Potential of Animal Bile as Bio-molluscicide against Land Snail Monacha cartusiana

Molluscs are the second-largest invertebrate category after arthropods in the animal kingdom and they make a substantial contribution to the overall biodiversity of the planet (Rosenberg 2014). Vegetables and ornamental plants are typically severely damaged by terrestrial snails, which are typically found in gardens and grassy areas (Capinera and White 2011). Both animals and humans are unable to consume these plants because of the unpleasant odour and taste that are left behind by the mucous secretions that snails leave behind on plants throughout their movements (Abd El-Atti et al., 2019). It has been determined by Iglesia et al., (2003) that snails and slugs are among the most dangerous agricultural pests, as they cause significant economic damage to field crops, vegetables and horticultural plants. Land snails are responsible for the transmission of bacteria, viruses and fungi to plants by the act of scratching plant parts when they are feeding (Raut and Barker, 2002). This affects plants in both a direct and indirect manner. According to Castle et al., (2017), the most common approach to controlling land snails comes in the form of chemical molluscicides. According to Khalil (2016), methomyl is the most widely used molluscicide because of its capacity to kill molluscs and its ease of application. This particular molluscicide, on the other hand, is toxic to beneficial invertebrates and poses a threat to humans, other animals, and the environment as a whole (Moustafa et al., 2016). According to Geasa et al., (2013), natural molluscicides are considered to be the most effective alternative to chemical molluscicides. This is because natural molluscicides are less expensive and pose fewer dangers to the general environment. Natural enemies, which include predators, diseases and parasites, have the potential to be a successful technique for the biological control of snails even over the long term. A few years ago, biological treatment with microbial agents against various land snails and slugs gained a lot of interest (Kramarz et al., 2007; Genena and Mostafa 2008; Shahawy 2018).
       
Several pharmacologically active chemicals have been derived from traditional medicinal plants, according to Yousif et al., (2007). The World Health Organization (WHO) estimates that between 65 and 80 percent of the population in developing countries relies on traditional medicine to cover their primary healthcare needs (Kumar et al., 2021). For this reason, a significant amount of work has been put into locating plant items that are safe for the environment, do not contain any poisonous substances and are selectively active for the integrated management of snails. Several of these plant products have demonstrated very encouraging results. Recently, for instance, we have conducted research that shows the potential for Trichoderma harzianum to act as a biocontrol agent against the land snail (Ahmed et al., 2023). Recent studies have demonstrated that certain natural compounds, like as extracts of Pulsatilla chinensis (Bunge) Regel, can kill O. hupensis. These extracts have been shown to have molluscicidal activity. According to Chen et al., (2012), they pose a lower risk of being detrimental to aquatic creatures that are not the intended targets of the molluscicide niclosamide. Additionally, Agave attenuata Salm-Dyck is poisonous to the snail Bulinus africanus, which is the target species, but it has fewer negative effects on fish and animals, or none at all (Brackenbury and Appleton 1997).
       
Traditional Chinese medicine (TCM) has utilized gallbladder liquid and various bile components derived from a wide variety of animals for several centuries (Wang and Carey, 2014). According to the biochemical compounds and pharmacological activities of bile, such as specific bile salts, the bile pigment bilirubin and its glucuronides, secondary bile ingredients such as Vitamins A, D, E, K and melatonin synthetic ingredients, animal bile, such as cow, sheep and others promising candidates for bear bile’s similar and alternative therapeutic purposes (Li et al., 2016). Animal bile has been shown to have anti-inflammatory, antioxidant, antipyretic, anodyne, anticonvulsive, antiallergic, anti-congestive, antidiabetic and antispasmodic characteristics (Wang and Carey 2014; Zehua 2015). Additionally, it has been shown to dissolve gallstones, improve liver function and suppress the reproduction of bacteria and viruses. Research conducted in the field of modern therapeutics has revealed that bear bile possesses a wide variety of pharmacological qualities. These properties include hepatoprotection, antibacterial, antiviral, anti-inflammatory, anti-gallstones, hypolipidemic and other actions (Kou et al., 2014; Zhao et al., 2015). According to Wang and Carey (2014), traditional Chinese medicine made use of goat bile as a medicinal agent since it was believed to be effective in the treatment of optic atrophy, acute hemorrhagic conjunctivitis and several infectious skin illnesses. Chicken bile has been used to control coccidiosis, as it has shown a destructive effect on the oocysts and their morphology of Eimeria papillate (Murshed et al., 2023).  The present study was the first to evaluate the potential activities of sheep bile against M. carutisiana.

