Australian Journal of Basic and Applied Sciences, 5(12): 3357-3365, 2011 ISSN 1991-8178

Cryptostegia Grandiflora Affecting Compatibility of Alexandrina and Biomphalaria Galabrata to Infection With With Emphasis on Some Hematological Effects

Kamelia, A. El Sayed Momeana, B. Mahmoud Hanan, S. Mossalem

Department of Medical Malacology, Theodor Bilharz Research Institute, P.O. BOX 30 Imbaba, Giza, Egypt.

Abstract: Cryptostegia grandiflora showed considerable mollluscicidal effect against Biomphalaria alexandrina and Biomphalaria galabrata intermediate host of Schistosoma mansoni. The effect of Cryptostegia grandiflora on the infection rate and morphology and number of haemocytes were studied under light and electron microscope. The sublethal concentrations (LC0, LC10, LC25) of the Latex aqueous solution of C. grandiflora were 0.88, 4.4 and 5.6 ppm for B. alexandrina and 3.5, 6.6 and 8.3 ppm for B. galabrata respectively. The susceptibility of B. alexandrina and B. galabrata , maintained at LC10 of Latex solution for 3-days perior miracidial exposure, to infection with S. mansoni was greatly reduced by 55.5 % and 58.9 % in comparison with control groups 66.7 % and 14.6 % respectively. Light microscopic investigation recorded that there are three types of haemocytes distributed in the haemolymph of B. alexandrina and B. galabrata snail. These cells are distinguished morphologically according to their shape, size and number. These cells are named granulocytes (50.9 %), hyalinocytes (19.1 %) and ameobocytes (30 %) in case of B. alexandrina and (28.4 %), (20.9 %) and (50.7 %) in case of B. galabrata respectively. After exposure to LC10 of C. grandiflora for three days cause a significante decrease in mean number of granulocytes in B. alexandrina and B. galabrata being (43 %) and (25 %) with a percentage reduction 15.5 % and 12.1 % respectively. This mean number of cells after exposure of snails to S. mansoni miracidia becomes 42 % and 26.3 % with a percentage reduction 17.4 % and 7.5 % in both snail respectively. There was a high percentage reduction in the number of granulocytes of B. alexandrina and B. galabrata snails after exposure to infection and LC10 of C. grandiflora at the same time being 29.6 % and 18.0 % respectively. While in the case of amoebocytes cells, the mean number showed a highly significant increase after exposure to S. mansoni miracidia being 47.0 % and 55.9 % with increasing percent 56.7 % and 10.1 % in B. alexandrina and B. galabrata snails respectively. This mean number reached to 44.1 % and 61.0 % when B. alexandrina and B. galabrata exposed to LC10 of C. grandiflora for three days then exposure to Schistosoma mansoni miracidia with increasing percent 47.1 % and 20.3 % respectively. Electron microscopic examination showed apoptotic effect, nuclear chromatin fragmentation vacuolated cytoplasm and fagocytosis characters are seen clearly in amoeboied cells.

Key words: Biomphalaria alexandrina, Biomphalaria galabrata, cryptostegia grandiflora, haemolymph, molluscicid, Schistosoma mansoni.

