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Schistosoma mansoni: Histochemical analysis of the postacetabular gland secretion of cercariae
EXPERIMENTAL PARASITOLOGY 33, 56-72
Schistosoma
mansoni:
(1973)
Histochemical Analysis of the Postacetabular Secretion of Cercariael
M. A. STIREWALT Naval
Biomedical
Medical
Research
Research
AND MILDRED WALTERS Institute,
Bethesda,
Institute, American Foundation Rockville, Maryland 20852
(Submitted
Gland
for publication,
6 April
Maryland
20014
for Biological
Research,
1972)
STIREWALT, M. A., AND WALTERS, MILDRED. 1972. Schistosoma mn.wni: Histochemical analysis of the postacetabular gland secretion of cercariae. Experimental Parasitology 32, 000-000. Contents of the funduses and ducts of the postacetabular glands of Schistosoma mansoni cercariae, the secreted deposits, and the surface film were compared by their histochemical reactions. Techniques for carbohydrate-containing substances, neutral and acid mucosubstance, proteins and amino acids, and enzymes were used. The secretion reacted differently before (within the glands) and after (in secreted deposits) emission, Before emission, the postacetabular gland contents reacted as a neutral mucosubstance containing periodate-engendered and periodate-reactive aldehydes rich in uic-glycols or their substituted amines, probably hexoses other than glucose, such as fucose or galactose. No reactions of significance were observed for acid groups or for glycogen or lipids. In this state, the secretion is termed mucigen. After emission, the secretion stained not only as mucigen, but also as acid mucosubstance, apparently sialomucin. After emission, it is termed mucin. It is probable that acid radicals were present in mucigen but were masked stearically by the presence of adjacent neutral radicals or basic proteins. The surface film reacted as both a neutral and an acid mucosubstance. Evidence suggested that the film itself was neutral and that the reaction for acid mucosubstance was from an overlay of mucin secreted from the postacetabular glands. Proteins and amino acids, especially arginine, and some tyrosine and tryptophan were indicated in mucigen and in mucin by the histochemical tests. There was no histochemical evidence of enzymes. Secretion of the postacetabular glands is concluded from histochemical reactions, as from earlier chromatographic data ( Stirewalt and Evans 1960), to be a carbohydrate-protein-lipid complex. Trematoda; INDEX DESCRIPTORS: Schistosomu mansoni; Schistosoma japonicum; Turbellaria; Planaria; Biomphalaria glabrata; Mice; Cercariae; Glands; Acetabular glands; Postacetabular glands; Preacetabular glands; Pericercarial envelope; Surface film; Mucus coat; Cercarienhiillen Reaktion (CHR); Skin; Lipids; Histochemistry; Naval service at large. The experiments reported herein were conducted according to the principles set forth in “Guide for Laboratory Animal Facilities and Care,” prepared by the Committee on the Guide for Laboratory Animal Resources, National Academy of Sciences-National Research Council.
1 From the Bureau of Medicine and Surgery, Navy Department, Research Task No. MF12.524. 009-1004B and the Office of Naval Research, Contract No. NOO14-70-C-0331. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the 56 Co yri ht 0 1973 by Academic Press, Inc. o f reproduction in any form reserved. Alfri&
SECRETION OF CERCARL4E
57
Techniques; Stains; Cryostat; Periodic acid Schiff (PAS); Periodic acid-N, N-d& methyl-p-phenylenediamine ( PAD ) ; Dimethyl-p-phenylenediamine; Mixed diamines; Low iron diamine (LID); High iron diamine (HID); Colloidal iron ( CI); Alcian blue (AB); CI-PAS: AB-PAS; Methylene blue; Toluidine blue; Azure A; Thionin; Mucicarmine; Aldehyde fuchsin; Best’s carmine; Eosinophilia; Acidophilia; Phenylhydrazine Schiff; Feulgen’s; Plasma1 reaction; Chromotrope BR-AB; Sakaguchi; Millon’s; DMAB-nitrite; DDD reaction; Ninhydrin; Coupled tetrazonium; MgCls; Methylation; Sulfation; Acetylation; Nitrosation; Carbohydrate-containing secretion; Mucosubstance; Neutral mucosubstance; Acidic mucosubstance; Mucopolysaccharide; Polysaccharide; Glycoprotein; Vie-glycols; Vie-OH groups; SH groups; Aldehydes; Hexose; Glucose; Fucose; Mannose; Galactose; Fucomucoid; Mannomucoid; Glycogen; Mucus; Mucin; Mucigen; Sialomucin; Sulfomucin; Mammalian epithelial mucin; Brunner’s gland; Carboxylated polyanions; Sulfated polyanions; Polyanions; Polypeptides; Enzymes; Amino acids; Alpha-ammo acids; Nucleic acids; Lysine; Arginine; Histidine; Tyrosine; Tryptophan; Aminopeptidase, Lipase; Esterase; Cholinesterase; Phosphatase; Diastase; Amylase; Calcium ions; Calcium salts; Enzymes.
Considerable attention over a long period of time has been given to the paired, unicellular acetabular glands of schistosome cercariae. This is partly because of their prominence when observed microscopically, and partly because their contents are secreted during the invasion of definitive host skin. In addition to histological and physiological studies (see review by Stirewalt and Kruidenier 1961), their contents and secretions have been partially analyzed chromatographically and immunologically (Stirewalt and Evans 1960). Results of preliminary tests on the contents of these glands have shown them to be distinguishable by their histochemical reactions into at least two different types. The preacetabular glands reacted positively with tests for calcium ions or salts (Stirewalt and K&denier 1961; Lewert et al. 1966), but were essentially negative in other histochemical tests. The preacetabular glands will not be dealt with in this report. Reactions of the secretions of the postacetabular glands to histochemical stains have been studied here in detail. The secretions have been tested for the presence of neutral and acid mucosubstances, aldehydes, glycogen, lipids, proteins, and specific amino acids, as well as for selected enzymes. Emitted deposits of secretion
from these glands, together with the surface film around cercariae, have been compared with the contents of the glands before emission. It is of importance to our clarification of the mechanisms of skin penetration to understand the composition of the contents and secretions of these acetabular glands which are involved in the penetration process. Histochemistry provides one approach; hence the investigation reported herein. MATERIALS AND METHODS
Both Schktosoma man.soni cercariae and their snail hosts, Biomphuluriu glubrata, used in these studies were of Puerto Rican origin. They have been reared in this laboratory since 1945. The vertebrate phase of the life cycle has been maintained in Webster strain Swiss albino mice (NMRI), or in hamsters. Cercariae were pooled from at least 10 infected snails from which the parasites emerged naturally between 8:30 and 11:30 AM into small volumes of tap water that had been dechlorinated by aging and warmed to 30 C in a lighted constant temperature box. Living cercaraiae were observed in dechlorinated tap water, physiological saline, serum, and purpurin-India ink solutions. The fixative used was one of the follow-
58
STTREWALT AND WALTERS
ing: Baker’s neutral buffered formalin, Tellyesniczky’s, Camoy’s, 95% ethanol, and cetyl pyridinium (Pearse 1968). Neutral formalin was the fixative of choice. Formolfixed cryostat and unfixed cryostat sections were also used. Cercariae were studied both as whole organisms and in sections of infected snail tissue. Tissues were embedded in paraffin and sectioned at 7-12 pm or quick frozen in liquid nitrogen and sectioned in the cryostat at the same thickness. Sections and whole cercariae were processed concurrently with the same histochemical procedures. Cercariae were affixed to slides or cover glasses by their own secreted mucus as follows. The center of the slide was coated with facial skin lipid from a finger which had been rubbed along the hair line or nose. Large numbers of cercariae were pipetted in a small drop of water onto the lipidized area. They were immediately attracted to the lipid (Stirewalt 1971) and were allowed to creep over it and attempt to penetrate until they had firmly attached the oral or ventral sucker in a mucus deposit. The water was drained off into absorbent toweling and the slide immersed in fixative, or the fixative was pipetted onto the slide. Cercariae handled in this manner adhered in large numbers, many in clumps, since creeping cercariae are attracted to a cercaria which is attempting to penetrate. After fixation, slides or cover glasses of whole cercariae and sections were transferred concurrently from medium to medium for histochemical processing in Coplin or Columbia jars. The histochemical tests used are listed with references in Tables I-VI. Reactions have been described separately for the material within the gland funduses and ducts, i.e., before emission; and for the secreted deposits and the surface film ensheathing the body and tail of the organism. Secreted deposits were examined adherent to the oral surfaces of the cercariae at the open-
ings of the ducts; within the ventral suckers into which they had been placed from the oral ends of the cercariae (Fig. 2 ) ( Stirewalt and Kruidenier 1961; Stirewalt 1965); and on the surfaces of glass slides after deposit thereon by creeping cercariae. Reactions of the cercarial surface film were recorded also. Reactions are tabulated separately for sections and whole cercariae when differences were noted but not otherwise. Technical controls were provided by the goblet cells of the snail gut in sections of infected snail tissue. BACKGROUND
Postacetabular gland secretion is used during the time-limited, free-living existence of cercariae, especially in the exploratory and initial entry phases of their penetration of skin (Stirewalt and Kruidenier 1961) . The postacetabular secretory apparatus consists of three pairs of unicellular glands, each made up of an expanded nucleated portion, the fundus, and a single, very long tubular process which serves as the duct. The funduses occupy most of the cercarial body aboral to the ventral sucker. In living organisms, they are in contrast to other cercarial tissues and especially to the macrogranular preacetabular glands in that the postacetabular secretory apparatus is translucent and light-refractive, as if filled with liquid containing a suspension of very fine granules. After fixation and histochemical processing, viewed either in intact whole cercariae or in cercarial sections, the contents of these glands appear as microgranules surrounded by a thin layer of cytoplasm lining the cell membrane. A nucleus, which is often necrotic, usually lies to one side of the fundus. The viscous surface film that ensheathes both the body and tail of free-living cercariae, as well as of cercariae within sporocysts in snail tissues, appears to be less sticky and viscous than acetabular secretion
59 deposits after emission, but its histochemical reactivity is generally similar. The source of the film has not been established. HISTOCIHEMICAL RESULTS Reactions to selected stains of the postacetabular gland secretion of well-developed cercariae in the various sites observed are tabulated in Tables I-VI. Selected reactions are illustrated in Figs. l-11.
Neutral Mucosubstance PAS (periodic acid-Schif ) ( Table I; Figs. 1, 2, 7). Magenta color with the PAS technique was intense in both funduses and ducts of the postacetabular glands. It varied in intensity in secretion deposits and the surface film (Figs. 1, 2, 7). The Schiff reagent without oxidation usually did not give a color reaction, although occasionally a weak pink color could be brought out in
TABLE Histochemical
Tests for Carbohydrate-Containing
Test
Postacetabular funduses and ducts
PAS” (Lillie 1954; Pearse 1968)
Magenta
Acetylation-PAS Acetylation-KOH-PAS (Pearse 1968)
I Substances in Schiitosoma mansoni Cercariae
Secretion deposits and surface film
To test for
Magenta
Magenta-staining mucosubstances (MS) (vie-glycols or their substituted amines)
NC”
NC
Magenta
Magenta
Colorless carbohydrates Magenta carbohydrate reacting groups
Phenylhydrazine-Schiff (Spicer 1961)
NC
NC
Colorless periodate (PA)engendered aldehydes
Schiff’s reagent (Pearse 1968)
NC
NC
Magenta-free aldehydes
Mixed diamines (48 hr) PA-mixed diaminea (Spicer and Henson 1967)
NC to gray-yellow Gray, gray-brown to lavender
Gray to purple Gray-brown deposits; purple surface film
Purple acidic mucosubstances Gray to gray-brown PA-reactive neutral and acidic MS; purple PA-unreactive acidic MS
PADc (5 hr) (Spicer and Jarrels 1961)
Orange-brown
Orange-brown
Periodate-reactive neutral MS containing fucose or hexoses other than glucose
NC
Reddish-purple aldehydes of nucleic acids
Feulgen’s (Pearse 1968) a-amylase-PAS a-amylase-carmine Diastaae-carmine (Lillie 1954; Pearse 1968)
Magenta Red Red
Magenta Red Red
Colorless glycogen
Acetone-PAS Ether-PAS (McManus and Mowry 1960)
Magenta Magenta
Magenta Magenta
Colorless lipid
NC
Reddish-purple acetal lipids
Plasmal reaction NC [Pearse (Hayes) 19681 0 Periodic acidSchiff b No color. c p-Diamine.
