Influence of Atrazine and Roundup pesticides on biochemical and molecular aspects of Biomphalaria alexandrina snails

Pesticide Biochemistry and Physiology 104 (2012) 9–18

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Influence of Atrazine and Roundup pesticides on biochemical and molecular aspects of Biomphalaria alexandrina snails Fayez A. Barky a,⇑, Hala A. Abdelsalam b, Momeana B. Mahmoud a, Salwa A.H. Hamdi b a b

Medical Malacology Department, Theodor Bilharz Research Institute, Giza, Egypt Zoology Department, Faculty of Science, Cairo University, Giza, Egypt

a r t i c l e

i n f o

Article history: Received 1 May 2012 Accepted 21 May 2012 Available online 23 June 2012 Keywords: Biomphalaria alexandrina Atrazine Roundup Some enzymatic activities Biochemical and molecular aspects

a b s t r a c t The excessive use of pesticides in agriculture has sparkled the interest of scientists in investigating the harmful effects of these compounds. The present study evaluates the pesticides Atrazine and Roundup (glyphosate) on biochemical and molecular aspects of Biomphalaria alexandrina snails. The results showed that LC10 of these two pesticides caused considerable reduction in survival rates and egg production of treated snails. Additionally, Atrazine proved to be more toxic to B. alexandrina snails than Roundup. In treated snails, glucose concentration (GL) in the hemolymph as well as lactate (LT) and free amino acid (FAA) in soft tissues of treated snails increased while glycogen (GN), pyruvate (PV), total protein (TP), nucleic acids (DNA and RNA) levels in snail’s tissues decreased. The activities of glycogen phosphorylase (GP), superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), succinic dehydrogenase (SDH), acetylcholinesterase (AChE), lactic dehydrogenase (LDH) and phosphatases (ACP and ALP) enzymes in homogenate of snail’s tissues were reduced in response to the treatment with the two pesticides while lipid peroxide (LP) and transaminases (GOT and GPT) activity increased (P < 0.001). The changes in the number, position and intensity of DNA bands induced by pesticides may be attributed to the fact that pesticide can induce genotoxicity through DNA damage. It was concluded that the pollution of the aquatic environment by Atrazine and Roundup pesticides, would adversely affect the metabolism of the B. alexandrina snails, and have adverse effects on its reproduction. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction The use of pesticides may contaminate the irrigation and drainage systems during agriculture activities and pests’ control, and then negatively affect the biotic and abiotic components of the polluted water course [1,2]. Some pesticides are highly persistent in the environment, and can affect and contaminate both aquatic and terrestrial species [3,4]. Atrazine is one of the most widely used pesticides for the control of both grasses and weeds in many crops and in non-agricultural situations such as on railways, highways and industrial sites. Due to high runoff potential, Atrazine is the pesticide most frequently detected in aquatic ecosystems and its concentration can exceed the general quality standard for surface water [5]. However, due to its persistence in aquatic environments, Atrazine may occasionally cause sublethal effects in various aquatic organ-

⇑ Corresponding author. E-mail address: [email protected] (F.A. Barky). 0048-3575/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2012.05.012

isms [5]. Furthermore, its physical and chemical properties allow its accumulation in the phytoplanktons, invertebrates and fish species. This process may induce chronic toxic effects to the aquatic species [6]. Acute and chronic toxicity of Atrazine in freshwater invertebrates is well documented [7]. Roundup is a commercial pesticide with active compound glyphosate used in a broad spectrum in agricultural applications for weed control [8]. Due to its high water solubility and extensive usage, especially in shallow water systems, the exposure of nontarget aquatic organisms to this pesticide is a concern [9]. It may be exceptionally dangerous for aquatic ecosystems through high water solubility [10]. Previous studies showed that glyphosate may affect plants, fishes, amphibians, arthropods and snails by causing physiological, immunological and biochemical alterations [11,12]. Certain pesticides persist as residues in the environment for only a few days, there may be a cumulative effect on aquatic organisms because the electrophonic nature of some pesticide that affects the various enzymes responsible for normal metabolic processes [13]. The aim of this study was to determine the effects of the pesticides, Atrazine and Roundup (glyphosate) on the biochemical and molecular aspects of Biomphalaria alexandrina snails.

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2. Materials and methods

2.4. Effect of exposure to LC10 from the two pesticides on mortality rate, egg normality and egg hatchability of snails

2.1. Snails Laboratory bred B. alexandrina snails (3–12 mm) from Medical Malacology Department, Theodor Bilharz Research Institute (TBRI), were used.

