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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 99 (2011) 96–104
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Biological and biochemical parameters of Biomphalaria alexandrina snails exposed to the plants Datura stramonium and Sesbania sesban as water suspensions of their dry powder Momeana B. Mahmoud a, Wafaa L. Ibrahim b, Basma M. Abou-El-Nour b, Mohamed A. El-Emam a,⇑, Alaa A. Youssef a a b
Medical Malacology Department, Theodor Bilharz Research Institute, Giza, Egypt Zoology Department, Faculty of Science, Al-Azhar University (Girls Branch), Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 4 October 2010 Accepted 9 November 2010 Available online 20 November 2010 Keywords: Biomphalaria alexandrina snails Schistosoma mansoni Plant mollucicides Transaminases and phosphatases enzymes
a b s t r a c t Four plant species, as a dry powder of their leaves, were tested against Biomphalaria alexandrina snails, the intermediate host of Schistosoma mansoni. The bioassay tests revealed the plants Datura stramonium and Sesbania sesban to be more toxic to the snails than the other two ones. Therefore, they were tested against snails’ fecundity (Mx), reproduction rate (Ro) and their infection with S. mansoni miracidia. In addition, total protein concentration and the activities of the transaminases (AsT and AlT) and phosphatases (AcP and AkP) enzymes in hemolymph and tissues of snails treated with these plants were determined. As well, glucose concentration in snails’ hemolymph was evaluated. Exposure of snails for 4 weeks to LC10 and LC25 of the plants D. stramonium and S. sesban dry powder markly suppressed their Mx and Ro. The reduction rates of Ro for snails exposed to LC25 of these plants were 62.1% and 76.4%, respectively. As well, a considerable reduction in the infection rates of snails exposed to these plants either during, pre- or post-miracidial exposure was recorded. Thus, infection rates of snails treated during miracidial exposure with LC10 of D. stramonium and S. sesban were 41.7% and 52.2%, respectively, compared to 92.6% for control group (P < 0.01). These plants, also, reduced the duration of cercarial shedding and cercarial production/snail. So, snails exposed to LC25 of these plants shed 372.8 and 223.2 cercariae/snail, respectively, compared to 766.3 cercariae/infected control snail (P < 0.01). The results, also, revealed that glucose and total protein concentrations in hemolymph of snails treated with LC10 and LC25 of these plants were decreased, meanwhile, the activities of the enzymes AsT, AlT, AcP and AkP were elevated (P < 0.01). However, the activity of AcP in tissues of treated snails was decreased compared to that of control ones. It is concluded that LC25 of the plants D. stramonium and S. sesban negatively interferes with biological and physiological activities of B. alexandrina snails, consequently it could be effective in interrupting and minimizing the transmission of S. mansoni. Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction There is no doubt that schistosomiasis is one of the major communicable diseases and it is second to the malaria with socio-economic and health importance in the developing world [1]. Controlling of the snail intermediate hosts of this parasite by molluscicides (synthetic and/or of natural origin) is still one of the most promising means in the battle against this parasitic disease [2]. Molluscicides of plant origin appear to be environmentally friendly having several advantages over the synthetic chemicals [3]. So far, more than 1500 plant species have been screened for ⇑ Corresponding author. E-mail address: [email protected] (M.A. El-Emam).
molluscicidal properties [4]. In Egypt, molluscicides of plant origin have received an increasing attention, so several plant species were screened in this concept [5–9]. The plant Sesbania aegyptiaca proved to have a molluscicial activity against Biomphalaria alexandrina snails as water suspensions from its dry powder [10] and that of Sesbania sesban exhibited a marked toxic effect against the snails Biomphalaria pfeifferi and Bulinus truncatus [11]. The methanol extract of Datura innoxia showed a promising molluscicidal potency against B. alexandrina, B. truncatus and Lymnaea caillaudi snails [12]. In addition, this extract of Datura stramonium has a strong antifungal property against Fusarium mangiferae fungus [13]. The amino transferases enzymes (AsT and AlT) are the most frequently measured for hepatic diseases. The enzymes may be released from hepatocytes into the circulation by necrosis or by
0048-3575/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2010.11.005
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M.B. Mahmoud et al. / Pesticide Biochemistry and Physiology 99 (2011) 96–104 Table 1 Molluscicidial activity of the dry powder from the tested plant species against adult Biomphalaria alexandrina snails (24 h exposure). Plant species
Family
LC0 (ppm)
LC10 (ppm)
LC25 (ppm)
LC50 (ppm)
LC90 (ppm)
Slope
Datura stramonium Sesbania sesban Cestrum diurnum Carissa carandas
Solanaceae Fabaceae Solanaceae Apocynaceae
2.5 5.11 189.3 >500
11.3 39.5 207.3
18.2 44.9 217.7
25.8 51.0 229.3
40.4 62.4 251.4
1.64 1.20 1.08
Table 2 Survival rate (Lx) and fecundity (Mx) of Biomphalaria alexandrina snails after 4 weeks of continuous exposure to sublethal concentrations of D. stramonium and S. sesban as dry powder water suspensions. Conc. (ppm)
Observation period (week, x) 1
Control
Ro R Lx Mx (reproductive rate)
2
3
% Reduction of Ro
4
Lx
Mx
Lx Mx
Lx
Mx
Lx Mx
Lx
Mx
Lx Mx
Lx
Mx
Lx Mx
1.0
17.1
17.1
1.0
13.4
13.4
0.95
14.3
13.6
0.95
21.9
20.8
64.9
D. stramonium
LC0 LC10 LC25
1.0 0.90 0.80
19.3 14.2 12.4
19.3 12.8 9.9
0.95 0.80 0.65
14.8 13.5 10.3
14.1 10.8 6.7
0.90 0.75 0.50
15.5 13.2 10.0
14.0 9.9 5.0
0.80 0.60 0.35
16.2 11.9 8.6
13.0 7.1 3.0
60.4 40.6 24.6
6.93 37.4 62.1
S. sesban
LC0 LC10 LC25
1.0 0.95 0.60
16.7 21.3 10.9
16.7 18.1 6.5
0.90 0.80 0.45
18.2 13.4 12.1
16.4 10.7 5.4
0.85 0.65 0.30
13.6 14.3 9.9
11.6 9.3 3.0
0.85 0.55 0.05
14.6 11.9 7
12.4 6.5 0.35
57.1 44.6 15.3
7.4 31.2 76.4
changes in cell membrane permeability that allows the enzymes to leak out of the cells [14]. AlT is more localized to the liver than AsT, so it is more specific to liver injury [15]. The phosphatases enzymes are capable of hydrolyzing organic phosphate esters. Acid phosphatase (AcP) is a lysosomal enzyme concerning with digestion of foreign substances and bacteria inside the cells [16] and is involved in the defense mechanisms of both vertebrates and invertebrates [17]. In addition, the deterioration in the activity of alkaline phosphatases can occur in all types of liver diseases [18]. The present work was designed to determine the molluscicidal and ovicidial potencies of the plants D. stramonium and S. sesban against B. alexandrina snails and their eggs. As well, the snails’ reproductive rate and their infection with Schistosoma mansoni miracidia were evaluated. In addition, the changes pattern in activities of transaminases (AsT and AlT) and phosphatases (AcP and AkP) enzymes and the total protein levels were assessed in tissues and hemolymph of treated snails.
2.3. Miracidia S. mansoni ova were obtained from Schistosomiasis Biological Supply Center (SPSC), TBRI. They were left in clean dechlorinated water for hatching under a desk lamp then fresh hatch miracidia were used in bioassay and infection tests. 2.4. Bioassay tests 2.4.1. Molluscicidal screening For each plant powder, a series of concentrations that would permit the computation of LC50 and LC90 was prepared on the basis of weight/volume as water suspensions. Three replicates, each of 10 snails/L, were prepared. Another three replicates in dechlorinated water were used as control. Exposure and recovery periods were 24 h each at 25 ± 1 °C [19,20]. Then, snails’ mortality was recorded and corrected according to Abbots’ formula [21].
