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Molluscicidal impacts of Anagallis arvensis aqueous extract on biological, hormonal, histological and molecular aspects of Biomphalaria alexandrina snails
Experimental Parasitology 192 (2018) 36–41
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Molluscicidal impacts of Anagallis arvensis aqueous extract on biological, hormonal, histological and molecular aspects of Biomphalaria alexandrina snails
T
Amina M. Ibrahim∗, Samah I. Ghoname Environmental Research and Medical Malacology Department, Theodor Bilharz Research Institute, Giza, Egypt
A R T I C LE I N FO
A B S T R A C T
Keywords: Biomphalaria alexandrina Anagallis arvensis Biological Histopathology Hormones activity Comet assay
Controlling of Biomphalaria alexandrina snails by plant molluscicides is the cornerstone in treating schistosomiasis in Egypt. The objective of this study is, to evaluate the molluscicidal activity of the aqueous leaves extract of Anagallis arvensis against B. alexandrina snails. The present results showed that this aqueous extract was lethal for B. alexandrina snails at (LC50 37.9 mg/l; LC90 48.3 mg/l), and caused reduction in survival; reproductive rates and hormonal activity (testosterone (T) and 17β-estradiol (E)) of these snails. Histopathological changes occurred in the hermaphrodite glands of snails exposed to the sub lethal concentrations of this aqueous extract are detected, where, there were degeneration in both eggs and sperms and there were losses of connective tissues between acini. The present investigation revealed that this plant had a genotoxic effect especially with its concentration (LC10 and LC25), where, the length of olive tail moment was significantly increased than control group. These observations prove the potent molluscicidal activity of aqueous leaves extract of A. arvensis against the intermediate hosts of Schistosoma mansoni and provide natural biodegradable resources for snails' molluscicidal agents.
1. Introduction Schistosomiasis is a widespread neglected tropical parasitic disease transmitted by snails (WHO, 2017). Freshwater snails of Biomphalaria genus are the intermediate hosts of Schistosoma mansoni in Egypt (Ibrahim and Abdalla, 2017) and several strategies have been used to control snail populations through breaking the life cycle (El-Ghany and El-Ghany, 2017). Manufactured molluscicides is an imperative part in the incorporated schistosomiasis control programs (Abdel-Ghaffar et al., 2016), but because they have high cost and being poisonous to creatures of land and water (WHO, 2014), have stimulated interest to find suitable plant molluscicides (Elsareh et al., 2016). Plant molluscicides are promising choices that may grow the scope of molluscicides accessible for controlling of B. alexandrina snails (Kiros et al., 2014), as these plants are cheaper, safer and having a high level of degradability (Salawu and Odaibo, 2011). A. arvensis is the name of Scarlet Pimpernel, with about 20–25 species of flowering plants in the family Myrsinaceae (Khoshkholgh-Pahlaviani et al., 2013). It is used in European traditional medicine for dermatological purposes (López et al., 2011). This plant is toxic to ruminants (Pande et al., 2016) at high doses and during long-term consumption (López et al., 2011). The bio
∗
active constituents of this plant have antibacterial, antifungal, antitumor, antidiabetic and hepatoprotective activities (Bruneton, 2001) and were found to be less toxic through the toxicity prediction tool. Also, A. arvensis has no toxicity on humans as it is used to treat diseases like Gout, Leprosy, Epilepsy and Urinary infection (Pande et al., 2016). In Aquaculture, studies aimed to find a plant-based remedy for Saprolegniasis (Caruana et al., 2012), which is a pathogen causing “water mold” threatens the fresh-water breeding facilities by infecting fish eggs and fry and is caused by Saprolegnia parasitica. In recent study by Wirth and Stadtlander (2016), they tested the alcoholic and aqueous extracts of 29 plants that had antimicrobial properties in vitro against S. parasitica, they found that aqueous extracts were ineffective and that the concentration of 5000 ppm 70% ethanol extracts of Scarlet Pimpernel (A. arvensis) and rosemary (Rosmarinus officinalis) showed fungistatic effects in the first 24 h. A. arvensis is considered one of the most promising molluscicidal plants and its saponin fraction has been reported to express high molluscicidal activity against schistosome intermediate snails (AbdelGawad et al., 2000; El-Sayed et al., 1990). The molluscicidal and cercaricidal properties of this plant are found in all its parts and the use of water suspension of its powder is an inexpensive means of controlling
Corresponding author. Environmental Research and Medical Malacology Department, Theodor Bilharz Research Institute, Imbaba, Giza, Egypt. E-mail address: [email protected] (A.M. Ibrahim).
https://doi.org/10.1016/j.exppara.2018.07.014 Received 7 December 2017; Received in revised form 2 June 2018; Accepted 20 July 2018 Available online 21 July 2018 0014-4894/ © 2018 Elsevier Inc. All rights reserved.
Experimental Parasitology 192 (2018) 36–41
A.M. Ibrahim, S.I. Ghoname
L) and covered with glass plates. Oven dried lettuce leaves and blue green algae (Nostoc muscorum) were used for feeding and water in the aquaria was changed weekly. Pieces of polyethylene sheets were put into the aquaria to collect egg masses.
both snails and schistosome free larval stages (Elkhyat and Gawish, 2006). (Ibrahim et al., 1994) stated that the LC90 of the dry powder of three local plants namely Anagallis arvensis, Agave lophantha and Bassia muruicata tested in the laboratory against B. alexandrina snails were 50,100 and 165 ppm, respectively. In two field trials carried out in Sharkia Governorate, Egypt (ElEmam et al., 1996), used relatively high concentrations of dry powder of A. arvensis to control vector snails of schistosomiasis and fascioliasis and used the concentrations of 125 and 100 ppm to induce death of B. alexandrina snails. Also (Mosta-Fa et al., 2005), carried out semi-field trials under stimulated natural conditions to evaluate different modes of exposure to A. arvensis and Calendula micrantha as plant molluscicides and chemical molluscicides like baylucide and they found that the pre exposure to sub lethal concentrations of A. arvensis (55 ppm) increases the snail mortality in all experiments of bayluscide and A. arvensis than the other plant. The biological response of an organism to xenobiotics starts with toxicant induced changes at the cellular and biochemical levels, leading to changes in the structure and function of the cells, tissues, physiology and behavior of the organism and these changes may affect the integrity of the population and ecosystem (Parvez and Raisuddin, 2005). The endocrine system regulates hormone-dependent physiologic functions necessary for survival of the organism and the species (Hontela, 1998). An endocrine disruptor is an exogenous substance that affects the function of the endocrine system and causes deleterious effects in an organism, or its progeny (WHO, 2002). Some substances either of natural or man-made can cause endocrine disruption both in vitro and in vivo (Schug et al., 2011). The degree of this disruption depends on some parameters including reproductive stage dependent changes in steroid action, and whether the steroid action is genomic or non-genomic (Thomas, 2000). The endocrine disruptions in terms of steroid levels (testosterone (T) and 17b-estradiol (E)) can be studied in B. alexandrina snail (Omran and Salama, 2016), because their hormonal system is comparable to that of vertebrates (Janer and Porte, 2007; Oehlmann et al., 2007). Certain compounds act as endocrine disrupters either by binding to the hormone receptors or modulating it, or by modulating endogenous hormone levels through interfering with biochemical processes associated with the production, availability, or metabolism of hormones (Oetken et al., 2004). Testosterone and estrogen hormones have an important role in the development of gonads in B. alexandrina (Omran, 2012). These snails can be used as a bio indicator for endocrine disrupters in terms of steroid levels (testosterone (T) and 17b-estradiol (E)), after exposure to sub lethal concentrations of any molluscicides (Omran and Salama, 2016). DNA damage produced by the androgens, testosterone (TES) and β-oestradiol in keratinocyte cell line can be examined by the Comet assay (Gopalan et al., 2006). Comet assay is a sensitive method for direct visualization of DNA damage on the level of a single cell (Azqueta et al., 2009). Some recent studies link DNA strand breaks in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics (Lee and Steinert, 2003; Shaldoum et al., 2016). So, in the current study, molluscicidal effects of A. arvensis plants were determined to study how it affects the biological system of B. alexandrina snails through evaluating its effects on biological, hormonal, histological and molecular parameters of these snails.
