Internarional Journalfor Primed in Greof Brirain
Parasitology
Vol. 20, No. 2. pp. 257-258,
1990 0
1990 Awtrolim
002&7519/90 $3.00 + 0.00 Pergamon Press plc Societyfor Parosiro/ogy
RESEARCH NOTE EXCYSTATION OF FIVE DIGENEAN METACERCARIAE IN VITRO AND IN THE PERITONEAL CAVITY OF THE MOUSE MOSES FON ASANJI* and BERNARDFRIED? * Department of Biological Sciences, Ecole Normale Superieure, Bambili, Mezam Division, North West Province, Republic of Cameroon, Africa t Department of Biology, Lafayette College, Easton, PA 18042, U.S.A. (Received 23 March 1989; accepted 22 November 1989)
M. F. and FRIEDB. 1990. Excystation of five digenean metacercariae in vitro and in the peritoneal cavity of the mouse. International Journal for Parasitology 20: 257-258. Excystation of metacercariae of Parorchis acanthus, Posthodiplostomum nanum, Posthodiplostomum sp., Posthodiplostomoides leonensis and Clinostomum tilupine in mammalian saline at 39°C was similar to their excystation in mouse peritoneal cavity. Extrinsic enzymes were not needed for C. tihzpiae excystation but were an absolute necessity for P. acanthus excystation. Extrinsic pepsin enhanced P. nanum excystation but was not obligatory. Acid pepsin was an absolute requirement for Posthodiplostomum sp. and Posthodiplostomoides leonensis excystation. It was noted that acid saline could not be substituted for acid pepsin and that there was diversity in the condition involved in digenean excystation. Abstract-AsANJr
INDEX KEY WORDS: Digenean; Parorchis acanthus; Posthodiplostomum nanum; Posthodiplostomum sp.; Posthodiplostomoides leonensis;Clinostomumtilapiae;metacercariae; excystment; mouse; peritoneal cavity; in vitro;enzymes.
IN a previous
study (Asanji & Williams, 1974) it was the experimental hosts of Parorchis acanthus, Posthodiplostomum sp., Posthodiplostomum nanum, Posthodiplostomoides leonensis and Clinostomum tilapiae were the piscivorous birds Stern sandvicensis, S. hirundo and the domestic chicken, Gallus gallus. In these experimental hosts P. acanthus establishes in the intestine while Posthodiplostomum sp. and P. leonensis establish in the duodenum and intestine. P. nanum and C. tilapiae are restricted to the stomach of their experimental hosts. Incidental observations in that study were made on the excystation of these species in the peritoneal cavity of mice, a site devoid of intestinal enzymes. Observations on the excystation of metacercariae of Fasciola hepatica in the mouse peritoneal cavity have been reported by Dawes (1961) and Dawes & Hughes (1964). Dixon (1966) explained the excystment of F. hepatica metacercariae in the peritoneal cavity as a response to stimulation from low concentrations of carbon dioxide. Boray (1969) noted excystment of F. hepatica metacercariae in the peritoneal cavity of sheep and suggested that excystment of this species is an active process not requiring host digestive enzymes. The present study reports observations on the excystation of the above named five species of digeneans in the mouse peritoneal cavity. These results are compared with controlled excystation studies done in vitro in mammalian saline (acidified Earle’s BSS pH 2.5) at 39°C. These studies allow for observations on the role of intrinsic (those of parasite origin) vs
noted
that
257
