Comp. Biochem. Physiol., 1971, Vol. 38B, pp. 35 to 42. Pergamon Press. Printed in Great Britain
VARIATIONS IN MALATE DEHYDROGENASE ISOENZYMES OF SCHISTOSOMA M A N S O N I G. C. C O L E S * Department of Zoology, Makerere University Colege, P.O. Box 7062, Kampala, Uganda (Received 12 M a y 1970)
Abstract--1. Malate dehydrogenase occurs as two or four isoenzymes in East African Schistosoma mamoni. 2. The presence or absence of the two faster moving malate dehydrogenase bands varies within samples from a small community and possibly even between worms in one host. 3. Non-specific esterase varies quantitatively between isolates and is affected in female worms by mating. INTRODUCTION ALTHOUGH malate dehydrogenase of Schistosoma mansoni from Puerto Rico has two isoenzymes (Conde-del-Pino et al., 1966), Coles (1970) found either two or four isoenzymes in African S . mansoni. T h e material used in the latter study came from three distinct sources: Egypt, West Nile District of Uganda and the northern shores of Lake Victoria, and it appeared possible that these differences might represent differences in the origin or strain of the parasite. Further samples were therefore examined by electrophoresis to see what variations occur in malate dehydrogenase of S. mansoni in Eastern Africa, and at the same time variations in the esterases of female worms were examined. T h e results of this study are reported in this paper. MATERIALS AND METHODS The sources of the infections used in the present study are shown in Table 1. "Strain" is used to denote samples from widely separated areas, e.g. Entebbe, West Nile or Sudan, and "isolate" to denote samples from within an area, e.g. West Nile 1, 2, etc. The samples from soldiers (West Nile 3-8) were from people whose home is in North Western Uganda, but who had recently been stationed in barracks at Jinja (see Fig. 1). Samples West Nile 9-11 were from different people in the same small village near Pachwach. Cercariae from infected snails were used to infect Clark OSl strain white mice by partial immersion. Mice were killed by a blow on the head at 8 weeks after infection, and theworms collected by perfusion and dissection. Worms were washed thoroughly in three changes of tyrode, and separated into males and females which were homogenized separately in tyrode in microglass homogenizers. Cell debris was removed by 5 rain centrifugation at 20,000 g, higher g and refrigeration for a * Present address : The Molteno Institute, Downing Street, Cambridge, CB2 3EE. 35
longer run being unavailable. Enzymes were separated by electrophoresis in cooled 2 mm polyacrylamide gels (photo polymerized) on microscope slides (Coles, 1969a) and the enzymes detected as in Coles (1969a, b). ~-Naphthyl acetate was used as substrate for all esterase patterns. All slides were scanned on a Joyce Loebl Chromoscan, except for some of the earlier samples (Entebbe, Kampala, West Nile 1 and 2) which were separated before any scanning equipment became available. Each isolate or strain was examined from worms from at least five mice. TABLE 1--ORIGINS OF S. mansoni
Strain or isolate
West Nile 1 West Nile 2 West Nile 3 West Nile 4 West Nile 5 West Nile 6 West Nile 7 West Nile 8 West Nile 9 West Nile 10 West Nile 11 Kampala Gaba 1 Gaba 2 Entebbe 1 Entebbe 2 Mwanza Wellcome
Infected snails Hospital patient Soldier Soldier Soldier Soldier Soldier Soldier Villager Villager Villager Infected snails Fishermen (mixed) Ferry operator Fisherman Fisherman Infected snails Infected snails
Infected snails Infected snails
Place or tribe Near Arua (Lugbara tribe) Near Arua (Lugbara tribe) Lugbara tribe Acholi tribe Kakwa tribe Lugbara tribe Lugbara tribe Jonam tribe Near Pachwach (Jonam tribe) Near Pachwach (Jonam tribe) Near Pachwach (Jonam tribe) Kampala Gaba near Kampala Gaba near Kampala Entebbe Entebbe Mwanza, Tanzania Originated from Egypt over 20 years ago Egypt Khartoum, Sudan
Snail intermediate host
B. pfeifferi B. p f eifferi
B. pfeifferi B. pfeifferi
B. pfeiffe~i B. pfeifferi B. pfeifferi B. pfeifferi B. pfeifferi B. pfeifferi B. pfeifferi B. pfeifferi B. sudanica B. sudanica B. sudanica B. sudanica B . p f eif f eri B. glabratus B. alexandrina B. p f eif f eri
(a) Malate dehydrogenase isoenzymes The usual patterns for malate dehydrogenase isoenzymes are two or four fractions (Fig. 2). In a few eases the slowest moving band appeared to split in two, but as this was not consistent within any one isolate the band was considered to be only one. I n two samples band 1 was so small that on scanning it was less than 1 per cent of the total--these samples were ignored for quantitative purposes. Table 2 gives the numbers of bands of malate dehydrogenase found in male and female worms in the various isolates and strains examined. I n one isolate of the West Nile Strain (West Nile 7) four mice were exposed to cercariae and 8 days later a further three mice were exposed (shortage of mice prevented larger numbers being used). I n the week between the infections most of the infected
MALATE DEHYDROGENASE ISOENZYMES OF S C H I S T O S O M A
snails had died, and on examination of the two mice that survived from the second exposure it was found that most of the worms were female. The female worms from both batches of mice had four malate dehydrogenase isoenzymes, but whereas the males from the first batch had four isoenzymes those from the second only had two. .........-'""" ....°-'- ........
