Schistosoma mansoni: calcium content of cercariae and its effects on protease activity in vitro

The localization and quantitation of calcium in the protease-containing preacetabular glands of Schistosoma mansoni cercariae were determined by electron probe analysis and atomic absorption spectroscopy. Levels of calcium in these glands appear to exceed 8 to 10 M. In vitro, these high levels of calcium inhibit the cercarial proteases reversibly. It is suggested that calcium functions to control protease activity in situ in these organisms. The infective stage of the human blood fluke, Schistosoma mansoni, infects its host by penetrating the skin. The penetration process is thought to be mediated by proteolytic enzymes present in the preacetabular glands of this organism (Lewert and Lee, 1956; Stirewalt and Fregeau, 1966; Gazzinelli et al., 1966; Dresden and Asch, 1972). Moreover, it has been demonstrated that these glands contain large amounts of calcium (Stirewalt, 1959; Lewert et al., 1966). Lewert and Lee (1956) originally reported that these proteases are stimulated at low concentrations of these cations and we have confirmed this observation (Dresden and Edlin, 1974). Recently we determined that the proteolytic enzymes in extracts of this organism are inhibited by high concentrations of calcium and magnesium (Dresden and Edlin, 1974). We suggested that calcium might play a significant role in controlling the activity of these proteases by acting as an inhibitor of protease activity in situ. The results described in this report provide qualitative and quantitative evidence on the levels of calcium in these glands and its effects on protease activity. MATERIALS AND METHODS Infected snails, Biomphalaria glabrata (Puerto Rican strain), were allowed to shed their cercariae at room temperature in beakers containing dechlorinated tap water or 1 mM Tris-HCl, pH 7.8. Using a depression slide and a dissecting microscope, individual cercariae were transferred by disposable pipette. For electron probe analysis single cercariae were dried on silica discs at room temperature as rapidly as possible, aided by withdrawal of water with a micropipette. For calcium analysis, individual cercariae (50 Received for publication 10 October 1974. t 200) were counted and transferred to appropriate tubes. Spectrophotometric analysis of calcium was by the method of Williams and Wilson (1961). CaCl2 and CaCO3 (0.5 to 100 ,ug) were used as standards. Atomic absorption spectroscopy for calcium was determined following extraction of 100 to 200 cercariae in 5% lanthanum chloride-1% HC1 at 100 C for 10 min. Calcium contributed by sources other than the cercariae was corrected for by using uninfected snails. Protease activity in cercarial extracts was measured using Azocoll substrate as described previously (Dresden and Edlin, 1974). RESULTS AND DISCUSSION The results of the electron probe X-ray analysis and scanning electron microscopy are shown in Figure la-c. These figures represent a single cercaria examined by scanning electron microscopy (Fig. la) and by X-ray probe analysis for calcium (Fig. lb) and phosphorus (Fig. lc). A number of individual cercariae were examined by these combined techniques. The results demonstrate dramatically a concentration of calcium in the region of the preacetabular glands, and a fairly uniform distribution of phosphorus throughout the body of the cercariae. Using the tail region as a control, scanning X-ray analysis was carried out for 20to 40-sec intervals at 25 kv with a 0.185-,uamp sample current. These experiments demonstrated that calcium is at least 1,000 to 2,000 times more abundant in the preacetabular gland region than in the tail region. Magnesium levels in the gland region were not significantly elevated over those in the tail region. While these experiments established the localization of calcium, it would have been necessary to determine the thickness and composition of the cercariae in order to determine