Zeitschriff fSr
Z. Parasitenk. 54, 165-173 (1977)
Parasitenkunde Parasitologic Research
© by Springer-Vedag 1977
Schistosoma mansoni: Localization of Calcium-Detecting Reagents in Electron-Lucent Areas of Specific Preaeetabular Gland Granules* C.H. D o r s e y ' and Margaret A. Stirewalt 2 ' Experimental Pathology Department, Naval Medical Research Institute, Bethesda, Maryland, and 2Biomedical Research Institute, Bethesda, Maryland
Summary. In an attempt to establish the exact location of calcium within the preacetabular glands of cercariae of Schistosoma mansoni, these larvae were exposed to reagents (potassium oxalate, potassium pyroantimonate, chloranilic acid, and silver nitrate) useful in the detection of calcium, and were subsequently observed with the aid o f light and electron microscopes. Cercariae incubated in potassium oxalate and viewed in polarized light showed birefringence only in the preacetabular gland funduses. At the ultrastructural level, the preacetabular glands of potassium oxalate-treated cercariae had no electron-dense precipitate, but instead had translucent, irregularly shaped inclusions, similar to space s left by volatilized calcium oxalate as described by others. Pyroantimonate treatment, on the other hand, localized the reaction in the electron-lucent areas of the lightspotted granules. The von Kossa silver nitrate procedure destroyed the secretory granules; therefore, an electron-dense precipitate was distributed throughout the gland. However, pretreatment with chloranilic acid before fixation preserved the granules, and subsequent exposure to the von Kossa silver nitrate gave a reaction identical to that obtained with the pyroantimonate alone. When viewed in polarized light, chloranilic acid-incubated cercariae showed birefringence in the fundus and duct areas. Introduction Since infection of definitive hosts by schistosomes follows cercarial penetration of skin, it is important that penetration mechanisms be understood. It is known that penetration requires both muscular and chemical activity by cercariae (Stirewalt, 1966). Movement through the horny layer of the skin is via openings made by disarticulation of the epithelial squames (Stirewalt and Dorsey, 1974). This is achieved by the probing action of the oral sucker, and is most likely facilitated by loosening of the squame interdigitations as a result oflysis of the * Supported by the Naval Medical Research and Development Command Work Unit No. MR041.05.01.0037, and Office of Naval Research Contract No. N 00014-76-C-0053. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Department of the Air Force, the Navy Department or the naval service at large
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intercellular ground substance by proteolytic enzyme(s) secreted by the preacetabular glands (Lewert and Lee, 1954; Stirewalt, 1973; Stirewalt and Dorsey, 1974). C alcium is secreted concurrently with the enzyme(s) (Stirewalt and Kruidenier, 1961), and may be involved in the enzyme activity. Preacetabular gland secretions of Schistosoma mansoni cercariae contain at least two types of secretory granules: type A, which has an electron-dense matrix spotted with electron-lucent areas; and type B, which is homogeneously granular (Dorsey and Sürewalt, 1971; Ebrahimzadeh and Kraft, 1971). The preacetabular glands have shown an affinity for dyes used in light microscopy to identify calcium: purpurin (Stirewalt and Kruidenier, 1961), glyoxal bis (2-hydroxyanil) (Lewert et al., 1966), and Luxol fast blue (Shanklin and Nassar, 1959). By using the electron microprobe and atomie absorption spectroscopy, Dresden and Edlin (1975) found an extremely high concentration of caleium in the preacetabular glands.
In our preliminary studies, staining with Luxol fast blue and counter-staining with periodic acid-Schiff according to Shanklin and Nassar's (1959) method produced a differential color in the granules, some staining blue, others pinkish. This suggests a difference in the cytochemical nature of the two types of secretory granules. The purpose of this work was to aseertain by cytochemical methods, at the ultrastructural level, the preacetabular granule in which calcium occurs and its location in the granule.
Materials and Methods Schistosoma mansoni cercariae were collected from snails (a Puerto Rican strain of Biomphalaria glabrata maintained in this laboratory) and trapped on a Gelman filter (Metricel, Alpha-8, 47 mm outer diameter) mounted on a Fisher Filtrator 9-788 (Fisher Scientific Co., Silver Spring, MD). They were immediately processed by one of the following calcium localization techniques. Oxalate Procedure of Costantin et al. (1965). Incubation in an aqueous mixture containing 0.2% pótassium oxalate and 1.2% potassium chloride for from 5 min through 1 h was followed by fixation in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), rinsing in the same buffer for 1 h, and postfixation for 60 min in 1% osmic acid in a 0.1 M cacodylate buffer (pH 7.4). Subsequent dehydration was in reagents saturated with potassium oxalate. Pyroantimonate Proeedure of Legato and Langer (1969). Cercariae were held in a 1% solution of osmic acid containing 2% potassium pyroantimonate in 0.01 M acetic acid (pH 7.5-7.8) for 2 h. Chloranilic A cid Procedure (Modification of Carr et aL 1961). Fixation was in 0.5% glutaraldehyde in 1% chloranilic acid buffered to pH 4 with 0.4% NaOH for I h, after which some eercariae were placed in 1% osmic acid (dissolved in deionized H20) for 1 la, while others were processed without osmicatiom Von Kossa's Silver Nitrate Procedure (According to Pearse, 1972). Cercariae were placed in an aqueous silver nitrate-dextrose solution (1% silver nitrate and 5% dextrose) for 2 h in the light of an ineandescent 60 W light bulb plaeed 15 cm away. They were rinsed briefly in tw0 changes of deionized watet, left overnight in 1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and proeessed for electron microscopy without osmication. Chloranilic Acid-Silver Nitrate Procedure. Incubation of cercariae in an aqueous solution of chloranilic acid (0.4% NaOH and 0.5% chloranilic acid) for 1 h preceded their fixation for 20 min in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.5). Some were then rinsed in deionized water and placed overnight in a 0.1% AgNO 3 solution in 95% ethanol containing 0.2% dextrose without osmication. Others were held in 0.5% chloranilic acid buffered to pH 4 with sodium hydroxide for 30 min, fixed in cacodylate-buffered 2.5% glutaraldehyde (0.1 M, pH 7.4) for 30 min, rinsed briefly in two changes of tertiary butyl alcohol, and kept overnight in 80% tertiary butyl alcohol containing 1% silver nitrate and 5% dextrose. For comparison, other cercariae were placed for 2 h in 2.5% glutaraldehyde (buffered as above), whieh contained 0.5% ehloranilic aeid, and maintained overnight in the dark in an aqueous solution of 2.5% dextrose and 1% osmic acid.
