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Isoenzyme patterns of phosphateses and esterases in Fasciola hepatica and Dicrocoelium dendriticum
Veterinary Parasitology, 30 (1989) 297-304 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
297
I s o e n z y m e P a t t e r n s of P h o s p h a t a s e s and Esterases in Fasciola hepatica and Dicrocoelium
dendriticum P. LEON, M. MONTEOLIVA and M. SANCHEZ-MORENO* Secci6n de Bioquimica del Instituto "Lopez-Neyra" de Parasitologia, C.S.I.C., C/Ventanilla 11, 18001 Granada (Spain)
(Accepted for publication 9 February 1988)
ABSTRACT Leon, P., Monteoliva, M. and Sanchez-Moreno, M., 1989. Isoenzyme patterns of phosphatases and esterases in Fasciola hepatica and Dicrocoelium dendriticum. Vet. Parasitol., 30: 297-304. A study was made of alkaline and acid phosphatase isoenzymes and non-specific esterases in homogenates of the trematodes Fasciola hepatica and Dicrocoelium dendriticum obtained from infected tissues of Capra hircus and Ovis aries using horizontal polyacrylamide gel electrophoresis. The esterase patterns in F. hepatica and D. dendriticum were different. Homogenate of F. hepatica from both hosts gave four enzyme bands. For D. dendriticum, however, while homogenates of parasites from C. hircus also gave four bands, those of O. aries gave only three bands. Acid and alkaline phosphatases in homogenates of both parasites showed three enzyme bands, but there was a host species difference between the enzyme patterns of specimens collected from C. hircus versus O. aries.
INTRODUCTION M u l t i p l e m o l e c u l a r f o r m s of e n z y m e s ( i s o e n z y m e s ) are recognized as a general p h e n o m e n o n of great biological i m p o r t a n c e . It has b e e n r e p o r t e d t h a t the m o s t i m p o r t a n t biological q u e s t i o n p o s e d b y i s o e n z y m e s is simply: w h y do t h e y exist? M a n y a u t h o r s have shown, b y b i o c h e m i c a l m e t h o d s , t h a t adult Fasciola hepatica c o n t a i n non-specific e s t e r a s e s ( E s t ) , acid p h o s p h a t a s e ( A c P ) a n d alkaline p h o s p h a t a s e (ALP), a n d have d e t e c t e d diverse m o l e c u l a r f o r m s ( P a n telouris, 1967; Alcaino et al., 1976; M o c z o n , 1983) using starch-gel e l e c t r o p h o r e s i s a n d isoelectric focusing p o l y a c r y l a m i d e gel techniques. Differences in t h e n u m b e r of i s o e n z y m e s a n d t h e i r e l e c t r o p h o r e t i c mobility as well *Author to whom correspondence should be addressed.
298 as quantitative differences could be very useful to characterize closely related species or strains of a certain parasite. Previous studies employing the same parasite species show differences in the isoenzyme profiles [lactate dehydrogenase for the same species from different hosts (Leon et al., 1986) ]. This work was designed to investigate acid and alkaline phosphatases and non-specific esterase isoenzymes of F. hepatica and DicrocoeIium dendriticum and to detect possible differences in the enzyme patterns of specimens from different hosts (Capra hircus and Ovis aries). MATERIALS AND METHODS Adult specimens of F. hepatica and D. dendriticum were obtained from livers of C. hircus and O. aries slaughtered in the local slaughterhouse; the time between collection and subsequent treatment in the laboratory was 2 h. The animals had been infected in naturally contaminated environments. The live specimens were washed several times with normal saline at 37°C until all traces of the host tissues were seen to be eliminated from the wash liquid after inspection against a strong light source. The final wash was in distilled water before cooling to 80 °C for preservation. Before homogenizing, they were thawed slowly to 4 ° C. Homogenization was by a Potter-Elvenjem glass teflon tissue grinder cooled in an ice bath using electrophoretic buffer diluted 