Autometallographic localization of protein-bound copper and zinc in the common winkle, Littorina littorea: a light microscopical study.

Copper (Cu), zinc (Zn) and calcium (Ca) were demonstrated histochemically by means of conventional stains (rubeanic acid for copper, dithizone 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 dithizone procedures exhibited poor sensitivity: there was no positive reaction 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 deposits in some key cell types. In winkles not exposed to either copper nor zinc, autometallographically demonstrated metals were found in the connective tissue pore cells, the lysosomes of digestive cells, the basal lamina of the digestive tubule epithelium, and cytoplasmic granules in the epithelial cells of the stomach wall. In addition, in winkles exposed to copper, metal deposits were present in some apical cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, and the circulating haemocytes. In winkles exposed to zinc, metal deposits were found in the basal cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, the apex and basal lamina of the nephrocytes in the kidney, and the connective tissue layer surrounding the blood vessels. Additionally, calcium was demonstrated histochemically in the cytoplasm 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 sperm 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 protein-bound copper and zinc in molluscan tissues, and its use in combination with conventional histochemical or chemical methods is highly recommended.