INTRODUCTION

Biomphalaria alexandrina and Biomphalaria galabrata are the molluscan intermediate host of Schistosoma mansoni which is considered the prime health problem in Egypt and in many tropical and subtropical countries (WHO,1992). Snail control is one of the most efficient methods to control these parasite. Plant origin molluscicides are gaining increased attention as they seem to be less expensive, more available, have low toxicity to non target organisms and have no side effect for other aquatic organisms. (Adewunmi et al., 1990; Vogg et al., 1999; Atlam, 2000 and Khodary, 2001). Cryptostegia grandiflora (Family: Asclepiadaceae) is a Latex producing plant (Neuwinger, 1996) and is known as Rubber vine and reputed to be toxic to humans and grazing (Zalabani et al., 2003). All living species have innate internal defense systems and the mollusks also, are therefore not defenseless against parasitic invaders or pathogens. They possess an internal defense system (IDS) which is different from the immune system of humans and higher vertebrate and this system comprises both cellular and humeral elements. The body cavity of fresh water snails () is filled with haemolymph which contains dissolved hemoproteins and several kinds of blood cells referred to as haemocytes which represent the primary mediators of cellular defense reactions and play an important role in recognition and elimination of invading foreign bodies through phagocytosis, encapsulation and release of cytotoxic mediators (Van Der Knaap and Locker 1990 and Bayne, 1990). Gastropods have an effective internal defence system consisting of cellular defens factor (Boehmler et al., 1996) and humeral defence factors (Johnston and Yoshino 1996; and Kofta 1997). Kamel et al., (2006 and 2007) evaluated that after 2 weeks of snails exposure to sublethal concentration (LC10) for (15 ppm) Anagallis arvensis and (85 ppm) for Calendula micrantha against B. alexandrina haemocytes causes a significant reduction in total number of haemocytes (p<0.01) LC10 in comparison with the control. The Corresponding Author: Kamelia, Department of Medical Malacology, Theodor Bilharz Research Institute, P. O. BOX 30 Imbaba, Giza, Egypt. 3357 Aust. J. Basic & Appl. Sci., 5(12): 3357-3365, 2011

reduction was more significant after exposure to sublethal concentration of LC25 and after 4 weeks than 2 weeks of exposure to the sublethal concentrations LC10 and LC25. El Sayed (2006) stated that exposure of B. alexandrina snails to sublethal concentrations ((LC0, LC10, LC25) of the dry powder of the plant Cupressus macrocorpa for three weeks significantly decreased the number of circulating hemocytes. The aim of the present study was to evaluate the effect of C. grandiflora as a plant origin on the compatibility of B. alexandrina and B. galabrata snails to infection with S. mansoni and on the count, morphological changes of their haemocytes.

MATERIALS AND METHODS

Snails: Snails used in this study were adult Biomphalaria alexandrina and Biomphalaria galabrata snails. Biomphalaria alexandrina were (8-10mm in diameter) collected from the River Nile and irrigation scheme near Cairo and brought to the laboratory, washed with dechlorinated water and examined for larval trmatodes. Unhealthy and infected snails were excluded, the healthy snails were maintained in plastic aquaria under standard conditions (25 ± 1oC, PH = 7.2) for three weeks before being used in molluscicidal tests. Snails were fed on boiled leaves and Blue green algae. Biomphalaria galabrata snails obtained from laboratory colony stock from Bayer Company (Germany).

Miracidia: Schistosoma mansoni miracidia were obtained from Schistosome Biological Supply Center (SBSC),TBRI,Egypt.

The Plant Material: Cryptostegia grandiflora were obtained from Orman Botanical Garden, Giza, Egypt. Aqueous solution of latex was released when stem was cut or injured. Astock solution of 1000 ppm concentration was prepared (1ml of the latex up to 1000 ml distilled water) After that a series of concentrations that would permit the computation of LC50 and LC90 values were prepared according to WHO (1965). Exposure and recovery periodes were 24 hour each. Thereafter, snails mortality was recorded. The lethal concentrations were calculated through a computer program (SPSS) employing the Probit analysis (Finney,1971). Sublethal concentrations (LC10 and LC25) were obtained from the toxicity lines and LC0 was obtained as 1/10 LC50 (WHO, 1965).

Effect of Sublethal Concentration Lc10 of Latex Aqueous Solution on Infection of Biomphalaria Alexandrina and Biomphalaria Galabrata Snails: Three groups (three replicates, each of 30 snails) of lab bred snails of each species (4-7mm in diameter) were maintained continuously for threee days in LC10 of latex aqueous solution of Cryptostegia and then the survived snails were exposed individually to Schistosoma mansoni miracidia with a dose of 10 miracidia / snail. A fourth snail group from each Spp. were served as control in which snails were exposed to miracidia and maintained in dechlorinated water under the same conditions. After 20 days examined snails for infection by crushing snails and examined under microscope for sporocyst to calculate the infection rate in both snails species. Four group of snails from each species (30 snails each) of all previous studies which mention above were used for haemolymph examination under light and electron microscope.

