Brevetoxins, like ciguatoxins, are potent ichthyotoxic neurotoxins that accumulate in fish☆
Introduction
Brevetoxins and ciguatoxins are potent marine neurotoxins. The source for the former is the planktonic red tide dinoflagellate Karenia brevis (McFarren et al., 1965; Baden et al., 1979) and for the latter is the epibenthic dinoflagellate Gambierdiscus toxicus (Yasumoto et al., 1977; Lewis et al., 2000). Both groups of toxins have similar chemical natures and similar biological activities. They are lipid-soluble polycyclic polyether compounds and are the only molecules known to activate voltage-sensitive sodium channels in mammals through a specific interaction with site 5 of the alpha subunit of the sodium channel (Poli et al., 1986; Lombet et al., 1987; Dechraoui et al., 1999). Both are toxic to mammals, and in humans the ingestion of brevetoxin-contaminated shellfish (Halstead, 1978; Steidinger, 1993) and of ciguatoxin-contaminated fish (Bagnis et al., 1979) results in severe forms of food poisoning—Neurotoxic Shellfish Poisoning (NSP) and Ciguatera Fish Poisoning (CFP), respectively. Because both toxins possess similar modes of action, the clinical manifestations following ingestion, although more severe, varied and longer-lasting for genuine ciguatera, are quite similar, with gastrointestinal, neurological and cardiovascular components (Bagnis et al., 1979; Baden et al., 1995; Poli et al., 2000; Kirkpatrick et al., 2004). The reversal of temperature discrimination, also known as paradoxical dysthesia, is probably the most characteristic symptom associated with ciguatera poisoning (Bagnis et al., 1979), but is also documented in severe cases of brevetoxin poisoning (Baden et al., 1995).
Despite these similarities, these toxins present obvious differences in their impacts on fish. Ciguatoxins are well known to accumulate in fish by trophic transfer in tropical fish food webs (Legrand, 1991), and have not been documented in association with fish mortalities. In contrast, K. brevis blooms (Florida red tides) characteristically result in massive fish kills (Steidinger et al., 1973; Landsberg, 2002). Because of the fish kills routinely observed along the west coast of Florida during red tides, it has been assumed that the ichthyotoxicity of brevetoxins precluded their vectoring or accumulation via live fish.
In 2004, a mass mortality of bottlenose dolphins (Tursiops truncatus) in the Florida panhandle clearly indicated that fish are not always killed by K. brevis red tides and that they have the potential to vector brevetoxins to higher trophic levels (Flewelling et al., 2005). Besides a coincident mortality of large redfish (Sciaenops ocellatus), no obvious indicators of a red tide were reported (i.e. there was no obvious bloom or other animal mortalities). However, high levels of brevetoxins were measured in multiple tissues of all bottlenose dolphins examined (n=36). Tissues from six undigested menhaden (Brevoortia sp.) recovered from the stomach contents of some dolphins contained excessive levels of brevetoxins, with concentrations reaching 33,200 ng g−1 in viscera and 1500 ng g−1 in muscle. At least eight other species of fish collected live from the area while the mortality was ongoing also contained elevated, but much lower, concentrations of brevetoxins in their tissues (Flewelling et al., 2005). During this event, we confirmed that bottlenose dolphins are susceptible to brevetoxicosis and that planktivorous fish can vector lethal concentrations of brevetoxins to higher trophic levels.
The goals of the present study were to experimentally identify pathways by which brevetoxins may accumulate in the tissues of live fish, and to determine whether the brevetoxin accumulation that we previously observed in live fish (Flewelling et al., 2005) was an exception or alternatively, an undocumented common occurrence.
Section snippets
Experimental exposure of omnivorous fish to contaminated shellfish
Hard clams (Mercenaria sp.) naturally contaminated with brevetoxins were collected from Charlotte Harbor, Florida during a red tide in March of 2003 and stored at −20 °C for 2 months prior to the experiments. Locally harvested nontoxic hard clams (M. mercenaria) were purchased in North Carolina immediately before the experiments began.
Adult pinfish (Lagodon rhomboides; 12–17 cm total length) and Atlantic croakers (Micropogonias undulatus; 15–22 cm total length) were collected using hook-and-line
Experimental exposure of omnivorous fish to toxic shellfish
A subset of hard clams used in the exposure studies was analyzed by LC-MS prior to exposures and was found to contain PbTx-3 and metabolites. Brevetoxin concentration by ELISA was 1800 ng g−1, and toxicity assessed by mouse bioassay was 30 mouse units (MU) per 100 g. No brevetoxins were detected by ELISA in the control clams.
While feeding toxic hard clams for 2 consecutive weeks, none of the pinfish or croakers died or exhibited any obvious signs of adverse effects. For comparison with the
Pathways for accumulation of brevetoxins in fish
Our experimental results demonstrate that fish can accumulate ichthyotoxic brevetoxins when exposed through their diet, and that toxins can be transferred through the food web by fish feeding on toxic prey such as toxic shellfish and K. brevis cells. However, fish in red tide-endemic areas are exposed to brevetoxins through other prey or forage items as well, including zooplankton, fish and seagrass with associated epiphytes (Fig. 6), all of which have the potential to accumulate brevetoxins (
Conclusions
Through experimental exposures we have demonstrated that fish have the potential to accumulate brevetoxins in their muscle and, at higher levels, their viscera when feeding on toxic prey; and our data from live-caught fish confirm that they do, in fact, accumulate brevetoxins in the wild. During a K. brevis bloom, brevetoxins were detected in the muscle and liver of 95% and 100% of the fish tested, respectively.
We have presented an overview of the levels of brevetoxins that we observed in fish
Acknowledgments
We thank Dr. Tomas Lankford and Dr. Carmelo Thomas (UNCW) for their assistance with exposure experiments and experimental K. brevis cell counts, Melissa Smith (UNCW) for artistic representation of brevetoxin transfer in the fish food web, Karen Atwood and Sheila O Dea (FWRI) for laboratory assistance, Earnest Truby (FWRI) for K. brevis cell counts and the Florida DEP St. Joseph Bay Aquatic Preserve staff for collection of monthly water samples. This work was supported by funds from the CDC and
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Ethical statement: The results presented in this manuscript are originals and are not being considered elsewhere. Regarding the use of vertebrate animals as described in the manuscript, experiments were performed according to the animal use policy (approved IUCAC protocol) at the corresponding author's institution.