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The unmodified von Kossa method destroyed the secretory granules, but gave an electron-dense reaction product when the silver nitrate solution contained dextrose, which functions as a silver reducing reagent. Pretreating in chloranilic acid and fixing briefly in glutaraldehyde before exposure to silver nitrate resulted in better preservation of the membrane integrity and secretory granules. All tissues were dehydrated, cleared in xylene, and processed for electron microscopy according to the methods of Luft (1961). The tissue was flat-embedded in a 60 mm diameter aluminum weighing tray (Fisher) under a 1 mm layer of Luft's mixture. For light-microscopic observation, whole cercariae were examined through the thin layer of epon in polarized light. For electron microscopy, groups of cercariae were cut from the embedding medium with a jeweler's saw, glued to a wooden rod, sectioned with an ultramicrotome, and examined in a Siemens Elmiskop lA electron microscope. Some of the thin sections (60-90 nm) were stained with aqueous uranyl acetate followedby lead citrate (Reynolds, 1963).
Results
Light Mieroseopy. Cercariae treated with potassium oxalate showed birefringence, but only in the gland fundus (Fig. 1). Cercariae incubated with chloranilic acid were birefringent throughout the area of the preacetabular glands, i.e., the fundus and duct regions (Fig. 2). When examined in nonpolarized light, the fundus area was a rusty red color.
Eleetron Mieroscopy. Two distinct types of secretory granules previously reported (Dorsey and Stirewalt, 1971; Ebrahimzadeh and Kraft, 1971) in the fundus of the preaeetabular glands are shown in Fig. 3. The granules with the electron-lucent areas have been designated type A; the homogeneous granules, type B.
Potassium Oxalate. Although the birefringence indicated the presence of a reaction produet (calcium oxalate) in the preacetabular glands, no granular electron-dense precipitates were observed (Fig. 4). However, some changes did occur in the birefringent area. It appeared that the electron-lucent areas in many of the type-A seeretory granules became brighter and progressively larger, finally coalescing into white, irregularly shaped inelusions. Type-B secretory granules did not coalesce. Pyroantimonate-Osmium. This method gave an electron-dense reaction product in the electron-lucent areas of type-A secretory granules of the preacetabular glands (Figs. 5, 6). N o reaction product was observed in type-B granules or free in the cytoplasm of the glands.
Von Kossa's Silver Nitrate. The reaction product was confined to the preacetabular glands. The secretory granules, however, had deteriorated to the extent that they could not be elearly identified and the reaction product was diffused throughout the cytoplasm (Fig. 7). Small amounts of the reaction product were present around the gland periphery. Chloranilie Acid-Silver Nitrate. This reaction gave results similar to those of the pyroantimonate-osmium in which the ele¢tron-dense product was present in the electron-lucent areas of the secretory granules (Figs. 8, 9). This was true if cercariae were exposed to chloranilic acid before fixation (Figs. 8, 9), or if the chloranilic acid was added to the fixative.
