Research Article

Mosquito larvicidal activity of solvent extracts of fruits of Acacia auriculiformis against Japanese encephalitis vector Culex vishnui group  

Mousumi Barik1 , Anjali  Rawani1, 2 , Goutam Chandra1
1 Mosquito, Microbiology and Nanotechnology Research Units, Parasitology laboratory, Department of Zoology, The University of Burdwan, Burdwan-713104, West Bengal, India
2 Department of Zoology, Gour Banga University, Malda, West Bengal, India
Author    Correspondence author
Journal of Mosquito Research, 2016, Vol. 6, No. 13   doi: 10.5376/jmr.2016.06.0013
Received: 01 Apr., 2016    Accepted: 09 May, 2016    Published: 24 Jun., 2016
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Barik M., Rawani A., and Chandra G., 2016, Mosquito larvicidal activity of solvent extracts of fruits of Acacia auriculiformis against Japanese encephalitis vector Culex vishnui group , Journal of Mosquito Research, 6(13): 1-8 (doi: 10.5376/jmr.2016.06.0013)


The purpose of present study was to determine the larvicidal activity of different solvent extracts of fruits of Acacia auriculiformis against the 3rd larval instar of Culex vishnui group. The solvent extracts of fruits of A. auriculiformis (concentrations were 100, 200, 300 ppm) showed varied results against 3rd larval instars with highest mortality at 300 ppm. The percent mortality was higher in chloroform: methanol (1:1 v/v) extract than other extracts. The order of efficacy of different solvent extracts was chloroform: methanol (1:1 v/v) > absolute alcohol > n-Hexane > acetone > petroleum ether. Results of regression analyses showed that percentage mortalities were positively correlated with the concentrations. Probit analysis revealed LC50 value and their LCL and UCL values at different time intervals and the lowest LC50 value was obtained at 72 h of exposures. There was no mortality of non-target organisms after 24 h of exposure to lethal concentration (LC50 value of chloroform: methanol (1:1 v/v) solvent extract at 24 h determined against 3rd instar larvae).

Culex vishnui group; Acacia auriculiformis; Larvicidal activity; Solvent extracts

1 Introduction                                                                  

Mosquito is one of the most notorious creatures of this world, causes nuisance by its bites and transmits deadly diseases like malaria, filariasis, yellow fever, dengue, Japanese encephalitis etc. and contribute significantly to poverty and social hindrance in tropical countries (Jang et al., 2002). Japanese encephalitis is a vector borne viral disease caused by JE virus (JEV). In a zoonotic cycle, JEV is maintained both in enzootic and epizootic cycles. In JEV life cycle, pigs are involved as the major amplifying reservoir host, water birds as carriers and mosquitoes as vectors. JEV is a major cause of viral encephalitis in South- East Asia, affecting more than 50,000 patients annually (Mackenzie et al, 2007). The Cx. vishnui groups of mosquitoes consisting of Cx. vishnui Theobald, Cx. pseudovishnui Colless and Cx. tritaeniorhynchus Giles have been implicated as major vectors of JE virus.  However from 16 species of mosquitoes JEV has been isolated in India (Solomon, 2007).


To combat the devastating scenario of fatal diseases, the control of mosquito population is essential. To the common people, killing of adult mosquitoes is the most common and easy way to control mosquito population. In ancient time, a wide range of insecticides were in a practice to control the mosquito population in adult stage. But now-a-days this attempt has been shifted towards larval control before they become able to transmit the serious diseases. So controlling mosquitoes in their immature stages is more rational than destroying adults and also easy since their breeding habitat is water. In the cosmo tropical areas public health authorities have been using hazardous synthetic insecticides as the principal means to control the high density of mosquito population. Biological control methods include the exploitation of natural enemies of targeted mosquitoes and also biological toxins to achieve effective vector management (Ansari et al., 2000). Among them medicinal plant products are the best alternative to synthetic chemical insecticides (Shaalan et al., 2005; Bhattacharya and Chandra, 2013; Singha and Chandra, 2011; Hossain et al., 2011). The plant products have several advantages as they showed their efficacy at very low test concentrations towards the target species, there is very less to no ill-effect on non-target organisms, field application is very easy, and there is no environmental pollution due to persistence as they are easily degradable (Rawani et al., 2012; Haldar et al., 2011; Bhattacharya and Chandra, 2014; Chowdhury et al., 2008a; Chowdhury et al., 2008b). Synergistic effect of two or more plant (Singha et al., 2011) or synthesized nano particles from plants (Haldar et al., 2013; Rawani et al., 2013a) also gives an alternating way in mosquito control programme. There is no or little evidence of resistance development in target mosquitoes.


