Differences in the toxicity of six Gambierdiscus (Dinophyceae) species measured using an in vitro human erythrocyte lysis assay

Abstract

This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ∼7-40% from log phase growth to late log - early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20-31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60-90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced by Gambierdiscus species. While the results from inhibition studies require validation by LC-MS analysis, the available data strongly suggest differences in hemolytic activity observed in this study reflect maitotoxicity.

Characterization of Gambierdiscus lapillus sp. nov. (Gonyaulacales, Dinophyceae): a new toxic dinoflagellate from the Great Barrier Reef (Australia)

“…, Holland et al. ), although LC‐MS analysis using known CTX analogs has not been conducted for this species as yet. A previous study found a possible CTX analog in a Gambierdiscus strain from Queensland, however, the production of the compound was unstable, and the strain precedes the discovery of species diversity in Gambierdiscus (Holmes et al.…”

Toxicity of benthic dinoflagellates on grazing, behavior and survival of the brine shrimp Artemia salina

“…A short stabilization of effects was observed after 3-h exposure, followed by a reduction in the percentage of individuals swimming on the water column as well as in the survival of the brine shrimps between 5 to 7-h exposure. Maitotoxins (MTX) alter ion transport systems causing an increase in free intracellular Ca 2+ [65] while ciguatoxins (CTXs) are neurotoxins that bind to the voltage-sensitive sodium channels on cell membranes [66–67]. The toxic effects of ciguatoxins seem to be similar to saxitoxins effects, the neurotoxins produced by the planktonic dinoflagellate Alexandrium spp.…”

Molecular phylogenetics and morphology of Gambierdiscus yasumotoi from tropical eastern Australia

“…However, a putative MTX analogue which had the same mass as MTX-3 Lewis et al, 1994) was found . A study using the human erythrocyte lysis assay (HELA) showed significant toxicity in Gambierdiscus ruetzleri (Holland et al, 2013). Experiments which used specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced in those studies (Holland et al, 2013).…”

“…A study using the human erythrocyte lysis assay (HELA) showed significant toxicity in Gambierdiscus ruetzleri (Holland et al, 2013). Experiments which used specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced in those studies (Holland et al, 2013). MTX was generally considered not to be related to CFP symptoms, which are thought to be solely caused by ciguatoxins (CTX) (Lewis and Holmes, 1993).…”