thereby enhancing the ability of the dinoflagellates to form massive blooms.     

HIGH BREVETOXIN CONCENTRATIONS IN GYMNODINIUM BREVE BLOOMS ALONG THE NORTHWEST FLORIDA COAST DURING 1999

Blooms of the dinoflagellate Gymnodinium breve (i.e. red tides) produce brevetoxins (PbTx) that negatively impact the Gulf of Mexico ecosystem, human health, and local economies. Characterizing and predicting bloom events and their impacts requires knowledge of G. breve abundance and PbTx concentrations in the water column. We report results from a bloom that occurred during the fall and winter of 1999 in NW Florida coastal waters. Data were collected from 16 stations on 3 sampling dates (29 Sept., 9 Nov., 1 Dec.), including basic hydrography, nutrient concentrations, G. breve abundances, and brevetoxin concentrations. G. breve cells were enumerated using flow cytometry and PbTx's were isolated from seawater using dichloromethane (DCM) partitioning. Brevetoxins were quantified by HPLC-DAD using a C-18 column and an acetonitrile-water gradient elution. Literature estimates of total PbTx concentration (PbTx's 1, 2, 3) of cultured and field-collected G. breve suggest a range in concentration from 7 to 17 pg cell⁻¹. We measured total PbTx levels that greatly exceeded these values [Sept., 47–67 pg cell⁻¹ (n=5); Nov., 59–126 pg cell⁻¹ (n=3), Dec., 12–63 pg cell⁻¹ (n=8)]. PbTx-2 was the predominant (67–75%) PbTx isomer found in these blooms. PbTx-1 and PbTx-3 were found at 11–22% and ND–28% of total PbTx, respectively.     

Florida red tide and brevetoxins: Association and exposure in live resident bottlenose dolphins (Tursiops truncatus) in the eastern Gulf of Mexico,U.S.A.

Bottlenose dolphins (Tursiops truncatus) along the Gulf of Mexico are frequently exposed to blooms of the toxic alga, Karenia brevis, and brevetoxins associated with these blooms have been implicated in several dolphin mortality events. Studies on brevetoxin accumulation in dolphins have typically focused on analyses of carcasses from large‐scale die‐offs; however, data are scarce for brevetoxin loads in live individuals frequently exposed to K. brevis blooms. This study investigated in vivo brevetoxin exposure in free‐ranging bottlenose dolphins resident to Sarasota Bay, Florida, utilizing samples collected during health assessments performed during multiple K. brevis blooms occurring from 2003 to 2005. Brevetoxins were detected by ELISA and LC‐MS in 63% of bottlenose dolphins sampled (n= 30) concurrently with a K. brevis bloom. Brevetoxins were present in urine and gastric samples at concentrations ranging from 2 to 9 ng PbTx‐3 eq/g, and in feces at concentrations ranging from 45 to 231 ng PbTx‐3 eq/g. Samples from individuals (n= 12) sampled during nonbloom conditions (≤1,000 cells/L) were negative for brevetoxin activity. Brevetoxin accumulation data from this study complement dolphin carcass and prey fish data from the same study area, and aid in evaluating impacts of harmful algal blooms on sentinel marine animal species along the west Florida coast.     

Review of Florida Red Tide and Human Health Effects

This paper reviews the literature describing research performed over the past decade on the known and possible exposures and human health effects associated with Florida red tides. These harmful algal blooms are caused by the dinoflagellate, Karenia brevis, and similar organisms, all of which produce a suite of natural toxins known as brevetoxins. Florida red tide research has benefited from a consistently funded, long term research program, that has allowed an interdisciplinary team of researchers to focus their attention on this specific environmental issue-one that is critically important to Gulf of Mexico and other coastal communities. This long-term interdisciplinary approach has allowed the team to engage the local community, identify measures to protect public health, take emerging technologies into the field, forge advances in natural products chemistry, and develop a valuable pharmaceutical product. The Review includes a brief discussion of the Florida red tide organisms and their toxins, and then focuses on the effects of these toxins on animals and humans, including how these effects predict what we might expect to see in exposed people.     

