Biocontrol potential of Arabidopsis thaliana against Biomphalaria glabrata, the intermediate host of Schistosoma mansoni, focusing on biological, physiological, histopathological, and genotoxic impacts

Abstract

Controlling Biomphalaria species with plant products is crucial in the treatment of schistosomiasis since it is eco-friendly and generally less harmful to non-target organisms. The goal of this study was to determine the molluscicidal activity of an aqueous extract of Arabidopsis thaliana leaves against Biomphalaria glabrata. A preliminary phytochemical screening test revealed the presence of flavonoids, terpenoids, steroids, anthraquinones, alkaloids, saponins, tannins, and carbohydrates. Following exposure to sub-lethal concentrations (LC₁₀ 75.62 mg/L and LC₂₅ 90.52 mg/L) of aqueous extract of A. thaliana, a reduction in survival, reproductive, and fecundity rates of B. glabrata were detected, as well as a substantial decrease in GSH, CAT, and SOD, while increasing MDA and NO levels. In addition, there was an increase in liver and renal functions as well as lipid profiles, compared to the control group. Histopathological examination of the digestive gland of treated B. glabrata showed a shrinkage of the tubules and an increase in the inter-tubular spaces as well as degeneration in the oocytes and spermatocytes of the hermaphrodite glands with a loss of connective tissues between the acini. Also, the comet assay revealed a genotoxic effect of aqueous extract of A. thaliana on B. glabrata, with a significant increase in the tail moment, tail length, and DNA percentage reflecting DNA damage compared to the control group. Conclusively, Arabidopsis thaliana aqueous extract acts as a natural molluscicidal agent against Biomphalaria glabrata.

Graphical abstract

1. Introduction

Schistosomiasis is a parasitic disease that is thought to be the most prevalent cause of morbidity globally and the infection is widespread in the tropics and subtropics, with considerable morbidity in parts of the Middle East, South America, Southeast Asia, and sub-Saharan Africa. It is also linked to poverty, leading to chronic ill health (Cando et al., 2022). It is brought on by helminths belonging to the genus Schistosoma. The prevalence rate of S. mansoni and S. haematobium is still high in endemic areas affecting 250 million people in 78 countries (LoVerde, 2019; El-Kassas et al., 2024; Wang et al., 2024), and Africans represent 85 % of the global infected population (Kappo and Okosun, 2017). All Schistosoma species have similar life cycles, requiring freshwater snails as intermediate hosts and infecting mammalian hosts through contact with free-swimming larvae released by the snails via waterborne transmission (Selbach et al., 2016). Biomphalaria glabrata (Say 1818) is widely distributed and easily infected by all strains of S. mansoni resulting in the release of several cercariae into the aquatic environment (Carvalho et al., 2018; McManus et al., 2018).

Chemical and biological control are among the numerous strategies that have been implemented to regulate the intermediate host and reduce transmission of schistosomiasis (Omobhude et al., 2017; Ibrahim, 2018). Chemical pest control has a detrimental effect on non-target organisms that can be transmitted to humans through the food chain (Barathinivas et al., 2022). Furthermore, they are costly and do not decompose in the environment (Ibrahim et al., 2022a, Ibrahim et al., 2022b; WHO, 2023). Consequently, recent research has focused on the development of an alternative to chemical molluscicides (Ibrahim and Ghoname, 2018).

Plant extracts, lectins, and essential oils (EOs) are thought to be significant sources of novel bioactive constituents such as saponins, flavonoids, and terpenoids (Nicoletti et al., 2016; Zulfiqar et al., 2017). These components are presently being evaluated for their antibacterial, fungicidal, herbicidal, molluscicidal, and pesticidal properties making them promising options for the management of schistosomiasis (Gomes et al., 2019; Abdel-Khalek et al., 2025).

Arabidopsis thaliana (Johannes Thal) is a dicotyledonous plant that is small and produces annual white flowers, also known as rockcress or thale cress, often considered to be a weed where it is found across Europe, Asia and Africa. It is a member of the Brassicaceae family, which comprises several important cultivated species such as cabbage, mustard and radish. Arabidopsis thaliana is easy to look after compared with animal model organisms. It grows quickly produces many small seeds with a small genome ∼114.5 Mb and is genetically well characterized, and life development cycle of roughly 6 weeks (Filipski and Kumar, 2005), and is widely used in the fields of plant science, genetics, and evolution and has helped further our understanding of germination and aspects of plant growth that are important in commercial crops (Hasegawa et al., 2016; Saeidfirozeh et al., 2018).

It has no agronomic value but significantly benefits fundamental genetic and molecular biology research (Taha et al., 2016). Previous studies have demonstrated that A. thaliana has even become a model organism for the study of the biochemical and molecular processes involved in human and is essential for the treatment of neurological disorders (Nemri, 2010). Also, Bömer et al. (2021) stated that the leaf of the non-medicinal plant Arabidopsis thaliana inhibiting cell growth in human breast cancer. Additionally, Taha et al. (2016) revealed the hypoglycemic and antioxidant effects of A. thaliana. Thus, the current study aimed to determine the molluscicidal properties of the aqueous extract of A. thaliana leaves against B. glabrata by evaluating its biological, biochemical, histopathological, and genotoxic alterations.