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Fig. 1. S. mansoni: Photomicrograph of cercariae exposed to potassium oxalate and viewed in polarized light. Birefringence (arrow heads) occurs only in the area of the fundus of the preaeetabular glands, x360
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Discussion The cytochemical techniques used herein are not specific for calcium, but for metallic cations. Dresden and Edlin (1975), however, demonstrated by electron probe analysis that calcium was the metallic cation present in very high concentration in the preacetabular glands of S. m a n s o n i cercariae. They theorized that the calcium would be in the secretory granules of these glands. With the aid of the electron-microscopic procedures used in this investigation, calcium, either ionic or as a salt, was found in specific granules of the preacetabular glands. It was in the type-A rather than the type-B granules or in the inter-granular region, and specifically in the electron-lucent areas of the type-A granules. Since these latter granules are located primarily in the fundus and the proximal zone of the ducts of free-swimming cercariae (Dorsey and Stirewalt, 1971), little if any of the electron-dense reaction product was observed in the more oral part of the ducts. Reaction products in penetrating cercariae and in the secretions in host skin (Stirewalt and Dorsey, 1974) are under study. The birefringence observed in cercariae examined in polarized light after oxalate treatment (Fig. 1) was apparently caused by the polymorphic eleetron-lucent areas seen with the electron microscope (Fig. 4). Mussini et al. (1972), who studied the criteria for recognizing calcium oxalate in sarcoplasmic reticulum at the ultrastruetural level, observed electron-lucent areas as tiny bubbles forming from calcium oxalate crystals similar to those in Fig. 4. They befieved that the reaetion was attributable to the decomposition of calcium oxalate to formic acid and other volatile substances caused by the heat of the electron beam. Volatilization of the oxalate crystals (calcium oxalate?) in this study could have resulted from the heat of polymerization of the embedding medium which caused the formation of the polymorphic electron-lucent bodies (Fig. 4). The pyroantimonate--osmium reaction (Figs. 5, 6), although nonspecific in that pyroantimonate combines with many metallic and organic cations (Clark and Ackerman, 1971), localized the activity in the electron-lucent areas of the type-A secretory granules in the preacetabular glands. The von Kossa reaction is a metal substitution method for identifying metalfic cations and depends upon the anionic part of the salt which reacts with silver. The silver is later reduced to metallic silver by light and dextrose. This was not a useful technique when used alone, since it caused extensive damage of the secretory granules. It was found that to preserve the structure of the secretory granules, pretreatment with chloranilic acid and a brief fixation in glutaraldehyde were necessary
Fig. 2. S. mansoni: Photomicrographof cercariaeexposedto chloranilicacid and viewedin polarized light. Birefringence,indicating the presence of calcium chloranilate,extends along various areas of the preacetabular glands (arrow heads). ×360 Fig. 3. S. mansoni: Electronmicrographshowingthe two types of secretorygranules commonlyfound in the fundus. Type A has electron-lucent areas (arrows)." Type B is homogeneously granular (asterisks). Granules of the postacetabular glands are indicated by arrow heads, x 27,000 Fig. 4. S. mansoni: Electronmicrographof the fundus of a preacetabular gland treated with potassium oxalate (cf. Fig. 1). Electron-lucentareas of type A secretorygranules (arrows) appear to be swollen and to coalesceinto larger amorphous bodies (asterisks). Type-Bsecretory granules do not show the coalescing electron-lucidareas (B). x21,600
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Fig. 5. S. mansoni: Eleetronmicrograph of the fundus of a preacetabular gland showing an eleetrondense precipitate resulting from the calcium-pyroantimonate reacüon concentrated in the electronlucent areas of type-A secretory granules (arrows). Compare with secretory granules in Figure 3. B, type-B secretory granules, x 31,500 Fig. 6. S. mansoni: Electronmicrograph with the electron-dense pyroantimonate reacüon product (arrows) in the duct region of the preacetabular gland proximal to the fundus shown in Figure 5. Note the decrease in concentration of the reaction product as compared with the granules of the fundus (cf. Fig. 5). x 31,500 Fig. 7. S. mansoni: Electronmicrograph of portions of pre- and postacetabular glands of a cercaria exposed to the von Kossa silver nitrate-dextrose solution. The secretory granules of the preaeetabular gland are deteriorated and misshapened and the eleetron-dense reaetion product is diffuse (arrows). The reaction product is absent from the postaeetabular glands (asterisks)~ × 23,400
Schistosoma mansoni: Localization of Calcium-Detecting Reagents
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Fig. 8. S. mansoni: Electronmicrograph of part of the fundus of a preacetabular gland treated sequentially with chloranilic acid, glutaraldehyde, and silver nitrate~lextrose solution in ethanol without postosmication or uranyl acetate-lead citrate staining. Note the electron-dense areas (arrows) containing the reaction product in type-A secretory granules (A). B, type-B secretory granule. × 31,500 Fig. 9. S. mansoni: Electronmicrograph of proximal portions of two ducts of an Unstained preacetabular gland, which was treated sequentially with chloranilic acid, glutaraldehyde, and silver nitrate-dextrose solution in tertiary butyl alcohol. Type-A secretory granules contain the electrondense reaction product (arrows). × 21,600
before exposing the cercariae to the von Kossa solution. Chloranilic acid replaces the anion of the calcium salt forming calcium chloranilate, an insoluble salt. Chloranilic acid also precipitates other metallic cations (Cu, Mn, Ba, Sn, Zn, and Ag), but these are not present in significant quantities in biologic material (Carr et al., 1961). In the combined chloranilic acid-von Kossa reaction the heavy aggregates of reduced silver probably are the result of the combining of the silver ion (von Kossa solution) with the chloranilate ion by replacing calcium, and of the reaction of silver with free