2.2. Plant materials A. arvensis (Family Myrsinaceae) plants were collected from the shores of water courses in Aboelkhawy, El-beheira Governorate, Egypt during the spring of 2016. It was identified by Prof. Dr. Shadia Mohamed El- Dafrawy, Theodor Bilharz Research Institute, Giza, Egypt. The plants were shade dried, finely powdered using an electrical grinder. The dry powder of the experimental plants was weighed and added directly to filtered water in individual beakers. 2.3. Molluscicidal screening To calculate LC50 and LC90, series of concentrations were prepared on the basis of weight/volume as water extract of leaves powder (50, 40, 30, 20, 10 mg/l) and ten B. alexandrina snails (8–10 mm) were placed in beakers for each concentration (Litchfield and Wilcoxon, 1949). Another snail group of the same size was dipped in dechlorinated water only as control. Three replicates were used, each of 10 snails, for each concentration. The exposure period was 24 h, then, the snails were removed from the experimental test solution, and washed thoroughly with dechlorinated tap water and transferred to containers with fresh dechlorinated tap water for another 24 h of recovery, and then, the percentages of observed mortalities were recorded. LC0 is the concentration of a toxicant, below which no measurable effects take place (Warren, 1900), and is estimated as 1/10 LC50 value (WHO, 1965). 2.3.1. Effect on snails' egg-laying capacity of adult snails B. alexandrina snails (8–10 mm) were exposed for 24 h/day for 2 weeks to the concentrations LC0, LC10, and LC25 of the herbicide. Three replicates, each of 10 snails/L, were prepared for each concentration, another group considered as control group was maintained in dechlorinated water, the following parameters were weekly recorded: Lx (the survival rate as a proportion of the correct one), Mx (the number of eggs/snail/week) and R0 (the reproductive rate which is the summation of LxMx during the experimental period) (El-Gindy et al., 1965). 2.3.2. Biochemical assays for steroid hormones Ten snails (8–10 mm) were subjected to each sub lethal concentrations (LC0, LC10 and LC25) of the tested plant for 24 h (exposure), followed by another 24 h of recovery for two weeks. Three replicates of each concentration were prepared. Unexposed snails (control) were assayed side by side with the experimented groups. Snail's shell was gently crushed between two glass slides and digestive gland was dissected out and pooled in 1 ml Eppendorf tube. The tissues of snails from each group were weighed and then homogenized in ice cold, twicedistilled water (1 g tissue/5 ml water) using a glass Dounce homogenizer. The homogenates were centrifuged at 3000 rpm for 10 min at 4 °C and the supernatants were stored at −80 C until used. Hormone concentrations (testosterone (T) and 17b-estradiol (E)) were assayed according to the manufacture instructions of T EIA kit (Enzo Life Science, Michigan, USA, ADI-900-065) and E EIA kit (Cayman Chemical Company, Michigan, USA, item no. 582251).
2. Materials and methods 2.3.3. Histological study Adult snails (8–10 mm) were exposed to the aqueous extract of plant leaves at (LC0, LC10 or LC25) for 24 hours/week for 2 successive weeks (exposure), then, the snails were removed from the experimental test solution, and washed thoroughly with dechlorinated tap water, and transferred to containers with fresh dechlorinated tap water for another 24 h of recovery, and this was done for two weeks. Changes in histology
2.1. Experimental animals (snails) B. alexandrina snails (8–10 mm) provided from Medical Malacology Laboratory, Theodor Bilharz Research Institute (TBRI), Giza, Egypt were used. Snails were kept in plastic aquaria (16 × 23 × 9 cm). The aquaria were provided with dechlorinated aerated tap water (10 snails/ 37
Experimental Parasitology 192 (2018) 36–41
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2.3.4. Comet assay Snails (8–10 mm) were subjected to LC0, LC10 and LC25 of the aqueous extract of plant leaves for 48 h. The hemolymphs of 10 snails from each group were collected by removing a small portion of the shell and inserting a capillary tube into the heart. Then hemolymph was pooled in a glass vial tube (1.5 ml) and stored at −80 C° until used and then DNA damage was measured by single cell gel assay (Grazeffe et al., 2008; Singh et al., 1988) as follows: Hemocytes suspensions are harvested by centrifugation and re-suspend cells at 1 × 105 cells/ml in ice cold 1X PBS (phosphate buffer saline). This suspension was stirred for 5 min and filtered, then, 100 μl of this cell suspension was mixed with 600 μl of low-melting agarose (0.8% in PBS). 100 μl of this mixture was spread on pre-coated slides. The coated slides were immersed in lyses buffer (0.045 M TBE (Tris/ Borate/EDTA)), pH 8.4, containing 2.5% SDS (sodium dodecyl sulphate) for 15 min. The slides were placed in the electrophoresis chamber containing the same TBE buffer, but devoid of SDS. The electrophoresis conditions were 2 V/cm for 2min and 100 mA, staining with ethidium bromide 20 μg/ml at 4 °C. The DNA fragment migration patterns of 100 cells for each dose level were evaluated with a fluorescence microscope at 510 nm. The comets tails lengths were measured from the middle of the nucleus to the end of the tail with 40× increase for the count and measure the size of the comet. For visualization of DNA damage, observations are made of EtBr-stained (Ethidium Bromide Staining) DNA using a 40× objective on a fluorescent microscope. Slides were coded independently and scored blindly.
negatively affected their fecundity (MX), i.e. the snails laid few eggs/ snail/week through the weeks of the experiment in comparison with that of control ones. Regarding the reproductive (Ro) of treated snails, it was extremely highly suppressed (p < 0.001) than control group. The percent of reduction is increased with increasing the sub lethal concentration being 99.27% at LC25. Testosterone (T) and 17β Estradiol (E) were determined in the tissue homogenate of exposed snails after 2 weeks of exposure. Levels of T and E were decreased significantly (p < 0.05) after exposure to sub lethal concentrations (LC10 and LC25) compared with control (Table 3). Histopathological effects were detected by light microscopy, where, the normal hermaphrodite gland of B. alexandrina comprised of the male reproductive cells and the female oogenic cells (Fig. 1. A). Exposing of snails to LC0 sub lethal concentration, caused a moderate effect such degeneration of some mature ova (Fig. 1. B). While, exposure of snails to LC10, showed degeneration and destruction in sperms and degeneration in eggs (Fig. 1. C), the great damage in gonadal cells occurred at LC25, where, eggs lost their shapes and degenerated. Sperms were degenerated and the connective tissue was dissolved and replaced by vacuoles (Fig. 1. D). The present results of alkaline comet assay demonstrated that the olive tail moment (OTM) of snails exposed for 48 h to sub lethal concentrations of the aqueous leaves extract of A. arvensis was highly increased (p < 0.01) (Fig. 2.) than control snails. Comets were classified and assigned to four categories (0–3) according to the extent of DNA migration. Fig. (3) shows that in snails exposed to LC10 of the aqueous leaves extract, took rank 2 (intermediate DNA migration), while, at LC25 took rank 3 (high DNA migration).