1:20, with a tissue/buffer ratio of 2.1 w/v. The homogenates of the whole parasites and specimens of non-infected host liver tissue were sonicated for three periods of 30 s at l-min intervals and then centrifuged at 6000 × g for 25 min at 4°C. The protein concentration was determined in aliquot fractions of the supernatant by the method of Lowry et al. (1951). Electrophoresis was carried out in an LKB 2117 Multiphor instrument equipped with an LKB 2209 Multitemp refrigeration unit to maintain the temperature at 4 °C during development. The polyacrylamide gel slabs were prepared according to FehrnstrSm and Moberg (1977) at concentrations of 5 and 7.5%. The compositions of the electrophoresis buffers used were: Buffer A, TrisGlycine (15.02 g l-i glycine, 0.5 g l-i sodium azide, adjusted with Tris-HCl) pH 8.3, Buffer B, Tris-citric (16.35 g l-I Tris-HCl, 0.04 g l-I citric acid) pH 6.5 and Buffer C, Tris-boric (6.057 g 1-1 Tris-HCl, 1.546 g I-I boric acid) pH 8.7. The gels were subjected to a pre-run electrophoresis for 30 min at 30 mA. After pre-electrophoresis, I0 ~tl of supernatant were applied to the gels with equivalent protein contents. The front was visualized with bromophenol blue. The samples were started for I0 min with a current of 15-20 mA and the field strength was increased in steps (see Table i). Esterases were visualized by using a modified version of the method of Shaw and Prasad (1970). The stain contained 8 mg of ~-naphthyl acetate and 8 mg
299
of ~-naphthyl acetate in 2 ml of acetone and 2 ml of distilled water, 25 ml of 0.1 M phosphate buffer pH 6.5 and 30 mg of Fast Blue Salt BB. The gels were incubated at room temperature. Alkaline phosphatase determinations were made using the modified method of Fletcher et al. (1981). The stain contained 30 mg of naphthyl phosphate, 500 mg of NaC1, 100 mg of polyvinyl-pyrrolidone, 1 mg of MgC12, 1 mg of MnC12 and 25 ml of 0.1 M Tris HC1 buffer pH 8.7. Just before use, 30 mg of Fast Blue Salt BB were added. AcP was measured using the techniques of Michelson and Dubois (1981). Both AcP and AlP were visualized by previous incubation of the gels at 37 ° C in the dark. The quantitative estimation of color in different parts of the stained gel was effected with a Cliniscan TM Helena densitometer at a wavelength of 645 nm. The relative concentrations of various isoenzymes were determined by expressing the peak areas as percentages of the total area. RESULTS
Table 1 shows the optimum electrophoretic conditions for each parasite species. F. hepatica homogenates all showed high esterase activities with a rapidly mobile band which was seen in the first few minutes of stain. Four isoenzymes were detected in homogenates of parasite specimens isolated from two hosts, C. hircus and O. aries; they all showed different enzyme patterns (Fig. 1 ) and these were confirmed by densitometric scanning. Specimens from C. hircus had the following percentage distribution: 6.51, 15.80, 19.24 and 58.45. For organisms from O. aries, the corresponding percentages were 4.12, 11.16, 13.42 and 71.30. After horizontal scanning of the A band, the percentages were ~ 33% for the first and ~ 67% for the second. TABLE1 Optimum conditions for electrophoresis developing for isoenzymes of esterase and phosphatase in F.
hepatica and D. dendriticum Enzyme
F. hepatica AlP
Protein concentrations (mg)
30-40
Development buffer
C
Gel concentration (%) Running conditions
7.5 35 mA 90 min
D. dendriticum AcP 20-40
Est. 16-33
AlP
AcP
Est.
6-12
3-6
4-9
B
A
B
B
A
5.0
5.0
5.0
5.0
5.0
35 mA 195 min
45 mA 150 min
60 mA 270 min
60 mA 270 min
45 mA 150 min
300
¢
A
a ) A .................
b)
B' :i~i!!iiiii[iiii[i!!iiiii
C D
Fig. 1. Esterase isoenzymes of F. hepatica by electrophoresis on polyacrylamide gel: (a) from C. hircus; (b) from O. aries. Fig. 2. Esterase isoenzymes of D. dendriticum by electrophoresis on polyacrylamide gel: (a) from C. hircus; (b) from O. aries.