extrinsic (those of host origin) enzymes on excystation. For most species of trematodes some combination of both intrinsic and extrinsic enzymes are involved in the complex process of excystation, although the exact mechanisms are obscure. For implantation procedures, mice were prepared for surgery as described by Asanji & Williams (1974). For all excystation experiments in the mouse peritoneal cavity and in vitro there were two groups of metacercariae for each of the five species of trematodes used, i.e. those pretreated in 1% acid pepsin and those untreated. A total of 12 mice was used for each species of metacercaria (six for pretreated and six for untreated). For P. acanthus a total of 1425 and 984 pretreated and untreated metacercariae, respectively were used while 615, 288, 594 and 294 pretreated and 542, 272, 641 and 247 untreated metacercariae of Posthodiplostomum sp., P. nanum, P. leonensis and C. tilapiae, respectively, were introduced into the peritoneal cavity of mice. Some cysts were treated for 2 h in 1% acid pepsin (1 g enzyme dissolved in 100 ml 0.01 N-NC) and pH adjusted with 1% NaHCO, to 2.5) prior to implantation and others in mammalian saline were not treated and then implanted in mice. Pepsin and Earle’s BSS were obtained from the British Drug House (BDH), U.K. All in vitro experiments were done in triplicate and the results are expressed as the mean per cent of the three trials. The in vitro cultures were done in 25 ml of either 1% acid pepsin in saline pH 2.5 or mammalian saline (pH 2.5) at 39°C. Each trial was repeated three times and the results were
M. F. ASANJIand B. FRIED
258
TABLEI-PER CENTEXCYSTATION AFTER6 h INMOUSE PERITONEAL CAVITY ORin vim In vitro
Mouse peritoneal cavity
1% acid pepsin Species
pretreatment Cysts/six
Parorchis acanthus Posthodiplostomum sp.
Mean
mice
f S.D.
1425 61.5
0 40 f 7.0 94 i9.6 46 f6.2 95 i8.8
Posthodiptostomum nanum
228
Po~thodiplostomaides leonensis
594
Cl~nos~orn~ titapiae
294
Cysts/six
Mean f SD.
Cysts/three trials
984 542
0 0
642 491
272
60 f 10.5 0
246
92 + 10.6
218
mice
expressed as described above. Excystation data were analysed for statistical significance by using Student’s t-test. The results of the excystation studies are summarized in Table 1. There were no differences in excystation in any of the species in mammalian saline at 39°C compared with those in the mouse peritoneal cavity. For at least one species, C. tiZu@e, intrinsic enzymes are probably involved in excystation, aided by elevated temperature and a physi~ochemical environment simulated by either the mouse peritoneal cavity or mammalian saline. Extrinsic enzymes are not needed for excystation of C. tilapiae. On the other hand, extrinsic enzymes are an absolute requirement for excystation of P. acanthus. In previous investigations Asanji & Williams (1975, 1985) successfully excysted the metacercariae of P. acanthus in 100 ml normal saline in which 1 g each of sodium bicarbonate and trypsin were dissolved. For Posthodiplostomum nanum the extrinsic enzyme pepsin enhances excystation but is not obligatory. For both Posthodipiosto~um sp. and Posthodiplostomoides feonensis, acid pepsin appears to be an absolute requirement. In these experiments acid saline could not be substituted for acid pepsin. For the four species of metacercariae that excysted in the peritoneal cavity of mice or in vitro (Table 1) the f-test did not reveal any significant difference. It is clear that there is great diversity in the conditions involved in the complex process of excystation in the Digenea and that each species must be examined individually. Also, the physicochemical environment of the mouse peritoneal cavity as an ectopic site for excystation appears no better than that of mammalian saline at 39”C, at least for the species of digeneans studied here. Although Howell (I 976) reviewed the suitability of the peritoneal cavity of vertebrates as a habitat for parasites, info~ation in this review is not relevant to the peritoneal cavity as a site for metacercarial excystation.
1% acid pepsin pretreatment
No treatment
641 247
No treatment
Mean Cysts/three f S.D. trails 0 36 f 8.3 93 f 9.7 41 f 9.8 87 f9.2
622
Mean If:S.D.
