The suppression, by Helisoma duryi, of the cercarial production of Schistosoma mansoni-infected Biomphalaria pfeifferi

The suppression, by -Helisoma duryi, of the cercarial production of Schistosoma mansoni-infected Biomphalaria pfeiferi FLEMMING FRANDSEN1 Biological control of the intermediate hosts of Schistosoma mansoni and S. haemato- bium by means of a competitor snail, Helisoma duryi, has been suggested. In the present laboratory study, the influence of H. duryi on the relationship between the parasite and the intermediate host was investigated. The results indicated that H. duryi behaves as a " decoy " if it is present when Biomphalaria pfeifferi is exposed to the miracidia of S. man- soni, and that the continued presence of H. duryi in the aquarium after B. pfeifferi has been exposed greatly reduces cercarial production. When these two species were present in equal numbers, cercarial production was reduced by 95.9VO in comparison with a control group. Biological control experiments so far have been based chiefly on the use of predators and diseases to limit the population of the undesired species. These methods have been reviewed by a number of authors, including Michelson (6), Radke et al. (7), and Chernin (2), and recently Berg (1) has described methods of controlling snail-borne diseases. A method of biological control that has not received much attention is based on the observation that closely related species with an almost identical way of life may compete with each other, with the result that one species thrives while the other declines. This may be due to a more efficient food uptake, better survival of the youngest phases, or the presence of population-limiting material. At the Danish Bilharziasis Laboratory, Dr G. Mandahl-Barth has initiated a series of experiments designed to investigate the feasibility of using the American planorbid Helisoma duryi as a competitor, for the biological control of Biomphalaria pfeifferi. The research reported here was carried out as part of that investigation. Its aim was to determine the influence of H. duryi on cercarial production in B. pfeifferi and its usefulness as a decoy-i.e., a snail that will take up miracidia but not become infected. The literature has been reviewed, and laboratory and field experiments relating to H. duryi have been 1 Assistant Director, Danish Bilharziasis Laboratory, Jaegersborg A116 1 D, 2920 Charlottenlund, Denmark. carried out, by 0. Rasmussen (unpublished observa- tions, 1976). MATERIALS AND METHODS Parasite strain Schistosoma mansoni from Pachwach, Uganda, was established in 1969 at the Danish Bilharziasis Laboratory. At the laboratory, B. pfeifferi and white mice (NMRI strain) have been used as the intermediate and definite hosts, respectively. Intermediate host and control snail B. pfeifferi from Tanzania (collected by 0. Ras- mussen in Kakonde, Nyanguge, and Ngudu in 1971) and from Cameroon (collected by Ngalle- Edimo in Yaounde in 1972) was used as the inter- mediate host. The control snail was H. duryi, imported from Florida, USA, by the Danish Aquarium together with aquatic plants in 1941, and maintained at the laboratory since 1964. Infection procedure The miracidia were obtained as described by Frandsen (4). Each snail was exposed to 5-12 mira- cidia. Exposure took place at 25-27°C for 13 h in the light, either in bowls containing 150 ml of pond water per 20 snails or in troughs (3) containing the same ratio of pond water to snails. After exposure, the snails were maintained in glass aquariums containing gravel, pond water, 3450 - 385 - BULL. WORLD HEALTH ORGAN., Vol. 53, 1976 F. FRANDSEN Table 1. Methods of exposure to miracidia and main- tenance of B. pfeifferi in relation to H. duryi Experimental Exposure of B. pfeifferi Maintenance of group to miracidia B. pfeifferi (control) alone in bowls alone 11 with H. duryi in bowls together with H. duryi III alone in bowls together with H. duryi 1 2 h after exposure IV with H. duryi in bowls alone V with H. duryi in troughs together with H. duryi Daphnia pulex, and Sagittaria natans. The size of the aquarium and the quantity of food were adjusted to the number of snails, 5 litres of water being allowed for every 20 snails. Experimental design The experimental design is presented in Table 1. In each experiment the snails were examined on the 25th day after exposure, to determine the number of infected specimens, the cercarial production for each individual snail, the number of dead B. pfeifferi and H. duryi, and the diameter of B. pfeifferi. These data were collected regularly every 7-10 days until all positive snails had died. The cercarial production per 100 exposed