A
B
a)+
b)
A
A
+A c
II
B
!
C
Fig. 3. (A) AlP isoenzymes ofF. hepatica: (a) from C. hircus; (b) from O. aries. (B) AcP isoenzymes ofF. hepatica: (a) from C. hircus; (b) from O. aries.
301
i!iii~ ~ i~
A
B
!ili!i!ii:ii: .....
iiiiii~
a)
~
~
~i~ili ~.....
~
iiiiiiiiiii!iiiiiiili~
ii?~i~
i
~i~!!II
b) ill! i!i!i ~i~ ~i~ii.......
A
~iii~ ~
i,~
B C ~i!!!
Fig. 4. (A) AlP isoenzymes olD. dendriticum: (a) from C. hircus; (b) from O. aries. (B) AcP isoenzymes ofD. dendriticum: (a) from C. hircus; (b) from O. aries.
i i~!~
~ !ii:iii
il
iiiili~ :ii~iiii!!ili ¸
Fig. 5. AlP isoenzymes of non-infected host tissues: (a) from C. hircus; (b) from O. aries. Fig. 6. AcP isoenzymes of non-infected host tissues: (a) from C. hircus; (b) from O. aries.
302
a)
~+
I'1 " ' ' " 1 ~
,..-,',,C b) rffr'm"~D ~ A ~_
i//
V////f.//H/A F
w / / / / / / / H ~ E)
.'lB
r~HHHH/~ E
....
[
....
F
Fig. 7. Esteraseisoenzymesof non-infectedhost tissues: (a) C. hircus; (b) O. aries. D. dendriticum also showed esterase activity; homogenates of specimens from C. hircus showed four different isoenzyme bands, those from O. aries only three bands (Fig. 2). Densitometric scanning gave the following percentage distributions: 28.35, 14.26, 27.43 and 30.06, and 42.73, 37.83 and 20.45, respectively. The enzyme patterns of both phosphatases are given in Figs. 3 and 4. F. hepatica homogenates gave 3 isoenzyme bands for both phosphatases. The densitometric scans of homogenates of specimens from both hosts showed the following percentage distributions: 43.27, 39.71 and 17.02 for AlP, and 39.78, 12.46 and 47.75 for AcP. D. dendriticum homogenate also showed three isoenzyme bands for the phosphatases. The enzyme profiles of specimens collected from both C. hircus and O. aries were very similar. Densitometric scans gave the following percentages: 36.93, 32.64 and 28.43 for AlP, and 22.60, 47.51 and 28.89 for AcP. Isoenzymatic bands from non-infected tissues had different mobilities from those obtained in the parasites (Figs. 5, 6 and 7).
DISCUSSION D. dendriticum homogenates exhibited more esterase and phosphatase activity than those of F. hepatica. This is revealed by the protein concentrations required to visualize the enzyme activities (Table 1 ). The esterase and phosphatase enzyme profiles obtained in the present study for F. hepatica and D. dendriticum are different from those published by other
303
authors. Thus, Mikulikova (1979), using polyacrylamide disc-gel electrophoresis, reports three esterase activity areas in both trematodes. Investigating phosphatase activities, Mikulikova (1979) reports a single AlP enzyme band. However, Pantelouris (1967), studying F. hepatica using starchgel electrophoresis, reports two AlP enzyme bands and one AcP band. In the present study, we have detected three different AcP and three AlP bands in homogenates of both parasites, using horizontal polyacrylamide gel electrophoresis. Densitometric scanning revealed clear host species differences in the esterase patterns of both F. hepatica and D. dendriticum. We can confirm that the A band ofF. hepatica homogenates from O. aries has a higher percentage value (71.30) than that of F. hepatica homogenates from C. hircus (58.45). The B band values, on the contrary, showed the higher figure in homogenates of specimens from C. hircus and the lower figure in those from O. aries. D. dendriticum esterase patterns differ with the host species; homogenates of specimens obtained from C. hircus had one more band than those of specimens from O. aries. These differences were confirmed by the densitometric scans. Finally, we should like to emphasize that parallel runs have confirmed that the electrophoretic profiles of hosts and parasites are clearly different. ACKNOWLEDGEMENTS
This study was supported by a grant from the CICYT (Spain). We thank D.W. Schofield for editing and translating the manuscript.