Haemolymph Collection: Haemolymph samples were collected according to the methods of Michelson (1966) by removing a small portion of the shell situated directly above the heart to insert a capillary tube into it. Two ml of haemolymph were pooled in a vial tube from each group and kept in ice-bath then centrifuged for 10 minutes at 3000 r.p.m. for electron microscope investigations. Hemolymph points were used for films to count number of haemocytes.

Staining of Haemocytes: Staining of haemocytes was performed by applying Leishman`s stain and Giemsa stain as follows:- a- Leishman's stain consists of :- 0.15 g Leishman`s stain 100 cm3 Methyl alcohol. The alcohol is added to the stain gradually till complete dissolving. The stain used after two weeks from preparation. b- Gemisa stain consists of:- 0.05 g Giemsa stain 33 cm3 Glycerin 33 cm3 of Methyl alcohol (free acetone) Giemsa stain was added to Glycerin and heat in an oven at 60◦C then the methanol alcohol was added to

3358 Aust. J. Basic & Appl. Sci., 5(12): 3357-3365, 2011 complete the solvent. Before use the stain was diluted with distilled water 1:10. It was kept in dark bottel away from sunlight according to Mossalem (2008).

Haemolymph Examination: By Light Microscopy: Blood films were done from the collected haemolymph of snails on clean glass slides, stained with Giemsa, examined and counted by light microscopy. Photographed using Agfa film RSX 100. By electron microscopy: The collected haemolymph samples were centrifuged and the sedimented cells were fixed in 4% glutaraldhyde with sodium cacodylate. Two hours later , the cells were post fixed in 2 % osmium tetraoxide, dehydrated with ascending concentration of alcohol and embedded in epoxy resin according to the technique of Grimaud et al. (1980). Semi-thin and ultra- thin sections were cut with a Leika ultra microtome. Ultra-thin sections were contrasted with uranyl acetate and lead citrate stains then examined by Phillips EM 208 Electron Microscope.

Statistical analysis: The data are presented as mean ± standard `deviation. The means of the different groups were compared globally using the student's t- test (Sokal and Rohlf, 1981). Molluscicidal activity of the tested plants and the tested chemicals were calculated by probit analysis Finney, (1971).

