Inter- and intraspecific variability of the ichthyotoxic activity of dinoflagellates of the Karlodinium genus towards cells of the Pacific oyster, Crassostrea gigas

Abstract

During the last decades, harmful algal blooms (HABs) of dinoflagellates of the genus Karlodinium, through their toxicity to marine fauna (ichthyotoxicity), were reported to be a threat to aquaculture in several region in the world (Europe, USA, Australia…). However, in France, despite the socio-economic importance of shellfish farming, the toxicity of French Karlodinium strains to shellfish remains unknown and needs to be expounded. This study aimed at assessing the ichthyotoxic potential of French strains of Karlodinium veneficum, K. armiger, K. ballantinum and K. gentienii in comparison with Karlodinium strains from other regions worldwide, which produce toxins and were involved in mortality events. In vitro bioassays were used, targeting hemocytes and gametes of the Pacific oyster Crassostrea gigas. These free cells, convenient to sample, are involved in important physiological functions, including immunity and reproduction, respectively, making them interesting target cell models to be used as indicator of toxicity to bivalves. Oyster hemocytes were exposed for two hours to concentrations ranging from 10^2 to 10^5 cells mL-1 of twelve strains of cultured Karlodinium spp.. Hemocyte viability was then measured by flow cytometry. Gametes (spermatozoa and oocytes) were exposed to eight strains of Karlodinium spp. at a concentration of 105 cells mL-1 for two hours, before viability, morphological changes, and ROS (Reactive Oxygen Species) production were assessed by flow cytometry. Results indicated that, at a concentration of 10^5 cells mL-1, the French strains of K. veneficum (DIA2C2, IFR-CC20-044 and MAR1F7) were more cytotoxic than strains from other countries known to be ichthyotoxic, with a dose-response effect and a high intraspecific variability. In particular, some K. veneficum strains, such as CBC7 and CB3B7, were non-cytotoxic, whereas DIA2C2, IFR-CC20-044 and MAR1F7 induced twice to 12 times higher mortality of hemocytes compared to control. French strains of K. ballantinum (KBAL DIA2 B2) and K. armiger (KAR-01D) were cytotoxic at lower concentrations, 10^4 and 10^3 cells mL-1 respectively. In gametes, the three strains of K. veneficum DIA2C2, IFR-CC20-044 and MAR1F7 caused a very high mortality rate in spermatozoa (88.26%, 99.87% and 99.2% respectivel and a milder mortality rate in oocytes (25.1%, 33.2%, and 27.8% respectively). The K. ballantinum strain also induced a high mortality in spermatozoa (98.76%) although it did not affect oocytes, suggesting the mechanism of toxicity is different from K. veneficum. This study highlights a cytotoxic effect of French Karlodinium spp. to Pacific oyster, which could particularly affect reproductive success. These results provide new findings on factors that can affect shellfish farming in France. Experiments at bloom realistic concentrations must be conducted to confirm the effects on reproduction, and in vivo experiments could be carried out to identify other effects on oyster physiology. These results will contribute to improve monitoring of ichthyotoxic microalgae in France, for a better risk management in the shellfish farming industry.