THE KURUME MEDICAL JOURNAL 1973 Vol.20, No.4, P.241-250

SUCCINATE DEHYDROGENASE ACTIVITY IN UNEMBRYONATED EGGS, EMBRYONATED EGGS, MIRACIDIA AND METACERCARIAE OF SOME FLUKES

MINORU AKUSAWA

Department of Parasitology, Kurume University School of Medicine, Kurume, Japan

KYOKO SAITO

Department of Chemistry, Nippon Veterinary and Zootechnical . College, Musashino, Tokyo, Japan

(Received for publication December 3, 1973)

The authors examined the activity of succinate dehydrogenase (SD) in and with the stages in develop- ment from cell division egg to metacercaria. As a result of this experiment, in the unicellular stages and miracidium stages of F. hepatica and P. wester- mani, a very slight SD activity was detected. However, in the miracidium formation stages of these flukes, the activity was very strong. Thus, it was clear that the reduction of methylene blue decreased suddenly from the highest value when metamorphosis occurred. It was suggested that respira- tory metabolism rises with the progress of development and ability to decolorize methylene blue in creases. In the metacercaria stages of F. hepatica, P. westermani, yokogawai, the ability to decolorize methylene blue was persistent, but it was not so strong. During the time, so called hibernation time, when metacercaria invades into final host, meta- cercaria keeps metabolism of respiration as low as possible, and saves consumption of energy for maintenance of life. Metacercaria has biological function for parasitism.

Biological studies have been made on has been already found in some flukes the development of the flukes as speci- (Huang and Chu, 1962; Bryant and Wil- mens. Accordingly, all the life history liams, 1962; Barry et al., 1968; Hamaji- has been clarified in these flukes up to ma, 1972, 1973). this time. Especially, morphological studies have been performed in detail MATERIALS AND METHODS on the forms of the asexual stage of flukes. At present, it is clear that the I) Liver fluke eggs flukes take a process of development Bile samples were harvested from the starting with egg through miracidium, gallbladder of a cow infected with sprocyst, redia, and cercaria to meta- Fasciola hepatica. Eggs were obtained cercaria. And succinate dehydrogenase from them by the sedimentation method.

241 242 AKUSAWA AND SAITO

II) Lung fluke eggs purpose, metacercariae of Metagonimus Metacercariae of Paragonimus wes- yokogawai were harvested from Gna- termani were isolated from Erioc heir thopogn elongates elongatus, a kind of fresh-water fish, collected from the japonicus, a kind of fresh-water crab, collected in Nagasaki Prefecture, and Tone River, and those of Heteroph yes used to cause lung fluke infection in a heteroph yes were isolated from Tribolo- dog. Adult worms were harvested from den hakonensis, another kind of fresh- the lungs of the infected dog. Then fluke water fish, produced in Hida, Oita eggs were obtained from the uterus of Prefecture. these adult worms. V) Estimation of succinate dehydro- III) Cultivation and observation of genase activity The Thunberg tube was used to esti- eggs mate succinate dehydrogenase (herein- Collected eggs were washed well, after referred to as SD) activity. The treated with 5% antiformin, and washed method of Tom and Wilson was applied again thoroughly with water. to the measurement of ability to de- The state of development of these colorize methylene blue. eggs was observed every 5 days during The reaction mixture and Thunberg the period of incubation. Materials were tube method is shown in Fig. 1. The collected at the time of observation. substrate is sodium succinate. The test IV) Metacercaria suspension is containing about 104 eggs Succinate dehydrogenase activity in and 1% of worms prepared in each the stage of metacercaria was compared stage of development. Eggs were col- with some species of flukes. For this lected by centrifugation, placed in the

