




































Agriculture and Food 

Sciences Research 
ISSN: 2411-6653 
Vol. 1, No. 1, 5-10, 2014 
http://www.asianonlinejournals.com/index.php/AESR 

  

 

* Corresponding Author 

 

 

5 

 

Evaluation of the Pesticide Emamectin and Methanol 

Extract of Wheat Bran against Biomphalaria Alexandrina 

Snails, Their Hemocytes and Their Infection with 

Schistosoma Mansoni 
 

Hanan S. Mossalem
1*

 --- Gehan L. ElEnain
2
 

 

1
Department of Environmental Research and Medical Malacology, Theodor Bilharz Research Institute (TBRI), Giza, Egypt 

2
Department of Parasitology, TBRI, Giza, Egypt; Natural Sciences, University College, Abu Dhabi University, UAE 

 

Abstract 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 
 

 

 

 

 

 

 
This work is licensed under a Creative Commons Attribution 3.0 License 

Asian Online Journal Publishing Group 

 

Contents 
1. Introduction ........................................................................................................................................................................... 6 

2. Materials and Methods .......................................................................................................................................................... 6 

3. Statistical Analysis ................................................................................................................................................................. 7 

4. Results .................................................................................................................................................................................... 7 

5. Discussion ............................................................................................................................................................................... 7 

References .................................................................................................................................................................................. 8 

 

 

The present study was carried out to evaluate the molluscicidal activity of the pesticide Emamectin (5% 

aqueous solution) and the methanol extract from the wheat bran (MEWB) against B alexandrina and their 

infection with Schistosoma mansoni was studied. The LC90 and LC50 values for Emamectin were 50.4 

and 22.3 ppm, respectively. Infection of snails with S. mansoni under the effect of the tested agents was 

evaluated via four experimental groups, each of 50 snails For three consecutive days, one group of snails 

was exposed to 9.08 ppm aqueous Emamectin solution, another group received 100 ppm methanol 

extract of wheat bran (MEWB), a third group was administered by a combination of 9.08 ppm 

Emamectin and 100ppm MEWB, The fourth group was control maintained under similar experimental 

conditions. After three days, all the experimental groups were infected with S. mansonimiracidia and 

observed till shedding of cercariae. The physiological and histological changes were assessed before and 

after the infection.The changes in hemocytes of infected snails after administration with LC25 of 

Emamectin or wheat bran was significantly (20% and 30%, respectively) suppressed compared to 75% 

for the control snails. However, the snails treated with joint of Emamectin and MEWB were the least 

infected snails (10%). On the other hand, the biochemical test results showed a remarkable reduction of 

GOT (p<0.01) GPT (p<0.05) and total protein (0.05) in the heamolyph  extracted from the snails treated 

with an aqueous Emamectin solution. Yet, the levels of GOT were significantly increased in the groups 

administered with the MEWB alone (p<0.01) or in combination with the Emamectin (p<0.001).  As for 

the total protein levels, there were slightly declining (p<0.05) in the group exposed to the aqueous 

Emamectin and to the contrary, theses levels were significantly increased (P<0.05) in the snails of the 

two other experimental groups when compared to the controls. Moreover, the haemocytes cells showed a 

differentiation which varied in number when detected under the microscope. Administration of the 

aqueous Emamectin solution resulted in a significant increase (p<0.05) of the amaebocytes and a 

considerable decrease (p<0.05) in the number of granulocytes and hyalinocytes compared to the control 

snails. On the contrary, a remarkable surge (P<0.05) of granulocytes and decrease of the amaebocytes 

was detected in the haemolyph of snails treated with MEWB. 

 
Keywords: Biomphalaria alexandrina, Emamectin, Wheat bran, Molluscicide, Antioxidant, Schistosoma mansoni. 

http://creativecommons.org/licenses/by/3.0/


Agriculture and Food Sciences Research, 2014, 1(1): 5-10 

 

 

 

 

6 

 

1. Introduction 
Schistosomiasis is the second major parasitic disease in the world after malaria [1]. It poses not only a health 

problem, but economic crisis as well. Relatively 200 million people are infected with Schistosoma and around 800 

millions are at risk of infection [2].  

