Introduction
Schistosomiasis is a serious communicable disease with public health implications and economic significance in the Eastern Mediterranean Region (Abuseir Reference Abuseir2023). More than 236 million people in 78 countries and territories worldwide are infected with schistosomiasis, an infectious parasitic disease caused by parasites of the genus Schistosoma spp. (WHO 2023). Schistosoma mansoni (S. mansoni) is a parasitic species found in various parts of Africa, including Egypt, the Middle East, and South America, where the intermediate host, a freshwater snail named Biomphalaria (phylum Mollusca, class Gastropoda), lives. Biomphalaria alexandrina (B. alexandrina) snails are abundant in Egypt, especially in the Nile Delta and along its tributaries (Ibrahim et al. Reference Ibrahim, El-Karim, Ali and Nasr2023). Even though schistosomiasis is one of the most common Neglected Tropical Diseases (NTDs), treatment and disease control are dependent on the use of a single drug, praziquantel (PZQ); however, there have been reports of PZQ schistosomal resistance (Mtemeli et al. Reference Mtemeli, Walter and Shoko2020). Control of schistosomiasis requires a combination of chemotherapy, health education, molluscicides, environmental management, safe water, and proper sanitation (WHO 2023). One of the most promising methods for preventing schistosomiasis is snail control (Kengne Fokam et al. Reference Kengne Fokam, Sumo, Bagayan, Nana-Djeunga, Kuete, Nganjou, Tchami Mbagnia, Djune-Yemeli, Wondji and Njiokou2022). Snail control using chemicals such as niclosamide has several drawbacks, including high production costs and toxicity to aquatic plants and other non-target creatures (Sarquis et al. Reference Sarquis, Pieri and dos Santos1997). Thus, it is essential to look for natural alternative therapies that work against schistosomiasis’ etiological agent and snails, especially in places where the disease is endemic (Pereira et al. Reference Pereira Moreira, Weber, Haeberlein, Mokosch, Spengler, Grevelding and Falcone2022). With the growing interest in green molluscicides and knowledge of these safe, specific, biodegradable, and eco-compatible components, numerous studies on botanicals’ potential as molluscicides are currently being conducted (Zheng et al. Reference Zheng, Deng, Zhong, Wang, Guo and Fan2021).
Pumpkin (Cucurbita spp.) seed oil (PSO) has long been recognized for its various health benefits and therapeutic properties. In recent years, there has been a growing interest in investigating the potential activities of pumpkin seed oil against various parasitic diseases. One such disease of significant concern in Egypt is schistosomiasis (Mtemeli et al. Reference Mtemeli, Walter and Shoko2020). Pumpkin seeds have been used in different parts of the world as a traditional medicine for treatments of gastrointestinal parasites such as anthelmintic, urinary dysfunctions, hyperplasia of the prostate, dysuria, cardiovascular disease, enuresis, and lowering blood glucose (Csikós et al. Reference Csikós, Horváth, Ács, Papp, Balázs, Dolenc, Kenda, Kočevar Glavač, Nagy, Protti and Mercolini2021). Pumpkin seed oil offers several advantages as a molluscicide, making it a promising option for controlling snail populations. It is characterized by its safety and non-toxic nature, as well as its wide availability, affordability, and ease of application. The key active component responsible for its molluscicidal properties is cucurbitin, which acts by binding to a specific receptor in the brains of mollusks. This binding event leads to muscular paralysis and eventual mortality. Notably, pumpkin seed oil has demonstrated superior efficacy compared to chemical molluscicides, achieving effective snail control even at lower concentrations. (Ayyildiz et al. Reference Ayyildiz, Topkafa and Kara2019). In the specific context of Egypt, several studies have investigated the effectiveness of botanical extracts in combating B. alexandrina snail populations (Mossalem et al. Reference Mossalem, Abdel-Hamid and El-Shinnawy2013; Mossalem and ElEnain Reference Mossalem and ElEnain2014; Ibrahim et al. Reference Ibrahim, Ghoname, Mansour and El-Dafrawy2020; Ali and Gad El-Karim Reference Ali and Gad El-Karim2023; Ibrahim et al. Reference Ibrahim, El-Karim, Ali and Nasr2023). However, data on the molluscicide effects of PSO on vector snails is scarce.
The current study aims to evaluate the activities of PSO against both B. alexandrina snails and the infective stages of S. mansoni. Furthermore, the study explored potential genotoxic and histopathological alterations induced by the oil, shedding light on its importance in the context of schistosomiasis control in Egypt.
The findings of this study have the potential to pave the way for further investigations and the development of innovative strategies to combat schistosomiasis in Egypt and other affected regions., potentially reducing the reliance on conventional treatment methods that often face challenges such as drug resistance and limited accessibility.