2. Materials and methods

2.1. Preparation of aqueous extract of A. thaliana

The leaves of A. thaliana were obtained from the Northwestern coast of Marsa Matrouh Governorate, Egypt in December 2024. It was identified by Prof. Dr. Heba Ahmed, Theodor Bilharz Research Institute, Giza, Egypt. The plants were shade-dried and finely powdered with an electric grinder. A stock solution of 5 % (5 g of leaf powder in 100 ml of distilled water) was soaked for 24 h in a flask. The mixtures were filtered, and the resulting extract was used for further analyses.

2.2. Phytochemical screening tests

Preliminary qualitative phytochemical screening tests were performed to determine the presence of various phytoconstituents, including alkaloids by Mayer's and Draggendorff's tests (Waldi, 1965), flavonoids (Shinoda, aluminum chloride, and potassium hydroxide tests), steroids and terpenoids (Salkowski and Libarman-Burchard's tests), and tannins (ferric chloride and gelatin tests) (Harborne, 1974). Saponins were tested using foaming and hemolytic tests (Elumalai et al., 2013), anthraquinones (Borntrager's test), carbohydrates (Molisch's and Barfoed's tests), and coumarins (Sodium hydroxide test) as described by Gul et al. (2017). The outcomes were measured visually as a change in hue or precipitation (Edeoga et al., 2005).

2.3. Experimental snails

Biomphalaria glabrata snails (8–10 mm long) were obtained from the Medical Malacology Laboratory of the Theodor Bilharz Research Institute (TBRI). The animals (10 snails / l) were housed in plastic aquaria (16 × 23 × 9 cm) with dechlorinated water (pH 7–7.5) at a temperature of 25 ± 2 °C, and lighting was provided by ceiling fluorescent lamps with a power of 80 W. Snails were fed oven-dried lettuce, blue-green algae (Nostoc muscorum), and dry flakes (TetraMin, Hannover, Germany), and aquaria were covered with glass plates.

2.4. Molluscicidal screening

A series of concentrations of aqueous extract of A. thaliana leaves powder were prepared (70, 50, 40, 30, and 20 mg/l) from stock solutions (1000 mg/l) to calculate LC₅₀ and LC₉₀ (Litchfield Jr and Wilcoxon, 1949). Three replicates were used, each of 10 snails; for each concentration, another group of snails was dipped in dechlorinated water as a control. Snails were subjected to the tested concentrations for 24 h; then they were removed and rinsed thoroughly with dechlorinated tap water and transferred to containers with fresh dechlorinated tap water for another 24 h of recovery (WHO, 1965). The sub-lethal concentrations were determined using probit analysis, and the percentages observed snail mortalities were recorded (Finney, 1971).

2.5. Effect of A. thaliana extract on the egg-laying capacity of B. glabrata

The snails (8–10 mm) were subjected to the sub-lethal concentrations (LC₀ (10.7, LC₁₀ 75.62, and LC₂₅ 90.52 mg/l) of the aqueous extract for 24 h/ week, followed by 6 days of recovery in clean dechlorinated water for four consecutive weeks. Three replicates of 10 snails/l for each concentration were prepared, while another set of snails served as a control in dechlorinated water. Water changed twice a week, and dead snails were eliminated every day. According to Chernin and Michelson (1957), the shell diameter was determined on a weekly basis using a caliper with a dissecting microscope. The egg mass was collected daily by placing tiny pieces of polyethylene film over the aquaria, as described by Pellegrino et al. (1965). The collected eggs were then preserved in small jars until they hatched (Liang et al., 1987).

The survival rate (Lx) was calculated by Frank (1963) using the following equation:

$$\text{Survival rate}=\frac{\text{live snails}}{\text{Total number of exposed snails}}\mathrm{x}100$$

The egg-laying capacity is expressed as (Mx), which is the number of eggs/snail/weeks, and the reproductive rate (R₀₎ is the sum of LxMx during the experimental period (El-Gindy and Radhawy, 1965).

2.6. Assessment of biochemical parameters

2.6.1. Tissue preparation

At the end of the experiment, 15 snails (5 from each experimental group) were used for biochemical analysis. Snails are cleaned and put between two slides then squeezed, and shells picked off, the soft tissues of both control and snails exposed to the sub-lethal concentrations of the aqueous extract (LC₁₀ 75.62, and LC₂₅ 90.52 mg/l) were removed, weighed, and homogenized in an ice-cold (0.1 M Tris HCl buffer, pH 7.4) using a glass Dounce homogenizer (Swedesboro, USA). The homogenates were centrifuged at 1764g for 15 min at 4 °C. The collected supernatant was preserved at −80 °C.

