Highlights in BioScience ISSN:2682-4043 DOI:10.36462/H.BioSci.202506 Research Article Open Access 1 Department of Zoology, Faculty of Sciences, Menoufia University. * To whom correspondence should be addressed: Hodaahmed83@sci- ence.menofia.edu.eg Editor: Muhammad M. Adeel, Arthritis Clinical Immunology program Oklahoma Medical Research Foundation, Oklahoma City, United States. Reviewer(s): Khrokalo Liudmyla, Physical Chemistry Department National Technical University of Ukraine "Igor Sikorsky Kyiv Polytechnic Institute" Kyiv 03056 Ukraine. Grace Magalhaes-Ghiotto, Department of Biotechnology, Genetics and Cell Biology, Biological Sciences Center, State University of Maringá, Maringá, Paraná 87020-900, Brazil. Received: May 11, 2025 Accepted: August 2, 2025 Published: August 12, 2025 Citation: AbdelAzeem HH, Osman GY, Sheir SK. Apoptotic and histopathological impacts of Moringa oleifera seed oil on the land snail Cornu aspersum (O. F. Müller, 1774). 2025 Aug. 12;8:bs202506 Copyright: © 2025 AbdelAzeem HH et al.. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and supplementary materials. Funding: The authors have no support or funding to report. Competing interests: The authors declare that they have no competing interests. Apoptotic and histopathological impacts of Moringa oleifera seed oil on the land snail Cornu aspersum (O. F. Müller, 1774) Hoda H. AbdelAzeem*1 >< �, Gamalat Y. Osman1 ><�, Sherin K. Sheir1 ><� Abstract Cornu aspersum (O. F. Müller, 1774) is among the most harmful pests for many crops. Using natural molluscicides is essential to reduce the adverse effects of chemical ones on the biosystem. The toxicity of Moringa oleifera seed oil was determined, and LC50 and LC90 were 20.06% and 29.5%, respectively, after 72 hr of exposure. Moringa oil diminished the survival rate of Cornu aspersum (O. F. Müller, 1774) by 50% compared to the control (98.6%). Moreover, Moringa oil significantly increased the apoptosis and necrosis of digestive and ovotestis gland cells (P ≤ 0.02). By the end of the experiment, the proportion of apoptotic cells rose dramatically to 62.6% and 50.3% in the digestive and ovotestis glands, respectively, compared to the control (10.8% and 12.3%). In addition, the percentage of necrotic cells significantly increased to 21.5% and 24.7% (P ≤ 0.05), while the control values were 5.6% and 4.5%, respectively, at the 8% concentration. Regarding the digestive gland, Moringa oil caused vacuolation, nuclear pyknosis, and haemocyte infiltration. Deficiency of mature ova and spermatozoa, fibrosis, degeneration, and necrosis were recorded in the ovotestis. Moringa oil has proved its effectiveness as a natural molluscicide. Keywords: Cornu aspersum (O. F. Müller, 1774), Moringa oleifera Seed Oil, survival rate, Apoptosis, Histology. Introduction Mollusca are characterized as the second-largest phylum in the animal kingdom due to their great reproductive capacity-especially slugs and snails-which enables them to spread quickly throughout agricultural fields, making population control extremely challenging [1; 2]. The damage caused by these snails is attributed to their rasping feeding habits and the deposition of waste products such as slime and feces. Crop losses of 50–90% in soybean and corn have been reported in India [3; 4]. Land snails are notorious pests that cause significant damage to various crops and vegetation throughout Egypt [5; 6; 7]. These Land snails (gastropods) can damage multiple plant species, leading to major economic losses in agricultural fields, gardens, orchards, and greenhouses [8; 9; 10; 11]. Several authors have recorded different snail species and investigated their harmful effects: Cornu aspersum (O. F. Müller, 1774), Achatina fulica, Helix vestalis, and Theba pisana and Monacha sp. and Oxychilus sp. [12; 13; 14; 15]. These pests have caused serious damage to vegetables, banana crops, tomatoes, ornamental plants, mulberries, grapevines, and germinated seeds [16]. Land mollusks also harm potatoes, grains, lettuce, cabbage, carrots, maize, clover, and other horticultural and field crops. They feed on roots, seedlings, seeds, and tubers of nearly every fruit, vegetable, oil