Methodology
Collection and maintenance of B. alexandrina snails
B. alexandrina snails were collected from the river Nile and irrigation channels located in Giza Governorate, Egypt. After collection, the snails were transported and maintained in the Medical Malacology Department at Theodor Bilharz Research Institute (TBRI) in Giza, Egypt, for four weeks as an acclimatization period and were examined weekly to exclude parasitic infections. The snails were placed in standard plastic aquaria, containing dechlorinated, aerated tap water with a pH of 7 ± 0.2 and a temperature of 25 ± 2ºC. To sustain their nourishment, the snails were given oven-dried lettuce leaves (ad libitum) (Eveland and Haseeb Reference Eveland, Haseeb, Toledo and Fried2011). Adult B. alexandrina snails (6–8 mm in diameter) were used in further investigations.
Preparation of pumpkin seed oil extract
The Egyptian pumpkin seeds (Cucurbita moschata, L. Family Curcubitaceae) used in this study were collected from a local market in Cairo, Egypt. The geographical coordinates of the collection site are 30.0444° N latitude and 31.2357° E longitude. The time of collection was during the afternoon, and the seeds were procured in the month of September, specifically in the year 2022. The PSO utilized in the study was obtained from the entomology laboratory at the Department of Entomology, Faculty of Science, Ain Shams University, following the method outlined by Al-Okbi et al. (Reference Al-Okbi, Mohamed, Kandil and Ahmed2014).
The seeds were thoroughly washed and subsequently dried in an air-circulated oven at a temperature of 40°C. Once dried, the seeds were crushed into a powder. Pre-extraction of powdered seeds used petroleum ether (60–80°C) for 24 hours at room temperature with occasional shaking. Afterward, the mixture was filtered using Whatman No. 1 filter paper, and the petroleum ether was evaporated under reduced pressure at a temperature below 40°C. Seeds were then soaked in 95% ethanol (v/v) for another 24 hours with occasional shaking to extract ethanol-soluble compounds. The ethanol mixture was also filtered using Whatman No. 1 filter paper, and the ethanol was evaporated under reduced pressure at a temperature below 40°C. The resulting oil was subsequently stored in a freezer at -20°C until further analysis within the study.
Gas chromatography-mass spectrometry (GC-MS)
The identification of the PSO specimen was carried out using a Shimadzu GC-MS instrument (model 5977 A, Japan) located in the central laboratory of the Faculty of Science, Ain Shams University. The GC-MS analysis was performed by injecting 0.5 μl of the ethanolic essential oil in split mode (15:1) at a temperature of 300°C. A capillary column (HP-5MS Capillary; 30 m x 0.25 mm I.D. x 0.25 μm film) was utilized. The carrier gas used was helium, with a flow rate of 1 ml/minute. The analysis program consisted of a column held at 60°C for 2 minutes after injection. The temperature was then increased to 200°C at a heating rate of 20°C/minute, followed by a 2.0-minute hold. Subsequently, the oven temperature was further increased to reach 300°C at a heating rate of 20°C/minute, again with a 2.0-minute hold. The GC-MS scan range encompassed 35–500 atomic mass units, utilizing electron impact ionization of 70 eV and a delay time of 4 minutes. The constituents of PSO were determined by comparing the fragmentation patterns of the mass spectra with the data available in the WILEY/NIST and Tutor Libraries (Moigradean et al. Reference Moigradean, Poiana, Alda and Gogoasa2013).
Molluscicidal activity of pumpkin seed oil
The molluscicidal activity of pumpkin seed oil was determined according to El-Gindy et al. (Reference El-Gindy, Rawi and Abdel-Kader1991), where a 1,000-ppm stock emulsion solution was formulated, and different concentrations of PSO (100, 200, 300, 400, 500, 600, and 800 ppm) were prepared in 100 ml water, using glass beakers. Each concentration was then used to expose 10 adult snails for four consecutive weeks, under controlled experimental conditions of temperature (25 ± 2°C) and pH (7.4), For each concentration, three replicates of 10 adult snails were used. Control groups of snails were kept in dechlorinated water under the same experimental conditions. At the end of the exposure period, the snails were removed from each test and thoroughly washed with dechlorinated water. Mortality rates were documented as the average of the three replicates.
Miracidicidal and cercaricidal activities
Five milliliters of dechlorinated tap water containing approximately 100 recently hatched miracidia or cercariae of S. mansoni were mixed with five milliliters of PSO solutions at double concentrations of 250, 500, 750, and 1,000 parts per million (ppm). A control was also included consisting of 10 milliliters of dechlorinated tap water containing 100 recently hatched miracidia or cercariae, as per the method described by Ritchie et al. (Reference Ritchie, Lorez and Cora1974). The mortality rates of stationary one were reported at time intervals (15, 30, 45, 60, and 120 minutes) since they were presumed to be dead as stated by WHO (1965).