2.6.2. Biochemical analysis

The obtained supernatants were utilized to assess renal function indicators (creatinine, urea, uric acid, and albumin) and hepatic markers (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total protein), in addition to total cholesterol (TC) and triglycerides (TG) using Bio Diagnostic kits from Egypt.

2.6.3. Oxidative stress markers

Lipid peroxidation was determined via the formation of malondialdehyde (MDA) according to Buege and Aust (1978). The stress-induced production of nitric oxide (NO) was analyzed as determined by Montgomery and Dymock (1961). Glutathione reduced (GSH) is the primary intracellular thiol used by cells to indicate antioxidant defense assessed as mentioned by Beutler (1963). Catalase activity (CAT) is used to protect against H₂O₂-induced stress was estimated according to Aebi (1984), and SOD was evaluated following the method established by Nishikimi et al. (1972) using Bio Diagnostic kits in Egypt.

2.7. Histopathological studies

The hermaphrodite and digestive glands of all experimental snails were carefully removed and stored in Bouin's solution, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin (Mohamed and Saad, 1990). The sections were investigated at magnifications of 200× and 400× using a Zeiss microscope (Carl Zeiss Micrography GmbH 07.745 Jena, Germany).

2.8. Comet assay

The hemolymph of ten snails from each group were obtained by removing a small part of the shell and introducing a capillary tube into the heart, then collected in a glass vial tube (1.5 ml) and stored at −80C° until used. DNA damage was assessed using a single cell gel assay according to Grazeffe et al. (2008) as follows:

Hemocytes suspensions are collected by centrifugation and resuspend cells at 1 × 105 cells/ml in ice cold 1× PBS. After stirring for 5 min and filtering, 100 μl of the cell suspension was combined with 600 μl of low-melting agarose (0.8 % in PBS). The mixture (100 μl) was applied to pre-coated slides, then immersed in lysis buffer (0.045 M TBE (Tris/Borate/EDTA)), pH 8.4, with 2.5 % SDS (sodium dodecyl sulphate) for 15 min. The slides were placed in an electrophoresis chamber containing the same TBE buffer but without SDS. The electrophoresis settings were 2 V/cm for 2 min and 100 mA, with staining with ethidium bromide (20 μg/ml) at 4 °C. A fluorescent microscope at 510 nm was used to assess the migratory patterns of DNA fragments in 100 cells at different concentrations. The tail lengths of comets were measured from the nucleus to the end, with a 40× increase to determine their size.

2.9. Statistical analysis

Data was analyzed by One-way ANOVA using Statistical Processor Systems Support, SPSS software, version 20 followed by Duncan post hoc test to compare group means. The obtained values were expressed as mean ± standard error of the mean (SEM). Values of P < 0.05 were considered statistically significant.

3. Results

3.1. Preliminary phytochemical screening of the aqueous extract of A. thaliana

Table 1 showed the presence of various active phytoconstituents in the aqueous extract of A. thaliana such as flavonoids, tannins, saponins, terpenoids, steroids, anthraquinones, alkaloids, and carbohydrates.

Table 1.

Preliminary phytochemical screening analysis of aqueous extract of A. thaliana.

Secondary metabolites	Phytochemical test	aqueous extract of A. thaliana
Flavonoids	i. Shinoda	+++
	ii. AlCl3	+++
Anthraquinones	i. Borntrager's	++
Alkaloids	i. Dragendorff's	++
	ii. Meyer's	++
Terpenoids & Steroids	i. Salkowski	+++
	ii.Libarman-Burchard's	+++
Tannins	i. FeCl3	++
Saponins	i. Frothing	++
Carbohydrates	i. Molisch's	++
	ii. Barfoed's	++
Coumarins	i. NaOH	−

−Not detected, +: Present in low concentration, ++: Present in moderate concentration, +++: Present in high concentration.

3.2. Total phenolic content and antioxidant capacity of the aqueous extract of A. thaliana

The aqueous extract of A. thaliana leaves has total phenolic content (120.5 ± 4.1) and antioxidant capacity (145.2 ± 3.9).

3.3. Toxicity of aqueous extract of A. thaliana against B. glabrata

The calculated lethal concentrations (LC₅₀ and L C₉₀) of the aqueous extract of A. thaliana leaves on B. glabrata snails after 24 h of exposure followed by another 24 h for recovery were 107 and 138.3 mg/L, respectively.

3.4. Effect of A. thaliana aqueous extract on survival rate (L_x) and fecundity (M_x) reproductive rate (R₀) of B. glabrata

Table 2 demonstrated that the survival rate of snails subjected to LC₀ (10.7 mg/L) was slightly affected, being 0.81 at the fourth week of exposure. After that, through the recovery of four weeks, the snails survived till the end of the experiment. Exposure of snails to LC₁₀ considerably reduced survival rate (Lx) to 0.41 in the 4th week of exposure, and snails died in the 8th week of recovery. At LC₂₅ a severe death among treated snails was observed through the first 4 weeks of the experiment with L_x value 0.16 compared to 0.95 for the control group. Later, these surviving snails could not withstand this treatment and died by the sixth week.