2.2. Pesticides 2.2.1. Atrazine Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine, 97.8% purity) was obtained from Cluzeau Info Labo (Ste Foy La Grande, France) (Fig. 1). With molecular formula C8H14ClN5, molecular weight of 215.68 g/mol, melting point 173–175 °C, boiling point 200 °C, 473 K, 392 °F and density 1.187 g/cm3. Solubility in water (33 ppm) (Fig. 1).

2.2.2. Roundup Roundup (glyphosate concentration 120 g/l in the form of glyphosateisopropylamine salt 162 g/l). Roundup herbicide was used in the liquid commercial form was supplied by Monsanto Company (St. Louis, MO, USA). With molecular formula C6H17N2O5P, molecular weight of 228.183 g/mol, melting point 200 °C and density 1.218 g/cm3 (Fig. 1).

2.3. Bioassays tests 2.3.1. Molluscicidal screening The efficiency of the pesticides (Atrazine and Roundup) against adult snails (6–12 mm) was determined according to WHO [14]. A stock solution (1000 ppm) was prepared using dechlorinated water on the basis of concentration/volume and a series of concentrations was prepared from each experimental pesticide that would permit the computation of LC10, LC50, and LC90. Exposure and recovery periods were 24 h each [15,16]. Mortality rates were recorded and SPSS was used to computer program under windows.

Fig. 1. Chemical structure of pesticides Atrazine and Roundup.

For studying the effect of exposure to LC10 for Atrazine and Roundup on mortality rate, egg normality and egg hatchability for 6 weeks, 150 healthy mature snails (8–12 mm diameter) were randomly divided into 3 groups (each of 50 snails). The 1st group was continuously exposed to LC10 of Atrazine and 2nd group was exposed to LC10 of Roundup. The 3rd group was left unexposed under the same laboratory conditions as control. Pesticide solutions were prepared every 24 h to avoid the effect of storage. Dead snails are removed daily from aquaria and the mortality rate was calculated. The containers of treated and untreated snails were provided by thin plastic sheets for egg deposition. In addition to lettuce, tetramine (fish food) was added twice weekly. The egg clutches were weekly collected and examined microscopically every week. Egg masses with deformed or dead embryos, masses containing different developmental stages, empty egg case or more than one embryo in one egg were considered as abnormal [17]. The egg clutches were transferred into container containing dechlorinated water and the control ones. The percentage of egg hatching and hatchability were recorded every week. 2.5. Effects of LC10 from the two pesticides on snails’ biochemical parameters Snails were randomly divided into three groups (50 snails each). The 1st and 2nd groups were continuously exposed to LC10 of Atrazine and Roundup pesticides for 4 weeks. The third group of snails was left unexposed under the same laboratory conditions as control. Surviving snails were subjected to withdrawal of their hemolymph. Hemolymph samples were collected [18] by removing a small portion of the shell and inserting a capillary tube into the heart. The hemolymph pooled from 10 snails was collected in a vial tube (1.5 ml) and kept in ice-box. For preparation of tissue homogenates of both exposed and unexposed snails, 1 g of snail’s soft tissues from each group was homogenized in 5 ml distilled water at pH 7.5. A glass homogenizer was used and the homogenate was centrifuged for 10 min at 3000 rpm, then the fresh supernatant was used. All physiological parameters were determined spectrophotometrically using kits purchased from BioMerieux Company, France. 2.5.1. Assay methods Glucose concentration (GL) (mg/ml hemolymph) in snails’ hemolymph was measured according to Trinder [19]. Lactate (LT) was estimated according to Barker and Summerson [20] as modified by Huckabee [21]. Lactate content was expressed as mg lactic acid/g of tissue. Total protein level (TP) was estimated according to the folin–phenol method of Lowry et al. [22], total protein content was expressed as lg protein/mg of tissue. Glycogen (GN) was measured according to Van der Vies [23]. Glycogen content was expressed as mg glycogen/g of tissue. The pyruvate level (PV) was measured according to Friedemann and Haugen [24]. The pyruvate content was expressed as lmol of pyruvate/g of tissue. Total free amino acid level (FAA) was measured according to Spies [25]. The homogenate (50 mg/ml, w/v) was prepared in 96%. Total free amino acid content was expressed as lg/mg of tissue. Nucleic acids (DNA and RNA) were estimated according to Schneider [26], using diphenylamine and ordinal reagents, respectively. Nucleic acids content was expressed as lg/mg of tissue. Glycogen phosphorylase (GP) [27], glucose-6-phosphatase (G6-Pase) [28], superoxide dismutase (SOD) [29], catalase (CAT) [30], glutathione reductase (GR) [31] lipid peroxides (LP) [32], lactic dehydrogenase (LDH) [33], succinic dehydrogenase (SDH) [34],

