Impact profenophos (pesticide) on infectivity of <i>Biomphalaria alexandrina</i> snails with <i>schistosoma mansoni</i> miracidiaand on their physiological parameters

doi:10.4236/oje.2011.12005

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Vol.1, No.2, 41-47 (2011)

http://dx.doi.org/10.4236/oje.2011.12005

Open Journal of Ecology

Impact profenophos (pesticide) on infectivity of

Biomphalaria alexandrina snails with schistosoma

mansoni miracidia and on their physiological

parameters

Ragaa Mohamed

Zoology Department, Faculty of Science, Fayoum University, Fayoum, Egypt. [email protected]

Received 22 May 2011; revised 22 June 2011; accepted 1 July 2011.

ABSTRACT

Profenophos is an organophosphoru s pesticide

which are used effectively against cotton in-

sects and mites.The present work was carried

out to evaluate the molluscicidal activity of

pesticides (Profenophos) against Biomphalaria

alexandrina snails. Also, the effect of sublethal

concentrations of pesticide on the infection of

B. alexandrina with Schistosoma mansoni and

some enzymes of energy metabolism were stu-

died. The results showed that the infection of B.

alexandrina with schistosoma mansoni was gr-

eatly reduced after exposure to LC0, LC10, LC25

of pesticide and also, reduction in number of

cercariae per snail during the patent period and

in the period of cercarial shedding. The present

study indicated that the increase in levels of

aminotransaminases, acid phosp hate-se and al-

kaline phosphatases enzymes in haemolymph

and soft tissue of snails and the activity level of

lactate dehydrogenase, hexoki-inase and py-

ruvatekinase was also significantly reduced in

response to treatmen t.

Keywords: Biomphalaria Alexandrina;

Profenophos; S chist osoma Mansoni; Physiology

1. INTRODUCTION

Excessive use of pesticides in agriculture has spar-

kled researchers’ interest in investigating the harmful

effects of these compounds. Consequently, there has

also been an increase in the number of studies aiming at

evaluating the action of the residues of such chemicals

on non-target organisms. Pesticides are ubiquitous con-

taminants of the environment and have been found in air,

soil, water, and human and animal tissues in samples

from all over the world. These cover a wide range of

compounds used in pest control, such as fungicides,

herbicides, molluscicides, insecticides, rodenticides and

others [1]. Pesticide may play an important role in the

disappearance of snail vectors on reaching water bodies

as residues from pesticide activities and consequently

hindering schistosomiasis transmission in these sites [2].

Profenophos is an organophosphorus pesticide which

are used effectively against cotton insects and mites.

Improper use of pesticides combined with their persis-

tence, volatilization and mobility lead to frequent detec-

tions of the pesticide residues in the atmosphere, soil, sur-

face water and in vegetable fruits and cereal grains [3, 4].

The use of pesticides may engender biological effects

beyond those for which they were originally manufac-

tured [5,6]. As example of these is the agricultural in-

secticides which may interfere in the life, reproduction

and infection of snail vectors of schistosomiasis when

they reach water bodies [7]. Many authors studied the

effect of agricultural pesticides that may reach water

courses as residues on different biological parameters of

snail vectors of schistosomiasis [8].

Organophosphate pestic i de re present one of the worl-

d’s most commonly used agrochemical. Consequently,

many of its residues are frequently found in the envi-

ronment. Of the organophosphates, Profenophos has

been extensively used because of its low toxicity on

non-targ organisms. The present work aimed to study

the mode of action of Profenophos as molluscicides

against Biomphalaria alexandrina as indicated by sub-

lethal concentrations action on infection of Biomphalar-

ia alexandrina to Schistosoma mansoni and some en-

zymes such as aminotransaminases, acid phosphatase

and alkaline phosphatases enzymes in haemolymph and

tissues of B. alexandrina. Moreover, lactate dehydro-

genase (LDH), pyruvatekinase (PK) and, Hexokinase

(HK) activity in soft tissue of treated snails.

R. Mohamed / Open Journal of Ecology 1 (2011) 41-47

2. MATERIAL AND METHODS

2.1. Snails

Laboratory bred Biomphalaria alexandrina snails (6 -

10 mm in shell diameter) were obtained from Schisto-

some Biological Supply Program (SBSP), Theodor Bil-

harz Research Institute (TBRI), Egypt.