2.4. Statistical analysis
4. Discussion
Lethal concentration values were defined by Probit analysis (Finney, 1971) and analysis of data was carried out by Student's t-test for comparing the means of experimental and control groups (Murray, 1981). Values were expressed as mean ± S.D., and the obtained data were analyzed using the Graph Pad Prism 6.04 software for Windows (Graph Pad Software, San Diego, California, U.S.A.; 1992–2014). A value of (P < 0.01) was considered significant.
The high cost and toxicity to non-target organisms of chemical molluscicides, have drawn much attention during recent years for the use of plant molluscicides (WHO, 2017). A. arvensis is one of the most promising molluscicidal plants and its saponin fraction has been reported to express high molluscicidal activity against schistosome intermediate hosts (Abdel-Gawad et al., 2000). The median lethal concentration (LC50) for any molluscicidal material must not exceed (100 ppm) (WHO, 1993). The present results showed the lethal concentrations LC50 and LC90 of the aqueous leaves extract of A. arvensis on B. alexandrina snails after 24 h of exposure were 37.9 and 48.3 mg/l, respectively. These outcomes are in congruity with the past investigation of Kamel et al. (2007), who, stated that the water suspension of A. arvensis plants has a molluscicidal activity against B. alexandrina snails. Also (Al-Snafi, 2015), confirmed that this plant has a molluscicidal activities. This molluscicidal activity is due to presence of two compounds called desgluco-anagalloside B and anagalloside B and their activities are comparable to that of the synthetic molluscicide, niclosamide (Abdel-Gawad et al., 2000). The present results showed that survival rates of adult B. alexandrina snails were markedly reduced post their exposure to sub lethal concentrations (LC0, LC10 or LC25) of aqueous leaves extract of A. arvensis. Comparable perceptions were recorded by Ibrahim and Abdalla (2017) who showed that survival rates of adult B. alexandrina snails were markedly reduced post their exposure to sub lethal concentrations of the aqueous seed extract of moringa oleifera and they linked this reduction by deformation in the digestive and hermaphrodite gland cells of the treated snails. Also, similar results were obtained after using the plants Panicum repens and Solanum nigrum against B. alexandrina snails (Ibrahim et al., 2004). The reproductive rate (R0) and fecundity (Mx) of adult B. alexandrina snails in the present study were significantly decreased by their exposure to the sub lethal concentrations (LC0, LC10 or LC25) of the aqueous leaves extract of A. arvensis, in comparison with the control group. Comparable perceptions were recorded by Hasheesh and
of hermaphrodite gland of treated snails compared with control snails were done according to Mohamed and Saad (1990).
3. Results From (Table 1), the calculated lethal concentrations (LC50 and LC90) on B. alexandrina snails after 24 h of exposure for the aqueous leaves extract of A. arvensis were 37.9 and 48.3 mg/l, respectively. Table 2 indicated that the survival rate of snails exposed the aqueous leaves extract of A. arvensis to LC0 (3.7 ppm) was slightly affected, being 0.55 at the 4th week of exposure. Thereafter, through the recovery four weeks, the snails survived till the end of the experiment. While, exposure of snails to LC10 (27.5 ppm) considerably reduced their survival rate (Lx) to be 0.42 at the 4th week of exposure compared to 0.95 for the control group. This group was died at 6th week of recovery. Raising the concentration to LC25 (32.4 ppm) caused a quick and severe death among treated snails through the first 4 weeks of the experiment as their LX was 0.1, then these survived snails could not tolerate this treatment as they died by the 5th week of the experiment. Also, the present data in (Table 2) indicated that snails exposed for 24 h/week to sub lethal concentration of plant (LC0, LC10 or LC25), Table 1 Toxicity of the tested plant on B. alexandrina snails after 24 h of exposure. Tested plant
LC50 (mg/l)
LC90 (mg/l)
Slope
LCo (mg/ l)
LC10 (mg/l)
LC25 (mg/ l)
A. arvensis
37.9
48.3
1.2
3.7
27.5
32.4
38
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Table 2 Survival rate (Lx) and fecundity (Mx) of B. alexandrina snails exposed for 24 h/week to sub lethal concentrations of the aqueous leaves extract of A. arvensis for 2 weeks followed by 2 weeks of recovery. Weeks
0 1 2 3 4 5 6 7 8 RO = Σ Lx Mx Reduction %
Control
LC0 (3.7 ppm)
LC10 (27.5 ppm)
LC25 (32.4 ppm)
LX
MX
LXMX
LX
MX
LXMX
LX
MX
LXMX
LX
MX
LXMX
1.00 0.99 0.98 0.95 0.95 0.90 0.85 0.85 0.85
2.89 2.94 3.72 5.62 9.63 4.43 3.86 3.37 3.40 33.97
2.89 2.910 3.645 5.339 9.14 3.96 3.28 2.864 2.89
1.00 0.85 0.82 0.71 0.55 0.36 0.10 0.10 0 1.231**** 96.37
2.89 0.82 0.30 0.24 0.15 0.10 0 0 0
2.89 0.697 0.246 0.170 0.082 0.036 0 0 0
1.00 0.80 0.65 0.61 0.42 0.31 0.10
2.89 0.88 0.08 0 0 0 0
2.89 0.704 0.052 0 0 0 0
1.00 0.60 0.43 0.30 0.10
2.89 0.41 0 0 0
2.89 0.246 0 0 0
0.756**** 97.77
0.246**** 99.27
Table 3 Hormonal level changes (T and E) in tissue homogenate of B. alexandrina after exposure to sub lethal concentrations of tested plant (LC0, LC10 or LC25). Values are mean ± S.D. n = 4. Groups
Testesterone (u/l)
Estrogen (u/l)
Control LC0 LC10 LC25
30.7 ± 0.3 28.22 ± 0.8 23.55 ± 0.9* 22.35 ± 1.4**
60.24 58.16 57.56 43.62
± 1.3 ± 0.98 ± 0.5* ± 0.63**
Fig. 2. Histogram showing the olive tail moment (OTM) of DNA of B. alexandrina snails exposed to sub lethal concentrations of A. arvensis for 48 h was highly increased (p < 0.01 and p < 0.001) than control snails. ** = highly significant compared to control at p < 0.01. *** = very highly significant compared to control at p < 0.001.
may be used as a biomarker for a molluscicide toxicity. The present results revealed that testosterone and 17-β estradiol (T and E) levels in the gonads' tissue, were significantly decreased (p < 0.05) after exposure to sub lethal concentrations of aqueous leaves extract of A. arvensis compared with control group, indicating the disrupter effect of the aqueous leaves extract of A. arvensis. These changes were compatible with the results of Rizk et al. (2012) who found that exposing the snails to chloroform extract of H. tuberculatum caused a decrease in the level of their sex hormones. They suggested that the tested plant might contain compounds which could inhibit the biosynthetic pathways of testosterone in the treated snails and correlated these results with the decrease in the reproductive rate and fecundity. In the present work, the histological examination of the hermaphrodite gland of exposed snails to the aqueous leaves extract of A. arvensis, showed losses of connective tissues, evacuation, detachment of some gonadal cells from acini. Ova and sperms lost their normal shape and some were degenerated. These results agree with (Al-Sharkawi and Rizk, 1996), who observed gametogenic maturity acceleration in the hermaphrodite gland of B. alexandrina snails after exposure to Ammi majus dry powder plant. These findings were mirroring the obtained fecundity, egg laying results and hormonal decrease after the exposure period. The fundamental building component of all living cells is DNA, where, it regulates the production of proteins via the ‘‘Central Dogma Theory’’ (Breithaupt, 2003), therefore, it was useful to study the effect of plant molluscicides on it (Bakry, 2009). DNA damage, such as DNA single-strand breaks (SSBs) was detected by comet analysis (Grazeffe et al., 2008). The results of alkaline comet assay of the present study
Fig. 1. Light micrograph showing sections in hermaphrodite glands of snails exposed to sub-lethal concentrations of A. arvensis for two weeks (H& E) (×40): (A) normal B. alexandrina snails (B) Snails exposed to LC0 of the extract (C) Snails exposed to LC10 (D) Snails exposed to LC25.
