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Biochemical changes in the transformation of Schistosoma mansoni cercariae to schistosomules
Comp. Biochem. PhysioL, 1975, VoL 51B, pp. 417 to 420. Pergamon Pre~s. Printed in Great Britain
BIOCHEMICAL CHANGES IN THE TRANSFORMATION OF SCHISTOSOMA MANSONI CERCARIAE TO SCHISTOSOMULES* C. C. DE OLIVEIRA,lt E. A. FIaUEmEDO,1 G. GAZZINELLI,t R. E. HOWELLS2~ AND J. PELLEGRINOa 1 Departamento de Bioquimica-Imunologia;u Departamento de Zoologia-Parasitologia; and 8 Grupo Interdepartamental de Estudos sobre Esquistossomose, Instituto de Ci6ncias Biol6gicas, C.P. 2486, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (Received 29 M a y 1974)
Abstract--1. In the transformation of Schistosoma mansoni cercariae to schistosomules there was an elimination of the ATEE-hydrolysing proteases to about 50 per cent of the original specific activity level, in 40-60 rain. 2. Histochemical examination of the pre- and postacetabular glands of cercariae stained by alizarin and PAS disclosed a similar loss of material as the larvae became schistosomules. 3. The ratio of carbohydrate/protein decreased to about 50 per cent of its original value during the transformation. 4. Extracts of cercariae, tails and bodies submitted to electrophoresis in deoxycholate-containing borate buffer, pH 9.5, disclosed three PAS-stained bands which were identified as post-acetabular gland content, surface coat and glycogen. 5. The loss of carbohydrate material from transforming cercariae was accompanied by changes in intensity of the PAS-stained bands obtained by electrophoresis of larval extracts. 6. The glycoprotein material of the cercarial glycocalyx was recovered from the incubation medium following transformation of the larvae to schistosomules.
INTRODUCTION DURING the penetration of mammalian skin the cercariae of Schistosoma mansoni undergo metamorphic changes to the schistosomules. These changes include extrusion of the contents of the acetabular glands, decaudation and the loss of a glycoprotein coat or glycocalyx from the cercarial body surface. Schistosomules additionally differ from cercariae in being sensitive to water and tolerant to normal guinea pig serum. Using an in vitro technique which induces in a stepwise manner the same sequence of changes that are observed in vivo (Ramalho-Pinto et al., 1974), we have investigated some of the biochemical events that occur during cercarial transformation. Coles * This work received support from Conselho Nacional de Pesquisas, Conselho de Pesquisas da UFMG, Banco Nacional para o Desenvolvimento Econ6mico (FUNTEC, 66), World Health Organization and the British Technical Co-operation Program. Some of the results given in this paper have been presented in the Meeting of the Sociedade Brasileira para o Progresso da CiSncia, held in Rio de Janeiro, Brazil, in July 1973. Contribution No. 53 from the Schistosomiasis Research Unit. 1"Fellow of the CAPES. Visiting Professor from the Liverpool School of Tropical Medicine, Liverpool, England.
(1973) observed that 32 per cent of the total nitrogen and 22 per cent of the cercarial body glycogen are lost during transformation, these data agreeing closely with those of Gazzinelli et al. (1973) on the loss of protein and total carbohydrate from the transforming cercariae. Gazzinelli et al. (1973) followed the elimination of proteases and reduction in carbohydrate content during this process and determined electrophoretically that two polysaccharide bands obtained from cercarial extracts were much reduced in schistosomule preparations. In this study we have further analysed the nature and sequence of carbohydrate loss from the transforming larvae. Of particular interest was the surface coat of the cercariae and evidence is presented to show that this material is lost from the cercaria and may be isolated from the incubation medium. The extent of glandular secretion has been determined by biochemical and histochemical methods and the results have been compared. MATERIALS AND METHODS Cercariae of S. mansoni (LE strain, B¢lo Horizonte) were obtained from laboratory reared Biomphalaria glabrata and concentrated to 7(g)--1000/mlas described by Gazzinelli et al. (1973). Ccrcariae were used 3-5 hr after emergence from the snails. Schistosomules and intermediate transformation stages, together with materials
417
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C . C . DE OLIVEIRA, E. A. FIGUEIREDO, G. GAZZINELLI, R. E. HOWELLSAND J. PELLEGRINO
secreted or liberated from the larvae, were obtained by the technique of Ramalho-Pinto et al. (1974) as follows.
Cercarial bodies and tails (Step 1) Ten-ml aliquots of the cercarial suspension were cooled in 15 ml glass conical centrifuge tubes for 10 min and centrifuged at low speed for 15 sec. Cercariae from five to six tubes were pooled by resuspending in a final volume of 2 ml of cold Hanks'-BSS pH 7.2-7.4. The cercarial tails were removed by stirring in a Vortex Jr. mixer (Scientific Industries, Inc., Queens Village, New York) for 1 rain. The volume was increased to 7 ml with the same medium and after standing for 8-10 min the supernatant containing the tails was removed from the gravity sedimented cercarial bodies. The bodies were resuspended in 7 ml of Hanks' and the procedure repeated twice. The resulting sediment of cercarial bodies, containing less than 5 per cent of tails, was suspended in 2 ml of Hanks' -BSS.