2.2. Ova

Schistosoma mansoni ova were obtained from the

Schistosome Biological Supply Program (SBSP), Theo-

dor Bilharz Research Institute (TBRI), Egypt.

2.3. Pesticides (Profenophos)

Organophosphorus pesticide, having the chemical

formula C11H15BrClO3PS [O-(4-Bromo-2-chlorophen-

yl)-O-ethyl-S-propy l phospho rothioate], Synonyms: Cu-

racron; Polycron; Selecron; Nonacron and properties

(M.Wt 373.6 and boiling point 110˚C). Profenophos

was obtained from the Ministry of Agriculture, Egypt.

2.4. Biological study

2.4.1. Molluscicidal Screening

The efficacy of pesticide (Profenophos) against adult

snails was determined according to the standard proce-

dure recommended by WHO (9). Stock solution (1000

ppm) were prepared using dechlorinated water and a

series of concentrations was prepared from each ex-

perimental pesticide on the basis of weight/volume that

would permit the computation of LC50 and LC90 values

were prepared [10]. Dead snails were counted and re-

corded. LC25 LC50 and LC90 values slop function and

95% confidence limits of compound were obtained from

the curve in which serial concentrations in ppm were

plotted against % mortality of snails [11]. While LC0

was determined as 1/10 LC50 [9].

2.4.2. Effect of Sublethal Concentration of Pes-

ticides on Infectivity of S. Mansoni Mirac-

idia to B. Alexandrina Snails.

Exposing 3 groups of snails (each of 50 snails) indi-

vidually to a dose of 10 miracidia/snail and maintained

in each sublethal of concentrations of Pesticide (LC0,

LC10 and LC25 of Profenophos) for 24 h under room

temperature (24  1˚C). After exposure to miracidia,

snails were maintained in their corresponding pesticide.

Another group of 50 snails was exposed to miracidia in

the absence of the tested pesticide an d maintained under

the same conditions (control group). The snails were

daily fed lettuce (Lactuca sativa plant) leaves and dead

ones were removed. Examination of snails for cercarial

shedding was carried out twice weekly, 25 days post

exposure, and the cercarial suspension was poured in a

graduated Petri dish, then a few drops of Bouin’s fluid

(Dye for coloring cercariae) were added and all cer-

cariae were counted, using a dissecting microscope.

Shedding snails were then isolated and kept in special

aquaria in complete darkness.

2.4.3. Effect of Sublethal Concentration of

Pesticides on Physiological Parameters

of B. Alexandrina Snails.

For studying some physiological parameters of B. al-

exandrina snails, four identical groups of snails (each of

six replicates) of which three groups of snails were ex-

posed for one month to sublethal of concentrations of

Pesticide (LC0, LC10 and LC25 of Profenophos). A four

group was left unexposed under the same laboratory

conditions as control. Snails surviving after exposure

was used to study selected enzymatic activities were

investigated both in treated and untreated snails. The

measured enzymes included aminotransaminases [As-

partate aminotransferase (AST) and alanine ami-

notransferase (ALT), (AST and ALT are the most fre-

quently measured for hepatic diseases, the enzymes may

be released from hepatocytes into the circulation by

necrosis [12]), acid phosphatase and alkaline phos-

phatases enzymes (these are a lysosomal enzymes con-

cerning with digestion of foreign substances and bacte-

ria inside the cells and is involved in the defense

mechanisms of both vertebrates and invertebrates [13])

in haemolymph and tissues of B. alexandrina. Moreover,

lactate dehydrogenase (LDH), pyruvatekinase (PK) and

Hexokinase (HK) activity [glycolytic enzymes] in soft

tissue of treated snails.

2.4.3.1. Biochemical Analysis

For preparation of tissue extracts of treated and con-

trol snails, one gram of the soft tissues of the snail was

homogenized in 5 ml distilled water, pH 7.5. A glass

homogenizer was then used to grind the tissue, and the

homogenate was centrifuged for 10 min at 300 rpm and

the fresh supernatant was decanted. Haemolymph of

infected and control snails was collected in accordance

with techniques described by Michelson [14]. The

haemolymph was obtained via small hole made in the

shell into which capillary tube was inserted then it was

drawn into tube by capillary suction. The haemolymph

was pooled from 10 snails collected in a vial tube (1.5

ml) and kept in ice-bath.