plant, and ornamental species in gardens, greenhouses, and fields. Damage includes killing seedlings (leading to poor stands) and destroying the leaves of young plants. Molluscs’ detrimental effects, however, differ depending on the crop. For example, some consume freshly planted wheat seeds, scrape strips of leaves from maize and many small grain plants, and make craters in the cotyledons and ragged holes in the leaves of soybean crops [17; 1; 18]. Pest control for snails requires a continuous and integrated approach. Physical methods such as hand collection and device innovation, the use of simple chemicals like sodium chloride, and the application of registered molluscicides such as methomyl are commonly employed, often in combination with biological control methods [18; 19; 20]. However, the extensive use of pesticides Highlights in BioScience Page 1 of 10 August 2025|Volume 8 https://doi.org/10.36462/H.BioSci.202506 https://creativecommons.org/licenses/by/4.0/ mailto:Hodaahmed83@science.menofia.edu.eg https://orcid.org/0000-0002-7599-8077 mailto: gamalat.osman@science.menofia.edu.eg https://orcid.org/0000-0001-9646-558X mailto:Shyreen.shaair@science.menofia.edu.eg https://orcid.org/0000-0002-0036-1514 http://bioscience.highlightsin.org/ AbdelAzeem HH et al., 2025 Apoptotic and histopathological impacts of Moringa oleifera seed oil by agricultural workers in many countries has raised seri- ous health concerns due to the toxicity of these chemicals [21]. Moreover, they often affect non-target organisms and disrupt the ecosystem [22]. Given the side effects associated with each method, there is an urgent need for safe and cost-effective alterna- tives. As a result, ongoing efforts are focused on discovering and evaluating promising, effective, and eco-friendly molluscicides against pest snail species. Natural plant derivatives, also known as botanical pesticides, have gained attention for their environ- mental safety. Oil extracts from these plants are excellent natural products-they are biodegradable into non-toxic compounds, easy to access and dispose of, and economically affordable [23]. Table 1. Physiochemical properties of Moringa seed oil Physical properties Refractive index 1.471 ±0.00 Specific gravity 0.903 ±0.2 Acid value (mg/g) 0.58 ±0.21 Peroxide value (Meq/kg) 2.37 ±0.06 Saponification value (mg KOH/g) 160.32 ±0.4 Iodine value (g/100g) 65.27 ±0.21 Chemical properties (Fatty acids composition) Name Concentration(%) Palimitic acid C 18:0 6.61 ±0.5 Stearic acid C18:0 5.30 ±0.5 Oleic acid C 18:1 66.12 ±0.04 Linolinic acid C 18:3 1.03 ±0.4 Palmitoleic acid C 16:1 1.78 ±0.02 Vaccinic acid C16:1 5.86 ±1 Linoleic acid C18:2 1.47 ±0.1 Arachidic acid C 20:0 3.46 ±0.04 Gadolic acidic C20:1 n9 2.75 ±0.1 Behenic acid 22:0 6.36 ±0.03 Total unsaturated fatty acids 79.01 ±1.2 Total saturated fatty acids 21.73 ±1 (n=3 replicates, data are mean ±SD) One of the most well-known and widely distributed species is Moringa oleifera [24]. The seed oil extract contains fatty acids similar to olive oil, with linoleic acid serving as a substitute [25]. It is rich in protein, and its leaves contain high levels of min- erals such as iron, vitamins, and calcium, making it useful for treating malnutrition [26]. Different parts of the tree including the root, leaf, fruit, and seedalong with their extracts or oils, possess various medicinal properties and have been used in both non-food products and traditional medicine [27]. It possesses antioxidant, anti-inflammatory, antianemic, and antidiabetic prop- erties, supports the immune system, and combats neurological, reproductive, cardiovascular, and bone illnesses. [28; 29]. Fur- thermore, the bioactive constituents of botanical molluscicides, such as flavonoids, saponins, and tannins found in Moringa spp., have been studied for their effects on snails [30; 31; 32]. Thus, the aim of this study is to evaluate the molluscicidal efficacy of Moringa oleifera seed oil against Cornu aspersum (O. F. Müller, 1774). Materials and Methods Experimental materials Moringa oleifera seed oil was purchased from the Moringa Unit at the National Research Center, Dokki, Egypt. The anal- ysis of the