Cercarial secretion (Step 2) The suspended cercarial bodies were packed by 1 min speed centrifugation and incubated at 30°C for 40 min unless otherwise stated. The supernatant was decanted and the pellet washed three times in cold Hanks'-BSS by resuspension and centrifugation. The supernatant and washing solutions were pooled.
S. mansoni infection and one drop of a suspension containing twenty to thirty larvae and incubating this preparation from 30 min to 12 hr at room temperature in a water vapour saturated atmosphere. Only larvae which completely lacked adventitious membranes (envelopes) were considered to be CHR negative. Protein, carbohydrate and esterolytic (proteolytic) activity These determinations were performed by the Lowry, anthrone and Hestrin methods as described by Gazzinelli et al. (1966).
Organism preparations Cercariae, cercarial bodies or tails, schistosomules or incompletely transformed organisms were sonicated as described by Gazzinelli et aL (1973). The suspending medium to prepare extracts for electrophoretic analysis was 1% deoxycholate (DOC) in 0.1 M borate buffer pH 9.5. For other determinations larvae were sonicated in deionized water.
Electrophoresis Electrophoresis was carried out in Cellogel strips (Chemetron, Milan) for 120 rain at 200 V using the same buffer as in the extracts. The strips were then PAS-stained as shown below and made transparent as described by Gazzinelli et al. (1973).
Cercarial coat (Step 3)
Polysaccharide and glycoprotein dyeing procedures
The larvae obtained in Step 2 were suspended in 5-10 ml of Minimal Eagle Medium (TC-MEM), transferred to 30 ml stoppered vials and incubated under continuous shaking at 37°C for 60 rain. The suspension was centrifuged and the coat was recovered from the supernatant by dialysis and lyophilization.
The cellogel strips were stained by a modification of the technique of Block et al. (1955): the strips were (1) fixed in ethanol for 10 min; (2) immersed in a 1% w/v aqueous solution of periodic acid (Merck) for 10 min; (3) rimed three to four times in distilled water; (4) immersed in Schiff's solution prepared according to de Tomazi as described by Pearse (1968) for 25--40 min; and (5) washed three to four times in sulfite water (sodium metabisulfiteconcentrated HC1--distilled water, 2 : 5 : 500).
Schistosomules The larvae collected in Step 3 are from now on called schistosomules.
Water sensitivity The organisms were exposed to deionized or distilled water for 10 min. Organisms which were motile after this time were considered water insensitive. A n alternative technique used was the methylene blue exclusion test of Clegg & Smithers (1968) in which the organisms were exposed to 0'03% methylene blue in deionized water for 10 rain and the water sensitivity of larvae calculated from the percentage which were blue stained. Controls were run in Hanks'.
Guinea-pig serum sensitivity This test for guinea-pig serum sensitivity was performed according to Ramalho-Pinto et aL (1974).
Glycogen standard Glycogen (Nutritional Biochemical Corporation) was dissolved in 1% DOC---containing 0.1 borate buffer pH 9-5--to a final concentration of 25 mg/ml.
Amylase treatment of cercarial preparations Saliva was diluted I : 5 with sterile saline and incubated with 10 vol. of cercarial preparation at 37°C for 30 min. Controls were run with heat denatured diluted saliva.
Staining of the larvae PAS and alizarin staining of intact organisms were performed as described by Gazzinelli et al. (1973). RESULTS AND DISCUSSION
Cercarienhiillen-Reaktion
( CHR) (Vogel & Minning, 1949)
The C H R technique was performed by placing on a glass slide one drop of serum from a patient with confirmed
U n l i k e cercariae, the larvae o b t a i n e d by c a r r y i n g o u t Steps 1, 2 a n d 3 outlined in Materials a n d M e t h o d s , were sensitive to water, insensitive to
INCUBATION
o
b
c
d
e
f
e÷f
0
I0
40
(÷;
(MIN)
80 (+)
P
P o c
0 C G
G
(-)
(-)
A
B
Fig. 1. PAS-stained bands in electrophoresis of S. mansoni extracts. Electrophoresis performed in cellogel strips with 0'1 M borate buffer, pH 9'5, containing 1 ~ sodium deoxycholate, for 120 rain at 200 V. O, Origin; P, post-acetabular gland material ; C, coat ; G, glycogen. A. Identification of the bands--tails (d), bodies (e) and coat (f) prepared as in the text; cercariae tails and bodies were washed with Hanks' solution and the extracts prepared in the cold, using the buffer described above. Part of the cercarial extract was used as such (a) and part was treated by amylase (b) prior to electrophoresis. Glycogen (c) was included for comparison. B. Electrophoresis of extracts of transforming S. mansoni cercarial bodies obtained as in Step 1 (zero time), incubated as in Step 2 (10 and 40 rain) and Step 3 (80 rain). (See text.) The extracts were adjusted to the same protein concentration and 50 p.g of protein were used in each spot.
