Mosquito larvicidal and phytochemical properties of Ageratina adenophora (Asteraceae) against three important mosquitoes.

insects wellknown for their public importance, since they act as vector for many tropical and subtropical disease such as dengue fever, yellow fever, chikungunya, malaria, filariasis and encephalitis of different types including, Japanese encephalitis1. Larviciding is a successful way of reducing mosquito densities in their breeding places before they emerge into adults. Larviciding largely depends on the use of synthetic chemical insecticides – organophosphates (e.g. temephos and fenthion), insect growth regulators (e.g. diflubenzuron and methoprene), etc. Although effective, their repeated use has disrupted natural biological control systems and sometimes resulting in the widespread development of resistance. These problems have warranted the need for developing alternative strategies using eco-friendly products2. These steadily growing problems demand an intensive search for new products that are environmentally safe, target specific and degradable. The above facts prompted us to undertake investigations of some plant species traditionally used as insecticidal agents, as well as other endangered plant species, with the aim of identifying lead compounds for the development of new plant based insecticidal agents3. Ageratina adenophora is a perennial herbaceous exotic shrub which may grow up to 1 or 2 m height. It has opposite trowell-shaped serrated leaves that are 6–10 cm long by 3–6 cm in width. The small compound flowers occur in late spring and summer, and are found in clusters at the end of branches. Each flower head is up to 0.5 cm in diam and creamy white in colour. They are followed by a small brown seed with a white feathery parachute. The mosquito larvicidal properties of Ageratina adenophora have not yet been reported. Therefore, the present study was carried out to determine the larvicidal efficacy of A. adenophora leaves extract against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi (Diptera: Culicidae). The leaves of A. adenophora were collected from hilly regions of the Nilgiris district, Tamil Nadu, India. It was authenticated by a plant taxonomist from the Department of Botany, Annamalai University. A voucher specimen is deposited at the herbarium of Plant Phytochemistry Division, Department of Zoology, and Annamalai University, India. Cx. quinquefasciatus, Ae. aegypti and An. stephensi were reared in the Vector Control Laboratory, Department of Zoology, Annamalai University. The larvae were fed on dog biscuits and yeast powder in the 3:1 ratio. Adults were provided with 10% sucrose solution and membrane feeding on goat blood. Mosquitoes were held at 28 ± 2°C, 70– 85% relative humidity (RH), with a photoperiod of 12 h light : 12 h dark. The dried leaves (1 kg) were extracted with methanol (5 L) by a soxhlet apparatus method and the extract was evaporated in a rotary vacuum evaporator to yield a dark greenish mass (195 g). Standard stock solutions were prepared at 1% by dissolving the residues in methanol, which was used for the bioassays. Phytochemical screening of the leaves of A. adenophora was carried out by using the standard protocols for the presence of carbohydrates, proteins, phenolic compounds, saponins, flavonoids, alkaloids, tannins, glycosides, phytosterols, oil and fats. The larvicidal activity of crude extract was evaluated as per the protocol previously described5. Early III instar larvae (20) were placed in 249 ml of water and 1 ml of methanol containing different experimental concentrations. The beaker containing the control larvae received 1 ml of methanol. Crude extract concentration ranging from 50 to 250 mg/l was tested. Each test was repeated five times. The larval mortality data were subjected to probit analysis6 for calculating LC50 and LC90 and chisquare values were calculated by using SPSS 13.0 for Windows. Significance level was set at p <0.05. Results of preliminary phytochemical analysis of the leaf extract of A. adenophora showed the presence of alkaloids, saponins, glycosides and coumarines (Table 1). The crude methanol extract, was tested in the concentraJ Vector Borne Dis 50, June 2013, pp. 141–143