2.4.3.2. Assay Methods

Aspartate aminotransferase (AST) activity in the

haemolymph and soft tissues of exposed and unexposed

R. Mohamed / Open Journal of Ecology 1 (2011) 41-47 43

snails was determined according to the method of

Reitman and Frankel [15], the oxaloacetate formed re-

acts with 2, 3-dinitrophenyl-hydrazine in alkaline solu-

tion(PH = 7.5 at 22˚C). The product of the reaction can

then be determi ned p hotometrically at 500 nm to560 nm

Alanine aminotransferase (ALT) activities in the

haemolymph and soft tissues of exposed and unexposed

snails were determined according to the method of

Reitman and Frankel [15], the pyruvate formed reacts

with 2, 4 dinitrophenyl-hydrazine in alkaline solu-

tion(PH = 7.5 at 22˚C). The product of the reaction can

then be determined spectrophometrically at 500 nm to

560 nm

Acid phosphatase (ADP) and alkaline phosphatase

(ALKP) activities were determined spectrophotometer

by measuring absorbance at 340 - 410 nm according to

Bessey et al. [16], Acid phosphatases have pH optima

below 7, whereas alkaline phosphatases are most active

above pH 7 at 22˚C.

Hexokinase (HK) was determined spectrophotometer

by measuring absorbance at 340 nm (PH = 7.5 at 22˚C)

according to the method of Uyeda & Racker [17] in

which glucose-6-phosphate formed by the hexokinase

reaction is measured by adding glucose-6-phosphate

dehydrogenase and NADP and following NADPH for-

mation.

Pyruvatekinase (PK) activity was measured spectro-

phometrically as the rate of decrease in extinction at 340

nm (PH = 7.5 at 22˚C) due to the oxidation of NADH

by coupling the system with excess of lactate dehydro-

genase [18].

Lactate dehydrogenase activity was measured spec-

trophometrically by measuring absorbance at 340 nm

((PH = 7.5 at 22˚C) according to the method of Cabaud

& Wroblewski [19]. In this method pyruvate is reduced

by incubation with the enzyme in the presence of coen-

zyme nicotinamide adenine dinucleotide (NADH).

All physiological parameters determined in this study

were determined spectrophotometrically, using reagent

kits purchased from BioMerieux Company, France.

2.5. Statistical Analysis

Analysis of data was carried out by student’s “t”-test

for comparing the means of experimental and control

groups Spiegel [20].

3. RESULTS

The molluscicidal activity of Profenophos on Biom-

phalaria alexandrina snails after 24 hours of exposure

under laboratory conditions is presented in Table 1. The

data obtained indicate that the recorded LC50 values for

this pesticide was 4.6 ppm and LC 90 values was 10.7

ppm for B. alexandrina. The sublethal concentrations

(LC0, LC10 and LC25) were found to be 0.46, 1.8 and 2.5

ppm for B. alexandr ina.

The effect of the tested sublethal concentrations of

Profenophos on infection of B. alexandrina with S.

mansoni miracidia was presented in Table 2. The infec-

tion rate was significantly (p < 0.001) lower than that of

control snails (55%) being 40%, 16.66% and 10% for

snails exposed to LC0, LC10 and LC25 of Profenophos

respectively with a reduction rate –27.7%, –69.70%

and 81.82% respectively.

There is no significant difference between prepatent

period of the snails exposed to LC0, LC10 and LC25 of

pesticide and the control group. Prepatent period (Table

3) of exposed snails to LC0, LC10 and LC25 of Profeno-

phos was prolonged to be 36.2 + 4.8, 31.6 + 2.6 and

33.2 + 3.2 days compared to 35.6 + 4.1 days for the

control group. Meanwhile, the duration of cercarial

shedding was significantly (p < 0.01) shortened among

these snails, being 8.2 + 1.2, 6.3 + 0.82 and 4.8 + 0.85

days for LC0, LC10 and LC25, respectively, compared

with 16.2 + 4.3 days for control snails. Highly signifi-

cant (p < 0.001) reductions of total cercarial production

per snails was also detected in experimental snails in

comparison with the control group.

The results showed that (Ta ble 4.) the concentration

of transaminase enzymes (AST and ALT) in haemo-

Table 1. Molluscicidal activity of profenophos (pesticide)

against adult Biomphalaria alexandrina snails after exposure

for 24 hours.

LC50

ppm*

Confidence

limit of LC50

ppm

LC90

ppm* Slope

function LC0

ppm LC10

ppm* LC25

ppm*

4.6 5.98 - 3.5410.71.64 - 3.54 0.46 1.8 2.5

*using SPSS computer program under windows.

Table 2. Effect of sublethal concentrations of profenophos

(pesticide) on infection of Biomphalaria alexandrina with

Schistosoma mansoni.

Survived snails

at first shedding Infected snails

Treatment Number of

exposed

snails Number% Number %

Reduction

Control50 40 80 22 55

LC0 50 30 60 12 40–27.7*

LC10 50 18 36 3 16.66–69.70***

LC25 50 10 20 1 1081.82***

p < 0.05, ** p < 0.01, *** p < 0.001.

R. Mohamed / Open Journal of Ecology 1 (2011) 41-47

OPEN ACCESS

Table 3. E ffect of sublethal concentrations of profe nophos ( p e s-

ticide) on cercarial production of Schistosoma mansoni from

infected snails.

Concentration

(ppm)

Prepatent

period

(days)

Duration of

shedding

(days)

Number of

cercariae/

snail

LC0 36.2 ± 4.8 8.2 ± 1.2* 621.5 ± 55.5 ***

LC10 31.6 ± 2.6 6.3 ± 0.82* 245.23 ± 44.2***

LC25 33.2 ± 3.2 4.8 ± 0.85*** 88.6 ± 12.4***

Control 35.6 ± 4.1 16.2 ± 4.3 2834.12 ± 213

lymph and soft tissue of experimental B. alexandrina

snails showed significant (p < 0.05) increase than that in

the control snails, The percentage of elevation 32.8%,

73.44% and 90.24% for AST and 37.11%, 41.41% and

61.71% for ALT in haemolymph and 29.33%, 83.47%

and 88% for AST, and 27.77%, 43.77%, 72.22% for

ALT in soft tissue of snails exposed to LC0, LC10 and

LC25 of pesticide, respectively than that in control

snails.

The present results (Table 5) indicated that there were

significant (p < 0.05) elevations in the level of acid

phosphatase 21.57%, 27.45% and 39.22% in haemo-

lymph and 27.27%, 31.82% and 40.91% in soft tissue of

snail treated LC0, LC10 and LC25 of pesticide, respec-

tively. The increase of alkaline phosphatase was 40%,

56% and 92% in haemolymph and 12.5%, 27.1% and

41.67% in soft tissue of LC0, LC10 and LC25 of pesticide,

respectively than that in control snails.

The levels of hexokinase (HK), pyruvatekinase (PK),

and lactate dehydrogenase (LDH) in the soft tissue in

normal and treated snails are displayed in Tab le 6. The

HK activity in snails exposed to sublethal concentra-

tions of the pesticide for one month was 21.4  1.4, 18.7

 1.1 and 13.5  1.6 mol/min/g, respectively. Such

reduced values were statistically significant than those

of the corresponding controls (30.2  1.7). The activity

levels of PK, LDH were also significantly reduced in

response to treatment with plant extract, those of PK

being 0.10  0.018, 0.082  0.011 and 0.066  0.016

mol/min/g wet tissue, as compared to the control being

0.12  0.042 mol/min/g.

4. DISCUSSION

Most of the chemical compounds residues found in

the environment, such as pesticides, end up being dis-

charged into water resources. In these environments, the

substances become more and more diluted and are,

therefore, usually found at low concentrations. In the

current study, residual/environmental concentrations of

commercial Profenophos were evaluated in order to

investigate their possible effects on biological and

physiological parameters of snails. The Profenophos

(pesticide) showed considerable molluscicidal effect

against B. alexandeina. The LC50 and LC90 were found

to be 4.6 ppm & 10.7 ppm respectively. This result is

supporting with Wafaa and Ragaa [8] indicated that

Chlorpyrifos and Profenophos have a considerable kill-

ing effect against B. truncatus with LC50, 1.32 ppm and

2.5 ppm of the two compounds, respectively. Similar

results were obtained by El-Fiki and Mohamed [21],

using the herbicides Gramaxone, Preforan and Trefla.