oil, including its components and physicochemical properties, was conducted at the same unit (Table 1). Methomyl (Copter 90% SP), a carbamate compound (S-methyl N-[(methyl- carbamoyl) oxy]thioacetimidate), with the molecular formula C5H10N2O2S, was used as a standard pesticide. It was obtained from Egyptchem International for Agrochemicals (Cairo, Egypt). The required concentrations of Moringa oil were prepared by dissolving the oil in a 1% Tween 80 solution (v/v) [33; 34]. The chemical structure of Tween 80 is 2-[2-[3,4-bis(2-hydroxyethoxy) oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate. Determination of LC50 and LC90 To determine the LC50 and LC90 of Moringa oleifera seed oil, acclimatized snails (mean weight: 4.4 ± 0.6 g) were divided into three replicates, with 10 snails in each group. Each group of 10 individuals was sprayed with 10 ml of its specific concen- tration. A range of concentrations (1%, 3%, 6%, 9%, 15%, 21%, 27%, and 30%) of Moringa oil was freshly prepared using a 1% Tween 80 solution (v/v) [35]. Snail mortality was observed and recorded daily for up to 72 hours. After counting and removing the dead individuals, the lethal concentrations (LC50 and LC90) were calculated using Probit analysis in the SPSS statistical soft- ware package (IBM Corp., Armonk, NY, USA). Two sub-lethal concentrations, 4% and 8%, were selected as LC10 and LC20, respectively. Collection of snails and experimental design Cornu aspersum (O. F. Müller, 1774) individuals were col- lected from garden plants and decorative trees, then transported in plastic boxes to the laboratory. The snails were maintained at a temperature of 23 ± 2◦C, under a 12 h photoperiod and relative humidity ranging from 75% to 85%. Each box contained a layer of soil at the bottom and was covered with muslin cloth. The snails were fed fresh lettuce leaves throughout the acclimatization and experimental periods. Snails were randomly divided into six groups, with three replicates per group, and each replicate consisting of 15 snails. The experimental groups were as follows: (a) Control group (unexposed). (b) Two groups exposed to 4% and 8% Moringa oleifera seed oil. (c) Two groups exposed to 4% and 8% methomyl. (d) One group exposed to 1% Tween 80 solution. Snails were sprayed daily with 1 ml of the respective treatment solution per snail (15 ml per replicate) over the course of three weeks. Survival rate was recorded as the percentage Highlights in BioScience Page 2 of 10 August 2025|Volume 8 http://bioscience.highlightsin.org/ AbdelAzeem HH et al., 2025 Apoptotic and histopathological impacts of Moringa oleifera seed oil Table 2. The effect of M. oleifera seed oil on the survival rate of Cornu aspersum (O. F. Müller, 1774). Exposure period Survival rate Control Methomyl Moringa Tween 80 4 8 4 8 1 Zero time 100 ±0 100 ±0 100 ±0 100 ±0 100 ±0 100 ±0 1st week 100 ±0 67.6 ±0.5 64.3 ±0.5 82 ±0.4 75.3 ±0.3 100 ±0 2nd week 100 ±0 54.3 ±1.5 47.6 ±1.1 72 ±0.5 63.3 ±1 98.9 ±0.5 3rd week 98.6 ±0.5 34.3 ±1.5a 30 ±1b 63.3 ±1 50 ±1ab 97.6 ±0.5 n = 3 replicates; data were reported as mean ±SD; a denotes a significant difference between the exposed and control groups and b vs methomyl-exposed group when P ≤ 0.05. of snails remaining alive at each observation point during the exposure period. At the end of the experiment, the digestive and ovotestis glands were dissected for apoptosis and necrosis assessment using cell cycle analysis and AnnexinV/PI staining, in addition to histological investigations. Cell cycle analysis Digestive and ovotestis glands (0.08 g) were homogenized in 1500 µl phosphate-buffered saline (cold) (PBS) and centrifuged at 1,000 rpm for 1 min at 4°C. The supernatant was collected for the assay. 