REFERENCES Alcaino, H.A., Baker, N.F. and Fish, R.A., 1976. Enzyme polymorphism in Fasciola hepatica L.: Esterases. Am. J. Vet. Res., 37: 1153-1157. Fehrnstriim, H. and Moberg, U., 1977. SDS and conventional polyacrylamide gel electrophoresis with LKE 2117 Multiphor. Application Note 306. Fletcher, M., Loverde, P.T. and Richards, C.S., 1981. Schistosoma mansoni. Electrophoresis characterization of strains selected for different levels of infectivity to snails. Exp. Parasitol., 52: 362-370. Leon, P., Hermoso, R. and Monteoliva, M., 1986. Isoenzymes of lactate dehydrogenase in Dicrocoelium dendriticum and Fasciola hepatica (Trematoda). Comp. Biochem. Physiol. 87B, ( 1 ): 159-161. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.S., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Michelson, E.H. and Dubois, L., 1981. An isoenzyme marker possibly associated with the susceptibility of Biomphalaria glabrata populations to Schistosoma mansoni. Acta Trop., 38: 419426. Mikulikova, L., 1979. Identification of helminth species by means of disc electrophoresis. Folia Parasitol., 26: 115-122.
304 Moczon, T., 1983. Oxidoreductase and phosphatases in miracidia of Fasciola hepatica as revealed by histochemical methods. A. Parasitol. Polonia, 28: 267-272. Pantelouris, E.M., 1967. Esterases of Fasciola hepatica. L. Res. Vet. Sci., 8: 157-159. Shaw, C.R. and Prasad, R., 1970. Starch gel electrophoresis of enzymes-- a compilation of recipes. Biochem. Gen., 4: 291-320.
297
I s o e n z y m e P a t t e r n s of P h o s p h a t a s e s and Esterases in Fasciola hepatica and Dicrocoelium
dendriticum P. LEON, M. MONTEOLIVA and M. SANCHEZ-MORENO* Secci6n de Bioquimica del Instituto "Lopez-Neyra" de Parasitologia, C.S.I.C., C/Ventanilla 11, 18001 Granada (Spain)
(Accepted for publication 9 February 1988)
ABSTRACT Leon, P., Monteoliva, M. and Sanchez-Moreno, M., 1989. Isoenzyme patterns of phosphatases and esterases in Fasciola hepatica and Dicrocoelium dendriticum. Vet. Parasitol., 30: 297-304. A study was made of alkaline and acid phosphatase isoenzymes and non-specific esterases in homogenates of the trematodes Fasciola hepatica and Dicrocoelium dendriticum obtained from infected tissues of Capra hircus and Ovis aries using horizontal polyacrylamide gel electrophoresis. The esterase patterns in F. hepatica and D. dendriticum were different. Homogenate of F. hepatica from both hosts gave four enzyme bands. For D. dendriticum, however, while homogenates of parasites from C. hircus also gave four bands, those of O. aries gave only three bands. Acid and alkaline phosphatases in homogenates of both parasites showed three enzyme bands, but there was a host species difference between the enzyme patterns of specimens collected from C. hircus versus O. aries.