Fig. 1. Outline of the Thunberg tube method. SUCCINATE DEHYDROGENASE OF SOME FLUKES 243

glass homogenizer with addition of TABLE I buffer solution, pH 9.0, and triturated Ability to decolorize (liver fluke) in cooled condition. Such amount of buffer solution was added to the result- ing sample as to make the total amount of the resulting suspension 5 ml. Then, 0.5 ml of this suspension was put in the main chamber of the Thunberg tube as the test suspension. Again, 2.5 ml of buffer solution was added to the content of the main chamber. In the side arm were placed 1.0 ml of 1/50,000 M methy- lene blue and then 1.0 ml of 1/20 M sodium succinate. After grease was painted, the tube was set with such care not to mix the content of the main for SD activity. As a result, the acti- chamber with that of the side arm. vity was first 0.578, as expressed in Then ventilation was induced by the terms of -log T, 0.55 after 3 minutes, vacum pump for about 3 minutes (at 4 0.541 after 6 minutes, and 0.541 after mg Hg). The tube was allowed to stand 9 minutes. In short, changes in SD in the thermostat at 30•Ž for 10 minu- activity were very slight. tes, so that it might react and remain b) Multicellular stage at a constant level of temperature. Eggs were incubated at 30 C for 5 Then the content of the side arm was days. A number of multicellular eggs mixed with that of the main chamber. were observed under the microscope. The intensity of decoloration of methy- The SD activity of these eggs showed lene blue was estimated by the photo- a mild increase in ability to decolorize electrocolorimeter immediately after 3, over a period from 6 to 9 minutes, as 6, and 9 minutes after mixing of the compared with the same activity of contents. unicellular eggs. c) Stage of miracidium formation (A) RESULTS AND DISCUSSION Some eggs presented the miracidium Experiment I. •kSuccinate dehydroge- formation during an incubation period nase activity in unembryonated eggs, of 10 days when it was observed by. embryonated eggs, miracidia and meta- microscopy. As shown in Fig. 2-C, the cercariae of liver fluke.•l ability to decolorize of the S D activity It was not always possible to prepare was 0.593, as expressed in terms of -log homogeneous materials for measure- T, immediately after mixing the two ment of the velocity of development of contents, 0.553, or 0.04 lower than the individual liver fluke eggs. Therefore, preceding value, after 3 minutes, and trials were repeated for this purpose. 0.553 after 6 minutes.. In other words, The results obtained are indicated in the activity was not reduced, and de- Table 1 and Fig. 2. coloration taken place in the same in- a) Unicellular stage clination was still sharp after 9 minutes. The extract obtained from 104 eggs The suspension retained only a very before cell division immediately after indistinct color of methylene blue after collection from the bile was examined 2 hours. 244 AKUSAWA AND SAITO

Fig. II Ability to decolorize (liver fluke) (a) : Unicellular stage (b) : Multicellular stage (c) : Stage of miracidium formation (A) (d) : Stage of miracidium formation (B) (e) : Miracidium stage (f) : Metacercaria stage

d) Stage of miracidium formation rease in this activity over a period from (B) 3 to 9 minutes. Some eggs were noticed to have f or- e) Miracidium stage med miracidia. Eggs at 15 days of in- A miracidium formed in the egg needs cubation are shown in Fig. II. Different photo-energy to leave the egg. There- from those at 10 days of incubation, fore, the eggs were taken out of the they exhibited SD activity rapidly for incubator and exposed to light in order the first 3 minutes, a little reduced to collect miracidia. After some time, activity after that, and a sudden dec- a number of miracidia could be recog- SUCCINATE DEHYDROGENASE OF SOME FLUKES 245

nized by the naked eye. When the cul- tures was confimed to contain miracidia released from eggs by microscopy, it was allowed to freeze rapidly in a freez- ing mixture composed of solid carbon dioxide and acetone, so that the mira- cidia might stop their movement. Then the frozen culture was thawed and centrifuged. The resulting material was used for the measurement. As shown in Figs. c and d, SD activity was very strong in eggs forming a miraci- Fig. III Difference of ability to decolorize dium. It was, however, extremely low (liver fluke) in the miracidia formed, as shown in (a) : Unicellular stage Fig. e. (b) : Multicellular stage f) Metacercaria stage (c) : Stage of miracidium Cercariae released from Lymnaea formation (A) (d) : Stage of miracidium snails were allowed to be settled on a formation (B) sheet of cellophane, and the resultant (e) : Miracidium stage metacercariae used as materials. Like (f) : Metacercaria stage miracidia, the metacercariae exhibited an ability hardly changeable to decolo- the stage of development are indicated rize methylene blue. This ability, as in Table II and Fig. III. expressed in terms of -log T, was re- corded to be 0.437-0.435-0.42-0.415 Experiment II.•kSuccinate dehydroge- with the lapse of time. In Fig. II, com- nase activity in unembryonated eggs, parison is made on the ability expressed embryonated eggs, miracidia and meta- in terms of-log T to decolorize methy- cercariae of lung fluke.•l lene blue measured sequentially at certin The lung fluke shows essentially the intervals over a period from the egg to same stages of development as the liver the metacercaria of the liver fluke. The results about the difference of TABLE III ability to decolorize (SD activity) with Ability to decolorize (lung fluke)