60
STIRFoWALT AND WALTERS
the fundus with very strongly staining leucofuchsin preparations. The magenta stain of strongly reactive PAS appeared as a smooth, diffuse agranular reaction throughout the glands and ducts and in secretion deposits and surface film. The preferred technique, with which gland contents and secretion stained selectively, colored the granules only, leaving the background essentially clear. It is obvious in the immature cercaria at center in Fig. 1 that the postacetabular gland cells begin to elaborate secretion early in the development of cercariae. The PAS reaction was positive in them and occasionally in the surface film, even before ducts were formed. PAD (Periodic acid-N, N-dimethyl-pphenykenediamine) (Table I; Fig. 3). With PAD after 5 hr of staining, the bright orange-brown color of the postacetabular gland secretion within cercariae was bril-
liant against a colorless background. In secreted deposits and surface film it tended to be browner and paler, as it was in the anlage of the postacetabular secretory cells in immature cercariae. Goblet cells of the snail gut and undifferentiated cells of developing cercariae were all colorless. A strong black color developed after sulfation (Fig. 12). *&I Mucosubstance-Baso~hiz~ PAD (Periodic acid-N, N-dimethyl-pphenylenediamine) (Table ZZ; Fig. 4). With PAD at longer staining intervals (24 or 48 hr), the orange-brown darkened in the secretory cells without any suggestion of purple (Fig. 4). The darker color might have been the result of some mixture with black since the granules in funduses sometimes appeared black. Color in secretion deposits and surface film was variably brown in some areas and purple in others.
FIG. 1. Section of infected snail tissue stained with PAS. A portion of a schistosome sporecyst (right) contains one well-developed (right) and one less-developed (left) cercaria. Postacetabular gland cells are magenta. The transverse section of the snail gut (lower left) contains goblet cells which are unstained. Color plate. (x400). FIG. 2. Whole cercaria stained with PAS. Ducts, secretion and surface film stain strongly, but most of the contents of the gland funduses have been secreted. Color plate. ( x500). FIG. 3. Section of infected snail tissue stained for 5 hr with PAD. Funduses and ducts of the postacetabular glands of cercariae are orange-brown. Color plate. (x430). FIG. 4. Whole cercaria stained for 20 hr with PAD. Glands, secreted deposits and surface film are brown, Color plate. ( x385). FIG. 5. Cercaria stained with HID-AB without oxidation. Glands are colorless. Secretion deposits and surface film are blue. Results were the same with LID-AB. Color plate. ( x640). FIG. 6. Whole cercaria stained with alcian blue (AB) pH 2.5. Glands are unstained, deposits and surface film blue. Color plate. (x320). FIG. 7. Whole cercaria stained with AB-PAS. Glands are magenta, deposits blue to purple, surface film magenta in midbody and tail, and blue to purple in the oral and aboral areas. Color plate. ( X430). FIG. 8. Section of infected snail tissue stained with azure A. Snail goblet cells (lower right) are dark purple. Glands in longitudinal section of a well-developed cercaria (upper left) are colorless. Color plate. ( x425). FIG. 9. Section of infected snail tissue stained with thionin. Snail goblet cells (upper right) are very dark purple. Undifferentiated cells of developing cercariae are pale purple, but the glands in the longitudinal section of a developed cercaria at center-left are colorless. Color plate. ( X200). FIG. 10. Cercarial body stained with eosin Y pH 7.0. Glands and secreted mucus are red. Color plate. ( X690). Fro. II. Cercarial body stained with mucicarmine. Red secretions show at oral end and in ventral sucker. Glands are colorless. Color plate. ( X360).
OF CER-
SEaTION
TABLE Histochemiud
Tests
61
II
for Acid Mucosubstances in Schistosoma mansoni Cercariae: Diamine Basophilia Postacetabular funduses and ducts
Test
Secretion deposits and surface film
To test for (Spicer and Henson 1967)
NO to pale graybrown with dark granules
Purple-black to black”
Black to purple-black acid mucosubstance (MS)
NC to pale gray-brown
Blue
Black to purple-black acid MS : blue acidic MS ; strong color slrggests sialomucins
NC to gray-brown
Purple
Dark gray-brown
Purple
Black to purple-black sulfomucins Bed, purple or violet compounds with ester sulfate groups
NC to pale gray-brown
Blue
Black to purple-black sulfated MS; blue acidic MS
Orange-brown
Orange-brown
Orange-brown PA-positive
24 hr
Blackish
brown
Blackish
brown
Orangebrown neutral black acid MS
MS ;
48 hr
Blackish
brown
Blackish
brown
Orange-brown neutral black acid MS
MS;
Low iron diamine (Spicer 1965)
(LID)
LID pH 2.5-Alcian blue (AR) (Spieer 1965) High iron diamine (HID) 107, FeCla (Spicer 1965) 377o Fe& pH 1.3 (Gad and Sylven 1969) HID-AB
(Spicer 1965)
Periodic acid-p-diamine (PAD) (Spicer and Jarrels 1961) 5 hr
PAD with UV light (Spicer and Jarrels
1961)
neutral MS
Orange fluorescence
Orange fluorescence
Orange fluorescent
Methylation (37 C)-PAD (Spicer 1960)
NC
Orange-brown
Colorless carboxyl groups ; orange-brown neutral MS
Sulfation-PAD (Spicer and Jarrels 1961)
Black
Black
Control
neutral
MS
: black sulfate groups
-
a No color. b Matrix of secreted deposits was colorless.
These were difficult colors to assess in these sites. Snail goblet cells and striated border were pale golden; undifferentiated cells of imm.ature cercariae and the sporocyst wall were purple. Under UV light, orange fluorescence was brilliant at all sites in which an orangebrown color was visualized with ordinary light, except that it was not seen constantly in the surface film. Tissues that had been stained for 5, 24, or 48 hr all fluoresced similarly.
As with PAS, neither digestion with 01amylase nor extraction with ether or acetone eliminated color development in the test material. A slight paling of the color after pretreatment with lipid solvents was noted in some cercariae, but not in others. Note in Table II that prestaining methylation eliminated color in glands but not after emission. Mixed acidic diamiws (Table I). The mixed diamines gave poor visualization of reactions in cercariae compared with those
62
STIRElWALT
AND
in mouse tissues used as controls and with the colors pictured by Spicer (1965) in mammalian tissues. Funduses and ducts of whole cercariae showed no significant color without oxidation. Variability characterized the color of the secretion after emission. The matrix of deposits was colorless to gray-purple, but gray-brown, gray-purple, and black granules were enmeshed in the deposited secretion. The surface film and control snail goblet cells were purple. LID
(low iron diumine)
and HID
(high
iron diumine) (Table II; Fig. 5). Variability was characteristic of colors in the glands with these techniques, but secreted mateTABLE Histochemical
Tests
for Acid
Mucosubstances
WALTERS
rial and the surface film were consistently and significantly stained. Treatment with LID or HID followed by alcian blue pH 2.5 (LID-AB) did not change the reaction in glands, but distinguished clearly the blue deposits of secretion and the surface film (Fig. 5). Goblet cells and striated border of the snail intestine were also blue. When exposed to iron diamines alone, however, snail golbet cells were gray-purple as were the undifferentiated cells of immature cercariae and the cercarial cell nuclei. The less differentiated the cells of the cercariae were, the darker the color reaction. It is necessary, therefore, to distinguish careIII
in Schistosoma mansoni Cercariae: Thiazine
Postacetabular funduses and ducts
Basophilia
Secretion deposits and surface fdm
To test for
Blue-green Blue-purple and magenta
Blue-green acid mucosubstance (MS) ; magenta hexosecontaining MS
Pale blue
NC NC Blue Blue
Blue weakly or strongly acidic sulfomucins at pH 1.0 and below; blue carboxylated MS at pH 2.5 and above
NC
Blue
NC NC
NC NC
Blue sulfated MS
Methylation 37 C + AB Methylation 60 C + AB hlethylation (60 C) + KOH + AB (Spicer and Henson 1967)
NC NC NC
NC NC Blue
Colorless sialomucins Colorless acid MS Blue carboxyl groups of acid hlS
AB-PAS (Mowry 1963)
Magenta
Blue-purple and magenta
Blue acid MS; magenta hexosecontaining MS
Methylene blue (Pearse 1968) > pH 4.0 < pH 4.0
Blue NC
Blue NC
Degree of bssophiiia ; carboxyl staining largely suppressed below pH 2.0
Test Colloidal iron (CI) CI-PAS (Mowry 1963) Alcian blue (1%) (AB) (Pearse 1968) pH 0.5 1.0 2.5 5.7 Sect. Cert. with MgClt (Scott et al. 1964) 0.1 M to 1.0 M Sect. Cert.
* No color.
NO
Magenta
NC NC NC
SECRFXION
OF
fully in snail sections containing parasites in sporocysts between the glands of mature and immature cercariae. No real difference was recognized between the reactions to LID and HID. Buffered formalin and cryostat sections gave similar results with diamines. Carnoy’s was not as suitable a fixative as formalin, because the reactions were too pale. Colloidal iron and al&n blue (Table IIl; Figs. 6, 7). The same difference in staining reaction of the secretion in different sites was noted with colloidal iron (CI) as with alcian blue (AB ) (Fig. 6). Color with both was unequivocally present in secretion in both sections and whole cercariae after emission, but there was no blue color before (Fig. 6). Within glands, the only basophilia demonstrable in either sections or whole cercariae with CI or with AB at pH 0.5 or 2.6 was in the oral ends of occasional ducts, indicating that the change which occurred in the staining reaction of secretion after emission could also take place within the distal ends of the ducts. Treatment of sections and whole cercariae with AB at pH 5.7 resulted in a weak and inconsistent reaction in the glands. When color developed, it was either a pale blue wash in all tissues with a darker concentration of dye in the gland funduses and ducts or a pale blue granular network in the funduses, depending on the length of the staining period. After emission, although the deposited secretions stained strongly with CI and with AB at pH 2.5 and 5.7, there was no color in either sections or whole cercariae at lower pH’s. Note that addition to the AB pH 5.7 ‘of 0.1 M to 1.0 M electrolyte, MgC&, eliminated the color. Combination techniques of CI-PAS and AB (pH 2.5)-PAS always produced magenta-colored glands and blue-magenta secreted deposits and surface fihns (Fig. 7). It may be sign&ant that while blue
CERCARIAE
63
predominated in the deposits, magenta showed more strongly in the surface film except in the vicinity of the suckers. Snail goblet cells contained bright blue or blue-green droplets after staining with either of these dyes. With AB, the reaction became more intense as the pH was increased. At pH 5.7, the striated border was also strongly stained. Addition of the electrolyte resulted in the same reaction in goblets as in the emitted ceroarial secretions. Results with these two cationic dyes were substantiated by the findings with methylene blue. Color within glands and after emission did not develop below pH 4.0. Acid mucosubstance - metachrorrwiu (Table IV; Figs. 8, 9). Metachromasia in secretion before emission was absent or so pale as to be essentially negative when compared with the control sections stained after sulfation. No metachromasia could be brought out in any of the sites with thionin in either form&n-paraffin (Fig. 9) or cryostat sections or in whole cercariae. Control snail goblet cells, red-purple after 5 min of stain (Fig. Q), were almost black after 15 min. With aqueous toluidine blue according to Kramer and Windrum (Pearse 1988), 0.1% in 30% alcohol, the surface film was strongly metachromatic and secretion at the oral sucker was diffuse pale purple before dehydration but colorless after. With alkaline toluidine blue (0.025% for 5 min) (Pearse 1968)) the faint purple color of the granules in gland funduses indicated a very weak reaction, while snail goblet cells were bright red-purple. Azure A, applied to both whole cercariae and formalin-paraffin and cryostat sections, produced no metachromasia below pH 3.0 in secretion before emission. The pale redpurple granules visualized infrequently in paraffin sections in funduses and ducts at pH 3.0 (Fig. 8) and above were probably
STLREXVALT AND WALTERS
64
TABLE Histochemical
Tests for Acid Mucosubstances
Postacetabular funduses and ducts
Test
IV
in Schiitosoma mansoni Cercariae: Secretion deposits and surface film
Metachromasia
To test for
NC NC
Red metachromasia of acid mucosubstance (MS)
NC to blue NC
Purple NC
Red to purple metachromasia of acid MS
NC to pale blue
NC
1.5 Sect. Cert.
NC to pale blue Pale blue
Not seen Blue
Purple metachromasia of strongly acid sulfated MS below pH 1.5; blue orthochromasia to purple
3.0 Sect.
NC to pale redpurple NC to pale blue
Not seen Red
Metachromasia of weakly acid MS below pH 4.5 ; blue to purple of sialomucins at pH 3.0 and above
NC to pale redpurple granules Pale blue
Not seen
Sect.a NC* Cert. NC
Thionin [Pearse (Schmorl’s) 1968; Gurr 19601
Toluidine blue Sect. [Gurr 1960; Cert. Pearse (Kramer and Windrum) 196S] Azure A (0.02%) (Pearse 1968 ; Spicer and Warren 1960) pH 0.5 Sect. Cert.