Mohamed (2011) who attributed the reduction in egg laying of Bulinus truncatus treated with methanol extract of the plant Sesbania sesban to severe histological damages to the snail's hermaphrodite gland cells and evacuations of some of its tubules from various gametogenetic stages. Similarly, the reproductive rate and fecundity of B. alexandrina decreased after treatment with chloroform extract of the plant Haplophyllum tuberculatum (Rizk et al., 2012). These results might be due to the harmful effect of such compounds on the regulation of the oviposition (Ragheb et al., 2018) and they supported these results by the decrease in the concentrations of testosterone and estradiol in the snails' tissues. Testosterone hormone which is a vertebrate-like hormone, is functional in the gonadal development in B. alexandrina (Omran, 2012) and 39
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1
2
3
4
Fig. 3. Showing ranks of comet according to the extent of DNA migration. 1) Control; 2, 3 and 4 snails exposed to 2) LC0 (Rank: 1); 3) LC10: (Rank: 2) and 4) LC25: (Rank: 3) of tested plant.
Compliance with ethical standards
demonstrated that the level of SSBs induced by exposure to the sub lethal concentration of the aqueous leaves extract of A. arvensis was significantly higher than that in the control group. These results reflected the decrease in survival, fecundity rates and hormonal activity. This was in accordance with (Ye et al., 2012), who stated that the relative amount of DNA strand breaks were higher after exposure to a standard well known DNA damaging chemicals compared with controls. These results were in agreement with (Abdel-Haleem, 2013) who studied the effects of methanol extracts of three plants, Euphorbia splendens, Ziziphus spinachristi and Ambrosia maritime on the protein patterns of digestive gland of two vectors of schistosomiasis; B. alexandrina and Bulinus truncatus. The results showed degradation of protein and high intensity of DNA after treatment with LC90 of each plant extract. Also, (Shaldoum et al., 2016), used comet assay to confirm the presence of genotoxic effect after using cuprous oxide nanoparticles (Cu2ONPs) and found a significant increase in both; tail moment and tail length of B. alexandrina snails DNA.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors. Funding This study was not funded by any organisations. Acknowledgment The authors would like to thank prof/Shadia Mohamed El- Dafrawy and prof/Huda Abu Taleb. References Abdel-Gawad, M.M., El-Amin, S.M., Ohigashi, H., Watanabe, Y., Takeda, N., Sugiyama, H., Kawanaka, M., et al., 2000. Molluscicidal saponins from Anagallis arvensis against schistosome intermediate hosts. Jpn. J. Infect. Dis. 53, 17–19. Abdel-Ghaffar, F., Ahmed, A.K., Bakry, F., Rabei, I., Ibrahim, A., 2016. The Impact of Three Herbicides on Biological and Histological Aspects of Biomphalaria Alexandrina. Intermediate Host of Schistosoma mansoni, Malacologia. https://doi. org/10.4002/040.059.0201. Abdel-Haleem, A.A., 2013. Molluscicidal impacts of some Egyptian plant extracts on protein and DNA-contents of two snail-vectors of schistosomiasis, using electrophoresis. J. Basic Appl. Zool. 66, 34–40. Al-Sharkawi, I.M., Rizk, E.T., 1996. Comparative study on the efficacy of Ammi majus water extract in the control of Biomphalaria alexandrina, Bulinus truncatus and Lymnaea caillaudi and its lethality to some non-target species. J. Union Arab Biol. 6, 577–597. Al-Snafi, A.E., 2015. The chemical contents and pharmacological effects of Anagallis arvensis-A review. Int. J. Pharm. 5, 37–41. Azqueta, A., Shaposhnikov, S., Collins, A.R., 2009. DNA oxidation: investigating its key role in environmental mutagenesis with the comet assay. Mutat. Res. Toxicol. Environ. Mutagen 674, 101–108. Bakry, F.A., 2009. Use of some plant extracts to control Biomphalaria alexandrina snails with emphasis on some biological effects. Pestic. Biochem. Physiol. 95, 159–165. https://doi.org/10.1016/j.pestbp.2009.08.007. Breithaupt, H., 2003. DNA and consumer confidence: DNA fingerprinting and DNA-based labelling systems are gaining importance in the security market to verify the authenticity of products. EMBO Rep. 4, 232–234. Bruneton, J., 2001. Farmacognosia, Fitoquímica, Plantas Medicinales. Acribia, Zaragoza, pp. 663–712.
5. Conclusion Scarlet pimpernel, A. arvensis, is a potent molluscicidal agent and is capable of inducing significant alterations in the biological, hormonal, histopathological and genotoxicological aspects in the freshwater snail B. alexandrina. Along these lines, new studies are expected to characterize the best possible system (s) for use of such tried agents in schistosomiasis control planning to limit water contamination and sparing the non-target organisms as it is inexpensive and eco-friendly.
Declarations of interest None.
Conflicts of interest On behalf of all the authors, I do declare that we do not have any conflict of interest. 40
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Caruana, S., Yoon, G.H., Freeman, M.A., Mackie, J.A., Shinn, A.P., 2012. The efficacy of selected plant extracts and bioflavonoids in controlling infections of Saprolegnia australis (Saprolegniales; Oomycetes). Aquaculture 358, 146–154. El-Emam, M., El-Amin, S., El-Sayed, M., El-Nahas, H., 1996. Field trials to control the snail vectors of schistosomiasis and fascioliasis by the plant Anagallis arvensis (Primulaceae). Egypt. J. Bilharz. 18, 89–100. El-Ghany, A.M.A., El-Ghany, N.M.A., 2017. Molluscicidal activity of Bacillus thuringiensis strains against Biomphalaria alexandrina snails. Beni-Suef Univ. J. Basic Appl. Sci. 6 (4), 391–393. El-Gindy, M.S., Radhawy, I.A., et al., 1965. Effect of low concentrations of sodium pentachlorophenate on the fecundity and egg viability of Bulinus truncatus from Central Iraq. Bull. Endem. Dis. (Baghdad) 7, 44–54. El-Sayed, M.M., El-Nahas, H.A., Refahy, L.A., et al., 1990. A study on the molluscicidal activity of Calendula micrantha (Officinals), Anagallis arvensis and Agave filifera. Egypt. J. Bilharz. 12, 207–216. Elkhyat, H.M.M., Gawish, F., 2006. Laboratory selection for resistance to Anagallis arvensisas as a plant molluscicide in Biomphalaria alexandrina snails. Egypt. J. Nat. Toxins 3, 97–117. Elsareh, F., Abdalla, R., Abdalla, E., 2016. The effect of aqueous leaves extract of Solenostemma argel (Del Hayne) on egg masses and neonates of Biomphalaria pfeifferi snails. J. Med. Plants 4, 271–274. Finney, D.J., 1971. Probit Analysis, third ed. Cambridge University Press. Gopalan, R., Nelson, L., Thornton, J.M., Anderson, D., 2006. Effect of the sex hormones and their modulators on DNA damage in skin cells measured in the comet assay. J. Prev. Med. 14, 14–31. Grazeffe, V.S., de Freitas Tallarico, L., de Sa Pinheiro, A., Kawano, T., Suzuki, M.F., Okazaki, K., de Bragança Pereira, C.A., Nakano, E., 2008. Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818). Mutat. Res. Toxicol. Environ. Mutagen 654, 58–63. Hasheesh, W.S., Mohamed, R.T., 2011. Bioassay of two pesticides on Bulinus truncatus snails with emphasis on some biological and histological parameters. Pestic. Biochem. Physiol. 