Transformation of Schistosoma mansoni cercariae to schistosomules normal guinea-pig serum and gave negative CHR. These larvae have been maintained in culture for up to 40 days (Tiba et al., 1974) and may be considered schistosomules according to the criteria of Stirewalt et al. (1966). Determinations of the rate of secretion of the preacetabular gland content, as measured by the decrease in the residual proteolytic activity of cercariae during incubation at Step 2 of the transformation process are in close agreement with direct observations, presented in Table 1, made on alizarinstained larval preparations. The selective affinity of the pre-acetabular gland for alizarin has already been conclusively demonstrated by Stirewalt & Kruidenier (1961). The depletion of the PAS-positive content of the post-acetabular glands of the transforming cercariae during Step 2 incubation, also presented in Table 1, Table 1. Glandular secretion of packed cercarial bodies incubated at 30°C in Hanks'-BSS Time of incubation (min)
Protease*
Alizarint
PASt
0 10 40 80:~
100 108 64 48
100 95 62 36
100 90 42 38
Residual glandular material (%)
* Specific activity of larval extracts on acetyl-tyrosineethyl ester. ,Cercariae showing no significant reduction in glandular contents. :1:The last 40 min in MEM at 37°C, under agitation. confirms the drastic reduction in carbohydrate content that occurs during the cerearial transformation, as observed by Gazzinelli et al. (1973) in comparative electrophoresis of cercariae and schistosomules. Following the addition of 1~o DOC to the cercarial extracts and to the electrophoresis buffer three welldefined PAS-stained bands were resolved, and not two as obtained by Gazzinelli e t aL (1973). These three bands have been identified as post-acetabular gland content, surface coat and glycogen, on the basis o f the evidence presented in Fig. 1A. The anodal band, band P, is considered to be derived from the post-acetabular glands since it is absent from preparations of tails. The cathodal band distal to the origin, band G, is cercarial glycogen since it is absent from cercarial preparations which have been treated with salivary amylase and it possesses the same electrophoretic mobility as a control glycogen preparation. F r o m the results obtained by concomitant running of whole cercarial extract, tail extract and coat preparations we consider that band C, the cathodal band proximal to the origin, is
419
material derived from the surface coat of the cercaria. Additional evidence for this identification was obtained from the observation that band C becomes reinforced when a mixture of body extract and the material obtained from the supernatant of Step 3 of the preparation procedure is electrophoresed. When Step 2 of the procedure is omitted and tail-less cercariae with replete glands are incubated as in Step 3, more than 90 per cent of the resulting larvae are water sensitive as determined by the methylene blue exclusion test. Electrophoretic analysis of these larvae, however, displays a band C less intense than that of the body prior to stage 3 incubation but stronger staining than would be expected if 90 per cent of the coat material had been removed. This may indicate that band C is not derived solely from coat material, but direct microscopical observation has demonstrated that complete removal of the coat material is not necessary for the appearance of water sensitivity (Stirewalt, 1963, confirmed by RamalhoPinto et al., 1974). Once the PAS-stained electrophoretic bands had been identified, it was possible to follow the main morphological events of the cercarial metamorphosis by electrophoretic analysis of extracts of larvae obtained at different stages of the transformation process. The results of such an analysis are shown in Fig. 1B, where it can be seen that most of the postacetabular material is voided during the first 10 rain. This observation contrasts markedly with the results obtained by examination of PAS-stained larvae and emphasizes the difficulty of attempting quantitauon by crude histochemieal observations. In this instance it is probable that a very large portion of the postacetabular gland content has been eliminated with no marked reduction in the visible content of the cells. Figure 1B also demonstrates that a significant reduction in band C is only apparent after 40 min incubation and that by 80 rain, during Step 3 of the induction process, there is very little of this material remaining in the larval preparations. The reduction in the intensity of band G, the cercarial glycogen, during the incubation period, indicates the utilization of this material by the larvae during their transformation. A further measure of the degree of change undergone by the cercaria is afforded by determinations of the non-dialysable carbohydrate and protein content of the body extracts. During the period of the transformation process in which glandular secretion occurs (Step 2), the ratio of carbohydrate to protein in the organisms decreases to about 50 per cent of its original value. In these experiments, depicted in Table 2, cerearial bodies were incubated at 30°C as described in Step 2. The various methods employed demonstrated that the glandular contents were eliminated from the larvae during Step 2 of the in vitro incubation procedure used. At the end of this phase the majority of the larvae were still insensitive to water and