INTRODUCTION M u l t i p l e m o l e c u l a r f o r m s of e n z y m e s ( i s o e n z y m e s ) are recognized as a general p h e n o m e n o n of great biological i m p o r t a n c e . It has b e e n r e p o r t e d t h a t the m o s t i m p o r t a n t biological q u e s t i o n p o s e d b y i s o e n z y m e s is simply: w h y do t h e y exist? M a n y a u t h o r s have shown, b y b i o c h e m i c a l m e t h o d s , t h a t adult Fasciola hepatica c o n t a i n non-specific e s t e r a s e s ( E s t ) , acid p h o s p h a t a s e ( A c P ) a n d alkaline p h o s p h a t a s e (ALP), a n d have d e t e c t e d diverse m o l e c u l a r f o r m s ( P a n telouris, 1967; Alcaino et al., 1976; M o c z o n , 1983) using starch-gel e l e c t r o p h o r e s i s a n d isoelectric focusing p o l y a c r y l a m i d e gel techniques. Differences in t h e n u m b e r of i s o e n z y m e s a n d t h e i r e l e c t r o p h o r e t i c mobility as well *Author to whom correspondence should be addressed.
298 as quantitative differences could be very useful to characterize closely related species or strains of a certain parasite. Previous studies employing the same parasite species show differences in the isoenzyme profiles [lactate dehydrogenase for the same species from different hosts (Leon et al., 1986) ]. This work was designed to investigate acid and alkaline phosphatases and non-specific esterase isoenzymes of F. hepatica and DicrocoeIium dendriticum and to detect possible differences in the enzyme patterns of specimens from different hosts (Capra hircus and Ovis aries). MATERIALS AND METHODS Adult specimens of F. hepatica and D. dendriticum were obtained from livers of C. hircus and O. aries slaughtered in the local slaughterhouse; the time between collection and subsequent treatment in the laboratory was 2 h. The animals had been infected in naturally contaminated environments. The live specimens were washed several times with normal saline at 37°C until all traces of the host tissues were seen to be eliminated from the wash liquid after inspection against a strong light source. The final wash was in distilled water before cooling to 80 °C for preservation. Before homogenizing, they were thawed slowly to 4 ° C. Homogenization was by a Potter-Elvenjem glass teflon tissue grinder cooled in an ice bath using electrophoretic buffer diluted 1:20, with a tissue/buffer ratio of 2.1 w/v. The homogenates of the whole parasites and specimens of non-infected host liver tissue were sonicated for three periods of 30 s at l-min intervals and then centrifuged at 6000 × g for 25 min at 4°C. The protein concentration was determined in aliquot fractions of the supernatant by the method of Lowry et al. (1951). Electrophoresis was carried out in an LKB 2117 Multiphor instrument equipped with an LKB 2209 Multitemp refrigeration unit to maintain the temperature at 4 °C during development. The polyacrylamide gel slabs were prepared according to FehrnstrSm and Moberg (1977) at concentrations of 5 and 7.5%. The compositions of the electrophoresis buffers used were: Buffer A, TrisGlycine (15.02 g l-i glycine, 0.5 g l-i sodium azide, adjusted with Tris-HCl) pH 8.3, Buffer B, Tris-citric (16.35 g l-I Tris-HCl, 0.04 g l-I citric acid) pH 6.5 and Buffer C, Tris-boric (6.057 g 1-1 Tris-HCl, 1.546 g I-I boric acid) pH 8.7. The gels were subjected to a pre-run electrophoresis for 30 min at 30 mA. After pre-electrophoresis, I0 ~tl of supernatant were applied to the gels with equivalent protein contents. The front was visualized with bromophenol blue. The samples were started for I0 min with a current of 15-20 mA and the field strength was increased in steps (see Table i). Esterases were visualized by using a modified version of the method of Shaw and Prasad (1970). The stain contained 8 mg of ~-naphthyl acetate and 8 mg
299
of ~-naphthyl acetate in 2 ml of acetone and 2 ml of distilled water, 25 ml of 0.1 M phosphate buffer pH 6.5 and 30 mg of Fast Blue Salt BB. The gels were incubated at room temperature. Alkaline phosphatase determinations were made using the modified method of Fletcher et al. (1981). The stain contained 30 mg of naphthyl phosphate, 500 mg of NaC1, 100 mg of polyvinyl-pyrrolidone, 1 mg of MgC12, 1 mg of MnC12 and 25 ml of 0.1 M Tris HC1 buffer pH 8.7. Just before use, 30 mg of Fast Blue Salt BB were added. AcP was measured using the techniques of Michelson and Dubois (1981). Both AcP and AlP were visualized by previous incubation of the gels at 37 ° C in the dark. The quantitative estimation of color in different parts of the stained gel was effected with a Cliniscan TM Helena densitometer at a wavelength of 645 nm. The relative concentrations of various isoenzymes were determined by expressing the peak areas as percentages of the total area. RESULTS
Table 1 shows the optimum electrophoretic conditions for each parasite species. F. hepatica homogenates all showed high esterase activities with a rapidly mobile band which was seen in the first few minutes of stain. Four isoenzymes were detected in homogenates of parasite specimens isolated from two hosts, C. hircus and O. aries; they all showed different enzyme patterns (Fig. 1 ) and these were confirmed by densitometric scanning. Specimens from C. hircus had the following percentage distribution: 6.51, 15.80, 19.24 and 58.45. For organisms from O. aries, the corresponding percentages were 4.12, 11.16, 13.42 and 71.30. After horizontal scanning of the A band, the percentages were ~ 33% for the first and ~ 67% for the second. TABLE1 Optimum conditions for electrophoresis developing for isoenzymes of esterase and phosphatase in F.