RESULTS AND DISCUSSION

The result in Table (1) indicates that the recorded LC50 and LC90 values for latex were 6.98 and 8.65 ppm for Biomphalaria alexandrina respectively and 10.29, 14.03 ppm for Biomphalaria galabrata respectively. The observed LC90 is lower than that recommended by WHO (1965) to an active aqueous plant extract kills 90% of the exposed snails for 24 hours which should less than 20 m g/L Mott ( 1987). In this respect, Sharaf El Din (2006) verfied that LC50 and LC90 values for Latex aqueous solutions of Cryptostegia grandiflora were 5.7 and 8.48 ppm respectively for Biomphalaria alexandrina and 4.7 and 7.55 ppm respectively for Lymnaea natalensis. Other investigators studied latex action from Euphorbia splendens Var. hislopii (Vasconcells and Schall, 1986) and verified that the LC90 was ranged between 1.07 and 4.04mg/L for Biomphalaria galabrata field snails. Also Schall et al. (1998) obtained a 90%lethal dose (LD90) ranging from 0.13ppm for Biomphalaria galabrata to 4.0ppm for tested with natural latex. Similar results were obtained by other authors using different plants as molluscicides Al-zanbagi, et al. (2000) used some Saudi Arabian euphorbiales (methanol and chloroform extracts of Jatropha glauca, Euphorbia helioscopia and Euphorbia Schimperiana) against the snail B. pfeifferi and found that the LD50 were in the range 10-100 ppm. Mantawy et al., (2004) found LC50 of the plants Capparis spinosa and Acacia Arabica 500 ppm and 66 ppm respectively and attributed the molluscicidal potency against Biomphalaria alexandrina through disturbance in metabolic pathways and protein contents. El-Sayed (2006) used dry poweder of Cupressus macrocapa against Biomphalaria alexandrina and found that the LC50 and LC90 values were 59.5 and 98.8 ppm respectively. El-Sayed et al., (2006) studied physalis angulata extracts against Biomphalaria alexandrinea snails and revealed that the 24-hour LC50 value was 65.4 and 62.2 ppm for water extract of leaves and fruits respectively. Gawish et al., (2008) recorded that methanolic extract of Callistemon citrinus leaves has a promising toxic effect against B. truncatus snails with LC50 and LC90 of 9.2 and 18.3 ppm respectively. The sublethal concentrations LC0, LC10, LC25 for Biomphalaria alexandrina snails were 0.88, 4.4, 5.6 ppm respectively while these sublethal concentrations were 3.5, 6.6, 8.3 ppm for Biomphalaria galabrata snails. Due to the moderate effect of LC10 it was selected for the next experiments. Table (2) shows that maintaining of Biomphalaria alexandrina and Biomphalaria galabrata snails at sublethal concentration LC10 (4.4 ppm and 6.6 ppm respectively) of latex aqueous solution of cryptostegia for 3 days perior to miracidial exposure led to a significant reduction in the infection rate with Schistosoma mansoni this reduction was 55.47 % and 58.9 % respectively. This may be explained by the deterioration of physiological parameters of snails making them unsuitable for the parasite development (Gawish et al, 2008). We noticed that, the infection rate of Biomphalaria galabrata with with Schistosoma mansoni miracidia is very low in comparison with Biomphalaria alexandrina being 14.6 % and 66.7 % respectively. This may be due to the less in number of amoebocytes which possess a phagocyting function performing intercellular digestion of non self materials (Furuta et al., 1986, 1987 and 1990). This agree with the results of Jeong et al.,(1983) which stated that the defense cells of Biomphalaria galabrata, the hemocytes, which are found circulating free in hemolymph and wandering about within connective tissues; take their origin from a special organ present in atiny area of the reno-pericardial zone. This organ has been designated "amebocyt forming organ " or APO and it is supposed to be the counterpart of the vertebrate bone marrow. Also Sullivan and Cheng (1984) and Sullivan et al., (2004) stated that when Biomphalaria galabrata snails are facing a schistosome infection, the outstanding presence of mitoses and cellular hyperplasia within the APO occur, indicating an active stage of new hemocyte production. Moreover Bezera et al., (1997) found that resistant Biomphalaria galabrata posses a natural defense system which protects them against S. mansoni larvae which generally die one to three days after penetrating.