Neurological illnesses associated with Florida red tide (Karenia brevis) blooms

Human respiratory and gastrointestinal illnesses can result from exposures to brevetoxins originating from coastal Florida red tide blooms, comprising the marine alga Karenia brevis (K. brevis). Only limited research on the extent of human health risks and illness costs due to K. brevis blooms has been undertaken to date. Because brevetoxins are known neurotoxins that are able to cross the blood-brain barrier, it is possible that exposure to brevetoxins may be associated with neurological illnesses. This study explored whether K. brevis blooms may be associated with increases in the numbers of emergency department visits for neurological illness. An exposure-response framework was applied to test the effects of K. brevis blooms on human health, using secondary data from diverse sources. After controlling for resident population, seasonal and annual effects, significant increases in emergency department visits were found specifically for headache (ICD-9 784.0) as a primary diagnosis during proximate coastal K. brevis blooms. In particular, an increased risk for older residents (≥55 years) was identified in the coastal communities of six southwest Florida counties during K. brevis bloom events. The incidence of headache associated with K. brevis blooms showed a small but increasing association with K. brevis cell densities. Rough estimates of the costs of this illness were developed for hypothetical bloom occurrences.     

Removal of harmful algal cells (Karenia brevis) and toxins from seawater culture by clay flocculation

Harmful algal blooms (HABs) occur worldwide causing serious threat to marine life, and to public health through seafood-borne illnesses and exposure to toxin-containing marine aerosol. This study was undertaken to assess the ability of phosphatic clay to remove the toxic dinoflagellate, Karenia brevis, and the potent neurotoxins (brevetoxins) produced by this species. Results showed that the addition of an aqueous slurry of 0.75 g (dry weight) clay to 3 l of K. brevis culture, containing 5×10⁶ and 10×10⁶ cells/l, removed 97±4% of brevetoxins from the water column within 4 h after the addition of clay. Clay flocculation of extra-cellular brevetoxins, released from cells ruptured (lyzed) by ultrasonication, removed 70±10% of the toxins. Addition of the chemical flocculant, polyaluminum chloride (PAC), removed all of the extra-cellular toxins. A 14 day study was undertaken to observe the fate of brevetoxins associated with clay flocculation of viable K. brevis cells. At 24 h following the clay addition, 90±18% of the toxins were removed from the water column, along with 85±4% of the cells. The toxin content of clay diminished from 208±13 μg at Day 1, to 121±21 μg at Day 14, indicating that the phosphatic clay retained about 58% of the toxins throughout the 14-day period. These studies showed the utility of natural clay as a means of reducing adverse effects from HABs, including removal of dissolved toxins, in the water column, although considerable work clearly remains before this approach can be used on natural blooms in open waters.     

Brevetoxin activation of voltage-gated sodium channels regulates Ca dynamics and ERK1/2 phosphorylation in murine neocortical neurons

Voltage-gated sodium channels (VGSC) are involved in the generation of action potentials in neurons. Brevetoxins (PbTx) are potent allosteric enhancers of VGSC function and are associated with the periodic 'red tide' blooms. Using PbTx-2 as a probe, we have characterized the effects of activation of VGSC on Ca(2+) dynamics and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling in neocortical neurons. Neocortical neurons exhibit synchronized spontaneous Ca(2+) oscillations, which are mediated by glutamatergic signaling. PbTx-2 (100 nm) increased the amplitude and reduced the frequency of basal Ca(2+) oscillations. This modulatory effect on Ca(2+) oscillations produced a sustained rise in ERK1/2 activation. At 300 nm, PbTx-2 disrupted oscillatory activity leading to a sustained increase in intracellular Ca(2+) ([Ca(2+)](i)) and induced a biphasic, activation followed by dephosphorylation, regulation of ERK1/2. PbTx-2-induced ERK1/2 activation was Ca(2+) dependent and was mediated by Ca(2+) entry through manifold routes. PbTx-2 treatment also increased cAMP responsive element binding protein (CREB) phosphorylation and increased gene expression of brain-derived neurotrophic factor (BDNF). These findings indicate that brevetoxins, by influencing the activation of key signaling proteins, can alter physiologic events involved in survival in neocortical neurons, as well as forms of synaptic plasticity associated with development and learning.     

