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Radioisotope labeling of Schistosoma mansoni miracidia for in vivo studies in Biomphalaria glabrata
JOURNAL
OF INVERTEBRATE
PATHOLOGY
33, 385-386
(1979)
Radioisotope Labeling of Schistosoma mansoni Miracidia for in Vivo Studies in Biomphalaria glabrata Radioisotopic labeling of larval trematodes has been reported by R. M. Lewert and B. J. Para (J. Znfec. Dis. 116, 171- 182, 1966), R. M. Lewert, B. J. Para, and M. A. Gzcel (Exp. Parasitol. 27, 273 -280, 1970; J. Parasitol. 56, 12501251, 1970), R. L. Stjernholm and K. S. Warren (Exp. Parasitol. 36, 222-232, 1974), P. Nansen, F. Frandsen, N. 0. Christensen (Parasitology 72, 163 - 17 1, 1976), G. P. Hoskin and T. C. Cheng (Exp. Parasitol. 35, 61-67, 1974), and W. A. Reid, S. M. Phillips, and R. J. Roscincki (Exp. Parasitol. 42, 331-342, 1977). The technique has provided useful information on the fate of certain metabolites incorporated by these parasites. Studies on defense mechanisms of the snail intermediate hosts to concurrent infections of different strains of the schistosome miracidia and/or sporocysts have been hampered by a lack of a suitable technique to differentiate between such strains in tissue sections of a single snail. We now report a radioisotopelabeling procedure that allows differentiation between labeled and unlabeled strains of Schistosotna rnansoni sporocysts and miracidia in individual Biomphalaria glabrata snails. Debris-free eggs, obtained from livers of routinely infected mice by the method of 0. 0. Kassim and D. E. Gilbertson (J. Pnrasitol. 62, 715-720, 1976) were transferred to a hatching medium containing 200 &i/ml of either [“Hlthymidine (sp act 20 Ci/mmol, New England Nuclear), [“Hlglucase (sp act 40 Ci/mmol, New England Nuclear), or [“Hlleucine (sp act 80 Ci/mmol, New England Nuclear). Eggs were also hatched in a control medium that contained no radioisotopic label. After 2 hr of incuba-
tion, actively swimming miracidia were pipetted out onto nylon screen cloth (with 28-pm openings), gently washed several times with water to remove any adhering label. Snails of groups of 6-8 mm B. gleebrata (NIH-M-line strain) were individually exposed to about 10 miracidia, which had earlier been incubated in one of the labeled compounds. A control group of snails were also exposed to unlabeled miracidia. In another set of experiments, individual snails were exposed to both label-free miracidia and those labeled with [“Hlleucine. The period of exposure in all the experiments was 1 hr. At 6, 24, and 48 hr post exposure, the snails were fixed in Bouin’s solution, embedded in paraffin, sectioned at 6 pm, and deparaffinized in ethanol. Sections were dipped in Kodak NTB-2 emulsion, air-dried for 1 hr, and stored under desiccation at 4°C for 8 to 10 weeks. Subsequently slides were developed for 4 min in Kodak D-19 developer, rinsed in water for 1 min, fixed for 4 min, and stained for 1 min each in Meyer’s hematoxylin and eosin. When sections were examined under the microscope, no grains were found either on control miracidia and sporocysts or on those incubated in [3H]thymidine. Miracidia and sporocysts labeled with [“Hlleucine showed optimal grain development, the grain density over those labeled in [“HIglucose being much less. In snails which were doubly exposed to leucinelabeled and unlabeled miracidia, grains were located on some sporocysts, but were absent over the others (Figs. 1,2,3). All the miracidia and sporocysts examined in tissue sections showed normal development, indicating that the amounts of radioisotopes used have no deleterious biological effects
385 0022-201
1/79/030385-02$01.00/O
Copyright 6:’ 1979 by Academic Prers. Inc All rights of reproduction in any form reserved.
