1 Introduction
Mosquito borne diseases are transmitted to human beings through mosquito bite as usual mode. Since there is no effective vaccine available for the control of these diseases, control of mosquito population and use of insect repellents to keep away mosquitoes from landing in order to take successful blood meal can provide a practical and economical means of preventing mosquito-borne diseases.

Synthetic pesticides viz. malathion, DDT, deltamethrin etc and bio-pesticides like Bacillus thuringiensis (Tabashnik et. al., 1994; Yang et. al., 2005) possess high environmental threats and charge huge operational cost. Besides, mosquitoes are developing fast adaptive resistances against these persisting synthetic chemicals. Insecticides of botanical origins are expected to be target specific in comparison to synthetic chemicals. They are also biodegradable, thus minimize the concern of biomagnifications of the harmful chemicals in the environment. Side effects on human and other non-target animals may also be reduced if green formulations in suitable form can be discovered and popularize (Jeyabalan et al., 2003; Prabhakar et al., 2004). Hence, control of adult mosquitoes with the use of plant derived agents has been highlighted for their instant effect at the time of need.

Insect repellents are not only important for permanent residents of disease prone areas, especially in tropical countries, but also for travelers who are vulnerable to the diseases spread by mosquito vectors. Various plants have long been familiar for their established insect repellent properties (Sukumar, 1991; Seyoum, 2002; Singha et al., 2011; Adhikari et al., 2012; Rawani et al., 2012) but a few among them have been studied more extensively during the past decades.

The common mosquito repellents available in Indian markets contain DEET (N,N-diethyl-3-methylbenzamide) as one of the chemical constituent which have shown excellent protection from mosquito bites (Thavara et al., 2001) and other biting insects (Coleman et al., 1993). Besides, there are reports of several toxicity problems on application of DEET ranged from mild effects, such as contact urticaria (Maibach and Johnson, 1975) and skin eruption (Reuveni and Yagupsky, 1982), to severe reactions, such as toxic encephalopathy (Edwards and Johnson, 1987). Many research groups are engaged to find and develop repellents of botanical origin mainly to overcome these adverse effects in addition to an environment-friendly remedy.

The Plant Drypetes roxburghii (Wall.) Hurus. is an evergreen tree found throughout India and it belongs to family Euphorbiaceae. It is generally upto 20 m in height, branches are usually pendent, and leaves are simple, alternate. Fruits are drupe type, ovoid-ellipsoid, seed normally one. Fruits have been used for many conventional medical applications such as treatment of ulcers of the mouth, stomach, hot swellings, small pox and also useful in burning sensation, ophthalmopathy, hyperdipsia, elephantiasis, constipation, strangury, azoospermia, habitual abortion, sterility (Sengupta et al., 1968, Varshney et al., 1973).

Though mosquito larvicidal studies with different plant extracts are prevalent in a vast volume of literature (Rawani et al., 2009; Mallick Haldar et al., 2012, 2013, 2014; Hossain et al., 2011; Kundu et al., 2013; Singha et al., 2011,2012; Bhattacharya et al., 2014; Singha Ray et al., 2014), similar reports on adult mosquito is substantially rare. In the present study, we reported for the first time the effect of crude and ethyl acetate extracts of mature fruits of D. roxburghii against adult Cx. quinquefasciatus Say and An. stephensi Liston. Adulticidal bioassay was performed to assess the efficacy of the extracts in causing mortality to adult mosquitoes in laboratory condition. To test the ability of the extracts to keep away mosquitoes, repellency test was conducted.

2 Results
Table 1 depicts adulticidal activity of fruit extract of D. roxburghii on Cx. quinquefasciatus and An. stephensi. In both the cases the mortality rate increased gradually with increased concentrations of ethyl acetate extract. LC₅₀ and LC₉₀ values suggested distinctly that adults of An. stephensi were more susceptible to death than Cx. quinquefasciatus. The higher R² values (near 1) in both the cases proved the significance of the tests. Crude extract of fruit also exerted some deleterious effect on both the species of mosquitoes.

Table 1 Adulticidal activity of fruit extracts of Drypetes roxburghii on Culex quinquefasciatus and Anopheles stephensi

Table 2 Embodies the results of repellency activity of D. roxburghii fruits in crude form (fruit juice) as well as in the form of ethyl acetate solvent extract of the same against the biting activity of Cx. quinquefasciatus and An. stephensi. The crude juice was used without any alteration whereas graded concentrations of the solvent extract were made for repellency test. The result reflected that the crude juice was hundred percent efficient against biting activity of Cx. quinquefasciatus up to 1 hour, but after that its efficiency slowly decreased and at the end of 150 minutes it became 73.33%. 1% ethyl acetate extract showed 5% repellency at 30 minutes of application but after 120 minutes its repellency became nil. 25% extract gave about 80% protection up to 30 minutes. Among the other concentrations while 50% concentration gave 100% protection up to 1 hour, 75% and 99 % concentrations conferred hundred percent protections up to 90 minutes and 2 hours respectively. 94.33 % protection was achieved from 99% concentration at 150 minutes after application. In case of An. stephensi 1% ethyl acetate extract showed 8% repellency at 30 minutes of application but at 150 minutes its repellency became nil.

