Pinnatoxin G is responsible for atypical toxicity in mussels (Mytilus galloprovincialis) and clams (Venerupis decussata) from Ingril, a French Mediterranean lagoon

Abstract

Following a review of official control data on shellfish in France, Ingril Lagoon had been identified as a site where positive mouse bioassays for lipophilic toxins had been repeatedly observed. These unexplained mouse bioassays, also called atypical toxicity, coincided with an absence of regulated toxins and rapid death times in mice observed in the assay. The present study describes pinnatoxin G as the main compound responsible for the toxicity observed using the mouse bioassay for lipophilic toxins. Using a well-characterised standard for pinnatoxin G, LC-MS/MS analysis of mussel samples collected from 2009 to 2012 revealed regular occurrences of pinnatoxin G at levels sufficient to account for the toxicity in the mouse bioassays. Baseline levels of pinnatoxin G from May to October usually exceeded 40 µg kg−1 in whole flesh, with a maximum in September 2010 of around 1200 µg kg−1. These concentrations were much greater than those at the other 10 sites selected for vigilance testing, where concentrations did not exceed 10 µg kg−1 in a 3-month survey from April to July 2010, and where rapid mouse deaths were not typically observed. Mussels were always more contaminated than clams, confirming that mussel is a good sentinel species for pinnatoxins. Profiles in mussels and clams were similar, with the concentration of pinnatoxin A less than 2% that of pinnatoxin G, and pteriatoxins were only present in non-quantifiable traces. Esters of pinnatoxin G could not be detected by analysis of extracts before and after alkaline hydrolysis. Analysis with a receptor-binding assay showed that natural pinnatoxin G was similarly active on the nicotinic acetylcholine receptor as chemically synthesized pinnatoxin G. Culture of Vulcanodinium rugosum, previously isolated from Ingril lagoon, confirmed that this alga is a pinnatoxin G producer (4.7 pg cell−1). Absence of this organism from the water column during prolonged periods of shellfish contamination and the dominance of non-motile life stages of V. rugosum both suggest that further studies will be required to fully describe the ecology of this organism and the accumulation of pinnatoxins in shellfish.

Cytotoxicity, Fractionation and Dereplication of Extracts of the Dinoflagellate Vulcanodinium rugosum, a Producer of Pinnatoxin G

“…PnTX-A, -E and -F have been shown to block neuromuscular transmission through the inhibition of nicotinic acetylcholine receptors subtypes [19,20]. These receptors also are the target of PnTX-G [11]. This mechanism of action seemed to be strongly related to the spiro-imine subunit [19].…”

“…In 2011, a new species belonging to a new benthic dinoflagellate genus was isolated from a French Mediterranean lagoon (Ingril), and has been named Vulcanodinium rugosum [10]. This isolate has been shown to produce PnTX-G [11]. By morphological and phylogenetic comparison with the isolates from Japan, New-Zealand and Australia, V. rugosum has been identified as a single species producing PnTX-E, -F and -G in these regions [12].…”

Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins

“…High maximum concentrations of pinnatoxin G were observed in mussels every year, e.g., 261 μg kg −1 (in 2009), 1244 μg kg −1 (2010), 568 μg kg −1 (2011) and 631 μg kg −1 (2012) with an overall maximum of 1244 μg kg −1 26 . So far, maximum concentrations have been reported to be below 110 μg kg −1 for whole shellfish flesh in Canada, 9 below 120 μg kg −1 in Norway, 121 and below 200 μg kg −1 in New Zealand.…”

“…On average, mussels contained seven- to eight-fold higher pinnatoxin G levels on the same sampling location (Ingril Lagoon, France), confirming that mussels are good sentinel species for pinnatoxins. 26 Spirolide uptake and depuration have been studied experimentally by exposing oysters ( Crassostrea gigas ) to cultures of the toxic dinoflagellate A. ostenfeldii producing 13,19-didesmethyl spirolide C, 13-desmethyl spirolide C, 13-desmethyl spirolide D, and traces of spirolide D. After a four-day exposure, 83% of the total initial spirolide content was found in the digestive glands, and only 17% in remaining tissues, as determined by LC-MS/MS. Inflammatory responses were observed in the intestinal tract of oysters, but no mortality was reported.…”

Spirolides and Cyclic Imines: Toxicological Profile

“…The recognition of the dinoflagellate producer of pinnatoxins was ultimately associated with the discovery of a new dinoflagellate species, named Vulcanodinium rugosum (Nézan and Chromérat 2011), in water samples of a Mediterranean lagoon in the French coast (Hess et al 2013), which produced pinnatoxin G (Table 5). Recently, a new pinnatoxin analogue, pinnatoxin H, was purified from a culture of the dinoflagellate Vulcanodinium rugosum from the South China Sea (Selwood et al 2014).…”