Molluscicidal Activity of Cannabis sativa , Acacia nilotica and Tinospora cordifolia Extracts Against Vector Snail Lymnaea acuminata

Background and Objective: Fresh water snails of family lymnaeidae serve as vectors for parasitic trematode Fasciola hepatica which causes a serious zoonotic disease fascioliasis. The study aimed to investigate the possibility of indigenous plants Cannabis sativa , Acacia nilotica and Tinospora cordifolia to serve as natural molluscicides to control the population of vector snails. Materials and Methods: Aqueous extracts were prepared from freshly collected aerial parts of these plants. Healthy and acclimatized snails were exposed against different concentrations of the plant extracts continuously up to 96 hrs to analyse the toxicity of these extracts. Results: Mortality of snails after exposure of different combinations of the extracts indicates that the extracts from indigenous plants C. sativa , A. nilotica and T. cordifolia shows very promising molluscicidal activity against Lymnaea acuminata . Leaves of Cannabis sativa showed highest toxicity. Conclusion: This study obviously indicates that these indigenous plant extracts can be used as a potent natural molluscicides.


INTRODUCTION
Fascioliasis is an important zoonotic disease caused by two trematodes, Fasciola hepatica and Fasciola gigantica [1][2][3] .These parasitic trematodes are of considerable veterinarian and medical importance.Fascioliasis has recently proved to be an important public health problem, with human cases reported in 91 countries and the global burden of fascioliasis estimated to be 2.7 million 4,5 .Population in the developing countries are likely to be most affected by facioliasis as the COVID-19 pandemic impacts their health symptoms and efforts to control Fasciola decline [6][7][8] .Yattoo et al. 9 reported two cases of human fascioliasis from Kashmir Valley in India.
Species of freshwater snails from the family Lymnaeidae are well known for their role as the intermediate host for Fasciola species.The most important host for F. hepatica is Lymnaea acuminata (Fig. 1 and 2) and Lymnaea rufescence in Indian subcontinent [8][9][10][11][12] and Lymnaea trunculata in Europe, Asia, Africa and North America 8,13,14 .The control of fascioliasis depends on the understanding of the ecology, biology and distribution of the intermediate host snails.Controlling the population of the host snails below the threshold level can be a rapid and effective means of reducing fascioliasis 3,14 .Various synthetic molluscicides have been widely used for effective control of harmful snails and such molluscicides includes copper sulphate, trifenmorph, Bayluscide, sodium pentachlorophenate, copper pentachlorophenate and niclosamide 15,16 .Significant success has been achieved by the use of these synthetic chemicals for the elimination of harmful snails.Nonetheless, the use of these synthetic molluscicides for controlling snails causes serious environmental pollution 17 .
There are large numbers of pharmacological effects of C. sativa, A. nilotica and T. cordifolia have been reported that C. sativa shows anti-oxidant activity 18,19 , larvicidal activity against mosquito larvae 20 and anti-microbial activity 21,22 .Revathi et al. 23 reported anti-cancer, anti-microbial and anti-oxidant properties of A. nilotica.Anti-cancer, anti-viral infection, inflammation, immunomodulatory role and neurological and anti-diabetes properties of T. cordifolia have already been reported 24 .
The search indigenous of plants with molluscicidal activity is considered more sustainable than the use of synthetic molluscicides.The present study aimed to evaluate the molluscicidal activity of Cannabis sativa, Acacia nilotica and Tinospora cordifolia to explore the full potential of these indigenous plants as potent molluscicides.

MATERIALS AND METHODS
The experiment was carried out in the Malacology Laboratory, Department of Zoology, D.D.U.Gorakhpur University, Gorakhpur in August to November, 2022.
Collection of plant materials: Aerial parts of Cannabis sativa (leaf, stem), Acacia nilotica (leaf, bark) and Tinospora cordifolia (leaf, stem) were collected freshly from local areas and identified by the Department of Botany, DDU Gorakhpur University, Gorakhpur, India.Animal collection: Adult Lymnaea acuminata (1.35±0.2cm in length) were collected from freshwater ponds of Gorakhpur District, Uttar Pradesh, India.The snails were acclimatized for 72 hrs in laboratory conditions.Ten snails were then allocated to each of the groups and immersed in 3 L untreated dechlorinated tap water at 23±1°C.The pH, free carbon dioxide, dissolved oxygen and bicarbonate alkalinity were 7.1-7.3,5.2-6.3,6.6-7.3 and 102-104 mg LG 1 , respectively.The dead snails were removed from the aquaria to avoid any contamination.