Table 2.

Survival rate (Lx) and fecundity (Mx) of adult B. glabrata snails exposed for 24 h/ week to sub-lethal concentrations of A. thaliana aqueous extract for 4 successive weeks followed by 4 weeks of recovery.

Weeks	Control			LC0 (10.7 mg/l)			LC10 (75.6 mg/l)			LC25 (90.5 mg/l)
	Lx	Mx	LxMx	Lx	Mx	LxMx	Lx	Mx	LxMx	Lx	Mx	LxMx
0	1.00	3.96	3.96	1.00	3.96	3.96	1.00	3.96	3.96	1.00	3.96	3.96
1	1.00	4.1	4.1	0.96	2.85	2.73	0.89	3.9	3.47	0.81	4.2	3.40
2	0.99	4.6	4.55	0.92	2.42	2.2	0.75	3.6	2.7	0.73	3.9	2.84
3	0.97	4.9	4.75	0.84	4.32	3.5	0.55	3.8	2.09	0.36	3.5	1.26
4	0.95	4.1	3.89	0.81	1.42	1.13	0.41	2.6	1.06	0.16	1.2	0.19
5	0.95	5.1	4.84	0.71	1.4	0.99	0.36	1.9	0.68	0.1	0	0
6	0.92	4.3	3.95	0.60	2.5	1.5	0.45	0.5	0.225
7	0.90	3.3	2.97	0.60	2.6	1.56	0.21	0	0
8	0.90	3.9	3.51	0.55	1.4	0.77
RO = ΣLx Mx		32.56		**14.38			⁎⁎⁎10.22			⁎⁎⁎7.69
Reduction %				55.83			68.61			76.38

^⁎⁎⁎Highly significant from control at P < 0.001, compared to control.

The fecundity of snails (M_x) was adversely affected at the LC₀ of A. thaliana aqueous extract, decreasing the number of laid eggs /snails /weeks throughout the experiment. At the 4th and 8th weeks, treated snails laid 1.42 and 1.4 eggs/snail/week compared to 4.1 and 3.9 eggs/control snail/week, respectively. At LC₁₀, the snail's fecundity was highly suppressed in the 4th week, 2.6 eggs/snail/ week, compared to 4.1 eggs/control snail/week. Moreover, the surviving snails stopped egg-laying in the 7th week till they died in the 8th week. The snail's fecundity at the 4th week was 1.2 eggs/snail/ week at LC₂₅ compared to 4.1 eggs/control snail/week. These snails stopped laying eggs at 5th week till they died at 6th week.

Regarding the reproductive rate (R₀) of treated snails, it was extremely highly suppressed (P < 0.001) by exposure to the tested concentrations than the control group. At LC₀, the reproductive rate (R_o) was 14.38, with a 55.83 % percent reduction. The R_o values of treated snails at LC₁₀ and LC₂₅ were 10.22 and 7.69, respectively, compared to 32.56 for the control group. The percentage of reduction increases with the increase in the sub-lethal concentration, which is 76.38 % at LC₂₅.

3.5. Biochemical analysis

Aqueous extract of A. thaliana leaves significantly increases (P < 0.05) liver (AST, ALT and ALP) and renal biomarkers (creatinine, urea, uric acid and albumin) as well as lipid profile parameters (total cholesterol, triglyceride) of treated B. glabrata snails, on the other hand, total protein was significantly decreased (P < 0.05) in treated groups compared to control one as shown in Table 3.

Table 3.

Effect of Arabidopsis thaliana on biochemical parameters of B. glabrata.

Groups	Control	LC10	LC25
AST (U/ml)	22.53 ± 3.082a	22.68 ± 2.37a	78.34 ± 2.598b
ALT (U/ml)	88.17 ± 1.089a	95.53 ± 2.017a	135.784 ± 4.523b
ALP (U/L)	26.508 ± 0.34a	122.45 ± 2.96b	34.584 ± 1.08c
Total protein(g/dl)	2.77 ± 0.174a	2.43 ± 0.109b	1.959 ± 0.02b
Uric acid (mg/dl)	0.073 ± 1.34a	0.044 ± 1.78b	0.1357 ± 1.497b
Creatinine (mg/dl)	0.184 ± 0.0475a	0.356 ± 0.067a,b	0.452 ± 0.0493b
Urea (mg/dl)	5.46 ± 0.308a	7.799 ± 0.253b	6.926 ± 0.2334b
Albumin (g/dl)	1.260 ± 0.09a	1.298 ± 0.039a	1.643 ± 0.037b
Triglycerides (mg/dl)	132.544 ± 1.113a	163.199 ± 1.253a,b	173.84 ± 16.89b
Total Cholesterol (mg/dl)	45.284 ± 0.982a	44.69 ± 0.74a	54.745 ± 2.23b

Values expressed as mean ± SEM (n = 10), data with different superscript letters are considered significant (P < 0.05).

