HABs as causes of physiological stress in shellfish

Lysosomal Destabilization

Lysosomes are regarded as valuable indicators of compromised biotic integrity.  Lysosomes, typically involved in cellular defense, tissue repair and nutrition, can become destabilized through exposure to a variety of stressors.  This assay is performed by incubating oyster hepatopancreas cells in neutral red dye, then counting the number of cells with dye present in the lysosomes (stable) or dye present in the cytosol (destabilized).  The leaking of neutral red reflects the efflux of lysosomal contents into the cytosol, which ultimately leads to cell death.

Stable hepatopancreas cell                                Destabilized hepatopancreas cell

Higher percentages of lysosomal destabilization indicate an adverse effect on the organism.

 P-glycoprotein Expression

P-glycoprotein (p-gp) is a membrane bound glycoprotein that acts as an efflux pump for organic xenobiotics.  P-gp has been found to be over-expressed in bivalves collected at sites with high levels of organic pollution.  Recent work has focused on the effects of natural xenobiotics on p-gp expression and activity (algal extracts have been shown to increase p-gp expression;  brevetoxin has been shown to affect p-gp activity).  This assay is performed using dot blot techniques and antibody visualization to determine differences in p-gp expression between individual oysters.

 Higher expression or activity of p-gp indicates an adverse effect on the organism.

RESULTS

In 2001, we found that lysosomal destabilization was significantly elevated in native oysters exposed to Kryptoperidinium sp. at the Kiawah 1 site.  In laboratory experiments, lysosomal destabilization was also significantly elevated in hatchery-reared oysters exposed to Kryptoperidinium bloom water for 4 days. 

An asterisk (*) indicates a significant difference from the control.

In 2002, hatchery-reared oysters were deployed at the Kiawah 1 site, and were collected every two weeks throughout the summer and fall.  Lysosomal destabilization rates were significantly related to Kryptoperidinium concentrations, as was P-gp expression.

In 2003, Heterosigma akashiwo blooms were collected from Bull’s Bay and Shem Creek.  Hatchery-reared oysters were exposed to this bloom water for 4 days, with results similar to those of 2001.  In both experiments, lysosomal destabilization rates were higher in oysters exposed to bloom water than control water.  P-gp expression was also elevated after exposure in the April 2003 experiment.

An asterisk (*) indicates a significant difference from the same experimental                               control.