420
C. C. DE OUVEmA, E. A. ~GtmmEDO, G. GAZZINELLI,R. E. HOWELLSAND J. PELLEGRINO Table 2. Carbohydrate--protein ratio of transforming S. mansoni larvae incubated as in Step 2* 100 x Carbohydrate/protein
(w/w) Experiment 1
2 3 4 5
Time of incubation (min)
Incubated
Non-incubated (controls)t
24 24 30 40 40
8.19 7.10 7.58 8.30 10"3
13.6 12.7 11.8 14.7 18"2
* Assays performed on dialysed extracts prepared according to Materials and Methods. The values for each experimental pair--incubated and controls--are the result of assays on dialysed extracts of 100,000-400,000 organisms obtained from cercaria¢ collected 3-5 hr after emergence from the B. glabrata snails. I" Controls: non-incubated cercarial bodies obtained as in Step 1. sensitive to guinea-pig serum, and retained the PASpositive surface coat or glycocalyx. The changes in water and complement sensitivities o f the larvae occurred during Step 3 incubation. The concomitant loss of the glycocalyx--as determined from PAS-stained histological sections--and the loss of band C material from the electrophoresed extracts is evidence that band C material is derived from the glycocalyx. The isolation of this material from the Step 3 incubation medium also demonstrates that the surface coat is indeed lost during cercarial transformation. These results are contrary to the suggestion of Curtis (1972) that the surface coat of the cercaria, as observed by electron microscope techniques, might represent an artifact of the preparation procedures employed. A further conclusion derived from these results is that the protease activity, the electrophoretic pattern of PAS-positive materials and the carbohydrateprotein ratio of the larvae can be used as indicators of the extent and nature of the metamorphic changes undergone by the cercaria in its transformation to schistosomule. Acknowledgement--We thank Mr. Jos6 de Souza Fitho for the technical assistance in obtaining the S. mansoni cercariae. REFERENCES
BLOCK R. J., DURRUME. L. & ZWEIO G. (1955) Paper Chromatography and Electrophoresis. Academic Press, New York. CLEGGJ. A. & SM1THERSS. R. (1968) Death of schistosome cercariae during penetration of the skin II. Penetration of mammalian skin by Schistosoma mansoni. Parasit. 58, 111-128. COLES G. C. (1973)The metabolism of schistosomes: a review. Int. J. Biochem. 4, 319-337. CORm A. S. G. (1972) Adhesive interactions between organisms. In Functional aspects of Parasite Surfaces (Edited by TAYLORA. E. R. and MUELLERR.), pp. 1-21. Blackwell Scientific, London.
GAZZINELLI G., DE OLIVEIRAC. C., FIGUEIREDOE. A., PEREIRAL. H., COELHOP. M. Z. & PELLEGRINOJ.(1973) Schistosoma mansoni: biochemical evidence for morphogenetic change from cercaria to schistosomule. Expl. Parasit. 34, 181-188. GAZZINELLI G., RAMALHO-PINTOF. J. & PELLEGRINOJ. (1966) Purification and characterization of the proteolytic enzyme complex of cercarial extract. Comp. Biochem. Physiol. 18, 689-700. PEARSE A. G. E. (1968) Histochemistry, Theoretical and Applied, 3rd edn., Vol. 1. Churchill, London. RAMALHO-PINTOF. J., GAZZINELLIG., HOWELLSR. E., MOTA-SANTOST. A., FIGUEIREDOE. A. & PELLEGRINO J. (1974) Schistosoma mansoni: a defined system for step-wise transformation of cercaria to schistosomule. Expl. Parasit. 36, 360-372. STIREWALT M. A. (1963) Cercariae vs Schistosomule (Schistosoma mansoni): absence of the pericercarial envelope in vivo and the early physiological and histological metamorphosis of the parasite. Expl. Parasit. 13, 395-406. STIREWALTM. A. & KRUIDENIERF. J. (1961) Activity of the acetabular secretory apparatus of cercariae of Schistosoma mansoni under experimental conditions. Expl. Parasit. 11, 191-211. STIREWALT M. A., MINNICK D. R. & FREGEAUW. A. (1966) Definition and collection in quantity of schistosomules of Schistosoma mansoni. Trans. R. Soc. trop. Med. Hyg. 60, 352-360. TIBA Y., HOLANDA J. C., RAMALHO-Pr~ro F. J., GAZZINELLIG. & PELLEGRINOJ. (1974) Schistosomula (Schistosoma mansoni) obtained in vitro. Viability in culture and infectivity for mice. Trans. R. Soc. trop. Med. Hyg. 68, 72. VOGEL M. & MINNING W. (1949) Hiillenbildung bei Bilharzia-Cercarien in Serum Bilharzia-infizierter Tiere und Menschen. Zenbl. Bakt. Paras#. Kde. 153, 91-105. Key Word lndex--Schistosoma mansoni; cercaria; electrophoresis; enzymes; polysaccharides; acetabular glands; cercarial coat; cercarial glycogen; histochemistry; alizarin; PAS; surface changes; Cercarienhiillen-Reaktion (CHR).