3.6. Oxidative stress markers

It was observed from data listed in Table 4 that sub-lethal concentrations (LC₁₀ and LC₂₅) of A. thaliana aqueous extract exhibited a significant increase (P < 0.05) in NO and MDA levels as well as a substantial decline in GSH, SOD, and CAT concentrations of B. glabrata compared to the control group.

Table 4.

Effect of A. thaliana on levels of oxidative stress markers of B. glabrata.

Groups	Control	LC 10	LC 25
CAT	0.376 ± 0.132a	0.276 ± 0.00118b	0.132 ± 0.0023c
GSH	19.6758 ± 0.534a	8.134 ± 0.82b	12.64 ± 0.55c
SOD	6492.118 ± 222.355a	10,564.13 ± 539.69b	5164 ± 17.07c
MDA	70.17 ± 5.74a	171.07 ± 17.69b	260.28 ± 35.12c
NO	19.26 ± 0.586a	14.94 ± 0.58b	49.76 ± 0.552c

Values expressed as mean ± SEM (n = 10), data with different superscript letters are considered significant (P < 0.05).

3.7. Histopathological observations

The digestive gland of the control snails is composed of tubules lined with a single layer of digestive cells and secretory cells (Fig. 1a, b). After exposure to LC₁₀ of A. thaliana_, most cells became vacuolated, degenerated, ruptured, and the lumen expanded (Fig. 1c, d). By raising the concentration to LC₂₅, cells lost their original shape due to their membrane's dissolution, and some digestive cells' tips ruptured (Fig. 1e, f).

Fig. 1.

Photomicrograph of the digestive gland of B. glabrata, a, b Control snails showing digestive tubules surrounded by connective tissue and composed of two cell types, digestive cells, secretory cells and lumen in the center. c, d Snails exposed to LC₁₀ of aqueous extract of A. thaliana showing degeneration and shrinkage of some tubules and cells lost their regular shapes and became vacuolated e, d Snails exposed to LC₂₅ showing degeneration and rupture of some tubules and increase in the central lumen with high damages in the digestive and secretory cells. Connective tissue CT, Digestive cells DC, Degenerated Digestive Cell DDC, Degenerated intestinal villi DIV, Degenerated Secretory Cells DSC, Digestive tubules DT, Inter tubular space IT, Lumen L, Secretory cells SC, vacuolated V, Vacuolated Digestive Cell VDC.

In the hermaphrodite gland of control snails, the male reproductive cells were differentiated into clusters formed of primary and secondary spermatocytes, the female oogenic cells occupied the acinar lumen, and the mature ova were surrounded by a follicular membrane (Fig. 2 a, b). Exposing snails to LC₁₀ of A. thaliana resulted in sperm shrinkage, destruction, and oocyte degeneration (Fig. 2 c, d). The sub-lethal concentration LC₂₅ caused great damage in gonadal cells where eggs lost their shape and degenerated, the number of sperms decreased, and connective tissue was replaced by vacuoles (Fig. 2 e, f).

Fig. 2.

Photomicrograph of the hermaphrodite gland of B. glabrata a, b Control snails displaying female acini with oocytes, mature ovum surrounded by a follicular membrane and follicular cavity and a large nucleus appeared, besides, spermatocytes and spermatids. c, d Snails exposed to LC₁₀ of A. thaliana revealing degenerations in oocytes, ova, and spermatocytes. e, f At LC₂₅ of A. thaliana, the severe reduction was represented by shrinkage of the acini and the tubules compacted together and degenerated. Acini A, Degenerated ovum DO, Degenerated Oocyte DOC, Degenerated spermatocytes DSPC, Follicular cavity FC, Follicular membrane FM, Mature ovum O, nucleus N, Oocytes OC, Spermatids SP, Spermatocytes SPC, Vacuole V.

3.8. Comet assay analysis

The comet assay was employed to assess DNA damage in B. glabrata exposed to sub-lethal concentrations (LC₁₀ and LC₂₅) of A. thaliana aqueous extract (Table 5 and Fig. 3). The assessment was based on four key parameters: tail length (TL), tail DNA percentage (TD), tail moment (TM), and olive tail moment (OTM). The parameters increased significantly (P < 0.05) in the group exposed to LC₂₅ of A. thaliana aqueous extract compared to the control one suggesting a higher degree of DNA fragmentation. The tailed length, which indicate cellular malformation, and the olive tail moment, a marker of DNA fragmentation, showed a significant increase (P < 0.05) in B. glabrata snails exposed to both sub-lethal concentrations compared to the control group. Additionally, there was a significant increase (P < 0.05) in the tail DNA percentage indicating migration from the head in the group exposed to LC₂₅ of A. thaliana aqueous extract. On the other hand, the snails exposed to the sib-lethal concentration LC₁₀ showed non-significant tail DNA% and tail moment as compared to control group.

