AUTOMETALLOGRAPHIC LOCALIZATION OF PROTEIN-BOUND COPPER AND ZINC IN THE COMMON WINKLE, LITTORINA-LITTOREA - A LIGHT-MICROSCOPIC STUDY

Copper (Cu), zinc (Zn) and calcium (Ca) were demonstrated histochemica lly by means of conventional stains (rubeanic acid for copper, dithizo ne for zinc, and cobalt nitrate for calcium) and by autometallography in various tissues of winkles (Littorina littorea) sublethally exposed to either copper or zinc dissolved in sea water. Rubeanic acid and di thizone procedures exhibited poor sensitivity: there was no positive r eaction after fixation tissues with Bouin's fixative, and only a weak reaction after ethanol fixation. Autometallography, however, produced a positive reaction with both fixatives in the form of black silver de posits in some key cell types. In winkles not exposed to either copper nor zinc, autometallographically demonstrated metals were found in th e connective tissue pore cells, the lysosomes of digestive cells, the basal lamina of the digestive tubule epithelium, and cytoplasmic granu les in the epithelial cells of the stomach wall. In addition, in winkl es exposed to copper, metal deposits were present in some apical cytop lasmic granules of ciliated cells in the gill epithelium, the mucous s ecretion of gill mucocytes, and the circulating haemocytes. In winkles exposed to zinc, metal deposits were found in the basal cytoplasmic g ranules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, the apex and basal lamina of the nephrocytes in th e kidney, and the connective tissue layer surrounding the blood vessel s. Additionally, calcium was demonstrated histochemically in the cytop lasm of digestive cells, the cytoplasm of the epithelial cells of the stomach wall, the mucocytes of gills, the basal lamina of the kidneys, the haemocytes, the calcium and pore cells of connective tissue, and the oocyte cytoplasm. Metals were not detected by any procedure in spe rm cells, in the cytoplasmic granules of oocytes, or in the basophilic cells in the digestive tubules. In conclusion, autometallography is a highly sensitive method and provides an excellent tool to localize pr otein-bound copper and zinc in molluscan tissues, and its use in combi nation with conventional histochemical or chemical methods is highly r ecommended.