Mosquito-borne diseases with an economic impact create a loss in commercial and labor outputs, particularly incountries with tropical and subtropical climates. Mosquito control is facing a threat because of the emergence ofresistance to synthetic insecticides. Insecticides of botanical origin may serve as suitable alternative biocontroltechniques in the future. In view of recently increased interest in developing plant origin insecticides as analternative to chemical insecticide, in the present study ovicidal efficacy of different solvent leaf and seed extractof Pithecellobium dulce against filariasis vector C. quinquefasciatus. The ovicidal efficacy of the crude leaf extractsof P. dulce with five different solvents, such as benzene, hexane, ethyl acetate, methanol, and chloroform, wastested against C. quinquefasciatus. Ovicidal activity was determined against C. quinquefasciatus mosquito tovarious concentrations ranging from 100 to 600 mg/L under the laboratory conditions. Among five solvent testedthe maximum efficacy was observed in the leaf and seed methanol crude extract was found to be most effectivefor ovicidal activity against vector mosquito. The extract of methanol exerted 100% mortality at 500-600 mg/Lagainst C. quinquefasciatus. From the result, it can be concluded the crude extract of P. dulce was a potentialcandidate to develop newer and safer ovicides against C. quinquefasciatus

Mohan Rajeswary, S. L. Hoti, Giovanni Benelli, Marimuthu Govindarajan

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116 Journal of Medicinal Herbs and Ethnomedicine ● Vol 1 ● 2015INTRODUCTIONMosquitoes not only cause nuisance by their bites but also transmit deadly diseases like malaria, filariasis, yellow fever, dengue, and Japanese encephalitis, contribute significantly to poverty and social debility in tropical countries (Jang et al., 2002; Benelli, 2015a). Lymphatic filariasis is a mosquito-borne disease caused by mosquito-transmitted filarial nematodes, including Wuchereria bancrofti and Brugia malayi. The infected people carry the nocturnally periodic W. bancrofti, which has Culex quinquefasciatus as the main mosquito vector. C. quinquefasciatus is a vector of lymphatic filariasis, which is a widely distributed tropical disease with around 120 million people infected worldwide, and 44 million people have common chronic manifestations (Bernhard et al., 2003). In India alone, 25 million people harbor microfilaria, and 19 million people suffer from filarial disease manifestations (Govindarajan et al., 2011). The major problems associated with the use of chemicals for the control of pests including mosquitoes include: The development of resistance to the chemicals, issues around the residues in animals and the environment, and their undesirable side effects (Peter et al., 2005). Extracts from plants may be alternative sources of mosquito control agents since they constitute a rich source of bioactive compounds that are biodegradable into nontoxic products and potentially suitable for use to control mosquitoes. Plant extracts, in general, have been recognized as an important natural resource of insecticides (Benelli, 2015b; Benelli et al., 2015).Phytochemicals derived from plant sources can act as larvicides, insect growth regulators, repellents, and oviposition attractants and can play an important role in the interruption of the transmission of mosquito-borne Ovicidal activity of Pithecellobium dulce (Family: Fabaceae) leaf and seed extracts against fi lariasis vector mosquito Culex quinquefasciatus (Diptera: Culicidae)Marimuthu Govindarajan1*, Mohan Rajeswary1, S. L. Hoti2, Giovanni Benelli31Department of Zoology, Phytochemistry and Nanotechnology, Unit of Vector Control, Annamalai University, Annamalainagar, Tamil Nadu, India, 2Regional Medical Research Centre (ICMR), Nehru Nagar, Belgaum, Karnataka, India, 3Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, ItalyReceived: 27.08.2015Accepted: 05.10.2015Published: 21.10.2015*Address for correspondence: Dr. Marimuthu Govindarajan, Unit of Vector Biology and Phytochemistry, Department of Zoology, Annamalai University, Annamalai Nagar - 