Molluscicidal potential of Lantana indica and Alstonia scholaris plants against freshwater snail Lymnaea acuminata
S Chauhan, A Singh
acetone extract, alstonia scholaris, fascioliasis, lantana indica, lymnaea acuminata, molluscicidal activity, schistosomiasis
S Chauhan, A Singh. Molluscicidal potential of Lantana indica and Alstonia scholaris plants against freshwater snail Lymnaea acuminata. The Internet Journal of Toxicology. 2009 Volume 7 Number 2.
Laboratory evaluation was made to assess the molluscicidal activity of acetone extracts of
From ancient time, plants are rich source of effective and safe compounds which are used for different purposes i.e. traditional medicines and control of pests and vectors (13). So control of vector snail through plant origin molluscicides is a very effective and new tool of integrated vector management programme.
Materials and methods
Plant: The leaves of the plant
Extraction of compounds: 50 gram powder of leaf of
Animal: The fresh water harmful snail
Toxicity Experiment: Toxicity experiment was performed by the method of Singh and Agarwal (17). Twenty animals were kept in glass aquaria containing 3L de-chlorinated tap water. Snails were exposed for 24h, 48h, 72h or 96h at four different concentrations of single or binary mixtures of both the plants extracts, control animals were kept in similar condition without any treatment. Each set of experiments, were replicated six times. Mortality was recorded after every 24h during the observation period of 96h. Contraction of the snail body within the shell and no response to a needle probe were taken as evidence of death of snails. Dead animals were removed to prevent the decomposition of body in experimental aquarium.
The effective doses (LC values), upper and lower confidence limits, slope value,‘t’ ratio and heterogeneity were calculated by the probit log method of Robertson et al., (18). Student’s’ test was applied to determine the significant (p<0.05) differences between treated and control animals. Product moment co-relation coefficient was applied in between exposure time and lethal concentrations (19).
Fecundity hatchability and Survivability Experiment
These experiments were performed according to the method of Presing, (20). In this experiment, fresh water adult
Lymnaeid snails attached ribbon like egg masses (spawns), containing variable number of eggs to the back surface of lotus leaf and inner wall of the aquarium when reproducing. The egg masses produced by the snail in the experiment were removed after every 24 hours up to 96 hours and the number of eggs counted under compound microscope. All the spawns of each group were transferred into separate petri-dishes containing one litter de- chlorinated tap water for hatching under the same exposure condition as above and kept at 25 ±1ºC for development of embryo in B.O.D. incubator. Hatched snails were counted and their survival rate was recorded for 28 days after hatching. Disintegration of embryos or absence of movement of the embryo was considered for calculating the percent mortality of eggs.
The experimental animals were treated with sub-lethal doses, i.e. 40% (2.33 mg/L) and 80% (4.67 mg/L) of LC50 of 24h of binary mixture of LILAE+ASLAE (in 1:1 ratio) for 96h exposure periods. Control groups were kept under similar conditions without any treatment, after completion of 96 hrs. the hepatopancreas and nervous tissue (brain tissue) of the treated as well as control group animal were quickly dissect out and used for bio-chemical estimations. In order to see the effect of 7th day of withdrawal, animals were exposed to sub lethal doses i.e. 80% of LC50 of 24h in binary mixture of LILAE+ASLAE in 1:1 ratio for 96h exposure periods. After 96h animals were transferred to freshwater free from any treatment, water was changed every 24h for the next six day. After completion of 7th day the nervous and hepatopancreas tissue was quickly dissect out and used for bio-chemical parameters.
Total free amino acid: Estimation of to total free amino acid was made according to the method of Spies 1957 (21). Homogenates (10 mg/mL, w/v) were prepared in 95% ethanol, centrifuged at 6000 xg and used for amino acid estimation.
Protein: Protein levels were estimated according to the method of Lowry et al., 1951 (22) using bovine serum albumin as standard. Homogenate (5 mg/mL, w/v) were prepared in 10% TCA.
Glycogen: Glycogen was estimated by the Anthrone method of Vander Vies 1954 (23) as modified by Mahendru and Agarwal 1982 (24) for snail
Nucleic acid: Estimation of nucleic acid (DNA and RNA) performed by the method of Schneider 1957 (25) using diphenylamine and orcinol reagents, respectively. Homogenates (1 mg/mL, w/v) were prepared in 5% TCA and centrifuged at 5000 xg for 20 minute and supernatant was prepared and used for estimation.
Activity of enzyme protease: Protease activity was measured according to the method of Moore and Stein 1954 (26); homogenate (50 mg/L, w/v) was prepared in cold distilled water (0o C) and optical density was measured at 570 nm. Protease activity is expressed in as micromoles of tyrosine equivalents per milligram of protein /hour.
Toxicological observations (Table1 and Fig. 1)
Behavioral change was seen after few minutes of exposure to the extracts. In initial 30-45 minutes snails were started to aggregating, and they started crawling on each others. As poison enters in the body of snails, there was a muscular twitching and snails become spirally twisted, which resulted ataxia, convulsion, paralysis and finally death of snails. In control groups, there were no such behavioural response and symptoms occur and there was no death also. The contraction of the body within the shell and no response to a niddle probe were taken as evidence of snail death.