Animal material
 
Bile liquid was acquired from the intact gallbladder of lambs from a slaughterhouse in Riyadh (Saudi Arabia). Gallbladders were isolated from 8 free-disease lambs. Gallbladders were sterilized with 80% methanol before eradicating the gallbladder, then Suctioning bile fluid with a syringe, transferred and collected to clean tubes and kept at 4°C until they were utilized.
 
Snails collection
 
The experimental design was carried out at the Zoology Department, Laboratory of Parasitology, Faculty of Science, King Saud University, for almost 2-3 months in September-November 2023. M. cartusiana adult snails were collected from the Al Ammariyah district of Riyadh, Kingdom of Saudi Arabia. Snails were transported in muslin bags, placed in plastic containers with sterilized, moist sandy loam soil in a 1:1 (V:V) ratio and given fresh lettuce (Lactuca sativa) leaves for 2 weeks to adapt to laboratory conditions.
 
Chemical analysis using (GC-MS)
 
Utilizing a Trace Ultra Gas Chromatographer in conjunction with a DSQII Mass Spectrometer (Thermo Scientific), an analysis of the volatile components of bile was carried out. To chromatographically separate the components according to the method of Adams (1995), the TR-5 MS capillary column was utilized. This column had a length of 30 m, a diameter of 0.25 mm and a length of 0.25 m. The substances were discovered by using pertinent data that was kept in databases from literature and equipment (Adams Book 07, Nist 98 and Xcalibur). A variety of n-alkanes, ranging from C8 to C24, were utilized in the calculation of the Relative Retention Index. Compounds’ relative percentages have been obtained by electronic means, using data on the percentage of the region.
 
TPC, TFC and TTC measurement
 
The phenolic and flavonoid, content of bile were determined according to the method by Ordonez et al., 2006. The blue color absorbance was measured at 765 nm with a UV-visible spectrophotometer. Based on the curve calibration equation:
   
The bile TPC and TFC, content was estimated as gallic acid equivalent and quercetin (mg/g DW), where (y) is absorbance and (x) is concentration.
       
The total tannin content was determined by utilizing this approach for the bile. An overall volume of 0.1 mL of the extract samples was put into an Eppendorf tube with a capacity of 2 mL, which already included 1.5 mL of Milli-Q water and 0.1 mL of the Folin-Ciocalteu phenol reagent. This mixture was incubated for 8 minutes. After that, 0.3 mL of a sodium carbonate solution containing 35 % was added to the mix. After that, the ingredients were thoroughly combined and then placed in a dark, ambient temperature area for twenty minutes. The measured value for the wavelength was 700 nm. The following equation, was utilized to determine the total tannin content that was present in the leaf extract. The total tannin content that was calculated was given in units of mg/g dry weight (DW).
   
Cytotoxicity evaluation (MTT assay)
 
Cell culture
 
The Hep-G2/2.2.15 Human Hepatoblastoma Cell Line was gained of (Sigma-Aldrich Chemie GmbH,Taufkirchen - Germany). The cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) that was supplemented with 10% fetal bovine serum (FBS) in an atmosphere that was humid and contained 5% CO₂ at 37°C.
 
Cell viability assay
  
The detection of cell viability and cell growth was performed using MTT Assay (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium Bromide, cat#475989-1GM, Sigma-Aldrich, Germany). Briefly, aliquots of 120 µl of the suspended cells (5 × 10⁴ mL^-1) were given to 60 µL of a serial dilution of the bile in a 96-well plate. After 3 days of incubations, 20 µl of MTT-solution was given to each well (Mosmann 1983) and the cells were further cultivated for an additional two hours. Formazan crystals were dissolved in isopropanol. The intensity of the resulting color was measured at 595 nm as described previously.  
Experimental design
 
The snails were kept at room temperature (23-28°C) and 75% relative humidity. Snails were divided into 5 groups, each of five snails. The first group was treated with sterilized water and served as a control group. The second to fifth groups were treated with the concentration (12.5, 25, 50 and 100%) of bile liquid.
 