100, 1–6. Hontela, A., 1998. Interrenal dysfunction in fish from contaminated sites: in vivo and in vitro assessment. Environ. Toxicol. Chem. 17, 44–48. https://doi.org/10.1002/etc. 5620170107. Ibrahim, A., Al-Zahaby, A.S., Shoeb, H., El Emam, M., AbdelKhalek, H., 1994. Molluscicidal activity of Anagallis arvensis, Agave lophantha and Bassia muricata plants against survival and fecundity of Biomphalaria alexandrina snails. Bull. Fac. Sci. Zagazig Univ. 16, 645–662. Ibrahim, A.M., Abdalla, A.M., 2017. Impact of Moringa oleifera seed aqueous extract on some biological, biochemical, and histological aspects of Biomphalaria alexandrina snails. Environ. Sci. Pollut. Res. 24. https://doi.org/10.1007/s11356-017-0397-0. Ibrahim, A.M., El-Emam, M.A., El-Dafrawy, S.M., Mossalem, H.S., 2004. Impact of certain plant species on Schistosoma mansoni Biomphalaria alexandrina system. In: Proceeding 3rd International Conference Science, pp. 390–413. Janer, G., Porte, C., 2007. Sex steroids and potential mechanisms of non-genomic endocrine disruption in invertebrates. Ecotoxicology 16, 145–160. Kamel, E.G., Refaat, S.W., El-Dafrawy, S.M., Mohamed, A.H., Mossalem, H.S., 2007. Toxicological effect of certain plants and synthetic molluscicides on ultra structural changes in haemocytes of Biomphalaria alexandrina. Egypt. J. Exper. Biol. (Zoology) 3, 135. Khoshkholgh-Pahlaviani, M.R.M., Massiha, A.R., Issazadeh, K., Bidarigh, S., Giahi, M., Ramtin, M., 2013. Evaluation of antifungal activity of methanol extract of Acacia (Anagalis arvensis) leaves and Nystatin against Candida Albicans in vitro. Zahedan J. Res. Med. Sci. 15. Kiros, G., Erko, B., Giday, M., Mekonnen, Y., 2014. Laboratory assessment of molluscicidal and cercariacidal effects of Glinus lotoides fruits. BMC Res. Notes 7, 1. Lee, R.F., Steinert, S., 2003. Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutat. Res. Mutat. Res. 544, 43–64. Litchfield, J.T. am, Wilcoxon, F., 1949. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Therapeut. 96, 99–113. López, V., Jäger, A.K., Akerreta, S., Cavero, R.Y., Calvo, M.I., 2011. Pharmacological properties of Anagallis arvensis L. (“scarlet pimpernel”) and Anagallis foemina Mill.
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Molluscicidal impacts of Anagallis arvensis aqueous extract on biological, hormonal, histological and molecular aspects of Biomphalaria alexandrina snails
T
Amina M. Ibrahim∗, Samah I. Ghoname Environmental Research and Medical Malacology Department, Theodor Bilharz Research Institute, Giza, Egypt
A R T I C LE I N FO
A B S T R A C T
Keywords: Biomphalaria alexandrina Anagallis arvensis Biological Histopathology Hormones activity Comet assay
Controlling of Biomphalaria alexandrina snails by plant molluscicides is the cornerstone in treating schistosomiasis in Egypt. The objective of this study is, to evaluate the molluscicidal activity of the aqueous leaves extract of Anagallis arvensis against B. alexandrina snails. The present results showed that this aqueous extract was lethal for B. alexandrina snails at (LC50 37.9 mg/l; LC90 48.3 mg/l), and caused reduction in survival; reproductive rates and hormonal activity (testosterone (T) and 17β-estradiol (E)) of these snails. Histopathological changes occurred in the hermaphrodite glands of snails exposed to the sub lethal concentrations of this aqueous extract are detected, where, there were degeneration in both eggs and sperms and there were losses of connective tissues between acini. The present investigation revealed that this plant had a genotoxic effect especially with its concentration (LC10 and LC25), where, the length of olive tail moment was significantly increased than control group. These observations prove the potent molluscicidal activity of aqueous leaves extract of A. arvensis against the intermediate hosts of Schistosoma mansoni and provide natural biodegradable resources for snails' molluscicidal agents.
1. Introduction Schistosomiasis is a widespread neglected tropical parasitic disease transmitted by snails (WHO, 2017). Freshwater snails of Biomphalaria genus are the intermediate hosts of Schistosoma mansoni in Egypt (Ibrahim and Abdalla, 2017) and several strategies have been used to control snail populations through breaking the life cycle (El-Ghany and El-Ghany, 2017). Manufactured molluscicides is an imperative part in the incorporated schistosomiasis control programs (Abdel-Ghaffar et al., 2016), but because they have high cost and being poisonous to creatures of land and water (WHO, 2014), have stimulated interest to find suitable plant molluscicides (Elsareh et al., 2016). Plant molluscicides are promising choices that may grow the scope of molluscicides accessible for controlling of B. alexandrina snails (Kiros et al., 2014), as these plants are cheaper, safer and having a high level of degradability (Salawu and Odaibo, 2011). A. arvensis is the name of Scarlet Pimpernel, with about 20–25 species of flowering plants in the family Myrsinaceae (Khoshkholgh-Pahlaviani et al., 2013). It is used in European traditional medicine for dermatological purposes (López et al., 2011). This plant is toxic to ruminants (Pande et al., 2016) at high doses and during long-term consumption (López et al., 2011). The bio
∗
active constituents of this plant have antibacterial, antifungal, antitumor, antidiabetic and hepatoprotective activities (Bruneton, 2001) and were found to be less toxic through the toxicity prediction tool. Also, A. arvensis has no toxicity on humans as it is used to treat diseases like Gout, Leprosy, Epilepsy and Urinary infection (Pande et al., 2016). In Aquaculture, studies aimed to find a plant-based remedy for Saprolegniasis (Caruana et al., 2012), which is a pathogen causing “water mold” threatens the fresh-water breeding facilities by infecting fish eggs and fry and is caused by Saprolegnia parasitica. In recent study by Wirth and Stadtlander (2016), they tested the alcoholic and aqueous extracts of 29 plants that had antimicrobial properties in vitro against S. parasitica, they found that aqueous extracts were ineffective and that the concentration of 5000 ppm 70% ethanol extracts of Scarlet Pimpernel (A. arvensis) and rosemary (Rosmarinus officinalis) showed fungistatic effects in the first 24 h. A. arvensis is considered one of the most promising molluscicidal plants and its saponin fraction has been reported to express high molluscicidal activity against schistosome intermediate snails (AbdelGawad et al., 2000; El-Sayed et al., 1990). The molluscicidal and cercaricidal properties of this plant are found in all its parts and the use of water suspension of its powder is an inexpensive means of controlling
Corresponding author. Environmental Research and Medical Malacology Department, Theodor Bilharz Research Institute, Imbaba, Giza, Egypt. E-mail address: [email protected] (A.M. Ibrahim).
https://doi.org/10.1016/j.exppara.2018.07.014 Received 7 December 2017; Received in revised form 2 June 2018; Accepted 20 July 2018 Available online 21 July 2018 0014-4894/ © 2018 Elsevier Inc. All rights reserved.
