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Research Article Toxicological Effect Of Egypt. J. Exp. Biol. (Zool.), 3: 135 – 143 (2007) © The Egyptian Society of Experimental Biology RESEARCH ARTICLE Erian G. Kamel Sanaa M. Wahba *Shadia M. El - D a f r a w y *Hanan, S. Mossalem TOXICOLOGICAL EFFECT OF CERTAIN PLANTS AND SYNTHETIC MOLLUSCICIDES ON ULTRASTRUCTURAL CHANGES IN HAEMOCYTES OF BIOMPHALARIA ALEXANDRINA SNAILS ABSTRACT: This study aimed to investigate the INTRODUCTION: effect of the water suspension of Anagallis The fresh water snail, B. alexandrina is arvensis or Calendula micrantha plants, as known to be the intermediate host for well as Bayluscide and copper sulphate on transmitting S. mansoni in Egypt. Application the haemocytes of Biomphalaria of molluscicides of either synthetic or of alexandrina snails at ultrastructural level at plant origin can confer a rapid and efficient three sublethal concentrations of LC 0, LC10, mean in reducing snail populations and and LC25. Examinations were performed consequently the transmission of this after 2 and 4 weeks of exposure. parasite. There is great interest in the use of The total number of the haemocytes molluscicidal plants by local communities in granulocytes, hyalinocytes and self-supporting system of schistosomiasis amoebocytes was found to be significantly control program (Taylor, 1986). In fact, plant reduced (P<0.01) after 2 weeks of snails molluscicides are gaining increased attention as they seem to be less expensive, exposure to LC 10 of the tested plants and synthetic molluscicides in comparison with more available, have low toxicity to non- the control. The reduction was more target organisms and may be applied significant after exposure to sublethal effectively by simple techniques that are more suitable for developing countries concentration of LC 25 and after 4 weeks than 2 weeks of exposure to the sublethal (Adewunmi et al., 1990; Wang and Song, concentrations LC and LC . 1995; Mengesha et al., 1997; Schall et al., 10 25 1998; Vogg et al., 1999; Atlam, 2000; El- Electron microscopic examination Khodary, 2001). Therefore, this study aimed showed apoptotic effect on exposure to the to evaluate the value of using plants plant derivatives and synthetic Anagallis arvensis and Calendula micrantha, molluscicides where snail haematocytes as well as the synthetic molluscicides were presented by nuclear chromatin Bayluscide or Copper sulphate against B. fragmentation and vacuolated cytoplasm. alexandrina haemocytes. Key words: MATERIALS AND METHODS: Plants, B. alexandrina, Haemolymph. 1- Snails Biomphalaria alexandrina snails used in this study were obtained from the laboratory- bred stock in Medical Malacology Laboratory, Theodor Bilharz Research Institute (TBRI) Egypt. 2-Plants and molluscicides Anagallis arvensis (Family Agavacae) and Calendula micrantha (Family Compositae) plants were collected from the shores of CORRESPONDANCE: water courses in El-Qanater El-Khayria, Erian G. Kamel Sanaa M. Wahba Qalubia Governorate, Egypt during the *Shadia M. El - D a f r a w y spring of 2006. The plants were shade dried, *Hanan, S. Mossalem finely powdered using an electrical grinder. Zoology Department, Faculty of Girls, The dry powder of the experimental plants Ain Shams University, Cairo, Egypt. was stored in clean, dark and dry cupboard *Medical Malacology,Theodor Bilharz till use. Research Institute, Egy p t h t t p : / / w w w . e g y p t s e b . o r g 136 Egypt. J. Exp. Biol. (Zool.), 3: 135 – 143 (2007) Bayluscide and Copper sulphate were haemolymph were pooled in a vial tube from kindly provided by Medicinal Chemistry each group. Laboratory, TBRI. 5- Haemolymph Examination: Screening and evaluation tests On the light microscopy level: Blood films The dry powder of each plant was used in were spread from the haemolymph of snails toxicity tests as aqueous suspensions. For on clean glass slides, stained with Giemsa, each plant or chemical used a series of examined and counted by light microscope concentrations that would permit the according to the methods described by computation of LC50 and LC90 values as Atlam (2000) then photographed using Agfa shown in Table (1) was prepared on basis of film R S X 100. weight /volume using dechlorinated tap On the electron microscopy level: The water. Three replicates were used each of collected haemolymph was centrifuged and 10 snails (8-10 mm/ L) for each plant the sedimented cells were fixed in 4% concentration. The