Table 5.

DNA damage parameters of B. glabrata after exposure to A. thaliana aqueous extract.

Groups	Tail length (TL)	Tail DNA (TD)	Tail moment (TM)	Olive Tail moment (OTM)
Control	2.92 ± 0.23a	24.09 ± 2.199a	0.68 ± 0.0065a	1.378 ± 0.172a
LC 10	3.7 ± 0.011b	27.58 ± 5.95a	0.97 ± 0.11a	17.73 ± 2.38b
LC 25	3.52 ± 0.046b	70.89 ± 4.25b	18.41 ± 3.05b	27.11 ± 6.06b

Values expressed as mean ± SEM (n = 10), data with different superscript letters are considered signific ant (P < 0.05).

Fig. 3.

Photomicrograph of comet assay showing DNA damage levels of B. glabrata exposed to sub-lethal concentration of A. thaliana. (a) Control (no damage), (b) LC₁₀ (intermediate damage), and (c) LC₂₅ (high damage).

4. Discussion

The World Health Organization studied several compounds to eradicate snails and found that molluscicides of plant origin are more promising due to their high biodegradability, safety, affordability, and eco-friendly (Salawu and Odaibo, 2011; Abou-Elnour et al., 2015; Jia et al., 2019). Medicinal plants have gained prominence due to their active secondary metabolites, which contain antioxidant, fungicidal, antimicrobial, molluscicidal, and herbicidal characteristics (Mungenge et al., 2014; Ibrahim and Sayed, 2021).

The current investigation revealed that the aqueous extract of A. thaliana leaves exhibited toxicity towards B. glabrata snails, with a lethal concentration (LC₅₀) of 107 mg/L. This is in agreement with the findings of Silva et al. (2013), who verified that M. oleifera possessed molluscicidal activity against the snails Biomphalaria glabrata and Physa marmorata. They also reported that the snails were retracted into the shell and experienced hemorrhage following treatment. This may be attributed to the impact of active phytoconstituents in plants, including flavonoids, tannins, saponins, terpenoids and steroids, anthraquinones, alkaloids, carbohydrates, and coumarins. Similarly, Bakir (1997) stated that the mechanism of action of Punica granatum on snails is related to the presence of tannins and phenolic compounds that can precipitate on the protein of the cell membrane after its penetration. Also, Lorent et al. (2014) and Bahgat et al. (2018) stated that the ability of saponin to perforate the cell membranes causes detergent effects on the soft tissues of snails. These compounds form hydrogen bonds with nitrogen-free and multi hydroxyl groups, inhibiting certain enzymes crucial for the organism's functioning (Reed, 1995; Covington, 1997).

The survival, reproductive (R_o), and fecundity (M_x) rates of B. glabrata were significantly reduced after exposure to the sub-lethal concentrations (LC₀, LC_10, and LC₂₅) of aqueous extract of A. thaliana with an increase in the mortality rate of the eggs and these changes were concentration dependent. These findings are consistent with Ibrahim and Sayed (2021), who verified that the methanolic extract of Ziziphus spina-christi and Carica papaya seeds had a molluscicidal action against B. alexandrina snails and attributed this activity to the presence of saponins. Ibrahim and Ghoname (2018) also found that treatment with an aqueous extract of Anagallis arvensis reduced the reproductive and fecundity rates of B. alexandrina. Ibrahim and Abdalla (2017) noticed that the survival rate of B. alexandrina and the hatchability rates of their eggs were significantly decreased following exposure to an aqueous extract of Moringa oleifera seeds. Similarly, Hasheesh and Mohamed (2011) recorded a decline in egg laying capacity of Bulinus truncatus treated with methanol extract of the plant Sesbania sesban resulted in severe histological damage of the snail's hermaphrodite gland, and evacuations of some tubules of various gametogenic stages.

AST and ALT are regarded as sensitive markers and critical enzymes for the assessment of hepatocellular damage in snails under stressful conditions (Tunholi et al., 2011). Alkaline phosphatase is beneficial in protein synthesis and other secretory activities in gastropods, its inhibition may result in a drop in protein levels (Ibrahim et al., 1977). Protein is the primary source of energy, mainly during stressful periods. Animals subjected to any toxicant material suffer protein loss due to proteolysis, cell necrosis, and increased metabolism (Yadav et al., 2003; Singh et al., 2012).