200 µl of cell suspension in citrate buffer was added, along with propidium iodide (PI). There were 10,000 assessed nuclei on average for each specimen, and 120 nuclei were scanned every second. Accuri™ C6 flow cytometer analysis (Becton Dickinson, United States) was used to assess apoptotic cells utilizing sub-G1 peak labeling with PI [36; 37]. • G0/G1 peak: Normal diploid cells. • S phase (Synthesis): Cells synthesizing DNA. • G2/M peak (Gap 2 /Mitosis): Cells ready to divide. • Sub-G1: Apoptotic cells with fragmented DNA. Assay of PI/annexin-V dual staining Discrimination of the apoptotic profile was determined using the Apoptosis Detection Kit I (Cat. No. 556547BD, Pharmin- gen™, Bioscience, New Zealand) according to the manufacturer’s instructions. The principle of this procedure relies on the inter- action between the membrane phospholipid phosphatidylserine of apoptotic cells and Annexin V, a phospholipid-binding pro- tein that requires calcium. To differentiate between viable and non-viable cells, propidium iodide (PI) was employed as a con- ventional flow cytometric viability probe. Non-viable or damaged cells with permeable membranes allow PI to enter, while viable cells with intact membranes exclude PI. The BD Accuri™ C6 flow cytometer was used to analyze the cells immediately. Four distinct cell populations were identified: (1) Early apoptotic cells (bound to Annexin V only), (2) Late apoptotic/necrotic cells (bound to both Annexin V and PI), (3) Necrotic cells (stained with PI only), (4) Viable cells (unstained). The percentage of fluorescent cells in each quadrant was calculated after the fluores- cence distribution was presented in a two-color dot plot analysis [38]. Histological investigation After three weeks, the digestive and ovotestis glands were dissected and fixed in Bouin’s fluid. After 24 hr of fixation, dehy- dration was performed through a series of alcohols and cleared in xylene. Paraffin blocks were sectioned at 5 µm thickness on glass slides for hematoxylin and eosin staining [39; 40]. A good wash with tap water was performed after staining. A photo-automated camera (Optika, Italy) was used to capture images of the histo- logical sections, which were then analyzed for histopathological syndromes. Statistical analysis The Statistical Package for the Social Sciences (SPSS; IBM Corp., Armonk, NY, USA) was used to analyze the data, which were presented as mean ± standard deviation. One-way ANOVA was performed, followed by the Least Significant Difference (LSD) post-hoc test to determine the significance between the control and different concentrations of the tested materials. A significance level of P < 0.05 was considered statistically signifi- cant. Results Toxical effect of Moringa oleifera seed oil The values (%) of LC50 and LC90 were 20.06 (95% confi- dence limit for log = 1.3) and 29.5 (95% confidence limit for log = 1.4), respectively, after 72 hr of exposure. The slopes of LC50 and LC90 were 2.5 and 3, respectively. The sublethal concen- trations used were 4% Moringa oil (40 mL/L of 1% Tween 80) and 8% Moringa oil (80 mL/L of 1% Tween 80) (v/v) solutions. Additionally, 4% methomyl (4 g / 100 mL) and 8% methomyl (4 g / 100 mL) (w/v) solutions were used. The survival rate of Cornu aspersum (O. F. Müller, 1774) under the effect of Moringa oil The survival rate of Cornu aspersum decreased gradually according to the concentration and exposure duration. The reduc- tion was more significant in methomyl-exposed snails, followed Highlights in BioScience Page 3 of 10 August 2025|Volume 8 http://bioscience.highlightsin.org/ AbdelAzeem HH et al., 2025 Apoptotic and histopathological impacts of Moringa oleifera seed oil Table 3. Percentage of cells in different cell cycle phases after treatment Concentration Exposure Period %Sub G1 % Cells/Cell Cycle G0/G1 S G2/M Control 1st week 12.8 ±0.1 91.9 ±0.1 51.03 ±1 4.4 ±0.1 24.5 ±0.8 2nd week 11.8 ±0.2 95.8 ±0.3 52 ±1 4.1 ±0.3 24.8 ±1 3rd week 11.7 ±0.4 96 ±0.4 52.1 ±1.2 4 ±0.2 24.3 ±0.5 4% Moringa oil 1st week 14.9 ±0.6 97.2 ±2.9 70.1 ±3.3 2.1 ±0.2 10.4 ±1.9 2nd week 15.02 ±0.1ab 83.2 ±1.4 40.1 ±0.7 15.03 ±0.05 15.5 ±0.7 3rd week 12.4 ±1.2 90.9 ±2.9 60.1 ±2.07 4.1 ±0 15.3 ±1.4 8% Moringa oil 1st week 18.2 ±0.3ab 99.2 ±1.3 78.3 ±1.4 1.8 ±0.2 0.9 ±0.1 2nd week 10.4 ±0.05 91.03 ±1.1 57.1 ±0.6 3.7 ±0.05 20.6 ±0.8 3rd week 13.3 ±0.9 90.2 ±2.03 59.5 ±2.4 4.03 ±0.1 14.5 ±3.2 4% Methomyl 1st week 21.3 ±0.3ab 96.3 ±1.1 64.3 ±1.06 1.3 ±0.05 9.93 ±0.1 2nd week 14.1 ±0.5b 92.1 ±0.8 51.4 ±1.1 6.5 ±0.05 21.1 ±1.6 3rd week 14.7 ±0.6 93.8 ±1.05 52.06 ±2.2 1.8 ±0.05 26.1 ±2.8 8% Methomyl 1st week 22.1 ±0.1ab 