Cert. 4.0 Sect.
Cert. 5.0 Sect. Cert. Sulfation-azure A Sect. (Spicer 1960)
Red-purple
Pale red-purple granules Pale blue
Not seen
Red-purple
Red-purple
Red-purple Control for sulfated mucosubstance
a Sections, cercariae. b No color.
in immature cercariae. No metachromasia was noted in these sites at any pH in emerged whole cercariae. In secreted deposits, red-purple metachromasia was visualized only at pH 3.0 and above. In the surface film, there was a spectrum of reaction from colorless through blue at pH 1.5 to red at pH 3.0 and finally red-purple from pH 3.0 to 5.0. Even when a 0.05% (Quintarelli 1963) or a 0.570 solution (McManus and Mowry 19sO) of azure A was used with formalinparaffin sections at pH 1.5, there was no
color anywhere in developed cercariae. At pH 3.0, results were erratic: in some cercariae, funduses of the glands were colorless; in others, pale red-purple. At pH 4.5, definite red-purple granules were present in gland funduses. Ducts were without color throughout. Control goblet cells of the snail gut were metachrom8atic at all pH’s. Prestaining sulfation made the test substance in all four sites strongly metachromatic and served as another control technique. Proteins and amino acids (Table V; Fig.
SECRETION OF CEXcARIAE
10). Color of the secretory material in the glands and after emission in secretion deposits, as well as in the surface film, was consistently present with all the techniques and after all the prestaining treatments listed in Table V. Granules were always darker red than the matrix of funduses and ducts, suggesting diffusion of the stain from them into the matrix. The data in Table V need no special comment except that with Adams’ DMABnitrite procedure, color intensity was variable within the glands, but reproducibly deep blue in the surface film and deposits. Our attempts to localize enzymes in TABLE Htitochemical
Tests
65
gland contents or after secretion failed, as did those of Dusanic ( 1959), Fripp ( 19&3), Sodeman et al. (19&3), and Ebrahimzadeh ( 1970). There was no histochemical evidence of amino-peptidase, lipase, nonspecific esterases, cholinesterase, or phosphatases in the sites studied herein. SIGNIFICANCE OF THE FIND~NC~ AND DIXXJSSION
Histochemical procedures are employed more often to demonstrate that a known substance can be visualized with the agents used than to provide tests for the presence of the substance. At the outset then, it is V
for Proteins and Amino Acids in Schistosoma mansoni Cercariae Postacetabular funduses and ducts
Test
Secretion Deposits and surface film
To test fora
Golden red Red Red
Red Red Red
An amino acid source of protein acidophilia
Eosinophilia aft.er acetylation (Pedersen 1959, 1963)
Red
Red
Guanidinium
group of arginine
Eosinophilia after nitrosation (Pedersen 1959, 1963)
Golden red
Red
Guanidinium
group of arginine
Chromotrope 2R-AB (Wagner and Shapiro 1957)
Red
Red to red and blue
Basic proteins
Sakaguchi (Baker (Pearse 1968)
Orange red
Red
Arginine
Xllon’s, Baker modification (Pearse 1968)
Red
Red
Tyrosine
DMAB-nitrite 1968]
NO to pale blue
Blue
Tryptophane
Red
Red
SH groups of a-amino (Pearse 1968)
Blue
Blue
Polypeptides
Red-brown
Red-brown
Tryosine,
Eosinophilia (Pedersen 1959, 1963)
pH
7.0 10.0 11.8
1947)
[Pearse
(Adams)
DDD reaction [Pearse (Barnett and Se&man) Ninhydrin
(McManus
19681
1968)
Coupled tetrazonium (Pearse 1968) o Color means probable b No color.
presence of indicated
protein
or amino acid.
acids
or protein
tryptophane
or histidine
66
STLREWALT
iND
FIG. 12. Section of infected snail tissue stained for 5 hr with PAD after sulfation. Glands and surface film of the cercaria are black. ( x340).
acknowledged that only a few of the reactions are specific enough to be used as proof of the presence of a chemical constituent. Nonetheless, histochemical techniques are invaluable as a means of demonstrating similarities and differences among substances in tissues and of indicating the probable presence and localization
of specific reacting substances. Finally, of course, the target material, in our case the secretions elaborated in the postacetabular glands of cercariae, should be identified by chemical means. Despite this, a great deal of information about the postacetabular gland secretion has been derived from histochemical tests. Of special importance was the finding that, with some tests, the secretion after emission reacted differently from the material within the secretory cells. Although secretory ceII contents, emitted secretion, and surface film all stained red or orange-red with Best’s, Orth’s, and lithium carmine, which color a variety of substances rich in &-OH groups, such as epithelial mucus secretions (Mowry 1963), this was not the case with mucicarmine and aldehyde fuchsin (Table VI). With both of the latter dyes, secreted deposits and the surface film reacted positively, in contrast to the material in the glands which was unstained in sections as well as whole cercariae (Fig. 11). While none of these stains has proven specificity, the differences noted in staining before and after emission indicate a change in the reacting substance. Similar staining of these glands of Schktowma japonicum cercariae with Best’s carmine and absence of stain with mucicarmine were described by Miura ( 1955).
TABLE Reaciions of Fully
WALTERS
VI
Developed Whole Cercariae and Sections of Schistosoma mansoni to Stains .for Mums
Test
Reference
Postacetabular funduses and ducts
Secreted deposits and surface film
Best’s carmine Orth’s lithium carmine
Pearse 1968 Lillie 1954
Red Orange-red
Red Orange-red
Mucicarmine (Southgate)
McManus and Mowry 1960
NO
Red
Aldehyde fuchsin (Gomori)
Pearse 1968
NC
Lavender
QNo color; color means reaction for mucus.
SECRETION
OF
Since mucicarmine stains mucin red, since the secreted material is viscous and sticky (Stirewalt 1965), since it has been shown chromatographically to be composed of amino acids, carbohydrates, and lipids ( Stirewalt and Evans 1960), and since its histochemical reactions are compatible with those of mucin, the material in the secreted deposits, and the surface film as well, is herein called mucin. On the other hand, because of the differences in reaction to histochemical procedures, the secretion in the glands before emission is termed mucigen. Mucigen may be characterized from histochemical evidence as follows. Its selective staining with PAS indicated it to be a hexose-containing mucosubstance rich in &c-l, 2 glycol groups or their substituted amines (Pearse 1968). The carbohydrate nature of the reacting substance was confirmed in all sites by prevention of the PAS reaction by acetylation and its restoration by the acetylation-KOH-PAS sequence (Table I; Pearse 1968; Spicer 1963; Quintarelli 1963). With the 5-hr PAD technique, it behaved as a neutral mucopolysaccharide, since it was selectively bright orange-brown (Table II; Spicer and Jarrels 1961; Spicer 1965; Pearce 1968). Spicer and Henson (1967) noted that fucose or hexoses other than glucose, such as galactose, are the most probable source of this kind of reaction, It may be recalled that glucoseamine and galactoseamine were observed in chromatographs of hydrolyzed secreted deposits (Stirewalt and Evans 1960). Orange fluorescence with PAD in UV light was compatible with the above. The findings were substantiated by the variable gray-brown to dark brown color of the mucigen and the absence of purpIe with aged p-diamine solution or a 24- ‘or 48-hr staining period. This, together with the magenta with PAS after 10 min of oxidation, the orange-brown color with PAD,
CEXCARIAE
67
and the absence of purple color with mixed diamines without oxidation, indicated its periodate-reactivity (Spicer and Henson 1967; Pearse 1968). The reactive mucigen was not glycogen, since it stained orange-brown with 5-hr PAD (Spicer and Jarrels 1961) and neither amylase nor diastase prevented color development with PAS or PAD (Table I). Neither aldehydes of lipids, free aldehydes, nor acetalphosphatides appeared to be responsible for the PAS reactivity (Table I; Stirewalt 1959). Mucigen was nonreactive for acid mucosubstance with most tests (Tables II-IV; Figs. 6-9). There was lack of convincing metachromasia in the glands with azure A, thionin, and toluidine blue; and there was essential failure of the glands to stain significantly with CI, AB, methylene blue below pH 4.0, 24- and 48-hr mixed diamines, phenylhydrazine-PAS, and acetylation-PAS. An occasional weak reaction for acidic groups was observed, however, with three of the stains for acid mucosubstances; a very pale-blue network in sections of funduses with AB at pH 5.7 with and without 0.1 M MgCla; occasional very weak metachromasia showing as pale red-purple granules with azure A at pH 3.0 and above; and variable brown to dark color with PAD after 24 hr (Tables III, IV). These reactions were positive only in sections in which control tissues, cells of immature cercariae, and the secretion droplets in snail goblet cells reacted strongly. No reaction developed in the glands of whole cercariae. These findings bring up the possibility that acidic groups were present in the mucigen, in addition to the neutral groups indicated by PAS and 5-hr PAD reactivity, but were masked by the close proximity of neutral groups or basic proteins, both of which have been shown herein to be present (Tables 1, V). The blue color in sec-
68
STIRF,WALT
tions with AB in the pH 5.7 buffer plus electrolyte is characteristic of the unmasking of acidic groups by basic proteins since this masking effect is said to be prevented by MgClz in pH 5.7 buffer (Mowry 1966). Neighboring periodate-engendered aldehydes and basic proteins have both been considered to be possible blocks to the staining of vicinal sulfates or carboxylates of acid mucins (Spicer 1965; Spicer and Henson 1967; Pearse 1968). Indeed, sialic acid carboxyls are, to a great extent, blocked by basic proteins (Quintarelli 1963). The term mu& has been used here for the material in secretion deposits adhering to the oral surface of cercariae, in the acetabulum, and on surfaces over which cercariae have crept. These deposits have been shown to contain polysaccharide, protein, and lipid (Stirewalt and Evans 1960). The surface film, which is viscous and sticky, as are the deposits, has also been considered here to be mucus although its source is not known. Mucin in secreted deposits gave reactions for neutral mucosubstance similar to those of mucigen with PAS and 5-hr PAD, but it alslo behaved histochemically as an acid mucosubstance with aged PAD and mixed diamines. Reactivity of the surface film was similar (Tables II-V). Reactive polyanions were, therefore, present in the mucin. It may be of value to comment that the magenta of the PAS and orange-brown of the 5-hr PAD reaction were weak in mucin in most cercariae compared with their intensity in mucigen. Possibly the less intense color in mucin simply reflected the thinness of the mucus layer in the surface film or dilution of the reactive substance in secretion deposits in water which the deposits are known to absorb and in which they swell (Stirewalt 1959). Alternatively, the explanation may be because in the mucin not only neutral but also acid radicals were
AND
WALTERS
reactive, and acidic mucins stain weakly or not at all with PAS. Our observations suggested that the surface film was a neutral mucosubstance and that the acid mucosubstance was an overlay of postacetabular gland secretion. There was overwhelming evidence for the presence in the mucin of carboxylated polyanions and little evidence for sulfated polyanions. The red color of mucicarminetreated deposits (Fig. 11) was characteristic of carboxylated groups. Metachromasia with azure A at pH 3.0 and above and AB staining of the mucin at pH 2.5 and 5.7 pointed to carboxylated mucosubstancesialomucins or other weakly acid mucosubstance-as the reacting material (Mowry 1963). Evidence from diamine techniques is as follows (Spicer and Henson 1967). Mucin after LID-AB and HID-AB was the blue (Table II) typical of sialomucins. The methylation-saponification-alcian blue sequence also indicated reactivity Iof carboxylated groups (Table III), for methylatedAB-treated cercariae and sections were colorless, while methylation-KOH-AB treatment restored the color (Spicer 1960). While these results indicated activity of carboxylated groups ( Spicer 1960)) they, of course, did not deny the possible presence of sulfated groups as well, although loss of AB stain at pH 5.7 after the addition of MgC& does so deny it. The lavender color of secreted deposits with aldehyde fuchsin was not a strong reaction, and thus has not been interpreted as indicative of sulfomucins. Basic proteins were visualized in both mucigen and mucin with eosin Y according to Pedersen ( 1963), whose technique was based on the statement by Singer (1952) that “groups in proteins undoubtedly form the fundamental basis for tissue acidophilia and the amino groups mainly responsible for acidophilia in general are the terminal amino groups of the polypeptide chains and