exposure period was 24 glutaraldhyde with sodium cacodylate. Two hours at room temperature (25± 1Co). At the hours later, the cells were post fixed in 2% end of exposure period, the snails were osmium tetraoxide, dehydrated with removed from each exposure concentration, ascending concentration of alcohol and washed thoroughly with dechlorinated water embedded in epoxy resin according to the and transferred to another container technique of Grimaud et al. (1980). Semi- containing fresh dechlorinated tap water for thin and ultra- thin sections were cut with a 24 hours of recovery. Thereafter, death of Leika ultra microtome. Ultra-thin sections snails was judged according to Nolan et al. were contrasted with uranyl acetate and lead (1953) and Jove (1956) which include the citrate stains then examined by Phillips EM odour of decayed animal matter given off, 208 Electron Microscope. lack of response to gentle prodding with a Statistical analysis curved blunt needle and no blood oozing out when the snail was crushed. The standard The data are presented as mean ± method of the same authors followed in the standard deviation. The means of the present study was the immersion of snails in different groups were compared using the a small amount of 15-20% sodium hydroxide student's t- test (Sokal and Rohlf, 1981). solution in a Petri dish, if bubbles or blood or both come out of the shell, or if snail retracts RESULTS: into the shell, it was recorded as alive and if The results are shown in Tables 2-5 and not it was recorded dead. The effectiveness Figures 1-5. of the tested plants has been expressed in Table 2. Effect of Anagallis arvensis on terms of LC50 and LC 90 via statistical Biomphalaria alexandrina haemocytes analysis according to the procedure of Granulocytes Amoebocytes Hyalinocytes Litchfield and Wilcoxon (1949). E xp er . C onc . p er iod (pp m) Mean % Mean ±S % Mean % Table 1. Molluscicidal activity of plant species ± S D R educ . D R educ . ± S D R educ . LC0 29. 2± 0.8 11.0± 0.7 9.8± 43.6 57.3 56. 4 and synthetic ( 5.3 *** *** 0. 8 LC) 26. 0± 0.7 6. 2± 0.5 7.8±*** Tested LC 5 0 Confidence LC 9 0 10 49.7 75.9 65. 3 material (ppm) 95% limits (ppm) ( 15) *** *** 0. 8 *** 2 w eeks LC 24. 4± 0.6 7. 8± 1.6 7.3± Anagallis 25 52.9 69.7 67. 8 53 34.2- 82. 2 188 ( 27) *** *** 1. 9 ar vensis LC0 25. 4± 0.6 9. 4± 0.6 5.2± Calendula 50.9 63.5 76. 9 135 116. 4- 156. 6 205 ( 5.3 *** *** 0. 5 micr ant ha LC) 24. 0± 0.7 5. 4± 0.9 3.6±*** 10 53.6 79.0 84. 0 ( 15) *** *** 0. 6 Cu SO4 0.6 0. 38-0.96 4 4 w eeks LC 17. 6± 1.1 4. 2± 0.5 3.***2± 25 65.9 83.7 85. 7 ( 27) *** *** 0. 8 22.5*** Bayluscide 0.1 0. 05 -0.21 0.6 C ontr ol 51. 8± 4.3 25.8± 8.1 ± 7.7 3-Effect of sublethal concetrations of the *** Highly significant compared to control at p<0.001. tested Plants &Chemicals on Table 3. Effect of Calendula micrantha on Biomphalaria alexandrina haemolymph:- Biomphalaria alexandrina haemocytes Granulocytes Amoebocytes Hyalinocytes Three sublethal concentrations (LC 0, LC10 E x p e r. C o n c . p e ri od ( p p m ) % M ea n % M e a n % and LC ) of the plants or chemicals were M ea n± S D 25 R e d uc . ± S D R e d uc ± S D R e d u c. used in this study. The snails were LC 0 25. 0± 0.7 13.2± 1.1 12. 2± 0.5 51. 6 48. 7 45.0 continuously exposed to the tested ( 13.5) *** *** *** LC 24. 8± 1.7 12.3± 0.5 11. 5± 1.9 1 0 52. 1 52. 0 48.0 concentrations for 2-days followed by 5-days ( 85) *** *** *** LC 2 5 20. 3± 0.5 10.5± 1.0 10. 0± 0.8 2 w eeks 60. 9 59. 2 56.0 recovery period in clean dechlorinated water ( 105) *** *** *** per week for two and four successive weeks. LC 0 22. 0± 0.8 11.5± 0.6 10. 5± 0.6 57. 0 55. 0 53.0 ( 13.5) *** *** *** LC 20. 5± 0.6 10.0± 0.8 8.0± 0. 8 4- Collection of haemolymph: 1 0 60. 0 61. 0 64.0 ( 85) *** *** *** Haemolymph samples were collected LC 2 5 17. 5± 1.3 7. 8±0. 5 6.8± 0.1. 0 4 weeks 66. 0 69. 0 70.0 according to the methods of Michelson ( 105) *** *** *** (1966) by removing a small portion of the C ontr ol 51. 8± 4.3 25.8± 8.1 22. 5± 7.7 shell situated directly above the heart to *** Highly significant compared to control at p<0.001. insert a capillary tube into it. Two ml of http://www.e g y p t s e b . o r g Kam el et al., Toxicological Effect of Certain Plants and Synthetic Molluscicides … 137 Table 4. Effect of Copper sulphate (CuSO4) are of small size and are found as groups on Biomphalaria alexandrina haemocytes collected together as shown by LM.
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