BIOCHEMICAL CHANGES IN THE TRANSFORMATION OF SCHISTOSOMA MANSONI CERCARIAE TO SCHISTOSOMULES* C. C. DE OLIVEIRA,lt E. A. FIaUEmEDO,1 G. GAZZINELLI,t R. E. HOWELLS2~ AND J. PELLEGRINOa 1 Departamento de Bioquimica-Imunologia;u Departamento de Zoologia-Parasitologia; and 8 Grupo Interdepartamental de Estudos sobre Esquistossomose, Instituto de Ci6ncias Biol6gicas, C.P. 2486, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (Received 29 M a y 1974)
Abstract--1. In the transformation of Schistosoma mansoni cercariae to schistosomules there was an elimination of the ATEE-hydrolysing proteases to about 50 per cent of the original specific activity level, in 40-60 rain. 2. Histochemical examination of the pre- and postacetabular glands of cercariae stained by alizarin and PAS disclosed a similar loss of material as the larvae became schistosomules. 3. The ratio of carbohydrate/protein decreased to about 50 per cent of its original value during the transformation. 4. Extracts of cercariae, tails and bodies submitted to electrophoresis in deoxycholate-containing borate buffer, pH 9.5, disclosed three PAS-stained bands which were identified as post-acetabular gland content, surface coat and glycogen. 5. The loss of carbohydrate material from transforming cercariae was accompanied by changes in intensity of the PAS-stained bands obtained by electrophoresis of larval extracts. 6. The glycoprotein material of the cercarial glycocalyx was recovered from the incubation medium following transformation of the larvae to schistosomules.
INTRODUCTION DURING the penetration of mammalian skin the cercariae of Schistosoma mansoni undergo metamorphic changes to the schistosomules. These changes include extrusion of the contents of the acetabular glands, decaudation and the loss of a glycoprotein coat or glycocalyx from the cercarial body surface. Schistosomules additionally differ from cercariae in being sensitive to water and tolerant to normal guinea pig serum. Using an in vitro technique which induces in a stepwise manner the same sequence of changes that are observed in vivo (Ramalho-Pinto et al., 1974), we have investigated some of the biochemical events that occur during cercarial transformation. Coles * This work received support from Conselho Nacional de Pesquisas, Conselho de Pesquisas da UFMG, Banco Nacional para o Desenvolvimento Econ6mico (FUNTEC, 66), World Health Organization and the British Technical Co-operation Program. Some of the results given in this paper have been presented in the Meeting of the Sociedade Brasileira para o Progresso da CiSncia, held in Rio de Janeiro, Brazil, in July 1973. Contribution No. 53 from the Schistosomiasis Research Unit. 1"Fellow of the CAPES. Visiting Professor from the Liverpool School of Tropical Medicine, Liverpool, England.
(1973) observed that 32 per cent of the total nitrogen and 22 per cent of the cercarial body glycogen are lost during transformation, these data agreeing closely with those of Gazzinelli et al. (1973) on the loss of protein and total carbohydrate from the transforming cercariae. Gazzinelli et al. (1973) followed the elimination of proteases and reduction in carbohydrate content during this process and determined electrophoretically that two polysaccharide bands obtained from cercarial extracts were much reduced in schistosomule preparations. In this study we have further analysed the nature and sequence of carbohydrate loss from the transforming larvae. Of particular interest was the surface coat of the cercariae and evidence is presented to show that this material is lost from the cercaria and may be isolated from the incubation medium. The extent of glandular secretion has been determined by biochemical and histochemical methods and the results have been compared. MATERIALS AND METHODS Cercariae of S. mansoni (LE strain, B¢lo Horizonte) were obtained from laboratory reared Biomphalaria glabrata and concentrated to 7(g)--1000/mlas described by Gazzinelli et al. (1973). Ccrcariae were used 3-5 hr after emergence from the snails. Schistosomules and intermediate transformation stages, together with materials
417
418
C . C . DE OLIVEIRA, E. A. FIGUEIREDO, G. GAZZINELLI, R. E. HOWELLSAND J. PELLEGRINO
secreted or liberated from the larvae, were obtained by the technique of Ramalho-Pinto et al. (1974) as follows.
Cercarial bodies and tails (Step 1) Ten-ml aliquots of the cercarial suspension were cooled in 15 ml glass conical centrifuge tubes for 10 min and centrifuged at low speed for 15 sec. Cercariae from five to six tubes were pooled by resuspending in a final volume of 2 ml of cold Hanks'-BSS pH 7.2-7.4. The cercarial tails were removed by stirring in a Vortex Jr. mixer (Scientific Industries, Inc., Queens Village, New York) for 1 rain. The volume was increased to 7 ml with the same medium and after standing for 8-10 min the supernatant containing the tails was removed from the gravity sedimented cercarial bodies. The bodies were resuspended in 7 ml of Hanks' and the procedure repeated twice. The resulting sediment of cercarial bodies, containing less than 5 per cent of tails, was suspended in 2 ml of Hanks' -BSS.