•..°.~ ° ........ •
Y /f :~Arua ~ ~ r
-o ~ ~
4 q Pachwach °°"..,..
Lake Kyoga Lake Albert
Fro. 1. Map to show sources of Ugandan S. mansoni. Tribal areas: 1, Acholi, 2, Kakwa, 3, Lugbara, 4, Jonam, 5, Baganda. Sometimes there were relatively large differences between the percentages of the two or four isoenzymes from different isolates. Quantitative figures for isoenzymes from some of the isolates are shown in Table 3. (b) Non-specific esterase The typical esterase pattern for male and female worms of S. mansoni is shown in Fig. 3. Due to the lack of a high speed refrigerated centrifuge for preparing
extracts there was often uneven blotching at the origin of the run, but this has not been displayed. Usually there were two bands of esterase activity in male worms and five in female worms, but in a few cases, e.g. West Nile 7 females,
Fic. 2. (a) Malate dehydrogenase isoenzymesof West Nile 4 S. mansoni. (b) Scan of above patterns. M, male; F, female; O, origin.
Fig. 4, there were from one to three extra slow moving bands. These extra bands could be found in worms from one mouse but not from worms from other mice in the same cage which were infected on the same day. The relative amounts of the esterase bands in male and female worms varied widely. Scans of typical female esterase patterns from 4 West Nile isolates are shown in Fig. 3. In the West Nile 7 isolate most of the snails had died by the second infection and the mice used produced mostly female worms. Extracts of mated and unmated worms from the same mouse were run side by side in the same gel, stained and then scanned. The result (Fig. 4) showed that considerable quantitative changes occurred when worms became sexually mature. Unfortunately only 3 mice were exposed to this infection and only two survived to 8 weeks, but worms from both mice gave similar results.
MALATEDEHYDROGENASEISOENZYMESOF S C H I S T O S O M 2 I M . 4 N S O N I
FIG. 3B FxG. 3. A. Non-specific ester~e isoer~ymes of S. mansoni. M, male; F, female; O, origin. B. Scan of non-specific esterase isoer~.ymes of female ,9. mansoni from four isolates of the West Nile strain. (a) West Nile 3, (b) West Nile 8, (e) West Nile 10, (d) West Nile l l . (X, extra band.)
..~ O !
FXG. 4. Scan of ester~e isoer~ymes of female S. ~ m o n / ( i s o l a t e West Nile 7)" ~ , mated females; . . . . . . , unmated females, both from the same mouse. T h e two slowest migrating bands (unnumbered) were not normally found in estemse patterns.
G . C . Cot,gs
OF BANDS OF MALATE DEHYDROGENASE F O U N D
No. of bands found Strain or isolate West Nile 1 West Nile 2 West Nile 3 West Nile 4 West Nile 5 West Nile 6 West Nile 7 West Nile 8 West Nile 9 West Nile 10 West Nile 11 Kampala Gaba I Gaba 2 Entebbe I Entebbe 2 Mwanza Wellcome Egypt Sudan
4 2 4 4 4 2 4 and 2 * 4 2 4 4 4 2 2 2 2 4 2 2 2
2 2 4 2 2 2 4 2 2 4 4 2 2 2 4 2 4 2 2 2
* For explanation see text. DISCUSSION Detailed examination of malate dehydrogenase isoenzymes from twenty samples of S. mansoni have shown that considerable qualitative and quantitative variations occur in this enzyme f r o m isolates within East Africa. T e n out of seventeen samples had patterns in which w o r m s from one or both sexes had four isoenzymes. By contrast none of three samples from outside East Africa had four isoenzymes. T h e occurrence of multiple forms of malate dehydrogenase could thus possibly be used to differentiate between strains of S. mansoni, but if this proved to be a valid method, it would clearly involve the examination of large n u m b e r s of isolates of each strain. Isolates West Nile 9, 10 and 1i came from three people living in the same small village of 200 people, near Pachwach in the West Nile district of Uganda where S. mansoni is prevalent. T w o of the isolates had four isoenzymes and one had only two isoenzymes in each sex of the worms. T h i s clearly indicates that the variation in isoenzyrne n u m b e r is a local occurrence. T h e two isolates f r o m E n t e b b e m a y indicate the same feature, b u t as m a n y migrant fishermen use the fishing village, and the history of the people from w h o m the faecal samples were collected was not recorded, it is not certain where they obtained their infections.