Regarding biochemical parameters, our study revealed that an aqueous extract of A. thaliana significantly increased (P < 0.05) liver biomarkers (AST, ALT, and ALP) and decreased protein levels. On the same line, Abdel-Hamid and Mekawey (2014) recorded that AST, ALT, and ALP activities of B. alexandrina snails were substantially increased after exposure to Paecilomyces variotii and Aspergillus niger silver nanoparticles. Also, Al-Sayed et al. (2014) found a significant elevation in the ALP activity of B. alexandrina post-exposure to B. aerius extract. In the same vein, Ibrahim and Bakry (2019) demonstrated that the protein levels of B. alexandrina snails reduced significantly when exposed to LC₂₅ of chlorophyllin. Additionally, Chaturvedi et al. (2017) stated that 80 % of LC₅₀ (264.80 mg/L) of chlorophyllin caused a maximum reduction in protein concentration of Lymnaea acuminata snails.

The kidney's principal function was to maintain osmotic balance between the snail and its environment, as well as to provide excretory processes (Larson et al., 2014; Dhara et al., 2022b; Dhara et al., 2022a). The current study demonstrated that the sub-lethal concentrations of aqueous extract of A. thaliana resulted in a substantial increase of creatinine, urea, uric acid, and albumin levels of B. glabrata. Similarly, Abd El-Rahman et al. (2019) observed substantial rise in urea and creatinine levels in the African catfish (Clarias gariepinus) following exposure to oxyfluorfen herbicide. They also related these alterations to the inhibition of protoporphyrinogen oxidase in the liver and kidney. Additionally, Ibrahim et al. (2023) demonstrated an increase in uric acid and creatinine activities of B. alexandrina snails following exposure to chitosan-capped gold nanocomposite.

In the current study, the total cholesterol and triglycerides levels of B. glabrata snails were elevated as a result of exposure to sub-lethal concentrations of aqueous extract of A. thaliana. In the same vein, Gabr et al. (1994) revealed that the levels of cholesterol and triglycerides in both B. glabrata and B. alexandrina snails were substantially elevated by saponin extracted from the fruit and root of the buffalo ground plant. Previous studies by Barlov (1966) demonstrated that snails' cholesterol levels increased in conjunction with their triglyceride levels.

Antioxidant enzymes were employed as a biomarker to assess stressors in marine and freshwater organisms (Tellez-Bañuelos et al., 2009). These enzymes were critical in removing reactive oxygen species (ROS) and controlling the response of living organisms to oxidative stresses (Pizzino et al., 2017). After being exposed to xenobiotics, cells produced antioxidants in response to oxidative damage induced by reactive oxygen species (ROS) (Abd El-Rahman et al., 2019; Impellitteri et al., 2023; Saha et al., 2023; Tresnakova et al., 2023). The results showed that the aqueous extract of A. thaliana altered antioxidant concentrations, leading to a significant decrease in SOD, CAT, and GSH activities of B. glabrata. On the other hand, NO and MDA levels were higher than in control snails. Similarly, Xu et al. (2009) demonstrated that saponin treatment reduced total antioxidant levels in B. truncatus tissues. This reduction was caused by acute oxidative stress and the release of reactive oxygen species (ROS). These factors can suppress the antioxidant enzyme and alter other parameters.

Histopathological examination is a reliable and useful tool for clarifying the harmful effects of contaminants on the organs of aquatic species (Lam, 2009). The digestive glands of molluscs are one of the target organs in toxicological investigations due to their role in pollutant material detoxification (Mello-Silva et al., 2006). The present study showed that A. thaliana caused a severe reduction in the size of the digestive tubules and loss of the connective tissue with an increase in the inter-tubular spaces in the digestive gland of B. glabrata. Also, the cells were greatly altered, losing their regular shape, and the lumen inside each tubule increased. Some digestive cells developed vacuoles, deteriorated, and ruptured and secretory cells became denser in color, and some disintegrated. Similarly, Ibrahim et al., 2022a, Ibrahim et al., 2022b showed that methanolic extract of Nerium oleander and Tecoma stans caused significant damage in the digestive glands of B. alexandrina snails, with some digestive cells ruptured and vacuolated, and the connective tissue between tubules degenerated, as well as a marked increase in the number of secretory cells. Also, Ibrahim and Abdel-Tawab (2020) exhibited that C. barbata algae caused histopathological changes in the digestive gland of B. alexandrina, such as rupture, vacuolization, and an increase in the number of secretory cells in the digestive tubules due to the presence of phenolic compounds as saponins, and alkaloids. Ibrahim et al. (2018) noticed histological changes in the intestinal wall of Archachatina marginata snails caused by an ethanolic extract of Carica papaya seeds, including crop vacuolization and a loss in submucosal fat. Furthermore, Saad et al. (2012) observed vacuolization, secretory cell degeneration, and necrotic alterations in digestive gland cells in response to the Cestrum diurnum plant's influence on B. alexandrina. This was explained by the presence of saponins, which have a distinctive detergent effect on snail epithelial tissues. In addition, El-Deeb and El-Nahas (2005) reported that Euphorbia nubica and Sesbania sesban plants caused epithelial necrosis and an aberrant rise in the ratio of secretory to digestive cells of B. alexandrina.