781 542
0 0
231
59 A9.4 0
584 204
86 f 8.9
Dawes (1961) and Dawes & Hughes (1964) reported successful excystation of F. hepatica in the peritoneal cavity of the mouse. For at least that digenean, the mouse peritoneal cavity must provide some factor(s) not present in warm saline since F. hepatica is incapable of excysting solely in saline maintained at an elevated temperature (Fried, 1978). Acknowledgements--We
thank the Department of Zoology, Fourah Bay College, University of Sierra Leone where this study was undertaken. We are particularly grateful to Professor M. 0. Williams who supervised this investigation and to the rest of the staff of the department for their material and moral encouragement without which this investigation would not have been realized. REFERENCES ASANJIM. F. LBWILLIAMSM. 0. 1974. Studies on the excystment of trematode metacercariae in vitro. Journal of Helminthology 48: 85-91. ASANJI M. F. & WILLIAMSM. 0.
excystment
of
trematode
1975. Studies on the metacercariae in vitro.
Parasitology Research 47: 15 l-l 63. ASANJIM. F. & WILLIAMSM. 0. 1985. Effect of age and environmental factors on the viability and excystment of metacercarial cysts of Pa~o~chjs acanthus in viva and in vitro. Parasitology Research 71: 595-601. &RAY
J.
C. 1969. Experimental fascioliasis in Australia. In: DAWES B.), pp, 95-210. Academic Press, London. DAWESB. 1961, Juvenile stages of Fasciofa hepatica in the liver of the mouse. Nature 190: 646-647. DAWESB. & HUGHESD. A. 1964. Fascioliasis: the invasive stages of Fasciola hepatica in mammalian hosts. In: Advances in Parasitology, Vol. II (Edited by DAWESB.), pp. 97-168. Academic Press, London. DIXONK. E. 1966. The physiology of excystment of the metacercariae of Fasciola hepatica L. Parasitology 56: 431Advances in Parasitology, Vol. VII (Edited by
456.
FRIED B. 1978. Trematoda. In: Methods of Cultivating Parasites in Vitro (Edited by TAYLOR A. E. R. and BAKER J. R.), pp. 151-192. Academic Press, London. HOWELL M. J. 1976. The peritoneal cavity of vertebrates, In: Ecological Aspects of Par~ito~0g.v (Edited by KENNEDY C. R.), pp. 243-268. North Holland, Amsterdam.
Parasitology
Vol. 20, No. 2. pp. 257-258,
1990 0
1990 Awtrolim
002&7519/90 $3.00 + 0.00 Pergamon Press plc Societyfor Parosiro/ogy
RESEARCH NOTE EXCYSTATION OF FIVE DIGENEAN METACERCARIAE IN VITRO AND IN THE PERITONEAL CAVITY OF THE MOUSE MOSES FON ASANJI* and BERNARDFRIED? * Department of Biological Sciences, Ecole Normale Superieure, Bambili, Mezam Division, North West Province, Republic of Cameroon, Africa t Department of Biology, Lafayette College, Easton, PA 18042, U.S.A. (Received 23 March 1989; accepted 22 November 1989)
M. F. and FRIEDB. 1990. Excystation of five digenean metacercariae in vitro and in the peritoneal cavity of the mouse. International Journal for Parasitology 20: 257-258. Excystation of metacercariae of Parorchis acanthus, Posthodiplostomum nanum, Posthodiplostomum sp., Posthodiplostomoides leonensis and Clinostomum tilupine in mammalian saline at 39°C was similar to their excystation in mouse peritoneal cavity. Extrinsic enzymes were not needed for C. tihzpiae excystation but were an absolute necessity for P. acanthus excystation. Extrinsic pepsin enhanced P. nanum excystation but was not obligatory. Acid pepsin was an absolute requirement for Posthodiplostomum sp. and Posthodiplostomoides leonensis excystation. It was noted that acid saline could not be substituted for acid pepsin and that there was diversity in the condition involved in digenean excystation. Abstract-AsANJr
INDEX KEY WORDS: Digenean; Parorchis acanthus; Posthodiplostomum nanum; Posthodiplostomum sp.; Posthodiplostomoides leonensis;Clinostomumtilapiae;metacercariae; excystment; mouse; peritoneal cavity; in vitro;enzymes.