TABLE II Difference of ability to decolorize (liver fluke) 246 AKUSAWA AND SAITO

Fig. IV Ability to decolorize (lung fluke) (a') : Unicellular stage (b') : Multicellular stage (A) (c') : Multicellular stage (B) (d') : Multicellular stage (C) (e') : Multicellular stage (D) (f') : Stage of miracidium formation (g') : Miracidium stage (h') : Metacercaria stage SUCCINATE DEHYDROGENASE OF SOME FLUKES 247

fluke, but it is not parasitic on the stage either when eggs were incubated same site as the latter. Essentially the for 20 days. A s shown in Fig. N e', the same experiment as the preceding one ability to decolorize, experessed to be was performed on lung flukes and gave 0.54•¨0.52•¨0.47•¨0.46. It decreased a the results presented in Table i and little over a period from 6 to 9 minutes Fig. N. As is clear from this figure, after the mixing of both contents. SD it was confirmed that lung flukes had activity, however, was manifested dis- shown SD activity similar to that of tinctly. liver flukes. f') Stage of miracidium formation a') Unicellular stage A large number of eggs forming Eggs were collected directly from miracidia were found in cultures on the the uterus of lung flukes and used as 23rd day of incubation. As shown in samples of the unicellular stage. The Fig. IV f', SD activity was very strong. results obtained from them are shown It was particularly strong over periods in Table III and Fig. IV a'. They showed from 3 to 6 minutes and from 6 to 9 perfectly the same tendency as those minutes. Therefore, SD activity was obtained from liver flukes of the uni- persistent. cellular stage. g') Miracidium stage b') Multicellular stage Fig. IV g' indicates the result obtain- Eggs were incubated at 30•Ž for 5 ed from the miracidium stage. Materi- days. The results obtained from them als for this stage were prepared by the are presented in Table III and Fig. IV b'. same procedure as employed in the ex- SD activity was shown very remarkably periment on the liver fluke. As shown over a period beginning immediately in Fig. IV g', eggs with formed miraci- after the mixing of both contents at dia exhibited the highest SD activity. lasting for 3 minutes. During this pe- Nevertheless, they presented such a riod, the activity expressed in -log T remakable decrease in ability to decolo- value was very strong, ranging from rize as liver fluke. 0.735 to 0.645, with a difference of 0.09. h') Metacercaria c') Multicellular stage (A) Fig. IV h' shows the results obtained Eggs were incubated for 10 days. The from metacercariae of the lung fluke results obtained from them are shown collected from a species of small fresh- in Table III and Fig. N c'. The ability water crabs as materials. The meta- to decolorize of these eggs was similar cercariae of the lung fluke exhibited a to that of liver fluke eggs incubated little stronger SD activity than those for 10 days. of the liver fluke, and stronger SD d') Multicellular stage (B) activity than the eggs of unicellular Miracidia were formed, without ex- stage, but they presented a low value ception, in the , liver fluke eggs incu- of ability to decolorize. This ability bated for 15 days, but not in any of was persistent, although it remained the lung fluke eggs incubated for 15 low. days. As shown in Fig. IV d', SD acti- The difference of abillity to decolo- vity was considerably stronger in the rize (SD activity) with the stages of multicellular stage of eggs (B) than in development is indicated in Table N and that of eggs (A). Fig. V. e') Multicellular stage (C) Miracidia, which were not observed in stage d', failed to appear in this 248 AKUSAWA AND SAITO

