Abstract
Tetrodotoxin (TTX) is one of the most potent neurotoxins known. It was originally thought to only occur in puffer fish but has now been identified in twelve different classes of freshwater and marine organisms, including bivalves. Despite being one of the world’s most studied biotoxins, its origin remains uncertain. There is contradictory evidence regarding the source of TTX and its pathway through food webs. To date, the distribution of TTX has not been examined in bivalves. In the present study, 48 Paphies australis, a TTX-containing clam species endemic to New Zealand, were collected. Thirty clams were dissected, and organs and tissues pooled into five categories (siphons, digestive gland, adductor muscles, and the ‘rest’) and analyzed for TTX using liquid chromatography-mass spectrometry (LC-MS). The micro-distribution of TTX was visualized in the remaining 18 individuals using an immunohistological technique incorporating a TTX-specific monoclonal antibody. The LC-MS analysis revealed that siphons contained the highest concentrations of TTX (mean 403.8 µg/kg). Immunohistochemistry analysis showed TTX in the outer cells of the siphons, but also in the digestive system, foot, and gill tissue. Observing TTX in organs involved in feeding provides initial evidence to support the hypothesis of an exogenous source in P. australis.
Highlights
Tetrodotoxin (TTX) is a lethal neurotoxin that selectively binds and blocks voltage-gated sodium channels [1]
Based on similar studies on the neurotoxin saxitoxin in clams Saxidomus gigantea [33] and in other Paphies species [34], we hypothesized that TTX would be located in the siphons where it would act as a defense mechanism, and in the reproductive system of P. australis where it would be transferred to larvae to aid in their protection post-spawning
Tetrodotoxin was the most abundant analogue in the matrices analyzed by liquid chromatography-mass spectrometry (LC-MS)/MS, accounting for >99%
Summary
Tetrodotoxin (TTX) is a lethal neurotoxin that selectively binds and blocks voltage-gated sodium channels [1]. Recent advances in chemical detection and quantification methods (i.e., Liquid ChromatographyTandem Quadrupole Mass Spectrometry; LC-MS/MS) have allowed the development of very specific, sensitive, and accurate methods to measure TTX concentrations [24,25] The use of these methods makes it possible to investigate and quantify the distribution of TTX in specific organs within bivalves. Based on similar studies on the neurotoxin saxitoxin (produced by planktonic marine dinoflagellates and freshwater cyanobacterial species) in clams Saxidomus gigantea [33] and in other Paphies species [34], we hypothesized that TTX would be located in the siphons where it would act as a defense mechanism, and in the reproductive system of P. australis where it would be transferred to larvae to aid in their protection post-spawning
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