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L) and covered with glass plates. Oven dried lettuce leaves and blue green algae (Nostoc muscorum) were used for feeding and water in the aquaria was changed weekly. Pieces of polyethylene sheets were put into the aquaria to collect egg masses.
both snails and schistosome free larval stages (Elkhyat and Gawish, 2006). (Ibrahim et al., 1994) stated that the LC90 of the dry powder of three local plants namely Anagallis arvensis, Agave lophantha and Bassia muruicata tested in the laboratory against B. alexandrina snails were 50,100 and 165 ppm, respectively. In two field trials carried out in Sharkia Governorate, Egypt (ElEmam et al., 1996), used relatively high concentrations of dry powder of A. arvensis to control vector snails of schistosomiasis and fascioliasis and used the concentrations of 125 and 100 ppm to induce death of B. alexandrina snails. Also (Mosta-Fa et al., 2005), carried out semi-field trials under stimulated natural conditions to evaluate different modes of exposure to A. arvensis and Calendula micrantha as plant molluscicides and chemical molluscicides like baylucide and they found that the pre exposure to sub lethal concentrations of A. arvensis (55 ppm) increases the snail mortality in all experiments of bayluscide and A. arvensis than the other plant. The biological response of an organism to xenobiotics starts with toxicant induced changes at the cellular and biochemical levels, leading to changes in the structure and function of the cells, tissues, physiology and behavior of the organism and these changes may affect the integrity of the population and ecosystem (Parvez and Raisuddin, 2005). The endocrine system regulates hormone-dependent physiologic functions necessary for survival of the organism and the species (Hontela, 1998). An endocrine disruptor is an exogenous substance that affects the function of the endocrine system and causes deleterious effects in an organism, or its progeny (WHO, 2002). Some substances either of natural or man-made can cause endocrine disruption both in vitro and in vivo (Schug et al., 2011). The degree of this disruption depends on some parameters including reproductive stage dependent changes in steroid action, and whether the steroid action is genomic or non-genomic (Thomas, 2000). The endocrine disruptions in terms of steroid levels (testosterone (T) and 17b-estradiol (E)) can be studied in B. alexandrina snail (Omran and Salama, 2016), because their hormonal system is comparable to that of vertebrates (Janer and Porte, 2007; Oehlmann et al., 2007). Certain compounds act as endocrine disrupters either by binding to the hormone receptors or modulating it, or by modulating endogenous hormone levels through interfering with biochemical processes associated with the production, availability, or metabolism of hormones (Oetken et al., 2004). Testosterone and estrogen hormones have an important role in the development of gonads in B. alexandrina (Omran, 2012). These snails can be used as a bio indicator for endocrine disrupters in terms of steroid levels (testosterone (T) and 17b-estradiol (E)), after exposure to sub lethal concentrations of any molluscicides (Omran and Salama, 2016). DNA damage produced by the androgens, testosterone (TES) and β-oestradiol in keratinocyte cell line can be examined by the Comet assay (Gopalan et al., 2006). Comet assay is a sensitive method for direct visualization of DNA damage on the level of a single cell (Azqueta et al., 2009). Some recent studies link DNA strand breaks in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics (Lee and Steinert, 2003; Shaldoum et al., 2016). So, in the current study, molluscicidal effects of A. arvensis plants were determined to study how it affects the biological system of B. alexandrina snails through evaluating its effects on biological, hormonal, histological and molecular parameters of these snails.
2.2. Plant materials A. arvensis (Family Myrsinaceae) plants were collected from the shores of water courses in Aboelkhawy, El-beheira Governorate, Egypt during the spring of 2016. It was identified by Prof. Dr. Shadia Mohamed El- Dafrawy, Theodor Bilharz Research Institute, Giza, Egypt. The plants were shade dried, finely powdered using an electrical grinder. The dry powder of the experimental plants was weighed and added directly to filtered water in individual beakers. 2.3. Molluscicidal screening To calculate LC50 and LC90, series of concentrations were prepared on the basis of weight/volume as water extract of leaves powder (50, 40, 30, 20, 10 mg/l) and ten B. alexandrina snails (8–10 mm) were placed in beakers for each concentration (Litchfield and Wilcoxon, 1949). Another snail group of the same size was dipped in dechlorinated water only as control. Three replicates were used, each of 10 snails, for each concentration. The exposure period was 24 h, then, the snails were removed from the experimental test solution, and washed thoroughly with dechlorinated tap water and transferred to containers with fresh dechlorinated tap water for another 24 h of recovery, and then, the percentages of observed mortalities were recorded. LC0 is the concentration of a toxicant, below which no measurable effects take place (Warren, 1900), and is estimated as 1/10 LC50 value (WHO, 1965). 2.3.1. Effect on snails' egg-laying capacity of adult snails B. alexandrina snails (8–10 mm) were exposed for 24 h/day for 2 weeks to the concentrations LC0, LC10, and LC25 of the herbicide. Three replicates, each of 10 snails/L, were prepared for each concentration, another group considered as control group was maintained in dechlorinated water, the following parameters were weekly recorded: Lx (the survival rate as a proportion of the correct one), Mx (the number of eggs/snail/week) and R0 (the reproductive rate which is the summation of LxMx during the experimental period) (El-Gindy et al., 1965). 2.3.2. Biochemical assays for steroid hormones Ten snails (8–10 mm) were subjected to each sub lethal concentrations (LC0, LC10 and LC25) of the tested plant for 24 h (exposure), followed by another 24 h of recovery for two weeks. Three replicates of each concentration were prepared. Unexposed snails (control) were assayed side by side with the experimented groups. Snail's shell was gently crushed between two glass slides and digestive gland was dissected out and pooled in 1 ml Eppendorf tube. The tissues of snails from each group were weighed and then homogenized in ice cold, twicedistilled water (1 g tissue/5 ml water) using a glass Dounce homogenizer. The homogenates were centrifuged at 3000 rpm for 10 min at 4 °C and the supernatants were stored at −80 C until used. Hormone concentrations (testosterone (T) and 17b-estradiol (E)) were assayed according to the manufacture instructions of T EIA kit (Enzo Life Science, Michigan, USA, ADI-900-065) and E EIA kit (Cayman Chemical Company, Michigan, USA, item no. 582251).
2. Materials and methods 2.3.3. Histological study Adult snails (8–10 mm) were exposed to the aqueous extract of plant leaves at (LC0, LC10 or LC25) for 24 hours/week for 2 successive weeks (exposure), then, the snails were removed from the experimental test solution, and washed thoroughly with dechlorinated tap water, and transferred to containers with fresh dechlorinated tap water for another 24 h of recovery, and this was done for two weeks. Changes in histology
2.1. Experimental animals (snails) B. alexandrina snails (8–10 mm) provided from Medical Malacology Laboratory, Theodor Bilharz Research Institute (TBRI), Giza, Egypt were used. Snails were kept in plastic aquaria (16 × 23 × 9 cm). The aquaria were provided with dechlorinated aerated tap water (10 snails/ 37
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2.3.4. Comet assay Snails (8–10 mm) were subjected to LC0, LC10 and LC25 of the aqueous extract of plant leaves for 48 h. The hemolymphs of 10 snails from each group were collected by removing a small portion of the shell and inserting a capillary tube into the heart. Then hemolymph was pooled in a glass vial tube (1.5 ml) and stored at −80 C° until used and then DNA damage was measured by single cell gel assay (Grazeffe et al., 2008; Singh et al., 1988) as follows: Hemocytes suspensions are harvested by centrifugation and re-suspend cells at 1 × 105 cells/ml in ice cold 1X PBS (phosphate buffer saline). This suspension was stirred for 5 min and filtered, then, 100 μl of this cell suspension was mixed with 600 μl of low-melting agarose (0.8% in PBS). 100 μl of this mixture was spread on pre-coated slides. The coated slides were immersed in lyses buffer (0.045 M TBE (Tris/ Borate/EDTA)), pH 8.4, containing 2.5% SDS (sodium dodecyl sulphate) for 15 min. The slides were placed in the electrophoresis chamber containing the same TBE buffer, but devoid of SDS. The electrophoresis conditions were 2 V/cm for 2min and 100 mA, staining with ethidium bromide 20 μg/ml at 4 °C. The DNA fragment migration patterns of 100 cells for each dose level were evaluated with a fluorescence microscope at 510 nm. The comets tails lengths were measured from the middle of the nucleus to the end of the tail with 40× increase for the count and measure the size of the comet. For visualization of DNA damage, observations are made of EtBr-stained (Ethidium Bromide Staining) DNA using a 40× objective on a fluorescent microscope. Slides were coded independently and scored blindly.