Plant extract preparation:
The extraction of plant parts was performed by the method of Singh and Agarwal 25 with some modifications.Freshly collected leaves and stems of C. sativa were washed thoroughly, then finely cut into small pieces and then separately mixed with water (50 g leaves/stem+50 mL water) then filtered with Whatman No. 1 filter paper.The filtered extract thus obtained was evaporated under a vacuum.The residues thus obtained were used for the determination of toxicity.The same procedure was performed with A. nilotica (leaf, bark) and T. cordifolia (leaf, stem) extraction.
Molluscicidal activity test: Molluscicidal evaluation of plant extracts was performed according to WHO guidelines as modified by Singh and Agarwal 25 .Ten uninfected snails were kept in a glass aquarium containing 3 L deionized water.In each setup, snails were prevented from crawling out of the aquaria employing a fine mesh placed above the water surface.Snails were challenged for 96 hrs with various concentrations of different plant part extracts singly and in various binary combinations (Table1).Six replicates were set up for each concentration.Control experiments were performed with deionized water without any treatment (negative control).These snails were neither fed nor disturbed during the exposure period.Mortality of snails was recorded at an interval of 24 up to 96 hrs.Death of the snails was determined and confirmed by lack of reaction to irritation of foot with needle prove to elicit typical withdrawal movements and contraction of their body in the shell (Fig. 2).

Statistical analysis (p<0.05):
The LC 50 values, Lower Confidence Limits (LCL), Upper Confidence Limits (UCL), t-ratio heterogeneity factor, slope values and g-values were calculated by polo software program (PoLo Plus LeOra software version 2.0).

Ethical consideration:
All applicable protocols of institutional, national or international guidelines for the care and use of animals were followed during this experiment.

RESULTS
The toxicity of extracts of C. sativa (leaf, stem), A. nilotica (leaf, bark) and T. cordifolia (leaf, stem) against Lymnaea acuminata was time and concentration-dependent.The LC 50 determined after 24 hrs treatment was highest in C. sativa leaf extract (304.31mg LG 1 ) (Table 2).Cannabis sativa stem extract was more toxic (24 hrs LC 50 = 404.39mg LG 1 ) than aerial part extracts of A. nilotica and T. cordifolia (Table 2).There was a significant (p<0.05)negative correlation between the LC 50 and the exposure period.The order of 24 hrs toxicity of different plant parts extracts singly against L. acuminata were C. sativa leaf>C.sativa stem>A.nilotica leaf>T.cordifolia leaf>A.nilotica bark>T.cordifolia stem (Table 2).
It is evident from the results that extracts of C. sativa (leaf, stem), A. nilotica (leaf, bark) and T. cordifolia (leaf, stem) are toxic against the snail Lymnaea acuminata.The toxicity of C. sativa leaf is highest in comparison to the stem and other parts.Binary combinations of extracts of plant pats show increased toxicity than they are used separately.
The slope values given in Tables 2-5 were steep.Based on each of the six replicates the separate estimates of LC values were found to be within 95% confidence limit of LC 50 .The t-ratio was higher than 1.96 and the heterogeneity factor was less than 1.0.The g-value was less than 0.5 at all the probability levels (90, 95 and 99).There was a significant negative regression (p<0.05) between exposure time and LC 50 of the exposures (  ----------------- Acacia nilotica, T. cordifolia: Tinospora cordifolia, LCL: Lower confidence limit, UCL: Upper confidence limit, Significant negative regression (p<0.05) was observed between exposure time and LC 50 of treatments Table 5: Toxicity of binary combinations (5:1) of crude extract (aqueous) of plant parts of C. sativa, A. nilotica and T. cordifolia against snail Lymnaea acuminata Limits -------------------Treatment Exposure period Effective dose (mg LG

Table 4 :
Toxicity of binary combinations (1:5) of crude extract (aqueous) of plant parts of C. sativa, A. nilotica and T. cordifolia against snail Lymnaea acuminata Limits - Mortality was determined every 24 up to 96 hrs, each set of experiment was replicate six times, C. sativa: Cannabis sativa, A. nilotica: