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 ALEXANDRINA

ABSTRACT: This study aimed to investigate the INTRODUCTION: effect of the water suspension of Anagallis The fresh water , 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 Research Institute, Egy p t till use.

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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 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 ± 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. EM Granulocytes Amoebocytes Hyalinocytes E xp er . C onc . study displayed abundant large and dense p er iod (pp m) Mea n % Mean % Mean % ± S D Reduc . ± SD R educ . ± S D R educ . granules in the cytoplasm of dense or heavy granulocytes that even overlap the nucleus. LC0 27. 8± 0.8 12.4± 1.1 9.2± 1. 6

46. 3 51. 8 59. 1 ( 0.06) *** *** *** While in light granulocytes, the nucleus is LC 25. 4± 1.1 11.4± 0.9 13. 2± 1.5 1 0 50. 9 55. 7 41. 3 small and eccentric and appears clearly (Fig. ( 0.095) *** *** *** w eeks 2a&b) 2 LC 23. 0± 1.6 10.8± 1.1 6.6± 1. 3 2 5 55. 6 58. 1 70. 6 ( 0.22) *** *** *** Amoebocytes: These cells are LC 24. 4± 0.6 11.6± 2.2 8.0± 3. 2 0 52. 8 55. 0 64. 4 characterized by the extending of many ( 0.06) *** *** *** LC 23. 6± 0.6 9. 6±0. 9 6.8± 1. 1 pseudopodia, central clear nucleus and their 1 0 54. 4 62. 7 69. 8 ( 0.095) *** *** *** sizes are larger than hyalinocytes and

4 w eeks LC 11. 6± 1.1 9. 4±0. 9 4.0± 1. 0 2 5 77. 6 63. 5 82. 2 granulocytes as shown by LM. EM displayed ( 0.22) *** *** *** their large irregular nuclei and vacuolated C ontr ol 51. 8± 4.3 25.8± 8.1 22. 5± 7.7 cytoplasm. The cytoplasm is filled with *** Highly significant compared to control at p<0. vacuoles that rush the nucleus to be eccentric (Fig. 2a&c). Table 5. Effect of Bayluscide on Biomphalaria Hyalinocytes: These Cells have circular alexandrina haemocytes Granulocytes Amoebocytes Hyalinocytes shape with faint cell membrane, few Ex per . C onc . granules and eccentric nucleus. They appear per iod ( pp m) Mean % Mean % Mean % ± S D R educ . ± S D R educ . ± S D R educ . as light violet. EM showed abundant

LC 0 26.1± 1.4 13. 4± 1.1 12.0± 1.2 organelles including mitochondria,

49.8 48. 0 46.7 ( 0. 01) *** *** *** endoplasmic reticulum and ribosomes in the LC 1 0 22.3± 2.1 11. 8± 1.2 9. 5± 1.6 56.9 54. 2 57.7 ( 0. 019) *** *** *** cytoplasm with faintly appearing cell

2 weeks LC 2 5 21.4± 1.1 8.2± 1. 3 7. 8± 0.8 membrane (Fig. 2a&d) 58.6 68. 2 65.0 ( 0. 039) *** *** ***

LC 0 24.2± 0.8 13. 0± 0.7 10.2± 0.8 53.2 49. 5 54.7 ( 0. 01) *** *** ***

LC 1 0 17.0± 4.1 9.2± 0. 8 7. 6± 1.1 67.1 64. 4 66.0 ( 0. 019) *** *** ***

4 w eeks LC 2 5 8.0± 0.7 3.0± 0. 7 4. 0± 1.0 84.5 88. 3 82.0 ( 0. 039) *** *** ***

C ontr ol 51.8± 4.3 25. 8± 8.1 22.5± 7.7 *** Highly significant compared to control at p<0.001.

Haemolymph of control B. Electron micrograph of normal alexandrina showing light granulocytes. Small number amoebocyte(A), hyalinocyte of dense granules and the (H) and a dark granulocyte nucleus could be seen (G). (100) clearly. (X 10.000)

