Evaluating the Effects of Biotoxins on Immune Cell Functions in Zebrafish

A variety of biological toxins can be present at harmful levels in the aquatic environment. Cyanobacteria are a diverse group of prokaryotic microorganisms that produce cyanotoxins in the aquatic environment. These biotoxins can be hepatotoxins, dermatoxins, or neurotoxins and can affect fish and mammals. At high levels, these compounds are fatal. At non-lethal levels, they act insidiously and affect immune cell functions. Algae-produced biotoxins include microcystin and anatoxin A. Aquatic animals can also ingest material contaminated with botulinum neurotoxin E (BoNT/E) produced by Clostridium botulinum, also resulting in death or decreased immune functions. Zebrafish can be used to study how toxins affect immune cell functions. In these studies, toxin exposures can be performed in vivo or in vitro. In vivo studies expose the zebrafish to the toxin, and then the cells are isolated. This method demonstrates how the tissue environment can influence leukocyte function. The in vitro studies isolate the cells first, and then expose them to the toxin in culture wells. The leukocytes are obtained by kidney marrow extraction, followed by density gradient centrifugation. How leukocytes internalize pathogens is determined by endocytic mechanisms. Flow cytometry phagocytosis assays demonstrate if endocytic mechanisms have been altered by toxin exposure. Studies using isolated leukocytes to determine how toxins cause immune dysfunction are lacking. The procedures described in this article will enable laboratories to use zebrafish to study the mechanisms that are impacted when an environmental toxin decreases endocytic functions of immune cells.