Burton J. Bogitsh

Acid phosphatase activity in spermatids of the snail Biomphalaria glabrata.

VISHNIAC, H. S. & WATSON, S. W. 1953. The steroid requirements of Labyrinthula vitellina var. pacifica. J. Gen. Microbiol., 8: 248-255. WATSON, S. W. 1957. Cultural and cytological studies on species of Labyrinthula. Ph.D. Dissertation, University of Wisconsin. 161 pp. University Microfilms, Ann Arbor, Michigan. WATSON, S. W. & ORDAL, E. J. 1951. Studies on Labyrinthula. Tech. Rep., Univ. Wash. Oceanogr. Lab., 3: 1-37. 1956. Techniques for the isolation of Labyrinthula and Thraustochytrium in pure culture. J. Bacteriol., 73: 589-590. WaTSON, S. W. & RAPER, K. B. 1957. Labyrinthula minuta sp. nov. J. Gen. Microbiol., 17: 868-877.

Ultrastructural localization of ATPases in the gastrodermis of Megalodiscus temperatus with additional observations on Schistosoma japonicum (Trematoda: Digenea).

The strontium-lead protocol was used to localize, ultrastructurally, a ouabainsensitive, potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) of the Na,KATPase complex in the gastrodermis of the frog rectal fluke Megalodiscus temperatus and the blood fluke Schistosoma japonicum. K-pNPPase activity was observed on the cytoplasmic side of the gastrodermal microvillus plasma membrane of Megalodiscus and extracellularly on the parenchymal side of the gastrodermal basal plasma membrane and its infoldings of Schistosoma. A ouabain-insensitive ATPase, possibly Mg-ATPase, and nonspecific acid phosphatase activities were observed extracellularly on the luminal sides of the gastrodermal amplifications of both organisms. Active transport of Na+ via Na,KATPase probably provides the driving force for the transport of solutes such as glucose, amino acids, etc. from the lumen into the gastrodermis of M. temperatus. This mechanism is either absent or much reduced in schistosomes, and the mediated transport of such molecules as glucose and amino acids is the function of their teguments. The ouabainsensitive ATPase in the basal plasma membrane of the schistosome gastrodermis may function in ionic regulation. The function of the tegument in the acquisition of nutrients is believed to determine the type of transport in the digestive tract. The final digestion of exogenous foodstuffs by digenetic trematodes occurs extracellularly in the cecal lumen. Morphological and cytochemical studies indicate that membrane-associated enzymes, as well as those secreted by the gastrodermis, participate in the final phases of food assimilation (for reviews, see Bogitsh, 1989; Smyth & Halton, 1983). Nutrients, including water and electrolytes, are carried from the lumen to various cells of the worm by way of the gastrodermis. The surface of the gastrodermis, amplified either by microvilli or lamellae, is well-suited morphologically for absorption of these nutrients. In a number of Digenea (e.g., the schistosomes), nutrients such as glucose and certain amino acids are transported across the tegument. In others (e.g., Megalodiscus temperatus), the tegument has limited ability to transport small molecules such as hexoses (Shannon & Bogitsh, 1971), and the digestive tract becomes the primary region for such transport. Also, because there is no evidence that endocytosis of materials from the cecal lumen to the gastrodermis is of any nutritional value to the Digenea, we may assume that nutritive benefits are derived by other means, such as passive diffusion and mediated transport. The presence of a Na,K-ATPase mechanism in the digestive tract of many 1Publication costs, in part, are being met by a grant from the Spencer-Tolles Fund of the American Microscopical Society. TRANS. AM. MICROSC. SOC., 109(3): 311-318. 1990. ? Copyright, 1990, by the American Microscopical Society, Inc. This content downloaded from 207.46.13.16 on Thu, 16 Jun 2016 06:14:56 UTC All use subject to http://about.jstor.org/terms animals indicates not only a means for Na+ absorption but also the possibility that the pump may be coupled to the active transport of Cl-, glucose, some amino acids, and other solutes into the digestive tract epithelium (Bonting, 1970; Castro, 1985; Lehninger, 1970; Skou, 1965). Cytochemical localization of a Na,K-ATPase in the digestive tract, therefore, not only presents strong evidence of a sodium transport mechanism but also may indicate a transport mechanism for other materials from the cecal lumen into the digestive tract epithelium (e.g., gastrodermis). The objective of the present paper is to document the cytochemical presence and distribution of Na,K-ATPase in the gastrodermis of Megalodiscus temperatus. Of the three protocols available for the localization of Na,K-ATPase (Firth, 1980), the method of choice for accuracy and resolution is the procedure for detecting potassium nitrophenylphosphatase (K-pNPPase) activity, one portion of the Na,K-ATPase reaction (Ernst, 1972a). MATERIALS AND METHODS Living adult specimens of Megalodiscus temperatus were removed from the rectal areas of commercially purchased specimens of Rana pipiens and rinsed thoroughly with 0.7% saline. Worms were sliced into 2-mm sections, fixed in 0.25% distilled glutaraldehyde and 1% paraformaldehyde in cacodylate buffer (0.1 M, pH 7.2) (Ernst, 1975) at room temperature for 10 min, rinsed in buffer for 30 min, and immersed in the incubation medium for 45 min at 37?C. The incubation medium consisted of MgCl (10 mM), KCI2 (10 mM), SrC12 (20 mM), para-nitrophenylphosphate-disodium (NPP) (5 mM) dissolved in tris-HCl buffer (10 ml, 0.1 M, pH 8.6). After 5 min of incubation, a faint yellow color, indicative of the enzymatic hydrolysis product nitrophenol, was discernible in the medium. Following incubation, the tissues were rinsed in three 5-min rinses with the tris-HCl buffer and then in two 5-min rinses with 2% lead nitrate to convert the strontium phosphate to lead phosphate. Two 5-min rinses with 5% sucrose to remove free lead were followed by a cacodylate buffer rinse (0.1 M, pH 7.2). After the final rinses, the tissue was postfixed in 2% osmium tetroxidecacodylate (0.1 M, pH 7.2) for 90 min at room temperature. Sections were rinsed in water, dehydrated in an ethanol series and embedded in Epon by way of propylene oxide. Sections for electron microscopy were made with a diamond knife on a LKB ultratome and viewed in a Hitachi HS-8 electron microscope. For controls, the incubating medium was varied as follows: (a) the 10 mM KCI was replaced by 10 mM choline chloride; (b) beta-glycerophosphate (GP) (5 mM) was used to replace NPP at pH 7.2 and 8.6; (c) ouabain (10 mM) was added; and (d) cysteine (10 mM) was added. The distribution of non-specific acid phosphatase was examined according to the method of Bogitsh (1973) using either cytosine monophosphate (CMP) or GP as substrate at pH 5.0. Selected sections also were tested for ATPase activity using the lead method of Wachstein & Meisel (1957). As a final control, some sections were incubated in a medium from which ATP was omitted. 312 TRANS. AM. MICROSC. SOC. This content downloaded from 207.46.13.16 on Thu, 16 Jun 2016 06:14:56 UTC All use subject to http://about.jstor.org/terms VOL. 109, NO. 3, JULY 1990