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 Fasciola hepatica and Paragonimus westermani 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, Metagonimus 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.