liver fluke, Fasciola sp., have been carried out by several workers (Itagaki and ... Fasciola hepatica and F, gigantica, several attempts have been made to charac-.
Japan. J. Med. Sci. Biol., 33, 249-254, 1980
ELECTROPHORETIC COMMON I.
ENZYME
STUDIES
ON
LIVER
FLUKE,
VARIATIONS
ENZYMES
IN THE
TAKESHI AGATSUMA Department
and
IN
FASCIOLA NATURAL
THE
JAPANESE
SP. POPULATION*
NORI JI SUZUKI
of Parasitology, Kochi Medical School, Kohasu, Oko-cho, Nankoku 781-51, Kochi, Japan
(Received, June 12, 1980. Accepted, August 28, 1980)
SUMMARY: A preliminary study on four enzymes, adenylate kinase, phospho glucomutase, phosphogluconate dehydrogenase and esterase, of Fasciola sp. found in Japan was carried out by horizontal starch gel electrophoresis. Among them, two enzymes, adenylate kinase and phosphoglucomutase, showed variations in the natural population, but the frequencies of the variants were considerably low; the remainders, phosphogluconate dehydrogenase and esterase, showed no variation.
INTRODUCTION Considerable studies on the taxonomical status of the Japanese common liver fluke, Fasciola sp., have been carried out by several workers (Itagaki and Akane, 1959; Ueno and watanabe, 1960; Oshima et al., 1969). However, more information on the liver fluke is still required to determine the status. Recently, Sakaguchi and Nakagawa (1975) performed karyological investigations of the liver fluke and found that the liver fluke population consisted of individuals, showing the chromosome number of 2n=20 and 3n=30. More recently, Moriyama et al. (1979) also made further investigations with respect to karyotypes of the liver fluke. They found not only the same two kinds of individuals as those reported by Sakaguchi and Nakagawa (1975), but also mosaic ones, which simultaneously had two kinds of cells showing the chromosome number of 2n=20 and 3n=30 in a single individual. These results suggest that the Japanese species of Fasciola may not be in an homogenous entity. Electrophoretical analyses of the enzymes in various organisms have provided a useful tool for the classification and characterization of the closely related species (Carter, 1978; Vrijenhoek, 1978; Kreutzer and Christensen, 1980). In Fasciola hepatica and F, gigantica, several attempts have been made to characterize their electrophoretic properties of enzymes (Probert and Lwin, 1977; * This study was supported in part by Scientific Research Grant (No. 577206)from the Ministry of Education, Science and Culture, Japan. 吾妻
健,鈴 木了司(高 知医科大学寄生虫学教室
南国市岡豊町小蓮)
249
250
AGATSUMA
et
SUZUKI
Vol.33
Probert and Durrani, 1977). With the Japanese common liver fluke, however, no electrophoretic study on the enzymes has been carried out. This paper presents preliminary results of electrophoretic studies on four enzymes of the Japanese species of Fasciola from the standpoint of their taxonomical status. MATERIALS AND METHODS Adults of Fasciola sp. were obtained from the cattle livers at a slaughter house in Kochi Prefecture, Japan, in 1979-1980. The liver flukes collected were washed with saline and stocked in a deep freezer at -80 C until use. The extracts were prepared by homogenizing each individual in 1.0 ml of 0.1 M phosphate buffer (pH 7.5) with a teflon homogenizer in an ice bath. Then, the homogenates were centrifuged at 3,000 rpm for 3 min at room temperature. The supernatants obtained were used in the present study. These procedures for preparation of extracts were based on the method employed by Ayala et al. (1973). During electrophoresis, the gels were maintained at about 4 C. As a control run of electrophoresis, samples of the cattle liver were also prepared in the same procedure as those of the parasites. Horizontal electrophoresis in 12% starch gel was carried out by Smith's method (1968). Four enzymes, adenylate kinase, phosphoglucomutase, phosphogluconate dehydrogenase and esterase, were examined in this study. Histochemical staining of the enzymes was done essentially by the method of Shaw and Prasad (1970). Since no band appeared when the substrates were removed from the reaction mixture, the bands observed were considered to be specific to the respective enzymes. The buffer systems and conditions of the electrophoresis (Shaw and Prasad, 1970; Nozawa et al., 1977) as well as the reaction mixtures for detection of the four enzymes are as follows: Buffer systems used in starch gel electrophoresis
1980
ISOZYME
PATTERN
OF
FASCIOLA
SP.