Total hemocyte count
To determine the total hemocyte count, 20 μl of hemolymph were collected individually from at least 5–10 snails per group at two different time points (24 hours and 7 days) after exposure to PSO. The hemolymph was diluted in a leucocyte count solution at a 1:20 ratio, and the number of cells in each experimental and control group was quantified using a Bürker-Turk hemocytometer. Three replicates were made for each group, and the mean number of circulating hemocytes was calculated (Cavalcanti et al. Reference Cavalcanti, Mendonça, Duarte, Barbosa, De Castro, Alves and Brayner2012).
Differential hemocyte count
Hemocyte monolayers were prepared by adhering 10 μl of hemolymph to a glass slide and incubating it in a moist chamber at room temperature for 15 minutes. The slide was then washed with snail Wringer buffer containing 10 mM Ca++ (SR) pH 7.3 and incubated for an additional 10 minutes. Hemocytes were dehydrated with methanol for 5 minutes at room temperature and then stained with 10% Giemsa stain in buffered distilled water (0.021M Na2HPO4/0.015M KH2PO4) pH 7.2 for 30 minutes. Differences in hemocyte counts were determined for each experimental and control group, and the mean ± standard error was calculated for each hemocyte population. (Abaza et al. Reference Abaza, Hamza, Farag, Abdel-Hamid and Moustafa2016).
Scanning electron microscopy study
S. mansoni miracidia and cercariae were exposed to 618.4 and 290.5 ppm LC50 of PSO for 2 hours. The specimens were then washed twice in phosphate buffer saline (PBS) and fixed for 24 hours in 2.5% glutaraldehyde and 0.2 Molar cacodylate buffer (pH 7.2). After fixation, the specimens were rinsed and dehydrated in an ascending series of ethanol (70–100%). The dehydrated specimens were immersed in acetone and isoamyl acetate and dried using a transitional medium of liquid carbon dioxide (Grimaud et al. 1980). Finally, the specimens were sputter-coated with gold-palladium and photographed by a scanning electron microscope (JSM-5200 LA, JOEL Company, USA) at the Applied Center for Entomo-nematodes (ACE) located in the Experimental Research Station at the Faculty of Agriculture, Cairo University, Giza, Egypt.
Histological study
For histological analysis, snails were randomly selected from both the control group and the continuously exposed group for four successive weeks. The shell of each snail was carefully crushed between two glass slides, and shell fragments were eliminated using pointed forceps under a dissecting microscope. The soft tissues of the snails were then routinely processed for histological examination following the protocol established by Romeis (Reference Romeis1989). Finally, the processed tissues were photographed using a Carl-Ziess microscope from Germany equipped with a digital camera.
Detection of DNA damage by comet assay
Comet assay was conducted at the Immunobiology and Immunopharmacology Unit of the Animal Reproduction Research Institute in Giza to assess DNA damage. The alkali method protocol developed by Singh et al. (Reference Singh, McCoy and Tice1988) was followed. B. alexandrina snails were exposed to a concentration of 1,000 ppm of oil for one month. After exposure, the snails were homogenized and centrifuged to obtain a pellet, which was then resuspended in a chilled buffer for nuclei preparation. Microgels were created on microscope slides using agarose, and isolated nuclei were mixed with low melting-point agarose and applied to the slides. After solidification, coverslips were removed, and the slides were treated with lysis buffer to break down cell structures. The microgels were then subjected to DNA unwinding and electrophoresis in a gel chamber. After electrophoresis, the microgels were neutralized. The slides were stained with ethidium bromide and incubated, and the excess stain was removed. They were then stored in a dark and humidified chamber for analysis. Using a fluorescence microscope and image analysis, comets consisting of a brightly fluorescent head and a tail formed by DNA strand breaks were visualized and documented. Parameters such as tail DNA percentage and tail length were used to evaluate the extent of DNA damage in the cells.
Statistical analysis
Results were expressed as mean ± SD, and the obtained data were statistically analyzed using the ‘t’ test (Spiegel Reference Spiegel1981) and ‘chi-square’ values of contingency tables to determine the significant differences in means between the control and the experimental groups. The median lethal concentration (LC50) value was determined by applying non-linear regression to obtain 50% lethal concentration (LC50), with 95% confidence intervals using the SPSS version 20 statistical program (SPSS, Inc., Chicago, IL) for Windows. Values were expressed as mean ± SE.
Results
Gas chromatography-mass spectrometry (GC-MS)
The result of the chemical compositions of PSO by GC-MS is shown in Table 1. Qualitative analysis was done to determine the constituents of Cucurbita moschata, by using GC-MS, which shows the main component was palmitic acid with an average rate (of 17.42%), and the minor component was 6-chlorohexanoic acid with an average rate (of 0.01%). Most of the compounds extracted with ethanol were fatty acids (98.7%), sesquiterpene hydrocarbons (0.72%), and phenyl propanoid (0.58%).
Table 1. The main components identified in pumpkin seed oil by using GC-MS
Molluscicidal activity of PSO
There was no significant effect of PSO emulsion on B. alexandrina adult snails concerning the mortality rates after continuous exposure for 4 successive weeks. Adult B. alexandrina snails managed to survive in both higher (1,000 ppm) and lower (100 ppm) concentrations as shown in Table 2.