608 002, Tamil Nadu, India. Tel.: 91-4144-238248.Fax: 91-4144-238080.E-mail: drgovindzoo@yahoo.comABSTRACTMosquito-borne diseases with an economic impact create a loss in commercial and labor outputs, particularly in countries with tropical and subtropical climates. Mosquito control is facing a threat because of the emergence of resistance to synthetic insecticides. Insecticides of botanical origin may serve as suitable alternative biocontrol techniques in the future. In view of recently increased interest in developing plant origin insecticides as an alternative to chemical insecticide, in the present study ovicidal efficacy of different solvent leaf and seed extract of Pithecellobium dulce against filariasis vector C. quinquefasciatus. The ovicidal efficacy of the crude leaf extracts of P. dulce with five different solvents, such as benzene, hexane, ethyl acetate, methanol, and chloroform, was tested against C. quinquefasciatus. Ovicidal activity was determined against C. quinquefasciatus mosquito to various concentrations ranging from 100 to 600 mg/L under the laboratory conditions. Among five solvent tested the maximum efficacy was observed in the leaf and seed methanol crude extract was found to be most effective for ovicidal activity against vector mosquito. The extract of methanol exerted 100% mortality at 500-600 mg/L against C. quinquefasciatus. From the result, it can be concluded the crude extract of P. dulce was a potential candidate to develop newer and safer ovicides against C. quinquefasciatus.KEY WORDS: Culex quinquefasciatus, eco-friendly control, filariasis vector mosquito, ovicides, plant-borne extractRegular ArticleJournal of Medicinal Herbs and Ethnomedicine 2015, 1: 116-119http://sciencefl ora.org/journals/index.php/jmhe/doi: 10.5455/jmhe.2015-10-024Govindarajan, et al.: Ovicidal activity of Pithecellobium dulceJournal of Medicinal Herbs and Ethnomedicine ● Vol 1 ● 2015 117diseases at the individual as well as at the community level (Govindarajan et al., 2008). For instance, the ovicidal activity of neem-borne azadirachtin against the mosquitoes Culex tarsalis and C. quinquefasciatus (Su and Mulla, 1998). The leaf extract of Cassia fi stula with different solvents, such as methanol, benzene, and acetone was studied for the larvicidal, ovicidal, and repellent activity against Aedes aegypti (Govindarajan, 2009). The toxicity of dichloromethane, petroleum ether, and methanol extracts from Vitex negundo seed and leaf to the second- and fourth-instar larvae showed oviposition-deterrent effects on Plutella xylostella (Yuan et al., 2006). The active components dymalol and triterpenes isolated from the extract of Dysoxylum malabaricum act as an oviposition repellent and/or deterrent to Anopheles stephensi (Govindachari, 1999). The leaf extracts of Pemphis acidula were evaluated for larvicidal, ovicidal, and repellent activities against C. quinquefasciatus and A. Aegypti (Samidurai et al., 2009).Furthermore, the larvicidal, ovicidal and repellent activities of crude benzene and ethyl acetate extracts of leaf of Ervatamia coronaria and Caesalpinia pulcherrima were assayed for their toxicity against three important vector mosquitoes (Govindarajan et al., 2011). Larvicidal, ovicidal, and repellent activities of marine sponge Cliona celata (Grant) extracts against C. quinquefasciatus say and A. aegypti (Reegan et al., 2013). Govindarajan and Karupannan, (2011) investigated the larvicidal and ovicidal activities of benzene, hexane, ethyl acetate, methanol and chloroform leaf extract of Eclipta alba against dengue vector, A. aegypti. The larvicidal, pupicidal, ovicidal and ovipositional deterrent activity of methanol leaves extract of Spathodea campanulata against A. aegypti (Karthika Devi et al., 2013). In this research, we evaluated the ovicidal potential of P. dulce leaf and seed extracts against the filariasis vector C. quinquefasciatus.MATERIALS AND METHODSCollection of PlantsFully developed leaves and seeds of the P. dulce were collected from Thanjavur District (between 9°50′ and 11°25′ of the north latitude and 78°45′ and 70°25′ of the east longitude), Tamil Nadu, India. It