Normal amoebocyte with Normal hyalinocytes extended pseudopodia showing clearly obvious (p). The cytoplasm is organelles and the cell filled with vacuoles (v) membrane appears faint. (X Fig. 1. Effect of tested compounds LC25 after 4weeks that push the nucleus to 10.000) on Biomphalaria alexandrina haemocytes. be eccentric. (X 10.000) 1- Light and Electron microscopic Fig. 2. The normal haemolymph of Biomphalaria alexandrina light and electron microscope. investigation of normal haemocytes of B.alexandrina snails: 2- Light and Electron microscopic investigation of treated haemocytes with Examination of Biomphalaria alexandrina molluscicdes on B.alexandrina snails: haemolymph by light and electron microscopy revealed three types of different Exposure of snails to sublethal cells classified according to their shape and concentrations of dry powder aqueous of the granular contents. These cells are granulo- plant Anagallis arvensis decreased the cytes, amoebocytes and hyalinocytes. The number of haemocytes as shown by light and total number of these cells were 51.8 ± 4.3, electron microscope. The reduction was 25.8 ± 8, and 22.5 ± 7.7, respectively. more evident with the highest concentration (LC25) after 2 weeks, being 52.8%, 69.7% Granulocytes: These cells are divided and 67.8% for granulocytes, amoebocytes into two types dense and light according to and hyalinocytes respectively. A still higher the density of the granular contents. They reduction was recorded after 4 weeks,

http://www.e g y p t s e b . o r g 138 Egypt. J. Exp. Biol. (Zool.), 3: 135 – 143 (2007)

reaching 65.9%, 83.7%, and 85.7% for level, granulocytes were shown to be granulocytes, amoebocytes and hyalinocytes fragmented and mostly of the dense type respectively. On the high microscopically (Fig.3).

A): Anagallis arvensis B): Calendula micrantha Granulocytes were fragmented and mostly of the dense type. The three types of haemocytes were fragmented where Amoebocytes degenerated; Hyalinocytes were presented with remnants of the cells were seen. (100) vacuolated cytoplasm. (100)

C: Copper sulphate D: Bayluscide Hyalinocytes with peripheral condensation of nuclear chromatin. Vacuolated granulocytes, amoebocytes and completely (100) degenerated halinocytes. (100)

Fig. 3. Effect of LC2 5 of the tested compounds on B. alexandrina haemocyts for 4 weeks under light microscope, Stained by Giemsa.

On the other hand amoebocytes appeared and LC25 for 2 weeks decreased the number degenerated while hyalinocytes were of haemocytes as shown by light and presented with vacuolated cytoplasm (Fig. electron microscope. The reduction 3). Morphological changes were clearly seen percentage in haemocytes increased after 4 by electron microscope especially after weeks from exposure to LC 25, being 66%, exposure to LC 25 for 4 weeks. Granulocytes 69% and 70% for granulocytes, amoebocytes showed condensed nuclear material on the and hyalinocytes respectively. The three inner side of the nuclear membrane. Cell types of cells were fragmented where organelles were partly ruptured (Fig. 4a). remnants of the cells were seen (Fig. 3). Similar features were seen in amoebocytes E.M. studies revealed granulocytes with and the nuclear membrane had irregular condensed nuclear materials (Fig. 4d). margin due to accumulation of vacuoles on Amoebocytes suffered from degenerative its outer membrane (Fig. 4b). Hyalinocites changes while nuclei were present with on the other hand were mostly degenerative vacuolated nuclear area and irregular where cellular organelles were difficult to nuclear boundries (Fig. 4e). Inspite of the identify (Fig. 4c). clear cellular membrane of hyalinocytes, yet Similarly, Calendula micrantha given at organelles were more or less necrotic. Again nuclear condensation was clearly identified the three sublethal concentrations LC 0, LCI0, (Fig.4f). Anagalis arvensis a b c

Granulocyte showed condensed nuclear Amoebocytes showed irregular margin of Hyalinocytes were mostly degenerated material. Cell organelles were partially the nuclear membrane due to accumulation where the cellular organelles were difficult ruptured. (X 10.000) of vacuoles on its outer membrane. (X to identify. (X 10.000) 10.000) Hyalinocytes Granulocytes Amoebocytes