Fresh water snails are the intermediate hosts that transmit the human schistosomiasis [3]. The intestinal 

Schistosomiasis is caused by Scistosomamansoni which is transmitted by the intermediate host Biomophalaria 

alexandrina, a fresh water snail found in Egypt [4].  

It is well-known that control of the freshwater snails might play a key role in the transmission of schistosomiasis. 

Snails are approved to be the most vulnerable link in the schistosome life cycle and consequently, targeting their 

populations is an important approach to prevent the transmission of shistosomes. Still comprehensive information 

about snails is required [5] to assist in developing a harmless molluscicide.  

Application of molluscicides (synthetic or of plant origin) can confer a rapid and efficient technique in tumbling 

the snail populations [6]. Attempts to reduce or eliminate populations of freshwater snails in Africa have been 

concentrated on the intermediate hosts of schistosomes with less attitude to use the chemicals for the purpose of 

reducing transmission of schistosomiasis [4]. The chemical molluscicides have not achieved the expected outcomes 

[7] and the synthetic ones were approved to be costly in relation to the restricted budgets [7] available for the control 

of communicable diseases in many countries [8]. The development of molluscicides from plant origin is currently of 

interest in measures for snail control because they are affordable, safe and useful in local community self-help 

projects [9]. There are some plants with antioxidant activity such as rice bran which play an important role in 

reducing the pollution of aquatic media and were proved to be safe for all manner of objects and the surrounding 

environment [10]. 

It possesses not only detect the molluscicidal activity of the aqueous Emamectin solution to assist in developing 

a candidate molluscicude, alone and combined with methanol extract from the wheat bran, as antioxidant agent, 

against B. alexandrina. 

 

2. Materials and Methods 
2.1. Snails 

Snails used in this study were adult Biomphalariaalexandrina (8-10mm) collected from the River Nile and 

irrigation scheme near Cairo and brought to the laboratory, washed with dechlorinated water and examined for larval 

trematodes. Unhealthy and infected snails were excluded, the healthy snails were maintained in plastic aquaria under 

standard conditions (25
o
C ± 1

o
C, PH = 7.2) for three weeks before being used in molluscicidal tests. Snails were 

daily fed boiled lettuce leaves and Blue green algae.  

 

2.2. Miracidia 
Schistosomamansonimiracidia were obtained from Schistosome Biological Supply Center (SBSC), TBRI, Egypt. 

 

2.3. Emamectin (Vetoken) 
Emamectin (Vetoken) is non-systemic insecticide which penetrates leaf tissues by translaminar movement. 

Paralyses the Lepidoptera, which stop feeding within hours of ingestion, and die 2-4 days.Uses For control of 

Lepidoptera on vegetables, brassicas, cotton and pine trees. The purchase company of Emamectin is Shanghai 

Agrochina, International, Trade, CO. LTD CHIN,The structural formula of Emamectin is (4″R) -4″-deoxy-4″-

(methylamino) avermectin B1 and Mode of Action: Chloride channel activator. Acts by stimulating the release of g-

aminobutyric acid, an inhibitory neurotransmitter, thus causing paralysis 

 

2.3.1. Extraction of Methanol Extract of Wheat Bran 
The wheat bran was dried in shade, then in an oven at 50

o
C. Ten grams powder was extracted with 250 ml 95% 

methanol at room temperature for one week. Methanol was evaporated under vacuum and the residues were used for 

the bioassay tests. 

 

2.4. The Molluscicidal Tests Using Aqueous Emamectin and Methanol Extract of Wheat Bran 

The potential molluscicidal activities of Emamectin and methanol extract of wheat bran were tested against adult 

B. alexandrina. Different concentrations in ppm were prepared on the basis of weight/volume. For each concentration 

three glass containers each containing one liter dechlorinated water to which the test material was added directly on 

the water surface and stirred properly then 10 snails were introduced. The glass containers were covered by porous 

plastic sheets and maintained for 24 hours of exposure at normal laboratory conditions (25
o
C ± 1

o
C). After that, the 

snails were washed thoroughly with dechlorinated water, transferred to jars containing fresh dechlorinated water for 

another 24 hours for recovery. Three replicates of control snails (10snails/l) were prepared in dechlorinated water. 