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carbonate or phosphate anions subsequent to its reduction. Postosmication did not enhance the fine structural qualities of chloranilic acid-silver nitrate-treated tissue. Carbonates and phosphates are the anions generally found in insoluble calcium salts in animal tissue (Pearse, 1972). The analysis of Dresden and Edlin (1975) showed an even distribution of phosphorus throughout the cercarial bodies, rather than high concentrations in the immediate area of the preacetabular glands. They suggested that carbonate, not phosphate, was the anion associated with caleium in the preacetabular glands. Insoluble calcium salts, however, appear eleetron-dense in the electron microscope, without coupling to electron-dense materials. Therefore, it is possible that high concentrations of ionic calcium are present in the electron-lucent areas of the type-A granules. It was demonstrated that at least some ionic calcium exists in the preacetabular glands by staining them with glyoxal bis which is specific for calcium under controlled conditions, e.g., the glands retained their color when subsequently immersed in a solution of carbonate and cyanide (Lewert et al., 1966). The high concentration of calcium in the preacetabular fundus was thought by Dresden and Edlin (1974) to inhibit the proteolytic activity of the secretory material in the cercariae. In in vitro studies they found that high concentrations of calcium carbonate inhibited the proteolytic activity of cercariae of S. mansoni, while small amounts enhanced it. This is in line with the thinking of Clemente and Meldolesi (1975), who suggested that calcium might regulate the activity of proteolytic zymogens within the granules of the exocrine glands of the guinea pig. Calcium is present in many secretory tissues, such as salivary glands, adrenal medulla, and exocrine pancreas (Clemente and Meldolesi, 1975). In summary, all the available data indicate that the metallic cation present in large concentrations in the preacetabular glands of S. mansoni cercariae is calcium, probably largely ionic. As indicated by the reactions reported here, it is localized in the electron-lucent areas of the type-A granules. Absence of reaction in type-B granules suggests that the A and B secretory granules are dissimilar cytochemically, as weil as morphologically. It is probable that further work will bring to light other differences between them. Acknowledgements. The authors wish to thank Mr. Gerald Armstrong for preparation of photo-
micrographs, and Mrs. M. Brown for editorial assistance.
References Carr, L.B., Rambo, O.N., Feichtmeir,T.V.: A method of demonstrating calciumin tissue sections using chloranilic acid. J. Histochem. Cytochem. 9, 415-417 (1961) Clark, M.A., Ackerman, G.A.: A histochemicalevaluation of the pyroantimonate-osmiumreaction. J. Histochem. Cytochern. 19, 727-737 (1971) Clemente, F., Meldolesi, J.: Calcium and pancreatic secretion. I. Subcellular distribution of calcium and magnesium in the exocrinepancreas of the guinea pig. J. Cell Biol. 65, 88-102 (1975) Costantin, L.L., Franzini-Armstrong, C., Podolsky, R.J.: Localization of calcium-accumulating structures in striated musclefibers. Science 147, 158-160 (1965) Dorsey, C.H., Stirewalt, M.A.: Schistosoma mansoni: Fine structure of cercarial acetabular glands. Exp. Parasit. 30, 199-214 (1971) Dresden, M.H., Edlin, E.M.: Schistosoma mansoni: Effects of some cations on the proteolyticenzymes of cercariae. Exp. Parasit. 35, 299-303 (1974) Dresden, M.H., Edlin, E.M.: Schistosoma mansoni: Calcium content of cercariae and its effects on protease activityin vitro. J. Parasit. 61, 398-402 (1975) Ebrahimzadeh, A., Kraft, M.: UltrastruktureUe Untersuchungen zur Anatomie der Cercarien von Schistosoma mansoni. III. Das Drüsensystem. Z. Parasitenk. 36, 291-303 (1971)
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Legato, M.J., Langer, G.A.: The subeellular localization of calcium ion in mammalian myocardium. J. Cell Biol. 41, 401-423 (1969) Lewert, R.M., Hopkins, D.R., Mandlowitz, S.: The role of caleium and magnesium ions in invasiveness of sehistosome eereariae. Amer. J. trop. Med. Hyg. 15, 314-323 (1966) Lewert, R.M., Lee, C.L.: Studies on the passage of helminth larvae through host tissues. J. Infect. Dis. 95, 13-51 (1954) Luft, J.H.: Improvements in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 409-414 (1961) Mussini, I., Margreth, A., Salviati, G.: On the efiteria for eharacterization of caleium oxalate in sareoplasmic reticulum fragments. J. Ultrastruct. Res. 38, 459-465 (1972) Pearse, A.G.E.: Histochemistry, theoretical and applied, 3rd ed., Vol. 2, pp. 1128-1170. Baltimore: Williams and Wilkins 1972 Reynolds, E.S.: The use of lead citrate at high pH as an eleetron-opaque stain in electron mieroseopy. J. Cell Biol. 17, 208-212 (1963) Shanklin, W.M., Nassar, T.K.: Luxol fast blue combined with the periodic acid-Schiff procedure for eytological staining of kidney. Stain Teehnol. 34, 257-260 (1959) Stirewalt, M.A.: Skin penetration meehanisms of helminths. In: Biology of parasites, E.J.L. Soulsby, ed., pp. 41-59. New York and London: A•ademie Press 1966 Stirewalt, M.A.: Sehistosoma mansoni: Histologieal loealization of gelatinase in the prea¢etabular glands of eereariae. Exp. Parasit. 34, 382-392 (1973) Stirewalt, M.A., Dorsey, C.H.: Sehistosoma mansoni: Cercarial penetration of host epidermis at the ultrastruetural level. Exp. Parasit. 35, 1-15 (1974) Stirewalt, M.A., Kruidenier, F.J.: Activity of the acetabular seeretory apparatus of eereariae of Sehistosoma mansoni under experimental eonditinns. Exp. Parasit. 11, 191-211 (1961) Received March 28, 1977
Z. Parasitenk. 54, 165-173 (1977)
Parasitenkunde Parasitologic Research
© by Springer-Vedag 1977
Schistosoma mansoni: Localization of Calcium-Detecting Reagents in Electron-Lucent Areas of Specific Preaeetabular Gland Granules* C.H. D o r s e y ' and Margaret A. Stirewalt 2 ' Experimental Pathology Department, Naval Medical Research Institute, Bethesda, Maryland, and 2Biomedical Research Institute, Bethesda, Maryland
