Abstract: The aim of the present investigation is to test the larvicidal activity of Azadirachta indica (A. Juss) and Datura metel (Linn.) leaf extract against the third instar larvae of C. quinquefasciatus (Say) (Diptera: Culicidae). A. indica and D. metel leaf extracted by hexane and chloroform extract method at various concentrations. The hexane extract of A. indica and D. metel at 62.5, 125, 250, 500 and 1000 ppm were showed 24, 36, 55, 64 and 72.50% mortality where second one shows 9, 17.50, 30, 42 and 57% mortality, respectively. The chloroform extract of A. indica was showed 12, 48.50, 56.50, 73 and 87% mortality where D. metel shows 13.75, 27, 32, 47 and 62% mortality respectively. The hexane and chloroform extract of A. indica and D. metel had significant larvicidal effect with LC50 values were 246.38, 198.82, 709.96 and 562.07 ppm respectively. At 24 h post-treatment against late third instar larvae, the chloroform extracts of A. indica and D. metel were found to be more effective than hexane extracts and caused a larval mortality of 87 and 62%, respectively at 1000 ppm concentration. The larvicidal effect of A. indica and D. metel against C. quinquefasciatus make these plant products are potential alternative to synthetic insecticide in mosquito control plans.
INTRODUCTION
Human beings have suffered from the activities of mosquito since time immemorial. It is believed that mosquitoes are ranked as the most important human health pests. They scourge him with their vicious biting and continuous singing, but most seriously they transmit malaria, filaria, japanese encephalitis, chikungunya and dengue fever to human beings. These diseases devastate Indian economy every year (Jaswanth et al., 2002). Worldwide, mosquitoes transmit diseases to more than 70,00,00,000 people annually and are responsible for one death for every seventeen people currently alive.
Since, prevention is better than cure, a major strategy of malarial control is to attack the vector with insecticides (Rajkumar and Jebanesan, 2009). The control of mosquito at the larval stage is necessary and efficient in integrated mosquitos management. During the immature stage, mosquitoes are relatively immobile; remaining more concentrated than they are in the adult stage (Elimam et al., 2009; Rutledge et al., 2003). Since the discovery of DDT, mosquito control approach has been almost completely based on synthetic organic insecticides. But the extensive use of synthetic organic insecticides during the last five decades have resulted in environmental pollution and also in the development of physiological resistance in major vector species in addition to the increased costs of insecticides. This has necessitated the need for search and development of environmentally safer, low cost, indigenous methods for vector control. During the last decade, various studies on natural plant products against mosquito vectors indicate them as possible alternatives to synthetic chemical insecticides (Elimam et al., 2009; Mittal and Subbarao, 2003; Rajkumar and Jebanesan, 2005a, b; Promsiri et al., 2006). Moreover to application as general toxicant against mosquito larvae, botanical insecticides also have potential uses as growth and reproduction inhibitors, repellents, growth regulation, fecundity suppression, male sterility, larvicidal, ovicidal and oviposition activity mostly as deterrence (Elimam et al., 2009). Several indigenous plants in India and subtropical parts of Asia, such as Ocimum basilicum, Ocimum santum, Azadirachta indica, Lantana camera, Vitex negundo and Cleome viscose (Senthil Nathan et al., 2006) were studied for their larvicidal action on the field which collected fourth instar larva of Culex quinquefasciatus (Alouani et al., 2009). To date, there is no report about the D. metel leaf extract against the mosquito. It grows in all the warmer parts of the world and is cultivated worldwide for its chemical and ornamental properties. Thereby, we decided to investigate the larvicidal activity of A. indica and D. metel leaf extract against the third instar larvae of C. quinquefasciatus.
MATERIALS AND METHODS
Collection and rearing of mosquitoes: The egg rafts of C. quinquefasciatus were collected from Coovam River (Chetpet) in Chennai, India during 2007. The egg rafts were placed in petridishes (10.5 diameters) containing aged tap water. Larvae were fed with finely ground mixture of yeast and dog biscuits in 3:1 ratio. The pupae were transferred into mosquito cage for emergence. Blood meal from a pigeon was given to adult mosquitoes after three days of emergency. After 3-4 days of blood feeding, adult mosquitoes were provided with petridishes filled with tap water inside the cage for oviposition. The egg rafts were separated and placed in glass petridishes for hatching (Verma and Rahman, 1986).