Regarding the hermaphrodite gland of B. glabrata, exposure to A. thaliana aqueous extract caused damage in both male and female gonadal cells including connective tissue loss, sperm distortion, shrinkage, and partial egg destruction. At LC₂₅, the majority of gonadal cells had detached from acini, they became severely degenerated and empty, with the absence of the developmental stages of oocytes and spermatogenic stages. The connective tissue between the acini was dissolved and replaced by vacuoles. On the same line, Ibrahim et al., 2022a, Ibrahim et al., 2022b reported histopathological alterations in the hermaphrodite glands of B. alexandrina subjected to the methanolic extract of N. oleander due to the presence of alkaloids, saponins, steroids, tannins, terpenoids, anthraquinones. Also, El-Khayat et al. (2018) revealed degeneration with severe deformation and destruction of male and female follicles of the hermaphrodite gland of B. alexandrina snails following exposure to the methanol extract of Anagallis arvensis and Viburnum tinus. Mossalem et al. (2013) observed that exposure to the anthelmintic plant derivative (Artemether) resulted in the destruction of gametogenic cells and severe damage to the hermaphrodite gland of B. alexandrina snails. Similarly, Abdalla et al. (2012) found degenerative changes in the hermaphrodite acini and their contents of ova and sperms of B. alexandrina snails after exposure to an ethanolic extract of Euphorbia aphylla, Ziziphus spina-christi, and Enterolobium contortisiliquum.

Gastropod snails, which are intermediate hosts for numerous parasitic digeneans (Grazeffe et al., 2008), have become popular models for assessing DNA damage in both laboratory and field settings. The majority of DNA damage studies focus on freshwater species, including, Lymnaea luteola (Ali et al., 2015), Biomphalaria glabrata (Grazeffe et al., 2008), Biomphalaria alexandrina (Ibrahim et al., 2018). Across these studies, a range of environmental pollutants, including heavy metals, pesticides, insecticides, nanomaterials, and radiation, have been found to cause significant DNA damage, often resulting in genotoxicity in various snail cell types. The comet test is a sensitive approach for detecting DNA damage on the level of a single cell (Azqueta et al., 2009). Recent research has linked DNA strand breaks in aquatic species to cause effect on immune system, reproduction, development, and population dynamics (Shaldoum et al., 2016). The present investigation showed a significant increase in tail length (TL), tail DNA percentage (TD), tail moment (TM), and olive tail moment (OTM) of B. glabrata exposed to sub-lethal concentrations (LC₁₀ and LC₂₅) of A. thaliana aqueous extract compared to the control snails suggesting a higher degree of DNA damage and fragmentation Also, (Ibrahim and Ghoname (2018), used comet assay to confirm the presence of genotoxic effect after using aqueous extract of Anagallis arvensis and found a significant increase in both; tail moment and tail length in DNA of B. alexandrina snails. Similarly, Abdel-Haleem (2013) reported the effects of methanol extracts of three plants, Euphorbia splendens, Ambrosia maritime, and Ziziphus spinachristi on the protein patterns of digestive gland of B. alexandrina and Bulinus truncatus.

5. Conclusion

Phytochemical screening test revealed the presence of various bioactive compounds in the aqueous extract of Arabidopsis thaliana such as flavonoids, saponins, terpenoids, tannins, steroids, anthraquinones, alkaloids, and carbohydrates. These compounds possess molluscicidal activities on the intermediate host, Biomphalaria glabrata snails through a decrease in survival, reproduction, and fecundity rates as well as a decline in antioxidant enzymes. Also, the levels of the digestive gland enzymes, AST and ALT and the kidney functions, creatinine, uric acid and albumin concentrations were raised. Furthermore, alterations in hermaphrodite and digestive glands were observed, as well as DNA damage causing genotoxicity using comet assay was detected. Hence, A. thaliana can be used as a low-cost, eco-friendly, and biodegradable molluscicide for controlling schistosomiasis with reducing water pollution, and saving non-target organisms.

Availability of data and materials

Data is available upon request from the corresponding author.

CRediT authorship contribution statement

Najla Y. Beit Elmal: Writing – original draft, Formal analysis, Data curation. Fathy A. Abdel Ghaffar: Writing – review & editing, Supervision, Methodology, Conceptualization. Salwa A.H. Hamdi: Writing – review & editing, Supervision, Investigation. Amina M. Ibrahim: Resources, Methodology, Formal analysis, Conceptualization. Mona F. Fol: Writing – review & editing, Writing – original draft, Visualization, Supervision, Methodology, Investigation, Formal analysis, Conceptualization. Nesma A. Mostafa: Writing – review & editing, Software, Methodology, Formal analysis.

Consent for publication

Not applicable.

Ethics approval and consent to participate

All procedures of using laboratory animals in this study agreed with the Ethics of Research Committee regulations at the Faculty of Science, Cairo University.

Funding

This research received no external funding.

Declaration of competing interest

The investigations were designed and conducted with the assistance of all authors and there were no conflicting interests that could have influenced the conduct and reporting of these studies.