Table 2. Effect of PSO on B. alexandrina adult stages
Cumulative Mortality of Schistosoma mansoni miracidia and cercariae
Mortality rate of miracidia: PSO exhibited a larvicidal activity at different concentrations, but the most noticeable effect was after exposure to 1,000 ppm, where, after 15 minutes of exposure, the mortality rate of S. mansoni miracidia was 2.9%, 0% for the control group. Furthermore, prolonging the miracidial exposure resulted in mortality rates of 35%, 62.9%, and 71.5% after 45, 60, and 120 min, respectively, compared to 20 and 45% for the control group. Increasing the concentration to LC50 and LC90 induced severe and rapid mortality of treated miracidia during short exposure times, with a 100% death rate after 10 minutes at the LC90 concentration and 15 minutes at the LC50 concentration (Table 3 and Figure 1).
Table 3. Effect of PSO on Schistosoma mansoni miracidia and cercariae
Figure 1. Cumulative mortality rate [%] [A] S. mansoni miracidia; [B] S. mansoni cercariae post-exposure to different concentrations of PSO.
The present results revealed that PSO has a larvicidal activity against S. mansoni miracidia after 2 hours of exposure at LC50 618.4 ppm. Also, it showed a convenient cercaricidal activity against S. mansoni cercariae after 2 hours of exposure at LC50 290.5 ppm ( Table 4).
Table 4. Larvicidal activity of PSO against S. mansoni miracidia and cercariae after 2 hours of exposure
Total hemocyte count
The results of total hemocyte count in the hemolymph of B. alexandrina snails are presented in Table 5 and Figure 2. From these results, it was obvious that exposure of B. alexandrina snails to 1.0 mg/ml of PSO for 24 hours and 7 days resulted in a significant increase in the total number of hemocytes, being 2683 ± 53.58 and 3433 ± 34.69 hemocyte/ml, respectively, compared to 2033 ± 25.46 hemocyte/ml for the control snails.
Table 5. Total hemocytes count/ml of Biomphilaria alexandrina snail’s hemolymph exposed to 1.0 mg/ml of pumpkin seed oil
Data expressed as mean ± SE, *& ** = significantly different from control at p<0.05 and p<0.01
Figure 2. Total hemocytes count/ml of Biomphalaria alexandrina snail’s hemolymph exposed to 1.0 mg/ml of pumpkin seed oil.
Differential hemocytes count
The classification scheme of B. alexandrina hemocytes presented in this study is based on the cell size and shape. Figure 3 shows that examination of hemocytes monolayer obtained from control and treated snails using a light microscope resulted in the observation of three cell types. The first type measured 20–25 μm in diameter, had plentiful cytoplasm with numerous pseudopodia and an irregular nucleus, and adhered to glass – the name ‘large granulocytes’ denoting their active role in phagocytosis and other defense mechanisms. The second type was designated as small granulocytes, which were not fully differentiated, measuring 8–10 μm in diameter, having a high cytoplasm-nucleus ratio, having few cytoplasmic granules, and did not adhere to glass or emit pseudopodia. The third type, hyalinocytes, was morphologically intermediate between large granulocytes and small granulocytes. Hyalinocytes resemble large granulocytes but differ in that they have a lower nuclear-cytoplasmic ratio, measuring about 12–15 μm in diameter, they have markedly basophilic cytoplasm with abundant dense granules and, in addition to their larger size, can form few and short pseudopodia.
Figure 3. Examples of Biomphalaria alexandrina circulating hemocytes following exposure to 1.0 mg/ml of pumpkin seed oil for 7 Days. [a] Control samples showing G: large granulocytes, S: small granulocytes, and H: hyalinocytes. [b–f] treated samples, [b] showing the formation of pseudopodia [arrows], [c] bi-nucleated hyalinocyte, [d] showing both formation of pseudopodia [arrows] and oil droplets [OD], [e] showing oil droplets [OD] and extracellular granules, [f] and [e] showing a granulocytes engulfing oil droplets [OD]. Scale bar = 5 μm.
The results in Table 6 and Figure 4 indicate that the three cell types were observed in all experimental groups but varied in their relative proportions according to the stressor. In normal conditions (control group), the small granulocytes recorded 20% of total hemocytes, large granulocytes presented the dominant cell type being 62% of total hemocytes, and hyalinocytes did not exceed 18% of total hemocytes.