Statistical analysis
 
An analysis of variance (ANOVA) with a single-way was performed on the data using SPSS (version 18) (SPSS Inc., Chicago, IL, USA). A two-tailed paired t-test and Duncan’s multiple ranges were utilized to investigate the statistical differences that existed between the means of the various groups.

GC/MS analysis was performed on the volatile contents of bile liquid, it was shown that bile contains 12 ingredients (Table 1), with oleic acid being the most prominent component (Fig 1). These compounds belong to the classes of monoterpenes, furanes, isocyanate metabolites, alkanes and alcohols, which are comparable to the compounds that were reported by Murshed et al., (2022). Because of the presence of multiple bioactive components, it is not possible to attribute the biocontrol actions of bile to a single bioactive constituent; rather, it is more likely to be attributed to the synergism among the various bioactive constituents.
 

 

       
Fig 2 revealed the findings of the phenolics TPC, flavonoids TFC and tannin TTC measurements taken of the bile liquid. The total potency concentrations of the bile were determined to be 50.65079365±0.809 mg GAE/g DW, 29.39351852±0.386mg QE/g DW, and 27.41669074±1.066 mg TAE/g DW, respectively (Fig 2).
 

       
The findings regarding the cytotoxicity of the bile liquid are presented in Fig 3, which shows the results of testing it at varying concentrations. The test included exposing Hep-G2 (hepatoblastoma cell line) cells to diverse concentrations of bile, namely 1, 12.5, 25, 50, 100, 200, 400 and 800 µg/mL. The survival ratio of the cells at these concentrations was found to be 99.76, 84.00, 73.19, 62.27, 53.11, 35.29, 18.81 and 5.19%, respectively. The IC50 value for Hep-G2 was calculated to be 151.569±8.5 µg/mL. As can be explained in Fig 3, the percentage of cells that managed to survive was reduced as the concentration of the bile was raised (Fig 3).
 

       
Molluscs are the second-largest invertebrate group in the animal kingdom, following arthropods (Rosenberg 2014). Due to snail resistance, great effort is being put into creating new, safe and effective treatments for M. cartusian.  After being submerged in concentrations, the snails generally displayed agitated behavior, moving in circles before slowing down and eventually dying. In the contact assay, bile at various doses demonstrated significant molluscicide activity against adult M. cartusian (Fig 4). Since it is believed to be ideal for pest management tactics and helps in avoiding the health and environmental concerns of chemical pesticides, the genus bile is now attracting a lot of research (Kumar et al., 2019). This is because it is supposed to be suitable for these strategies. To combat a variety of plant infections and insect pests, bile is widely utilized as a biocontrol agent (Napitupulu et al., 2019). The development of several secondary metabolites that are volatile, non-volatile, antifeedant and repellant, such as pyrones, sesquiterpenes and peptaibols, was found to be connected with its potential for utilization (Reino et al., 2008). In light of this, the purpose of this research was to determine the chemical components of bile and to assess the efficacy of bile as a molluscicide against M. cartusiana in a laboratory setting. The bile is efficacious in preventing it due to its inhibitory capability and influence on the sporulation of coccidian oocysts of E. stiedae (Murshed et al., 2022).
 

       
The results showed that the bile molluscicidal effect against the M. cartusian snail was concentration-dependent. It was discovered that as bile concentration increased, the survival rate of M. cartusian snails significantly decreased (Fig 5). The highest mortality (97%) was attained at the dose of 100% of bile.
 

 
The data revealed substantial differences between the mortality means of 100% concentration after 40 minutes, 50% concentration of bile after 20 minutes and the remaining mortality means (Table 2). There were also significant changes between the control and 100% concentrations after 40 minutes (Fig 6). The maximum mortality occurred after 40 minutes at 50 and 100% doses. The lowest mortality was seen after 5 minutes at all doses (Table 2). Significant differences (P<0.05) were seen in M. cartusian. Adult death rates by bile concentration (12.5, 25, 50 and 100%) and control (Fig 7). These findings demonstrated that bile has activity against snails.
 

 

 

Bile is a very promising candidate that could be used to control land snails, or exploited as a starting point to develop new, effective and environmentally friendly molluscicides. Bile showed molluscicide activity almost comparable to that of the recommended chemical molluscicides and in contrast, bile is less harmful to the environment and health. The exact mode of action should be investigated.

We thank the Project of Supporting Researchers No. RSP2024R94, at King Saud University, Saudi Arabia.

The authors declare that there are no conflicts of interest.