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Calendula micrantha d e f

Granulocytes with condensed nuclear Amoebocytes suffered from degenerative In spite of the clear cellular membrane of material. (X 10.000) changes, nuclei were with vacuolated hyalinocytes, the organelles were more or less nuclear area and irregular nuclear necrotic. (X 10.000) boundaries. (X 10.000) Fig. 4. Effect of LC25 of Anagallis arvensis and Calendula micrantha on B. alexandrina haemocyts treated for 4 weeks. (Stained by uranyl acetate and lead citrate). Exposure to sublethal concentrations of exposure to the highest concentration of either copper sulphate (CuSO4) or LC25. Decreased figures reached 55.6%, Bayluscide caused more pronounced 58.1%, and 70.6%, after 2 weeks while decrease in the number of haemocytes 77.6%, 63.5% and, 82.2% after 4 weeks for especially after 4 weeks of exposure. granulocytes, amoebocytes and hyalinocytes respectively. The morphological changes by In CuSO4 the percentage reduction for light microscope were seen as peripheral different haemocytes following exposure to condensation of nuclear chromatin (Fig. 3). LC is recorded to be 46.3%, 51.8% and 0 In Ultrastructure changes granulocytes did 59.1% after 2 weeks while 52.8%, 54.95% not have any cellular membrane where and 64.4% after 4 weeks for granulocytes, nuclear material disappeared (Fig. 5a). amoebocytes and hyalinocytes respectively. Changes were clearly noticed as a ring of A more significant decrease was seen at condensed nuclear chromatin in LC reaching 50.9%, 55.7% and 41.3% after 10 amoebocytes and many vacuoles formed 2 weeks while 54.4%, 62.7% and 69.8% after from cell membrane in hyalinocytes (Fig. 4 weeks for granulocytes, amoebocytes and 5b&c). hyalinocytes, respectively. Still higher significant decreases were recorded after

CuSO4 b c a

Granulocytes didn’t have any cellular Changes were clearly noticed as a ring of Many vacuoles formed from cell membrane where nuclear material condensed nuclear chromatin in amoebocytes. membrane in hyalinocytes. (X 10.000) disappeared. (X 10.000) (X 10.000) Granulocytes Amoebocytes Hyalinocytes Bayluscide d e f

Granulocytes showing partial absence Amoebocytes showing peripheral nuclear Hyalinocytes showing peripheral nuclear nuclear material and degenerated chromatin condensation and fragmentation. (X chromatin condensation and organelles. (X 10.000) 10.000) fragmentation. (X 10.000) Fig. 5. Effect of LC25 of Bayluscide and copper sulphate on B.alexandrina haemocyts treated for 4 weeks (Stained by uranyl acetate and lead citrate). Continues exposure of snails to the three haemocytes especially evidenced after 4 weeks of exposure. The reduction sublethal concentrations of Bayluscide LC 0, percentage in LC 0 is 49.8%, 48%, 46.7%, LC10 and LC25 for 2 and 4 weeks caused more decreases in the number of and 53.2%, 49.5% 54.7% for granulocytes,