Table 6. Differential hemocyte count of Biomphalaria alexandrina snail’s hemolymph exposed to 1.0 mg/ml of pumpkin seed oil
Data expressed as mean ± SE, *& ** = sign
Figure 4. Differential hemocytes count/ml of Biomphalaria alexandrina snail’s hemolymph exposed to 1.0 mg/ml of pumpkin seed oil
Exposure of B. alexandrina snails to 1.0 mg/ml of PSO for 24 hours resulted in a significant increase in the large granulocytes proportion recording 74%, and this elevation was associated with a significant decline in the hyalinocyte proportion of 14%. The examination of the hemolymph monolayer films of B. alexandrina exposed to 1.0 mg/ml of PSO revealed the presence of numerous abnormalities shown in Figure 1. Some examples of these abnormalities are the formation of multiple pseudopodia, bi-nucleated cells, the occurrence of extracellular granules, and the existence of some granulocytes engulfing oil droplets.
Scanning electron microscopy (SEM)
The present study showed that PSO has a biocidal activity against miracidia and cercariae of S. mansoni. Exposure to LC50 (290.5 ppm) of PSO for 2 hours led to the reduction in movement of miracidia and sinking of the cercariae to the bottom of the container. This was followed by the death of both organisms. SEM studies showed that the normal unexposed miracidium of S. mansoni is covered with cilia (Figure 5a). The apical papillae showed a characteristic honeycomb pattern, whereas the miracidium exposed to LC50 (290.5 ppm) of PSO for 2 hours showed distinct loss of cilia from its surface (Figure 5b) and the protruded apical papillae showed swollen edematous corrugated areas.
Figure 5. Scanning electron microscopy [SEM] of Schistosoma mansoni miracidia, unexposed miracidia showing [a] cilia and tegumental folds covering the surface of the miracidium [dashed arrows] and the apical papillae of the miracidium with its characteristic honeycomb [arrow]. Miracidium exposed to LC50 [290.5 ppm] of pumpkin seed oil for 2 hours showing [b] loss of cilia and tegumental folds from the surface of miracidium with surface blubbing [dashed arrow], protruded apical papillae with swollen edematous corrugated areas [arrow].
In the present study, SEM of unexposed cercariae (Figure 6a) showed that the cercarial glycocalyx envelops the whole organism, the most anterior part of the head is provided with spiny tegmental folds, and the body region surface was irregular showing invagination and unfolding of the tegument forming frequent tubular profiles. Also, the cercarial tail and its furculae are covered by the glycocalyx similar to that seen on the body.
Figure 6. Scanning electron microscopy [SEM] of Schistosoma mansoni cercariae. [a] normal cercaria showing cercarial head [H], body [B], and tail [T] with its two furculae [F], covered with glycocalyx [arrow]. Cercariae exposed LC50 [290.5 ppm] of pumpkin seed oil for 2 hours showing [b] thickening of the tegument with marked loss of the glycocalyx [arrow]. [c] detachment of the body from the tail with marked loss of the glycocalyx [arrows] and surface blubbing [dashed arrow] and [d] deformation of the cercarial body [dashed arrow] and the two furculae are twisted around each other [arrow].
Cercariae exposed to LC50 (290.5 ppm) of PSO for 2 hours showed thickening of the tegument with marked loss of the glycocalyx (Figure 6b) and detachment of the body from the tail with marked loss of the glycocalyx and surface blubbing (Figure 6c). Some other exposed cercariae also showed deformation of the cercarial body, and the two furculae are elongated and twisted around each other (Figure 6d).
Histological studies
The digestive gland of B. alexandrina snails consists of multiple digestive tubules that are connected by connective tissue. Each tubule consists of two main cell types: the digestive and secretory cells with different shapes and functions (Figure 7a–d).
Figure 7. Light micrographs showing the effect on the digestive gland of Biomphalaria alexandrina snails. [a] Control: digestive tubule [DT], digestive cell [DC], secretory cell [SC], lumen [L]. [b–d]: Snails exposed to 1,000 ppm of pumpkin seed oil for 4 successive weeks showed the presence of cellular blebs [CB], vacuolation [V], dilated lumens [DL], enlarged interstitial spaces [IS], and tissue degeneration [arrow] and necrosis.
Histopathological investigations of the digestive glands of B. alexandrina snails showed that chronic exposure to 1,000 ppm of PSO for 4 successive weeks led to multiple pathological changes such as the degeneration of connective tissue between digestive tubules leading to an increase in the interstitial spaces, cellular blebbing, and excessive vacuolation. The digestive tubules became damaged, deformed, and necrotic (Figure 7a–d).
The hermaphrodite gland of B. alexandrina snails normally comprises simple branched acini that are connected by a thin layer of connective tissue. The acini contain different developmental stages of spermatogenesis and oogenesis (Figure 8a).
Figure 8. Light micrographs showing the effect on the hermaphrodite gland of Biomphalaria alexandrina snails. [a] Control: sperms [SP], lumen [L], different stages of oogenesis [arrows]. [b–d]: Snails exposed to 1,000 ppm of pumpkin seed oil for 4 successive weeks showed degenerated acini [DA], degenerated spermatozoa [DS], necrotic ova [NO], and necrosis [NE].