Dead snails were distinguished and counted. Further experiments were carried out such that Laboratory bred B. 

alexandrina snails (8-10 mm) were deployed into four groups, each of 50 snails. They were continuously treated for 

3 days with the tested agents one group Emamectin solution was treated with the LC50 of Emamectin, another group 

was treated with MEWB, the third was exposed to a combination of 9.08ppm Emamectin and 100ppm WEMB, 

whereas the fourth group was maintained at the same conditions in dechlorinated water as a control group. Dead 

snails were removed daily from all experimental groups. Snails were considered dead if they probed and remained 

motionless or if the shell looked discolored. After 3 days of treatment, the survived snails at all groups were 

subjected to to infection with S. mansonimiracidia and kept till shedding of cercaria. The extent of the survival of the 

experimental snails were estimated by relating them to their counterparts of the control snails. Determination of total 

protein, Haemocytes number, glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transminase (GPT) in 

snails' hemolymph. 

 



Agriculture and Food Sciences Research, 2014, 1(1): 5-10 

 

 

 

 

7 

 

2.5. Heamatocyte Count 
Haemolymph samples were collected according to Michelson [11] by removing a small portion of the shell and 

inserting a capillary tube into the heart. The haemolymph pooled from 10 snails were collected in a vial tube (1.5ml) 

and kept in an ice-box.  

 

2.5.1. Total Protein, Glutamic Oxaloacetic Transaminase (GOT) and Glutamic Pyruvic 

Transaminase (GPT) 

Colorimetric estimation of total protein content in haemolymph of B. alexandrina snails was estimated according 

to the principle of Biuret-tartrate method [12]. that of GOT and GPT was detected as stated by Reitman and Frankel 

[13]. 

 

3. Statistical Analysis 
The means of the different groups were compared globally using the student's t- test Sokal and Rohlf [14]. The 

Molluscicidal activity of the tested chemical was calculated by Probit analysis, SPSS Computer Program version19, 

Window7, MW Office 2007. 

 

4. Results 
4.1. Molluscicidal Activity of Aqueous Emamectin and Methanol Extract of Wheat Bran 

The effect of various concentrations of the aqueous Emamectin (5%) and MEWB on adult B. alexandrina snails 

after 24hr exposure was evaluated (Table 1 and Fig. 1). All MEWB concentrations up to 100ppm were harmless to 

the snails. The LC25, LC50 and LC90 obtained with the aqueous Emamectin solution against B. alexandrina snails 

were 9.08 ppm, 22.3ppm and 50.4 ppm, respectively. Surprisingly, administration of joint 100ppm MEWB and 

aqueous Emamectin solution changed the activity of the Emamectin against B. alexanderia snails to be 22.916ppm at 

LC25, 31.8 ppm at LC50 and 48.816 ppm in LC90, as depicted in table 2. 

 

4.2. Moullscicidal Efficacy on the B. Alexandrina Infectivity withS. Mansoni 
The percent S. mansoni infection to the experimental snails was remarkably lower (p<0.001) than that detected in 

the infected negative control snails. The lowest infection (10%) was determined by the group of snails administered 

by a joint of aqueous Emamectin and MEWB. 

 

4.3. The Effect of Emamectin and MEWB on B. Alexanderina Physiologic and Histologic Activity 
The enzyme activity measured by the levels of GOT, GPT and total protein showed variable responses to the 

administered substances. In the 9.08ppm Emamectin treated snails, there was a significant decline in the levels of the 

three types of protein, whereas administration of MEWB alone or in combination with the aqueous Emamectin 

solution to snails increased the levels of  GOT, GPT and total proteins in the haemolymph, considerably (Table 2). 

Administration of the Emamectin resulted in a significant increase (p<0.05) of the amaebocytes and a considerable 

decrease (p<0.05) in the number of granulocytes and hyalinocytes compared to the control snails.    On the contrary, 

a remarkable surge (P<0.05) of granulocytes and decrease of the amaebocytes was detected in the haemolyph of 

snails treated with MEWB (Table 3 & Fig. 1). 