negatively affected their fecundity (MX), i.e. the snails laid few eggs/ snail/week through the weeks of the experiment in comparison with that of control ones. Regarding the reproductive (Ro) of treated snails, it was extremely highly suppressed (p < 0.001) than control group. The percent of reduction is increased with increasing the sub lethal concentration being 99.27% at LC25. Testosterone (T) and 17β Estradiol (E) were determined in the tissue homogenate of exposed snails after 2 weeks of exposure. Levels of T and E were decreased significantly (p < 0.05) after exposure to sub lethal concentrations (LC10 and LC25) compared with control (Table 3). Histopathological effects were detected by light microscopy, where, the normal hermaphrodite gland of B. alexandrina comprised of the male reproductive cells and the female oogenic cells (Fig. 1. A). Exposing of snails to LC0 sub lethal concentration, caused a moderate effect such degeneration of some mature ova (Fig. 1. B). While, exposure of snails to LC10, showed degeneration and destruction in sperms and degeneration in eggs (Fig. 1. C), the great damage in gonadal cells occurred at LC25, where, eggs lost their shapes and degenerated. Sperms were degenerated and the connective tissue was dissolved and replaced by vacuoles (Fig. 1. D). The present results of alkaline comet assay demonstrated that the olive tail moment (OTM) of snails exposed for 48 h to sub lethal concentrations of the aqueous leaves extract of A. arvensis was highly increased (p < 0.01) (Fig. 2.) than control snails. Comets were classified and assigned to four categories (0–3) according to the extent of DNA migration. Fig. (3) shows that in snails exposed to LC10 of the aqueous leaves extract, took rank 2 (intermediate DNA migration), while, at LC25 took rank 3 (high DNA migration).
2.4. Statistical analysis
4. Discussion
Lethal concentration values were defined by Probit analysis (Finney, 1971) and analysis of data was carried out by Student's t-test for comparing the means of experimental and control groups (Murray, 1981). Values were expressed as mean ± S.D., and the obtained data were analyzed using the Graph Pad Prism 6.04 software for Windows (Graph Pad Software, San Diego, California, U.S.A.; 1992–2014). A value of (P < 0.01) was considered significant.
The high cost and toxicity to non-target organisms of chemical molluscicides, have drawn much attention during recent years for the use of plant molluscicides (WHO, 2017). A. arvensis is one of the most promising molluscicidal plants and its saponin fraction has been reported to express high molluscicidal activity against schistosome intermediate hosts (Abdel-Gawad et al., 2000). The median lethal concentration (LC50) for any molluscicidal material must not exceed (100 ppm) (WHO, 1993). The present results showed the lethal concentrations LC50 and LC90 of the aqueous leaves extract of A. arvensis on B. alexandrina snails after 24 h of exposure were 37.9 and 48.3 mg/l, respectively. These outcomes are in congruity with the past investigation of Kamel et al. (2007), who, stated that the water suspension of A. arvensis plants has a molluscicidal activity against B. alexandrina snails. Also (Al-Snafi, 2015), confirmed that this plant has a molluscicidal activities. This molluscicidal activity is due to presence of two compounds called desgluco-anagalloside B and anagalloside B and their activities are comparable to that of the synthetic molluscicide, niclosamide (Abdel-Gawad et al., 2000). The present results showed that survival rates of adult B. alexandrina snails were markedly reduced post their exposure to sub lethal concentrations (LC0, LC10 or LC25) of aqueous leaves extract of A. arvensis. Comparable perceptions were recorded by Ibrahim and Abdalla (2017) who showed that survival rates of adult B. alexandrina snails were markedly reduced post their exposure to sub lethal concentrations of the aqueous seed extract of moringa oleifera and they linked this reduction by deformation in the digestive and hermaphrodite gland cells of the treated snails. Also, similar results were obtained after using the plants Panicum repens and Solanum nigrum against B. alexandrina snails (Ibrahim et al., 2004). The reproductive rate (R0) and fecundity (Mx) of adult B. alexandrina snails in the present study were significantly decreased by their exposure to the sub lethal concentrations (LC0, LC10 or LC25) of the aqueous leaves extract of A. arvensis, in comparison with the control group. Comparable perceptions were recorded by Hasheesh and
of hermaphrodite gland of treated snails compared with control snails were done according to Mohamed and Saad (1990).
3. Results From (Table 1), the calculated lethal concentrations (LC50 and LC90) on B. alexandrina snails after 24 h of exposure for the aqueous leaves extract of A. arvensis were 37.9 and 48.3 mg/l, respectively. Table 2 indicated that the survival rate of snails exposed the aqueous leaves extract of A. arvensis to LC0 (3.7 ppm) was slightly affected, being 0.55 at the 4th week of exposure. Thereafter, through the recovery four weeks, the snails survived till the end of the experiment. While, exposure of snails to LC10 (27.5 ppm) considerably reduced their survival rate (Lx) to be 0.42 at the 4th week of exposure compared to 0.95 for the control group. This group was died at 6th week of recovery. Raising the concentration to LC25 (32.4 ppm) caused a quick and severe death among treated snails through the first 4 weeks of the experiment as their LX was 0.1, then these survived snails could not tolerate this treatment as they died by the 5th week of the experiment. Also, the present data in (Table 2) indicated that snails exposed for 24 h/week to sub lethal concentration of plant (LC0, LC10 or LC25), Table 1 Toxicity of the tested plant on B. alexandrina snails after 24 h of exposure. Tested plant
LC50 (mg/l)
LC90 (mg/l)
Slope
LCo (mg/ l)
LC10 (mg/l)
LC25 (mg/ l)
A. arvensis
37.9
48.3
1.2
3.7
27.5
32.4
38
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Table 2 Survival rate (Lx) and fecundity (Mx) of B. alexandrina snails exposed for 24 h/week to sub lethal concentrations of the aqueous leaves extract of A. arvensis for 2 weeks followed by 2 weeks of recovery. Weeks
0 1 2 3 4 5 6 7 8 RO = Σ Lx Mx Reduction %
Control
LC0 (3.7 ppm)
LC10 (27.5 ppm)
LC25 (32.4 ppm)
LX
MX
LXMX
LX
MX
LXMX
LX
MX
LXMX
LX
MX
LXMX
1.00 0.99 0.98 0.95 0.95 0.90 0.85 0.85 0.85
2.89 2.94 3.72 5.62 9.63 4.43 3.86 3.37 3.40 33.97
2.89 2.910 3.645 5.339 9.14 3.96 3.28 2.864 2.89
1.00 0.85 0.82 0.71 0.55 0.36 0.10 0.10 0 1.231**** 96.37
2.89 0.82 0.30 0.24 0.15 0.10 0 0 0
2.89 0.697 0.246 0.170 0.082 0.036 0 0 0
1.00 0.80 0.65 0.61 0.42 0.31 0.10
2.89 0.88 0.08 0 0 0 0
2.89 0.704 0.052 0 0 0 0
1.00 0.60 0.43 0.30 0.10
2.89 0.41 0 0 0
2.89 0.246 0 0 0
0.756**** 97.77
0.246**** 99.27
Table 3 Hormonal level changes (T and E) in tissue homogenate of B. alexandrina after exposure to sub lethal concentrations of tested plant (LC0, LC10 or LC25). Values are mean ± S.D. n = 4. Groups
Testesterone (u/l)
Estrogen (u/l)
Control LC0 LC10 LC25
30.7 ± 0.3 28.22 ± 0.8 23.55 ± 0.9* 22.35 ± 1.4**
60.24 58.16 57.56 43.62
± 1.3 ± 0.98 ± 0.5* ± 0.63**
Fig. 2. Histogram showing the olive tail moment (OTM) of DNA of B. alexandrina snails exposed to sub lethal concentrations of A. arvensis for 48 h was highly increased (p < 0.01 and p < 0.001) than control snails. ** = highly significant compared to control at p < 0.01. *** = very highly significant compared to control at p < 0.001.