Preparation of plant extract: The leaves of A. indica and D. metel were collected from Chetpet area in Chennai, India. The leaves were washed with tap water, shade dried at room temperature and then powdered by using electrical blender. The leaf powder (250 g) of neem and D. metel was sequentially extracted with hexane and chloroform after soaking for 48 h in each solvent. The excess solvent in the filtrate was evaporated under reduced pressure in rotary vacuum evaporator. One percent stock solution (1000 ppm) was prepared by dissolving 100 mg of crude extract in 100 mL of acetone. As of the stock, different concentrations viz., 62.5, 125, 250, 500 and 1000 ppm were prepared according to the procedures of WHO (1996).
Larvicidal bioassay: About 50 numbers of late third instar larvae of C. quinquefasciatus were introduced into 250 mL of dechlorinated water contained different concentrations of plant extracts in 500 mL of plastic containers. For control, 1.0 mL of acetone dissolved in 249 mL of dechlorinated water was used. Three replicates for each concentration and control were maintained. The mortality counts were made by using Abbott’s formula (Abbott, 1925) after 24 h of the treatment and the LC50 was calculated by Probit analysis (Finney, 1971).
Statistical analysis: One way Analysis of Variance (ANOVA) was worked out to find out the significance of the treatments. The treatments were separated by Least Significant Difference (LSD) at p = 0.05 level.
RESULTS
The hexane and chloroform leaf extract of A. indica and D. metel have been studied for use as eco-friendly insecticides instead of eco-enemy synthetic insecticides. Results on the larvicidal effect of leaf extracts were reported in the present study, confirm their potential for control of larval population and management of mature C. quinquefasciatus mosquito population. Figure 1 shows the results on percent mortality of larvae. After 24 h of exposure, the 5 different concentrations tested 62.5, 125, 250, 500 and 1000 ppm, the hexane extract of A. indica at above concentration produced 24, 36, 55, 64 and 72.50% and D. metel produced 09.00, 17.50, 30.00, 42.00 and 57.00% larval mortality, respectively. The chloroform extract produced 12.25, 48.50, 56.50, 73.00 and 87.00% mortality in A. indica and 13.75, 27.00, 32.00, 47.00 and 62.00% mortality in D. metel, respectively. Among the two plants, the chloroform extract exhibited higher activity. The LC50 of hexane and chloroform extracts of A. indica was calculated as 246.38 and 198.82 whereas LC10 were 17.06 and 34.44 and LC90 3557.71 and 1147.5, respectively. The LC50 of hexane and chloroform extracts of D. metel was calculated as 709.96 and 562.07 whereas LC10 were 65.96 and 38.59 and LC90 7641.69 and 8186.26, respectively (Table 1). No mortality was recorded in the control (data not shown). These results suggest that D. metel was less effective than A. indica to the larvae of C. quinquefasciatus. As the concentration of the plant extracts increased, the total larval mortality of the mosquitoes was also found to be increased.
| Fig. 1: | Larvicidal activity of A. indica and D. metel leaf extracts against the C. quinquefasciatus |
| Table 1: | Median lethal concentration (ppm) of hexane and chloroform extracts of A. indica and D. metel against C. quinquefasciatus larva |
| Significant at p≤0.05 level | |
In A. indica, 125 ppm of chloroform gave a mortality of nearly 50% (48.5%), but D. metel extracts needed a concentration of more than 500 ppm (Fig. 1).