2. Materials and methods 2.1. Snails B. alexandrina snails (6–8 mm) from laboratory bred colony in Medical Malacology Dept., Theodor Bilharz Research Institute (TBRI), Giza, Egypt were used.
2.2. Plants The four plant species used are Carissa carandas (Apocynaceae) and S. sesban (Fabaceae) from the garden of Faculty of Agriculture, Al-Azhar University, Cairo, Cestrum diurnum and D. stramonium (Solanaceae) from Al-Orman garden, Giza, Egypt (spring 2007). They were kindly identified by Botany Dept., Faculty of Agriculture, Al-Azhar University. Their leaves were shade dried, then powdered by an electric mill. The dry powder was stored in clean dry dark glass bottle till use in biological tests.
Fig. 1. Reduction rate (%) of reproductive rate (Ro) for B. alexandrina snails after 4 weeks of continuous exposure to Datura stramonium and Sesbania sesban as dry powder water suspension.
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Due to the high effect of the plants D. stramonium and S. sesban, they were chosen for detailed tests as follows: 2.4.1.1. Effect on snails’ egg-laying capacity. B. alexandrina snails (6– 8 mm) were continuously exposed for 4 weeks to the sublethal concentrations LC0, LC10 and LC25 from dry powder of each plant [19,22]. Three replicates, each of 10 snails/L, were prepared for each concentration, another three ones of control snails were maintained in dechlorinated water (25 ± 1 °C). The concentrations were weekly changed by fresh ones. The snails were daily fed oven dried lettuce leaves. Each aquarium was provided with one piece of polyethylene sheet for oviposition. The eggs laid on these sheets and on the wall of the aquaria were weekly counted using a
stereomicroscope and a hand lens (10). Dead snails were removed daily and the following parameters were weekly recorded [23]: Lx (the survival rate as a proportion of the correct one), Mx (the number of eggs/snail/week) and Ro (the reproductive rate which is the summation of Lx Mx). 2.4.1.2. Effect on infection of B. alexandrina snails with S. mansoni miracidia. The snails (6–8 mm) were exposed for 24 h to the sublethal concentrations LC0, LC10 and LC25 of the plants’ dry powder during miracidial exposure and at 1 day either pre- or post-miracidial exposure. Snails were exposed to miracidia in mass, 10 fresh hatched miracidia/snail. For each concentration, three replicates were
Table 3 Effect of Datura stramonium dry powder on survival rate at 1st shedding, infection rate, prepatent period, life span, duration of shedding and cercarial production of Biomphalaria alexandrina exposed to Schistosoma mansoni miracidia. Parameter
D. stramonium
Control
Pre-exposure
*
Post-exposure
LC0
LC10
LC25
LC0
LC10
LC25
LC0
LC10
LC25
Survival rate at 1st shedding
No. exposed No. survived %
30 26 86.7
30 22 73.4**
30 18 60.0**
30 27 90.0
30 24 80.0
30 19 63.3**
30 26 86.7
30 23 76.7*
30 19 63.3**
30 27 90
Infection of snails
Number %
23 88.5
19 86.4
16 88.9
18 66.7**
10 41.7**
6 31.6**
22 84.6
12 52.2**
4 21.1**
25 92.6
Prepatent period (days)
Range Mean ±S.E. Range Mean ±S.E.
21–27 21.6 1.7 7–34 17.8 1.7
21–27 22.3 2.1 4–22 12.2 1.3
21 21* 0.0 4–31 13.2 2.1
21–24 21.5 1.2 4–25 14.2 2.0
21–24 21.3 0.9 7–28 11.2 1.9
21–24 23 2.4 4–22 12.5 3.0
21–27 22.1 0.87 4–22 13.6 1.0
21–24 22.6 1.35 4–31 11.3 2.1
21 21* 0.0 4–22 12.3 3.8
12–27 22 1.9 4–43 15.5 1.8
Life span (days)
Range Mean ±S.E.
27–54 37.8 1.7
24–42 32.2 1.3
24–51 33.2 2.1
24–45 34.2 2.0
27–48 31.2 1.9
24–42 32.5 3.0
24–42 33.6 1.0
24–51 31.3 2.1
24–42 32.3 3.8
24–54 35.5 1.8
Total cercariae/snail
Range Mean ±S.E.
17–1486 710.4 111.2
16–1041 634.2 102.3
28–1400 788.6 131.2
9–1088 674.1 116.7
20–891 501.0** 79.8
10–660 372.8** 78.6
80–1010 619.9 103.3
10–731 679.3* 94.6
5–790 432.0** 65.4
14–1674 766.3 132.9
Duration of shedding
**
During exposure
P < 0.05. P < 0.01.
Table 4 Effect of Sesbania sesban dry powder on survival rate at 1st shedding, infection rate, prepatent period, life span, duration of shedding and cercarial production of Biomphalaria alexandrina exposed to Schistosoma mansoni miracidia. Parameter
S. sesban
Control
Pre-exposure
* **
During exposure
Post-exposure
LC0
LC10
LC25
LC0
LC10
LC25
LC0
LC10
LC25
Survival rate at 1st shedding
No. exposed No. survived %
30 25 83.3
30 20 66.7**
30 12 40.0*
30 27 90.0
30 23 76.7*
30 13 43.3**
30 27 90.0
30 21 70.1**
30 12 40.0**
30 27 90
Infection of snails
Number %
23 92.0
17 85.0
10 83.3
20 74.1**
12 52.2**
7 53.8**
24 88.9
14 66.7**
3 25.0**
25 92.6
Prepatent period (days)
Range Mean ±S.E.
21–24 21.5 1.2
21–24 21.7 1.3
21–24 22 1.5
21–27 22.2 2.0
21–27 22.1 1.9
21–27 21.9 2.3
21–27 21.3 0.62
21–24 21.2 0.0
21 21 0.0
12–27 22 1.9
Duration of shedding
Range Mean ±S.E.
4–19 11.6 1.1
4–22 9.5** 1.5
7–16 11.2* 0.9
7–22 11.4 1.0
7–22 11.8 1.4
4–16 7.0** 1.6
4–34 16.9 2.0
4–22 12.4 2.3
4–10 6.0** 2.0
4–43 15.5 1.8
Life span (days)
Range Mean ±S.E.
24–39 31.6 1.1
24–42 29.5** 1.5
27–36 31.2* 0.9
27–42 31.4 1.0
27–42 31.8 1.4
24–36 27.0** 1.6
24–54 36.9 2.0
24–48 32.4 2.3
24–30 26.0** 2.0
24–54 35.5 1.8
Total cercariae/snail
Range Mean ±S.E.
26–1354 652.2 134.6
40–748 412.6 88.6**
21–370 228.9 76.5**
20–990 671.1 100.9
18–810 592.3 78.3**
25–396 223.3 65.3**
23–1317 748.6 126.3
10–831 561.3 87.2**
14–376 272.5 61.3*
14–1674 766.3 132.9
P < 0.05. P < 0.01.
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prepared, each 10 snails/L in plastic aquaria. Thereafter, the snails were transferred to clean dechlorinated water (25 ± 1 °C) and daily supplied with oven dried lettuce leaves throughout the prepatent and patent periods. A control group of three replicates was exposed to miracidia concurrently with the experimental snails and treated similarly till cercarial emergence. Dead snails were daily removed and surviving ones were individually examined once weekly for cercarial shedding 20 days post-miracidial exposure. Thereafter, the cercarial production/infected snail was recorded [24].
2.5. Effect on biochemical parameters in snails’ hemolymph and tissues Adult B. alexandrina snails (6–8 mm) were continuously exposed for 4 weeks to the concentrations LC0, LC10 and LC25 from the powder of each plant. For each concentration, a group of 30 snails was used, and another one was maintained in dechlorinated water (25 ± 1 °C) as control. The concentrations were renewed weekly. For collection of hemolymph samples from survived snails, a small portion of the snails’ shell situated above the heart was removed and a capillary tube was inserted into the heart to collect hemolymph [25]. The hemolymph pooled from eight snails was collected in an Eppendorf vial (1.5 ml) and kept in an ice-box. As well, tissue homogenates were prepared by dissecting out the snails’ soft parts from their shells, then 1 g was homogenized in 10 ml cold distilled water in glass homogenizer and centrifuged for 10 min at 3000 rpm, then the fresh supernatant was used.