The toxicity of both plant extracts were time and dose-dependent. There was a significant negative correlation between LC values and exposure periods. Thus with increase in exposure periods the LC50 values decreased from 25.05 mg/L (24h) to 15.32 mg/L (96h) and 06.01 mg/L (24h) to 01.31 mg/L (96h) in case of ASLAE and LILAE
The steep slope value given in the toxicity table was steep and the heterogeneity factor was less than 1.0, which indicates that the results found fall within 95% confidence limits of LC values. The regression test (‘t’ ratio) was greater than 1.96 and the potency estimation test (‘g’ value) was greater than 0.5 at all probability levels
Batches of twenty snails were exposed to four different concentrations of the extract. Concentrations given are the final concentration (w/v) in the aquarium water containing de-chlorinated tap water. Each set of experiment was replicated six times. Mortality was recorded after every 24h. Regression coefficient showed that there was significant (P<0.05) negative correlation between exposure time and different LC values. LCL-Lower confidence limit; UCL-Upper confidence limit. There was no mortality in the control group.
Fecundity, hatchability and survivability Experiment (Table 2, 3 and 4)
The results of the fecundity, hatchability and survivability experiment on the freshwater snail
Treatment of snail with sub lethal doses (20% and 40% of LC50 of 24h) of LILAE (Table 2), fecundity was reduced to 88.77% to 83.30% of control after 96h exposure periods, and the number of hatched eggs was reduced to 80.33% to 69.63% of control. The survival rate of the hatched snails was greatly reduced to 73.48% to 62.91% of control after 7 days of hatching and it was further reduced to 17.99% to 5.86% of control after 28 days of hatching, respectively.
Treatment of snail with sub-lethal doses 20% and 40% of LC50 (24h) of
All experiments were replicated six times. Values are means ± SE of six replicates. Values in parentheses are percentages of the corresponding value with control taken as 100%.*, Significant (P<0.05), when Student’s ‘t’ test was applied between control and treated groups.
Other details are as given in Table 2.
Similarly the binary mixture of extract i.e. 20% and 40% of LC50 (24h) LILAE + ASLAE in 1:1 ratio (Table 4) also caused reduction in the fecundity of snail
-, No survivability was recorded. Other details are as given in Table 2.
Biochemical estimations: (Fig. 2)
Sub-lethal doses (40% and 80% of LC50 of 24h) of binary mixture of LILAE + ASLAE in 1:1 ratio, caused significant alterations in the level of total protein, total free amino acids, glycogen and nucleic acids and activity of enzyme protease in nervous and hepatopancreas tissue of the snail
Students ‘t’ test showed that these biochemical changes were significantly (p<0.05) time and dose dependent. Seven days withdrawal experiment shows, there was highly significant (p<0.05) recovery in all the above biochemical parameters in both the tissues of snail
It is evident from results section that
From result section it is also clear that there was a positive correlation between exposure period and mortality. The increases in mortality with increase in exposure period could be due to several factors, which may be acting separately are conjointly. The uptake of the active moiety of acetone extract of both plant
Statistical analysis of the data on toxicity brings out several important points. The X2 test for goodness of fit demonstrated that the mortality counts were not found to be significantly heterogeneous and other variables e.g. resistance etc. do not significantly affect the LC50 values, as these were found to lie within the 95% confidence limits. The dose mortality graph exhibits steep slope values. The steepness of slope line indicates that there is a large increase in the mortality of vectors population with relatively small increase in the toxicant. The slope is thus an index of the susceptibility of the target animal to the pesticides used. A steep slope is also indicative of rapid absorption and onset of effects. Even though the slope alone is not a very reliable indicator of toxicological mechanism, yet it is a useful parameter (29) for such a study. Since the LC50 of the plant extracts lay within the 95% confidence limits, it is obvious that in replicate test of random samples, the concentration response lines would fall in the same range (29).
It is also clear from the result section that acetone extract of both the plants in binary combination are more effective than individual extract towards snail. It seems that action of binary mixture is non-interactive as the components do not affect the transport and final concentration of each other at the site of action. Moreover they also do not influence the changes induced by each other at the site of action (30,31 ).
In the developmental study of snail it was found that number of laid egg (fecundity), number of hatched eggs, survival rate and development of the hatchlings reduced after exposure to all the sub-lethal doses of the acetone extracts of both the plants (Table 2, 3 and 4). There was a significant reduction in the fecundity (egg laying) was observed in comparison to control groups, it may be due to the significant reduction in the level of glycogen and protein content in the different body tissues of snail
The hatchability of eggs were also significantly decreased (Table 2, 3, and 4) after exposure to the acetone extract than control group. A similar trend of observations was reported by Mostafa and Tantawy, 2000 (37), they observed that
The survivability of the hatched young snails were significantly decreased than the control group after exposure to sub lethal doses of acetone extracts of
From biochemical results it is clear that these extract alters the different biochemical parameters. Total protein level, glycogen and nucleic acid level were significantly reduced while total free amino acid and protease level was significantly increased. The depletion of protein fraction in nervous and hepatopanceas tissue of snail may be due to their degradation and possible utilization of degraded products for metabolic purposes. The enzyme protease functions in hydrolyzing proteins to free amino acids and small peptides. Increased protease activity in the body tissue of both groups was evidence that proteins had undergone degradation processes such as proteolysis and used the degrades products for increased energy metabolism. Similar trend in protease has been reported by several workers in different animals as
Withdrawal experiment indicates that the toxicity of leaf extract of both the plant against snail was significantly reversible at 7 days of withdrawal from treatment. The reversibility of the action of plant extract despite of the high toxicity would be an added advantage in their use.
It is our belief that the use of
Acknowledgments: One of the authors (Saroj Chauhan) is thankful to Indian Council of Medical Research New Delhi, for financial support.