SECRETION
OF
the side groups from lysine, histidine and arginine.” Following Pedersen’s eosin Y procedure, the secretion was red at all three recommended pH’s in all sites. Ebrahimzadeh (1970) also found the postacetabular glands to be eosinophilic. Acetylation and nitrosation, which block the terminal amino groups and the e-amino groups of lysine, had little, if any, effect on the color of either mucin or mucigen in these cercariae. Basic groups other than the guanidinium group from arginine are not available for binding to eosin at pH 11.8 (Wislocki et al. 1957), at which pH the cercarial secretion stained strongly in all sites. On this basis, the guanidinium group from arginine may be considered to be responsible for the eosin binding by the secretion. In confirmation, the secretion in all sites gave a strong Sakaguchi reaction for a&nine. Furthermore, as assayed chromatographically, a&nine was present in a far greater percentage than any other amino acid residue identified in acid hydrolysates of cercarial secretion deposits (Stirewalt and Evans 1960). Histochemical reactions were also positive in both mucigen and mucin, though less strongly, for tyrosine ( Millon’s ), tryptophane ( DMAB-nitrite) and the SH groups of alpha amino acids (DDD) (Table VI). There was no evidence of tryptophane or SH-containing amino acids in the chromatographic pattern of cercarial secretion (Stirewalt and Evans 1960), but tryptophane, of course, is not identifiable in acid hydrolyzates and the chromatographic techniques used may not have been sensitive enough to test for the SH groups. Since the secretion was Feulgen-negative in fully developed cercariae, nuclei acids were not involved. As with the secretory cells of planarians described by Pedersen (1963), however, in early stages of elaboration the secretory cells reacted positively with stains for both neutral and acidic mucosubstances
CER-
69
and nucleic acids. These results are oompatible with the abundant ergastoplasm in cells active in the production of secretion. Results of these investigations place the postacetabular gland secretion in the same general histochemical category with certain mammalian epithelial mucins, especially with that of human Brunner’s gland (Spioer and Henson 1967). Both the cercarial mucin and mammalian epithelial mu&s are strongly reactive with PAS, rich in vicinal hydroxyl groups, and they are acidic, reliably colorable with either colloidal iron or alcian blue (Mowry 1963). Histochemical evidence thus fits well with the chromatographic finding that deposits of secretion contained polysaccharides in firm chemical union with proteins; 15 amino acid residues, glucose and galactose-amine, and some steroid spots were demonstrated ( Stirewalt and Evans 1960). Several important problems are unresolved. One is the presence or absence of acid radicals in the mucigen. The second involves the chemical change responsible for the histochemically demonstrable difference in secretion in glands and after emission, for the reaction of mucin in deposits (and in surface films) was unquestionably characteristic of acid as well as of neutral mucosubstance (Tables II-IV). If stearic hindrance by proximal neutral radicals and/or basic proteins is responsible for the lack of staining of acid radicals in the mucigen, perhaps it is removed when the secretion is hydrolyzed in the water into which it is secreted. These uncertainties make it impossible to classify postacetabular gland secretion at this time according to the scheme proposed by Spicer et al. ( 1965), although it is obvious that it contains neutral periodate-reactive mucosubstance, either neutral glycoprotein or fuco- or mannomucoid. The strong orange-brown color with 5-hr PAD suggests that it is fucose- or galactose-con-
70
STIREWALT
AND WALTERS
taining and places it in this regard with the secretion of human Brunner’s gland and gastric surface epithelium. Further classification depends not only on its demonstrated periodate reactivity but also on the presence of sulfomucins as well as sialomucins. Postacetabular gland secretion of cercariae did not react similarly to the subcuticular unicellular mucus glands of those planarians described by Pedersen ( 1963), despite the close phylogenetic relationship usually assumed between Turbellaria and Trematoda. For example, the cercarial mucus was both basophilic and eosinophilic. E’osinophilic and basophilic granules were found in separate secretory cells in planarians (Pedersen 1959). Quintarelli (1963) stated that such plurality of mucus has been encountered regularly, and Spicer (1963) found that exactly similar mucins were seldom encountered. The results reported here agree with those of Smith et al. (1969) for the surface film of cercariae which they have called a pericercarial envelope or mucoid coat. The term “pericercarial envelope” has previously been used to designate the product of the interaction of the cercarial surface film or mucus coat with globulin in the fast-moving gamma fraction of antischistosome serum (Evans and Stirewalt 1959) in the Cercarienhiillen Reaktion of Vogel and Minning. Since Smith and his co-workers did not expose their cercariae to antischistosome serum, the term mucus coat or surface film is preferable to avoid confusion. Our histochemical results, however, disagree substantially with those tabulated by Smith et al. (1969) for the acetabular glands, no matter whether their reactions were with the pre- or postacetabular glands. Unfortunately, they did not distinguish, either in table or figures, between these two types of secretory cells which differ dramatically both in function ( Stire-
walt and Kruidenier 1961) and in histochemical reaction. Since their Table I lists the glands as PAS-reactive, we have assumed that the postacetabular glands were indicated because the preacetabular glands are negative with the PAS reaction, as well as with most of the other tests used. As reported herein (Table V), both the postacetabular glands and the surface films of our cercariae were positive for protein (eosin, ninhydrin, and Chromotrope 2RAB ), arginine ( Sakaguchi) , tyrosine ( Millon’s ) , tryptophane ( DMAB-nitrite), and SH groups (DOD). Smith et al. ( 1969) found them to be negative for protein, tryptophane, and SH groups. Conversely, they reported the presence in both glands and surface film of acid mucosubstance (Hale’s colloidal iron) which we have found only in the surface film and secreted deposits as judged by reactions for acidic groups with Hale’s colloidal iron and other tests depending on basophilia (Tables II, III), and metachromasia (Table IV), while our contents within the glands gave histochemical evidence of neutral mucosubstance only. As discussed earlier, however, acid radicals may be present but masked by neutral radicals or proteins. REFERENCES D. G. 1959. Histochemical observations of alkaline phosphatase in Schistosoma man-
DUSANIC,
soni. Journal
of Infectious
Diseases
105, 1-8.
A. 1970. Beitrgge zur Entwicklung, Histologie und Histochemie des Driisen-
EBRAHIMZADEH,
systems der Cercarien von Schistosomu mansoni Sambon ( 1907). Zeitschrift fiir Parasitenkunde 34, 319-342. EVANS, A. S., AND STIREWALT, M. A. 1959. Serologic reactions in Schistosoma munsoni infections. V. Localization of CHR and cercarial agglutinating factors in electrochromatographically fractionated human sera. Experimental Parasitology 8, l-9. FRIPP, P. J. 1966. Histochemical localization of B-glucuronidase in schistosomes. Experimental Parasitology 19, 254-263.
SECRETION
OF
GAD, A., AND SELWYN, B. 1969. On the nature of the high iron diamine method for sulfomucins. Journal of Histochemisty and Cytochemistry 17, 156-160. GURR, E. 1960. “Methods of Analytical Histology and Histochemistry.” The Williams and Williams CO., Baltimore, Maryland. LEWERT, R. M., HOPKINS, D. R., AND MANDLOwrr~, S. 1966. The role of calcium and magnesium ions in invasiveness of schistosome cercariae. American Journal of Tropical Medicine and Hygiene 15, 314-323. LILLIE, R. D. 1954. “Histopathologic Technic and Practical Histochemistry.” The Blakiston Co., New York. MCMAMJS, J. F. A., AND MOWRY, R. W. 1960. “Staining Methods Histologic and Histochemical.” Paul B. Hoeber, Inc., New York. MNRA, M. 1955. Histochemical studies on Schistosomu japonicum. 3. Histochemical studies of cercariae (Japanese). Kumumoto Igakkwai Zasshi 29, 99-109. MOWRY, R. W. 1963. The special value of methods that color both acidic and vicinal hydroxyl groups in the histochemical study of mu&s. With revised directions for the colloidal iron stain, the use of alcian blue 8GX and their combinations with the periodic acid-Schiff reaction. Annals of the New York Academy of Sciences 106( 2), 402-423. MOWRY, R. W. 1966. The coloration of polyanions by alcian blue 8GX in buffered 70% ethanol with electrolyte (magnesium chloride). JOWna2 of Histochemisty and Cytochemistry 14, 800-801. PEARSE, A. G. E. 1968. “Histochemistry: Theoretical and Applied.” Little, Brown and CO., Boston. PEDERSEN, K. J. 1959. Some features of the fine structure and histochemistry of planarian subepidennal gland cells. Zeitschrift fiir Zellforschung 50, 121-142. PEDERSEN, K. J. 1963. Slime-secreting cells of planarians. Annals of the New York Academy of Sciences 106(2), 424-442. QUINTAFIELLX, G. 1963. Histochemical identification of salivary mucins. Annals of the New York Academy of Sciences 106(Z), 339-363. SCOTT, J. E., DORLING, J., AND QUINTARELLI, G. 1964. Differential staining of acid glycosaminoglycans by alcian blue in salt solutions. Biochemical Journal 91, 4P-5P. SINGER, M. 1952. Factors which control the staining of tissue sections with acid and basic dyes. International Review of Cytology 1, 211-255.
CXRCARIAE
71
SMI’nx, J. H., REYNOLDS, E. S., AND VON LICHTENBERG, F. 1969. The integument of Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 18, 28-49. SODEMAN, W. A., BERRY, E. G., AND FORS, M. B. 1968. Schistosomal phosphatases. Histochemical localization of alkaline and acid phosphatase in cercariae of Schistosoma munsoni, Schistosomu haematobium and Schistosomu japonicum. American Journal of Tropical Medicine and Hygiene 17, 851-857. SPICER, S. S. 1960. A correlative study of the histochemical properties of rodent acid mucopolysaccharide. Journal of Histochemistry and Cytochemisty 8, 18-35. SPICER, S. S. 1961. The use of various cationic reagents in histochemical difFerentiation of mucopolysaccharides. American Journal of Clinical Pathology 5, 393467. SPICER,S. S. 1963. Histochemical differentiation of mammalian mucopolysaccharides. Annals of the New York Academy of Sciences 106(2), 379-388. SPICER, S. S. 1965. Diamine methods for differentiating mucosubstances histochemically. Journal of Histochemisty and Cytochemistry 13, 211-234. SPICER, S. S., AND HENSON, J. G. 1967. Methods for localizing mucosubstances in epithelial and connective tissues. In “Methods and Achievement in Experimental Pathology” (Ed. E. Bajusz and G. Jasmin, eds.), Vol. 2, pp. 78112. S. Karger, Basel/New York. SPICER, S. S., AND JARRELS, M. H. 1961. Histochemical reaction of an aromatic diamiue with acid groups and periodate engendered aldehydes in mucopolysaccharides. Journal of Histochemistry and Cytochemistry 9, 368-379. SPICER, S. S., AND WARREN, L. 1960. The histochemistry of sialic acid containing mucoproteins. Journal of Histochemisty and Cytochemistry 8, 135-137. SPICER, S. S., LEPPI, T. J., AND STOWARD, P. J. 1965. Suggestions for a histochemical terminology of carbohydrate-rich tissue components. Journal of Histochemistry and Cytochemistry 13, 599-603. STIREWALT, M. A. 1959. Isolation and characterization of deposits of secretion from the acetabular gland complex of cercariae of Schistosomu mansoni. Experimental Parasitology 8, 199-214. STIREWALT, M. A. 1965. Mucus in schistosome cercariae. Annals of the New York Academy of Sciences 118(24), 966-968.
72 STIREWALT, M. A. 1971. Penetration
STIRF,WALT
stimuli for schistosome cercariae. In “The Biology of Symbiosis” (T. C. Cheng, ed. ), 327 pp. University Park Press, Baltimore, Maryland. STIREWALT, M. A., AND EVANS, A. S. 1960. Chromatographic analysis of secretions from the acetabular glands of cercariae of Schistosomu mans&. Experimental Parasitology 10, 75-80. STIREWALT, M. A., AND KRUIDENIER, F. J. 1961. Activity of the acetabular secretory apparatus of cercariae of Schistosomu munsoni under
AND WALTERS
experimental conditions. Experimental Parasitology 11, 191-211. WAGNER, B. M., AND SHAPIRO, S. H. 1957. Application of alcian blue as a histochemical method. Laboratory Investigation 6, 47M77. WISLOCKI, A. B., WEISS, L. P., BURGOS, M. H., AND ELLIS, Ft. A. 1957. The cytology, histochemis&y and electron microscopy of the granular cells of the metrial gland of the gravid rat. Journal of Anatomy 91, 130-140.