The biochemical parameters evaluated in this study were total protein concentration [26,27], activities of transaminases AsT and AlT [28,29] and phosphatases AcP [30] and AkP [31] enzymes. In addition, glucose concentration in snails’ hemolymph was, also, determined [32]. These parameters were spectrophotometrically evaluated using kits from Quimica clinica Aplicada S.A. (QCA) Ltd., Span. 2.6. Statistical analysis Snails’ mortality and infection rates were analyzed by Chi-square values of contingency tables [33]. The mean values of prepatent and patent periods, cercarial production/snail and life span of infected snails in the tested and control groups were compared using student ‘‘t’’ test [34]. Statistical analysis was performed with the aid of the SPSS computer program (version 13.0 windows). 3. Results It is seen from Table 1 that the dry powder of the plants D. stramonium and S. sesban as water suspensions has an acceptable molluscicidal activity with LC90’s of 40.4 and 62.4 ppm, respectively. Meanwhile, that of the plant C. carandas has no effect up to 500 ppm after 24 h of exposure. However, the plant C. diurnum exhibited a moderate toxic effect against B. alexandrina snails as the LC90 of its dry powder was 251.4 ppm. Therefore, the plants D. stramonium and S. sesban were selected to evaluate their effect on fecundity of B. alexandrina snails, their
AsT& AlT
T. Protein 25
60 50
20
40
15
30 10
20
5
10 0
0 control
LC0
LC10
LC25
AcP
control
LC0
LC25
LC10
LC25
AkP
16
60
LC10
14
50
12
40
10
30
8 6
20
4
10
2
0
0 control
LC0
LC10
LC25
control
LC0
Fig. 2. Effect of Datura stramonium dry powder on glucose and protein levels and activities of AsT, AlT, AkP and AcP enzymes in hemolymph of adult Biomaphalaria alexandrina snails.
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infection with S. mansoni miracidia and the changes in some biochemical parameters of snails’ hemolymph and tissues. From Table 2, its is clear that the survival rate (Lx) of snail groups continuously exposed for 4 weeks to LC0 from the dry powder of each plant were slightly reduced in comparison with that of control snails. Meanwhile, increasing the concentration of the plant D. stramonium to LC25 caused a continuous and marked reduction in Lx values of treated snails, being 0.35 at the 4th week compared to 0.95 for control group (P < 0.001). Moreover, few snails could survive after 4 weeks of exposure to LC25 S. sesban, as Lx was 0.05 at the 4th week. Table 2, shows also, that exposure of snails for 4 weeks to LC0, LC10 and LC25 of both plant species obviously reduced their fecundity (Mx) in comparison with that of control group. Thus, Mx of snails exposed to LC25 of D. stramonium and S. sesban at the 4th week were 8.6 and 7 eggs/snail, respectively, compared to 21.9 eggs/control snail. This reduction in fecundity of treated snails has a negative reflect on their reproductive rate (Ro) throughout the experimental period (4 weeks). Thus Table 2 and Fig. 1 declare that Ro of snails exposed to LC25 from the plants D. stramonium and S. sesban were reduced by 62.1% and 76.4%, respectively, in proportion to that of control group. The data in Table 3 revealed that survival rates at the first shedding of snail groups exposed for 24 h to LC10 and LC25 of D. stramonium dry powder either during, pre- or post-miracidial exposure were less than that of control group. The rates for snails treated with these concentrations pre-miracidial exposure were 73.4% and 60%, respectively, compared to 90% for control ones (P < 0.01). The same trend was seen for infection rates of treated snails. So, the rates of snails exposed to LC25 either during or
AsT&AlT
T. Protein 50 45 40 35 30 25 20 15 10 5 0
post-miracidial exposure were 31.6% and 21.1%, respectively, compared to 92.6% for control group (P < 0.01). It was, also, noticed that the prepatent period, duration of cercarial shedding and life span of infected snails from the treated groups, generally, did not significantly different from those of control snails. For cercarial production/snail, it was reduced by snails’ exposure to the tested concentrations of the plant D. stramonium. Thus, this parameter for snail groups exposed to LC10 and LC25 either during or post-miracidial exposure was significantly different from that of control snails. At LC25, it was 372.8 and 432 cercariae/snail, respectively, compared to 766.3 cercariae/control snail (P < 0.01). It is clear from Table 4 that the survival rates at 1st shedding of snail groups exposed to LC10 and LC25 of S. sesban dry powder either during, pre-, and post-miracidial exposure were less than that of control ones. The rates for snails exposed to LC25 during or post-miracidial exposure were 43.3% and 40%, respectively, compared to 90% for control ones (P < 0.01). Similar observation was recorded for the infection rates of treated snails, as they were 53.8% and 25% for groups exposed to LC25 during or post-miracidial exposure, respectively, compared to 92.6% for control snails (P < 0.01). The duration of S. mansoni cercarial shedding and life span of infected snails from groups treated with LC10 and LC25 either during, pre- and post-miracidial exposure were shorter than those of control group. So, durations of cercarial shedding for snail exposed to LC25 during or post-miracidial exposure were 7 and 6 days, respectively, compared to 15.5 days for control snails (P < 0.01). This decrease in the periods of cercarial shedding has a negative reflect on the total cercarial production/infected snail from the
30 25 20 15 10 5
control
LC0
LC10
LC25
control
LC0
LC10
LC25
LC10
LC25
AkP
AcP
60
0
18
50
16 14
40
12 10
30
8 20
6 4
10
2 0
0 control
LC0
LC10
LC25
control
LC0
Fig. 3. Effect of Datura stramonium dry powder on protein levels and activities of AsT, AlT, AkP and AcP enzymes in tissue homogenate of adult Biomaphalaria alexandrina snails.
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groups treated with LC10 and LC25 from S. sesban dry powder. Thus, it was 223.3 and 272.5 cercariae/snail, for snail exposed to LC25 during or post-miracidial exposure, respectively, in comparison with 766.3 cercariae/control snail (P < 0.01). For the biochemical parameters in hemolymph and tissues of snails treated with D. stramonium dry powder. The data in Fig. 2 indicated that glucose concentrations in hemolymph of snails exposed to LC10 and LC25 were slightly less than that of control ones, being 33.8, 29.8 mg/100 ml, respectively, compared to 43 mg/100 ml for control group. The reduction rates in this case were 21.4% and 30.7%, respectively (P < 0.01). The hemolymph total protein concentrations of snails exposed to the same concentrations (LC10 and LC25) revealed, also, slight variations from that of control group. On the other hand, the activities of the enzymes AsT, AlT, AcP and AkP in hemolymph of snails treated with LC10 and LC25 markedly elevated in comparison with those of control group. So, the activities of these enzymes for snails treated with LC25 were 91.7,
Glucose Glucose
50
5 4
30
3
20
2
10
1 control
LC0
LC10
0
LC25
control
AcP
9 7
300
6
250
5
200
4
LC0
LC10
LC25
LC10
LC25
AkP
350
8
150
3
100
2
50
1 0
T. Protein
6
40
0
127.3, 8.9 and 292.8 U/L, respectively, compared to 30, 33, 5.6 and 117.1 U/L for control ones (P < 0.01). The biochemical parameters evaluated in tissues of snails treated with the same plant dry powder (Fig. 3) indicated that they exhibited similar pattern as those of snails’ hemolymph, except the activity of AcP enzyme, as it was slightly decreased, being 30.4 U/g protein for snails treated with LC25 compared 44.3 U/g protein for control group (P < 0.05). Concerning the effect of the plant S. sesban on biochemical parameters of treated B. alexandrina snails, the data in Fig. 4 show a slight reduction of glucose concentrations in hemolymph of snails exposed to LC0, LC10 and LC25 of the plant dry powder. So, this parameter of snails exposed to LC25 was 34.5 mg/100 ml, compared to 43 mg/100 ml for control snails (only 19.8% reduction, P < 0.05). A similar pattern of slight reduction was observed for the total protein concentrations in hemolymph of treated snails. Thus, its values for snails exposed to LC25 was less than that of control group by 26.9% (P < 0.01).
control
LC0
LC10
0
LC25 AsT
120
control
LC0
AlT
100 80 60 40 20 0
control
LC0
LC10
LC25
Fig. 4. Effect of Sesbania sesban dry powder on glucose and protein levels and activities of AsT, AlT, AkP and AcP enzymes in heamolymph of adult Biomaphalaria alexandrina snails.