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activities of phosphatases [acid and alkaline phosphatase (ACP7ALP) [35], and aminotransaminases [glutamic oxalic transaminase and glutamic pyruvic transaminase (GOT and GPT)] [36] activity were assayed in the tissue by measuring the liberated end products. Enzyme activity is expressed as lmol/min/mg protein. Acetylcholinesterase activity (AChE) was measured in nerve tissue of treated and control snails using the method of Singh and Singh [37]. Soft parts of snails were dissected out from shells after gentle crushing, the cerebral ganglia around the buccal mass were dissected and collected in isotonic saline and freezed at 15 °C. Nerve tissue (50 mg). The fresh supernatant was used as enzyme source. 2.6. Molecular assay 2.6.1. Genomic DNA extraction DNA was extracted from the treated snails according to the method described by Sharma et al. [38]. The concentration of DNA and its relative purity were determined using a spectrophotometer based on absorbance at 260 and 280 nm, respectively. The integrity of extracted genomic DNA was checked by electrophoresis in 0.8% agarose gels using DNA molecular weight marker (Eurobio, Paris, France). 2.6.2. PCR conditions Inter Simple Sequence Repeat (ISSR) analysis was performed using three different primers listed in Table 1. The RAPD primers codes and sequences were listed in Table 2. PCR for both analyses (RAPD and ISSR) was performed in 25 ll volume containing 2.5 mM MgCl2, 0.2 mM dNTPs, 20 lM primer, 50 ng genomic DNA and 1 unit Taq DNA polymerase (Bioron, Germany). All reactions were performed in a Perkin Elmer 2400 thermal cycler. ISSR program was performed as 1 cycle of 94 °C for 4 min and 35 cycles of 94 °C for 30 s, the annealing temperature for each primer 72 °C for 1.5 min. Then, a final extension step of 72 °C for 10 min was done. For each primer, the annealing temperature was chosen after different trials with different temperatures (Table 1). RAPD Program was performed as 1 cycle of 94 °C for 4 min and 40 cycles of 94 °C for 1 min, 35 °C for 1 min, and 72 °C for 2 min. To visualize the PCR products, 15 ll of each reaction was loaded on 1.2% agarose gel. The gel was run at 90 V for 1 h and visualized with UV transilluminator and photographed using UVP gel documentation system (GelWorks 1D advanced software, UVP). For amplification, a negative control reaction without DNA template was included. PCR reactions, those which generated high level of polymorphism across both types of analyses, were repeated twice in order to verify the reproducibility of polymorphic bands scored. This procedure allowed only those bands present in all replicated experiments to be scored as markers. 2.7. Statistical analysis The results obtained in the present work are represented as means ± standard deviation (SD), and were analyzed using analysis

Table 1 ISSR primer codes and sequences used for ISSR–PCR and their annealing temperature.

Table 2 RAPD primer codes and sequences. Primer set

Primer code

Primer sequence

1 2 3

A 12 A 16 A 18

50 TCGGCGATAG 30 50 AGCCAGCGAA 30 50 AGGTGACCGT 30

of variance (ANOVA). The significance of difference between means was calculated using the Duncan Multiple Range Test [39].

3. Results The molluscicidal activity of Atrazine and Roundup on B. alexandrina snails after 24 h of exposure under the present laboratory conditions is presented in Table 3. The data obtained clearly indicate that the recorded LC50 and LC90 values were 1.25 and 4.75 ppm for Atrazine and 3.15 and 12.6 ppm for Roundup, respectively. The results in Table 4 showed that a rapid increase in mortality rates of snails exposed to LC10 of Atrazine and Roundup which are significantly higher than those of control group (P > 0.01). This data indicated that no B. alexandrina snails could survive more than 4 and 5 weeks after exposure to LC10 of Atrazine and Roundup, respectively. Table 4 showed that the least percentage of abnormal egg masses was seen in control snails (12.9%). The percentage of abnormal egg masses laid by treated B. alexandrina snails was more significantly elevated in case of Atrazine (88.4%) and Roundup (85.6%) after 2 weeks of experiment, in comparison with the control snails. The greatest number of eggs (egg/snails) was seen in case of control B. alexandrina, however, this number was declined significantly in case of all the treated snails compared to the control snails. The present investigation showed the effect of continuous exposure for 6 weeks to LC10 of Atrazine completely inhibited egg production after 3 weeks while Roundup stopped snail’s egg lying after 4 weeks. All tested pesticides induced marked increases in the percentage of abnormal laid eggs, compared to controls. The present investigation showed that the used pesticides caused a marked reduction in egg hatchability after 2 weeks. Atrazine (11.6%) and Roundup (14.47%) caused hatchability reduction compared to controls (58.1%) after 2 weeks. Sub-lethal concentrations were enough to alter the biochemical parameters in soft tissues of the snail. Glycogen, pyruvate, total protein, and nucleic acid (DNA and RNA) levels were reduced after exposure, while the glucose concentration in the hemolymph, lactate and free amino acid levels increased in tissues of B. alexandrnia after exposure to the two pesticides for 4 weeks (Table 5). Table 5 and Fig. 2 showed that GL concentration was increased to 43.88% and 21.79% in hemolymph, LT content was increased to 85.87% and 43.48% and FAA content was increased to 78.87% and 28.53% in the tissues of the snails exposed to LC10 of Atrazine and Roundup pesticides. TP content was reduced to 40.82% and 15.96%, GN content was reduced to 52.65% and 28.78%, PV level was reduced to 40.54% and 12.43%, DNA level was reduced to 28.53% and 20.79%, and RNA

