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Keywords:

  • molluscicide;
  • Jatropha curcas;
  • Biomphalaria;
  • Bulinus;
  • Schistosoma;
  • toxicity;
  • attenuation;
  • cercariae;
  • miracidia;
  • nontarget organisms

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Summary The aim of studies on plant molluscicides is to complement methods for controlling snails acting as intermediate hosts of schistosomes. We report on the toxic activity of extracts from Jatropha curcas L. (Euphorbiaceae) against snails transmitting Schistosoma mansoni and S. haematobium. We studied different extracts' effects on infectious larvae, cercariae and miracidia of S. mansoni. Compared to aqueous extract, methanol extract showed the highest toxicity against all tested organisms with LC100-values of 25 p.p.m. for cercariae and the snail Biomphalaria glabrata and 1 p.p.m. for the snails Bulinus truncatus and B. natalensis. Attenuation of cercariae leading to reduced infectivity in mice could be achieved in concentrations below those exerting acute toxicity. In view of our results and the ongoing exploitation of J. curcas for other purposes, this plant could become an affordable and effective component of an integrated approach to schistosomiasis control.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Schistosomiasis is the second most important parasitic disease after malaria in terms of overall morbidity burden. For the control of the disease, multifaceted approaches are desirable ( El Khoby et al. 1998 ), including control of the intermediate host snails. At present niclosamide (Bayluscide®, Bayer, Leverkusen, Germany) is the only molluscicide applied on a large scale ( WHO 1993). Since this synthetic molluscicide is also toxic to fish ( Andrews et al. 1983 ) and frequently not affordable in poor, schistosomiasis-endemic areas, alternative possibilities for snail control need to be evaluated. Plants with molluscicidal activity may be exploited to contribute to schistosomiasis control, particularly if they are already grown locally for other purposes.

  Jatropha curcas L. (Euphorbiaceae) is widespread in tropical and subtropical areas, planted as ‘living fences’ around fields and settlements. It can easily be grown on almost any soil and therefore prevents erosion. Most parts of the plant and their extracts are used in traditional medicine, e.g. for their antimicrobial properties ( Thomas 1989). Oil from the seeds can be used as a substitute for diesel and for producing soap ( Henning & von Mitzlaff 1995), and press cake from the seeds provides organic manure ( Sherchan et al. 1989 ). As J. curcas is available and used for various purposes in many tropical areas, exploitation of its molluscicidal properties appears to be viable.

Our previous studies in small test volumes indicated toxic activity of J. curcas against intermediate host snails of S. mansoni and S. japonicum ( Liu et al. 1997 ; Rug et al. 1997 ). Therefore, we decided to study the effects of extracts from J. curcas on snails following WHO (1965) guidelines. Here we report on tests with Biomphalaria glabrata (intermediate host of S. mansoni which causes intestinal schistosomiasis), Bulinus truncatus and B. natalensis (intermediate hosts of S. haematobium, the agent of urinary schistosomiasis). We also tested extracts for possible toxic activity against larval stages of S. mansoni to further explore the potential of J. curcas to interfere with the life cycle of schistosomes. Finally, we determined the toxicity of the plant against selected other aquatic animals.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant material and extracts

Plant extracts were prepared and analysed at the Institute of Pharmaceutical Biology, University of Heidelberg. Briefly, aqueous extracts of ground seeds were prepared according to Liu et al. (1997) and Rug et al. (1997) ; crude oil was obtained by mechanical pressing of ground seeds, and a methanol extract of the crude oil was prepared to enrich for phorbol esters ( Adolf et al. 1984 ; Hirota et al. 1988 ). Crude oil and methanol extract showed the typical phorbol ester pattern of J. curcas in high pressure liquid chromatography (HPLC) ( Makkar et al. 1997 ). This was absent in the aqueous extract. The solvents were removed in a rotavapor.

For molluscicidal and larvicidal testing, all preparations, except for the aqueous extract, were first homogenously suspended in dimethylsulfoxide (DMSO; 1 : 10 w/v); further dilutions were prepared in water.