B. pfeifferi was calculated as follows. The total cercarial production per test group at each counting was multiplied by the number of days between countings, the product being multiplied by 100 and divided by the number of snails in the exposed group. RESULTS The results of two of the experiments (groups II and V) are presented in Table 2. A similar pattern of results was observed in subsequent experiments and, since no difference was observed in the results whether the snails were maintained in bowls or in troughs, further experiments were conducted using only bowls. The use of bowls for exposure to miracidia made it possible to simulate natural con- ditions, in which different snail species are mixed. The results of these experiments are summarized in Table 3. It was observed that the infection rate in the con- trol group was very high as regards both the total number of exposed snails and the number of snails surviving after the 30th day following exposure to miracidia. The infection rate in the control group was three times as high as that in the group in which the two species were exposed and maintained together in equal numbers (group II), and it was noted that the infection rate decreased when the number of H. duryi present increased. In addition, there was a remarkable increase in the death rate in group II, in comparison with that in the control group, both at 30 and at 60 days after exposure. Again, the death rate increased with increasing numbers of H. duryi. The average diameter of B. pfeifferi, measured at 30 and at 60 days after exposure, was smaller for all the experimental groups than it was for the control Table 2. Results of exposing B. pfeifferi (Kakonde, Tanzania) and H. duryi together, in various proportions, to miracidia in bowls and troughs (12 miracidia per B. pfeifferi) Cecrar-Longest dur- B.pfiffe No. of Cercarianl pro ation ofExperiment B. pdeuffer: B. pfeifferi exposed cercarial pro-yi posit ductionExperiment H. duryi positive ~~snails (days) B. pfeifferi and H. duryi exposed together in 10: 0 7 236 160 47 a bowl (150 ml) 10:3 3 9440 19 10:10 1 520 13 H. duryi as - decoy - between the miracidia 10:0 7 475 680 57 and B. pfeifferi in trough (1 50 ml) 10:3 4 31240 27 10:10 0 0 0 386 SCHISTOSOMIASIS: SNAIL CONTROL Table 3. Summarized results of exposing B. pfeifferi (B.p.) and H. duryi (H.d.) separately and together to miracidia of S. mansoni natoSnails Infected Deathe Diameter Cercarial productionNo.ofSninfete survivors rat () (mm) ofExperimental group No otf NH of B.d: iNfected after of exposed Bp after- Total per Maxi- LongestExperimenalg B.p. H.d. H. d. No / 30 days Snails after: ______ 100 mum per duration No. % (% 30d 0 30 d 60 d snails snail (days)30d60d ~~~exposed 1. B.p. alone 165 0 1: 0 104 63.4 75.4 1 6.4 42.4 6.5 7.5 486 656 495 48 II. B.p. and H.d. ex- 250 250 1: 1 47 18.8 26.7 29.6 57.2 5.3 5.9 19 923 162 25 posed and maintained 35 70 1: 2 3 8.6 16.6 48.5 82.8 4.3 4.5 7 920 53 13 together 25 75 1: 3 4 16.0 23.5 32.0 88.0 4.0 4.6 5 150 46 12 Ill. B.p. exposed and 85 85 1: 1 27 31.8 51.9 39.0 67.0 5.4 6.7 76 692 196 35 1 2 h later maintained with H.d. IV. B.p. and H.d. ex- 45 45 1: 1 16 35.5 64.0 44.4 60.0 7.6 8.3 564 790 660 61 posed together and H.d. removed after 12 h. group. In the experiment in which there were three times as many H. duryi as B. pfeifferi, the average diameters of the latter at days 30 and 60 were only 61.5% and 61.3%, respectively, of those of the control snails. Where H. duryi and B. pfeifferi were present in the same proportions, the cercarial production per 100 snails was 4.10% of that of the control group. When the two species were present in the proportions of 2: 1 and 3:1, the cercarial production was 1.6 % and 1.1%, respectively, of that of the control group. Maximum cercarial production per snail per day was found to be 495, for the control group. This number decreased with an increase in the number of H. duryi present. A similar observation was made for the duration of cercarial production by the snails. When B. pfeifferi were placed in the aquarium together with H. duryi 12 h after the B. pfeifferi had been exposed alone to miracidia (group III), the infection rate for snails present in equal numbers was higher than that observed for the group in which they were exposed and maintained together (group II), but lower (by 500O) than that of the control group. A corresponding pattern was found for the remaining data. Similar results for the infection rate were obtained for group IV, in which B. pfeifferi was exposed to miracidia together with H. duryi, removed 12 h later, and subsequently maintained alone. However, for this group, the average diameter