hepatica and D. dendriticum Enzyme
F. hepatica AlP
Protein concentrations (mg)
30-40
Development buffer
C
Gel concentration (%) Running conditions
7.5 35 mA 90 min
D. dendriticum AcP 20-40
Est. 16-33
AlP
AcP
Est.
6-12
3-6
4-9
B
A
B
B
A
5.0
5.0
5.0
5.0
5.0
35 mA 195 min
45 mA 150 min
60 mA 270 min
60 mA 270 min
45 mA 150 min
300
¢
A
a ) A .................
b)
B' :i~i!!iiiii[iiii[i!!iiiii
C D
Fig. 1. Esterase isoenzymes of F. hepatica by electrophoresis on polyacrylamide gel: (a) from C. hircus; (b) from O. aries. Fig. 2. Esterase isoenzymes of D. dendriticum by electrophoresis on polyacrylamide gel: (a) from C. hircus; (b) from O. aries.
A
B
a)+
b)
A
A
+A c
II
B
!
C
Fig. 3. (A) AlP isoenzymes ofF. hepatica: (a) from C. hircus; (b) from O. aries. (B) AcP isoenzymes ofF. hepatica: (a) from C. hircus; (b) from O. aries.
301
i!iii~ ~ i~
A
B
!ili!i!ii:ii: .....
iiiiii~
a)
~
~
~i~ili ~.....
~
iiiiiiiiiii!iiiiiiili~
ii?~i~
i
~i~!!II
b) ill! i!i!i ~i~ ~i~ii.......
A
~iii~ ~
i,~
B C ~i!!!
Fig. 4. (A) AlP isoenzymes olD. dendriticum: (a) from C. hircus; (b) from O. aries. (B) AcP isoenzymes ofD. dendriticum: (a) from C. hircus; (b) from O. aries.
i i~!~
~ !ii:iii
il
iiiili~ :ii~iiii!!ili ¸
Fig. 5. AlP isoenzymes of non-infected host tissues: (a) from C. hircus; (b) from O. aries. Fig. 6. AcP isoenzymes of non-infected host tissues: (a) from C. hircus; (b) from O. aries.
302
a)
~+
I'1 " ' ' " 1 ~
,..-,',,C b) rffr'm"~D ~ A ~_
i//
V////f.//H/A F
w / / / / / / / H ~ E)
.'lB
r~HHHH/~ E
....
[
....