Test organisms

Snails

Biomphalaria glabrata (Puerto Rican strain) were reared in our laboratory, and used at a uniform size of 8–10 mm. Laboratory-bred Bulinus natalensis were obtained from the University of Wales, UK and Bulinus truncatus were collected in the field in Egypt.

Schistosome larvae

The life-cycle of S. mansoni was maintained by passage through mice (NMRI-strain) and B. glabrata snails. Infected snails were exposed to light for 30 min and the cercariae shed during this period were pooled and counted (6 × 50 μl). Livers of mice infected with S. mansoni were passed through a sieve and schistosome eggs were collected after sedimentation in saline solution. The eggs were suspended in tap water and hatched miracidia collected after 30 min. All larvae were diluted to approximately 1000 per ml.

Control organisms

Crustaceans belonging to the orders Onychura (Polyphemus spec.) and Ostracoda (unidentified species) were collected from ponds of the Botanical Garden in Heidelberg. Individuals of the same genus were selected under a dissecting microscope. Tubifex tubifex (Annelida) were collected from our snail breeding tanks and taken as representative inhabitants of eutrophic water bodies.

Test systems

All tests were carried out in deionized water. Stock solutions of plant extracts were prepared in DMSO (10%; w/v) and further diluted in water. Toxicity is expressed as LC50 and LC100, referring to concentrations killing 50% and 100% of the organisms, respectively.

Toxicity tests with snails

Tests with B. glabrata, B. natalensis and B. truncatus were performed according to WHO guidelines (WHO 1965). Groups of 10 snails were placed in glass containers with 400 ml of water containing the test substances. Tests were carried out at 26 °C. Snails were prevented from crawling out of the containers by a fine stainless steel mesh suspended just above the water surface. After 24 h of incubation the snails were transferred to deionized water and maintained for another 48 h. Death of the snails was determined by absent heartbeat and lack of reaction to irritation of the foot with a needle. Control experiments were performed with DMSO alone and with Bayluscide®. All tests were independently repeated three times.

Toxicity tests with schistosome larvae

Tests were performed in 24-well plates (Greiner, Nürtingen, Germany) with 100 μl of test solution per well to which 100 μl of water containing cercariae or miracidia were added. The plates were incubated at room temperature with slow horizontal rotation (15 r.p.m.). Every 20 min the number of dead larvae was recorded by absence of movement (miracidia) and a protruding sucker (cercariae) ( Haas 1984). After 2 h of incubation, a fixative (Bouin) was added and the total number of larvae was determined for each well. Tests were conducted at least three times in duplicate. Control experiments were set up as described for snails.

Toxicity tests with control organisms

Tests were carried out in Petri dishes (9 cm diameter; Greiner, Nürtingen, Germany) containing a determined number of control organisms in a minimal amount of water. Test solutions (10 ml) were added using samples from the containers wherein the snail tests were subsequently performed. After incubation for 24 h, the number of dead individuals was determined by irritating their foot with a needle. Each test was performed three times independently.

Attenuation of cercariae

To determine the attenuating effect of J. curcas methanol extract on cercariae, the larvae were exposed to sublethal concentrations under conditions for toxicity tests described above. For each concentration of the extract, six wells were prepared. After 2 h of incubation, the cercariae of three wells were used to immediately infect three mice (the number of dead larvae had been counted previously). The remaining three wells were used to determine the total number of cercariae per well (mean ± SD), i.e. the number of cercariae given to each mouse. Infections were performed with the ring method ( Smithers & Terry 1965), i.e. leaving the cercarial suspension on the shaved abdominal skin of the anaesthetized (sodium pentobarbiturate) mice for 45 min. Control infections were either done with cercariae exposed to the highest concentration of DMSO used to dilute extracts or with cercariae kept in water for 2 h. Six weeks after infection the mice were killed by a lethal injection of sodium pentobarbiturate, perfused, and the number of worms was counted. Livers and guts were squashed between glass plates to reveal unperfused worms.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Toxic activity of J. curcas against snails was determined for crude oil, methanol and aqueous extracts. With B. glabrata the methanol extract of the oil showed the highest activity with values for LC50 = 5 p.p.m. and LC100 = 25 p.p.m. ( Fig. 1). By comparison, for crude oil we obtained values of LC50 = 50 p.p.m. and LC100 = 100 p.p.m., while the aqueous extract performed poorly with an LC50 of 5000 p.p.m. ( Fig. 1). Bayluscide® was used as a reference substance and killed all B. glabrata at 1 p.p.m.; control assays with DMSO showed no effect on the snails (data not shown).