Summary. In an attempt to establish the exact location of calcium within the preacetabular glands of cercariae of Schistosoma mansoni, these larvae were exposed to reagents (potassium oxalate, potassium pyroantimonate, chloranilic acid, and silver nitrate) useful in the detection of calcium, and were subsequently observed with the aid o f light and electron microscopes. Cercariae incubated in potassium oxalate and viewed in polarized light showed birefringence only in the preacetabular gland funduses. At the ultrastructural level, the preacetabular glands of potassium oxalate-treated cercariae had no electron-dense precipitate, but instead had translucent, irregularly shaped inclusions, similar to space s left by volatilized calcium oxalate as described by others. Pyroantimonate treatment, on the other hand, localized the reaction in the electron-lucent areas of the lightspotted granules. The von Kossa silver nitrate procedure destroyed the secretory granules; therefore, an electron-dense precipitate was distributed throughout the gland. However, pretreatment with chloranilic acid before fixation preserved the granules, and subsequent exposure to the von Kossa silver nitrate gave a reaction identical to that obtained with the pyroantimonate alone. When viewed in polarized light, chloranilic acid-incubated cercariae showed birefringence in the fundus and duct areas. Introduction Since infection of definitive hosts by schistosomes follows cercarial penetration of skin, it is important that penetration mechanisms be understood. It is known that penetration requires both muscular and chemical activity by cercariae (Stirewalt, 1966). Movement through the horny layer of the skin is via openings made by disarticulation of the epithelial squames (Stirewalt and Dorsey, 1974). This is achieved by the probing action of the oral sucker, and is most likely facilitated by loosening of the squame interdigitations as a result oflysis of the * Supported by the Naval Medical Research and Development Command Work Unit No. MR041.05.01.0037, and Office of Naval Research Contract No. N 00014-76-C-0053. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Department of the Air Force, the Navy Department or the naval service at large
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intercellular ground substance by proteolytic enzyme(s) secreted by the preacetabular glands (Lewert and Lee, 1954; Stirewalt, 1973; Stirewalt and Dorsey, 1974). C alcium is secreted concurrently with the enzyme(s) (Stirewalt and Kruidenier, 1961), and may be involved in the enzyme activity. Preacetabular gland secretions of Schistosoma mansoni cercariae contain at least two types of secretory granules: type A, which has an electron-dense matrix spotted with electron-lucent areas; and type B, which is homogeneously granular (Dorsey and Sürewalt, 1971; Ebrahimzadeh and Kraft, 1971). The preacetabular glands have shown an affinity for dyes used in light microscopy to identify calcium: purpurin (Stirewalt and Kruidenier, 1961), glyoxal bis (2-hydroxyanil) (Lewert et al., 1966), and Luxol fast blue (Shanklin and Nassar, 1959). By using the electron microprobe and atomie absorption spectroscopy, Dresden and Edlin (1975) found an extremely high concentration of caleium in the preacetabular glands.
In our preliminary studies, staining with Luxol fast blue and counter-staining with periodic acid-Schiff according to Shanklin and Nassar's (1959) method produced a differential color in the granules, some staining blue, others pinkish. This suggests a difference in the cytochemical nature of the two types of secretory granules. The purpose of this work was to aseertain by cytochemical methods, at the ultrastructural level, the preacetabular granule in which calcium occurs and its location in the granule.
Materials and Methods Schistosoma mansoni cercariae were collected from snails (a Puerto Rican strain of Biomphalaria glabrata maintained in this laboratory) and trapped on a Gelman filter (Metricel, Alpha-8, 47 mm outer diameter) mounted on a Fisher Filtrator 9-788 (Fisher Scientific Co., Silver Spring, MD). They were immediately processed by one of the following calcium localization techniques. Oxalate Procedure of Costantin et al. (1965). Incubation in an aqueous mixture containing 0.2% pótassium oxalate and 1.2% potassium chloride for from 5 min through 1 h was followed by fixation in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), rinsing in the same buffer for 1 h, and postfixation for 60 min in 1% osmic acid in a 0.1 M cacodylate buffer (pH 7.4). Subsequent dehydration was in reagents saturated with potassium oxalate. Pyroantimonate Proeedure of Legato and Langer (1969). Cercariae were held in a 1% solution of osmic acid containing 2% potassium pyroantimonate in 0.01 M acetic acid (pH 7.5-7.8) for 2 h. Chloranilic A cid Procedure (Modification of Carr et aL 1961). Fixation was in 0.5% glutaraldehyde in 1% chloranilic acid buffered to pH 4 with 0.4% NaOH for I h, after which some eercariae were placed in 1% osmic acid (dissolved in deionized H20) for 1 la, while others were processed without osmicatiom Von Kossa's Silver Nitrate Procedure (According to Pearse, 1972). Cercariae were placed in an aqueous silver nitrate-dextrose solution (1% silver nitrate and 5% dextrose) for 2 h in the light of an ineandescent 60 W light bulb plaeed 15 cm away. They were rinsed briefly in tw0 changes of deionized watet, left overnight in 1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and proeessed for electron microscopy without osmication. Chloranilic Acid-Silver Nitrate Procedure. Incubation of cercariae in an aqueous solution of chloranilic acid (0.4% NaOH and 0.5% chloranilic acid) for 1 h preceded their fixation for 20 min in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.5). Some were then rinsed in deionized water and placed overnight in a 0.1% AgNO 3 solution in 95% ethanol containing 0.2% dextrose without osmication. Others were held in 0.5% chloranilic acid buffered to pH 4 with sodium hydroxide for 30 min, fixed in cacodylate-buffered 2.5% glutaraldehyde (0.1 M, pH 7.4) for 30 min, rinsed briefly in two changes of tertiary butyl alcohol, and kept overnight in 80% tertiary butyl alcohol containing 1% silver nitrate and 5% dextrose. For comparison, other cercariae were placed for 2 h in 2.5% glutaraldehyde (buffered as above), whieh contained 0.5% ehloranilic aeid, and maintained overnight in the dark in an aqueous solution of 2.5% dextrose and 1% osmic acid.