96.9 ±0.6 63.6 ±1.5 1.6 ±0.05 10.1 ±1.7 2nd week 12.8 ±0.6 90.7 ±3.03 41.9 ±2.4 13.4 ±0.3 23.6 ±3.9 3rd week 14.1 ±0.5b 92.6 ±1.5 45.4 ±1.2 5.2 ±0.1 28.9 ±2.4 1% Tween 1st week 12.8 ±2.7 94.03 ±1.7 76.1 ±1.2 2.23 ±0.2 3.5 ±0.1 2nd week 9.2 ±0.7 87.8 ±4.9 65.6 ±4.2 13.3 ±0.1 0.8 ±0.1 3rd week 14.02 ±1 86.3 ±4.4 54.3 ±3.9 3.7 ±0.4 16.1 ±0.2 Data are stated as mean ± SD, n = 3 replicates; significant differences are represented as a when compared with the control and b when compared with methomyl at P ≤ 0.05. Figure 1. Representative flow cytometry histogram showing Moringa oil’s effect on cell cycle distribution of Cornu aspersum (O. F. Müller, 1774) after the third week of exposure. The digestive gland is repre- sented by panels (a), (b), and (c): (a) control; (b) 8% Moringa oil; (c) 8% methomyl. The ovotestis gland is represented by panels (d), (e), and (f): (d) control; (e) 8% Moringa oil; (f) 8% methomyl. by those exposed to M. oleifera seed oil. A substantial decrease (P = 0.01) was observed in the third week of exposure, with survival rates of 50% for M. oleifera oil and 30% for methomyl, compared to 98.6% in the control. Methomyl exposure signifi- cantly reduced the survival rate compared to M. oleifera seed oil (Table 2). Cell cycle distribution after Moringa oil exposure The proportion of cells in the G0/G1, S, and G2/M phases of the cell cycle was determined in both digestive and ovotestis glands after 1, 2, and 3 weeks. Exposure to 4% and 8% M. oleifera seed oil resulted in a significant increase in fragmented cells (sub-G1) compared to the control (P ≤ 0.003) and methomyl (P ≤ 0.01). Notably, the number of G0/G1 phase cells increased to 78.3 ± 1.4% in the 8% Moringa oil group, compared to 51.03 ± 1% in the control and 63.6 ± 1.5% in the methomyl group. Consequently, the proportions of S and G2/M phase cells decreased at the same concentration (Table 3, Figure 1). In the digestive gland, 8% Moringa oil significantly increased sub-G1 phase cells compared to the control (P ≤ 0.001) and Highlights in BioScience Page 4 of 10 August 2025|Volume 8 http://bioscience.highlightsin.org/ AbdelAzeem HH et al., 2025 Apoptotic and histopathological impacts of Moringa oleifera seed oil Table 4. Percentage of cells in different cell cycle phases after treatment Concentration Exposure Period %Sub G1 % Cells/Cell Cycle G0/G1 S G2/M Control 1st week 9.3 ±1 96.5 ±1.3 52.2 ±0.1 3.1 ±0.2 11.4 ±1 2nd week 10.4 ±0.3a 95.4 ±0.8 52.2 ±0.1 3.5 ±0.3 10.9 ±1 3rd week 10.4 ±0.5 95.3 ±0.4 52.5 ±0.2 3 ±0.2 10.8 4% Moringa oil 1st week 15.4 ±0.4 93.6 ±0.5 74.1 ±1.1 10.5 ±0.1 1.1 ±0.1 2nd week 17.06 ±0.5a 88.5 ±3.2 74.1 ±2.4 10.5 ±0.2 0.9 ±0.1 3rd week 15.2 ±0.05 93.6 ±0.2 75.1 ±0.5 10.3 ±0.05 1.2 ±0.3 8% Moringa oil 1st week 16.2 ±0.04 92 ±0 67.1 ±0.2 9.1 ±0.05 2.9 ±0.2 2nd week 30.2 ±0.1a 82.2 ±0.5 67.1 ±0.4 14.1 ±0.1 3 ±0.1 3rd week 15.7 ±0.7 96.1 ±1.4 67.4 ±1.5 13.1 ±0.2 2.8 ±2.4 4% Methomyl 1st week 18.1 ±0.1a 91.5 ±0.5 63.6 ±0.7 17.9 ±0.05 14 ±0.8 2nd week 12.9 ±0.1 86.2 ±0.2 63.7 ±0.5 17.6 ±0.1 14.1 ±0.9 3rd week 15.1 ±0.1 90.4 ±0.8 63.6 ±0.2 12 ±0.05 14.2 ±1.05 8% Methomyl 1st week 18.07 ±0.8a 91.3 ±1.4 68.6 ±3 12 ±0.05 13.4 ±1.8 2nd week 14.6 ±0.05 93.3 ±2.8 68.9 ±2.7 12.1 ±0.05 13.3 ±0.1 3rd week 17.9 ±0.4a 94.7 ±1.4 68.8 ±2.3 11.9 ±0.1 13.4 ±0.7 1% Tween 1st week 11.2 ±0.1 96.3 ±0.4 56 ±0.2 25.3 ±0.3 0.6 ±0.05 2nd week 11.5 ±0.2 96.6 ±0.7 59 ±0.2 25.7 ±0.7 0.7 ±0.1 3rd week 14.6 ±0.2 87.3 ±0.8 52.5 ±0.5 5.3 ±0.1 16.5 ±0.8 Data are expressed as mean ± SD, n = 3 replicates; significant differences are represented by a vs. control when P ≤ 0.05. methomyl (P ≤ 0.005). The G0/G1 phase cell counts increased to 75.1 ± 0.5% (4% Moringa) and 67.4 ± 1.5% (8% Moringa) versus 52.5 ± 0.2% in the control. Furthermore, G2/M phase per- centages decreased with both Moringa oil concentrations (Table 4, Figure 1). Detection of apoptosis by annexin-V/PI Using flow cytometry, apoptotic cells were identified by incu- bating them with FITC-labeled annexin V and PI. To differentiate between necrotic and apoptotic cells, PI was utilized. The ex- posure of snails to Moringa oil caused a significant increase in apoptotic cells (early and late apoptosis) in