of B. pfeifferi; the cercarial pro- duction per 100 exposed snails; the maximum cercarial production for one snail; and the longest duration of cercarial production were nearly Table 4. Results of an experiment in which B. pfeifferi (Nyanguge, Tanzania) was exposed to miracidia together with H. duryi in various proportions (group 11; 6 miracidia per B. pfeifferi) Cecaia po Longest dur-B. pfeifferi: No. of snails drcarin/al p0re- ation of cer-H. duryi positive posed snailscrxalpodno -tion (days) 10:0 8 230 980 32 10:10 2 50140 29 10:20 2 720 4 10:30 0 0 0 identical, if not higher, than in the control group. On the other hand, there was an increase in the death rate. Detailed results from two experiments are pre- sented in Tables 4 and 5. The individual observations are slightly higher than those reported in Table 3, but a similar pattern of results may be observed. The results for the four strains of B. pfeifferi from Nyanguge, Kakonde, Kanami (all in Tanzania), and Yaounde (Cameroon) showed the same pattern of infection rates and suppression of cercarial produc- tion. Some of the results for these strains are pre- sented in Tables 2 and 4 and in Fig. 1, 2, and 3. The rate of cercarial production in three experi- ments in which different strains of B. pfeifferi were used are presented in Fig. 1, 2, and 3. Fig. 1 indicates the results obtained for the control group and for 387 F. FRANDSEN Table 5. Results of an experiment in which the exposure and maintenance of B. pfeifferi (Kakonde, Tanzania) and H. duryi were varied (6 miracidia per B. pfeifferi) Longest dur- B.pfeifferi: No. of snails Cercarial pro- ation of cer-Experiment H. duryi positive duction/100 carial pro-H.duryi positive exposed snails duction (days) control group 10: 0 8 734 910 70 B. pfeifferi and H. duryi exposed together 10: 10 5 16 830 35 and maintained in the same aquarium H. duryi removed from B. pfeifferi 12 h after 10: 10 6 727 090 75 exposure H. duryi together with B. pfeifferi 12 h after 10: 10 4 71 750 56 exposure two groups in which H. duryi and B. pfeifferi were present in proportions of 1: 1 and 3: 1, respectively. Fig. 2 and 3 show the results obtained for the A Control group * B. pfeifferi: H. duryi (1:1) * B. pfeifferi: H. duryi (1:3) Cercarial production per batch - Cercarial production per positive snail -A I/ ' tI A\ IV '*A/.x 10 20 30 40 50 60 70 80 90 100 110 120 Days after exposure W'HO 7,602 control group and for the group in which the two snail species were present in equal proportions. The snails were exposed and maintained together and, as in the previous experiments, the control group showed the highest cercarial production and the longest duration of production. Variations in the results were probably due to external physical factors, such as the influence of light. DISCUSSION The results show that the growth rate, infection rate, and cercarial production of B. pfeifferi are highly influenced by the presence of H. duryi. Inhibition of the growth of B. pfeifferi could indicate a crowding effect, as described by Wright (8), although the size of the aquarium and the amount of food placed in it were adjusted to the number of snails. The results of the experiment in which H. duryi was exposed to miracidia together with B. pfeifferi, the two species being subsequently maintained together, and the experiment in which H. duryi was removed 12 h after exposure, show not only that H. duryi functions as a decoy snail, as described by Chernin (2) but also that this snail somehow influences the development of miracidia to sporo- cysts and, finally, the production of cercariae. These experiments have been concerned primarily with determining cercarial production and not with investigating the development of the sporocyst. However, the decreased growth rate and increased death rate of B. pfeifferi in the presence of H. duryi indicate that H. duryi must have influenced the metabolism of the snail in some way. This aspect requires closer investigation. 'D ._ 4) Z 200 0 m ._; 'u 400 .0 .= 200 cJ9 z O Fig. 1. Cercarial production for one of the experiments with B. pfeifferi (Kanami, Tanzania) exposed and maintained with H. duryi. 