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T. Protein
AsT& AlT 25
60 50
20
40
15
30 10 20 5
10 0
control
LC0
LC10
LC25
AcP
0
LC0
LC10
LC25
AkP
16
60
14
50
12
40
10
30
8 6
20
4
10 0
control
2 control
LC0
LC10
LC25
0
control
LC0
LC10
LC25
Fig. 5. Effect of Sesbania sesban dry powder on protein levels and activities of AsT, AlT, AkP and AcP enzymes in tissue homogenate of adult Biomaphalaria alexandrina snails.
Meanwhile, a marked elevation in the activities of AsT, AlT, AcP and AkP enzymes in hemolymph of snail groups exposed to LC10 and LC25 from the plant dry powder was recorded. The activities of these enzymes for snails exposed to LC25 were 88.7, 93, 7.4 and 291 U/L, respectively, compared to 30, 33, 5.6 and 117.1 U/L for control ones (P < 0.01). For snails’ tissues (Fig. 5), the total protein concentrations and enzymes activities of treated snails exhibited a similar pattern as those of their hemolymph, except the activity of AcP enzyme that was markedly reduced, being 14.2 U/g protein for the snail group exposed to LC25, compared to 44.3 U/g protein for control snails (67.9% reduction, P < 0.01).
4. Discussion The dry powder of the plants D. stramonium and S. sesban exhibited and acceptable toxic effect to B. alexandrina snails according to WHO [35] recommendations on plant molluscicides. This is coincide with the activity of S. sesban against B. pfeifferi and B. trancatus snails [11]. This effect could be due to the presence of three glucuronide derivatives of oleanolic acid in this plant species that have a high molluscicidal activity [36,10]. As well, methanol extract from D. innoxia exhibited a remarkable toxic effect against the snails B. alexandrina, B. trancatus and L. caillaudi, and this could be due to the presence of a compound from the coronaridine glycoside derivatives [12]. The egg-laying capacity (Mx) and the reproductive rate (Ro) of B. alexandrina snails were markedly reduced by their continuous exposure for 4 weeks to LC10 and LC25 from the dry powder of
the plants D. stramonium and S. sesban. This observation was supported by the disturbance in activities of the transaminases AsT and AlT and phosphatases AcP and AkP enzymes in hemolymph and tissues of the snails treated with these plant species, that means a damage to the snails’ tissues and interrupt their physiological activities, consequently their Mx and Ro. These data are in accordance with the effect of methanol extract from the plants Agave angustifolia and Syzygium jambos on Mx and Ro of B. alexandrina snails [37,24]. As well, the severe damage in the hermaphrodite gland cells of B. alexandrina snails treated with the plants Plumatella repens and S. nigrum has a negative reflect on their Mx and Ro [38]. In addition, there is a correlation between the activity of phenol oxidase in tissues of B. alexandrina treated with the plant Callistemon lanceolatus and Mx values of these snails [39]. The infection rates of B. alexandrina with S. mansoni miracidia were reduced by 24 h of exposure to LC10 and LC25 of the tested plants dry powder. This was supported by the interruptions in total protein and glucose concentrations, as well, the activities of AsT, AlT, AcP and AkP enzymes in the hemolymph of treated snails, that render their physiological processes unsuitable for the parasite development and reduce cercarial production. Comparable results were recorded in the literature [24,38, 40,41] using the plants Fagonia cretica, Solanum nigrum, P. repens, Callistemon citrinus and S. jambos. Although, the prepatent period of B. alexandrina snails exposed to the experimental plants was not significantly different from that of control group, yet the duration of cercarial shedding, life span and cercarial production of infected snails were significantly reduced post exposure to LC25 of S. sesban dry powder. These phenomena were stated by
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many authors using different plant species as molluscicidal agents. Thus Badawy [42], Gawish [24] and Bakry [9] found that the plants Viburnum tinus, S. jambos, Euphorbia splendens and Atriplex stylosa have a remarkable decrease in the duration of cercarial shedding and cercarial production/snail from B. alexandrina infected with S. mansoni miracidia. The authors attributed this, probably, to the disturbances in the activities of snails’ enzyme system, and the total protein concentration in their hemolymph that negatively affects the developmental stages of the parasite within their tissues. It was stated that the enzyme AcP has an important role in the defense system of snails [43], so elevation of its activity in hemolymph of B. alexandrina snails infected with S. mansoni miracidia and treated with the plant Calendula micrantha may decrease the development of the parasite larval stages within snails’ tissues that resulted in low production of cercariae/infected snail [44]. In addition, the success or failure of S. mansoni infection in Biomphalaria glabrata snails depends on mobilization of their internal defense system, mainly by hemocytes and plasma factors [45–47]. The current results indicated a reduction of total protein concentrations in the hemolymph and tissues of B. alexandrina snails exposed to the tested plants. Similar observations were noticed in the lymph and tissues of Lymnaea natalensis [48], B. alexandrina and Lanistes carinatus [49] snails treated with the plants C. micrantha and Euphorbia peplus as molluscicial agents. This harmful effect could be attributed to enhancement of energy utilization and/or destruction of cells’ organelles of treated snails that may led to inhibition of protein synthesis [48,49]. The present data revealed a significant reduction of glucose concentration in hemolymph of B. alexandrina snails exposed to LC25 of the tested plants’ dry powder. This may be due to the rapid direct utilizing of hemolymph glucose as an energy fuel for snails’ physiological and biological activities as a trial to overcome the effect of such harmful agents. This was stated in the serum of rats after intraperitonial injection with methanol extract of the plant Salvia officinalis [50]. However, methanol extract of E. splendens [9] and the dry powder of Furcrea celloa marginata [51] elevated glucose level in hemolymph of treated B. alexandrina snails. The transaminases enzymes AsT and AlT are not solely located in hepatocytes, but rather are, also, in many body organs and elevation in their activities could be due to a variety of conditions including muscle damage, intestinal and hepatic injury and toxic hepatitis [18]. As well, the increase in serum alkaline phosphatase (AkP) activity in patients could be attributed to hepatocytes injury and interruptions in their natural activities [18]. Accordingly, the present elevations or reductions in the activities of AsT, AlT, AcP and AkP enzymes in hemolymph and tissues of B. alexandrina snails treated with the tested plants could be, partially, due to cells injury of their different organs. This was previously stated that application of pesticides against snail species may led to disturbances in their enzymatic systems [52]. In addition, elevations in the activities of AsT, AlT and AkP in hemolymph and tissues of B. alexandrina snails were recorded by several authors using the plants S. jambos [24], Dracaena fragrans [37], Asparagus densiflours and Orepanax guatemalensis [53]. Meanwhile, the plants S. officinalis and Lantana camara reduced the activities of these enzymes in tissues of this snail species [54]. From the forgoing results, it is concluded that the sublethal concentrations LC10 and LC25 of the plants D. stramonium and S. sesban have deleterious effects on the biological and physiological activities of B. alexandrina snails, hence markedly deteriorate their susceptibility to infection with S. mansoni miracidia and production of cercariae that consequently interrupt and minimize the transmission of this parasite. However, comprehensive investigations are required to define the proper technique(s) for applications of such agents in molluscicidal operations aiming to minimize water
103
pollution with chemicals and saving the non-target organisms in the treated water bodies.