was authenticated by a plant taxonomist from the Department of Botany, Annamalai University. Voucher specimens were deposited at the Herbarium of Plant Phytochemistry Division, Department of Zoology, Annamalai University.Plant ExtractsThe leaves and seeds were washed with tap water, shade-dried, and finely ground. The finely ground plant leaf powder (0.8 kg/solvent) was loaded in soxhlet apparatus and was extracted with five different solvents, namely hexane, benzene, chloroform, ethyl acetate, and methanol, individually. The solvents from the extracts were removed using a rotary vacuum evaporator to collect the crude extract. Standard stock solutions were prepared at 1% by dissolving the residues in ethanol. From this stock solution, different concentrations were prepared, and these solutions were used for ovicidal bioassays.Mosquito RearingC. quinquefasciatus was 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 1-week-old chick for blood meal. Mosquitoes were held at 28 ± 2°C, 70-85% relative humidity, with a photoperiod of 14 h light, 10 h dark.Ovicidal ActivityThe ovicidal activity, slightly modified method of Su and Mulla (1998) was performed. The eggs of C. quinquefasciatus were collected from vector control laboratory, Annamalai University. The leaf and seed extracts were diluted in the ethanol to achieve various concentrations ranging from 100 to 600 mg/L. Eggs of these mosquito species (100 numbers of 12-18 h old eggs) were exposed to each concentration of leaf and seed extracts. After treatment, the eggs from each concentration were individually transferred to distilled water cups for hatching assessment after counting the eggs under microscope. Each experiment was replicated six times along with appropriate control. The hatch rates were assessed 48 h post-treatment by the following formula.%. of mortality No of hatched larvaeTotal no  of eggs= ×100RESULTS AND DISCUSSIONThe ovicidal activity of crude hexane, benzene, chloroform, ethyl acetate, and methanol solvent extracts of leaf, and seed of P. dulce against the vector mosquito C. quinquefasciatus are presented in Table 1. Among extracts tested for ovicidal activity against C. quinquefasciatus the leaf and seed methanol extract of P. dulce exerted 100% mortality (zero hatchability) at 500 and 600 mg/L, respectively. The leaf extract of P. dulce was found to be most effective than seed against eggs of vector mosquito. Control eggs showed the 100% hatchability.Govindarajan, et al.: Ovicidal activity of Pithecellobium dulce118 Journal of Medicinal Herbs and Ethnomedicine ● Vol 1 ● 2015Plants are rich sources of bioactive compounds that can be used to develop environmentally safe vector and pest managing agents. Phytoextracts are emerging as potential mosquito control agents, with low-cost, easy-to-administer, and risk-free properties. Our findings are comparable to previous research by Al-Doghairi et al. (2004), which reported that the ovicidal effect of Solenostemma argel was low. However, concentrations of 0.05% and 0.1% exhibited significant effects, producing 65% and 75%; 62.9%, and 62.9%, respectively, on the 1st and 2nd day after treatment, respectively, the 0.1% concentration reduced egg hatch by 33.7%, compared with the control and 100% mortality values were evident in concentrations as low as 0.025% at 2 days post-hatching against C. pipiens. The oviposition deterrent effectiveness (76-100% repellency) against A. albopictus of 21 commercial insect repellent products (at 0.1% concentration), including 12 botanical, six deet-based, and three synthetic organics (Xue et al., 2006). All essential oil showed moderate ovicidal effects. However, the leaf ethanolic extract of Cassia obtusifolia had significant larvicidal effect with LC50 and LC90 values of 52.2 and 108.7 mg/l, respectively, at the higher concentration (400 mg/l) that showed 92.5% effective repellency against late third instar larvae of A. stephensi (Rajkumar and Jebanesan, 2009). The benzene extracts of Citrullus vulgaris exerted 100% mortality (zero hatchability) at 250 ppm, a very low hatchability (11.8%) at 200 ppm, and complete ovicidal activity at 300 