image

Figure 1. Toxicity of different extracts from Jatropha curcas seeds against intermediate host snails of schistosomes. Snails were exposed to the following test substances for 24 h under standard conditions (WHO 1965): ●, methanol extract; ▪, crude oil; ▴, aqueous extract. Open symbols (○): (-–-) Bulinus truncatus and (—) Bulinus natalensis; closed symbols: Biomphalaria glabrata; Each curve represents data pooled from three independent experiments.

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Methanol extract showed even higher toxic activity against snails of the genus Bulinus than B. glabrata. LC50 and LC100 values for both B. truncatus and B. natalensis were 0.2 p.p.m. and 1 p.p.m., respectively ( Fig. 1).

Cercariae and miracidia were also exposed to the three extracts. The dose and time dependency was determined for each of the extracts and with both types of larvae ( Fig. 2). As an example for a full set of data obtained with all types of extracts and larvae, Fig. 2(a) shows the toxicity of oil for cercariae: 250 p.p.m. killed all cercariae in 80 min, while 100 p.p.m. were sufficient to kill all larvae within 2 h. To compare directly the results for all extracts tested against cercariae and miracidia, we selected the toxicity values obtained after 2 h for the subsequent condensed presentations ( Fig. 2b,c). As observed with snails, methanol extract was most active followed by oil and aqueous extract. With each preparation, miracidia were at least 10 times less sensitive than cercariae. These results show that snails and cercariae are both highly sensitive to 25 p.p.m. of the methanol extract with the cercariae being killed in a much shorter period of time. By comparison, Bayluscide® was slightly more toxic to cercariae than the methanol extract (LC100 = 5 p.p.m.) and showed the same toxic activity against miracidia (LC100 = 1000 p.p.m.). DMSO had no toxic effect on either type of larvae at concentrations used in the assays (data not shown).

image

Figure 2. Time- and dose-dependent toxic activity of different extracts from Jatropha curcas seeds against larvae of Schistosoma mansoni. Larvae were exposed to test substances and dead organisms counted every 20 minutes for 2 h. (a) Time dependent toxicity against cercariae was determined for crude oil at (— - -) 250 ppm, (——) 100 ppm, (– – –) 50 ppm and (- - - -) 25 ppm. Dose dependent toxicity against (b) cercariae and (c) miracidia was measured for (●) methanol extract, (▪) crude oil or (▴) aqueous extract as shown in (a), but only values obtained after 2 h of incubation were plotted. Each curve represents pooled data from three independent experiments in duplicates.

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Dramatic surface alterations were induced in both types of larvae by incubation with methanol extracts of J. curcas. We observed vesiculation of the cercarial surface ( Fig. 3a), and vesiculation and shedding of plates from the surfaces of miracidia ( Fig. 3b).

image

Figure 3. Surface alterations on larvae of S. mansoni after incubation with methanol extract from J. curcas seeds. (A) Head of cercaria with vesicles (arrowheads) budding from the surface; (B) Miracidium with vesicles (arrowheads) and shed epidermal plates (arrows). Note: Cilia of the epidermal plates appear as a halo. Bar = 25 μm.

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A possible attenuating effect on cercariae was tested with methanol extract, as this was the most active. Cercariae which had been exposed to various concentrations of this extract were used to infect mice. Six weeks after infection the resulting worms were counted. As shown in Fig. 4, the extract exerted a significant attenuating effect on the cercariae. Treatment of cercariae with 10 or 15 p.p.m. reduced their ability to develop to worms by approximately 50%, although acute toxicity to cercariae was negligible at this concentration.

image

Figure 4. Reduced infectivity of S. mansoni cercariae after treatment with methanol extract from J. curcas seeds. Cercariae were exposed to the indicated concentrations of the methanol extract for 2 h and used to infect mice. Infectivity of cercariae was determined 6 weeks after infection from the worm counts in mice (□) and toxicity of the extract against cercariae was recorded immediately after 2 h incubation (▪). Mean values (±SD) were obtained from three mice or cercarial counts for each extract concentration and tests were performed two times independently. Data are expressed percentages of values derived from untreated cercariae. Note that infectivity of cercariae was significantly (*) reduced at 10 or 15 p.p.m., although acute toxicity of the extract against the larvae was only marginal.