Furthermore, the chronic exposure had a dramatic impact on the hermaphrodite gland of B. alexandrina snails and altered the histological structure of ovotestis with an increase in the number of necrotic sperms, atrophy, deformation of male or female gametocytes, and degeneration of the acini (Figure 8b–d). It was also highly noticeable in multiple sections the shift towards either spermatogenesis or oogenesis as in Figure 8c.
Values of DNA damages detected by comet assay after exposure of tested substance for one month to 1,000 ppm PSO (results are expressed as means)
The study examined the impact of a concentration of 1,000 ppm of PSO on snail DNA for 4 weeks. The results revealed a remarkable and statistically significant (p<0.05) increase in DNA damage compared to the control group. Specifically, the tested concentration led to a noticeable increase in the percentage of tailed cells, with a value of 33.6 ± 0.5%, whereas the control group only exhibited 9.2 ± 0.3%. Additionally, the olive tail moment, which represents DNA damage, was significantly higher at 2.1 ± 0.05 units compared to the control group’s value of 1.4 ± 0.001 units as shown in Figures 9 and 10.
Figure 9. Values of DNA damages detected by comet assay after exposure of tested substance for one month to 1,000 ppm PSO. Data represent as mean ± SD of three identical experiments made in triplicates, and significance is ascribed as *p≤0.05. In all cases, significance was tested concerning control using a t-test. Tail DNA [%], % tailed [DNA damage], and olive tail moment were significantly increased [p≤0.05] with increased concentrations of tested botanical material.
Figure 10. DNA comet assay after exposure of tested substances after one month to 1,000 ppm PSO comet assay records show [a] Control group; [b] Adult B. alexandrina snails after exposure to 1,000 ppm of PSO for 4 successive weeks.
Discussion
Schistosomiasis is a devastating parasitic disease that affects millions of people worldwide. The control of this disease is largely based on the use of molluscicides to eliminate the snail vectors that transmit the parasite. However, the use of synthetic chemicals for this purpose has raised concerns about their potential toxicity to humans and the environment (Wang et al. Reference Wang, He, Juma, Kabole, Guo, Dai, Li and Yang2019).
In recent years, there has been increasing interest in identifying natural compounds that have molluscicidal and schistosomicidal activities, with a lower risk of harmful effects (Coelho and Caldeira Reference Coelho and Caldeira2016; Mossalem Reference Mossalem2018; Silva et al. Reference Silva, Siqueira, Sá, Silva, Martins, Aires, Amâncio, Pereira, Albuquerque, Melo and Silva2018).
Pumpkin seeds are considered a medicinal plant and have widely been used in traditional treatment worldwide for the prevention of several diseases (Hussein and Shukur Reference Hussein and Shukur2020).
Pumpkin seed oil is extracted from the seeds of the pumpkin plant (Cucurbita moschata) and has been traditionally used in food, cosmetics, and medicine. It was obtained by ethanol extraction, and its chemical constituents were 24 constituents as detected by GC-MS. The major constituent was fatty acids (98.7%) as palmitic acid with an average rate (17.42%).
GC-MS analysis is a powerful tool for identifying and quantifying the chemical compounds in natural products. The GC-MS composition of PSO varies depending on the geographical origin and extraction method. However, most studies have identified linoleic acid, oleic acid, and palmitic acid as the major fatty acids in the oil, which have numerous health benefits. Moreover, the oil has been reported to possess molluscicidal activity against snail vectors of schistosomiasis, a parasitic disease that affects millions of people worldwide (Mtemeli et al. Reference Mtemeli, Walter and Shoko2020; Mtemeli et al. Reference Mtemeli, Walter, Tinago and Shoko2021; Šamec et al. Reference Šamec, Loizzo, Gortzi, Çankaya, Tundis, Suntar, Shirooie, Zengin, Devkota, Reboredo‐Rodríguez and Hassan2022).
A previous study by Selvi and Santhanam (Reference Selvi and Santhanam2016) dedicated that palmitic acid has antioxidant, hypocholesterolemic, nematicidal, pesticidal, lubricant, and hemolytic properties which may be the main effector in the larvicidal impact of the tested oil. Also, it was previously mentioned that the Cucurbitaceae plant family, collectively known as cucurbits, exhibit berberine and palmatine demonstrated for their inhibitory effects against Toxoplasma gondii, antimalarial, anti-leishmaniasis, and anti-schistosomiasis properties (Grzybek et al. Reference Grzybek, Kukula-Koch, Strachecka, Jaworska, Phiri, Paleolog and Tomczuk2016).
Several studies have identified the major compounds present in PSO using GC-MS analysis. For instance, a study conducted by Prommaban et al. (Reference Prommaban, Kuanchoom, Seepuan and Chaiyana2021) reported that the major fatty acids in PSO were linoleic acid, oleic acid, and palmitic acid. Other minor components identified included stearic acid, linolenic acid, and myristic acid. Moreover, the study found that PSO also contained various phytosterols and tocopherols.