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Biological and biochemical parameters of Biomphalaria alexandrina snails exposed to the plants Datura stramonium and Sesbania sesban as water suspensions of their dry powder Momeana B. Mahmoud a, Wafaa L. Ibrahim b, Basma M. Abou-El-Nour b, Mohamed A. El-Emam a,⇑, Alaa A. Youssef a a b
Medical Malacology Department, Theodor Bilharz Research Institute, Giza, Egypt Zoology Department, Faculty of Science, Al-Azhar University (Girls Branch), Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 4 October 2010 Accepted 9 November 2010 Available online 20 November 2010 Keywords: Biomphalaria alexandrina snails Schistosoma mansoni Plant mollucicides Transaminases and phosphatases enzymes
a b s t r a c t Four plant species, as a dry powder of their leaves, were tested against Biomphalaria alexandrina snails, the intermediate host of Schistosoma mansoni. The bioassay tests revealed the plants Datura stramonium and Sesbania sesban to be more toxic to the snails than the other two ones. Therefore, they were tested against snails’ fecundity (Mx), reproduction rate (Ro) and their infection with S. mansoni miracidia. In addition, total protein concentration and the activities of the transaminases (AsT and AlT) and phosphatases (AcP and AkP) enzymes in hemolymph and tissues of snails treated with these plants were determined. As well, glucose concentration in snails’ hemolymph was evaluated. Exposure of snails for 4 weeks to LC10 and LC25 of the plants D. stramonium and S. sesban dry powder markly suppressed their Mx and Ro. The reduction rates of Ro for snails exposed to LC25 of these plants were 62.1% and 76.4%, respectively. As well, a considerable reduction in the infection rates of snails exposed to these plants either during, pre- or post-miracidial exposure was recorded. Thus, infection rates of snails treated during miracidial exposure with LC10 of D. stramonium and S. sesban were 41.7% and 52.2%, respectively, compared to 92.6% for control group (P < 0.01). These plants, also, reduced the duration of cercarial shedding and cercarial production/snail. So, snails exposed to LC25 of these plants shed 372.8 and 223.2 cercariae/snail, respectively, compared to 766.3 cercariae/infected control snail (P < 0.01). The results, also, revealed that glucose and total protein concentrations in hemolymph of snails treated with LC10 and LC25 of these plants were decreased, meanwhile, the activities of the enzymes AsT, AlT, AcP and AkP were elevated (P < 0.01). However, the activity of AcP in tissues of treated snails was decreased compared to that of control ones. It is concluded that LC25 of the plants D. stramonium and S. sesban negatively interferes with biological and physiological activities of B. alexandrina snails, consequently it could be effective in interrupting and minimizing the transmission of S. mansoni. Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction There is no doubt that schistosomiasis is one of the major communicable diseases and it is second to the malaria with socio-economic and health importance in the developing world [1]. Controlling of the snail intermediate hosts of this parasite by molluscicides (synthetic and/or of natural origin) is still one of the most promising means in the battle against this parasitic disease [2]. Molluscicides of plant origin appear to be environmentally friendly having several advantages over the synthetic chemicals [3]. So far, more than 1500 plant species have been screened for ⇑ Corresponding author. E-mail address: [email protected] (M.A. El-Emam).
molluscicidal properties [4]. In Egypt, molluscicides of plant origin have received an increasing attention, so several plant species were screened in this concept [5–9]. The plant Sesbania aegyptiaca proved to have a molluscicial activity against Biomphalaria alexandrina snails as water suspensions from its dry powder [10] and that of Sesbania sesban exhibited a marked toxic effect against the snails Biomphalaria pfeifferi and Bulinus truncatus [11]. The methanol extract of Datura innoxia showed a promising molluscicidal potency against B. alexandrina, B. truncatus and Lymnaea caillaudi snails [12]. In addition, this extract of Datura stramonium has a strong antifungal property against Fusarium mangiferae fungus [13]. The amino transferases enzymes (AsT and AlT) are the most frequently measured for hepatic diseases. The enzymes may be released from hepatocytes into the circulation by necrosis or by
0048-3575/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2010.11.005
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M.B. Mahmoud et al. / Pesticide Biochemistry and Physiology 99 (2011) 96–104 Table 1 Molluscicidial activity of the dry powder from the tested plant species against adult Biomphalaria alexandrina snails (24 h exposure). Plant species
Family
LC0 (ppm)
LC10 (ppm)
LC25 (ppm)
LC50 (ppm)
LC90 (ppm)
Slope
Datura stramonium Sesbania sesban Cestrum diurnum Carissa carandas
Solanaceae Fabaceae Solanaceae Apocynaceae
2.5 5.11 189.3 >500
11.3 39.5 207.3
18.2 44.9 217.7
25.8 51.0 229.3
40.4 62.4 251.4
1.64 1.20 1.08
Table 2 Survival rate (Lx) and fecundity (Mx) of Biomphalaria alexandrina snails after 4 weeks of continuous exposure to sublethal concentrations of D. stramonium and S. sesban as dry powder water suspensions. Conc. (ppm)
Observation period (week, x) 1
Control
Ro R Lx Mx (reproductive rate)
2
3
% Reduction of Ro
4
Lx
Mx
Lx Mx
Lx
Mx
Lx Mx
Lx
Mx
Lx Mx
Lx
Mx
Lx Mx
1.0
17.1
17.1
1.0
13.4
13.4
0.95
14.3
13.6
0.95
21.9
20.8
64.9
D. stramonium
LC0 LC10 LC25
1.0 0.90 0.80
19.3 14.2 12.4
19.3 12.8 9.9
0.95 0.80 0.65
14.8 13.5 10.3
14.1 10.8 6.7
0.90 0.75 0.50
15.5 13.2 10.0
14.0 9.9 5.0
0.80 0.60 0.35
16.2 11.9 8.6
13.0 7.1 3.0
60.4 40.6 24.6
6.93 37.4 62.1
S. sesban
LC0 LC10 LC25
1.0 0.95 0.60
16.7 21.3 10.9
16.7 18.1 6.5
0.90 0.80 0.45
18.2 13.4 12.1
16.4 10.7 5.4
0.85 0.65 0.30
13.6 14.3 9.9
11.6 9.3 3.0
0.85 0.55 0.05
14.6 11.9 7
12.4 6.5 0.35
57.1 44.6 15.3
7.4 31.2 76.4
changes in cell membrane permeability that allows the enzymes to leak out of the cells [14]. AlT is more localized to the liver than AsT, so it is more specific to liver injury [15]. The phosphatases enzymes are capable of hydrolyzing organic phosphate esters. Acid phosphatase (AcP) is a lysosomal enzyme concerning with digestion of foreign substances and bacteria inside the cells [16] and is involved in the defense mechanisms of both vertebrates and invertebrates [17]. In addition, the deterioration in the activity of alkaline phosphatases can occur in all types of liver diseases [18]. The present work was designed to determine the molluscicidal and ovicidial potencies of the plants D. stramonium and S. sesban against B. alexandrina snails and their eggs. As well, the snails’ reproductive rate and their infection with Schistosoma mansoni miracidia were evaluated. In addition, the changes pattern in activities of transaminases (AsT and AlT) and phosphatases (AcP and AkP) enzymes and the total protein levels were assessed in tissues and hemolymph of treated snails.
2.3. Miracidia S. mansoni ova were obtained from Schistosomiasis Biological Supply Center (SPSC), TBRI. They were left in clean dechlorinated water for hatching under a desk lamp then fresh hatch miracidia were used in bioassay and infection tests. 2.4. Bioassay tests 2.4.1. Molluscicidal screening For each plant powder, a series of concentrations that would permit the computation of LC50 and LC90 was prepared on the basis of weight/volume as water suspensions. Three replicates, each of 10 snails/L, were prepared. Another three replicates in dechlorinated water were used as control. Exposure and recovery periods were 24 h each at 25 ± 1 °C [19,20]. Then, snails’ mortality was recorded and corrected according to Abbots’ formula [21].