251
Specific staining systems for detection of the enzyme activities
RESULTS As shown in Fig. 1, the electrophoretic patterns of the four enzymes were different between the parasite and the host. Two electrophoretic enzyme forms of adenylate kinase (AK) were identified in the liver fluke samples from the natural population (Fig . 1). One form (AK-1) showed three major bands with minor ones, while the other (AK-2) possessed four bands with minor ones. The slowest band of each form showed the identical mobility. Minor bands of both forms were faint and not always reproducible. As shown in Table I, AK-1 was predominant in the frequency , while AK-2 was found in only one sample so far examined . The liver flukes possessed also two enzyme forms of phosphoglucomutase (PGM), which showed anodally-migrating bands, and tended to produce a series of sub-bands running in the anodal direction from the major band. These two forms were distinguishable owing to their differential mobilities of anodal bands (Fig. 1). Although this difference was small, the patterns observed were highly reproducible . The remaining two enzymes, phosphogluconate dehydrogenase (6PGD) and esterase
AGATSUMA
252
Fig.1.
TABLE I Frequencies
et
SUZUKI
Vol.33
Starch gel electrophoretic patterns of four enzymes in the Japanese common liver fluke, Fasciola sp., and the host, cattle. 0; origin, H; host. For other abbreviations, see RESULTS. of enzyme
forms
in natural
population
1980
ISOZYME
PATTERN
OF FASCIOLA
SP.
253
(EST), showed no electrophoretic variations; 6PGD of all parasites sampled indicated the same three banded pattern, and EST was detected as only a single band in all samples. DISCUSSION As a preliminary study, four enzymes of the Japanese common liver fluke, Fasciola sp., were examined by starch gel electrophoresis. With regard to the Japanese common liver fluke, this is the first attempt to show the electrophoretic properties of the enzymes. Up to date, in Japan, little information has been available on the isozymes of parasitic helminths. Recently, Agatsuma and Suzuki (1980) studied the electrophoretic properties of six enzymes in the Oriental lung flukes, Paragonimus ohirai and P. miyazakii in Japan. They reported that these two Paragonimus species were distinguished each other in the electrophoretic profiles of their enzymes, acid that, therefore, this technique was useful for classification and characterization of the two species of the genus. It was of interest that the band patterns of the three enzymes, AK, 6PGD and EST, found in this study showed good similarities with those in the two Paragonimus species. Probert and Durrani (1977) demonstrated that the whole-worm homogenates of F, hepatica showed six bands of cholinesterase using acetylthiocholine as substrate in acrylamide gel electrophoresis. Thus, it is noticeable that only a single band of EST was detected in the present materials, Fasciola sp. It is not known yet if this discrepancy was due to the difference in species or in the method of experiments. In the present electrophoretic systems, AK and PGM of the liver fluke formed sub-bands running in the anodal direction. A similar phenomenon has been found in the same enzymes of Plasmodium spp. (Carter, 1978) and Paragonimus spp. (Agatsuma and Suzuki, 1980) in the same electrophoretic system. Probably it can be assumed that these sub-bandings are due to polymerization of the enzyme protein molecules. From the present study, it was obvious that there was little variation in the enzymes of the liver fluke in the natural population so far examined. This fact might be explained by the possible occurrence of their reproducing system (parthenogenesis); the population examined may have been composed of descendants of a single individual. To know overall enzyme variations of the liver fluke, therefore, more individuals including members of other remote populations should be surveyed with regard to these enzymes. We believe that these electrophoretic studies may provide a basis for determination of the taxonomical status of the Japanese common liver fluke.