386
NOTES
on the intramolluscan parasites. The absence of grains over miracidia and sporocysts incubated with [3H]thymidine is probably due to nonincorporation of the metabolite for DNA synthesis by mature S. IIZLIIZsorzi miracidia. Also the lesser grain density over glucose-labeled sporocysts may be due to the lower specific activity of the radioisotope sugar. [“H]Leucine seems to be an optimal metabolite for radioisotope labeling of S. nzansorzi miracidia. The technique described in this report is especially suitable for studying host -parasite interactions of two different strains of S. r~zansorri early larval stages in individual vector snails, one strain being labeled and the other unlabeled. The scintillation counting procedure of P. Nansen, F. Frandsen, and N. 0. Christensen (lot. cit.) does not allow for strain differentiation in snail hosts, while the incubation of S. r?rnrz.sorzi eggs at 37°C in 0.9% saline and 200 mCi C3H] glucose for 3 hr, as described by R. M. Lewert, M. J. Para, and M. A. &cel (lot. cit.), would have had a deleterious effect on the ability of miracidia to penetrate the snails. Snails readily incorporate sugars and amino acids which are in turn metabolized and retained by the developing cercariae in the snails’s digestive gland (W. A. Reid, S. M. Phillips, and R. J. Roscincki, lot. cit.). Results from our preliminary experiments showed that the release of the vacuolar fluid during the hatching of labeled S. mansoni eggs led to the incorporation of radioactive labels into snail tissues and made differentiation between labeled and unlabeled sporocysts impossible. KEY WORDS: Autoradiography, [“HISchistosoma manleucine, trematode, soni. miracidia, sporocysts, intermediate glabr.ata. host snails, Biomphaluria OLAKUNLE FIGS l-3. Autoradiographs of sections of Schi.crosoma mansor~i sporocysts in tissues of the snail intermediate host Biomphalovia glnbrcrttr. All sporocysts are 48 hr old. FIG. 1. Sporocyst labeled with [:‘H]leucine. Note the abundance of grains over the sporocyst. FIG. 2. Sporocyst labeled with [“H]glucose. FIG. 3. Sporocyst incubated in [“Hlthymidine. Note the absence of grains over the sporocyst. 250x.
CHARLES
Received
August
1. 1978
0. KASSIM S. RICHARDS
OF INVERTEBRATE
PATHOLOGY
33, 385-386
(1979)
Radioisotope Labeling of Schistosoma mansoni Miracidia for in Vivo Studies in Biomphalaria glabrata Radioisotopic labeling of larval trematodes has been reported by R. M. Lewert and B. J. Para (J. Znfec. Dis. 116, 171- 182, 1966), R. M. Lewert, B. J. Para, and M. A. Gzcel (Exp. Parasitol. 27, 273 -280, 1970; J. Parasitol. 56, 12501251, 1970), R. L. Stjernholm and K. S. Warren (Exp. Parasitol. 36, 222-232, 1974), P. Nansen, F. Frandsen, N. 0. Christensen (Parasitology 72, 163 - 17 1, 1976), G. P. Hoskin and T. C. Cheng (Exp. Parasitol. 35, 61-67, 1974), and W. A. Reid, S. M. Phillips, and R. J. Roscincki (Exp. Parasitol. 42, 331-342, 1977). The technique has provided useful information on the fate of certain metabolites incorporated by these parasites. Studies on defense mechanisms of the snail intermediate hosts to concurrent infections of different strains of the schistosome miracidia and/or sporocysts have been hampered by a lack of a suitable technique to differentiate between such strains in tissue sections of a single snail. We now report a radioisotopelabeling procedure that allows differentiation between labeled and unlabeled strains of Schistosotna rnansoni sporocysts and miracidia in individual Biomphalaria glabrata snails. Debris-free eggs, obtained from livers of routinely infected mice by the method of 0. 0. Kassim and D. E. Gilbertson (J. Pnrasitol. 62, 715-720, 1976) were transferred to a hatching medium containing 200 &i/ml of either [“Hlthymidine (sp act 20 Ci/mmol, New England Nuclear), [“Hlglucase (sp act 40 Ci/mmol, New England Nuclear), or [“Hlleucine (sp act 80 Ci/mmol, New England Nuclear). Eggs were also hatched in a control medium that contained no radioisotopic label. After 2 hr of incuba-