388 SCHISTOSOMIASIS: SNAIL CONTROL Fig. 3. Cercarial production for one of the experiments with B. pfeifferi (Nyanguge, Tanzania) exposed and maintained with H. duryi. Fig. 2. Cercarial production for one of the experiments with B. pfeifferi (Kakonde, Tanzania) exposed and maintained with H. duryi. The influence of other snail species on cercarial production by B. pfeifferi has not been thoroughly investigated, but experiments in this field would be highly relevant and, in fact, are needed for a closer evaluation of these results. Preliminary experiments with infected B. pfeifferi and noninfected B. pfeifferi as a competitor snail did not give the same pattern of results, whereas the results of preliminary experi- ments with B. glabrata (F. Frandsen, unpublished observations, 1975), and H. duryi resembled those reported in this paper. In this investigation, the most emphasis has been placed on the total cercarial production in a group of exposed snails (taking 100 snails as a standard), because this aspect of the problem is of the greatest epidemiological importance. The effect of H. duryi on cercarial production when a larger volume of water is used, and its effect in the field, have not yet been investigated. If the results of field experiments bear out those of the laboratory experiments, it means that it is not necessary to aim at total competition, since a halving of the B. pfeifferi population resulted in a 95%/ reduction in cercarial production. It would appear from the results of a field experiment by Rasmussen (unpublished observations, 1976), carried out in Tanzania with H. duryi, that a very high reduction a) co 0. m 600 0 0 0 6 Z 400 200 0 WHO 76604 10 20 30 40 50 60 70 Days after exposure 389 390 F. FRANDSEN of the B. pfeifferi population occurred after the introduction of the competitor. According to the schistosomiasis model of MacDonald (5), a certain reduction in the " snail factor" may cause the population of schistosomes to reach a critical number-the " break-point "- where the parasite population can no longer be sustained. The observations made in our laboratory indicate that it may be possible to reach the break- point by using a competitor snail that has an influence on the number of intermediate hosts and on cercarial production. ACKNOWLEDGEMENTS I thank Dr G. Mandahl-Barth for his criticism of the manuscript. I am very grateful to Mrs L. Svendsen for help with the preparation of the manuscript. Thanks are due also to Mrs B. Rasmussen and Mrs B. Fredsted for their technical assistance. This work was supported by a grant from DANIDA, Ministry for Foreign Affairs, Denmark. RI2SUMt L'INHIBITION, PAR HELISOMA DURYI, DE LA PRODUCTION DE CERCAIRES CHEZ BIOMPHALARIA PFEFFERI INFECTES PAR SCHISTOSOMA MANSON1 Les resultats de cette etude montrent que, lorsque Helisoma duryi et Biomphalaria pfeifferi, presents en pro- portions egales, sont exposes aux miracidiums de Schisto- soma mansoni, la presence de la premiere espece entraine chez la seconde une reduction des taux d'infection qui passe a 18,8 %, contre 63,4% chez un groupe temoin de B. pfeifferi expose seul. En outre, le taux de mortalite chez B. pfeifferi s'accroit, alors que le diametre moyen diminue. L'observation la plus remarquable a e une reduction correspondante, chez B. pfeifferi, dans la production de cercaires calculee par groupe de 100 mollusques exposes, cette production passant de 486 000 pour le groupe temoin a 19 000 pour le groupe expose en presence d'un nombre egal de mollusques de I'autre espece. La pro- duction s'abaissait encore jusqu'au chiffre de 7900 dans le groupe oui H. duryi et B. pfeifferi etaient presents dans la proportion de 2:1. Les resultats de cette experience indiquent que, de quelque maniere, H. duryi influe sur le metabolisme de F'h6te intermediaire, B. pfeifferi, inhibant ainsi le deve- loppement du sporocyste en cercaires. REFERENCES 1. BERG, C. 0. Biological control of snail-borne diseases: a review. Experimental parasitology, 33: 318-330 (1973). 2. CHERNIN, E. Interference with the capacity of Schistosoma mansoni to infect the molluscan host. Journal ofparasitology, 54: 509-516 (1968). 3. CHERNIN, E. A system for studying "target-finding" by schistosome miracidia and other motile organisms. Experientia, 24: 973 (1968). 4. FRANDSEN, F. Host-parasite relationship of Bulinus forskalii (Ehrenberg) and Schistosoma intercalatum Fisher, 1934 from Cameroon. Journal ofhelminthology, 49: 73-84 (1975). 5. MAcDONALD, G. The dynamics of helminth infec- tions with special reference to schistosomes. Trans- actions of the Royal Society of Tropical Medicine and Hygiene, 59: 489-506 (1965). 6. MICHELSON, E. H. Studies on the biological control of schistosome-bearing snails, predators, and parasites of fresh-water snails: a review of the literature. Parasitology, 47: 413-426 (1957). 7. RADKE, H. G. ET AL. Demonstrated control of Australorbis glabratus by Marisa cornuarietis under field conditions in Puerto Rico. American journal of tropical medicine and hygiene, 10: 370-373 (1961). 8. WRIGHT, C. A. The crowding phenomenon in labo- ratory colonies of freshwater snails. Annals of tropical medicine and parasitology, 54: 224-232 (1960).