A. auriculiformis, belongs to fabaceae family is raised as an ornamental plant, as a shade tree and it is also raised on plantations for fuel wood throughout southeast Asia, Oceania and in Sudan. Its wood is good for making paper, furniture and tools. It contains tannin useful in animal hide tanning. In India, its wood and charcoal are widely used for fuel. Gum from the tree is sold commercially, but it is said not to be as useful as gum arabic. The tree is used to make an analgesic by indigenous Australians. Extracts of A. auriculiformis heartwood inhibit fungi that attack wood. The fruit is irregular in shape, 1 to 3 inches in length, dry and hard covering, brown in colour. Seeds are rich source of triterpenoid saponin especially Acaciaside A (Ac-A) and Acaciacide-B (Ac-B). Ac-B isolated from seed extract shows spermicidal activity (anti fertility) in lower concentration (Pakrashi et al., 1991; Pal et al., 2009) and most importantly inhibits transmission of HIV (Kabir et al., 2008) without any mutagenic effect. Bark extract of   A. auriculiformis also shows some pesticidal activity.


The purpose of the present study was to evaluate the mosquitocidal activity of five solvent extracts of fruits of A. auriculiformis against Cx. vishnui group.


2 Material and Method          

2.1 Collection of Plant material

Fresh and matured fruits of A. auriculiformis were harvested randomly during April – June, 2011, at outskirts of Burdwan (23°16'N, 87°54'E). The plant was identified and submitted as herbarium (Voucher No: GCMB-06) in the Department of Zoology, The University of Burdwan. Fruits were cleaned with tap water, washed with distilled water and soaked on a paper towel.


2.2 Collection and rearing of larvae

The eggs of Cx. vishnui group were collected from rice field near the University campus. They were reared and maintained at 28 ± 2°C temperature and 85% RH in Mosquito Microbiology and Nanotechnology Research units, Parasitology laboratory, Department of Zoology, The University of Burdwan. The eggs were immersed in dechlorinated tap water in enamel tray of 30 cm diameter. The hatched larvae were fed with artificial food (dried yeast powder and powder of dog biscuits in the ratio of 1:3). They were maintained at 28 ± 2ºC. The transformed pupae were separated manually with a glass dropper into a 500 ml beaker with water and introduced into adult cages of 12.6” X 10” X 6” for adult emergence. A cotton ball soaked in 10% glucose solution was used for glucose meal of adult mosquitoes and was periodically blood fed on immobilized pigeon.


The adults were identified by Barraud (1934), Christophers (1933) and Chandra (2000).  Eggs laid were similarly reared and 1st generation laboratory breed larvae were used for bioassay and control experiments.


2.3 Preparation of different solvent extracts

Air dried 250 g mature fruits of A. auriculiformis were crushed and put in the thimble of Soxhlet apparatus and five different solvents, namely petroleum ether, n-hexane, chloroform: methanol (1:1, v/v), acetone and absolute alcohol were applied one after another (extraction period 72 h for each solvent with 8 h maximum a day). The extracts were collected separately, and the column of the Soxhlet apparatus was washed with 200 ml of water and 100 ml of a similar solvent as an eluent after each type of solvent extraction procedure. The eluted material of each type of extract was concentrated by evaporation in a rotary evaporator.