2. Materials and methods 2.1. Snails B. alexandrina snails (6–8 mm) from laboratory bred colony in Medical Malacology Dept., Theodor Bilharz Research Institute (TBRI), Giza, Egypt were used.
2.2. Plants The four plant species used are Carissa carandas (Apocynaceae) and S. sesban (Fabaceae) from the garden of Faculty of Agriculture, Al-Azhar University, Cairo, Cestrum diurnum and D. stramonium (Solanaceae) from Al-Orman garden, Giza, Egypt (spring 2007). They were kindly identified by Botany Dept., Faculty of Agriculture, Al-Azhar University. Their leaves were shade dried, then powdered by an electric mill. The dry powder was stored in clean dry dark glass bottle till use in biological tests.
Fig. 1. Reduction rate (%) of reproductive rate (Ro) for B. alexandrina snails after 4 weeks of continuous exposure to Datura stramonium and Sesbania sesban as dry powder water suspension.
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Due to the high effect of the plants D. stramonium and S. sesban, they were chosen for detailed tests as follows: 2.4.1.1. Effect on snails’ egg-laying capacity. B. alexandrina snails (6– 8 mm) were continuously exposed for 4 weeks to the sublethal concentrations LC0, LC10 and LC25 from dry powder of each plant [19,22]. Three replicates, each of 10 snails/L, were prepared for each concentration, another three ones of control snails were maintained in dechlorinated water (25 ± 1 °C). The concentrations were weekly changed by fresh ones. The snails were daily fed oven dried lettuce leaves. Each aquarium was provided with one piece of polyethylene sheet for oviposition. The eggs laid on these sheets and on the wall of the aquaria were weekly counted using a
stereomicroscope and a hand lens (10). Dead snails were removed daily and the following parameters were weekly recorded [23]: Lx (the survival rate as a proportion of the correct one), Mx (the number of eggs/snail/week) and Ro (the reproductive rate which is the summation of Lx Mx). 2.4.1.2. Effect on infection of B. alexandrina snails with S. mansoni miracidia. The snails (6–8 mm) were exposed for 24 h to the sublethal concentrations LC0, LC10 and LC25 of the plants’ dry powder during miracidial exposure and at 1 day either pre- or post-miracidial exposure. Snails were exposed to miracidia in mass, 10 fresh hatched miracidia/snail. For each concentration, three replicates were
Table 3 Effect of Datura stramonium dry powder on survival rate at 1st shedding, infection rate, prepatent period, life span, duration of shedding and cercarial production of Biomphalaria alexandrina exposed to Schistosoma mansoni miracidia. Parameter
D. stramonium
Control
Pre-exposure
*
Post-exposure
LC0
LC10
LC25
LC0
LC10
LC25
LC0
LC10
LC25
Survival rate at 1st shedding
No. exposed No. survived %
30 26 86.7
30 22 73.4**
30 18 60.0**
30 27 90.0
30 24 80.0
30 19 63.3**
30 26 86.7
30 23 76.7*
30 19 63.3**
30 27 90
Infection of snails
Number %
23 88.5
19 86.4
16 88.9
18 66.7**
10 41.7**
6 31.6**
22 84.6
12 52.2**
4 21.1**
25 92.6
Prepatent period (days)
Range Mean ±S.E. Range Mean ±S.E.
21–27 21.6 1.7 7–34 17.8 1.7
21–27 22.3 2.1 4–22 12.2 1.3
21 21* 0.0 4–31 13.2 2.1
21–24 21.5 1.2 4–25 14.2 2.0
21–24 21.3 0.9 7–28 11.2 1.9
21–24 23 2.4 4–22 12.5 3.0
21–27 22.1 0.87 4–22 13.6 1.0
21–24 22.6 1.35 4–31 11.3 2.1
21 21* 0.0 4–22 12.3 3.8
12–27 22 1.9 4–43 15.5 1.8
Life span (days)
Range Mean ±S.E.
27–54 37.8 1.7
24–42 32.2 1.3
24–51 33.2 2.1
24–45 34.2 2.0
27–48 31.2 1.9
24–42 32.5 3.0
24–42 33.6 1.0
24–51 31.3 2.1
24–42 32.3 3.8
24–54 35.5 1.8
Total cercariae/snail
Range Mean ±S.E.
17–1486 710.4 111.2
16–1041 634.2 102.3
28–1400 788.6 131.2
9–1088 674.1 116.7
20–891 501.0** 79.8
10–660 372.8** 78.6
80–1010 619.9 103.3
10–731 679.3* 94.6
5–790 432.0** 65.4
14–1674 766.3 132.9
Duration of shedding
**
During exposure
P < 0.05. P < 0.01.
Table 4 Effect of Sesbania sesban dry powder on survival rate at 1st shedding, infection rate, prepatent period, life span, duration of shedding and cercarial production of Biomphalaria alexandrina exposed to Schistosoma mansoni miracidia. Parameter
S. sesban
Control
Pre-exposure
* **
During exposure
Post-exposure
LC0
LC10
LC25
LC0
LC10
LC25
LC0
LC10
LC25
Survival rate at 1st shedding
No. exposed No. survived %
30 25 83.3
30 20 66.7**
30 12 40.0*
30 27 90.0
30 23 76.7*
30 13 43.3**
30 27 90.0
30 21 70.1**
30 12 40.0**
30 27 90
Infection of snails
Number %
23 92.0
17 85.0
10 83.3
20 74.1**
12 52.2**
7 53.8**
24 88.9
14 66.7**
3 25.0**
25 92.6
Prepatent period (days)
Range Mean ±S.E.
21–24 21.5 1.2
21–24 21.7 1.3
21–24 22 1.5
21–27 22.2 2.0
21–27 22.1 1.9
21–27 21.9 2.3
21–27 21.3 0.62
21–24 21.2 0.0
21 21 0.0
12–27 22 1.9
Duration of shedding
Range Mean ±S.E.
4–19 11.6 1.1
4–22 9.5** 1.5
7–16 11.2* 0.9
7–22 11.4 1.0
7–22 11.8 1.4
4–16 7.0** 1.6
4–34 16.9 2.0
4–22 12.4 2.3
4–10 6.0** 2.0
4–43 15.5 1.8
Life span (days)
Range Mean ±S.E.
24–39 31.6 1.1
24–42 29.5** 1.5
27–36 31.2* 0.9
27–42 31.4 1.0
27–42 31.8 1.4
24–36 27.0** 1.6
24–54 36.9 2.0
24–48 32.4 2.3
24–30 26.0** 2.0
24–54 35.5 1.8
Total cercariae/snail
Range Mean ±S.E.
26–1354 652.2 134.6
40–748 412.6 88.6**
21–370 228.9 76.5**
20–990 671.1 100.9
18–810 592.3 78.3**
25–396 223.3 65.3**
23–1317 748.6 126.3
10–831 561.3 87.2**
14–376 272.5 61.3*
14–1674 766.3 132.9
P < 0.05. P < 0.01.
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prepared, each 10 snails/L in plastic aquaria. Thereafter, the snails were transferred to clean dechlorinated water (25 ± 1 °C) and daily supplied with oven dried lettuce leaves throughout the prepatent and patent periods. A control group of three replicates was exposed to miracidia concurrently with the experimental snails and treated similarly till cercarial emergence. Dead snails were daily removed and surviving ones were individually examined once weekly for cercarial shedding 20 days post-miracidial exposure. Thereafter, the cercarial production/infected snail was recorded [24].
2.5. Effect on biochemical parameters in snails’ hemolymph and tissues Adult B. alexandrina snails (6–8 mm) were continuously exposed for 4 weeks to the concentrations LC0, LC10 and LC25 from the powder of each plant. For each concentration, a group of 30 snails was used, and another one was maintained in dechlorinated water (25 ± 1 °C) as control. The concentrations were renewed weekly. For collection of hemolymph samples from survived snails, a small portion of the snails’ shell situated above the heart was removed and a capillary tube was inserted into the heart to collect hemolymph [25]. The hemolymph pooled from eight snails was collected in an Eppendorf vial (1.5 ml) and kept in an ice-box. As well, tissue homogenates were prepared by dissecting out the snails’ soft parts from their shells, then 1 g was homogenized in 10 ml cold distilled water in glass homogenizer and centrifuged for 10 min at 3000 rpm, then the fresh supernatant was used.