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To obtain a rough estimate of the ecological tolerance to J. curcas extracts, their toxicity against a few other freshwater organisms was tested. We chose genera occurring in snail breeding sites in Mali which could also be obtained in Heidelberg. Using crude oil, tested animals were not affected at concentrations which killed all cercariae and snails. Aqueous extract had the same, relatively low toxic effect on target and nontarget organisms (data not shown). Methanol extract, again the most potent preparation, only affected Polyphemus spec. at concentrations which killed both snails and cercariae ( Fig. 5). Ostracoda ( Fig. 5) and Tubifex tubifex (not shown) were unaffected at concentrations lethal for the target organisms.

image

Figure 5. Toxicity of different extracts from J. curcas seeds against crustaceans. Open symbols: Polyphemus spec.; closed symbols: ostracods; (○, ●) methanol extract, (□, ▪) crude oil.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The results demonstrate molluscicidal and larvicidal activity of all tested preparations from seeds of J. curcas. We extended our previous work on Biomphalaria glabrata ( Liu et al. 1997 ; Rug et al. 1997 ) by determining the toxic activity according to standards required for the screening of molluscicides ( WHO 1965) and by measuring the toxicity of J. curcas against snails of the genus Bulinus and against miracidia and cercariae of S. mansoni.

Irrespective of whether snails or schistosome larvae were tested, the toxic activity of J. curcas was highest in methanol extracts and lowest in aqueous extracts of the seeds. With methanol extract, LC50 values were extremely low for both Bulinus species (0.1 p.p.m.) compared to those for B. glabrata (5 p.p.m.; this report) and Oncomelania hupensis (intermediate host of S. japonicum; 2.5 p.p.m.; Liu et al. 1997 ; Rug et al. 1997 ). Similarly, cercariae were more sensitive to all tested extracts than miracidia. At present, we have no obvious explanation for these differences.

Whereas we used extracts from seeds of J. curcas, roots of this plant were extracted in an earlier study with ethanol or water and tested against B. truncatus from Sudan ( El Kheir & El Tohami 1979). LD50 values for ethanol root extract were much higher (60 p.p.m.) than the values we obtained for any snail using the seed extract. Whether this different level of toxicity is related to the geographical origin of J. curcas (Mali vs. Sudan), the extract source (seed vs. roots), or other reasons remains to be clarified.

The most important toxic compounds in extracts of J. curcas are likely to be phorbol esters, which are enriched as hydrophobic molecules by methanol extraction ( Hirota et al. 1988 ). This hypothesis is supported by two observations. First, we demonstrated the toxicity of commercially available phorbol esters against B. glabrata and O. hupensis ( Liu et al. 1997 ). Second, formation of vesicles on the surfaces of schistosomes occurred after incubating miracidia and cercariae with methanol extracts, which can be induced by phorbol esters ( Wiest et al. 1994 ).

Phorbol esters are known to directly activate protein kinase C (PKC) ( Castagna et al. 1982 ). This key enzyme of signalling cascades plays a critical role in maintaining the integrity of the schistosome surface ( Wiest et al. 1994 ). Activation of PKC by phorbol esters may lead to phosphorylation of different proteins and a consequent reorganization of the cell cytoskeleton ( Bershadsky et al. 1990 ). PKC also regulates the activity of ion channels, which may lead to vesicle formation on the parasite surface, as observed for schistosomes treated with praziquantel ( Xiao et al. 1984 ) or with a pore-forming toxin ( Ruppel et al. 1987 ). Thus we hypothesize that the phorbol esters in extracts of J. curcas induce osmolaric instability, surface vesiculation and subsequent death of both cercariae and miracidia.