3359 Aust. J. Basic & Appl. Sci., 5(12): 3357-3365, 2011

Sharaf El-Din (2006) recorded that the susceptibility of Biomphalaria alexandrina snails, maintained at LC0, LC10 and LC25 of latex solution of Cryptostegia grandiflora for one week perior miracidial exposure, to infection with Schistosoma mansoni miracidia led to a significant reduction in the infection rate with Schistosoma mansoni by 22.8 %, 29.7 % and 53.5 % respectively. El-Ansary et al., ( 2001) recorded that the sublethal concentrations of plant molluscicides were effective in reducing the compatibility of Biomphalaria alexandrina to with Schistosoma mansoni through reduction in cercarial shedding and elongation of the prepatent period. Gawish and El Bardicy (2002) found that exposure of snails to LC10of the dry powder of solnum nigrum and A. maritima plant reduced their infection rate with Schistosoma mansoni by 54.3 % and 47.8 % respectively. Gawish et al, (2008) was recorded that exposure of B. truncatus to sublethal concentration (LC10) of C. citrinus for one week reduced the susceptibility of snails to infection with Schistosoma haematobium from 40 % to 4.4 %, Mahmoud et al, (2011) was recorded a reduction in infection rate of Biomphalaria alexandrina snails treated during miracidial exposure of Schistosoma mansoni with LC10 of Datura stramonium and sesbania sesban being 41.7 % and 52.2 % respectively compared with control group 92.6 %. No doubt that the haemolymph of mollusks is of critical importance as the haemocytes react against foreign biotic and abiotic bodies, digest and transport nutrients, accumulate various substances such as heavy metals, pestisides and molluscicides (Malek and Cheng, 1977). The use of plants having molluscicidal properties were found to suppress the total number of snails' haemocytes. In the present study light microscopic investigation recorded that there are three types of haemocytes distributed in the haemolymph of Biomphalaria alexandrina and Biomphalaria glabrata snails. These cells are named granulocytes (50.9 %), hyalinocytes (19.1 %) and amoebocyte (30 %) in the case of Biomphalaria alexandrina and (28.4 %), (20.9 %) and (50.7 %) in the case of Biomphalaria glabrata snails respectively (Table 3) and (Fig. 1. a, b). After exposure to LC10 of Cryptostegia grandiflora for three days cause a significant decrease in mean number of granulocytes in Biomphalaria alexandrina and Biomphalaria galabrata being 43 % and 25 % with a percentage reduction 15.5 % and 12.1 % respectively (Table 3 and Fig.1. c, d). This reduction may be due to a direct action of the plant on these cells. This results agree with the findings of Sharaf El-Din (2003), Martin et al., (2006), Souza and Andrade (2006), Kamel et al., (2006, 2007) and Gawish et al., (2008). The results of the study indicated that, the mean number of granulocytes after exposure of snails to Schistosoma mansoni miracidia becomes 42% and 26.3% with apercentage reduction 17.4% and 7.5% in both snail species respectively (Table 3 and Fig.1. e, f). This decrease may be due to migration of haemocytes out of the haemolymph circulation to participate in the incapsulation of the parasite or take part in the repaire of tissue damage caused by the parasite (Stumpf and Gilbertson, 1980; Loker et al., 1982; Grantath and Yoshino, 1983). This results agree with the finding of Kamel et al., (2006) which reported that, the number of granulocytes cells decrease frome 51.8% in control group to 36.3% after 14 days from exposed Biomphalaria alexandrina snails to Schistosoma mansoni miracidia. The results of the study indicate that the maintainance of snails in LC10 of latex solution for 3-days perior miracidial exposure, to infection with Schistosoma mansoni results a high percentage reduction in the number of granulocytes of Biomphalaria alexandrina and Biomphalaria glabrata being 29.6% and 18% respectively with a percentage reduction 41.9% and 36.7% respectively (Table 3 and Fig. 1, g, h). These observation are in agreement with Kamel et al., (2007) which stated that, the cause of this decrease in the number of haemocytes may be due to inhibition in transaminase enzyme activity.These enzymes are considered good sensitive tools for detection of any variations in the physiological process of living organisms (Nevo et al., 1978; Tolba et al., 1997). While in the case of amoebocytes, the study revealed increasing percent with 14.8%, 56.7% and 47.1% in the case of Biomphalaria alexandrina and 13%, 10.1% and 20.3% in the case of Biomphalaria glabrata in all experimental groups respectively (Table 3 and Fig.1 from a to h). These results confirms the data of Sharaf El- Din (2003) who reported that, exposure of snails to Schistosoma mansoni and Echinostoma liei miracidia could stimulate the hematopoietic organ to produce a number of new undifferentiated haemocytes but reduced the hemoglobin content in their hemolymph during first, second and third week post miracidial exposure. On the other hand, El-Sayed (2006) found, the percentage of different haemocyte categories of Biomphalaria alexandrina snails was changed when treated with dry powder of the plant Cupressus macrocarpa. Also stated, the maintainance of Biomphalaria alexandrina in sublethal concentration (LC25) reduced protein, hemoglobin content and enzymes activities in the hemolymph and tissues of treated snails. The enzymes were pyruvate kinase (PK), lactate dehydrogenase (LDH), hexokinase (HK) and phosphoenol pyruvate carboxy kinase (PEPCK) which are very important in metabolism of the protein and carbohydrate. Examination of Biomphalaria alexandrina and Biomphalaria glabrata haemolymph by electron microscopy revealed three types of different cells classified according to their shape and granular contents. These cells are Granulocytes (G), Hyalinocytes (H) and Amoebocytes (A). Normal Biomphalaria alexandrina haemocytes showing (G) granulocyte with condensed granules (g) and (H) hyalinocytes with clearly obvious organelles (n) nucleus; (ga) Gologi apparatus;( m) Mitochondria) and the cell membrane appears faint (A)amoebocyte with large and clear pseudopodia(p) Fig. (2.a). Biomphalaria alexandrina haemocytes after exposure to LC10 for 3 days showing(G) granulocyte with condensed granules(g) and large phagolysosmes (ph); (H) hyalinocytes with clearly obvious condencsed cell membrane (cm) and (A)amoebocyte with large and clear peudopodia(p) and activated eccentric nucleous(n)) Fig. (2.b). Biomphalaria alexandrina haemocytes after exposed to Schistosma