The biochemical parameters evaluated in this study were total protein concentration [26,27], activities of transaminases AsT and AlT [28,29] and phosphatases AcP [30] and AkP [31] enzymes. In addition, glucose concentration in snails’ hemolymph was, also, determined [32]. These parameters were spectrophotometrically evaluated using kits from Quimica clinica Aplicada S.A. (QCA) Ltd., Span. 2.6. Statistical analysis Snails’ mortality and infection rates were analyzed by Chi-square values of contingency tables [33]. The mean values of prepatent and patent periods, cercarial production/snail and life span of infected snails in the tested and control groups were compared using student ‘‘t’’ test [34]. Statistical analysis was performed with the aid of the SPSS computer program (version 13.0 windows). 3. Results It is seen from Table 1 that the dry powder of the plants D. stramonium and S. sesban as water suspensions has an acceptable molluscicidal activity with LC90’s of 40.4 and 62.4 ppm, respectively. Meanwhile, that of the plant C. carandas has no effect up to 500 ppm after 24 h of exposure. However, the plant C. diurnum exhibited a moderate toxic effect against B. alexandrina snails as the LC90 of its dry powder was 251.4 ppm. Therefore, the plants D. stramonium and S. sesban were selected to evaluate their effect on fecundity of B. alexandrina snails, their
AsT& AlT
T. Protein 25
60 50
20
40
15
30 10
20
5
10 0
0 control
LC0
LC10
LC25
AcP
control
LC0
LC25
LC10
LC25
AkP
16
60
LC10
14
50
12
40
10
30
8 6
20
4
10
2
0
0 control
LC0
LC10
LC25
control
LC0
Fig. 2. Effect of Datura stramonium dry powder on glucose and protein levels and activities of AsT, AlT, AkP and AcP enzymes in hemolymph of adult Biomaphalaria alexandrina snails.
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infection with S. mansoni miracidia and the changes in some biochemical parameters of snails’ hemolymph and tissues. From Table 2, its is clear that the survival rate (Lx) of snail groups continuously exposed for 4 weeks to LC0 from the dry powder of each plant were slightly reduced in comparison with that of control snails. Meanwhile, increasing the concentration of the plant D. stramonium to LC25 caused a continuous and marked reduction in Lx values of treated snails, being 0.35 at the 4th week compared to 0.95 for control group (P < 0.001). Moreover, few snails could survive after 4 weeks of exposure to LC25 S. sesban, as Lx was 0.05 at the 4th week. Table 2, shows also, that exposure of snails for 4 weeks to LC0, LC10 and LC25 of both plant species obviously reduced their fecundity (Mx) in comparison with that of control group. Thus, Mx of snails exposed to LC25 of D. stramonium and S. sesban at the 4th week were 8.6 and 7 eggs/snail, respectively, compared to 21.9 eggs/control snail. This reduction in fecundity of treated snails has a negative reflect on their reproductive rate (Ro) throughout the experimental period (4 weeks). Thus Table 2 and Fig. 1 declare that Ro of snails exposed to LC25 from the plants D. stramonium and S. sesban were reduced by 62.1% and 76.4%, respectively, in proportion to that of control group. The data in Table 3 revealed that survival rates at the first shedding of snail groups exposed for 24 h to LC10 and LC25 of D. stramonium dry powder either during, pre- or post-miracidial exposure were less than that of control group. The rates for snails treated with these concentrations pre-miracidial exposure were 73.4% and 60%, respectively, compared to 90% for control ones (P < 0.01). The same trend was seen for infection rates of treated snails. So, the rates of snails exposed to LC25 either during or
AsT&AlT
T. Protein 50 45 40 35 30 25 20 15 10 5 0
post-miracidial exposure were 31.6% and 21.1%, respectively, compared to 92.6% for control group (P < 0.01). It was, also, noticed that the prepatent period, duration of cercarial shedding and life span of infected snails from the treated groups, generally, did not significantly different from those of control snails. For cercarial production/snail, it was reduced by snails’ exposure to the tested concentrations of the plant D. stramonium. Thus, this parameter for snail groups exposed to LC10 and LC25 either during or post-miracidial exposure was significantly different from that of control snails. At LC25, it was 372.8 and 432 cercariae/snail, respectively, compared to 766.3 cercariae/control snail (P < 0.01). It is clear from Table 4 that the survival rates at 1st shedding of snail groups exposed to LC10 and LC25 of S. sesban dry powder either during, pre-, and post-miracidial exposure were less than that of control ones. The rates for snails exposed to LC25 during or post-miracidial exposure were 43.3% and 40%, respectively, compared to 90% for control ones (P < 0.01). Similar observation was recorded for the infection rates of treated snails, as they were 53.8% and 25% for groups exposed to LC25 during or post-miracidial exposure, respectively, compared to 92.6% for control snails (P < 0.01). The duration of S. mansoni cercarial shedding and life span of infected snails from groups treated with LC10 and LC25 either during, pre- and post-miracidial exposure were shorter than those of control group. So, durations of cercarial shedding for snail exposed to LC25 during or post-miracidial exposure were 7 and 6 days, respectively, compared to 15.5 days for control snails (P < 0.01). This decrease in the periods of cercarial shedding has a negative reflect on the total cercarial production/infected snail from the
30 25 20 15 10 5
control
LC0
LC10
LC25
control
LC0
LC10
LC25
LC10
LC25
AkP
AcP
60
0
18
50
16 14
40
12 10
30
8 20
6 4
10
2 0
0 control
LC0
LC10
LC25
control
LC0
Fig. 3. Effect of Datura stramonium dry powder on protein levels and activities of AsT, AlT, AkP and AcP enzymes in tissue homogenate of adult Biomaphalaria alexandrina snails.
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groups treated with LC10 and LC25 from S. sesban dry powder. Thus, it was 223.3 and 272.5 cercariae/snail, for snail exposed to LC25 during or post-miracidial exposure, respectively, in comparison with 766.3 cercariae/control snail (P < 0.01). For the biochemical parameters in hemolymph and tissues of snails treated with D. stramonium dry powder. The data in Fig. 2 indicated that glucose concentrations in hemolymph of snails exposed to LC10 and LC25 were slightly less than that of control ones, being 33.8, 29.8 mg/100 ml, respectively, compared to 43 mg/100 ml for control group. The reduction rates in this case were 21.4% and 30.7%, respectively (P < 0.01). The hemolymph total protein concentrations of snails exposed to the same concentrations (LC10 and LC25) revealed, also, slight variations from that of control group. On the other hand, the activities of the enzymes AsT, AlT, AcP and AkP in hemolymph of snails treated with LC10 and LC25 markedly elevated in comparison with those of control group. So, the activities of these enzymes for snails treated with LC25 were 91.7,
Glucose Glucose
50
5 4
30
3
20
2
10
1 control
LC0
LC10
0
LC25
control
AcP
9 7
300
6
250
5
200
4
LC0
LC10
LC25
LC10
LC25
AkP
350
8
150
3
100
2
50
1 0
T. Protein
6
40
0
127.3, 8.9 and 292.8 U/L, respectively, compared to 30, 33, 5.6 and 117.1 U/L for control ones (P < 0.01). The biochemical parameters evaluated in tissues of snails treated with the same plant dry powder (Fig. 3) indicated that they exhibited similar pattern as those of snails’ hemolymph, except the activity of AcP enzyme, as it was slightly decreased, being 30.4 U/g protein for snails treated with LC25 compared 44.3 U/g protein for control group (P < 0.05). Concerning the effect of the plant S. sesban on biochemical parameters of treated B. alexandrina snails, the data in Fig. 4 show a slight reduction of glucose concentrations in hemolymph of snails exposed to LC0, LC10 and LC25 of the plant dry powder. So, this parameter of snails exposed to LC25 was 34.5 mg/100 ml, compared to 43 mg/100 ml for control snails (only 19.8% reduction, P < 0.05). A similar pattern of slight reduction was observed for the total protein concentrations in hemolymph of treated snails. Thus, its values for snails exposed to LC25 was less than that of control group by 26.9% (P < 0.01).
control
LC0
LC10
0
LC25 AsT
120
control
LC0
AlT
100 80 60 40 20 0
control
LC0
LC10
LC25
Fig. 4. Effect of Sesbania sesban dry powder on glucose and protein levels and activities of AsT, AlT, AkP and AcP enzymes in heamolymph of adult Biomaphalaria alexandrina snails.