Table 3 Molluscicidal activity of Atrazine and Roundup pesticides on Biomphalaria alexandrina after 24 h of exposure under laboratory conditions.

Primer set

Primer code

Primer sequence

Annealing T °C

LC50

1 2 3

HB10 HB11 HB12

50 (GA) 6 CC 30 50 (GT) 6 CC 30 50 (CAC) 3 GC 30

48 48 48

LC50 (ppm)

Confidence limit of LC50 (ppm)

Slope function

LC90 (ppm)

LC10 (ppm)

Atrazine Roundup

1.25 3.15

0.83–1.88 0.89–4.82

2.48 2.16

4.75 12.6

0.33 0.84

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Table 4 Effect of continuous exposure for 6 weeks to LC10 of Atrazine and Roundup on egg hatchability and normality of B. alexandrina snails. Exposure period (weeks)

Control

Atrazine

Roundup

Week

Mortality rate of snails Number of eggs Number of abnormality eggs Hatchability Abnormality % Hatchability %

5% 420 42 378 10% 90%

16% 184 122 62 66.3% 33.7%

11% 288 222 66 57.2% 22.92%

2 weeks

Mortality rate of snails Number of eggs Number of abnormality eggs Hatchability Hatchability % Abnormality %

16% 310 40 180 58.1% 12.9%

38% 86 76 10 11.6% 88.4%

28% 152 130 22 14.47% 85.6%

3 weeks

Mortality rate of snails Number of eggs Number of abnormality eggs Hatchability Hatchability % Abnormality %

24% 260 40 220 84.6% 15.3%

55% 0 0 0 0 0

37% 36 32 4 11.2% 88.8%

4 weeks

Mortality rate of snails Number of eggs Number of abnormality eggs Hatchability Hatchability % Abnormality %

48% 190 50 140 73.7% 26.32%

100% 0 0 0 0 0

66% 0 0 0 0 0

5 weeks

Mortality rate of snails Number of eggs Number of abnormality eggs Hatchability Hatchability % Abnormality %

66% 55 22 32 53.3% 46.7%

100% 0 0 0 0 0

100% 0 0 0 0 0

6 weeks

Mortality rate of snails Number of eggs Number of abnormality eggs Hatchability Hatchability % Abnormality %

92 0 0 0 0 40%

100% 0 0 0 0 0

100% 0 0 0 0 0

Table 5 The effect of prolonged exposure to LC10 of Atrazine and Roundup pesticides for 4 weeks on different biochemical parameters in the hemolymph and tissues of Biomphalaria alexandrina snails.

Glucose (GL) Lactate (LT) Total protein (TP) Glycogen content (GN) The pyruvate content (PV) Total free amino acid (FAA) DNA RNA

Hemolymph In tissues of snails

Control

Atrazine

Mean ± SD

Mean ± SD

33.5 ± 2.3 1.84 ± 0.21 38.22 ± 1.32 2.64 ± 0.45 1.85 ± 0.33 13.11 ± 2.21 36.32 ± 2.11 19.16 ± 2.21

Roundup

**

48.2 ± 1.6 3.42 ± 0.21*** 22.62 ± 1.68** 1.25 ± 0.84** 1.1 ± 0.12** 23.45 ± 1.68*** 21.21 ± 2.31** 12.11 ± 2.31**

% Change 43.88 85.87 40.82 52.65 40.54 78.87 28.53 36.80

Mean ± SD *

40.8 ± 2.1 2.64 ± 0.22** 32.12 ± 1.17* 1.88 ± 0.31* 1.62 ± 0.25* 16.85 ± 2.11* 28.77 ± 2.14* 15.11 ± 0.88*