 

5. Discussion 
The aqueous Emamectin (5%) is one of the most chemical pesticides used in cotton agriculture and causes 

pollution in the aquatic environment. The molluscicidal activity of aqueous Emamectin (5%) alone and joint with the 

methanol extract from wheat bran was evaluated against B. alexandrina snails. The lethal concentrations of this 

molluscicide obtained against B.alexandrina snails LC90 and LC50 values were 50.4ppm and 22.3ppm.The current 

data showed a severe decrease (20% and 10%) in the infection rate of snails infected with S.mansoni after being 

continuously exposed to the sublethal dose LC25 of Emamectin (5%) alone and in combination with the MEWB, 

respectively. This may be explained by the deteriorations of physiological parameters of snails making them 

incongruous for the parasite development [15]. Mahmoud, et al. [16] found that exposure of B. alexandrina and B. 

glabrata snails to sublethal concentration of Cryptostegiagrandiflora 3 days pre-miracidial exposure led to a 

significant reduction in the infection rate with S. mansoni by 55.47% and 58.9%, respectively. In another study [17]  

,administration of LC10 of Daturastramonium and Sesbaniasesban during S. mansonimiracidial exposure  reduced 

the infection rate of B. alexandrina snails  by 41.7% and 52.2%, respectively compared to the control group. All the 

recorded results in the present study concerning total protein levels, GOT and GPT activities are mostly in agreement 

with  with similar research work conducted by many authors [18-21]. 

The present investigation showed that haemocytes in the experimental snails were significant different in number 

when compared to the control ones.  Three types of haemocytes were detected unevenly tribute in the haemolymph 

of B. alexandrina snails. They ranged from Granulocytes 60%, Amoebocytes 25% to Hyalinocytes 15% in the lymph 

of the control snails, which is supported by the results obtained in different studies [15, 22-26]. Administration of 

Emamectin (5%) reduces the number of Granulocytes and, Hyalinocytes (40% and 10%, respectivel) but increased 

the  number of Amoebocytes by 50%. This observed surge in the number and size of Amoebocytes might be due to 

their main role in encountering any invaders/foe that attack the snails' tissue and being activated by engulfing such 

administered harmful pesticide.  Previous similar observations were detected when snails were subjected to the 

molluscicideArtemether for 3 days and obvious abnormalities were detected in haemocytes morphology [10] In the 

present study, exposure to the wheat bran alone or in combination with the Emamectin has increased the number of 

haematocytes which might be due to the highly effective role of all extracts of wheat bran in inhibiting the Linoleic 

acid peroxidation as demonstrated by Laokuldilok, et al. [27]. It has been approved that rice bran has antioxidant 



Agriculture and Food Sciences Research, 2014, 1(1): 5-10 

 

 

 

 

8 

 

activity due to the high content of phytochemicals Norhaizan, et al. [28] which may indicate that the wheat bran also 

have antioxidants that improve the performance of the Emamectin and reduce its harmful pesticidal activity.  

This work vigorously urge more researches on all types of bran that could be anti-pollutant agents and friendly 

environment substances. 

 

References 
[1] World Health Organization (WHO), African network for drug/diagnostics discovery and innovation (ANDI). Abuja, Nigeria: World 

Health Organization (WHO), 2008. 

[2] P. Steinmann, J. Keiser, R. Bos, M. Tanner, and J. Utzinger, "Schistosomiasis and water resources development: Systematic review, 
meta-analysis, and estimates of people at risk," Lancet Infect Dis., vol. 6, pp. 411-425, 2006. 

[3] W. Lotfy, R. Dejong, A. Abdel-Kader, and E. Loker, "A molecular survey of biomphalaria in Egypt: Is B. Glabrata present?," Am. J. 

Trop. Med. Hyg., vol. 73, pp. 131-139, 2005. 

[4] World Health Organization WHO, "Mollusciciding in schistosomiasis control. WHO/Schisto/92.107," 1992. 
[5] G. M. El-Khodary, "Comparative ultrastructural and immunological studies on the effect of factors of variable nature on schistosoma 

and intermediate hosts," Ph.D. Thesis, Fac. Science, Tanta University, Egypt, 2001. 

[6] A. Ibrahim, M. El-Emam, S. El-Dafrawy, and H. Mossalem, "Impact of certain plant species on schistosoma mansoni biomphalaria 

alexandrina system," Proceeding 3rd International Conference Science, vol. 3, pp. 390-413, 2004. 
[7] W. Wu, Y. Chen, Z. Zhai, S. Xiao, and W. Y. Yu-Lin, "Study on the mechanism of action of artemether against schistosomes: The 

identification of cysteine adducts of both carbon-centred free radicals derived from artemether," Bioorg Med. Chemist Letters, vol. 