3360 Aust. J. Basic & Appl. Sci., 5(12): 3357-3365, 2011 mansoni miracidia for two weeks showing activation for all cells as appearance of vacuolated cytoplasm (vc), with many pseudopodia (p), thick cell membrane (cm) and dense nuclear chromatine (n) Fig. (2.c). Biomphalaria haemocytes after exposure to LC10 for 3 days then exposed to S. mansoni miracidia for two weeks showing activation for all cells as many pseudopodia (P); many vacuoles (v) filled with antiparasite enzymes; thick cell membrane (cm) and dense nuclear chromatine (n) (Fig. 2. d).

Fig. 1: Photograph of haemolymph showing granulocyte (G), hyalinocyte (H), and amoebocyte (A) (400). (a) Control haemolymph of Biomphalaria alexandrina. (b) Control haemolymph of Biomphalaria glabrata showing collection of light granulocytes, dark granulocytes(G), an amoebocyte (A) and hyalinocyte (H). (c) Treated Biomphalaria alexandrina after exposure to LC10 for 3-days. (d) Treated Biomphalaria glabrata after exposure to LC10 for 3-days. (e) Infected Biomphalaria alexandrina. (f) Infected Biomphalaria glabrata. (g) Infected Biomphalaria alexandrina after exposure to LC10 for 3-days plan. (h) Infected Biomphalaria galabrata after exposure to LC10 for 3-days plants.

Normal Biomphalaria glabrata haemocytes classified to granulocytes (G) one with abundant dense granules (g) in the cytoplasm and other have few granules. Hyalinocytes (H) with transparent cytoplasm. Amoebocyte (A) with clear psedopodia (P) Fig. (3.a). Biomphalaria glabrata haemocytes after exposure to LC10 for 3 days showing (A) amoebocyte with extended pseudopodia (p). The cytoplasm is filled with granules (g) that push the nucleus to be eccentric as in (G) granulocyte; many large filled vacuoles(v) and chromatine condensation(ch) in their nucleus as in hyalinocytes (H) ) Fig. (3.b). Haemcytes of Biomphalaria glabrata after exposed to Schistosoma mansoni miracidia for two weeks showing many vacuoles in cytoplasm (v), pseudopodia (p) and activated nucleus (n) and cell membrane (cm) become ready with their tentacles against parasite) Fig. (3.c). Biomphalaria glabrata haemocytes after exposed to LC10 for 3 days then exposed to Schistosoma mansoni miracidia showing many extended pseudopodia (p) and the cytoplasm is filled with large number of phagolysosomes (ph) with many distributed vacuoles in their cytoplasm (v) and condensed nucleus (n) ) Fig. (3.d). Ultrastructure changes observed in the present study Agree with Tolba (1997) who observed the toxic effect of Anagallis arvensis against the foot, of Monacha sp. and showed that the plant was more effective and caused complete destruction of the foot tissue. Also Atlam (2000) studied the effect of molluscicidal activity of Euphorbia pepus and Sesbania sesban on the haemocytes of B. alexandrina. She observed ruptured cell membranes, degenerated nuclei, swollen, ruptured mitochondria, increase of lysosomal vesicles and the appearance of many fine filopodia and autolysis of vacuoles. Kamel et al., (2006 and 2007) revealed remarkable activation represented by vacuolated cytoplasm, extened pseudopodia and condensed nuclear chromatin. Such alterations could be due to a direct toxic effect of the molluscicidal of plant used.