Schistosoma
mansoni:
(1973)
Histochemical Analysis of the Postacetabular Secretion of Cercariael
M. A. STIREWALT Naval
Biomedical
Medical
Research
Research
AND MILDRED WALTERS Institute,
Bethesda,
Institute, American Foundation Rockville, Maryland 20852
(Submitted
Gland
for publication,
6 April
Maryland
20014
for Biological
Research,
1972)
STIREWALT, M. A., AND WALTERS, MILDRED. 1972. Schistosoma mn.wni: Histochemical analysis of the postacetabular gland secretion of cercariae. Experimental Parasitology 32, 000-000. Contents of the funduses and ducts of the postacetabular glands of Schistosoma mansoni cercariae, the secreted deposits, and the surface film were compared by their histochemical reactions. Techniques for carbohydrate-containing substances, neutral and acid mucosubstance, proteins and amino acids, and enzymes were used. The secretion reacted differently before (within the glands) and after (in secreted deposits) emission, Before emission, the postacetabular gland contents reacted as a neutral mucosubstance containing periodate-engendered and periodate-reactive aldehydes rich in uic-glycols or their substituted amines, probably hexoses other than glucose, such as fucose or galactose. No reactions of significance were observed for acid groups or for glycogen or lipids. In this state, the secretion is termed mucigen. After emission, the secretion stained not only as mucigen, but also as acid mucosubstance, apparently sialomucin. After emission, it is termed mucin. It is probable that acid radicals were present in mucigen but were masked stearically by the presence of adjacent neutral radicals or basic proteins. The surface film reacted as both a neutral and an acid mucosubstance. Evidence suggested that the film itself was neutral and that the reaction for acid mucosubstance was from an overlay of mucin secreted from the postacetabular glands. Proteins and amino acids, especially arginine, and some tyrosine and tryptophan were indicated in mucigen and in mucin by the histochemical tests. There was no histochemical evidence of enzymes. Secretion of the postacetabular glands is concluded from histochemical reactions, as from earlier chromatographic data ( Stirewalt and Evans 1960), to be a carbohydrate-protein-lipid complex. Trematoda; INDEX DESCRIPTORS: Schistosomu mansoni; Schistosoma japonicum; Turbellaria; Planaria; Biomphalaria glabrata; Mice; Cercariae; Glands; Acetabular glands; Postacetabular glands; Preacetabular glands; Pericercarial envelope; Surface film; Mucus coat; Cercarienhiillen Reaktion (CHR); Skin; Lipids; Histochemistry; Naval service at large. The experiments reported herein were conducted according to the principles set forth in “Guide for Laboratory Animal Facilities and Care,” prepared by the Committee on the Guide for Laboratory Animal Resources, National Academy of Sciences-National Research Council.
1 From the Bureau of Medicine and Surgery, Navy Department, Research Task No. MF12.524. 009-1004B and the Office of Naval Research, Contract No. NOO14-70-C-0331. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the 56 Co yri ht 0 1973 by Academic Press, Inc. o f reproduction in any form reserved. Alfri&
SECRETION OF CERCARL4E
57
Techniques; Stains; Cryostat; Periodic acid Schiff (PAS); Periodic acid-N, N-d& methyl-p-phenylenediamine ( PAD ) ; Dimethyl-p-phenylenediamine; Mixed diamines; Low iron diamine (LID); High iron diamine (HID); Colloidal iron ( CI); Alcian blue (AB); CI-PAS: AB-PAS; Methylene blue; Toluidine blue; Azure A; Thionin; Mucicarmine; Aldehyde fuchsin; Best’s carmine; Eosinophilia; Acidophilia; Phenylhydrazine Schiff; Feulgen’s; Plasma1 reaction; Chromotrope BR-AB; Sakaguchi; Millon’s; DMAB-nitrite; DDD reaction; Ninhydrin; Coupled tetrazonium; MgCls; Methylation; Sulfation; Acetylation; Nitrosation; Carbohydrate-containing secretion; Mucosubstance; Neutral mucosubstance; Acidic mucosubstance; Mucopolysaccharide; Polysaccharide; Glycoprotein; Vie-glycols; Vie-OH groups; SH groups; Aldehydes; Hexose; Glucose; Fucose; Mannose; Galactose; Fucomucoid; Mannomucoid; Glycogen; Mucus; Mucin; Mucigen; Sialomucin; Sulfomucin; Mammalian epithelial mucin; Brunner’s gland; Carboxylated polyanions; Sulfated polyanions; Polyanions; Polypeptides; Enzymes; Amino acids; Alpha-ammo acids; Nucleic acids; Lysine; Arginine; Histidine; Tyrosine; Tryptophan; Aminopeptidase, Lipase; Esterase; Cholinesterase; Phosphatase; Diastase; Amylase; Calcium ions; Calcium salts; Enzymes.
Considerable attention over a long period of time has been given to the paired, unicellular acetabular glands of schistosome cercariae. This is partly because of their prominence when observed microscopically, and partly because their contents are secreted during the invasion of definitive host skin. In addition to histological and physiological studies (see review by Stirewalt and Kruidenier 1961), their contents and secretions have been partially analyzed chromatographically and immunologically (Stirewalt and Evans 1960). Results of preliminary tests on the contents of these glands have shown them to be distinguishable by their histochemical reactions into at least two different types. The preacetabular glands reacted positively with tests for calcium ions or salts (Stirewalt and K&denier 1961; Lewert et al. 1966), but were essentially negative in other histochemical tests. The preacetabular glands will not be dealt with in this report. Reactions of the secretions of the postacetabular glands to histochemical stains have been studied here in detail. The secretions have been tested for the presence of neutral and acid mucosubstances, aldehydes, glycogen, lipids, proteins, and specific amino acids, as well as for selected enzymes. Emitted deposits of secretion
from these glands, together with the surface film around cercariae, have been compared with the contents of the glands before emission. It is of importance to our clarification of the mechanisms of skin penetration to understand the composition of the contents and secretions of these acetabular glands which are involved in the penetration process. Histochemistry provides one approach; hence the investigation reported herein. MATERIALS AND METHODS
Both Schktosoma man.soni cercariae and their snail hosts, Biomphuluriu glubrata, used in these studies were of Puerto Rican origin. They have been reared in this laboratory since 1945. The vertebrate phase of the life cycle has been maintained in Webster strain Swiss albino mice (NMRI), or in hamsters. Cercariae were pooled from at least 10 infected snails from which the parasites emerged naturally between 8:30 and 11:30 AM into small volumes of tap water that had been dechlorinated by aging and warmed to 30 C in a lighted constant temperature box. Living cercaraiae were observed in dechlorinated tap water, physiological saline, serum, and purpurin-India ink solutions. The fixative used was one of the follow-
58
STTREWALT AND WALTERS
ing: Baker’s neutral buffered formalin, Tellyesniczky’s, Camoy’s, 95% ethanol, and cetyl pyridinium (Pearse 1968). Neutral formalin was the fixative of choice. Formolfixed cryostat and unfixed cryostat sections were also used. Cercariae were studied both as whole organisms and in sections of infected snail tissue. Tissues were embedded in paraffin and sectioned at 7-12 pm or quick frozen in liquid nitrogen and sectioned in the cryostat at the same thickness. Sections and whole cercariae were processed concurrently with the same histochemical procedures. Cercariae were affixed to slides or cover glasses by their own secreted mucus as follows. The center of the slide was coated with facial skin lipid from a finger which had been rubbed along the hair line or nose. Large numbers of cercariae were pipetted in a small drop of water onto the lipidized area. They were immediately attracted to the lipid (Stirewalt 1971) and were allowed to creep over it and attempt to penetrate until they had firmly attached the oral or ventral sucker in a mucus deposit. The water was drained off into absorbent toweling and the slide immersed in fixative, or the fixative was pipetted onto the slide. Cercariae handled in this manner adhered in large numbers, many in clumps, since creeping cercariae are attracted to a cercaria which is attempting to penetrate. After fixation, slides or cover glasses of whole cercariae and sections were transferred concurrently from medium to medium for histochemical processing in Coplin or Columbia jars. The histochemical tests used are listed with references in Tables I-VI. Reactions have been described separately for the material within the gland funduses and ducts, i.e., before emission; and for the secreted deposits and the surface film ensheathing the body and tail of the organism. Secreted deposits were examined adherent to the oral surfaces of the cercariae at the open-
ings of the ducts; within the ventral suckers into which they had been placed from the oral ends of the cercariae (Fig. 2 ) ( Stirewalt and Kruidenier 1961; Stirewalt 1965); and on the surfaces of glass slides after deposit thereon by creeping cercariae. Reactions of the cercarial surface film were recorded also. Reactions are tabulated separately for sections and whole cercariae when differences were noted but not otherwise. Technical controls were provided by the goblet cells of the snail gut in sections of infected snail tissue. BACKGROUND
Postacetabular gland secretion is used during the time-limited, free-living existence of cercariae, especially in the exploratory and initial entry phases of their penetration of skin (Stirewalt and Kruidenier 1961) . The postacetabular secretory apparatus consists of three pairs of unicellular glands, each made up of an expanded nucleated portion, the fundus, and a single, very long tubular process which serves as the duct. The funduses occupy most of the cercarial body aboral to the ventral sucker. In living organisms, they are in contrast to other cercarial tissues and especially to the macrogranular preacetabular glands in that the postacetabular secretory apparatus is translucent and light-refractive, as if filled with liquid containing a suspension of very fine granules. After fixation and histochemical processing, viewed either in intact whole cercariae or in cercarial sections, the contents of these glands appear as microgranules surrounded by a thin layer of cytoplasm lining the cell membrane. A nucleus, which is often necrotic, usually lies to one side of the fundus. The viscous surface film that ensheathes both the body and tail of free-living cercariae, as well as of cercariae within sporocysts in snail tissues, appears to be less sticky and viscous than acetabular secretion
59 deposits after emission, but its histochemical reactivity is generally similar. The source of the film has not been established. HISTOCIHEMICAL RESULTS Reactions to selected stains of the postacetabular gland secretion of well-developed cercariae in the various sites observed are tabulated in Tables I-VI. Selected reactions are illustrated in Figs. l-11.
Neutral Mucosubstance PAS (periodic acid-Schif ) ( Table I; Figs. 1, 2, 7). Magenta color with the PAS technique was intense in both funduses and ducts of the postacetabular glands. It varied in intensity in secretion deposits and the surface film (Figs. 1, 2, 7). The Schiff reagent without oxidation usually did not give a color reaction, although occasionally a weak pink color could be brought out in
TABLE Histochemical
Tests for Carbohydrate-Containing
Test
Postacetabular funduses and ducts
PAS” (Lillie 1954; Pearse 1968)
Magenta
Acetylation-PAS Acetylation-KOH-PAS (Pearse 1968)
I Substances in Schiitosoma mansoni Cercariae
Secretion deposits and surface film
To test for
Magenta
Magenta-staining mucosubstances (MS) (vie-glycols or their substituted amines)
NC”
NC
Magenta
Magenta
Colorless carbohydrates Magenta carbohydrate reacting groups
Phenylhydrazine-Schiff (Spicer 1961)
NC
NC
Colorless periodate (PA)engendered aldehydes
Schiff’s reagent (Pearse 1968)
NC
NC
Magenta-free aldehydes
Mixed diamines (48 hr) PA-mixed diaminea (Spicer and Henson 1967)
NC to gray-yellow Gray, gray-brown to lavender
Gray to purple Gray-brown deposits; purple surface film
Purple acidic mucosubstances Gray to gray-brown PA-reactive neutral and acidic MS; purple PA-unreactive acidic MS
PADc (5 hr) (Spicer and Jarrels 1961)
Orange-brown
Orange-brown
Periodate-reactive neutral MS containing fucose or hexoses other than glucose
NC
Reddish-purple aldehydes of nucleic acids
Feulgen’s (Pearse 1968) a-amylase-PAS a-amylase-carmine Diastaae-carmine (Lillie 1954; Pearse 1968)
Magenta Red Red
Magenta Red Red
Colorless glycogen
Acetone-PAS Ether-PAS (McManus and Mowry 1960)
Magenta Magenta
Magenta Magenta
Colorless lipid
NC
Reddish-purple acetal lipids
Plasmal reaction NC [Pearse (Hayes) 19681 0 Periodic acidSchiff b No color. c p-Diamine.