ppm. The ovicidal effect of S. argel was low; however, concentrations of 0.05% and 0.1% exhibited significant effects (P < 0.05), producing 65% and 75% and 62.9% and 62.9% on the 1st and 2nd day after treatment, respectively.The essential oil from leaves and stems of Piper marginatum exhibited an oviposition deterrent effect against A. aegypti at 50 and 100 ppm in that significantly lower numbers of eggs (<50%) were laid in glass vessels containing the test solutions compared with the control solution (Autran et al., 2009). The benzene extracts of C. vulgaris exerted 100% mortality (zero hatchability) at 250 ppm and at 200 ppm a very low hatchability (11.8%), complete ovicidal activity at 300 ppm and the Fraction I at 80 ppm exerted a very low hatchability rate of 3.2% followed by Fraction II (6.9%), Fraction III, and IV afforded 4.9 and 5.3% hatchability recorded against A. stephensi and A. Aegypti, respectively, (Mullai et al., 2008). The leaf methanol extract of C. fi stula was tested for larvicidal and ovicidal activity of against C. quinquefasciatus and A. stephensi with the LC50 values of 17.97 and 20.57 mg/l, respectively, (Govindarajan et al., 2008). The complete ovicidal activity (100% mortality) was attained at 300 ppm for methanol, benzene, petroleum ether, and ethyl acetate extracts of Citrullus pubescens against C. quinquefasciatus (Mullai and Jebanesan, 2006).CONCLUSIONOur findings highlighted that the plant P. dulce exhibits moderate ovicidal activity against filariasis vector mosquito C. quinquefasciatus. These results could encourage the search for new active natural compounds offering an alternative to synthetic insecticides from other medicinal plant. P. dulce extracts may contribute to reduce environmental pollution and to an overall reduction in the population density of mosquito vectors of medical and veterinary importance.ACKNOWLEDGMENTThe authors are grateful to the University Grants Commission (UGC) (F.No.39-646/2010 (SR)), New Delhi, India for providing financial assistance for the present investigation and thankful to the Professor and Head, Department of Zoology, Annamalai University for the laboratory facilities provided. We acknowledge the staff members of the VCRC (ICMR), Pondicherry for their cooperation.Table 1: Ovicidal activity of P. dulce plant leaf and seed extracts against the filariasis vector C. quinquefasciatusPlant partSolvent Egg hatchability (%)Control 100 mg/L 200 mg/L 300 mg/L 400 mg/L 500 mg/L 600 mg/LLeaf Hexane 100±0.0a 81.8±1.6b 71.9±1.1c 58.6±1.5d 37.8±1.3e 28.4±1.4f 16.8±1.3gBenzene 100±0.0a 78.2±1.2b 63.4±1.3c 48.7±1.3d 31.1±1.4e 24.3±1.2f NHChloroform 100±0.0a 70.4±1.8b 54.6±1.5c 42.4±1.8d 28.6±1.2e 20.2±1.4f NHEthyl acetate 100±0.0a 65.3±1.1b 49.5±1.6c 38.7±1.4d 25.4±1.9e 18.5±1.1f NHMethanol 100±0.0a 56.1±2.0b 41.8±1.4c 29.8±2.0d 19.1±1.0e NH NHSeed Hexane 100±0.0a 92.6±1.6b 78.5±1.9c 65.9±1.2d 50.7±1.4e 33.6±1.6f 26.8±1.1gBenzene 100±0.0a 88.9±1.1b 67.2±1.2c 56.5±1.8d 41.3±1.6e 28.7±1.4f 20.6±0.9gChloroform 100±0.0a 81.6±1.0b 61.4±1.4c 49.7±1.4d 35.1±1.2e 24.2±1.8f 18.2±1.3gEthyl acetate 100±0.0a 73.7±1.7b 57.6±1.3c 44.1±0.9d 30.4±1.9e 19.3±0.9f NHMethanol 100±0.0a 62.5±2.0b 51.6±1.9c 39.3±1.4d 23.5±1.7e 17.8±1.2f NHValues are means±SD, Within each row, different letters indicate significant differences (Tukey’s HSD, P<0.05), NH: No hatchability, SD: Standard deviation, HSD: Honestly standard deviation, C. quinquefasciatus: Culex quinquefasciatus, P. dulce: Pithecellobium dulceGovindarajan, et al.: Ovicidal activity of Pithecellobium dulceJournal of Medicinal Herbs and Ethnomedicine ● Vol 1 ● 2015 119REFERENCESAl-Doghairi M, El-Nadi A, Elhag E, Al-Ayedh H. 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Ovicidal activity of Pithecellobium dulce (Family: Fabaceae) leaf and seed extracts against fi lariasis vector mosquito Culex quinquefasciatus (Diptera: Culicidae)

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Ovicidal activity of Pithecellobium dulce (Family: Fabaceae) leaf and seed extracts against fi lariasis vector mosquito Culex quinquefasciatus (Diptera: Culicidae)

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