Brevetoxin exposure,superoxide dismutase activity and plasma protein electrophoretic profiles in wild-caught Kemp's ridley sea turtles (Lepidochelys kempii) in southwest Florida

Because of their vulnerable population status, assessing exposure levels and impacts of toxins on the health status of Gulf of Mexico marine turtle populations is critical. From 2011 to 2013, two large blooms of the red tide dinoflagellate, Karenia brevis, occurred along the west coast of Florida USA (from October 2011 to January 2012 and October 2012 to April 2013). Other than recovery of stranded individuals, it is unknown how harmful algal blooms affected the Kemp's ridley sea turtles (Lepidochelys kempii) inhabiting the affected coastal waters. It is essential to gather information regarding brevetoxin exposure in these turtles to determine if it poses a threat to marine turtle health and survival. From April 2012 to May 2013, we collected blood from 13 immature Kemp's ridley turtles captured in the Pine Island Sound region of the Charlotte Harbor estuary. Nine turtles were sampled immediately after or during the red tide events (bloom group) while four turtles were sampled between the events (non-bloom group). Plasma was analyzed for total brevetoxins (reported as ng PbTx-3 eq/mL), superoxide dismutase (SOD) activity, total protein concentration and protein electrophoretic profiles (albumin, alpha-, beta- and gamma-globulins). Brevetoxin concentrations ranged from 7.0 to 33.8 ng PbTx-3 eq/mL. Plasma brevetoxin concentrations in the nine turtles sampled during or immediately after the red tide events were significantly higher (by 59%, P = 0.04) than turtles sampled between events. No significant correlations were observed between plasma brevetoxin concentrations and plasma proteins or SOD activity, most likely due to the small sample size; however alpha-globulins tended to increase with increasing brevetoxin concentrations in the bloom group. Smaller (carapace length and mass) bloom turtles had higher plasma brevetoxin concentrations than larger bloom turtles, possibly due to a growth dilution effect with increasing size. The research presented here improves the current understanding of potential impacts of environmental brevetoxin exposure on marine turtle health and survival.     

Antifouling properties of 10beta-formamidokalihinol-A and kalihinol A isolated from the marine sponge Acanthella cavernosa

Many soft-bodied sessile marine invertebrates such as sponges and soft corals defend themselves against fouling directly through the production of antifouling compounds, or indirectly through regulating the epibiotic microbes that affect larval settlement. In this study, 10beta-formamidokalihinol-A and kalihinol A were isolated and purified from the marine sponge Acanthella cavernosa (Dendy). The results indicated that both compounds inhibited the growth of bacteria isolated from the natural environment whereas kalihinol A suppressed larval settlement of a major fouling polychaete, Hydroides elegans with an EC50 of 0.5 microg ml(-1). Kalihinol A was incorporated in Phytagel that was exposed to the bacterial consortia in natural seawater for biofilm formation. Biofilms that developed on the Phytagel surfaces were analysed for bacterial abundance and bacterial species composition using a DNA fingerprinting technique, terminal restriction fragment length polymorphism (T-RFLP). The results showed that kalihinol A only slightly reduced bacterial abundance (t-test, p = 0.0497), but modified the bacterial species composition of the biofilms. Inhibition of H. elegans larval settlement was observed when biofilms developed under the influence of kalihinol A were exposed to larvae, suggesting that compounds like kalihinol A from the sponge A. cavernosa may change bacterial community composition on the sponge surface, which in turn, modulates larval settlement of fouling organisms.     

