Indian J Med Res 138, July 2013, pp 129-134 Larvicidal & ovicidal efficacy ofPithecellobium dulce (Roxb.) Benth. (Fabaceae) against Anopheles stephensi Liston & Aedes aegypti Linn. (Diptera: Culicidae) M. Govindarajan, M. Rajeswary & R. Sivakumar Department of Zoology, Annamalai University, Annamalainagar, Tamil Nadu, India Received February 24, 2012 Background & objectives: In view of the recently increased interest in developing plant origin insecticides as an alternative to chemical insecticide, this study was undertaken to assess the larvicidal and ovicidal potential of the crude hexane, benzene, chloroform, ethyl acetate and methanol solvent extracts from the medicinal plant Pithecellobium dulce against the mosquito vectors, Anopheles stephensi and Aedes aegypti (Diptera: Culicidae). Methods: Larvicidal activity of P. dulce plant extracts was studied in the range of 60 to 450 mg/l against early third instar larvae of An. stephensi and Ae. aegypti in the laboratory. The larval mortality was observed after 24 h of exposure. The ovicidal activity was determined against An. stephensi and Ae. aegypti to various concentrations ranging from 100 to 750 mg/l under the laboratory conditions. Mean per cent hatchability of the eggs were observed after 48 h post treatment. Results: All leaf and seed extracts showed moderate larvicidal and ovicidal effects; however, the highest larval mortality was found in methanol extract of leaf of P. dulce against the larvae of An. stephensi and Ae. aegypti with the LC50 and LC90 values 145.43, 155.78 mg/l and 251.23, 279.73 mg/l, respectively. The per cent hatchability was inversely proportional to the concentration of extract and directly proportional to the eggs. Zero hatchability was observed at 400 mg/l for leaf methanol extract and 625 mg/l for seed methanol extract of P. dulce against An. stephensi and Ae. aegypti, respectively. Compared to leaf extracts, seed extracts have low potency against the two mosquitoes. Interpretation & conclusions: The present results suggest that the leaf and seed extracts of P. dulce have the potential to be used as an ideal eco-friendly approach for the control of mosquitoes. Key words Aedes aegypti - Anopheles stephensi - larvicidal-ovicidal - Pithecellobium dulce Mosquitoes are the major vector for the transmission in humans1. Aedes aegypti (L.) is generally known of malaria, dengue fever, yellow fever, chikungunya, as a vector for an arbovirus responsible for dengue filariasis, schistosomiasis, and Japanese encephalitis. fever, which is endemic to Southeast Asia, the Pacific Mosquitoes also cause allergic responses that include island area, Africa, and the Americas, and also for local skin and systemic reactions such as angioedema yellow fever in Central and South America and West 129 130 INDIAN J MED RES, JULy 2013 Africa2. In the past decade, chikungunya - a virus Turraea wakefieldii and Turraea floribunda against transmitted by Aedes spp mosquitoes - has re-emerged third-instar larvae14 and extracts of Pelargonium in Africa, southern and southeastern Asia, and the citrosa leaf have been tested for their biological, Indian Ocean Islands as the cause of large outbreaks larvicidal, pupicidal, adulticidal, antiovipositional of human disease3. Malaria is a protozoan infection of activity, repellency, and biting deterrency15 against erythrocytes caused in human beings by five species of An. stephensi. Hexane extract obtained from leaves of the genus Plasmodium (P. falciparum, P. vivax, P. ovale, Eucalyptus citriodora was tested against larvae of An. P. malariae, and P. knowlesi). In most cases, malaria is stephensi, Culex quinquefasciatus, and Ae. aegypti to transmitted via the bite of an infected female anopheline assess its toxicity and growth-inhibiting activity16. mosquito, but congenital malaria and acquisition Pithecellobium dulce Benth. (Fabaceae) is a small to through infected blood transfusion are well described4. medium sized, evergreen, spiny tree up to 18 m height, More than 40 per cent of the world’s population - native of tropical America and cultivated throughout approximately 3 billion people - are exposed to malaria 5 the plains of India (Vilayati babul). The presence of in 108 endemic countries . About one million cases of steroids, saponins, lipids, phospholipids, glycosides, malaria are reported in India every year. In 2010 an glycolipids and polysaccharides has been reported in estimated 219 million (range 154-289 million) cases the seeds. The bark contains 37 per cent of tannins occurred worldwide and 660,000 people died (range of catechol type. Quericitin, kaempferol, dulcitol and 610 000-971 000), mostly in children under five years 17 6 afezilin have been reported from the leaves . The plant of age . Presently, organochlorine, organophosphate, has been shown to have potential in treating a number and synthetic pyrethroid insecticides are being used of ailments where the free radicals have been reported for mosquito control. Successive changes in the to be the major factors contributing to the disorders. insecticides result in multiple insecticide resistant However, no information was available on the ovicidal malaria vectors. Malaria vectors in India are resistant and larvicidal activities of this plant species against to several insecticides (DDT, HCH, malathion, and 7 An. stephensi and Ae. aegypti. Therefore, the aim of deltamethrin) . this study was to investigate the mosquito ovicidal and There has been an increasing interest in anti- larvicidal activities of the different solvent extracts of mosquito products derived from natural origin because P. dulce. the continued applications of synthetic compounds have some drawbacks, including the widespread Material & Methods development of insecticide resistance8. Another Collection of plants: Fully developed leaves and drawback with the use of chemical insecticides is seeds of the P. dulce were collected from Thanjavur that these are non-selective and could be harmful to district, Tamil Nadu, India. It was authenticated by other organisms in the environment. The toxicity a plant taxonomist from the Department of Botany, problem, together with the growing incidence of insect Annamalai University, Annamalainagar. A voucher resistance, has called attention to the need for novel specimen was deposited at the herbarium of plant insecticides, and for more detailed studies of naturally- phytochemistry division, Department of Zoology, occurring insecticides9. It is, therefore, necessary to Annamalai University. develop new materials for controlling mosquitoes in an environmentally safe way, using biodegradable and Extraction: The healthy leaves and seeds were washed target-specific insecticides against them10. with tap water, shade-dried, and finely ground. The finely ground plant leaf and seed powder (1.0 kg/solvent) was The larvicidal activity of crude acetone, hexane, loaded in Soxhlet extraction apparatus and was extracted ethyl acetate, methanol, and petroleum ether extracts with five different solvents, namely, hexane, benzene, of the leaf of Centella asiatica, Datura metal, Mukia chloroform, ethyl acetate and methanol, individually. scabrella, Toddalia asiatica, extracts of whole plant of The solvents from the extracts were removed using a Citrullus colocynthis and Sphaeranthus indicus11; the rotary vacuum evaporator to collect the crude extract. leaf benzene, chloroform, ethyl acetate, and methanol Standard stock solutions were prepared at 1 per cent extracts of Acalypha indica12; the methanol extracts of by dissolving the residues in ethanol. From this stock leaves of Dysoxylum malabaricum have been tested solution, different concentrations were prepared and against mature and immature stages of An. stephensi these solutions were used for larvicidal and ovicidal under laboratory conditions13; root bark extracts of bioassays. GOVINDARAJAN et al: MOSQUITO LARVICIDAL & OVICIDAL EFFICACy OF P. DULCE 131 Test organisms: The laboratory-bred pathogen free was replicated six times along with appropriate control. strains of mosquitoes were reared in the vector control The hatch rates were assessed 48 h post treatment by laboratory, Department of Zoology, Annamalai following formula: University. The larvae were fed on dog biscuits and yeast No. of hatched larvae % hatchability = 100 powder in the 3:1 ratio. At the time of adult feeding, Total no. of eggs these mosquitoes were 3-4 days old after emergences (maintained on raisins and water) and were starved for Statistical analysis: The average larval mortality data 12 h before feeding. Each time 500 mosquitoes per were subjected to probit analysis for calculating LC50, cage were fed on blood using a feeding unit fitted with LC90, and other statistics at 95% confidence limits of parafilm as membrane for 4 h.Ae. aegypti feeding was upper confidence limit (UCL) and lower confidence done from 1200 to 1600 h, and An. stephensi were fed limit (LCL) values were calculated using the SPSS12.0 during 1800 to 2200 h. A membrane feeder with the (Statistical Package of Social Sciences Inc., USA) bottom end fitted with parafilm was placed with 2.0 software. ml of the blood sample (obtained from a slaughter house by collecting in a heparinized vial and stored Results at 4 °C) and kept over a netted cage of mosquitoes. The results of the larvicidal activity of crude The blood was stirred continuously using an automated hexane, benzene, chloroform, ethyl acetate, and stirring device, and a constant temperature of 37 °C methanol solvent leaf and seed extracts