Similarly, another study conducted by Benalia et al. (Reference Benalia, Djeridane, Gourine, Nia, Ajandouz and Yousfi2015) reported the GC-MS composition of PSO. The study identified linoleic acid (18.4–39.6%), oleic acid (18.4–39.6%), and palmitic acid (13.9–20.0%) as the major fatty acids in the oil. Additionally, the study identified various other minor compounds such as stearic acid, linolenic acid, and myristic acid.
The proceeding data showed that PSO had no significant effect on B. alexandrina adults after chronic exposure for 4 successive weeks in both higher (1,000 ppm) and lower (100 ppm) concentrations. Even though there is a dearth of literature on the molluscicidal effects of PSO on vector snails, numerous studies show that using pumpkin seed extracts has no detrimental effects on the health of rats and pigs used as models. According to Grzybek et al. (Reference Grzybek, Kukula-Koch, Strachecka, Jaworska, Phiri, Paleolog and Tomczuk2016), feeding pumpkin seed extract to swine and rats over an extended period produced no fatal consequences. From a significant point of view, this result is considered logical enough as pumpkins are generally known not only for their edible fruits and seeds but also for their several health benefits and thus have been used for a long time in traditional medicine in many countries (Mtemeli et al. Reference Mtemeli, Walter, Tinago and Shoko2021).
PSO exhibited larvicidal activity at different concentrations against Schistosoma mansoni miracidia and cercariae with LC50 (618.4 and 290.5 ppm), respectively, after 2 hours of exposure. Such a noticeable effect comes in line with many studies that have been done on pumpkin seeds and stated their anthelmintic potential, which proved to be a success on S. mansoni and nematodes as well (Beshay et al. Reference Beshay, Rady, Afifi and Mohamed2019; Ježek et al. Reference Ježek, Mirtič, Rešetič, Hodnik and and Rataj2021).
The current findings clearly showed that acute and chronic exposure of B. alexandrina snails to 1.0 mg/ml of PSO for 24 hours and 7 days, respectively, resulted in a significant increase in the total number of hemocytes compared to the control snails (p<0.05).
Based on cellular and humoral defense components, mollusks have an effective internal defense system (Le Clec’h et al. Reference Le Clec’h, Anderson and Chevalier2016; Wang et al. Reference Wang, Tang, Li, Wu, Qiao, Wan, Qian and Liu2023). Circulating hemocytes are the primary regulator of cellular defense responses against any external stressor in B. alexandrina snails (Fried Reference Fried2016). Some other studies reported also that immune stimulation of B. alexandrina snails with sodium alginates and β-glucans led to an obvious elevation in hemocyte count in a time-dependent manner (El Sayed et al. Reference El Sayed, Soliman, Elfekky and Ouf2017; Soliman et al. Reference Soliman, El Sayed, Abou Ouf, El Fekky and Gad2017).
Granulocytes are widely documented to play a crucial part in the immune system’s defense mechanism against biotic and abiotic foreign invaders in snails (Al-Khalaifah Reference Al-Khalaifah2022). Granular (spreading) hemocytes were the most impacted cell type following treatment with pumpkin seed oil, which was corroborated by the current findings.
The increase in the total number of hemocytes and granulocytes portion in the present study was also associated with some abnormalities indicating the immune response where some of B. alexandrina snails’ hemocytes tended to form pseudopodia, bi-nucleated hyalinocyte, and extracellular granules, and they exhibited some granulocytes engulfing oil droplets following chronic exposure to 1.0 mg/ml of PSO for 7 days.
These consequences are consistent with several studies that have demonstrated the effectiveness of snail hemocytes’ innate immune responses and their capacity to identify, cling to, encircle, and ultimately eradicate foreign objects and invasive parasites in a crucial defense-related process known as phagocytosis (Oliveira et al. Reference Oliveira, Levada, Zanotti-Magalhaes, Magalhães and Ribeiro-Paes2010; Gad El-Karim et al. Reference El-Karim2022).
Scanning electron microscopy showed that PSO has a larvicidal activity against miracidia and cercariae of Schistosoma mansoni. The impact was highly related to surface blubbing, edematous corrugated areas, and obstruction of means of movement in both stages. These structural changes may account for the inability of miracidia to find the compatible host or cercariae to infect the final host. Moreover, surface blebbing is considered an indicator of stress and has been observed in previous SEM studies evaluating anti-schistosomal drugs (Manneck et al. Reference Manneck, Haggenmüller and and Keiser2010). The current study agrees with the investigation of the morphological alterations and motility of cercariae and miracidia both prior to and following exposure to antioxidants where scanning electron microscopy analysis revealed that the morphological alterations and lack of mobility of cercariae and miracidia increased with exposure time and concentration. (Mossalem Reference Mossalem2018).