In this study, the infection of B. alexandrina with S.

mansoni miracidia was greatly reduced by the tested

sublethal concentrations of Profenophos. The reduction

of infection rate was found to increase with the increase

of sublethal concentrations of Profenophos. Thia agree

with Wafaa and Ragaa, [8] found that increasing reduc-

tion of infection of snails maintained in LC25 of Chlor-

pyrifos and Profenophos pesticide. Similar inhibitory

effects were seen in the results were obtained in litera-

ture by several authors working on various che- mical

and plant molluscicides [22] using copper sulphate and

Tributylin fluoride; [23] using Bayluscide; [24] using

the plant Calendula micranth a officinalis; [25] using the

Table 4. Aspartate amino transferase (AST)), Alanine amino transferase (ALT) in Biomphalaria alexandrina exposed to sublethal

concentrations of profenophos (pesticide).

Aspartate amino transferase concentrations

(/mg protein) % increase Alanine amino transferase concentrations

(/mg protein) % increase

Parameter

Treatment Haemolymph

mg/ml % of

change Soft tissue

mg/g % of

change Haemolymph

mg/ml % of

charge Soft tissue

mg/g % of

charge

Unexposed

(control) 18.45  2.3 37.5  3.2 25.6 ± 4.1 50.4  3.5

lC0 24.5  1.1 32.8% 48.5  4.3* 29.33% 35.1  2.4* 37.11% 64.4  8.2* 27.77%

LC10 32  5.1* 73.44% 68.8  4.5**83.47% 36.2  3.2* 41.41% 72.43  10 .1* 43.77%

LC25 35.1  3.2** 90.24% 70.5  4.6**88% 41.4  2.7* * 61.71% 86.8  6.3** 72.22%

Values are expressed as means  SD of 4 independent experiments;Enzymatic activities expressed as  m ole NADH reacted/min/g wet. Tissue; *p < 0.05,**& p

< 0.01

R. Mohamed / Open Journal of Ecology 1 (2011) 41-47 45

Table 5. Acid phosphatase (ADP), Alkaline phosphatase (ALKP) in Biomphalaria alexandrina exposed to sublethal concentrations

of profenophos (pesticide).

ADP concentration

( mg protein) ALKP concentrations ( mg protein)

Parameters

Treatments Haemolymph

mg/ml % of

change Soft tissue

mg/g % of

charge Haemolymph

mg/m % of

change Soft tissue mg/g% of

charge

Unexposed

(control) 0.051  0.05 0.44  0.042 0.25 0.34 0.048  0.046

LC0 0.062  0.07* 21.57% 0.56  0.12* 27.27% 0.35  0.17** 40% 0.054  0.05* 12.5%

LC10 0.065  0.06* 27.45% 0.58  0.33* 31.82% 0.39  0.13** 56% 0.061  0.086*27.1%

LC25 0.071  0.048** 39.22% 0.62  0.55**40.91% 0.48  0.64*** 92% 0.068  0033**41.67%

Values are exp ress ed as means  SD of 4 independent experiments; Enzymatic activities expressed as  mole NADH reacted/min/g wet. Tissue; *p < 0.05,**&

p < 0.01

Tabl e 6 . Levels of lactate dehydrogenase (LDH), hexokinase (HK) and glucose phosphate isomerase (GPI) in the tissue of Biom-

phalaria alexandrina exposed to sublethal concentrations of profenophos (pesticide).

Enzyme activity ( U/mg tissue)

6 LDH % of change HK % of changePyruvate kinase (PK) % of change

Unexposed (control) 44.2  1.2 30.2  1.7 0.12  0.042

LC0 36.2  2.1* –18.1% 21.4  1.4 *–29.14% 0.10  0.018* –16.67%

LC10 24.4  1.8* –44.80% 18.7  1.1* –38.1% 0.082  0.011* 31.67%

LC25 17.5  2.1**–60.41% 13.5  1.6**–55.30% 0.066  0.016** –45%

Values are exp ressed as means  SD of 4 independent experiment; Enzymatic activities expressed as  mole NADH reacted/min/g wet. Tissue; *p< 0.05,**&

p< 0.01.