Cercarial secretion (Step 2) The suspended cercarial bodies were packed by 1 min speed centrifugation and incubated at 30°C for 40 min unless otherwise stated. The supernatant was decanted and the pellet washed three times in cold Hanks'-BSS by resuspension and centrifugation. The supernatant and washing solutions were pooled.
S. mansoni infection and one drop of a suspension containing twenty to thirty larvae and incubating this preparation from 30 min to 12 hr at room temperature in a water vapour saturated atmosphere. Only larvae which completely lacked adventitious membranes (envelopes) were considered to be CHR negative. Protein, carbohydrate and esterolytic (proteolytic) activity These determinations were performed by the Lowry, anthrone and Hestrin methods as described by Gazzinelli et al. (1966).
Organism preparations Cercariae, cercarial bodies or tails, schistosomules or incompletely transformed organisms were sonicated as described by Gazzinelli et aL (1973). The suspending medium to prepare extracts for electrophoretic analysis was 1% deoxycholate (DOC) in 0.1 M borate buffer pH 9.5. For other determinations larvae were sonicated in deionized water.
Electrophoresis Electrophoresis was carried out in Cellogel strips (Chemetron, Milan) for 120 rain at 200 V using the same buffer as in the extracts. The strips were then PAS-stained as shown below and made transparent as described by Gazzinelli et al. (1973).
Cercarial coat (Step 3)
Polysaccharide and glycoprotein dyeing procedures
The larvae obtained in Step 2 were suspended in 5-10 ml of Minimal Eagle Medium (TC-MEM), transferred to 30 ml stoppered vials and incubated under continuous shaking at 37°C for 60 rain. The suspension was centrifuged and the coat was recovered from the supernatant by dialysis and lyophilization.
The cellogel strips were stained by a modification of the technique of Block et al. (1955): the strips were (1) fixed in ethanol for 10 min; (2) immersed in a 1% w/v aqueous solution of periodic acid (Merck) for 10 min; (3) rimed three to four times in distilled water; (4) immersed in Schiff's solution prepared according to de Tomazi as described by Pearse (1968) for 25--40 min; and (5) washed three to four times in sulfite water (sodium metabisulfiteconcentrated HC1--distilled water, 2 : 5 : 500).
Schistosomules The larvae collected in Step 3 are from now on called schistosomules.
Water sensitivity The organisms were exposed to deionized or distilled water for 10 min. Organisms which were motile after this time were considered water insensitive. A n alternative technique used was the methylene blue exclusion test of Clegg & Smithers (1968) in which the organisms were exposed to 0'03% methylene blue in deionized water for 10 rain and the water sensitivity of larvae calculated from the percentage which were blue stained. Controls were run in Hanks'.
Guinea-pig serum sensitivity This test for guinea-pig serum sensitivity was performed according to Ramalho-Pinto et aL (1974).
Glycogen standard Glycogen (Nutritional Biochemical Corporation) was dissolved in 1% DOC---containing 0.1 borate buffer pH 9-5--to a final concentration of 25 mg/ml.
Amylase treatment of cercarial preparations Saliva was diluted I : 5 with sterile saline and incubated with 10 vol. of cercarial preparation at 37°C for 30 min. Controls were run with heat denatured diluted saliva.
Staining of the larvae PAS and alizarin staining of intact organisms were performed as described by Gazzinelli et al. (1973). RESULTS AND DISCUSSION
Cercarienhiillen-Reaktion
( CHR) (Vogel & Minning, 1949)
The C H R technique was performed by placing on a glass slide one drop of serum from a patient with confirmed
U n l i k e cercariae, the larvae o b t a i n e d by c a r r y i n g o u t Steps 1, 2 a n d 3 outlined in Materials a n d M e t h o d s , were sensitive to water, insensitive to
INCUBATION
o
b
c
d
e
f
e÷f
0
I0
40
(÷;
(MIN)
80 (+)
P
P o c
0 C G
G
(-)
(-)
A
B
Fig. 1. PAS-stained bands in electrophoresis of S. mansoni extracts. Electrophoresis performed in cellogel strips with 0'1 M borate buffer, pH 9'5, containing 1 ~ sodium deoxycholate, for 120 rain at 200 V. O, Origin; P, post-acetabular gland material ; C, coat ; G, glycogen. A. Identification of the bands--tails (d), bodies (e) and coat (f) prepared as in the text; cercariae tails and bodies were washed with Hanks' solution and the extracts prepared in the cold, using the buffer described above. Part of the cercarial extract was used as such (a) and part was treated by amylase (b) prior to electrophoresis. Glycogen (c) was included for comparison. B. Electrophoresis of extracts of transforming S. mansoni cercarial bodies obtained as in Step 1 (zero time), incubated as in Step 2 (10 and 40 rain) and Step 3 (80 rain). (See text.) The extracts were adjusted to the same protein concentration and 50 p.g of protein were used in each spot.