13, pp. 1645–1647, 2003. 

[8] H. Abdel-Hamid, "Effect of ethanol extracts from the plants, anagallis arvensis and zingiber officinale and their mixtures against 
lymnaea natalensis, the snail vector of fasciola gigantica," New Egypt J Med., vol. 38, pp. 109-116, 2008. 

[9] F. Bakry, K. El-Hommossany, and H. Mossalem, "Immunological and physiological parameters of biomphalaria alexandrina snails 

exposed to azadirachta indica plant," Euro Rev Med Pharmacol Sci., vol. 16, pp. 133-143, 2012. 

[10] S. M. Hanan, H. Abdel-Hamid, and N. A. El-Shinnawy, "Impact of artemether on some histological and histochemical parameters in 
biomphalaria alexandrina," African Journal of Pharmacy and Pharmacology, vol. 7, pp. 2220-2230, 22 2013. 

[11] A. Michelson, "Specificity of haemolymph antigens in taxonomic discrimination of medically important snails," J Parasitol., vol. 52, 

pp. 466-472, 1966. 

[12] T. E. Weichselbaum, "An accurate and rapid method for the determination of proteins in small amounts of blood and serum," Am. J. 
Clin. Pathol., vol. 10, pp. 40-49, 1946. 

[13] S. Reitman and S. Frankel, "A colorimetric method for the determination of glutamic–oxaloacetic and glutamic–pyruvic 

transaminases," Am. J. Clin. Pathol., vol. 28, pp. 56–63, 1957. 

[14] R. R. Sokal and F. J. Rohlf, "Taxonomic congruence in the leptopodo morpha re-examined," Systematic Zool., vol. 30, pp. 309-325, 
1981. 

[15] K. El Sayed, M. Mahmoud, and H. Mossalem, "Cryptostegia grandiflora affecting compatibility of biomphalaria alexandrina and 

biomphalaria galabrata to infection with schistosoma mansoni with emphasis on some hematological effects," Austr J Basic Appl 

Sci., vol. 5, pp. 3357-3365, 2011. 
[16] M. B. Mahmoud, W. L. Ibrahim, B. M. Abou- EL-Nour, M. A. EL-Emam, and A. A. Youssif, "Biological and biochemical 

parameters of biomphalaria alexandrina snails exposed to the plants datura stramonium and sesbania sesban as water suspensions of 

their dry powder," Pesticide Biochemistry and Physiology, vol. 99, pp. 96-104, 2011. 

[17] A. El Ansary, S. El Bardicy, M. S. Soliman, and N. Zayed, "Sublethal concentration of ambrosia maritime (Damsissa) affecting 
compatibility of biomphalaria alexandrina snails to infection with schistosoma mansoni through disturbing the glycolytic," Journal of 

the Egyptian Society of Parasitology Pathway, vol. 30, pp. 809-819, 2000. 

[18] A. A. Tantawy, A. T. Sharaf El-Din, and F. A. Bakry, "Mollusciciding effect of solanum dubium (Solanaceae) against biomphalaria 

alexandrina snails under laboreatory conditions," Proc. Int. Conf. Biol. Sci., vol. 1, pp. 307-318, 2000. 
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haematobium," J Egypt Soc Parasitol, vol. 35, pp. 859-874, 2005. 

[20] F. Bakry, H. Abdel-Hamid, and H. Abu El Einin, "Effect of neem plant (Azadirachta Indica) on some biological and histological 

parameters of non-infected biomphalaria alexandrina and infected with schistosoma mansoni," J. Egypt. Ger. Soc. Zool., vol. 54, pp. 
51-68, 2007. 

[21] W. Hasheesh, R. Mohamed, and S. El-Monem, "Biological and physiological parameters of bulinus truncatus snails exposed to 

methanol extract of the plant sesbania sesban plant," Adv Biol Chem., vol. 1, pp. 65-73, 2011. 

[22] A. T. Sharaf El-Din, "Effects of double infection with echinostoma liei and schistosoma mansoni on hemoglobin content and 
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vol. 25, pp. 41- 52, 2003. 