% Change 21.79 43.48 15.96 28.79* 12.43 28.53 20.79 21.14

Values are expressed as: mg/ml for GL, lg/mg for TP, FAA, DNA, and RNA; mg/g for GN and LT and lmol/g for PV. Values are means ± SD of six replicates. P < 0.05. ** P < 0.01. *** P < 0.001. *

level was reduced to 36.80% and 21.14%, respectively (Table 5 and Fig. 2). Table 6 and Fig. 3 showed that the maintenance of Biomphalaria snails in LC10 of Atrazine and Roundup pesticides for 4 weeks induced a clear inhibitory effect in activities of glycogen phosphorylase, G-6-Pase, lactic dehydrogenase, succinic dehydrogenase, acetylcholinesterase, acid phosphatase, and alkaline phosphatase

in snail’s soft tissues. Meanwhile the activities of, glutamic oxalic transaminase and glutamic pyruvic transaminase were increased after the exposure to LC10 of Atrazine and Roundup pesticides. In the tissues, of snail exposed to LC10 of Atrazine and Roundup pesticides, GP activity was decreased to 56.38% and 34%, respectively. G-6-Pase activity was decreased to 51.16% and 20.93%, LDH activity was decreased to 34.38% and 15.63%, SDH activity was inhibited

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Fig. 2. Changes (%) of glucose (GL) in the hemolymph, lactate (LT), total protein (TP), glycogen content (GN), pyruvate content (PV), total free amino acid (FAA) and nucleic acids (DNA and RNA) in soft tissues of Biomphalaria alexandrina exposed to LC10 of Atrazine and Roundup pesticides for 4 weeks.

to 48.91% and 24.35%, AChE activity was decreased to 85.52% and 43.45%, ACP activity was decreased to 49.59% and 16.94% and ALP activity was decreased to 62.81% and 34.69%. Meanwhile, GOT activity was increased to 29.16% and 14.1%, GPT activity was increased to 40.18% and 25.17%. Table 7 and Fig. 4 showed that the effect of LC10 of Atrazine and Roundup pesticides, on some antioxidant enzymes SOD, CAT, GR, LP. The data demonstrated that, there was a significant inhibition in SOD, CAT and GR and a significant elevation in LP in treated snails compared to the normal snails. SOD activity was reduced to 46.15% and 24.35%, CAT activity was reduced to 41% and 23.4% and GR was reduced to 55.26% and 31.58%. Meanwhile LP was increased to 55.26% and 36.9%. ISSR analysis was performed on DNA extracted from soft tissue of snails after treatment with Atrazine and Roundup pesticides. Three anchor primers (HB10, HB11 and HB12) were used in the present study to analyze the genetic variation among the tested groups (Table 8). The molecular size of amplified bands ranged from 190 to 1350-bp. Animals treated with Atrazine showed the highest lost bands (total bands were 15 compared to 23 for control group), which could be a clear indication for the high genotoxic effect of Atrazine due to losses of alleles compared with groups treated with Roundup (total bands were 19 compared to 23 for control group) (Table 9). These results suggested that snails treated with Roundup had less genotoxic effect compared with snails treated with Atrazine. The obtained results of ISSR gave remarkable distinct characterization of each tested group (Fig. 5–7). A total number of 36 RAPD bands separated by electrophoresis on agarose gel across the experimental groups were obtained. The highest number of amplicons was generated from snails treated

Fig. 3. Changes (%) of activities of some glycogen phosphorylase (GP), glucose-6phosphatase (G-6-Pase), lactic dehydrogenase (LDH), succinic dehydrogenase (SDH), acid phosphatase (ACP), alkaline phosphatase (ALP), glutamic oxalic transaminase (GOT) and glutamic pyruvic transaminase (GPT) enzymes in soft tissues of Biomphalaria alexandrina exposed to LC10 of Atrazine and Roundup pesticides for 4 weeks.

with Roundup (22 amplicons) while snails treated with Atrazine generated the lowest (15 amplicons) (Table 9). The molecular size of amplified bands ranged from 200 to 1000-bp. The obtained results affected DNA banding pattern in all treatments compared with control group (Figs. 8–10). The results of RAPD and ISSR showed substantial differences between control and snails treated with Atrazine and Roundup with apparent changes in the number and size of amplified DNA fragments for different primers. The disappearing of a normal band is the obvious changes in the RAPD and ISSR patterns generated by pesticides.

4. Discussion The present study showed that Atrazine and Roundup have a considerable killing effect against B. alexandrina snails with LC50 1.25 and 3.15 ppm, respectively. Similar results were obtained by Bakry et al. [4] using Deltamethrin and Malathion pesticide, Hasheesh and Mohamed [40], using Chlorpyrifos and Profenophos pesticides and El-Fiki and Mohamed [41], using the herbicides Gramaxone, Preforan and Treflan. The mortality rate of B. alexandrina treated with LC10 of Atrazine and Roundup were higher than that of control snails. The mortality rate of B. alexandrina snails treated with LC10 of Atrazine is significantly higher than those treated with LC10 of Roundup. These results coincide with those of Bakry et al. [4] the mortality rate of Helisoma duryi snails treated with LC10 of Malathion is significantly higher than those treated with LC10 of Deltamethrin. Similar effect of sublethal concentrations of different biocides on survival of snails was reported by several authors such as [40] using Chlorpyrifos and Profenophos [42] herbicides, dithiopyridine carboxylic acid.