plants Thymus capitatus and Piper nigrum; [26], [11]

using the plant Synadenium grantii. Tantawy [27] re-

ported that the herbicides, Butachlor and Fluazi-

fop-p-butyl reduced the survival and infection rates of

B. alexandrina snail s. Bakry [28] using plant extract.

However, there was no difference between the pre-

patent period of the snails exposed to sublethal concen-

tration of Profenophos pesticide and the control. De-

spite that, a h ighly redu ction in th e duration of cercarial

shedding and total cercarial production per infected

snails were reported. This reduction in cercarial shed-

ding period and total cercarial production per snail is

probably due to rupture of snails’ tissues through

miracidial penetration in the presence of those pesti-

cides which increased the harmful effects of these

plants [8]. These observations are in accordance with

many authors using different plant species as mollus-

cicides. Thus, Ibrahim et al. [29] stated that low con-

centrations of the organophosphorus pesticides Chlro-

pyrifos (Dursban) caused blockage of cercarial shed-

ding of infected of B alexandrina snails. Sharaf El-Din

et al. [30] obtained similar reduction in cercarial shed-

ding and cercarial production from B. alexandrina

treated with sublethal concentrations of aqueous sus-

pension of Zygophyllum simplex. This supports other

authors on various molluscicid es, e.g.

El-Ansary et al. [31] recorded longer prepatent pe-

riod in B. alexandrina infected with S. mansoni in

presenc e of Ambrosia maritima, and Gawish [32] found

that the period of cercarial shedding in snails treated

with the experimental molluscicides during their expo-

sure to miracidia are significantly short than that in

control snails. The results also indicated that treatment

of snails continuously with the methanol extract of

E.soongerica plant resulted in highly significant reduc-

tion of total cercarial production per snails in compari-

son with control.

The activity of aspartate aminotransferase (AST) and

alanine aminotransferase (ALT) enzymes in haemo-

lymph and tissue of B. alexandrina snails were signifi-

cantly increased than that in the control snails. This

result is in accordance with Bakry et al. [33] who re-

ported that the levels of AST and ALT in haemolymph

of Bulinus truncatus are increased significantly when

exposed to oreopanax reticulum and Furcraea selloea

plants. El-mam and Ebeid [34] reported that the activity

of AST and ALT of the heamolymph of B. alexand rina

were significantly decreased by S. mansoni infection.

Acid phosphatase enzyme plays an important role in

the defense mechanism of host snails Sabry et al. [35].

R. Mohamed / Open Journal of Ecology 1 (2011) 41-47

The present results indicated that there are signifi-

cant elevations in the level of acid phosphatase and

alkaline phosphatase which can be explained by the

destruction of internal snail cells.

This finding agrees with Bakry et al. [33] using ore-

opanax reticulum and Furcraea selloea plants. Also,

Michelson and Dubois [36] found an increase in alka-

line phosphatase levels in both the heamolymph and

digestive gland from infected B. glabrata with S. man-

soni. El-Emam and Ebeid [34] reported that the acid

phosphatase activity in the heamolymph of B. alexan-

drina was increased by S. mansoni infection. The pre-

sent study showed a significant decrease in LDH activ-

ity in the whole tissue extract of Bulinus truncatus in

response to treatment with the methanol extract of

E.soongerica plant. Several authors have reported a

significant decline in LDH activity of tissues of

vari]ous molluscs in response to some molluscicides

[Aboul-Zahab & El-Ansary, [351 and Bakry et al. [38].

4. CONCLUSIONS

It is concluded that Profenophos pesticide are toxic

to the intermediate snail host of S. mansoni. and there-

fore may have adverse effects on natural populations.

Sublethal concentration of this pesticide play also a

role in suppressing transmission of schistosomiasis by

reducing the infection of snails with schistosomes. In

addition, pesticides caused reduction in number of cer-

cariae per snail during the patent period and in the pe-

riod of cercarial shedding. The present study indicated

that the increase in levels of aminotransaminases, acid

phosphatase and alkaline phosphatases enzymes in

haemolymph and soft tissue of snails and the activity

level of lactate dehydrogenase, hexokinase and pyru-

vatekinase was also significantly reduced in response to

treatment. According to the results obtained, we can

observe that the Low concentrations of Profenophos

tested (residues found in the environment) induced

toxic to biological and physiological of snails.

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