IN a previous
study (Asanji & Williams, 1974) it was the experimental hosts of Parorchis acanthus, Posthodiplostomum sp., Posthodiplostomum nanum, Posthodiplostomoides leonensis and Clinostomum tilapiae were the piscivorous birds Stern sandvicensis, S. hirundo and the domestic chicken, Gallus gallus. In these experimental hosts P. acanthus establishes in the intestine while Posthodiplostomum sp. and P. leonensis establish in the duodenum and intestine. P. nanum and C. tilapiae are restricted to the stomach of their experimental hosts. Incidental observations in that study were made on the excystation of these species in the peritoneal cavity of mice, a site devoid of intestinal enzymes. Observations on the excystation of metacercariae of Fasciola hepatica in the mouse peritoneal cavity have been reported by Dawes (1961) and Dawes & Hughes (1964). Dixon (1966) explained the excystment of F. hepatica metacercariae in the peritoneal cavity as a response to stimulation from low concentrations of carbon dioxide. Boray (1969) noted excystment of F. hepatica metacercariae in the peritoneal cavity of sheep and suggested that excystment of this species is an active process not requiring host digestive enzymes. The present study reports observations on the excystation of the above named five species of digeneans in the mouse peritoneal cavity. These results are compared with controlled excystation studies done in vitro in mammalian saline (acidified Earle’s BSS pH 2.5) at 39°C. These studies allow for observations on the role of intrinsic (those of parasite origin) vs
noted
that
257
extrinsic (those of host origin) enzymes on excystation. For most species of trematodes some combination of both intrinsic and extrinsic enzymes are involved in the complex process of excystation, although the exact mechanisms are obscure. For implantation procedures, mice were prepared for surgery as described by Asanji & Williams (1974). For all excystation experiments in the mouse peritoneal cavity and in vitro there were two groups of metacercariae for each of the five species of trematodes used, i.e. those pretreated in 1% acid pepsin and those untreated. A total of 12 mice was used for each species of metacercaria (six for pretreated and six for untreated). For P. acanthus a total of 1425 and 984 pretreated and untreated metacercariae, respectively were used while 615, 288, 594 and 294 pretreated and 542, 272, 641 and 247 untreated metacercariae of Posthodiplostomum sp., P. nanum, P. leonensis and C. tilapiae, respectively, were introduced into the peritoneal cavity of mice. Some cysts were treated for 2 h in 1% acid pepsin (1 g enzyme dissolved in 100 ml 0.01 N-NC) and pH adjusted with 1% NaHCO, to 2.5) prior to implantation and others in mammalian saline were not treated and then implanted in mice. Pepsin and Earle’s BSS were obtained from the British Drug House (BDH), U.K. All in vitro experiments were done in triplicate and the results are expressed as the mean per cent of the three trials. The in vitro cultures were done in 25 ml of either 1% acid pepsin in saline pH 2.5 or mammalian saline (pH 2.5) at 39°C. Each trial was repeated three times and the results were
M. F. ASANJIand B. FRIED
258
TABLEI-PER CENTEXCYSTATION AFTER6 h INMOUSE PERITONEAL CAVITY ORin vim In vitro
Mouse peritoneal cavity
1% acid pepsin Species
pretreatment Cysts/six
Parorchis acanthus Posthodiplostomum sp.
Mean
mice
f S.D.
1425 61.5
0 40 f 7.0 94 i9.6 46 f6.2 95 i8.8
Posthodiptostomum nanum
228
Po~thodiplostomaides leonensis
594
Cl~nos~orn~ titapiae
294
Cysts/six
Mean f SD.