Table 6 The effect of prolonged exposure to LC10 of Atrazine and Roundup pesticides for 4 weeks on Enzyme activities in tissues of Biomphalaria alexandrina snails.

Glycogen phosphorylase (GP) Glucose-6-phosphatase (G-6-Pase) Lactic dehydrogenase (LDH) Succinic dehydrogenase (SDH) Acetylcholinesterase AChE Acid phosphatase ACP Alkaline phosphatase ALP Glutamic oxalic transaminase GOT Glutamic pyruvic transaminase GPT

Control

LC10 of atrazine

Mean ± SD

Mean ± SD

% Change

Mean ± SD

% Change

9.4 ± 0.82 0.86 ± 0.16 0.064 ± 0.02 35.43 ± 3.11 0.145 ± 0.036 0.242 ± 0.082 0.441 ± 0.062 7.82 ± 0.81 8.66 ± 0.31

4.1 ± 0.41** 0.42 ± 0.18** 0.042 ± 0.022* 18.1 ± 3.21** 0.021 ± 0.019*** 0.122 ± 0.052** 0.164 ± 0.044*** 10.10 ± 0.41** 12.14 ± 0.65**

56.38 51.16 34.38 48.91 85.52 49.59 62.81 29.16 40.18

6.2 ± 0.34* 0.68 ± 0.15* 0.054 ± 0.015* 26.8 ± 2.53* 0.082 ± 0.052** 0.201 ± 0.028* 0.288 ± 0.038* 8.92 ± 0.62* 10.84 ± 1.68*

34 20.93 15.63 24.35 43.45 16.94 34.69 14.1 25.17

Values are means ± SD of six replicates. Values are expressed in lmol/min/mg protein. P < 0.05. ** P < 0.01. *** P < 0.001. *

LC10 of Roundup

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Table 7 The effect of prolonged exposure to LC10 of Atrazine and Roundup pesticides for 4 weeks on some antioxidant enzymes in tissues of Biomphalaria alexandrina snails. Parameters

Superoxide dismutase (SOD) Catalase (CAT) Glutathione reductase (GR) Lipid peroxides (LP)

Control

LC10 of Atrazine

LC10 of Roundup

Mean ± SD

Mean ± SD

% Change

Mean ± SD

% Change

0.078 ± 0.08 88.8 + 10.2a 0.38 + 0.12a 8.62 + 0.82

0.042 + 0.023** 52.4 + 5.8** 0.17 + 0.046** 14.3 + 0.62**

46.15 41 55.26 55.26

0.059 ± 0.062* 68 + 8.2 ± 0.045* 0.26 + 0.082* 11.8 + 0.48*

24.35 23.4 31.58 36.9

Values are means ± SD of six replicates. Values are expressed in lmol/min/mg protein while lipid peroxide is expressed in lg/g tissue. P < 0.05. P < 0.01.

*

**

The present investigation showed the effect of continuous exposure to LC10 of Atrazine and Roundup completely inhibited egg production after 4 weeks while LC10 of Atrazine stopped snails’ egg lying after 5 weeks. All tested pesticides induced marked increases in the percentage of abnormal laid eggs, compared to controls. This may be due to the active substances present in the two pesticides which could affect the internal mechanism inside the snails to lay eggs. Similar results were obtained by Bakry et al. [4] using Deltamethrin and Malathion with H. duryi. Hasheesh and Mohamed [40] found that the egg production of B. truncatus treated with LC25 of Chlorpyrifos and Profenophos pesticides was significantly reduced compared to that of the control group. Ibrahim et al. [43] stated that low concentrations of the organophosphorus pesticide Chlorpyrifos (Dursban) suppressed the egg production of B. alexandrina snails. Also, Abdel Kader et al. [44] studied the interruption of several synthetic and natural molluscicides on egg production, egg abnormality and egg masses of B. alexandrina. They found that the long term exposure to low concentration of different mollusciciding agents markedly induced inhibition in egg production and increased abnormal eggs and egg masses rates. The present investigation showed Atrazine and Roundup pesticides caused marked reduction in egg hatchability. This agreed with Abdel Kader et al. [44] who found that the mollusciciding agents caused marked reduction in eggs’ hatchability especially with Bayluscide and Uccmaluscide. Agave filifera and Agave attenuate plants caused hatchability reduction compared to controls after 4 and 5 weeks, respectively. The fall in glycogen content in the body tissues of B. alexandrina snails and glucose increased in hemolymph indicates its rapid