digestive gland cells at both concentrations (P ≤ 0.001) and in the two concentrations of methomyl (P ≤ 0.01) compared with the control group. At the end of the experiment, the percentages of apoptotic cells were 50.3% and 63.9% at 8% Moringa oil and methomyl, respectively, while that of the control was 12.3%. Depending on the material, the increase in apoptotic cells in both concentrations of Moringa oil was significant compared with methomyl (P ≤ 0.001). In ovotestis gland cells, a significant increase in apoptotic cells was observed at both concentrations of Moringa oil (P ≤ 0.003) and at the two concentrations of methomyl (P ≤ 0.02). At the third week of exposure, the percentages of apoptotic cells were 62.6% and 50.8% at 8% Moringa oil and methomyl, respec- tively, while the control was 10.8%. Depending on the time of exposure, the increase in apoptotic cells in the third week was significant when compared with that in the first week (P = 0.01). Cells that were stained with PI were identified as necrotic, and they significantly increased in the digestive gland cells after ex- posure to both Moringa oil concentrations (P ≤ 0.005) and methomyl (P ≤ 0.02) compared with the control. The percent- ages were 21.5% and 14.9% at 8% Moringa oil and methomyl, respectively, while that of the control was 5.6%. In the ovotestis gland, a significant increase was noticed at both Moringa oil concentrations and methomyl (P ≤ 0.03) compared with the con- trol. Regarding exposure time, the increase in the third week was 24.7% and 31.1% (P = 0.02) at the 8% concentration of Moringa oil and methomyl, respectively, compared with their values in the first week (5.5% and 3.7%), when the control was 4.5%. As a result, the number of viable cells was significantly decreased in Moringa oil and methomyl (70.03% and 60.2%, respectively) compared with the control (84.6%) in the third week of exposure Highlights in BioScience Page 5 of 10 August 2025|Volume 8 http://bioscience.highlightsin.org/ AbdelAzeem HH et al., 2025 Apoptotic and histopathological impacts of Moringa oleifera seed oil Figure 2. Fluorocytograms of one representative experiment from three separate trials following a three-week exposure to M. oleifera seed oil. (Figure S1, Figure S2, Figure 2). Histopathological signs after exposure to Moringa oil Histological sections of the normal (unexposed) digestive gland of Cornu aspersum (O. F. Müller, 1774) revealed that it is mainly composed of digestive tubules, each lined with columnar epithelial cells of various types, including digestive and excretory cells. The digestive cells are typically characterized by numerous green and yellow cytoplasmic granules. These tubules enclose a distinct central lumen and are separated by interlobular con- nective tissue containing hemolymphatic sinuses rich in actively circulating hemocytes. Externally, the tubules are encased by a distinct circular muscle layer. Exposure to 8% M. oleifera oil caused pronounced pathologi- cal alterations in the digestive gland, including marked vacuoliza- tion and pyknosis of nuclei. Hemocytic infiltration, a clear sign of inflammation, was also observed. Severe epithelial damage, in- cluding extensive vacuolization, degeneration, and necrosis, was evident following exposure to both 4% and 8% concentrations of methomyl. Additionally, Moringa oil induced progressive degen- eration of muscle fibers (Figure 3). The ovotestis of unexposed snails consists of numerous small follicles (acini) lined with ger- minal epithelium, which is clearly differentiated into primary and secondary spermatogonia/oogonia and mature spermatozoa/ova. After three weeks of exposure, the high concentration (8%) of Moringa oil caused notable alterations in the histological archi- tecture of the ovotestis. Histopathological signs included degen- eration of ova and spermatozoa, inhibition of ova maturation, appearance of dense fibrous tissue, and necrosis. Methomyl (8%) exposure led to a substantial reduction or complete absence of mature ova and degeneration of spermatozoa (Figure 4). Discussions Figure 3. Light photomicrographs of sections through the digestive gland of Cornu aspersum (O. F. Müller, 1774) after three weeks of exposure. (a & b) Control, (c, d, e, & i) exposed snails to 4% and 8% M. oleifera seed oil, and (f, g, h, & j) exposed snails to 4% and 8% methomyl. Dt, digestive tubule; Dc, digestive cell; Se, secretory cell; Ct, connective tissue; L, lumen; Py, pyknosis; V, vacuolization; Dg, degeneration; Uf, undigested food; IG, increasing the granules in digestive cells; N, necrosis; Hi, hemocytic infiltration; Mf, muscle fibers. Effect of Moringa oleifera seed oil on the survival rate of Cornu aspersum (O. F. Müller, 1774) The present study recorded the influential role of Moringa oil in reducing the survival rate of Cornu aspersum snails. As primary consumers, land snails serve as models for rapid physio- logical responses to dietary habits and breeding conditions. Pre- vious research [41; 42] confirmed the sensitivity of Mollusca to minimal dietary changes due to their rapid metabolism. Moringa oil is composed of unsaturated and saturated fatty acids. Expo- sure of snails to this oil may alter their fatty acid profile, which is sufficient to change the physiological state of gastropods [43]. A previous study also indicated that fluctuations in polyunsaturated fatty acids can serve as a valuable ecotoxicological test in snails [44]. Benzylamine extracted from M. oleifera has demonstrated molluscicidal potency by reducing survival and reproductive rates in Biomphalaria alexandrina snails [45]. Plant extracts contain- ing essential oils, flavonoids, terpenes, saponins, and tannins have been reported as effective agents in snail control [46; 47]. The negative impact of such extracts on the land snail Monacha obstructa has also been documented [48]. Apoptotic effect of M. oleifera seed oil on Cornu aspersum The findings indicated the apoptotic potential of M. oleifera oil on both digestive and ovotestis gland tissues. This can be explained by the fact that mollusks possess a unique composi- tion of fatty acids. Given this, previous studies have shown that unsaturated and saturated fatty acids exert different effects on steatosis and cell death, influencing apoptosis in a variety of ex- perimental systems and being identified as the most harmful lipid types [49; 50; 51]. The presence of polyunsaturated fatty acids in cellular membranes influenced membrane permeability. Previ- ous research showed that apoptosis was caused by palmitic acid Highlights in BioScience Page 6 of 10 August 2025|Volume 8 http://bioscience.highlightsin.org/ AbdelAzeem HH et al., 2025 Apoptotic and histopathological impacts of Moringa oleifera seed oil Figure 4. Light photomicrographs of sections through the ovotestis gland of Cornu aspersum (O. F. Müller, 1774) after three weeks of exposure. (a) Control; (b, c, & f) exposed snails to 8% M. oleifera oil; (d & e) exposed snails to 8% methomyl. f, follicle; fw, follicular wall; ov, ovum; sp, spermatozoa; dv, developing oocyte; ss, stages of spermatogonia; d, degeneration; ft, fibrous tissue; n, necrosis; vo, vitellogenic oocyte. Reduction/lack of mature ova and spermatozoa (black arrows). through caspase-dependent Beclin 1 cleavage. The researchers concluded that essential fatty acids induce apoptosis of tumor cells by overexpressing cytochrome P450 [52]. Mitochondria play a major role in apoptosis by releasing cytochrome c and caspase 3, which activate apoptotic enzymes [53]. Fatty acids that stimulate lipotoxicity were also reported to play a crucial role in pathogenesis [54]. Moreover, the toxicity of metals has been linked to disturbances in lipid membranes [55]. The observed changes in fatty acid levels represent metabolic adjustments initi- ated by external stimuli [56]. A recent study explored the ability of Moringa oleifera leaf extract to promote apoptosis in adipocyte cells (3T3-L1 cells) by increasing caspase 3 activity and regu- lating the expression of BAX and BCL2 genes [57]. Moringa seed extract has been shown to induce apoptosis and cell cycle arrest in cancer cells, with fatty acids like caprylic acid, oleic acid, and stearic acid contributing to apoptosis in lung, leukemia, and ovarian cancer cells [58]. Autophagy and hepatic steatosis were reported in