2.4 Dose response larvicidal bioassay

The larvicidal bioassay followed the World Health Organization (1981) standard protocols. Twenty five 3rd instars larvae of Cx. vishnui group were put in separate glass Petri-dishes (9 cm diameter/150 mL capacity) containing 100 mL of tap water. Three concentrations of each solvent extracts (100 ppm, 200 ppm and 300 ppm) were applied into separate Petri-dishes to investigate the mortality. Dried yeast powder (920 mg) was added in each Petri dish as larval food. A similar type of bioassay was conducted with only distilled water without any of the solvent extracts of the matured fruits against 3rd larval instars of Cx. vishnui group separately as control. Larval mortalities were recorded after 24 h, 48 h and 72 h of exposure. The experiments were repeated thrice and maintained at 25-30°C and 80-90% relative humidity.


2.5 Effect on non-target organism

The effect of ethyl acetate solvent extract was also studied on non-target organisms such as Daphnia sp, Diplonychus annulatum and Chironomus circumdatus larvae. Non targets were exposed to lethal concentration (LC50 value of chloroform: methanol (1:1v/v) solvent extract at 24h against 3rd instar larvae) to observe mortality and other abnormalities such as sluggishness and reduced swimming activity up to 72 h of post exposure.


2.6 Statistical analyses

The percentage mortality observed (% M) was corrected using Abbott’s formula (Abbott, 1925). Statistical analysis include the LC50, LC90 regression equation (Y=mortality; X=concentration) and regression coefficient value. ANOVA was carried out using different concentration, different instars and hours as variable to justify the significance between the above parameter and mortality rate.


3 Result

Among the five solvent extracts, the highest mortality (76% mortality) was observed in chloroform: methanol (1:1v/v) solvent extract at 300 ppm concentration against 3rd larval instars and was significantly (p<0.05) higher than 100 ppm, 200 ppm concentrations at 24 h, 48 h and 72 h of exposure (Table 1).



Table 1 Mean mortality of 3rd instar larvae of Culex vishnui group mosquito at different concentrations of different solvent extracts of fruits of Acacia auriculiformis


The order of efficacies of different solvent extracts on 3rd instars larvae of Cx. vishnui group mosquito were chloroform: methanol (1:1 v/v) > absolute alcohol > n-Hexane > acetone > petroleum ether. The result of log probit analysis and regression analysis showed that LC50 values gradually decreased with exposure periods and mortality rate (Y) was positively correlated with concentration of exposure (X) having  regression coefficient (R) value close to one (Table 2).



Table 2 Log probit analysis and regression analysis of larvicidal activity of different solvent extracts of fruits of Acaccia auriculiformis against 3rd instar larvae of Culex vishnui group


Result of three way ANOVA analyses using different solvent extract, concentration and hours of exposures as variable are presented in Table 3.



Table 3 Three way ANOVA of mortality rates of different solvent extracts, different hours of exposures and concentrations as variables


The Tukey’s test revealed significant difference in mortality rates in different solvent extracts (Table 4) indicating significant difference in mortality rates between all three days of exposures in all solvent extracts (Table 5). No mortality and changes in swimming activity of the non-target organisms, e.g. Daphnia sp., D. annulatum and C. circumdatus were observed within 72 h of post-exposure to a certain concentration of chloroform: methanol (1:1v/v) solvent extract.



Table 4 Multiple comparisons by Tukey’s HSD on mortality of different solvent extracts of Acacia auriculiformis fruit extracts on mortality rates of Culex vishnui group



Table 5 Multiple comparisons on mortality of Culex vishnui group larvae at different hours of exposures