may be used as a biomarker for a molluscicide toxicity. The present results revealed that testosterone and 17-β estradiol (T and E) levels in the gonads' tissue, were significantly decreased (p < 0.05) after exposure to sub lethal concentrations of aqueous leaves extract of A. arvensis compared with control group, indicating the disrupter effect of the aqueous leaves extract of A. arvensis. These changes were compatible with the results of Rizk et al. (2012) who found that exposing the snails to chloroform extract of H. tuberculatum caused a decrease in the level of their sex hormones. They suggested that the tested plant might contain compounds which could inhibit the biosynthetic pathways of testosterone in the treated snails and correlated these results with the decrease in the reproductive rate and fecundity. In the present work, the histological examination of the hermaphrodite gland of exposed snails to the aqueous leaves extract of A. arvensis, showed losses of connective tissues, evacuation, detachment of some gonadal cells from acini. Ova and sperms lost their normal shape and some were degenerated. These results agree with (Al-Sharkawi and Rizk, 1996), who observed gametogenic maturity acceleration in the hermaphrodite gland of B. alexandrina snails after exposure to Ammi majus dry powder plant. These findings were mirroring the obtained fecundity, egg laying results and hormonal decrease after the exposure period. The fundamental building component of all living cells is DNA, where, it regulates the production of proteins via the ‘‘Central Dogma Theory’’ (Breithaupt, 2003), therefore, it was useful to study the effect of plant molluscicides on it (Bakry, 2009). DNA damage, such as DNA single-strand breaks (SSBs) was detected by comet analysis (Grazeffe et al., 2008). The results of alkaline comet assay of the present study
Fig. 1. Light micrograph showing sections in hermaphrodite glands of snails exposed to sub-lethal concentrations of A. arvensis for two weeks (H& E) (×40): (A) normal B. alexandrina snails (B) Snails exposed to LC0 of the extract (C) Snails exposed to LC10 (D) Snails exposed to LC25.
Mohamed (2011) who attributed the reduction in egg laying of Bulinus truncatus treated with methanol extract of the plant Sesbania sesban to severe histological damages to the snail's hermaphrodite gland cells and evacuations of some of its tubules from various gametogenetic stages. Similarly, the reproductive rate and fecundity of B. alexandrina decreased after treatment with chloroform extract of the plant Haplophyllum tuberculatum (Rizk et al., 2012). These results might be due to the harmful effect of such compounds on the regulation of the oviposition (Ragheb et al., 2018) and they supported these results by the decrease in the concentrations of testosterone and estradiol in the snails' tissues. Testosterone hormone which is a vertebrate-like hormone, is functional in the gonadal development in B. alexandrina (Omran, 2012) and 39
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A.M. Ibrahim, S.I. Ghoname
1
2
3
4
Fig. 3. Showing ranks of comet according to the extent of DNA migration. 1) Control; 2, 3 and 4 snails exposed to 2) LC0 (Rank: 1); 3) LC10: (Rank: 2) and 4) LC25: (Rank: 3) of tested plant.
Compliance with ethical standards
demonstrated that the level of SSBs induced by exposure to the sub lethal concentration of the aqueous leaves extract of A. arvensis was significantly higher than that in the control group. These results reflected the decrease in survival, fecundity rates and hormonal activity. This was in accordance with (Ye et al., 2012), who stated that the relative amount of DNA strand breaks were higher after exposure to a standard well known DNA damaging chemicals compared with controls. These results were in agreement with (Abdel-Haleem, 2013) who studied the effects of methanol extracts of three plants, Euphorbia splendens, Ziziphus spinachristi and Ambrosia maritime on the protein patterns of digestive gland of two vectors of schistosomiasis; B. alexandrina and Bulinus truncatus. The results showed degradation of protein and high intensity of DNA after treatment with LC90 of each plant extract. Also, (Shaldoum et al., 2016), used comet assay to confirm the presence of genotoxic effect after using cuprous oxide nanoparticles (Cu2ONPs) and found a significant increase in both; tail moment and tail length of B. alexandrina snails DNA.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors. Funding This study was not funded by any organisations. Acknowledgment The authors would like to thank prof/Shadia Mohamed El- Dafrawy and prof/Huda Abu Taleb. References Abdel-Gawad, M.M., El-Amin, S.M., Ohigashi, H., Watanabe, Y., Takeda, N., Sugiyama, H., Kawanaka, M., et al., 2000. Molluscicidal saponins from Anagallis arvensis against schistosome intermediate hosts. Jpn. J. Infect. Dis. 53, 17–19. Abdel-Ghaffar, F., Ahmed, A.K., Bakry, F., Rabei, I., Ibrahim, A., 2016. The Impact of Three Herbicides on Biological and Histological Aspects of Biomphalaria Alexandrina. Intermediate Host of Schistosoma mansoni, Malacologia. https://doi. org/10.4002/040.059.0201. Abdel-Haleem, A.A., 2013. Molluscicidal impacts of some Egyptian plant extracts on protein and DNA-contents of two snail-vectors of schistosomiasis, using electrophoresis. J. Basic Appl. Zool. 66, 34–40. Al-Sharkawi, I.M., Rizk, E.T., 1996. Comparative study on the efficacy of Ammi majus water extract in the control of Biomphalaria alexandrina, Bulinus truncatus and Lymnaea caillaudi and its lethality to some non-target species. J. Union Arab Biol. 6, 577–597. Al-Snafi, A.E., 2015. The chemical contents and pharmacological effects of Anagallis arvensis-A review. Int. J. Pharm. 5, 37–41. Azqueta, A., Shaposhnikov, S., Collins, A.R., 2009. DNA oxidation: investigating its key role in environmental mutagenesis with the comet assay. Mutat. Res. Toxicol. Environ. Mutagen 674, 101–108. Bakry, F.A., 2009. Use of some plant extracts to control Biomphalaria alexandrina snails with emphasis on some biological effects. Pestic. Biochem. Physiol. 95, 159–165. https://doi.org/10.1016/j.pestbp.2009.08.007. Breithaupt, H., 2003. DNA and consumer confidence: DNA fingerprinting and DNA-based labelling systems are gaining importance in the security market to verify the authenticity of products. EMBO Rep. 4, 232–234. Bruneton, J., 2001. Farmacognosia, Fitoquímica, Plantas Medicinales. Acribia, Zaragoza, pp. 663–712.
5. Conclusion Scarlet pimpernel, A. arvensis, is a potent molluscicidal agent and is capable of inducing significant alterations in the biological, hormonal, histopathological and genotoxicological aspects in the freshwater snail B. alexandrina. Along these lines, new studies are expected to characterize the best possible system (s) for use of such tried agents in schistosomiasis control planning to limit water contamination and sparing the non-target organisms as it is inexpensive and eco-friendly.
Declarations of interest None.
Conflicts of interest On behalf of all the authors, I do declare that we do not have any conflict of interest. 40
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