Fig. 4. Changes (%) of activities of some antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and lipid peroxides (LP)] in soft tissues of Biomphalaria alexandrina exposed to LC10 of Atrazine and Roundup pesticides for 4 weeks.

utilization by the respective tissues as a consequence of pesticide toxic stress. Under hypoxic conditions, animals derive their energy from anaerobic breakdown of glucose, which is available to the cells by increased glycogenolysis [45]. Nakano and Tomlinson [46] have suggested that catecholamine levels rise under stressful environmental conditions, enabling the increased utilization of glycogen for energy production. Glycogen levels appear to be related, at least to some extent, to the detoxification mechanisms, essential for metabolism or degradation and elimination of pesticides from the body [47]. The decrease in pyruvate levels is due to the higher energy demand during pesticidal exposure, and suggests the possibility of a shift towards anaerobic dependence due to a notable drop in the aerobic segment. The decrease in pyruvate could be due to

Table 8 Number of obtained bands using ISSR analysis in snails treated with Atrazine and Roundup pesticides with compared with control group. Marker type

2 3 4 Total

Analysis type

ISSR

Marker name

HB 10 HB11 HB12

Total band number

13 12 12

Mobility range bp

220–1350 190–900 300–1000

Number of bands Control

Snails treated with Atrazine

Snails treated with Roundup

10 7 6 23

5 6 4 15

7 7 5 19

Table 9 Number of obtained bands using RAPD analysis in snails treated with Atrazine and Roundup pesticides compared with control group. Marker type

2 3 4 Total

Analysis type

RAPD

Marker name

A12 A16 A18

Total band number

12 12 12

Mobility range bp

350–1000 200–1000 200–800

Number of band Control

Snails treated with Atrazine

Snail treated with Roundup

8 9 8 25

6 5 4 15

7 7 8 22

F.A. Barky et al. / Pesticide Biochemistry and Physiology 104 (2012) 9–18

Fig. 5. ISSR–PCR fragments for genomic DNA. Groups C, A and B with primer HB10. M represents DNA marker, A represents snails treated with Atrazine, B represents snails treated with Roundup.

Fig. 6. ISSR–PCR fragments for genomic DNA. Groups C, A and B with primer HB11. M represents DNA marker, A represents snails treated with Atrazine, B represents snails treated with Roundup.

its conversion to lactate, or its mobilization to form amino acids, lipids, triglycerides, and glycogen synthesis in addition to its role in detoxification [48]. The increase in lactate also suggests a shift towards anaerobiosis as a consequence of hypoxia created from pesticide toxic impact leading to respiratory distress [49]. The depletion of the protein fraction in the body tissues of the snails in this experiment may have been due to protein degradation for metabolic purposes. Under stress conditions, the dietary protein consumed by snails is not stored in the body tissue [50] and hence the treated snails met their extra energy requirements from body proteins which are mobilized to produce glucose, the instant energy of which is made available for the snail by the process of gluconeogenesis [51]. Thus, the decreased protein content may be attributed to the destruction/necrosis of cells and consequent impairment in protein synthesis machinery [52].

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Fig. 7. ISSR–PCR fragments for genomic DNA. Groups C, A and B with primer HB12. M represents DNA marker, A represents snails treated with Atrazine, B represents snails treated with Roundup.

Fig. 8. RAPD fingerprinting profiles of genomic DNA. Groups C, A and B represent PCR products with primers A12 M represents DNA marker, A represents snails treated with Atrazine, B represents snails treated with Roundup.

The increment in free amino acid level was also the result of the breakdown of proteins for energy use and the subsequent impaired incorporation of amino acids in protein synthesis [53]. It also attributed to the reduced use of amino acids in the maintenance of the acid–base balance [54]. Pesticide appears as potential inhibitor of DNA synthesis, which might also result in reduction of the RNA level. It might also be noted that deprivation of food possibly caused some nutritional deficiency in the test snails, leading to lower concentration of nucleic acids and aberrations in DNA-directed RNA formation and protein synthesis, consequently limiting growth and adding to the metabolic stress of the snails [55]. In the present study, the levels of GP and G-6-Pase in the soft tissues of normal and treated snails were also significantly reduced in response to treatment with the two pesticides. With respect to

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Fig. 9. RAPD fingerprinting profiles of genomic DNA. Groups C, A and B represent PCR products with primers A16 M represents DNA marker, A represents snails treated with Atrazine, B represents snails treated with Roundup.