4 Discussion

Use of synthetic chemical insecticides brought many problems like persistence in the environment, development of resistance in the insect, bioaccumulations etc. More a function of frequency of use and persistence there is more development of resistance. So, there is an urge to find an alternative way that can substitute the use of chemical insecticides, which are effective, biodegradable, reducing the likelihood of resistance development. To combat with this problem, medicinal plants now a day are in more demands. They are extensively used in traditional medicine practices worldwide because of availability, low cost, proven efficiency and negligible side effects (Martin-Bettolo, 1980; Singh et al., 2015). Phytochemical based research works have been effectively published in many well established journals (Bhattacharya et al., 2014; Singh Ray et al., 2014; Mukherjee et al., 2015). These plants are not only used as a biocontrol agent but known to contain physiologically active principles that have been explored, incorporated and exploited in traditional medicine for the treatment of various diseases including ulcers in human and animals also (Sokmen et al., 1999; Kelmanson et al., 2000; Srinivasan et al., 2001; Chen et al., 1989). The beneficial medicinal effects of plant materials typically result from the combination of secondary products present in the plant. The present study evaluates the larvicidal activity of five solvent extracts against Cx. vishnui group. Highest mortality was observed at 300 ppm concentration of chloroform: methanol (1:1 v/v) solvent extract with a LC50 value of 117.27 ppm. The activity of chloroform: methanol (1:1 v/v) extract of many other plants has been reported by several authors. Chowdhury et al., (2009) reported activity of Chloroform: methanol extract (1:1) of leaf of Solanum villosum against Anopheles subpictus. All the graded concentrations (30, 50, 100 and 200 ppm) of chloroform: methanol extracts (1:1) showed significant (P < 0.05) larval mortality. LC50 values for all instars were between 24.20 and 33.73 ppm after 24 h and between 23.47 and 30.63 ppm after 48 h of exposure period. Similarly the activity of chloroform: methanol extracts (1:1) of leaf of Cestrum diurnum was tested against An. stephensi by Ghosh and Chandra (2006). The LC50 value of the active ingredient was determined as 0.70, 0.89, 0.90 and 1.03 mg/100mL for 1st, 2nd, 3rd and 4th instars larvae respectively in 24 h study period against An. stephensi. Ghosh et al., (2008) reported the efficacy of chloroform: methanol extract (1:1) of leaf of C. diurnum against Cx. quinquefasciatus. The LC50 value calculated was 0.29, 0.35, 0.57 and 0.65 % for 1st, 2nd, 3rd and 4th instar larvae at 24 h exposure period. Chowdhury et al., (2008b) also studied the activity of chloroform: methanol extract (1:1) of berry of Solanum villosum against Ae. aegypti. The lowest LC50 value calculated was 5.97 ppm at 72 h of bioassay experiment. Rawani et al., (2013b) studied the crude and chloroform: methanol (1:1 v/v) extracts of fresh, mature, green berries of S. nigrum against Cx. quinquefasciatus. In the chloroform: methanol (1:1, v/v) solvent extract, the maximum mortality was recorded at a concentration of 120 μg/mL. The log probit analysis (95% confidence level) recorded lowest LC50 value at 72 h of exposure. Singha and Chandra, (2011) evaluated efficacy of chloroform: methanol (1:1 v/v) extract of four vegetable waste viz. Allium sativum, Cuminum cyminum, Zingiber offinale, Curcuma longa and Solanum tuberosum germinated tuber against the 3rd  instar larvae of Cx. quinquefasciatus and An. stephensi larvae. At 75 ppm concentration of chloroform: methanol (1:1 v/v) extract of Curcuma longa showed highest mortality to 3rd instars larvae of both Cx. quinquefasciatus and An. stephensi. The practice of only mosquito control would  not bring out the desired results against vector control, importance should be given to the broad aspect of mosquito control approaches comprises of the use of insecticides, biocontrol agents and environmental managements.


In conclusion, fruit of A. auriculiformis offers a potential larvicidal agent against Cx. vishnui group larvae. However, further studies are required to know the final active ingredient and also evaluate their mode of action. Field trials are also needed to recommend A. auriculiformis as a potent mosquitocidal agent.


Conflict of interest statement

We declare that we don’t have any conflict of interest.



The authors are indebted to Professor Dr. A. Mukhopadhyay, Department of Botany, The University of Burdwan, for his kind help in plant species identification.



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