Cysts/three trials
984 542
0 0
642 491
272
60 f 10.5 0
246
92 + 10.6
218
mice
expressed as described above. Excystation data were analysed for statistical significance by using Student’s t-test. The results of the excystation studies are summarized in Table 1. There were no differences in excystation in any of the species in mammalian saline at 39°C compared with those in the mouse peritoneal cavity. For at least one species, C. tiZu@e, intrinsic enzymes are probably involved in excystation, aided by elevated temperature and a physi~ochemical environment simulated by either the mouse peritoneal cavity or mammalian saline. Extrinsic enzymes are not needed for excystation of C. tilapiae. On the other hand, extrinsic enzymes are an absolute requirement for excystation of P. acanthus. In previous investigations Asanji & Williams (1975, 1985) successfully excysted the metacercariae of P. acanthus in 100 ml normal saline in which 1 g each of sodium bicarbonate and trypsin were dissolved. For Posthodiplostomum nanum the extrinsic enzyme pepsin enhances excystation but is not obligatory. For both Posthodipiosto~um sp. and Posthodiplostomoides feonensis, acid pepsin appears to be an absolute requirement. In these experiments acid saline could not be substituted for acid pepsin. For the four species of metacercariae that excysted in the peritoneal cavity of mice or in vitro (Table 1) the f-test did not reveal any significant difference. It is clear that there is great diversity in the conditions involved in the complex process of excystation in the Digenea and that each species must be examined individually. Also, the physicochemical environment of the mouse peritoneal cavity as an ectopic site for excystation appears no better than that of mammalian saline at 39”C, at least for the species of digeneans studied here. Although Howell (I 976) reviewed the suitability of the peritoneal cavity of vertebrates as a habitat for parasites, info~ation in this review is not relevant to the peritoneal cavity as a site for metacercarial excystation.
1% acid pepsin pretreatment
No treatment
641 247
No treatment
Mean Cysts/three f S.D. trails 0 36 f 8.3 93 f 9.7 41 f 9.8 87 f9.2
622
Mean If:S.D.
781 542
0 0
231
59 A9.4 0
584 204
86 f 8.9
Dawes (1961) and Dawes & Hughes (1964) reported successful excystation of F. hepatica in the peritoneal cavity of the mouse. For at least that digenean, the mouse peritoneal cavity must provide some factor(s) not present in warm saline since F. hepatica is incapable of excysting solely in saline maintained at an elevated temperature (Fried, 1978). Acknowledgements--We
thank the Department of Zoology, Fourah Bay College, University of Sierra Leone where this study was undertaken. We are particularly grateful to Professor M. 0. Williams who supervised this investigation and to the rest of the staff of the department for their material and moral encouragement without which this investigation would not have been realized. REFERENCES ASANJIM. F. LBWILLIAMSM. 0. 1974. Studies on the excystment of trematode metacercariae in vitro. Journal of Helminthology 48: 85-91. ASANJI M. F. & WILLIAMSM. 0.
excystment
of
trematode
1975. Studies on the metacercariae in vitro.
Parasitology Research 47: 15 l-l 63. ASANJIM. F. & WILLIAMSM. 0. 1985. Effect of age and environmental factors on the viability and excystment of metacercarial cysts of Pa~o~chjs acanthus in viva and in vitro. Parasitology Research 71: 595-601. &RAY
J.
C. 1969. Experimental fascioliasis in Australia. In: DAWES B.), pp, 95-210. Academic Press, London. DAWESB. 1961, Juvenile stages of Fasciofa hepatica in the liver of the mouse. Nature 190: 646-647. DAWESB. & HUGHESD. A. 1964. Fascioliasis: the invasive stages of Fasciola hepatica in mammalian hosts. In: Advances in Parasitology, Vol. II (Edited by DAWESB.), pp. 97-168. Academic Press, London. DIXONK. E. 1966. The physiology of excystment of the metacercariae of Fasciola hepatica L. Parasitology 56: 431Advances in Parasitology, Vol. VII (Edited by
456.
FRIED B. 1978. Trematoda. In: Methods of Cultivating Parasites in Vitro (Edited by TAYLOR A. E. R. and BAKER J. R.), pp. 151-192. Academic Press, London. HOWELL M. J. 1976. The peritoneal cavity of vertebrates, In: Ecological Aspects of Par~ito~0g.v (Edited by KENNEDY C. R.), pp. 243-268. North Holland, Amsterdam.