TABLE IV TABLE V Difference of ability to decolorize Comparison of metacercaria stage (lung fluke) ability to decolorize of flukes

Fig. V Difference of ability to decolorize (lung fluke) (a') : Unicellular stage (b') : Multicellular stage (A) (c') : Multicellular stage (B) (d') : Multicellular stage (C) (e') : Multicellular stage (D) (f') : Stage of miracidium formation) (g') : Miracidium stage (h') : Metacercaria stage

Fig. VI Comparison of metacercaria stage ability to decolorize of flukes. (k) : Fasciola hepatica (l) : Paragonimus westermani (m) : Metagonimus yokogawai SUCCINATE. DEHYDROCENASE OF SOME FLUKES 249

Experiment III. •kSuccinate dehydro- the other hand, a lack of cytochrome genase activity in metacercariae of oxidase activity was reported for Para- Metagonimus yokogawai•l gonimus unembryonated eggs (Hama- It was observed that there was a jima 1973), and similar results were decrease in SD activity when metamor- obtained from low 02 uptake and utili- phosis occurred to the liver and lung zation of substrates in the homogenates flukes in the course of development. of unembryonated eggs, miracidia, re- Metacercariae of Metagonimus yokoga- diae and cercariae, and metacercariae wai were collected from scales of dece of Paragonimus (Hamajima 1972). And or Leuciscus hakonensis. The ability to also, metacercariae of Paragonimus decolorize methylene blue was measured resisted to cyanide inhibition (Hamaji- in them and results obtained are indi- ma 1973). Thus these results indicate cated in Table V and Fig. VI. It was that the physiological role of succinate persistent, though low in value, in the oxidation is minor important in matacer- metacercariae of both species. caria stage. Moreover, it is suggested that the energy metabolism probably Succinate dehydrogenase is impor- proceeds to the direction of the forma- tant for oxidative metabolism. The tion of pyruvate, phosphoenol pyruvate activity is indicative of presence of and succinate by malic enzyme, phosp- tricarboxylic acid cycle and succinate hoenol pyruvate carboxylase and f uma- oxidation. In this experiment, these rate reductase. On the contrary, the results indicate that succinate dehydro- result obtained in this experiment for genase is present in unembryonated succinate dehydrogenase seems to eggs, embryonated eggs, miracidia and indicate that the physiological role of metacercariae of these flukes as found succinate oxidation is more important in various life cycle stages of several to the miracidium formation stage eggs trematode species (unembryonated eggs, than unicellular stage eggs, miracidia embryonated eggs of P, westermani, and metacercariae becuase of the deve- Hamajima 1972, 1973; miracidia of F. lopment and differentiation required hepatica, Bryant and Williams 1962; large amount of energy. The activity rediae and cercariae of Paragonimus in metacerccaria of lung fluke was ohirai, Hamajima 1973; metacercariae strong as compared with other flukes. of Paragonimus miyazaki, Hamajima, This will be caused that metacercaria of 1972, 1973; adults of F. hepatica, Barry lung fluke lives in Eriocheir .japonicus. et al. 1968 and Bryant and Williams 1962; adults of , ACKNOWLEGDEMENT Huang and Chu 1962; adults of P. wes- We wish to thank Prof. K. Okabe for many termani, Hamajima 1973). In adition valuable suggestions made during the course to the above mentioned, Bruce et al of this work and also Dr. F. Hamajima for (1969) have shown a result of the ex- helpful advice and discussion. periment, suggesting presence of the dehydrogenase in cercariae of S. man- REFERENCES soni. Furthermore, in this experiment 1) BARRY, D. H., MAWCLESLEY-THOMAS,L. E., of F. hepatica and P, westermani, the and MALONE,J. C.: Enzyme histochemistry rate of methylen blue reduction was of the adult liver fluke, Fasciola hepa- higher in the miracidium formation tica. Experimental Parasitology 23, 355- stage eggs than in the unicellular stage 360, 1968. eggs, miracidia and metacercariae. On 2) BRUCE, J. I., WEISS, E., STIREWALT, M. A., 250 AKUSAWA AND SAITO

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