http://www.e g y p t s e b . o r g 140 Egypt. J. Exp. Biol. (Zool.), 3: 135 – 143 (2007) ameoebocytes and hyalinocytes after 2 and living organisms (Nevo et al., 1978; Tolba et 4 weeks respectively. In LC 25 highly al., 1997). significant as compared with control The effect of Anagallis arvensis and appeared after 2 and 4 weeks recording Calendula micrantha on the B.alexandrina figures 58.6%, 68.2% 65% and 84.5%, were studied by El- Emam et al. (1986), El- 88.3%, 82% for granulocytes, amoebocytes Emam and Ebied (1989), El-Emam et al. and hyalinecytes, respectively. By light (1996), Mostafa and Tantawy (2000), Abd El- microscope, vacuolated granulocytes and Gawad et al. (2000), Tantawy (2002), and amoebocytes appeared and completely Abd El-Kader et al.(2005).They found that degenerated the survival rate of B.alexandrina in aqueous Hyalinocytes (Fig. 3). Ultrastructure solution of Anagallis arvensis and Calendula changes can be observed as partial absence micrantha increased the role in the defense of nuclear material in granulocytes and system inside the snails but reduced the degenerated organelles (Fig.5d). Amoebo- cercarial production and the survival rate cytes and hyalinocytes showing peripheral when the snail is still alive. nuclear chromatin condensation and Also, The effect of copper sulphate and fragmentation (Fig. 5e&f). Bayluscide on B.alexandrina were observed by El Gindy (1969a&b; 1975), Gawish DISCUSSION: (1997), Rawi et al. (1994&1996), Ragab et No doubt that the haemolymph of al. (1998), Bakry et al. (2000), Bakry and mollusks is of critical importance as the Sharaf El- Din (2000), Mohamed et al. haemocytes react against foreign biotic and (2000), and El-Ansary et al. (2001). They abiotic bodies, digest and transport reported a marked reduction in the survival nutrients, accumulate various substances rate and in hatchability of eggs of B. such as heavy metals, pesticides and alexandrina after treatment of snails with molluscicides (Malker and Chong, 1974). In Bayluscide and copper sulphate. Similar addition, snail's haemocytes are being used findings were reported by Sturrock (1966), in determining the prepatency period of Mohamed et al. (1981), Gawish (1997), and schistosomiasis infection (Kamel et al., Ibrahim et al. (1997). They found that the 2006). rate of infection of B. alexandrina and Three types of haemocytes were cercarial production decreased when snails identified in the haemolymph of B. were treated with sublethal concentrations of alexandrina snails. These are granulocytes, CuSO4. hyalinocytes and amobocytes. This agrees On the other hand, Stumpf and Gilbertson with the finding of many authors (Vander (1980), Loker et al (1982), and Granath and Knaap&Loker, 1990; Atlam, 2000; Sharaf El- Yoshino (1983) found a dramatic decrease in Din, 2003; Kamel et al., 2006). the number of haemocytes after infection The present results indicated that the due to migration of haemocytes out of the prolonged exposure of snails to aqueous haemolymph to participate in the suspension of the plants Anagallis arvensis encapsulation of the parasite or for the and Calendula micrantha and the tested repair of tissue damage caused by the molluscicidal copper sulphate and parasite. Bayluscide for 2 and 4 weeks at Ultrastructure changes observed in the concentrations of LC 10, and LC25 of both present study as the effect of sublethal tested plants decreased the total numbers of concentrations of the tested plants haemocytes of B. alexandrina. The cause of &chemicals on B. alexandrina haemolymph this decrease in the number of haemocytes appeared as degenerative changes in the may be due to inhibition in transaminase cells where cellular organelles were difficult enzyme activity; (asparatate and alanine to identify. These findings agree with Tolba aminotransferase AST, ALT).These (1997) who observed the toxic effect of observations are in agreement with those of Anagallis arvensis against the foot, of El-Gindy et al.(1991); Rawi et al.(1996) and Monacha sp. and Theba sp, and showed that Mantawy et al. (2004) who studied the toxic the plant was more effective and caused effect of tested plants against B. complete destruction of the foot tissue. Also alexandrina. The studies showed that Atlam (2000) studied the effect of prolonged exposure to the sublethal molluscicidal activity of Euphorbia pepus and concentration of these plants inhibited Sesbania sesban on the haemocytes of B. transaminase enzymes activity; (AST, alexandrina. She observed ruptured cell ALT).The aminotransferase (AST, ALT) membranes, degenerated nuclei, swollen, present an important link between ruptured mitochondria, increase of lysosomal carbohydrate and aminoacid metabolic vesicles and the appearance of many fine pathways (Christie and Michelson, filopodia and autolysis of vacuoles. Kamel et 1975).Also these enzymes are considered al. (2006) revealed remarkable activation good sensitive tools for detection of any represented by vacuolated cytoplasm, variation in the physiological process of extended pseudopodia and condensed 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 … 141 nuclear chromatin. 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اﻟﺘﺎﺛﯿﺮ اﻟﺴﻤﻰ ﻟﺒﻌﺾ اﻟﻤﺒﯿﺪات اﻟﻄﺒﯿﻌﯿﺔ واﻟﻤﺨﻠﻘﺔ ﻋﻠﻰ ﺧﻼﻳﺎ ھﯿﻤﻮﻟﯿﻤﻒ ﻗﻮاﻗﻊ ﺑﯿﻮﻣﻔﻼرﻳﺎ اﻟﻜﺴﻨﺪرﻳﻨﺎ واﻟﺘﻐﯿﺮات اﻟﻤﺠﮫﺮﻳﺔ اﻟﺘﻰ ﺗﺤﺪث ﻟﮫﺎ ﻋﺮﻳﺎن ﺟﻮرج ﻛﺎﻣﻞ- ﺳﻨﺎء ﻣﺤﻤﺪوھﺒﺔ- ﺷﺎدﻳﺔ ﻣﺤﻤﺪ اﻟﺪﻓﺮاوى*- ﺣﻨﺎن ﺷﺤﺎت ﻣﺴﻠﻢ* ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﯿﻮان ﺑﻜﻠﯿﺔ اﻟﺒﻨﺎت- ﺟﺎﻣﻌﺔ ﻋﯿﻦ ﺷﻤﺲ *ﻣﻌﻤﻞ اﻟﺮﺧﻮﻳﺎت اﻟﻄﺒﯿﺔ ﺑﻤﻌﮫﺪ ﺗﯿﻮدور ﺑﻠﮫﺎرس ﻟﻼﺑﺤﺎث