Transformation of Schistosoma mansoni cercariae to schistosomules normal guinea-pig serum and gave negative CHR. These larvae have been maintained in culture for up to 40 days (Tiba et al., 1974) and may be considered schistosomules according to the criteria of Stirewalt et al. (1966). Determinations of the rate of secretion of the preacetabular gland content, as measured by the decrease in the residual proteolytic activity of cercariae during incubation at Step 2 of the transformation process are in close agreement with direct observations, presented in Table 1, made on alizarinstained larval preparations. The selective affinity of the pre-acetabular gland for alizarin has already been conclusively demonstrated by Stirewalt & Kruidenier (1961). The depletion of the PAS-positive content of the post-acetabular glands of the transforming cercariae during Step 2 incubation, also presented in Table 1, Table 1. Glandular secretion of packed cercarial bodies incubated at 30°C in Hanks'-BSS Time of incubation (min)
Protease*
Alizarint
PASt
0 10 40 80:~
100 108 64 48
100 95 62 36
100 90 42 38
Residual glandular material (%)
* Specific activity of larval extracts on acetyl-tyrosineethyl ester. ,Cercariae showing no significant reduction in glandular contents. :1:The last 40 min in MEM at 37°C, under agitation. confirms the drastic reduction in carbohydrate content that occurs during the cerearial transformation, as observed by Gazzinelli et al. (1973) in comparative electrophoresis of cercariae and schistosomules. Following the addition of 1~o DOC to the cercarial extracts and to the electrophoresis buffer three welldefined PAS-stained bands were resolved, and not two as obtained by Gazzinelli e t aL (1973). These three bands have been identified as post-acetabular gland content, surface coat and glycogen, on the basis o f the evidence presented in Fig. 1A. The anodal band, band P, is considered to be derived from the post-acetabular glands since it is absent from preparations of tails. The cathodal band distal to the origin, band G, is cercarial glycogen since it is absent from cercarial preparations which have been treated with salivary amylase and it possesses the same electrophoretic mobility as a control glycogen preparation. F r o m the results obtained by concomitant running of whole cercarial extract, tail extract and coat preparations we consider that band C, the cathodal band proximal to the origin, is
419
material derived from the surface coat of the cercaria. Additional evidence for this identification was obtained from the observation that band C becomes reinforced when a mixture of body extract and the material obtained from the supernatant of Step 3 of the preparation procedure is electrophoresed. When Step 2 of the procedure is omitted and tail-less cercariae with replete glands are incubated as in Step 3, more than 90 per cent of the resulting larvae are water sensitive as determined by the methylene blue exclusion test. Electrophoretic analysis of these larvae, however, displays a band C less intense than that of the body prior to stage 3 incubation but stronger staining than would be expected if 90 per cent of the coat material had been removed. This may indicate that band C is not derived solely from coat material, but direct microscopical observation has demonstrated that complete removal of the coat material is not necessary for the appearance of water sensitivity (Stirewalt, 1963, confirmed by RamalhoPinto et al., 1974). Once the PAS-stained electrophoretic bands had been identified, it was possible to follow the main morphological events of the cercarial metamorphosis by electrophoretic analysis of extracts of larvae obtained at different stages of the transformation process. The results of such an analysis are shown in Fig. 1B, where it can be seen that most of the postacetabular material is voided during the first 10 rain. This observation contrasts markedly with the results obtained by examination of PAS-stained larvae and emphasizes the difficulty of attempting quantitauon by crude histochemieal observations. In this instance it is probable that a very large portion of the postacetabular gland content has been eliminated with no marked reduction in the visible content of the cells. Figure 1B also demonstrates that a significant reduction in band C is only apparent after 40 min incubation and that by 80 rain, during Step 3 of the induction process, there is very little of this material remaining in the larval preparations. The reduction in the intensity of band G, the cercarial glycogen, during the incubation period, indicates the utilization of this material by the larvae during their transformation. A further measure of the degree of change undergone by the cercaria is afforded by determinations of the non-dialysable carbohydrate and protein content of the body extracts. During the period of the transformation process in which glandular secretion occurs (Step 2), the ratio of carbohydrate to protein in the organisms decreases to about 50 per cent of its original value. In these experiments, depicted in Table 2, cerearial bodies were incubated at 30°C as described in Step 2. The various methods employed demonstrated that the glandular contents were eliminated from the larvae during Step 2 of the in vitro incubation procedure used. At the end of this phase the majority of the larvae were still insensitive to water and