Aqueous extract of J. curcas seeds was much less toxic against intermediate host snails and schistosome larvae, possibly due to hydrophilic components in J. curcas such as saponins, curcin, phytates and protease inhibitors ( Morgue et al. 1961 ; Stirpe et al. 1976 ; Makkar et al. 1997 ). Saponins are known to possess molluscicidal properties ( Lemma 1965; Hostettmann et al. 1982 ) and may contribute to toxicity, as this remained stable after boiling the aqueous extract for 5 min (unpublished). However, the high dose of aqueous extract required for killing snails and larvae, which also affects nontarget organisms, does not favour its use in the field.

Crude oil and methanol extract of J. curcas, however, have most of the characteristics desirable in a molluscicide ( Kloos & McCullough 1982): Acute toxic activity against infective larvae of the parasite was observed at molluscicidal concentrations. Cercariae lost infectivity at even lower concentrations. By using J. curcas in the field, the reduced infectious potential of cercariae might not only diminish the risk of infection, but attenuated cercariae might also be able to induce resistance against infection in analogy to cercariae attenuated by radiation ( Shi et al. 1993 ).

Secondly, toxicity to nontarget organisms may be limited as suggested by our present data, by nontoxicity of crude oil to fish (Cyprinus carpio) at molluscicidal concentrations in preliminary experiments (Y.L. Li, personal communication). Similarly, no mortality was observed among C. carpio fed on a diet containing up to 1000 p.p.m. of phorbol esters from J. curcas seeds for 7 days ( Becker & Makkar 1998). Phorbol esters of J. curcas decompose completely within 6 days (C. Koschmieder; personal communication).

Effective exploitation of a plant-derived compound depends on a sufficient supply of plant material. In the case of J. curcas, which is abundant in tropical and subtropical areas and adapted to arid climates ( Heller 1996), exploitation seems to be feasible. The perennial J. curcas is easily propagated, grows quickly and, since only the seeds are needed for snail control, the plant would not need to be destroyed to obtain molluscicidal preparations.

The supply of J. curcas could be ensured by multipurpose exploitation of the plant. The seeds are used as a source for combustibles (substitute for diesel oil), as organic manure or in traditional soap production ( Sherchan et al. 1989 ; Henning & von Mitzlaff 1995). Extracts of J. curcas show insecticidal effects against crop pests such as Helicoverpa armigera and Sitophilus zeamays ( Solsoloy 1995). They also proved to be toxic against vectors of filariae such as Culex quinquefasciatus ( Karmegam et al. 1997 ), which occur in the same habitats as schistosome intermediate host snails and larvae.

Since all parts of the plant are used in traditional medicine, e.g. for their antibacterial and wound-healing properties ( Thomas 1989; Nath & Dutta 1992), cultural acceptance to use the plant for curing or controlling diseases is given. However, seeds and oil have been reported to be toxic to humans, rodents and livestock after ingestion ( Adam 1974; Adam & Magzoub 1975; Ahmed & Adam 1979; Joubert et al. 1984 ) and phorbol esters are cocarcinogens ( Horiuchi et al. 1987 ). Therefore, decomposition of these components under field conditions must be evaluated before applying extracts of J. curcas in the field.

Large-scale cultures of J. curcas for industrial use have already been established in several tropical countries ( Henning & von Mitzlaff 1995). Techniques for sustainable local production of a molluscicide from J. curcas remain to be developed. We believe that the molluscicidal and larvicidal activities observed so far justify further research with the aim of using this plant in schistosomisis control.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank the following colleagues for kindly providing various materials: R. Henning (Projet Pourghère; Bamako; Mali) for the crude oil and R. Metzler (FAKT; Furtwangen; Germany) for the seeds from Mali; F. Sporer and C. Koschmieder (Institute for Pharmaceutical Biology, University of Heidelberg; Germany) for preparing and analysing the seed extracts; Dr M. Doenhoff (School of Biological Sciences; University of Wales; UK), Dr A. Hassan and Prof R. Ramzy (Ain Shams University; Cairo; Egypt) for providing Bulinus natalensis and B. truncatus, respectively. We also thank Dr M. Doenhoff, Dr R. Snow and an anonymous referee for their helpful comments on the manuscript. This work received financial support from the Deutsche Gesellschaft für Technische Zusammenarbeit (GTZ).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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