Abstract

Harmful algal blooms (HABS, red tides) are increasing worldwide and occur nearly annually in the Gulf of Mexico. The dinoflagellate Karenia brevis is one organism responsible for blooms that severely impact marine life including dolphins, manatees, and sea turtles. K. brevis produces a suite of neurotoxins referred to as brevetoxins (PbTx). PbTx-3 is known to bind to voltage-gated sodium channels (VGSCs), affecting cell permeability and leading to a cascade of events resulting in cell death.¹ Exposed animals suffer from altered neurological and immune function and induced inflammation, with long-term effects due to bioaccumulation and biomagnification.^2,3 In 2005, at least 109 loggerhead sea turtles in Florida were affected by red tides with over 70 impacted during a 2006 bloom.⁴ Brevetoxicosis is difficult to treat in sea turtles as the physiological impacts have not been investigated and the magnitude and duration of brevetoxin exposure are generally unknown. Due to their threatened and endangered status, experimental exposures cannot be performed to determine the fate of PbTx-3 in sea turtle tissues, making it difficult to design appropriate treatments. We used the freshwater turtle Trachemys scripta as a model for brevetoxin exposure. Turtles were exposed to either intratracheal instillation (10.53 µg/kg) or oral dosing (33.48 µg/kg) 3x weekly over a period of 2–4 weeks. Tissues including the heart, lungs, kidneys, brain, fat, intestine, liver, spleen and trachea were collected for ELISA to investigate PbTx-3 uptake and distribution, route of excretion and rates of clearance (1h–1wk post-exposure). Tissues were also preserved for histopathology and blood samples were collected for immune studies. Primary turtle neuronal cell cultures were also exposed to PbTx-3 in the presence and absence of various agonists and antagonists to determine the toxin's mode of action. PbTx-3 was widely distributed in all tissues and fluids following both intratracheal and oral exposures, but was largely cleared from the system within 24 hours; concentrations were higher in orally exposed animals than in those receiving the toxin intratracheally. PbTx-3 moved into the bile and feces over 48 h post exposure indicating that this is the main route of excretion. While exposed animals showed clear clinical symptoms of toxicity including partial paralysis, swimming in circles, and ataxia, there was no evident tissue pathology. Immune system effects were variable, with intratracheal exposure increasing SOD activity, and both intratracheal and oral exposures decreasing LPS-induced lymphocyte proliferation. Despite the evident clinical effects, turtle neurons are surprisingly resistant to PbTx-3; while cell viability decreased in a dose dependent manner across PbTx concentrations of 100–2000 nM, the LC50 was significantly higher than is seen in mammalian neurons. PbTx-3 exposure resulted in significant Ca²⁺ influx, which can trigger a cascade of excitotoxic events eventually leading to cell death. Tetrodotoxin (TTX), MK-801, and tetanus toxin prevent Ca²⁺ influx when added with PbTx-3 confirming that the mechanism of action is through VGSCs. We are currently testing treatment strategies that can be implemented to reduce the number of sea turtle deaths from PbTx-3 exposure, including those aimed at reducing neurological symptoms and increasing toxin clearance rates.

Acknowledgements

The authors wish to thank the NOAA-ECOHAB Program for funding this project. This work was funded by the ECOHAB grant: NOAA-NOS-NCCOS-201102002577.

* Presenting author
⁺ Student presenter

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