60
STIRFoWALT AND WALTERS
the fundus with very strongly staining leucofuchsin preparations. The magenta stain of strongly reactive PAS appeared as a smooth, diffuse agranular reaction throughout the glands and ducts and in secretion deposits and surface film. The preferred technique, with which gland contents and secretion stained selectively, colored the granules only, leaving the background essentially clear. It is obvious in the immature cercaria at center in Fig. 1 that the postacetabular gland cells begin to elaborate secretion early in the development of cercariae. The PAS reaction was positive in them and occasionally in the surface film, even before ducts were formed. PAD (Periodic acid-N, N-dimethyl-pphenykenediamine) (Table I; Fig. 3). With PAD after 5 hr of staining, the bright orange-brown color of the postacetabular gland secretion within cercariae was bril-
liant against a colorless background. In secreted deposits and surface film it tended to be browner and paler, as it was in the anlage of the postacetabular secretory cells in immature cercariae. Goblet cells of the snail gut and undifferentiated cells of developing cercariae were all colorless. A strong black color developed after sulfation (Fig. 12). *&I Mucosubstance-Baso~hiz~ PAD (Periodic acid-N, N-dimethyl-pphenylenediamine) (Table ZZ; Fig. 4). With PAD at longer staining intervals (24 or 48 hr), the orange-brown darkened in the secretory cells without any suggestion of purple (Fig. 4). The darker color might have been the result of some mixture with black since the granules in funduses sometimes appeared black. Color in secretion deposits and surface film was variably brown in some areas and purple in others.
FIG. 1. Section of infected snail tissue stained with PAS. A portion of a schistosome sporecyst (right) contains one well-developed (right) and one less-developed (left) cercaria. Postacetabular gland cells are magenta. The transverse section of the snail gut (lower left) contains goblet cells which are unstained. Color plate. (x400). FIG. 2. Whole cercaria stained with PAS. Ducts, secretion and surface film stain strongly, but most of the contents of the gland funduses have been secreted. Color plate. ( x500). FIG. 3. Section of infected snail tissue stained for 5 hr with PAD. Funduses and ducts of the postacetabular glands of cercariae are orange-brown. Color plate. (x430). FIG. 4. Whole cercaria stained for 20 hr with PAD. Glands, secreted deposits and surface film are brown, Color plate. ( x385). FIG. 5. Cercaria stained with HID-AB without oxidation. Glands are colorless. Secretion deposits and surface film are blue. Results were the same with LID-AB. Color plate. ( x640). FIG. 6. Whole cercaria stained with alcian blue (AB) pH 2.5. Glands are unstained, deposits and surface film blue. Color plate. (x320). FIG. 7. Whole cercaria stained with AB-PAS. Glands are magenta, deposits blue to purple, surface film magenta in midbody and tail, and blue to purple in the oral and aboral areas. Color plate. ( X430). FIG. 8. Section of infected snail tissue stained with azure A. Snail goblet cells (lower right) are dark purple. Glands in longitudinal section of a well-developed cercaria (upper left) are colorless. Color plate. ( x425). FIG. 9. Section of infected snail tissue stained with thionin. Snail goblet cells (upper right) are very dark purple. Undifferentiated cells of developing cercariae are pale purple, but the glands in the longitudinal section of a developed cercaria at center-left are colorless. Color plate. ( X200). FIG. 10. Cercarial body stained with eosin Y pH 7.0. Glands and secreted mucus are red. Color plate. ( X690). Fro. II. Cercarial body stained with mucicarmine. Red secretions show at oral end and in ventral sucker. Glands are colorless. Color plate. ( X360).
OF CER-
SEaTION
TABLE Histochemiud
Tests
61
II
for Acid Mucosubstances in Schistosoma mansoni Cercariae: Diamine Basophilia Postacetabular funduses and ducts
Test
Secretion deposits and surface film
To test for (Spicer and Henson 1967)
NO to pale graybrown with dark granules
Purple-black to black”
Black to purple-black acid mucosubstance (MS)
NC to pale gray-brown
Blue
Black to purple-black acid MS : blue acidic MS ; strong color slrggests sialomucins
NC to gray-brown
Purple
Dark gray-brown
Purple
Black to purple-black sulfomucins Bed, purple or violet compounds with ester sulfate groups
NC to pale gray-brown
Blue
Black to purple-black sulfated MS; blue acidic MS
Orange-brown
Orange-brown
Orange-brown PA-positive
24 hr
Blackish
brown
Blackish
brown
Orangebrown neutral black acid MS
MS ;
48 hr
Blackish
brown
Blackish
brown
Orange-brown neutral black acid MS
MS;
Low iron diamine (Spicer 1965)
(LID)
LID pH 2.5-Alcian blue (AR) (Spieer 1965) High iron diamine (HID) 107, FeCla (Spicer 1965) 377o Fe& pH 1.3 (Gad and Sylven 1969) HID-AB
(Spicer 1965)
Periodic acid-p-diamine (PAD) (Spicer and Jarrels 1961) 5 hr
PAD with UV light (Spicer and Jarrels
1961)
neutral MS
Orange fluorescence
Orange fluorescence
Orange fluorescent
Methylation (37 C)-PAD (Spicer 1960)
NC
Orange-brown
Colorless carboxyl groups ; orange-brown neutral MS
Sulfation-PAD (Spicer and Jarrels 1961)
Black
Black
Control
neutral
MS
: black sulfate groups
-
a No color. b Matrix of secreted deposits was colorless.
These were difficult colors to assess in these sites. Snail goblet cells and striated border were pale golden; undifferentiated cells of imm.ature cercariae and the sporocyst wall were purple. Under UV light, orange fluorescence was brilliant at all sites in which an orangebrown color was visualized with ordinary light, except that it was not seen constantly in the surface film. Tissues that had been stained for 5, 24, or 48 hr all fluoresced similarly.
As with PAS, neither digestion with 01amylase nor extraction with ether or acetone eliminated color development in the test material. A slight paling of the color after pretreatment with lipid solvents was noted in some cercariae, but not in others. Note in Table II that prestaining methylation eliminated color in glands but not after emission. Mixed acidic diamiws (Table I). The mixed diamines gave poor visualization of reactions in cercariae compared with those
62
STIRElWALT
AND
in mouse tissues used as controls and with the colors pictured by Spicer (1965) in mammalian tissues. Funduses and ducts of whole cercariae showed no significant color without oxidation. Variability characterized the color of the secretion after emission. The matrix of deposits was colorless to gray-purple, but gray-brown, gray-purple, and black granules were enmeshed in the deposited secretion. The surface film and control snail goblet cells were purple. LID
(low iron diumine)
and HID
(high
iron diumine) (Table II; Fig. 5). Variability was characteristic of colors in the glands with these techniques, but secreted mateTABLE Histochemical
Tests
for Acid
Mucosubstances
WALTERS
rial and the surface film were consistently and significantly stained. Treatment with LID or HID followed by alcian blue pH 2.5 (LID-AB) did not change the reaction in glands, but distinguished clearly the blue deposits of secretion and the surface film (Fig. 5). Goblet cells and striated border of the snail intestine were also blue. When exposed to iron diamines alone, however, snail golbet cells were gray-purple as were the undifferentiated cells of immature cercariae and the cercarial cell nuclei. The less differentiated the cells of the cercariae were, the darker the color reaction. It is necessary, therefore, to distinguish careIII
in Schistosoma mansoni Cercariae: Thiazine
Postacetabular funduses and ducts
Basophilia
Secretion deposits and surface fdm
To test for
Blue-green Blue-purple and magenta
Blue-green acid mucosubstance (MS) ; magenta hexosecontaining MS
Pale blue
NC NC Blue Blue
Blue weakly or strongly acidic sulfomucins at pH 1.0 and below; blue carboxylated MS at pH 2.5 and above
NC
Blue
NC NC
NC NC
Blue sulfated MS
Methylation 37 C + AB Methylation 60 C + AB hlethylation (60 C) + KOH + AB (Spicer and Henson 1967)
NC NC NC
NC NC Blue
Colorless sialomucins Colorless acid MS Blue carboxyl groups of acid hlS
AB-PAS (Mowry 1963)
Magenta
Blue-purple and magenta
Blue acid MS; magenta hexosecontaining MS
Methylene blue (Pearse 1968) > pH 4.0 < pH 4.0
Blue NC
Blue NC
Degree of bssophiiia ; carboxyl staining largely suppressed below pH 2.0
Test Colloidal iron (CI) CI-PAS (Mowry 1963) Alcian blue (1%) (AB) (Pearse 1968) pH 0.5 1.0 2.5 5.7 Sect. Cert. with MgClt (Scott et al. 1964) 0.1 M to 1.0 M Sect. Cert.
* No color.
NO
Magenta
NC NC NC
SECRFXION
OF
fully in snail sections containing parasites in sporocysts between the glands of mature and immature cercariae. No real difference was recognized between the reactions to LID and HID. Buffered formalin and cryostat sections gave similar results with diamines. Carnoy’s was not as suitable a fixative as formalin, because the reactions were too pale. Colloidal iron and al&n blue (Table IIl; Figs. 6, 7). The same difference in staining reaction of the secretion in different sites was noted with colloidal iron (CI) as with alcian blue (AB ) (Fig. 6). Color with both was unequivocally present in secretion in both sections and whole cercariae after emission, but there was no blue color before (Fig. 6). Within glands, the only basophilia demonstrable in either sections or whole cercariae with CI or with AB at pH 0.5 or 2.6 was in the oral ends of occasional ducts, indicating that the change which occurred in the staining reaction of secretion after emission could also take place within the distal ends of the ducts. Treatment of sections and whole cercariae with AB at pH 5.7 resulted in a weak and inconsistent reaction in the glands. When color developed, it was either a pale blue wash in all tissues with a darker concentration of dye in the gland funduses and ducts or a pale blue granular network in the funduses, depending on the length of the staining period. After emission, although the deposited secretions stained strongly with CI and with AB at pH 2.5 and 5.7, there was no color in either sections or whole cercariae at lower pH’s. Note that addition to the AB pH 5.7 ‘of 0.1 M to 1.0 M electrolyte, MgC&, eliminated the color. Combination techniques of CI-PAS and AB (pH 2.5)-PAS always produced magenta-colored glands and blue-magenta secreted deposits and surface fihns (Fig. 7). It may be sign&ant that while blue
CERCARIAE
63
predominated in the deposits, magenta showed more strongly in the surface film except in the vicinity of the suckers. Snail goblet cells contained bright blue or blue-green droplets after staining with either of these dyes. With AB, the reaction became more intense as the pH was increased. At pH 5.7, the striated border was also strongly stained. Addition of the electrolyte resulted in the same reaction in goblets as in the emitted ceroarial secretions. Results with these two cationic dyes were substantiated by the findings with methylene blue. Color within glands and after emission did not develop below pH 4.0. Acid mucosubstance - metachrorrwiu (Table IV; Figs. 8, 9). Metachromasia in secretion before emission was absent or so pale as to be essentially negative when compared with the control sections stained after sulfation. No metachromasia could be brought out in any of the sites with thionin in either form&n-paraffin (Fig. 9) or cryostat sections or in whole cercariae. Control snail goblet cells, red-purple after 5 min of stain (Fig. Q), were almost black after 15 min. With aqueous toluidine blue according to Kramer and Windrum (Pearse 1988), 0.1% in 30% alcohol, the surface film was strongly metachromatic and secretion at the oral sucker was diffuse pale purple before dehydration but colorless after. With alkaline toluidine blue (0.025% for 5 min) (Pearse 1968)) the faint purple color of the granules in gland funduses indicated a very weak reaction, while snail goblet cells were bright red-purple. Azure A, applied to both whole cercariae and formalin-paraffin and cryostat sections, produced no metachromasia below pH 3.0 in secretion before emission. The pale redpurple granules visualized infrequently in paraffin sections in funduses and ducts at pH 3.0 (Fig. 8) and above were probably
STLREXVALT AND WALTERS
64
TABLE Histochemical
Tests for Acid Mucosubstances
Postacetabular funduses and ducts
Test
IV
in Schiitosoma mansoni Cercariae: Secretion deposits and surface film
Metachromasia
To test for
NC NC
Red metachromasia of acid mucosubstance (MS)
NC to blue NC
Purple NC
Red to purple metachromasia of acid MS
NC to pale blue
NC
1.5 Sect. Cert.
NC to pale blue Pale blue
Not seen Blue
Purple metachromasia of strongly acid sulfated MS below pH 1.5; blue orthochromasia to purple
3.0 Sect.
NC to pale redpurple NC to pale blue
Not seen Red
Metachromasia of weakly acid MS below pH 4.5 ; blue to purple of sialomucins at pH 3.0 and above
NC to pale redpurple granules Pale blue
Not seen
Sect.a NC* Cert. NC
Thionin [Pearse (Schmorl’s) 1968; Gurr 19601
Toluidine blue Sect. [Gurr 1960; Cert. Pearse (Kramer and Windrum) 196S] Azure A (0.02%) (Pearse 1968 ; Spicer and Warren 1960) pH 0.5 Sect. Cert.