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T. Protein
AsT& AlT 25
60 50
20
40
15
30 10 20 5
10 0
control
LC0
LC10
LC25
AcP
0
LC0
LC10
LC25
AkP
16
60
14
50
12
40
10
30
8 6
20
4
10 0
control
2 control
LC0
LC10
LC25
0
control
LC0
LC10
LC25
Fig. 5. Effect of Sesbania sesban dry powder on protein levels and activities of AsT, AlT, AkP and AcP enzymes in tissue homogenate of adult Biomaphalaria alexandrina snails.
Meanwhile, a marked elevation in the activities of AsT, AlT, AcP and AkP enzymes in hemolymph of snail groups exposed to LC10 and LC25 from the plant dry powder was recorded. The activities of these enzymes for snails exposed to LC25 were 88.7, 93, 7.4 and 291 U/L, respectively, compared to 30, 33, 5.6 and 117.1 U/L for control ones (P < 0.01). For snails’ tissues (Fig. 5), the total protein concentrations and enzymes activities of treated snails exhibited a similar pattern as those of their hemolymph, except the activity of AcP enzyme that was markedly reduced, being 14.2 U/g protein for the snail group exposed to LC25, compared to 44.3 U/g protein for control snails (67.9% reduction, P < 0.01).
4. Discussion The dry powder of the plants D. stramonium and S. sesban exhibited and acceptable toxic effect to B. alexandrina snails according to WHO [35] recommendations on plant molluscicides. This is coincide with the activity of S. sesban against B. pfeifferi and B. trancatus snails [11]. This effect could be due to the presence of three glucuronide derivatives of oleanolic acid in this plant species that have a high molluscicidal activity [36,10]. As well, methanol extract from D. innoxia exhibited a remarkable toxic effect against the snails B. alexandrina, B. trancatus and L. caillaudi, and this could be due to the presence of a compound from the coronaridine glycoside derivatives [12]. The egg-laying capacity (Mx) and the reproductive rate (Ro) of B. alexandrina snails were markedly reduced by their continuous exposure for 4 weeks to LC10 and LC25 from the dry powder of
the plants D. stramonium and S. sesban. This observation was supported by the disturbance in activities of the transaminases AsT and AlT and phosphatases AcP and AkP enzymes in hemolymph and tissues of the snails treated with these plant species, that means a damage to the snails’ tissues and interrupt their physiological activities, consequently their Mx and Ro. These data are in accordance with the effect of methanol extract from the plants Agave angustifolia and Syzygium jambos on Mx and Ro of B. alexandrina snails [37,24]. As well, the severe damage in the hermaphrodite gland cells of B. alexandrina snails treated with the plants Plumatella repens and S. nigrum has a negative reflect on their Mx and Ro [38]. In addition, there is a correlation between the activity of phenol oxidase in tissues of B. alexandrina treated with the plant Callistemon lanceolatus and Mx values of these snails [39]. The infection rates of B. alexandrina with S. mansoni miracidia were reduced by 24 h of exposure to LC10 and LC25 of the tested plants dry powder. This was supported by the interruptions in total protein and glucose concentrations, as well, the activities of AsT, AlT, AcP and AkP enzymes in the hemolymph of treated snails, that render their physiological processes unsuitable for the parasite development and reduce cercarial production. Comparable results were recorded in the literature [24,38, 40,41] using the plants Fagonia cretica, Solanum nigrum, P. repens, Callistemon citrinus and S. jambos. Although, the prepatent period of B. alexandrina snails exposed to the experimental plants was not significantly different from that of control group, yet the duration of cercarial shedding, life span and cercarial production of infected snails were significantly reduced post exposure to LC25 of S. sesban dry powder. These phenomena were stated by
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many authors using different plant species as molluscicidal agents. Thus Badawy [42], Gawish [24] and Bakry [9] found that the plants Viburnum tinus, S. jambos, Euphorbia splendens and Atriplex stylosa have a remarkable decrease in the duration of cercarial shedding and cercarial production/snail from B. alexandrina infected with S. mansoni miracidia. The authors attributed this, probably, to the disturbances in the activities of snails’ enzyme system, and the total protein concentration in their hemolymph that negatively affects the developmental stages of the parasite within their tissues. It was stated that the enzyme AcP has an important role in the defense system of snails [43], so elevation of its activity in hemolymph of B. alexandrina snails infected with S. mansoni miracidia and treated with the plant Calendula micrantha may decrease the development of the parasite larval stages within snails’ tissues that resulted in low production of cercariae/infected snail [44]. In addition, the success or failure of S. mansoni infection in Biomphalaria glabrata snails depends on mobilization of their internal defense system, mainly by hemocytes and plasma factors [45–47]. The current results indicated a reduction of total protein concentrations in the hemolymph and tissues of B. alexandrina snails exposed to the tested plants. Similar observations were noticed in the lymph and tissues of Lymnaea natalensis [48], B. alexandrina and Lanistes carinatus [49] snails treated with the plants C. micrantha and Euphorbia peplus as molluscicial agents. This harmful effect could be attributed to enhancement of energy utilization and/or destruction of cells’ organelles of treated snails that may led to inhibition of protein synthesis [48,49]. The present data revealed a significant reduction of glucose concentration in hemolymph of B. alexandrina snails exposed to LC25 of the tested plants’ dry powder. This may be due to the rapid direct utilizing of hemolymph glucose as an energy fuel for snails’ physiological and biological activities as a trial to overcome the effect of such harmful agents. This was stated in the serum of rats after intraperitonial injection with methanol extract of the plant Salvia officinalis [50]. However, methanol extract of E. splendens [9] and the dry powder of Furcrea celloa marginata [51] elevated glucose level in hemolymph of treated B. alexandrina snails. The transaminases enzymes AsT and AlT are not solely located in hepatocytes, but rather are, also, in many body organs and elevation in their activities could be due to a variety of conditions including muscle damage, intestinal and hepatic injury and toxic hepatitis [18]. As well, the increase in serum alkaline phosphatase (AkP) activity in patients could be attributed to hepatocytes injury and interruptions in their natural activities [18]. Accordingly, the present elevations or reductions in the activities of AsT, AlT, AcP and AkP enzymes in hemolymph and tissues of B. alexandrina snails treated with the tested plants could be, partially, due to cells injury of their different organs. This was previously stated that application of pesticides against snail species may led to disturbances in their enzymatic systems [52]. In addition, elevations in the activities of AsT, AlT and AkP in hemolymph and tissues of B. alexandrina snails were recorded by several authors using the plants S. jambos [24], Dracaena fragrans [37], Asparagus densiflours and Orepanax guatemalensis [53]. Meanwhile, the plants S. officinalis and Lantana camara reduced the activities of these enzymes in tissues of this snail species [54]. From the forgoing results, it is concluded that the sublethal concentrations LC10 and LC25 of the plants D. stramonium and S. sesban have deleterious effects on the biological and physiological activities of B. alexandrina snails, hence markedly deteriorate their susceptibility to infection with S. mansoni miracidia and production of cercariae that consequently interrupt and minimize the transmission of this parasite. However, comprehensive investigations are required to define the proper technique(s) for applications of such agents in molluscicidal operations aiming to minimize water
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pollution with chemicals and saving the non-target organisms in the treated water bodies.
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