F
Fig. 7. Esteraseisoenzymesof non-infectedhost tissues: (a) C. hircus; (b) O. aries. D. dendriticum also showed esterase activity; homogenates of specimens from C. hircus showed four different isoenzyme bands, those from O. aries only three bands (Fig. 2). Densitometric scanning gave the following percentage distributions: 28.35, 14.26, 27.43 and 30.06, and 42.73, 37.83 and 20.45, respectively. The enzyme patterns of both phosphatases are given in Figs. 3 and 4. F. hepatica homogenates gave 3 isoenzyme bands for both phosphatases. The densitometric scans of homogenates of specimens from both hosts showed the following percentage distributions: 43.27, 39.71 and 17.02 for AlP, and 39.78, 12.46 and 47.75 for AcP. D. dendriticum homogenate also showed three isoenzyme bands for the phosphatases. The enzyme profiles of specimens collected from both C. hircus and O. aries were very similar. Densitometric scans gave the following percentages: 36.93, 32.64 and 28.43 for AlP, and 22.60, 47.51 and 28.89 for AcP. Isoenzymatic bands from non-infected tissues had different mobilities from those obtained in the parasites (Figs. 5, 6 and 7).
DISCUSSION D. dendriticum homogenates exhibited more esterase and phosphatase activity than those of F. hepatica. This is revealed by the protein concentrations required to visualize the enzyme activities (Table 1 ). The esterase and phosphatase enzyme profiles obtained in the present study for F. hepatica and D. dendriticum are different from those published by other
303
authors. Thus, Mikulikova (1979), using polyacrylamide disc-gel electrophoresis, reports three esterase activity areas in both trematodes. Investigating phosphatase activities, Mikulikova (1979) reports a single AlP enzyme band. However, Pantelouris (1967), studying F. hepatica using starchgel electrophoresis, reports two AlP enzyme bands and one AcP band. In the present study, we have detected three different AcP and three AlP bands in homogenates of both parasites, using horizontal polyacrylamide gel electrophoresis. Densitometric scanning revealed clear host species differences in the esterase patterns of both F. hepatica and D. dendriticum. We can confirm that the A band ofF. hepatica homogenates from O. aries has a higher percentage value (71.30) than that of F. hepatica homogenates from C. hircus (58.45). The B band values, on the contrary, showed the higher figure in homogenates of specimens from C. hircus and the lower figure in those from O. aries. D. dendriticum esterase patterns differ with the host species; homogenates of specimens obtained from C. hircus had one more band than those of specimens from O. aries. These differences were confirmed by the densitometric scans. Finally, we should like to emphasize that parallel runs have confirmed that the electrophoretic profiles of hosts and parasites are clearly different. ACKNOWLEDGEMENTS
This study was supported by a grant from the CICYT (Spain). We thank D.W. Schofield for editing and translating the manuscript.
REFERENCES Alcaino, H.A., Baker, N.F. and Fish, R.A., 1976. Enzyme polymorphism in Fasciola hepatica L.: Esterases. Am. J. Vet. Res., 37: 1153-1157. Fehrnstriim, H. and Moberg, U., 1977. SDS and conventional polyacrylamide gel electrophoresis with LKE 2117 Multiphor. Application Note 306. Fletcher, M., Loverde, P.T. and Richards, C.S., 1981. Schistosoma mansoni. Electrophoresis characterization of strains selected for different levels of infectivity to snails. Exp. Parasitol., 52: 362-370. Leon, P., Hermoso, R. and Monteoliva, M., 1986. Isoenzymes of lactate dehydrogenase in Dicrocoelium dendriticum and Fasciola hepatica (Trematoda). Comp. Biochem. Physiol. 87B, ( 1 ): 159-161. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.S., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Michelson, E.H. and Dubois, L., 1981. An isoenzyme marker possibly associated with the susceptibility of Biomphalaria glabrata populations to Schistosoma mansoni. Acta Trop., 38: 419426. Mikulikova, L., 1979. Identification of helminth species by means of disc electrophoresis. Folia Parasitol., 26: 115-122.
304 Moczon, T., 1983. Oxidoreductase and phosphatases in miracidia of Fasciola hepatica as revealed by histochemical methods. A. Parasitol. Polonia, 28: 267-272. Pantelouris, E.M., 1967. Esterases of Fasciola hepatica. L. Res. Vet. Sci., 8: 157-159. Shaw, C.R. and Prasad, R., 1970. Starch gel electrophoresis of enzymes-- a compilation of recipes. Biochem. Gen., 4: 291-320.