254
AGATSUMA
et
SUZUKI
Vol.33
ACKNOWLEDGEMENTS The authors are greatly indebted to Drs. K. Nozawa and T. Shotake, Primate Research Institute, Kyoto University, for their encouragement and advice , and to Mr. Y. Kawamoto, Kyoto U niversity, for his technical assistance in the procedure of electrophoresis . They are also grateful to Dr. Y. Hashiguchi, Kochi Medical School, for his critical reading of the manuscript , and to M r. H. Kumazawa and Miss K. Nishimura, Kochi Medical School , for their supports throughout this study.
REFERENCES AGATSUMA,T. AND SUZUKI,N. (1980): Electrophoretic studies on enzyme in Paragonimus spp . I . Comparison of isozyme patterns between P. ohirai and P . miyazakii. Japan. J. Parasitol., in press. AYALA,F. J., HEDGECOCK, D., ZUMWALT,G. S. ANDVALENTINE , J. W. (1973): Genetic variation in Tridacna maxima, an ecological analog of some unsuccessful evolutionary lineages . Evolu tion, 27, 177-191. CARTER,R. (1978): Studies on enzyme variation in the murine malaria parasites Plasmodium berghei, P. yoelii, P. vinckei and P. chaubaudi by starch gel electrophoresis . Parasitology, 76, 241-267. ITAGAKI,H. ANDAKANE,S. (1959) : Morphological study on the Japanese liver fluke , compared with the African specimens. Bull. Azabu Vet. Coll. , 6, 115-123.K REUTZER, R. D. ANDCHRISTENSEN, H. A. (1980): Characterization of Leishinania spp . by isozyme electrophoresis. Am. J. Trop. Med. Hyg., 29, 199-208. MORIYAMA,N., TSUJI, M. AND SETO, T. (1979): Three karyotypes and their phenotypes of Japanese liver flukes (Fasciola sp.). Japan. J. Parasitol., 28, 23-33. NOZAWA,K., SHOTAKE,T., OHKURA,Y. AND TANABE , Y. (1977): Genetic variations within and between species of Asian macaques. Japan. J. Genetics , 52, 15-30. OSHIMA, T., AKAHANE,H. AND SHIMAZU,T. (1969): Patterns of the variation of the common liver fluke (Fasciola sp.) in Japan. I. Variations in the sizes and shapes of the worms and eggs. Japan. J. Parasitol., 17, 97-105. PROBERT,A. J. AND DURRANI,M. S. (1977): Fasciola hepatica and Fasciola gigantica: Total cholinesterase, characteristic, and effects of specific inhibitors . Exptl. Parasitol., 42,203-210. PROBERT,A. J. ANDLWIN, T. (1977): Fasciola hepatica: The subcellular distribution and kinetic and electrophoretic properties of malate dehydrogenase. Exptl . Parasitol., 41, 89-97. SAKAGUCHI, Y. ANDNAKAGAWA, C. (1975): A note on the chromosomes of the common liver fluke (Fasciola sp.) from Japan. Chromosome Information Service, 19, 20-21. SHAW, C. R. AND PRASAD,R. (1970): Starch gel electrophoresis of enzymes-A compilation of recipes. Biochem. Genet., 4, 297-320. SMITH,I. (1968): Starch gel electrophoresis. p. 217-324. In I. SMITH [ed.], Chromatographic and Electrophoretic Techniques. Heinemann Medical Books Ltd., New York. UENO, H. AND WATANABE,S. (1960): Ecological studies on the common liver fluke in Japan . Kachiku Eisei Shikenjo Hokoku, 38, 167-181 (in Japanese). VRIJENHOEK,R. C. (1978): Genetic differentiation among larval nematodes infecting fishes . J. Parasitol., 64, 790-798.