On the histopathological level, various observations were noticed in the digestive gland of B. alexandrina snails following chronic exposure to 1.0 mg/ml of pumpkin seed oil for 4 successive weeks, represented mainly in tissue damage, deformation, and necrotic changes. The involvement of the digestive glands in homeostasis, pollutant uptake, digestion, metabolism, and the detoxification process is closely related to their high sensitivity (Abdel-Azeem et al. Reference Abdel-Azeem, Mohamed and Habib2023). The tested oil may have accumulated directly in the digestive gland cells, or it may have done so indirectly through oxidative damage brought on by the generation of reactive oxygen species (ROS). Tissue damage involving a range of physiological functions and environmental factors, such as necrotic and apoptotic cell death, has been connected to high oxidative stress (Habib et al. Reference Habib, Ghoname, Ali, El-Karim, Youssef, Croll and Miller2020). Furthermore, B. alexandrina snails’ hermaphrodite gland suffered collateral damage as a result of contact with the oil under consideration. These alterations are probably going to decrease snails’ capacity for reproduction and, consequently, their level of fitness within the aquatic ecosystem (Larson et al. Reference Larson, Greenwood, Flanigan and Krist2023).
The molluscicidal activity of PSO against snail vectors of schistosomiasis is attributed to its high content of fatty acids. These compounds may disrupt the snail’s cell membranes, leading to its damage (Zhang and Zou Reference Zhang and Zou2020). Moreover, the oil’s toxicity against the snail varies depending on the geographical origin, extraction method, and dosage. The specific mechanism by which pumpkin oil acts as a molluscicide is not fully elucidated.
So, in the present study, we investigated the potential genotoxic effects of PSO on the DNA of B. alexandrina snails using the comet assay. The comet assay or single-cell gel electrophoresis is a reliable and sensitive technique that is used to detect DNA damage in individual cells. The assay involves embedding cells in agarose, lysing the cells to release the DNA, and subjecting it to electrophoresis. The damaged DNA fragments of the cells can then be visualized as comet-like structures under a microscope. The comet assay is widely used as a biomonitoring tool to evaluate the genotoxic effects of various agents (Nandhakumar et al. Reference Nandhakumar, Parasuraman, Shanmugam, Rao, Chand and Bhat2011).
A concentration of 1,000 ppm of PSO was applied to adult snails for 4 weeks. The results demonstrated a significant increase (p≤0.05) in DNA damage compared to the control group, as indicated by changes in comet parameters such as the percentage of tailed cells, tail DNA percentage, and olive tail moment. These findings emphasize the genotoxic potential of PSO highlighting its impact on DNA integrity in B. alexandrina snails.
The cells of the control showed almost rounded nuclei in cells exposed to tested botanical material. The nuclei with a clear tail-like extension were observed, indicating that cells of adult snails were damaged and DNA strand breaks had occurred. In damaged cells, breaks appear as fluorescent tails extending from the core towards the anode. Therefore, the migrated nuclear DNA was considered damaged DNA (Collins et al. Reference Collins, Møller, Gajski, Vodenková, Abdulwahed, Anderson, Bankoglu, Bonassi, Boutet-Robinet, Brunborg and Chao2023).
Conclusion
The present study demonstrated the potential effects of pumpkin seed oil (PSO) as a molluscicide, miracidicide, and cercaricide. PSO exhibited remarkable miracidicidal and cercaricidal activities against Schistosoma mansoni larval stages, which is an important focus in infection control strategies, and it caused multiple pathological changes in the histological examination of B. alexandrina snails. Furthermore, PSO had morphological changes in snail hemocytes and showed genotoxic effects. These results suggest that PSO could be considered as a possible alternative to traditional molluscicides for the control of schistosomiasis under appropriate field conditions. However, further studies are necessary to evaluate the efficacy and safety of PSO in field conditions and to optimize its application. Moreover, studies should also aim to investigate the mechanism of action of PSO against different schistosome species and other aquatic organisms. Identification of active compounds present in PSO and their efficacy in inhibiting schistosome development and growth may also provide valuable insights into the development of new anti-schistosomal drugs. Overall, this study has provided valuable data on the potential of PSO in controlling schistosomiasis, but further research is needed to explore these findings.
Data availability
All data generated or analyzed during this study are included in this published article.
Author contribution
SM, RE, and SF conducted practical work and contributed to the data collection. AA and RE wrote the initial draft of the paper. HM revised and edited the manuscript. AA collected and organized the research material and finalized the manuscript.
Funding
No funding was received to assist with the preparation of this manuscript.
Competing interest
The authors declare no competing interests.
Ethics approval
The study was performed following the recommendations of the Ethics Committee of Theodor Bilharz Research Institute (TBRI), Imbaba, Giza governorate, Egypt, and found that the research work is exempted from review as the research does not involve human or experimental animals.
Consent for publication
The authors declare that this manuscript is not published elsewhere. All the co-authors meet the criteria for authorship and ensure appropriate acknowledgements made in the manuscript. All the authors have read the manuscript and approved it entirely.
Financial interest
The authors have no relevant financial or non-financial interests to disclose.