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The unmodified von Kossa method destroyed the secretory granules, but gave an electron-dense reaction product when the silver nitrate solution contained dextrose, which functions as a silver reducing reagent. Pretreating in chloranilic acid and fixing briefly in glutaraldehyde before exposure to silver nitrate resulted in better preservation of the membrane integrity and secretory granules. All tissues were dehydrated, cleared in xylene, and processed for electron microscopy according to the methods of Luft (1961). The tissue was flat-embedded in a 60 mm diameter aluminum weighing tray (Fisher) under a 1 mm layer of Luft's mixture. For light-microscopic observation, whole cercariae were examined through the thin layer of epon in polarized light. For electron microscopy, groups of cercariae were cut from the embedding medium with a jeweler's saw, glued to a wooden rod, sectioned with an ultramicrotome, and examined in a Siemens Elmiskop lA electron microscope. Some of the thin sections (60-90 nm) were stained with aqueous uranyl acetate followedby lead citrate (Reynolds, 1963).
Results
Light Mieroseopy. Cercariae treated with potassium oxalate showed birefringence, but only in the gland fundus (Fig. 1). Cercariae incubated with chloranilic acid were birefringent throughout the area of the preacetabular glands, i.e., the fundus and duct regions (Fig. 2). When examined in nonpolarized light, the fundus area was a rusty red color.
Eleetron Mieroscopy. Two distinct types of secretory granules previously reported (Dorsey and Stirewalt, 1971; Ebrahimzadeh and Kraft, 1971) in the fundus of the preaeetabular glands are shown in Fig. 3. The granules with the electron-lucent areas have been designated type A; the homogeneous granules, type B.
Potassium Oxalate. Although the birefringence indicated the presence of a reaction produet (calcium oxalate) in the preacetabular glands, no granular electron-dense precipitates were observed (Fig. 4). However, some changes did occur in the birefringent area. It appeared that the electron-lucent areas in many of the type-A seeretory granules became brighter and progressively larger, finally coalescing into white, irregularly shaped inelusions. Type-B secretory granules did not coalesce. Pyroantimonate-Osmium. This method gave an electron-dense reaction product in the electron-lucent areas of type-A secretory granules of the preacetabular glands (Figs. 5, 6). N o reaction product was observed in type-B granules or free in the cytoplasm of the glands.
Von Kossa's Silver Nitrate. The reaction product was confined to the preacetabular glands. The secretory granules, however, had deteriorated to the extent that they could not be elearly identified and the reaction product was diffused throughout the cytoplasm (Fig. 7). Small amounts of the reaction product were present around the gland periphery. Chloranilie Acid-Silver Nitrate. This reaction gave results similar to those of the pyroantimonate-osmium in which the ele¢tron-dense product was present in the electron-lucent areas of the secretory granules (Figs. 8, 9). This was true if cercariae were exposed to chloranilic acid before fixation (Figs. 8, 9), or if the chloranilic acid was added to the fixative.