Cert. 4.0 Sect.
Cert. 5.0 Sect. Cert. Sulfation-azure A Sect. (Spicer 1960)
Red-purple
Pale red-purple granules Pale blue
Not seen
Red-purple
Red-purple
Red-purple Control for sulfated mucosubstance
a Sections, cercariae. b No color.
in immature cercariae. No metachromasia was noted in these sites at any pH in emerged whole cercariae. In secreted deposits, red-purple metachromasia was visualized only at pH 3.0 and above. In the surface film, there was a spectrum of reaction from colorless through blue at pH 1.5 to red at pH 3.0 and finally red-purple from pH 3.0 to 5.0. Even when a 0.05% (Quintarelli 1963) or a 0.570 solution (McManus and Mowry 19sO) of azure A was used with formalinparaffin sections at pH 1.5, there was no
color anywhere in developed cercariae. At pH 3.0, results were erratic: in some cercariae, funduses of the glands were colorless; in others, pale red-purple. At pH 4.5, definite red-purple granules were present in gland funduses. Ducts were without color throughout. Control goblet cells of the snail gut were metachrom8atic at all pH’s. Prestaining sulfation made the test substance in all four sites strongly metachromatic and served as another control technique. Proteins and amino acids (Table V; Fig.
SECRETION OF CEXcARIAE
10). Color of the secretory material in the glands and after emission in secretion deposits, as well as in the surface film, was consistently present with all the techniques and after all the prestaining treatments listed in Table V. Granules were always darker red than the matrix of funduses and ducts, suggesting diffusion of the stain from them into the matrix. The data in Table V need no special comment except that with Adams’ DMABnitrite procedure, color intensity was variable within the glands, but reproducibly deep blue in the surface film and deposits. Our attempts to localize enzymes in TABLE Htitochemical
Tests
65
gland contents or after secretion failed, as did those of Dusanic ( 1959), Fripp ( 19&3), Sodeman et al. (19&3), and Ebrahimzadeh ( 1970). There was no histochemical evidence of amino-peptidase, lipase, nonspecific esterases, cholinesterase, or phosphatases in the sites studied herein. SIGNIFICANCE OF THE FIND~NC~ AND DIXXJSSION
Histochemical procedures are employed more often to demonstrate that a known substance can be visualized with the agents used than to provide tests for the presence of the substance. At the outset then, it is V
for Proteins and Amino Acids in Schistosoma mansoni Cercariae Postacetabular funduses and ducts
Test
Secretion Deposits and surface film
To test fora
Golden red Red Red
Red Red Red
An amino acid source of protein acidophilia
Eosinophilia aft.er acetylation (Pedersen 1959, 1963)
Red
Red
Guanidinium
group of arginine
Eosinophilia after nitrosation (Pedersen 1959, 1963)
Golden red
Red
Guanidinium
group of arginine
Chromotrope 2R-AB (Wagner and Shapiro 1957)
Red
Red to red and blue
Basic proteins
Sakaguchi (Baker (Pearse 1968)
Orange red
Red
Arginine
Xllon’s, Baker modification (Pearse 1968)
Red
Red
Tyrosine
DMAB-nitrite 1968]
NO to pale blue
Blue
Tryptophane
Red
Red
SH groups of a-amino (Pearse 1968)
Blue
Blue
Polypeptides
Red-brown
Red-brown
Tryosine,
Eosinophilia (Pedersen 1959, 1963)
pH
7.0 10.0 11.8
1947)
[Pearse
(Adams)
DDD reaction [Pearse (Barnett and Se&man) Ninhydrin
(McManus
19681
1968)
Coupled tetrazonium (Pearse 1968) o Color means probable b No color.
presence of indicated
protein
or amino acid.
acids
or protein
tryptophane
or histidine
66
STLREWALT
iND
FIG. 12. Section of infected snail tissue stained for 5 hr with PAD after sulfation. Glands and surface film of the cercaria are black. ( x340).
acknowledged that only a few of the reactions are specific enough to be used as proof of the presence of a chemical constituent. Nonetheless, histochemical techniques are invaluable as a means of demonstrating similarities and differences among substances in tissues and of indicating the probable presence and localization
of specific reacting substances. Finally, of course, the target material, in our case the secretions elaborated in the postacetabular glands of cercariae, should be identified by chemical means. Despite this, a great deal of information about the postacetabular gland secretion has been derived from histochemical tests. Of special importance was the finding that, with some tests, the secretion after emission reacted differently from the material within the secretory cells. Although secretory ceII contents, emitted secretion, and surface film all stained red or orange-red with Best’s, Orth’s, and lithium carmine, which color a variety of substances rich in &-OH groups, such as epithelial mucus secretions (Mowry 1963), this was not the case with mucicarmine and aldehyde fuchsin (Table VI). With both of the latter dyes, secreted deposits and the surface film reacted positively, in contrast to the material in the glands which was unstained in sections as well as whole cercariae (Fig. 11). While none of these stains has proven specificity, the differences noted in staining before and after emission indicate a change in the reacting substance. Similar staining of these glands of Schktowma japonicum cercariae with Best’s carmine and absence of stain with mucicarmine were described by Miura ( 1955).
TABLE Reaciions of Fully
WALTERS
VI
Developed Whole Cercariae and Sections of Schistosoma mansoni to Stains .for Mums
Test
Reference
Postacetabular funduses and ducts
Secreted deposits and surface film
Best’s carmine Orth’s lithium carmine
Pearse 1968 Lillie 1954
Red Orange-red
Red Orange-red
Mucicarmine (Southgate)
McManus and Mowry 1960
NO
Red
Aldehyde fuchsin (Gomori)
Pearse 1968
NC
Lavender
QNo color; color means reaction for mucus.
SECRETION
OF
Since mucicarmine stains mucin red, since the secreted material is viscous and sticky (Stirewalt 1965), since it has been shown chromatographically to be composed of amino acids, carbohydrates, and lipids ( Stirewalt and Evans 1960), and since its histochemical reactions are compatible with those of mucin, the material in the secreted deposits, and the surface film as well, is herein called mucin. On the other hand, because of the differences in reaction to histochemical procedures, the secretion in the glands before emission is termed mucigen. Mucigen may be characterized from histochemical evidence as follows. Its selective staining with PAS indicated it to be a hexose-containing mucosubstance rich in &c-l, 2 glycol groups or their substituted amines (Pearse 1968). The carbohydrate nature of the reacting substance was confirmed in all sites by prevention of the PAS reaction by acetylation and its restoration by the acetylation-KOH-PAS sequence (Table I; Pearse 1968; Spicer 1963; Quintarelli 1963). With the 5-hr PAD technique, it behaved as a neutral mucopolysaccharide, since it was selectively bright orange-brown (Table II; Spicer and Jarrels 1961; Spicer 1965; Pearce 1968). Spicer and Henson (1967) noted that fucose or hexoses other than glucose, such as galactose, are the most probable source of this kind of reaction, It may be recalled that glucoseamine and galactoseamine were observed in chromatographs of hydrolyzed secreted deposits (Stirewalt and Evans 1960). Orange fluorescence with PAD in UV light was compatible with the above. The findings were substantiated by the variable gray-brown to dark brown color of the mucigen and the absence of purpIe with aged p-diamine solution or a 24- ‘or 48-hr staining period. This, together with the magenta with PAS after 10 min of oxidation, the orange-brown color with PAD,
CEXCARIAE
67
and the absence of purple color with mixed diamines without oxidation, indicated its periodate-reactivity (Spicer and Henson 1967; Pearse 1968). The reactive mucigen was not glycogen, since it stained orange-brown with 5-hr PAD (Spicer and Jarrels 1961) and neither amylase nor diastase prevented color development with PAS or PAD (Table I). Neither aldehydes of lipids, free aldehydes, nor acetalphosphatides appeared to be responsible for the PAS reactivity (Table I; Stirewalt 1959). Mucigen was nonreactive for acid mucosubstance with most tests (Tables II-IV; Figs. 6-9). There was lack of convincing metachromasia in the glands with azure A, thionin, and toluidine blue; and there was essential failure of the glands to stain significantly with CI, AB, methylene blue below pH 4.0, 24- and 48-hr mixed diamines, phenylhydrazine-PAS, and acetylation-PAS. An occasional weak reaction for acidic groups was observed, however, with three of the stains for acid mucosubstances; a very pale-blue network in sections of funduses with AB at pH 5.7 with and without 0.1 M MgCla; occasional very weak metachromasia showing as pale red-purple granules with azure A at pH 3.0 and above; and variable brown to dark color with PAD after 24 hr (Tables III, IV). These reactions were positive only in sections in which control tissues, cells of immature cercariae, and the secretion droplets in snail goblet cells reacted strongly. No reaction developed in the glands of whole cercariae. These findings bring up the possibility that acidic groups were present in the mucigen, in addition to the neutral groups indicated by PAS and 5-hr PAD reactivity, but were masked by the close proximity of neutral groups or basic proteins, both of which have been shown herein to be present (Tables 1, V). The blue color in sec-
68
STIRF,WALT
tions with AB in the pH 5.7 buffer plus electrolyte is characteristic of the unmasking of acidic groups by basic proteins since this masking effect is said to be prevented by MgClz in pH 5.7 buffer (Mowry 1966). Neighboring periodate-engendered aldehydes and basic proteins have both been considered to be possible blocks to the staining of vicinal sulfates or carboxylates of acid mucins (Spicer 1965; Spicer and Henson 1967; Pearse 1968). Indeed, sialic acid carboxyls are, to a great extent, blocked by basic proteins (Quintarelli 1963). The term mu& has been used here for the material in secretion deposits adhering to the oral surface of cercariae, in the acetabulum, and on surfaces over which cercariae have crept. These deposits have been shown to contain polysaccharide, protein, and lipid (Stirewalt and Evans 1960). The surface film, which is viscous and sticky, as are the deposits, has also been considered here to be mucus although its source is not known. Mucin in secreted deposits gave reactions for neutral mucosubstance similar to those of mucigen with PAS and 5-hr PAD, but it alslo behaved histochemically as an acid mucosubstance with aged PAD and mixed diamines. Reactivity of the surface film was similar (Tables II-V). Reactive polyanions were, therefore, present in the mucin. It may be of value to comment that the magenta of the PAS and orange-brown of the 5-hr PAD reaction were weak in mucin in most cercariae compared with their intensity in mucigen. Possibly the less intense color in mucin simply reflected the thinness of the mucus layer in the surface film or dilution of the reactive substance in secretion deposits in water which the deposits are known to absorb and in which they swell (Stirewalt 1959). Alternatively, the explanation may be because in the mucin not only neutral but also acid radicals were
AND
WALTERS
reactive, and acidic mucins stain weakly or not at all with PAS. Our observations suggested that the surface film was a neutral mucosubstance and that the acid mucosubstance was an overlay of postacetabular gland secretion. There was overwhelming evidence for the presence in the mucin of carboxylated polyanions and little evidence for sulfated polyanions. The red color of mucicarminetreated deposits (Fig. 11) was characteristic of carboxylated groups. Metachromasia with azure A at pH 3.0 and above and AB staining of the mucin at pH 2.5 and 5.7 pointed to carboxylated mucosubstancesialomucins or other weakly acid mucosubstance-as the reacting material (Mowry 1963). Evidence from diamine techniques is as follows (Spicer and Henson 1967). Mucin after LID-AB and HID-AB was the blue (Table II) typical of sialomucins. The methylation-saponification-alcian blue sequence also indicated reactivity Iof carboxylated groups (Table III), for methylatedAB-treated cercariae and sections were colorless, while methylation-KOH-AB treatment restored the color (Spicer 1960). While these results indicated activity of carboxylated groups ( Spicer 1960)) they, of course, did not deny the possible presence of sulfated groups as well, although loss of AB stain at pH 5.7 after the addition of MgC& does so deny it. The lavender color of secreted deposits with aldehyde fuchsin was not a strong reaction, and thus has not been interpreted as indicative of sulfomucins. Basic proteins were visualized in both mucigen and mucin with eosin Y according to Pedersen ( 1963), whose technique was based on the statement by Singer (1952) that “groups in proteins undoubtedly form the fundamental basis for tissue acidophilia and the amino groups mainly responsible for acidophilia in general are the terminal amino groups of the polypeptide chains and