4 6 8 8 9 10 11
M , m a l e ; F, female.
W e s t Nile W e s t Nile W e s t Nile W e s t Nile W e s t Nile W e s t Nile W e s t Nile Gaba 2 Mwanza Sudan
Strain or isolate
M M M F M F M M M F
Sex 21 20 23 38 34 34 30 13 23 34
+ S.D. + S.D. _ S.D. + S.D. _ S.D. _+ S . D . _+ S . D . _+S . D . _+S . D . + S.D.
1 6 7 8 7 7 8 9 5 10 8
51 + S . D . 80 + S.D. 43 _+ S.D. 62 _+ S.D. 66 _+ S . D . 34 _+ S . D . 50 _+ S.D. 87 _ S.D. 43 _+ S.D. 66 + S . D .
2 4 7 7 7 7 8 6 5 6 8
3 21 + S . D . -26 _+ S.D. --21 + S . D . 18 + S . D . -25 _ S . D . --
P e r c e n t a g e o f total
7 + S.D. -8 _+ S.D. --10 +_ S.D. 2 + S.D. -9 _+ S . D . --
T A B L E 3 - - P E R C E N T A G E C O M P O S I T I O N OF THE BANDS OF MALATE DEHYDROGENASE ISOENZYMES FROM SOME SAMPLES OF
T h e results from isolate West Nile 7 in which the first batch of male worms had four isoenzymes and the second only two strongly suggests that worms in any one person may have either two or four isoenzymes, and that in this particular case the snails transmitting the parasites with four isoenzymes had died. This result may be more likely to be detected with transmission of S. mansoni by Biomphalaria pfeifferi, rather than with other species of snail as the B. pfeifferi receive only three miracidia per snail whilst others receive ten miracidia per snail, and therefore the chances of obtaining a single miracidial infection are greater in B. pfeifferi. It is unfortunate in the West Nile 7 isolate that larger numbers of infected mice had not been available. If worms with different isoenzyme patterns do occur in the same host, it could explain why there are differences in the relative amounts of the isoenzymes in different isolates from East Africa. T h e variation in esterase isoenzymes was not studied in such detail as that of malate dehydrogenase, but it was shown that there were large quantitative variations in the relative amounts of the bands present. Neither the significance of this finding nor that of the occurrence of occasional additional bands is known. But the comparison of esterase patterns from mated and unmated female worms makes it clear that mating does affect the patterns. In a study of testes n u m b e r in East African S. mansoni, Coles & T h u r s t o n (1970) showed that the number of testes varies significantly, even within the isolates called West Nile 9, 10 and 11 in the present paper. It is clear that malate dehydrogenase isoenzymes and quantitative aspects of esterase isoenzymes must now join testes n u m b e r as being factors that vary between isolates of the same strain of schistosome.
.4cknowledgements--I wish to thank the following for samples of S. mansoni. West Nile 2, Dr. E. Williams, West Nile 3-11, Dr. V. Ongom, Mwanza, Dr. V. M. Eyakuze, Egypt, Dr. K. U. Chu, Sudan, Dr. M. F. A. Saoud, Wellcome, Dr. O. D. Standen. I am grateful to Professor W. Banage for facilities in his laboratory, to Dr. B. A. Newton for reading the manuscript and to the Wellcome Trust for generous financial support. REFERENCES COLES G. C. (1969a) Isoenzymes of snail livers--I. Hydrolysing enzymes and peroxidase. Comp. Biochem. Physiol. 29, 403-411. Coitus G. C. (1969b) Isoenzymes of snail livers--II. Dehydrogenases. Comp. Biochem. Physiol. 31, 1-14. COL~S G. C. (1970) A comparison of some isoenzymes of Schistosorna mansoni and Schistosoma haematobium. Comp. Biochem. Physiol. 33, 549-558. COLES G. C. & THURSTONJ. P. (1970) Testes number in East African Schistosoma mansoni. ft. Helrninth. 44, 69-73. CONDE-DEL-PINo E., PEREZ-VILLAR M., CINTRON-RIVERA A. A. & SENERIZ R. (1966) Studies on Schistosoma rnansoni--I. Malicand lactic dehydrogenase of adult worms and cercariae. Expl Parasit. 18, 320-326.
Key Word Index--Biomphalaria pfeifferi; isoenzymes; malate dehydrogenase; nonspecific esterase; Schlstosoma mansoni; variation.