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Fig. 1. S. mansoni: Photomicrograph of cercariae exposed to potassium oxalate and viewed in polarized light. Birefringence (arrow heads) occurs only in the area of the fundus of the preaeetabular glands, x360
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Discussion The cytochemical techniques used herein are not specific for calcium, but for metallic cations. Dresden and Edlin (1975), however, demonstrated by electron probe analysis that calcium was the metallic cation present in very high concentration in the preacetabular glands of S. m a n s o n i cercariae. They theorized that the calcium would be in the secretory granules of these glands. With the aid of the electron-microscopic procedures used in this investigation, calcium, either ionic or as a salt, was found in specific granules of the preacetabular glands. It was in the type-A rather than the type-B granules or in the inter-granular region, and specifically in the electron-lucent areas of the type-A granules. Since these latter granules are located primarily in the fundus and the proximal zone of the ducts of free-swimming cercariae (Dorsey and Stirewalt, 1971), little if any of the electron-dense reaction product was observed in the more oral part of the ducts. Reaction products in penetrating cercariae and in the secretions in host skin (Stirewalt and Dorsey, 1974) are under study. The birefringence observed in cercariae examined in polarized light after oxalate treatment (Fig. 1) was apparently caused by the polymorphic eleetron-lucent areas seen with the electron microscope (Fig. 4). Mussini et al. (1972), who studied the criteria for recognizing calcium oxalate in sarcoplasmic reticulum at the ultrastruetural level, observed electron-lucent areas as tiny bubbles forming from calcium oxalate crystals similar to those in Fig. 4. They befieved that the reaetion was attributable to the decomposition of calcium oxalate to formic acid and other volatile substances caused by the heat of the electron beam. Volatilization of the oxalate crystals (calcium oxalate?) in this study could have resulted from the heat of polymerization of the embedding medium which caused the formation of the polymorphic electron-lucent bodies (Fig. 4). The pyroantimonate--osmium reaction (Figs. 5, 6), although nonspecific in that pyroantimonate combines with many metallic and organic cations (Clark and Ackerman, 1971), localized the activity in the electron-lucent areas of the type-A secretory granules in the preacetabular glands. The von Kossa reaction is a metal substitution method for identifying metalfic cations and depends upon the anionic part of the salt which reacts with silver. The silver is later reduced to metallic silver by light and dextrose. This was not a useful technique when used alone, since it caused extensive damage of the secretory granules. It was found that to preserve the structure of the secretory granules, pretreatment with chloranilic acid and a brief fixation in glutaraldehyde were necessary
Fig. 2. S. mansoni: Photomicrographof cercariaeexposedto chloranilicacid and viewedin polarized light. Birefringence,indicating the presence of calcium chloranilate,extends along various areas of the preacetabular glands (arrow heads). ×360 Fig. 3. S. mansoni: Electronmicrographshowingthe two types of secretorygranules commonlyfound in the fundus. Type A has electron-lucent areas (arrows)." Type B is homogeneously granular (asterisks). Granules of the postacetabular glands are indicated by arrow heads, x 27,000 Fig. 4. S. mansoni: Electronmicrographof the fundus of a preacetabular gland treated with potassium oxalate (cf. Fig. 1). Electron-lucentareas of type A secretorygranules (arrows) appear to be swollen and to coalesceinto larger amorphous bodies (asterisks). Type-Bsecretory granules do not show the coalescing electron-lucidareas (B). x21,600
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Fig. 5. S. mansoni: Eleetronmicrograph of the fundus of a preacetabular gland showing an eleetrondense precipitate resulting from the calcium-pyroantimonate reacüon concentrated in the electronlucent areas of type-A secretory granules (arrows). Compare with secretory granules in Figure 3. B, type-B secretory granules, x 31,500 Fig. 6. S. mansoni: Electronmicrograph with the electron-dense pyroantimonate reacüon product (arrows) in the duct region of the preacetabular gland proximal to the fundus shown in Figure 5. Note the decrease in concentration of the reaction product as compared with the granules of the fundus (cf. Fig. 5). x 31,500 Fig. 7. S. mansoni: Electronmicrograph of portions of pre- and postacetabular glands of a cercaria exposed to the von Kossa silver nitrate-dextrose solution. The secretory granules of the preaeetabular gland are deteriorated and misshapened and the eleetron-dense reaetion product is diffuse (arrows). The reaction product is absent from the postaeetabular glands (asterisks)~ × 23,400
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Fig. 8. S. mansoni: Electronmicrograph of part of the fundus of a preacetabular gland treated sequentially with chloranilic acid, glutaraldehyde, and silver nitrate~lextrose solution in ethanol without postosmication or uranyl acetate-lead citrate staining. Note the electron-dense areas (arrows) containing the reaction product in type-A secretory granules (A). B, type-B secretory granule. × 31,500 Fig. 9. S. mansoni: Electronmicrograph of proximal portions of two ducts of an Unstained preacetabular gland, which was treated sequentially with chloranilic acid, glutaraldehyde, and silver nitrate-dextrose solution in tertiary butyl alcohol. Type-A secretory granules contain the electrondense reaction product (arrows). × 21,600
before exposing the cercariae to the von Kossa solution. Chloranilic acid replaces the anion of the calcium salt forming calcium chloranilate, an insoluble salt. Chloranilic acid also precipitates other metallic cations (Cu, Mn, Ba, Sn, Zn, and Ag), but these are not present in significant quantities in biologic material (Carr et al., 1961). In the combined chloranilic acid-von Kossa reaction the heavy aggregates of reduced silver probably are the result of the combining of the silver ion (von Kossa solution) with the chloranilate ion by replacing calcium, and of the reaction of silver with free
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carbonate or phosphate anions subsequent to its reduction. Postosmication did not enhance the fine structural qualities of chloranilic acid-silver nitrate-treated tissue. Carbonates and phosphates are the anions generally found in insoluble calcium salts in animal tissue (Pearse, 1972). The analysis of Dresden and Edlin (1975) showed an even distribution of phosphorus throughout the cercarial bodies, rather than high concentrations in the immediate area of the preacetabular glands. They suggested that carbonate, not phosphate, was the anion associated with caleium in the preacetabular glands. Insoluble calcium salts, however, appear eleetron-dense in the electron microscope, without coupling to electron-dense materials. Therefore, it is possible that high concentrations of ionic calcium are present in the electron-lucent areas of the type-A granules. It was demonstrated that at least some ionic calcium exists in the preacetabular glands by staining them with glyoxal bis which is specific for calcium under controlled conditions, e.g., the glands retained their color when subsequently immersed in a solution of carbonate and cyanide (Lewert et al., 1966). The high concentration of calcium in the preacetabular fundus was thought by Dresden and Edlin (1974) to inhibit the proteolytic activity of the secretory material in the cercariae. In in vitro studies they found that high concentrations of calcium carbonate inhibited the proteolytic activity of cercariae of S. mansoni, while small amounts enhanced it. This is in line with the thinking of Clemente and Meldolesi (1975), who suggested that calcium might regulate the activity of proteolytic zymogens within the granules of the exocrine glands of the guinea pig. Calcium is present in many secretory tissues, such as salivary glands, adrenal medulla, and exocrine pancreas (Clemente and Meldolesi, 1975). In summary, all the available data indicate that the metallic cation present in large concentrations in the preacetabular glands of S. mansoni cercariae is calcium, probably largely ionic. As indicated by the reactions reported here, it is localized in the electron-lucent areas of the type-A granules. Absence of reaction in type-B granules suggests that the A and B secretory granules are dissimilar cytochemically, as weil as morphologically. It is probable that further work will bring to light other differences between them. Acknowledgements. The authors wish to thank Mr. Gerald Armstrong for preparation of photo-
micrographs, and Mrs. M. Brown for editorial assistance.