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the side groups from lysine, histidine and arginine.” Following Pedersen’s eosin Y procedure, the secretion was red at all three recommended pH’s in all sites. Ebrahimzadeh (1970) also found the postacetabular glands to be eosinophilic. Acetylation and nitrosation, which block the terminal amino groups and the e-amino groups of lysine, had little, if any, effect on the color of either mucin or mucigen in these cercariae. Basic groups other than the guanidinium group from arginine are not available for binding to eosin at pH 11.8 (Wislocki et al. 1957), at which pH the cercarial secretion stained strongly in all sites. On this basis, the guanidinium group from arginine may be considered to be responsible for the eosin binding by the secretion. In confirmation, the secretion in all sites gave a strong Sakaguchi reaction for a&nine. Furthermore, as assayed chromatographically, a&nine was present in a far greater percentage than any other amino acid residue identified in acid hydrolysates of cercarial secretion deposits (Stirewalt and Evans 1960). Histochemical reactions were also positive in both mucigen and mucin, though less strongly, for tyrosine ( Millon’s ), tryptophane ( DMAB-nitrite) and the SH groups of alpha amino acids (DDD) (Table VI). There was no evidence of tryptophane or SH-containing amino acids in the chromatographic pattern of cercarial secretion (Stirewalt and Evans 1960), but tryptophane, of course, is not identifiable in acid hydrolyzates and the chromatographic techniques used may not have been sensitive enough to test for the SH groups. Since the secretion was Feulgen-negative in fully developed cercariae, nuclei acids were not involved. As with the secretory cells of planarians described by Pedersen (1963), however, in early stages of elaboration the secretory cells reacted positively with stains for both neutral and acidic mucosubstances
CER-
69
and nucleic acids. These results are oompatible with the abundant ergastoplasm in cells active in the production of secretion. Results of these investigations place the postacetabular gland secretion in the same general histochemical category with certain mammalian epithelial mucins, especially with that of human Brunner’s gland (Spioer and Henson 1967). Both the cercarial mucin and mammalian epithelial mu&s are strongly reactive with PAS, rich in vicinal hydroxyl groups, and they are acidic, reliably colorable with either colloidal iron or alcian blue (Mowry 1963). Histochemical evidence thus fits well with the chromatographic finding that deposits of secretion contained polysaccharides in firm chemical union with proteins; 15 amino acid residues, glucose and galactose-amine, and some steroid spots were demonstrated ( Stirewalt and Evans 1960). Several important problems are unresolved. One is the presence or absence of acid radicals in the mucigen. The second involves the chemical change responsible for the histochemically demonstrable difference in secretion in glands and after emission, for the reaction of mucin in deposits (and in surface films) was unquestionably characteristic of acid as well as of neutral mucosubstance (Tables II-IV). If stearic hindrance by proximal neutral radicals and/or basic proteins is responsible for the lack of staining of acid radicals in the mucigen, perhaps it is removed when the secretion is hydrolyzed in the water into which it is secreted. These uncertainties make it impossible to classify postacetabular gland secretion at this time according to the scheme proposed by Spicer et al. ( 1965), although it is obvious that it contains neutral periodate-reactive mucosubstance, either neutral glycoprotein or fuco- or mannomucoid. The strong orange-brown color with 5-hr PAD suggests that it is fucose- or galactose-con-
70
STIREWALT
AND WALTERS
taining and places it in this regard with the secretion of human Brunner’s gland and gastric surface epithelium. Further classification depends not only on its demonstrated periodate reactivity but also on the presence of sulfomucins as well as sialomucins. Postacetabular gland secretion of cercariae did not react similarly to the subcuticular unicellular mucus glands of those planarians described by Pedersen ( 1963), despite the close phylogenetic relationship usually assumed between Turbellaria and Trematoda. For example, the cercarial mucus was both basophilic and eosinophilic. E’osinophilic and basophilic granules were found in separate secretory cells in planarians (Pedersen 1959). Quintarelli (1963) stated that such plurality of mucus has been encountered regularly, and Spicer (1963) found that exactly similar mucins were seldom encountered. The results reported here agree with those of Smith et al. (1969) for the surface film of cercariae which they have called a pericercarial envelope or mucoid coat. The term “pericercarial envelope” has previously been used to designate the product of the interaction of the cercarial surface film or mucus coat with globulin in the fast-moving gamma fraction of antischistosome serum (Evans and Stirewalt 1959) in the Cercarienhiillen Reaktion of Vogel and Minning. Since Smith and his co-workers did not expose their cercariae to antischistosome serum, the term mucus coat or surface film is preferable to avoid confusion. Our histochemical results, however, disagree substantially with those tabulated by Smith et al. (1969) for the acetabular glands, no matter whether their reactions were with the pre- or postacetabular glands. Unfortunately, they did not distinguish, either in table or figures, between these two types of secretory cells which differ dramatically both in function ( Stire-
walt and Kruidenier 1961) and in histochemical reaction. Since their Table I lists the glands as PAS-reactive, we have assumed that the postacetabular glands were indicated because the preacetabular glands are negative with the PAS reaction, as well as with most of the other tests used. As reported herein (Table V), both the postacetabular glands and the surface films of our cercariae were positive for protein (eosin, ninhydrin, and Chromotrope 2RAB ), arginine ( Sakaguchi) , tyrosine ( Millon’s ) , tryptophane ( DMAB-nitrite), and SH groups (DOD). Smith et al. ( 1969) found them to be negative for protein, tryptophane, and SH groups. Conversely, they reported the presence in both glands and surface film of acid mucosubstance (Hale’s colloidal iron) which we have found only in the surface film and secreted deposits as judged by reactions for acidic groups with Hale’s colloidal iron and other tests depending on basophilia (Tables II, III), and metachromasia (Table IV), while our contents within the glands gave histochemical evidence of neutral mucosubstance only. As discussed earlier, however, acid radicals may be present but masked by neutral radicals or proteins. REFERENCES D. G. 1959. Histochemical observations of alkaline phosphatase in Schistosoma man-
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systems der Cercarien von Schistosomu mansoni Sambon ( 1907). Zeitschrift fiir Parasitenkunde 34, 319-342. EVANS, A. S., AND STIREWALT, M. A. 1959. Serologic reactions in Schistosoma munsoni infections. V. Localization of CHR and cercarial agglutinating factors in electrochromatographically fractionated human sera. Experimental Parasitology 8, l-9. FRIPP, P. J. 1966. Histochemical localization of B-glucuronidase in schistosomes. Experimental Parasitology 19, 254-263.
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GAD, A., AND SELWYN, B. 1969. On the nature of the high iron diamine method for sulfomucins. Journal of Histochemisty and Cytochemistry 17, 156-160. GURR, E. 1960. “Methods of Analytical Histology and Histochemistry.” The Williams and Williams CO., Baltimore, Maryland. LEWERT, R. M., HOPKINS, D. R., AND MANDLOwrr~, S. 1966. The role of calcium and magnesium ions in invasiveness of schistosome cercariae. American Journal of Tropical Medicine and Hygiene 15, 314-323. LILLIE, R. D. 1954. “Histopathologic Technic and Practical Histochemistry.” The Blakiston Co., New York. MCMAMJS, J. F. A., AND MOWRY, R. W. 1960. “Staining Methods Histologic and Histochemical.” Paul B. Hoeber, Inc., New York. MNRA, M. 1955. Histochemical studies on Schistosomu japonicum. 3. Histochemical studies of cercariae (Japanese). Kumumoto Igakkwai Zasshi 29, 99-109. MOWRY, R. W. 1963. The special value of methods that color both acidic and vicinal hydroxyl groups in the histochemical study of mu&s. With revised directions for the colloidal iron stain, the use of alcian blue 8GX and their combinations with the periodic acid-Schiff reaction. Annals of the New York Academy of Sciences 106( 2), 402-423. MOWRY, R. W. 1966. The coloration of polyanions by alcian blue 8GX in buffered 70% ethanol with electrolyte (magnesium chloride). JOWna2 of Histochemisty and Cytochemistry 14, 800-801. PEARSE, A. G. E. 1968. “Histochemistry: Theoretical and Applied.” Little, Brown and CO., Boston. PEDERSEN, K. J. 1959. Some features of the fine structure and histochemistry of planarian subepidennal gland cells. Zeitschrift fiir Zellforschung 50, 121-142. PEDERSEN, K. J. 1963. Slime-secreting cells of planarians. Annals of the New York Academy of Sciences 106(2), 424-442. QUINTAFIELLX, G. 1963. Histochemical identification of salivary mucins. Annals of the New York Academy of Sciences 106(Z), 339-363. SCOTT, J. E., DORLING, J., AND QUINTARELLI, G. 1964. Differential staining of acid glycosaminoglycans by alcian blue in salt solutions. Biochemical Journal 91, 4P-5P. SINGER, M. 1952. Factors which control the staining of tissue sections with acid and basic dyes. International Review of Cytology 1, 211-255.
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SMI’nx, J. H., REYNOLDS, E. S., AND VON LICHTENBERG, F. 1969. The integument of Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 18, 28-49. SODEMAN, W. A., BERRY, E. G., AND FORS, M. B. 1968. Schistosomal phosphatases. Histochemical localization of alkaline and acid phosphatase in cercariae of Schistosoma munsoni, Schistosomu haematobium and Schistosomu japonicum. American Journal of Tropical Medicine and Hygiene 17, 851-857. SPICER, S. S. 1960. A correlative study of the histochemical properties of rodent acid mucopolysaccharide. Journal of Histochemistry and Cytochemisty 8, 18-35. SPICER, S. S. 1961. The use of various cationic reagents in histochemical difFerentiation of mucopolysaccharides. American Journal of Clinical Pathology 5, 393467. SPICER,S. S. 1963. Histochemical differentiation of mammalian mucopolysaccharides. Annals of the New York Academy of Sciences 106(2), 379-388. SPICER, S. S. 1965. Diamine methods for differentiating mucosubstances histochemically. Journal of Histochemisty and Cytochemistry 13, 211-234. SPICER, S. S., AND HENSON, J. G. 1967. Methods for localizing mucosubstances in epithelial and connective tissues. In “Methods and Achievement in Experimental Pathology” (Ed. E. Bajusz and G. Jasmin, eds.), Vol. 2, pp. 78112. S. Karger, Basel/New York. SPICER, S. S., AND JARRELS, M. H. 1961. Histochemical reaction of an aromatic diamiue with acid groups and periodate engendered aldehydes in mucopolysaccharides. Journal of Histochemistry and Cytochemistry 9, 368-379. SPICER, S. S., AND WARREN, L. 1960. The histochemistry of sialic acid containing mucoproteins. Journal of Histochemisty and Cytochemistry 8, 135-137. SPICER, S. S., LEPPI, T. J., AND STOWARD, P. J. 1965. Suggestions for a histochemical terminology of carbohydrate-rich tissue components. Journal of Histochemistry and Cytochemistry 13, 599-603. STIREWALT, M. A. 1959. Isolation and characterization of deposits of secretion from the acetabular gland complex of cercariae of Schistosomu mansoni. Experimental Parasitology 8, 199-214. STIREWALT, M. A. 1965. Mucus in schistosome cercariae. Annals of the New York Academy of Sciences 118(24), 966-968.
72 STIREWALT, M. A. 1971. Penetration
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stimuli for schistosome cercariae. In “The Biology of Symbiosis” (T. C. Cheng, ed. ), 327 pp. University Park Press, Baltimore, Maryland. STIREWALT, M. A., AND EVANS, A. S. 1960. Chromatographic analysis of secretions from the acetabular glands of cercariae of Schistosomu mans&. Experimental Parasitology 10, 75-80. STIREWALT, M. A., AND KRUIDENIER, F. J. 1961. Activity of the acetabular secretory apparatus of cercariae of Schistosomu munsoni under
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experimental conditions. Experimental Parasitology 11, 191-211. WAGNER, B. M., AND SHAPIRO, S. H. 1957. Application of alcian blue as a histochemical method. Laboratory Investigation 6, 47M77. WISLOCKI, A. B., WEISS, L. P., BURGOS, M. H., AND ELLIS, Ft. A. 1957. The cytology, histochemis&y and electron microscopy of the granular cells of the metrial gland of the gravid rat. Journal of Anatomy 91, 130-140.