tion, actively swimming miracidia were pipetted out onto nylon screen cloth (with 28-pm openings), gently washed several times with water to remove any adhering label. Snails of groups of 6-8 mm B. gleebrata (NIH-M-line strain) were individually exposed to about 10 miracidia, which had earlier been incubated in one of the labeled compounds. A control group of snails were also exposed to unlabeled miracidia. In another set of experiments, individual snails were exposed to both label-free miracidia and those labeled with [“Hlleucine. The period of exposure in all the experiments was 1 hr. At 6, 24, and 48 hr post exposure, the snails were fixed in Bouin’s solution, embedded in paraffin, sectioned at 6 pm, and deparaffinized in ethanol. Sections were dipped in Kodak NTB-2 emulsion, air-dried for 1 hr, and stored under desiccation at 4°C for 8 to 10 weeks. Subsequently slides were developed for 4 min in Kodak D-19 developer, rinsed in water for 1 min, fixed for 4 min, and stained for 1 min each in Meyer’s hematoxylin and eosin. When sections were examined under the microscope, no grains were found either on control miracidia and sporocysts or on those incubated in [3H]thymidine. Miracidia and sporocysts labeled with [“Hlleucine showed optimal grain development, the grain density over those labeled in [“HIglucose being much less. In snails which were doubly exposed to leucinelabeled and unlabeled miracidia, grains were located on some sporocysts, but were absent over the others (Figs. 1,2,3). All the miracidia and sporocysts examined in tissue sections showed normal development, indicating that the amounts of radioisotopes used have no deleterious biological effects
385 0022-201
1/79/030385-02$01.00/O
Copyright 6:’ 1979 by Academic Prers. Inc All rights of reproduction in any form reserved.
386
NOTES
on the intramolluscan parasites. The absence of grains over miracidia and sporocysts incubated with [3H]thymidine is probably due to nonincorporation of the metabolite for DNA synthesis by mature S. IIZLIIZsorzi miracidia. Also the lesser grain density over glucose-labeled sporocysts may be due to the lower specific activity of the radioisotope sugar. [“H]Leucine seems to be an optimal metabolite for radioisotope labeling of S. nzansorzi miracidia. The technique described in this report is especially suitable for studying host -parasite interactions of two different strains of S. r~zansorri early larval stages in individual vector snails, one strain being labeled and the other unlabeled. The scintillation counting procedure of P. Nansen, F. Frandsen, and N. 0. Christensen (lot. cit.) does not allow for strain differentiation in snail hosts, while the incubation of S. r?rnrz.sorzi eggs at 37°C in 0.9% saline and 200 mCi C3H] glucose for 3 hr, as described by R. M. Lewert, M. J. Para, and M. A. &cel (lot. cit.), would have had a deleterious effect on the ability of miracidia to penetrate the snails. Snails readily incorporate sugars and amino acids which are in turn metabolized and retained by the developing cercariae in the snails’s digestive gland (W. A. Reid, S. M. Phillips, and R. J. Roscincki, lot. cit.). Results from our preliminary experiments showed that the release of the vacuolar fluid during the hatching of labeled S. mansoni eggs led to the incorporation of radioactive labels into snail tissues and made differentiation between labeled and unlabeled sporocysts impossible. KEY WORDS: Autoradiography, [“HISchistosoma manleucine, trematode, soni. miracidia, sporocysts, intermediate glabr.ata. host snails, Biomphaluria OLAKUNLE FIGS l-3. Autoradiographs of sections of Schi.crosoma mansor~i sporocysts in tissues of the snail intermediate host Biomphalovia glnbrcrttr. All sporocysts are 48 hr old. FIG. 1. Sporocyst labeled with [:‘H]leucine. Note the abundance of grains over the sporocyst. FIG. 2. Sporocyst labeled with [“H]glucose. FIG. 3. Sporocyst incubated in [“Hlthymidine. Note the absence of grains over the sporocyst. 250x.
CHARLES
Received
August
1. 1978
0. KASSIM S. RICHARDS