In vitro Incorporation of Precursors of RNA into the Digestive Gland of Normal and of Schistosoma-Infected Biomphalaria glabrata

Abstract

Digestive glands of normal Biomphalaria glabrata were incubated in a balanced salt solution containing 3H-uridine, 3H-cytidine, or 'H-orotic acid. RNA was then extracted and its specific activity measured. Greatest activity was obtained from organs incubated in the nucleosides; incorporation of radioactive orotic acid was low. In addition, digestive glands from Schistosoma mansoni-infected snails were incubated in tritiated uridine. Specific activity of digestive gland RNA from infected snails was higher than that from uninfected controls. In vitro incorporation of exogenous uridine was accelerated in the digestive gland of infected snails before the sporocysts reached this organ. This suggests that infection in anterior organs may decrease the rate of RNA breakdown, decrease the host's uridine pool, or increase the rate of RNA synthesis in the still-unparasitized digestive gland. The molluscan digestive gland, or hepatopancreas, is the site of intense reproductive activity of many trematode larvae. In Biomphalaria glabrata the architecture of the digestive gland becomes extensively altered during the course of infection by Schistosoma mansoni. In fact, the schistosome larvae replace a large amount of the host's tissue. Structural changes in the hepatopancreas of trematode-infected snails are accompanied by various metabolic alterations (see reviews by Cheng and Snyder, 1962; Wright, 1966). However, no published accounts deal with the influence of larval parasites on the nucleic acid metabolism of snails. This study was designed to investigate incorporation of exogenous precursors of RNA into the isolated digestive gland of Biomphalaria glabrata, and to determine if the incorporation of these precursors is altered by parasitism. MATERIALS AND METHODS Biomphalaria glabrata (strain PR-2'; Pan, 1965), maintained on romaine lettuce at 26 1 C, was used in all experiments. Infections were obtained by mass-exposing snails to miracidia of a Puerto Rican strain of Schistosoma mansoni. Infected snails were identified by the presence of visible Received for publication 30 August 1968. * This investigation was supported by Grants AI00513 and AI-00046 from the NIAID, U. S Public Health Service. t Present address: Department of Zoology, College of Biological Sciences, University of Minnesota, Minneapolis, Minn. 55455. mother sporocysts in the tentacles or head-foot. Digestive glands were examined by press preparations (Chernin and Dunavan, 1962) to determine if daughter sporocysts were present. Snails were 13 ? 1 mm in shell diameter when used in experiments. Each snail was gently crushed between glass slides, removed from its shell, and the digestive gland excised. The outer epithelium (tunica propria) was stripped away from each gland to provide a more uniform surface, and adjacent organs were carefully removed. The pooled glands were washed in a balanced salt solution (Chernin, 1963), and used within 10 min. The incubation medium consisted of the balanced salt solution, penicillin (200 units/ml), and phenol red, plus 1 A/c/ml of tritiated uridine, cytidine, or orotic acid (specific activity = 2 Ci, 2 Ci and 1.64 Ci per m Mole, respectively). All labeled compounds were obtained from New England Nuclear Corp., Boston, Mass. Groups of three digestive glands were placed in 25-ml Erlenmeyer flasks containing 10 ml of the medium. After incubation at 25 ? 1 C on a rotary shaker the digestive glands were washed and each pool of three organs was immediately placed in 2 ml of cold sucrose (0.25 M) containing 5% citric acid. The tissue was homogenized with a Ten Broeck grinder, and 1 ml of the homogenate was withdrawn for nucleic acid extraction according to the modification by Scott, Fraccastoro, and Taft (1956) of the SchmidtThannhauser method. The extraction procedure was further modified by using 0.3 N NaOH for the hydrolysis of RNA. RNA was measured by ultraviolet spectrophotometry (Scott, Fraccastoro, and Taft, 1956) in a Zeiss PM QII spectrophotometer. For counting, 0.1-ml aliquots of supernatant fraction were added to 15 ml of a scintillation fluid consisting of 42 ml of Liquifluor? per liter of toluene-ethanol (3:1). Radioactivity was measured in a NuclearChicago Unilux liquid scintillation counter.