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.     

Brevetoxin persistence in sediments and seagrass epiphytes of east Florida coastal waters

A bloom of Karenia brevis Davis developed in September 2007 near Jacksonville, Florida and subsequently progressed south through east Florida coastal waters and the Atlantic Intracoastal Waterway (ICW). Maximum cell abundances exceeded 10⁶ cells L⁻¹ through October in the northern ICW between Jacksonville and the Indian River Lagoon. The bloom progressed further south during November, and terminated in December 2007 at densities of 10⁴ cells L⁻¹ in the ICW south of Jupiter Inlet, Florida. Brevetoxins were subsequently sampled in sediments and seagrass epiphytes in July and August 2008 in the ICW. Sediment brevetoxins occurred at concentrations of 11–15 ng PbTx-3 equivalents (g dry wt sediment)⁻¹ in three of five basins in the northern ICW during summer 2008. Seagrass beds occur south of the Mosquito Lagoon in the ICW. Brevetoxins were detected in six of the nine seagrass beds sampled between the Mosquito Lagoon and Jupiter Inlet at concentrations of 6–18 ng (g dry wt epiphytes)⁻¹. The highest brevetoxins concentrations were found in sediments near Patrick Air Force Base at 89 ng (g dry wt sediment)⁻¹. In general, brevetoxins occurred in either seagrass epiphytes or sediments. Blades of the resident seagrass species have a maximum life span of less than six months, so it is postulated that brevetoxins could be transferred between epibenthic communities of individual blades in seagrass beds. The occurrence of brevetoxins in east Florida coast sediments and seagrass epiphytes up to eight months after bloom termination supports observations from the Florida west coast that brevetoxins can persist in marine ecosystems in the absence of sustained blooms. Furthermore, our observations show that brevetoxins can persist in sediments where seagrass communities are absent.     

Concurrent exposure of bottlenose dolphins (Tursiops truncatus) to multiple algal toxins in Sarasota Bay, Florida, USA

Sentinel species such as bottlenose dolphins (Tursiops truncatus) can be impacted by large-scale mortality events due to exposure to marine algal toxins. In the Sarasota Bay region (Gulf of Mexico, Florida, USA), the bottlenose dolphin population is frequently exposed to harmful algal blooms (HABs) of Karenia brevis and the neurotoxic brevetoxins (PbTx; BTX) produced by this dinoflagellate. Live dolphins sampled during capture-release health assessments performed in this region tested positive for two HAB toxins; brevetoxin and domoic acid (DA). Over a ten-year study period (2000–2009) we have determined that bottlenose dolphins are exposed to brevetoxin and/or DA on a nearly annual basis (i.e., DA: 2004, 2005, 2006, 2008, 2009; brevetoxin: 2000, 2004, 2005, 2008, 2009) with 36% of all animals testing positive for brevetoxin (n = 118) and 53% positive for DA (n = 83) with several individuals (14%) testing positive for both neurotoxins in at least one tissue/fluid. To date there have been no previously published reports of DA in southwestern Florida marine mammals, however the May 2008 health assessment coincided with a Pseudo-nitzschia pseudodelicatissima bloom that was the likely source of DA observed in seawater and live dolphin samples. Concurrently, both DA and brevetoxin were observed in common prey fish. Although no Pseudo-nitzschia bloom was identified the following year, DA was identified in seawater, fish, sediment, snails, and dolphins. DA concentrations in feces were positively correlated with hematologic parameters including an increase in total white blood cell (p = 0.001) and eosinophil (p<0.001) counts. Our findings demonstrate that dolphins within Sarasota Bay are commonly exposed to two algal toxins, and provide the impetus to further explore the potential long-term impacts on bottlenose dolphin health.