Assessing the occurrence of a stress syndrome in mussels (Mytilus edulis) using a combined biomarker/gene expression approach

A combination of biomarkers and gene expression analyses was used to investigate the occurrence of a stress syndrome in mussels (Mytilus edulis) caged along a copper pollution gradient in the Visnes fjord, Norway. The stress level in mussels, as calculated by a novel algorithm (the “Expert System”) from a set of seven biomarkers, was compared with gene expression changes utilising a low-density oligonucleotide microarray, employing 24 different genes involved in both cellular homeostasis and stress-related responses. The biomarker battery included lysosomal membrane stability, lysosomal accumulation of neutral lipids and lipofuscin, lysosomal/cytoplasm volume ratio, Ca2+-ATPase and catalase activities, and total metallothionein content. Integration of the biomarkers into the Expert System ranked individuals sampled at site 2 as unstressed, mussels sampled at site 3 as being subject to low stress, and those from site 4, which is adjacent to what used to be a copper mine, as being highly stressed, with respect to specimens sampled from the reference site. Microarray analyses demonstrated that at the two innermost and mostly polluted sites, gene expression patterns where severely altered. In particular, some genes exhibited a linear activation response along the copper gradient, e.g. metallothioneins mt 20 and mt 10, and catalase. In addition, stress responsive kinase (krs), glutathione transferase (gst), major vault protein and histones (h1, h2a and h4) were significantly up-regulated at the innermost site. In conclusion, these results demonstrated that sites could be discriminated using both a physiological and a molecular approach. The development of a stress syndrome along the pollution gradient was evidenced with a novel mussel microarray, both in terms of numbers of regulated genes and level of gene response.