[23] R. Martins-Souza, C. Pereira, and O. Martins-Filho, "Differential lectin labeling of circulating haemocytes from biomphalaria 
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[24] S. Souza and Z. Andrade, "On the origin of the biomphalaria glabrata haemocytes," Mem Inst Oswaldo Cruz, vol. 101, pp. 213-218, 

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[25] E. Kamel, S. Refaat, S. El-Dafrawy, A. Mohamed, and H. Mossalem, "The effect of schistosoma mansoni infection on biomphalaria 

alexandrina haematocytes at ultra structural level," in Proceeding of the 4th International Conference of Biological Science 

(Zoology), 219, 2006. 

[26] E. Kamel, S. Refaat, S. El-Dafrawy, A. Mohamed, and H. Mossalem, "Toxicological effect of certain plants and synthetic 
molluscicides on ultra structural changes in haemocytes of biomphalaria alexandrina," The Egyptian Journal of Experimental Biology 

(Zoology), vol. 3, p. 135, 2007. 

[27] T. Laokuldilok, C. Shoemaker, S. Jongkaewwattana, and V. Tulyathan, "Antioxidants and antioxidant activity of several pigmented 

rice brans," Journal of Agricultural and Food Chemistry, vol. 12;59, pp. 193-9. 
[28] M. E. Norhaizan, A. K. Khairul-Kamilah, A. Zulkhairi, and A. Azrina, "Improving the lipid profile in hypercholesterolemia-induced 

rabbit by supplementation of germinated brown rice," J Agric Food Chem 2011 Jul 27, vol. 59, pp. 7985-91, 2011. 

 

 

 

 

 

 

 

 

 

 

 



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Table-1. Mortality rate of Vetoken in comination with methanol extract of wheat bran against Biomophalaria alexandrina 
snails 

Total no. 

of dead snails 

Total no. 

of snails 

Concentration 

(ppm) 

40 48 45 

35 48 40 

30 48 35 

22 48 30 

11 48 25 

8 48 15 

1 48 10 

 

Table-2.Molluscicidal activity of Vetoken (5%) alone and joint with Wheat bran against B. alexandrina 

PROBIT Aqueous Vetoken (5%) Vetoken (5%) combined with 100 ppm MEWB 

95% Confidence Limits for Concentration 

Lower Bound Upper Bound Lower Bound Upper Bound 

LC90 50.4ppm 48.816ppm 

39.480 79.538 45.275 53.865 

LC50 22.3ppm 31.847 

16.748 31.370 29.804 33.977 

LC25 9.08ppm 22.916 

-5.032 15.834 19.937 25.235 

 

Fig-1. Dose/Probit regression line of joint Vetoken/MEWB on Biomophalaria alexandrina 

 
 

Table-3. The average percentage of S. mansoni infection to B. alexandrina in the experimental groups 

Concentrations % infection GOT (g/dl) GPT (g/dl) Total protein (g/dl) 

Mean ± SD 

9.08ppm Vetoken 20%*** 0.318667±0.006351** 0.348667±0.005508* 5.44±0.1* 

100ppm WEMB 30%*** 0.415667±0.005508** 0.392333±0.005508* 6.776667±0.01527* 

9.08ppm Vetoken + 

100ppm WEMB 

10%*** 0.542667±0.009018*** 0.415667±0.005508* 5.95±0.01* 

Control 75% 0.350667±0.000577 0.353±0.001 5.748± 0.001 
           ***high significant at p<0.001; ** moderate significant at <0.01; *slightly Significant at p<0.05 

 
 

Table-4.The haematocytes countbefore and after three-consecutive days’ exposure to aqueous vetoken and MEWB 

Concentration Granulocyte Amoebocyte Hyalinocyte 

Control 60% 25% 15% 

Vetoken 40%* 50%* 10%* 

MEWB 70%* 15%* 15% 
              *significant at P<0.05 

 



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Fig-2. Haemocytes under light microscope showing G granulocyte, A amoebocyte, and H hyalinocyte a. Control;   b. 
LC25Vetoken-treated snails showed decreasing in the number of granulocytes;  c. Haemocytes from 100ppm  MEWB-

treated snails showing an increase in the  number and size of granulocytes. A is denoted for amaebocytes, G stands for  

granulocytes and H refers to hylinocytes (Light microscope, 200X) 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
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