Histol Histopath (1 993) 8: 537-546 Light microscopic catalase histochemistry in mussel digestive gland tissue M.P. Cajaraville, J.A. Uranga and E. Angulo Laboratory of Cytology and Histology, Department of Cell Biology and Morphological Sciences, University of the Basque Country, Bilbao, Spain Summary. Different light microscopical procedures for the histochemical demonstration of catalase were tested in cryostat sections of mussel digestive gland tissue by using both benzidine and diaminobenzidine (DAB) as hydrogen donors. The selected procedure, which was also applied to mouse liver for comparative purposes, consisted of incubation in media containing 0.2% DAB and 0.3% H202 at pH 10.4 for 35 min at 42 "C. Addition of 0.01 M imidazole to the incubation medium increased the staining intensity of the histochemical procedure. The positive reaction product was localized in epithelial cells lining the digestive tubules and the ducts. The histochemical reaction was inhibited partially by aminotriazole or sodium azide and disappeared completely by omission of H202 from the incubation medium. On the other hand, heat resistant non- enzymatic reactions were observed in sites known to contain lipofuscins such as epithelial cells of the gastrointestinal tract and connective tissue brown cells. Key words: Catalase, Histochemistry, Benzidine, Diaminobenzidine, Imidazole, Mussel, Digestive tissue, Light Microscopy Introduction The digestive gland of bivalve molluscs consists of primary ducts arising from the stomach, followed by thinner, branched secondary ducts opening into the digestive tubules (Morton, 1983). The digestive tubules are comprised of digestive cells, involved in the intracellular digestion of food material, and basophilic cells, whose functions are still not well understood (Owen, 1970, 1972b, 1973; Pal, 1971 ; Morton, 1983; Cajaraville et al., 1990a). In addition to duct cells, both digestive and basophilic cells contain peroxisomes identified by using the alkaline 3,3'-diaminobenzidine Offprint requests to: Dr. M.P. Cajaraville, Zitologi eta Histologi Laborategia, Biologia Zelularra eta Zientzia Morfologikoen Saila, Euskal Herriko Uniberlsitatea,644 P.K., 48080 Bilbo, Spain Histology and Histopathology (DAB) technique (Fahimi, 1969; Novikoff and Goldfischer, 1969) in the bivalves Nucula sulcata (Owen, 1972a), Ruditapes decussatus (Henry, 1987) and Mytilus sp (Cajaraville et al., 1992b). The brief report of Yokota (1970) also indicates that DAB-positive peroxisomes are found in a number of marine bivalve molluscs. Within the gastropod molluscs, Dannen and Beard (1977) find peroxisomes, identified by morphological criteria, in the kidney of two terrestrial pulmonate gastropods, but while peroxisomes of Arion ater are reactive to DAB cytochemistry, peroxisomes of Ariolimax columbianus are not. Finally, peroxisomes have been detected cytochemically in the nervous system of the marine gastropod Aplysia californica (Beard and Holtzman, 1985). The data quoted above illustrates that few investigations have been coducted on the cyto- and histochemical localization of catalase, the marker enzyme for peroxisomes, in molluscs. The aim of the present study was to apply the methodology of catalase histochemistry to the digestive gland tissue of mussels, Mytilus galloprovincialis Lmk. This seems particularly interesting in view of the world-wide use of mussels in pollution monitoring programmes (Bayne, 1989) and preliminary studies have already been conducted on the use of mussel catalase activity as an indicator of damage caused by petroleum hydrocarbon exposure (Di Giulio et al., 1989; Cajaraville et al., 1990b, 1992a; Livingstone et al., 1990). Materials and methods Animals Mytilus galloprovincialis individuals were collected from Mefiakoz, Biscay (43"24'N, 2"93'W) and transferred to the laboratory. Individuals (2.5-3.5 cm shell length) were maintained in 25-1 polyethylene- covered tanks in aerated thermostatized (15-16 "C) semicontinuous water flow system with activated charcoal and glass-wool filtered natural seawater (Zierbena, Biscay). Mussels were maintained unfed for