References Carr, L.B., Rambo, O.N., Feichtmeir,T.V.: A method of demonstrating calciumin tissue sections using chloranilic acid. J. Histochem. Cytochem. 9, 415-417 (1961) Clark, M.A., Ackerman, G.A.: A histochemicalevaluation of the pyroantimonate-osmiumreaction. J. Histochem. Cytochern. 19, 727-737 (1971) Clemente, F., Meldolesi, J.: Calcium and pancreatic secretion. I. Subcellular distribution of calcium and magnesium in the exocrinepancreas of the guinea pig. J. Cell Biol. 65, 88-102 (1975) Costantin, L.L., Franzini-Armstrong, C., Podolsky, R.J.: Localization of calcium-accumulating structures in striated musclefibers. Science 147, 158-160 (1965) Dorsey, C.H., Stirewalt, M.A.: Schistosoma mansoni: Fine structure of cercarial acetabular glands. Exp. Parasit. 30, 199-214 (1971) Dresden, M.H., Edlin, E.M.: Schistosoma mansoni: Effects of some cations on the proteolyticenzymes of cercariae. Exp. Parasit. 35, 299-303 (1974) Dresden, M.H., Edlin, E.M.: Schistosoma mansoni: Calcium content of cercariae and its effects on protease activityin vitro. J. Parasit. 61, 398-402 (1975) Ebrahimzadeh, A., Kraft, M.: UltrastruktureUe Untersuchungen zur Anatomie der Cercarien von Schistosoma mansoni. III. Das Drüsensystem. Z. Parasitenk. 36, 291-303 (1971)
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Legato, M.J., Langer, G.A.: The subeellular localization of calcium ion in mammalian myocardium. J. Cell Biol. 41, 401-423 (1969) Lewert, R.M., Hopkins, D.R., Mandlowitz, S.: The role of caleium and magnesium ions in invasiveness of sehistosome eereariae. Amer. J. trop. Med. Hyg. 15, 314-323 (1966) Lewert, R.M., Lee, C.L.: Studies on the passage of helminth larvae through host tissues. J. Infect. Dis. 95, 13-51 (1954) Luft, J.H.: Improvements in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 409-414 (1961) Mussini, I., Margreth, A., Salviati, G.: On the efiteria for eharacterization of caleium oxalate in sareoplasmic reticulum fragments. J. Ultrastruct. Res. 38, 459-465 (1972) Pearse, A.G.E.: Histochemistry, theoretical and applied, 3rd ed., Vol. 2, pp. 1128-1170. Baltimore: Williams and Wilkins 1972 Reynolds, E.S.: The use of lead citrate at high pH as an eleetron-opaque stain in electron mieroseopy. J. Cell Biol. 17, 208-212 (1963) Shanklin, W.M., Nassar, T.K.: Luxol fast blue combined with the periodic acid-Schiff procedure for eytological staining of kidney. Stain Teehnol. 34, 257-260 (1959) Stirewalt, M.A.: Skin penetration meehanisms of helminths. In: Biology of parasites, E.J.L. Soulsby, ed., pp. 41-59. New York and London: A•ademie Press 1966 Stirewalt, M.A.: Sehistosoma mansoni: Histologieal loealization of gelatinase in the prea¢etabular glands of eereariae. Exp. Parasit. 34, 382-392 (1973) Stirewalt, M.A., Dorsey, C.H.: Sehistosoma mansoni: Cercarial penetration of host epidermis at the ultrastruetural level. Exp. Parasit. 35, 1-15 (1974) Stirewalt, M.A., Kruidenier, F.J.: Activity of the acetabular seeretory apparatus of eereariae of Sehistosoma mansoni under experimental eonditinns. Exp. Parasit. 11, 191-211 (1961) Received March 28, 1977