Fig. 10. RAPD fingerprinting profiles of genomic DNA. Groups C, A and B represent PCR products with primers A18 M represents DNA marker, A represents snails treated with Atrazine, B represents snails treated with Roundup.

G-6-Pase as glycogenolytic enzyme, it showed a reduced activity in treated snails which was attributed to either synthesis and/or degradation of glycogen [56] increasing the glucose concentration stimulated glycogen synthesis and decreased the activity of glycogen phosphorylase. Glucose was incorporated into glycogen during period of net glycogen breakdown, and vice versa; glycogen degradation occurred during periods of net glycogen synthesis which depends on glucose concentration [57]. Also, in the present study, LDH and SDH activities in the snail tissues showed variable decrease after exposure to LC10 of Atrazine and Roundup. The decrease in LDH activity in the tissues of B. alexandrina in response to the tested pesticides may be attributed to the release of the enzyme from the tissues as a result of cellular damage caused by the toxic effect of the pesticides. The enzyme lactic dehydrogenase LDH forms the centre for a delicately balanced equilibrium between catabolism and anabolism of carbohydrates and is associated with cellular metabolic activity [58]. LDH is present in most animal tissues and is involved in the interconversion of pyruvic acid and lactic acid. It serves as a pivotal enzyme between the glycolytic pathway and the tricarboxylic acid cycle. The action of pesticide in the respiratory chain is between FAD

and co-enzyme Q, and SDH containing one molecule of covalently bound FAD is inhibited [59]. In the present study, SDH presented the lowest activity after exposure to the two tested pesticides. This could be due to swollen, disrupted mitochondrial membranes and fewer crystal with subsequent leakage of enzymes as reported by Aboul-Zahab and El-Ansary [60]. SDH is an important active regulatory enzyme of the tricarboxylic acid cycle (TCA), the common pathway for carbohydrates Gonzaler and Tejedor [61]. GOT and GPT are two key enzymes known for their role in utilization of protein and carbohydrates. The studies of Harper et al. [62] showed that transamination and transdeamination reactions are prominent under stress conditions. GOT and GPT functions at the junction between protein and carbohydrate metabolism by interconverting ketoglutarate, oxalacetate, and pyruvate on one side and alanine, aspartate, and glutamate on the other side [62]. In the present study, the activities of, GOT and GPT were increased after the exposure to LC10 of Atrazine and Roundup pesticides. This result is supporting by Christensen et al. [63] suggested that toxic chemical pollutants affect the activity of enzymes to varying degrees, and hence enzymes are logical candidates to be used as bio-monitors. AChE is involved in the synaptic transmission of nerve impulses. Inhibition of AChE results in the accumulation of acetylcholine which causes twitching of muscle and leads to tetanus and eventual paralysis of the muscle. Paralysis of respiratory muscles may lead to death. Due to their neurotoxicity nature the physiology of several systems may be affected, resulting in disturbances of the metabolic system. At 20–70% inhibition of AChE, adverse effects become more suitable and can include reproduction problems [64]. It has also been shown that alkaline phosphate, through the process of phosphorylation of carbohydrates and fats, plays an important role in the active transport of chemicals across cell membranes [65]. Since the active sites of alkaline phosphatase [66] contain a serine residue, it is possible that the inhibition of alkaline phosphatase observed in this study could be due to the phosphorylation of the active sites of alkaline phosphatase. Acid phosphatase is a lysosomal enzyme and plays an important role in catabolism, pathological necrosis, autolysis, and phagocytosis [67]. The data obtained in the present study showed that, significant reduction in SOD, CAT and GR with significant increase in LP was noticed in the tissue treated with the two pesticides. Since the complex mechanism of lipid peroxidation is known to require the participation of highly reactive oxygen and other reactive metabolites in the chain of biochemical reaction. Thus, in any part of the body where these free radicals are produced, lipid peroxides are in turn increased. Such phenomenon was previously reported by Botros et al. [68]. The exposure of B. alexandrina LC10 of Atrazine and Roundup pesticides, affects various physiological mechanisms. The toxic effects of the pesticide were not fully neutralized, and there was evidence of protein denaturation, disturbance of cellular metabolic activities and impairment in neural transmission. Therefore, pollution of the aquatic environment by Atrazine and Roundup pesticides, would adversely affect the metabolism of the snail, and have adverse effects on its reproduction. So the use of LC10 of Atrazine and Roundup pesticides in the water bodies or fields adjoining the water bodies should be undertaken only after careful consideration of less harmful alternatives. The molecular studies resulting from cluster and genetic diversity analysis were performed based on the changes of the ISSR and RAPD bands in the pattern. The changes in ISSR and RAPD pattern between control and treated groups were obvious, including the changes in the number, the position and the intensity of the bands. The disappearance of bands possibly resulted from the presence of

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