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C. C. DE OUVEmA, E. A. ~GtmmEDO, G. GAZZINELLI,R. E. HOWELLSAND J. PELLEGRINO Table 2. Carbohydrate--protein ratio of transforming S. mansoni larvae incubated as in Step 2* 100 x Carbohydrate/protein
(w/w) Experiment 1
2 3 4 5
Time of incubation (min)
Incubated
Non-incubated (controls)t
24 24 30 40 40
8.19 7.10 7.58 8.30 10"3
13.6 12.7 11.8 14.7 18"2
* Assays performed on dialysed extracts prepared according to Materials and Methods. The values for each experimental pair--incubated and controls--are the result of assays on dialysed extracts of 100,000-400,000 organisms obtained from cercaria¢ collected 3-5 hr after emergence from the B. glabrata snails. I" Controls: non-incubated cercarial bodies obtained as in Step 1. sensitive to guinea-pig serum, and retained the PASpositive surface coat or glycocalyx. The changes in water and complement sensitivities o f the larvae occurred during Step 3 incubation. The concomitant loss of the glycocalyx--as determined from PAS-stained histological sections--and the loss of band C material from the electrophoresed extracts is evidence that band C material is derived from the glycocalyx. The isolation of this material from the Step 3 incubation medium also demonstrates that the surface coat is indeed lost during cercarial transformation. These results are contrary to the suggestion of Curtis (1972) that the surface coat of the cercaria, as observed by electron microscope techniques, might represent an artifact of the preparation procedures employed. A further conclusion derived from these results is that the protease activity, the electrophoretic pattern of PAS-positive materials and the carbohydrateprotein ratio of the larvae can be used as indicators of the extent and nature of the metamorphic changes undergone by the cercaria in its transformation to schistosomule. Acknowledgement--We thank Mr. Jos6 de Souza Fitho for the technical assistance in obtaining the S. mansoni cercariae. REFERENCES
BLOCK R. J., DURRUME. L. & ZWEIO G. (1955) Paper Chromatography and Electrophoresis. Academic Press, New York. CLEGGJ. A. & SM1THERSS. R. (1968) Death of schistosome cercariae during penetration of the skin II. Penetration of mammalian skin by Schistosoma mansoni. Parasit. 58, 111-128. COLES G. C. (1973)The metabolism of schistosomes: a review. Int. J. Biochem. 4, 319-337. CORm A. S. G. (1972) Adhesive interactions between organisms. In Functional aspects of Parasite Surfaces (Edited by TAYLORA. E. R. and MUELLERR.), pp. 1-21. Blackwell Scientific, London.
GAZZINELLI G., DE OLIVEIRAC. C., FIGUEIREDOE. A., PEREIRAL. H., COELHOP. M. Z. & PELLEGRINOJ.(1973) Schistosoma mansoni: biochemical evidence for morphogenetic change from cercaria to schistosomule. Expl. Parasit. 34, 181-188. GAZZINELLI G., RAMALHO-PINTOF. J. & PELLEGRINOJ. (1966) Purification and characterization of the proteolytic enzyme complex of cercarial extract. Comp. Biochem. Physiol. 18, 689-700. PEARSE A. G. E. (1968) Histochemistry, Theoretical and Applied, 3rd edn., Vol. 1. Churchill, London. RAMALHO-PINTOF. J., GAZZINELLIG., HOWELLSR. E., MOTA-SANTOST. A., FIGUEIREDOE. A. & PELLEGRINO J. (1974) Schistosoma mansoni: a defined system for step-wise transformation of cercaria to schistosomule. Expl. Parasit. 36, 360-372. STIREWALT M. A. (1963) Cercariae vs Schistosomule (Schistosoma mansoni): absence of the pericercarial envelope in vivo and the early physiological and histological metamorphosis of the parasite. Expl. Parasit. 13, 395-406. STIREWALTM. A. & KRUIDENIERF. J. (1961) Activity of the acetabular secretory apparatus of cercariae of Schistosoma mansoni under experimental conditions. Expl. Parasit. 11, 191-211. STIREWALT M. A., MINNICK D. R. & FREGEAUW. A. (1966) Definition and collection in quantity of schistosomules of Schistosoma mansoni. Trans. R. Soc. trop. Med. Hyg. 60, 352-360. TIBA Y., HOLANDA J. C., RAMALHO-Pr~ro F. J., GAZZINELLIG. & PELLEGRINOJ. (1974) Schistosomula (Schistosoma mansoni) obtained in vitro. Viability in culture and infectivity for mice. Trans. R. Soc. trop. Med. Hyg. 68, 72. VOGEL M. & MINNING W. (1949) Hiillenbildung bei Bilharzia-Cercarien in Serum Bilharzia-infizierter Tiere und Menschen. Zenbl. Bakt. Paras#. Kde. 153, 91-105. Key Word lndex--Schistosoma mansoni; cercaria; electrophoresis; enzymes; polysaccharides; acetabular glands; cercarial coat; cercarial glycogen; histochemistry; alizarin; PAS; surface changes; Cercarienhiillen-Reaktion (CHR).