the mouse liver after high-fat diets [59]. An- other study demonstrated the harmful effects of Moringa extract (containing saponins, flavones, and flavonols) on Biomphalaria glabrata embryos, adults, and Schistosoma mansoni adult worms [60]. Histopathological effect of M. oleifera seed oil on Cornu aspersum Exposure to seed oil induced histopathological changes in both digestive and ovotestis glands. This observation aligns with previous findings [61], where damage such as fragmentation and vacuolization was reported in the digestive and excretory cells of B. alexandrina and B. truncatus snails following treatment with Egyptian wild plant extracts. Exposure to aqueous seed extract of M. oleifera caused severe damage in digestive cells, including tip loss, degeneration, and increased numbers of secretory cells [62]. Additional histopathological features included vacuolation of digestive and secretory cells. Degeneration and rupture of sperm and ova in B. truncatus were observed following expo- sure to cerium oxide nanoparticles synthesized using Moringa seeds [63]. Chlorophyllin from deep-frozen M. oleifera leaves caused deformation of secretory cells and rupture of connective tissue between tubules, leading to degeneration of digestive cells in B. truncatus. When combined with magnesium or copper, this compound also caused histological malformations due to photosensitization [64; 65]. In Cornu aspersum, degeneration of digestive tubules and damage to the basement membrane and hep- atopancreas were reported following exposure to thiamethoxam (200 mg/L) [66]. Conclusions Moringa oleifera seed oil has significant molluscicidal po- tency, exhibiting both apoptotic and histopathological effects on the ovotestis and digestive glands of Cornu aspersum (O. F. Müller, 1774). This study investigated the effects of Moringa oil as a whole against the snail Cornu aspersum. Further studies will aim to isolate the main active constituents of the oil and assess their effects on non-target organisms to ensure greater eco-friendliness compared with conventional chemical molluscicides. List of Abbreviations • PBS: Phosphate-buffered saline • PI: Propidium iodide • COI: Cytochrome oxidase subunit I • ND1: NADH dehydrogenase subunit 1 • DNA: Deoxyribonucleic acid Supplementary Figure S1: Exposure to M. oleifera seed oil increased cell apoptosis and necrosis of the digestive gland of Cornu aspersum (O. F. Müller, 1774) after exposure for3 weeks: (a) first week, (b) second week, and (c) third week. Values expressed as mean ±SD. c The significant difference in necrosis between control and exposed snails when P ≤ 0.02. The significant difference in apoptosis was indicated as follows: a,b, between Moringa exposed groups vs. control and methomyl exposed groups when P ≤ 0.05, ANOVA. Figure S2: Exposure to M. oleifera seed oil increased cell apoptosis and necrosis of the ovotestis gland of Cornu aspersum (O. F. Müller, 1774) after exposure 3 weeks: (a) first week; (b) second week; and (c) third week. 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Histological changes and biochemical parameters in the hepatopancreas of terrestrial gastropod Cornu aspersum as biomarkers of neonicotinoid insecticide exposure. Afr J Biotechnol. 2012;11(96):16277-83. Highlights in BioScience Page 10 of 10 August 2025|Volume 8 http://bioscience.highlightsin.org/ Abstract Introduction Materials and Methods Experimental materials Determination of LC50 and LC90 Collection of snails and experimental design Cell cycle analysis Assay of PI/annexin-V dual staining Histological investigation Statistical analysis Results Toxical effect of Moringa oleifera seed oil The survival rate of Cornu aspersum (O. F. Müller, 1774) under the effect of Moringa oil Cell cycle distribution after Moringa oil exposure Detection of apoptosis by annexin-V/PI Histopathological signs after exposure to Moringa oil Discussions Effect of Moringa oleifera seed oil on the survival rate of Cornu aspersum (O. F. Müller, 1774) Apoptotic effect of M. oleifera seed oil on Cornu aspersum Histopathological effect of M. oleifera seed oil on Cornu aspersum Conclusions List of Abbreviations Supplementary