ﺗﮫﺪف ھﺬة اﻟﺪراﺳﺔ ﻟﻤﻌﺮﻓﺔ ﺗﺎﺛﯿﺮ اﺳﺘﺨﺪام اﻧﻮاع ﻣﻦ ھﺬة اﻟﺨﻼﻳﺎ وﻳﺰداد اﻧﺨﻔﺎﺿﺎ ﺑﻌﺪ 4 اﺳﺎﺑﯿﻊ وذﻟﻚ اﻟﻤﺒﯿﺪات ﺳﻮاء اﻟﻤﺴﺘﺨﻠﺼﺔ ﻣﻦ اﻟﻨﺒﺎﺗﺎت ﻣﺜﻞ ﻣﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺔ اﻟﻀﺎﺑﻄﺔ اﻟﺘﻰ ﻟﻢ ﺗﺘﻌﺮض ﻻى ﻣﻦ Anagallis arvensis or Calendula) اﻟﻤﺒﯿﺪات وذﻟﻚ ﺑﺎﺳﺘﺨﺪام اﻟﻤﯿﻜﺮﺳﻜﻮب اﻟﻀﻮﺋﻰ. (micranthaاواﻟﻤﺮﻛﺒﺎت اﻟﻜﯿﻤﯿﺎﺋﯿﺔ Bayluscide or) وﺑﻔﺤﺺ ﺧﻼﻳﺎ ھﯿﻤﻮﻟﯿﻤﻒ اﻟﻘﻮاﻗﻊ ﺑﺎﻟﻤﯿﻜﺮوﺳﻜﻮب (copper sulphateﻋﻠﻰ ﺧﻼﻳﺎ ھﯿﻤﻮﻟﯿﻤﻒ ﻗﻮاﻗﻊ اﻻﻟﻜﺘﺮوﻧﻰ اﺗﻀﺢ وﺟﻮد ﺗﮫﺘﻚ ﻓﻰ ﻣﻜﻮﻧﺎت ھﺬة اﻟﺨﻼﻳﺎ ﺑﯿﻮﻣﻔﻼرﻳﺎ اﻟﻜﺴﻨﺪرﻳﻨﺎ وﻗﺪ اﺳﺘﺨﺪم ﺛﻼث ﺗﺮﻛﯿﺰات LC0, وﺧﺎﺻﺔ ﻓﻰ اﻟﺸﺒﻜﺔ اﻟﻜﺮوﻣﺎﺗﯿﻨﯿﺔ ﻣﻊ وﺟﻮد ﻓﺠﻮات LC25 , LC10.وﻗﺪ ﺗﻢ اﺧﺬ ﻋﯿﻨﺎت ﻣﻦ ھﯿﻤﻮﻟﯿﻤﻒ ھﺬة ﺳﯿﺘﻮﺑﻼزﻣﯿﺔ. اﻟﻘﻮاﻗﻊ ﺑﻌﺪ اﺳﺒﻮﻋﯿﻦ وارﺑﻊ اﺳﺎﺑﯿﻊ ﻣﻦ اﻟﺘﻌﺮﻳﺾ ﻟﻠﻤﺒﯿﺪات ووﺟﺪ ان ﻋﺪد اﻟﺨﻼﻳﺎ اﻟﻤﺤﺒﺒﺔ و اﻟﺨﻼﻳﺎ اﻻﻣﯿﺒﯿﺔ اﻟﻤﺤﻜﻤﻮن: و اﻟﺨﻼﻳﺎ اﻟﺰﺟﺎﺟﯿﺔ ﻳﻨﺨﻔﺾ ﻣﻌﻨﻮﻳﺎ ﺑﻌﺪ اﺳﺒﻮﻋﯿﻦ ﻣﻦ أ.د. ﻣﺤﻤﺪ ﺣﺴﻦ ﻣﻨﺎ ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﯿﻮان، ﻋﻠﻮم طﻨﻄﺎ اﻟﺘﻌﺮﻳﺾ ﻟﻠﻤﺒﯿﺪ ﻓﻰ ﺗﺮﻛﯿﺰات LC25 & LC10 ﻓﻰ اﻟﺜﻼﺛﺔ أ.د. اﺳﻤﺎﻋﯿﻞ اﻟﺸﺮﻗﺎوي ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﯿﻮان، ﻋﻠﻮم طﻨﻄﺎ

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