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Fig. 2: Biomphalaria alexandrina haemocytes (10000 X). a- Normal haemocytes. b- Haemocytes after exposed to LC10 of plant. c- Haemocytes after exposed to S. mansoni miracidia. d- Haemocytes after exposed to LC10 of plant and S. mansoni miracidia.

Table 1: Molluscicidal activities of latex aqueous solutions of Cryptostegia grandiflora against Biomphalaria alexandrina and Biomphalaria galabrata snails. Snails species Lethal concentrations (ppm) LC50 LC90 Slope Sublethal concentrations (ppm) LC0 LC10 LC25 B. alexandrina 6.98(5.51-8.02) 8.65(8.81-12.7) 2.46 0.88 4.4 5.6 B. galabrata 10.29(8.19-11.8) 14.03(12.4-17.9) 3.3 3.5 6.6 8.3

Table 2: Infection rate with Schistosoma mansoni in Biomphalaria alexandrina and Biomphalaria galabrata before and post exposed to sublethal concentration (LC10) of Cryptostegia grandiflora. Snails species Snails exposed to Schistosoma Snails exposed to %Reduction mansoni miracidia Schistosoma mansoni miracidia and LC10 of C. grandiflora B. alexandrina 66.7 29.7 55.47 B. galabrata 14.6 6.0 58.9

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Fig. 3: Biomphalaria glabrata Haemocytes (10000 X). a- Normal haemocytes. b- Haemocytes after exposed to LC10 of plant. c- Haemocytes after exposed to S. mansoni miracidia. d- Haemocytes after exposed to LC10 of plant and S. mansoni miracidia.

Table 3: Number and percentage reduction or increasing in haemocytes of Biomphalaria alexandrina and Biomphalaria glabrata snails before and after exposed to Schistosoma mansoni miracidia and treated with (LC10) of Cryptostegia grandiflora under light microscope. Types of cells Normal group Infected group Treated with C. grandiflora Infected and treated (control) B.alexandrina B.glabrata B.alexandrina B.glabrata B.alexandrina B.glabrata B.alexandrina B.glabrata

Granulocytes 50.86± 28.43± 42± 3.74 26.29± 43.00± 6.32 25.00± 3.37 29.57± 18.00± Mean ± S.D. 3.72 2.23 7.46 * 3.31 2.00 *** *** % Reduction (-) or increasing (+) -17.42 -7.53 -15.45 -12.06 -41.86 -36.69

Hyalinocytes 19.14 20.86 11.00 17.86 22.57 17.71 ± 26.29 ± 21.00 Mean ± S.D. ± ± ± ± ± 1.98 3.55 ± 1.07 1.46 1.15 1.77 2.37 ** *** 2.24 *** ** *** %Reduction (-) or -42.52 -14.38 +17.92 -15 +37.35 +0.67 increasing (+) Amoebocytes 30.00 50.71 47.00 55.85 34.43 57.29 44.14 61.00 Mean ± S.D. ± ± ± ± ± ± ± ± 3.79 0.95 3.83 2.19 7.39 5.06 3.24 2.71 *** *** *** *** %Reduction (-) or +56.66 +10.13 +14.77 +12.96 +47.13 +20.29 increasing (+) * Significant at p< 0.05 ** highly significant p< 0.01 *** very highly significant p< 0.001

3363 Aust. J. Basic & Appl. Sci., 5(12): 3357-3365, 2011

Conclusion: Exposed of tested snails to LC10 of the latex aqueous solution of Cryptostegia grandiflora showed a highly significant increase of snail's amoebocytes that is a component of the internal defense system, hence their compatability to Schistosoma mansoni was decreased. It is evident that, Cryptostegia grandiflora may be used for snail control which is one of the efficient methods to control this parasite. ACKNOWLEDGMENT

This research was founded by the internal project NO. 78C, Theodor Bilharz Research Institute (TBRI).

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