DISCUSSION
Today indiscriminate and continuous use of various insecticides has resulted
in development of resistance in mosquitoes. Tikar et
al. (2008) have recorded development of resistance in A. aegypti
larvae against temephos, fenthion, malathion and DDT from different locations
in India. The decrease in insecticidal susceptibility or development of insecticide
resistance in populations of C. quinquefasciatus has been reported against
temephos, fenthion, cypermethrin, α-cypermethrin and λ-cyhalothrin
from Bathinda, Bikaner, Jodhpur and Jamnagar in India indicting need of search
for some safe and effective alternative eco-safe control measures. Besides development
of insecticides resistance, insecticides are toxic to other non-target organisms
(Suman et al., 2010). In contract to insecticides,
the use of plant products against mosquito control is studied by Mehlhorn
et al. (2005), Amer and Mehlhorn (2006) and
Govindarajan et al. (2008) etc. Pharmaceutical
companies tend to focus on single active anti-malarial drugs for revenue and
profit including those from botanical sources (Rajkumar
and Jebanesan, 200
In the present investigation, the hexane extract of A. indica and D. metel at 62.5, 125, 250, 500 and 1000 ppm were showed 24, 36, 55, 64 and 72.50% mortality where second one shows 9, 17.50, 30, 42 and 57% mortality, respectively. The chloroform extract of A. indica was showed 12, 48.50, 56.50, 73 and 87% mortality where D. metel shows 13.75, 27, 32, 47 and 62% mortality, respectively. The hexane and chloroform extract of A. indica and D. metel had significant larvicidal effect with LC50 values were 246.38, 198.82, 709.96 and 562.07 ppm, respectively. At 24 h post-treatment against late third instar larvae, the chloroform extracts of A. indica and D. metel were found to be more effective than hexane extracts and caused a larval mortality of 87 and 62%, respectively at 1000 ppm concentration. The result is consistent with earlier works by Wandscheer et al. (2004) and Tonk et al. (2006), who separately reported that neem seed kernel extracts that are effective against mosquitoes were prepared with hexane, ethyl ether, acetone, ethanol and methanol. The most potent larvicide is 0.71% neem seed kernel oil obtained with hexane extract. This dosage is higher than that of Sinniah et al. (1994), who reported that 0.02% neem seed oil extracts caused 100% mortality in Aedes aegypti and Culex quinquefasciatus where the present investigation showed chloroform extracts of A. indica and D. metel larval mortality of 87 and 62% at 1000 ppm. Ambrose (1995) used much lower concentrations and showed that the larvicidal effects of neem oil on 3rd and 4th instar larvae of Culex quinquefasciatus were having the LC50 values of 0.99 and 1.20 ppm for neem oil for the two larvae respectively and 0.55 and 0.72 ppm for the deoiled neem cake. The present study shows that the chloroform extract of A. indica and D. metel had significant larvicidal effect with LC50 values were 198.82 and 562.07 ppm, respectively. These variations in susceptibility of extracts/oil to mosquito larvae may be due to variations in extraction solvents, mosquito species or exposure periods (Umar et al., 2002). It reveals as the concentration of the plant extracts increased, the total larval mortality of the mosquitoes was also found to be increased. In A. indica, 125 ppm of chloroform gave a mortality of nearly 50% (48.5%), but D. metel extracts needed a concentration of more than 500 ppm (Fig. 1).
In comparison with the results of earlier studies, it was noticeable that leaf extract tested in this study exerted promising mosquito larvicidal potential. The mode of action of these leaf extract on mosquito larvae are not known, but previous studies demonstrated that phytochemicals interfered with the proper functioning of mitochondria more specifically at the proton transferring sites (Usta et al., 2002) and other studies by Rey et al. (1999) and David et al. (2000) found that phytochemicals primarily affect the midgut epithelium and secondarily affect the gastric caeca and the malpighian tubules in mosquito larvae. Furthermore, the crude extracts may be more effective compared to the individual active compounds, due to natural synergism that discourages the development of resistance in the vectors (Rajkumar and Jebanesan, 2009; Maurya et al., 2007).
CONCLUSION
In conclusion, leaves extract of A. indica and D. melei can be suggested as a natural larvicidal for controlling mosquitoes in India. Since, the both plants are economically safe and less expensive to control mosquito and the histological studies to be performed to recognize the mode of action between leaf extract and mosquito.