Table 2 Repellency activity of fruit extracts of Drypetes roxburghii against Culex quinquefasciatus and Anopheles stephensi

3 Discussion
An. stephensi and Cx. quinquefasciatus are the major vectors of urban malaria and lymphatic filariasis respectively. Among different methods one of the approaches for control of these mosquito-borne diseases is the interruption of disease transmission by killing or preventing mosquitoes from biting human beings. Many plant based formulations though have potential larvicidal activity failed to demonstrate their adulticidal efficiencies. Lee and Chiang (1994) reported a good larvicidal property of Stemona tuberose, however found no adulticidal effect. (Choochote et al., 1999) also tried to demonstrate the adulticidal property of Kaempferia galanga, however it only gave a knockdown effect at the initial stage of exposure but after transferring to the holding tube, the mosquito restored from the knocked down state. Therefore they inferred that perhaps K. galanga could be useful as a repellent instead. However, (Rohani et al., 1997) reported that Litsea elliptica and Piper aduncum had potentials as adulticides against Ae. aegypti . (Jeyabalan et al., 2003) reported the adulticidal effect of Pelargonium citrosa on An. stephensi, with LC₅₀ and LC₉₀ values of 1.56% and 5.22% respectively. Similar result was obtained from root extract of Valeriana jatamansi which established its adulticidal efficacy against An. stephensi, An. culicifacies, Aedes aegypti, An. albopictus, and Cx. quinquefasciatus (Dua et al., 2008). Results of our study showed that the crude and ethyl acetate fraction of D. roxburghii possessed marked adulticidal activity against Cx. quinquefasciatus with LC₅₀ and LC₉₀ values of 109.62 and 189.01ppm and against An. stephensi with LC₅₀ and LC₉₀ values of 95.41 and 181.96 ppm which were comparable to other reported botanical formulations having adulticidal activity on mosquitoes. The Probable cause of exhibiting adulticidal effect on mosquitoes might be due to the presence of different secondary metabolites in the fruit extract of D. roxburghii. Alkaloids are responsible for toxic effect in adult mosquitoes if kept in contact or ingested (Gakuru and Foua, 1996). Flavonoids, saponins are also reported to be toxic to mosquitoes (Bouchelta et al., 2005). As the fruits of D. roxburghii contain alkaloids, flavonoids, saponins (Mallick Haldar et al., 2013), their individual or combined action might be responsible for causing mortality in mosquitoes as different compounds involve different mechanisms of action which reduce the effectiveness of detoxification mechanism of those compounds in mosquito (Shama and Dhiman, 1993; Corbel et al., 2004).

Herbal products with proven potential as repellents can play an important role in the interruption of the transmission of mosquito-borne diseases at the individual as well as at the community level. (Hebbalkar et al., 1992) documented that the essential oil obtained from Vitex negundo leaves showed repellency ranged from 1 to 3 hour (Hebbalkar et al., 1992). Essential oil extracted from Tagetes minuta, provide 90% protection for 2 hour against Ae. aegypti , An. stephensi and Cx. quinquefasciatus (Tyagi et al., 1994). Repellency activity of methanol extract of Ferronia elephantum leaves against Ae. aegypti mosquitoes was reported by Venkatachalam and Jebanesan (2001). According to them the extract at 1.0 mg/cm² and 2.5 mg/cm² concentrations was responsible for 100% protection up to 2.14±0.16 hour and 4.00±0.24 hour respectively, while 45.8% and 59.0% protection was achieved up to10 hours at 1 mg/ cm² and 2.5 mg/cm² concentrations. Tawatsin et al., (2001) reported repellency activity of essential oil of Curcuma longa against Ae. aegypti, An. dirus and Cx. quinquefasciatus was improved due to addition of 5% vanillin with it. The same research group reported repellant activity of essential oils extracted from 18 other plant species, of 11 families (Tawatsin et al., 2006). 10% solutions of the oils in absolute ethanol with additives exhibited excellent repellency of 4.5-8 hour against night-biting mosquitoes like Cx. quinquefasciatus, An. dirus and Ae. albopictus though their effects on Ae. aegypti were not so commendable (0.3-2.8 hour). DEET and ethyl butylactylamino-propionate (IR3535) showed excellent repellency against An. dirus, Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus (repellency 6.7-8 hour). Amer and Mehlhorn (2006) described five most effective oils, Cajeput (Melaleuca leucadendron), Litsea (Litsea cubeba), Catnip (Nepeta cataria), Niaouli (Melaleuca quinquenervia) and Violet (Viola odorata) which induced 100% repellency against An. stephensi, Cx. quinquefasciatus and Ae. aegypti for 8 hour of maximum protection time. The essential oil of Zingiber officinalis provided 100% protection through repellent activity at 4.0 mg/ cm²up to 120 min against Cx. quinquefasciatus (Pushpanathan et al., 2008). It was revealed from our study that the efficacy of the ethyl acetate extract of D. roxburghii was quite comparable with the above mentioned study reports since results showed that 100% protection was achieved up to 2 hour continuously from 99 % concentration of the ethyl acetate extract against both species of mosquitoes. Pandey et al., (2009) described that the thymol compound isolated from the seed of Trachyspermum ammi, at the dose of 25.0 mg/mat imparted complete repellency for 1 hour duration against adult form of An. stephensi, whereas the same degree of repellency was obtained by the essential oil of the seeds at the dose of 55.0 mg/mat. Double-fold activity of the isolated thymol compound over the oil was reported in that study.  In our study the coils containing crude extract exhibited complete protection for 1 hour and the efficiency gradually diminished with time whereas the 75% and 99% ethyl acetate extract containing coils conferred 100% protection from bites of Cx. quinquefasciatus and An. stephensi with duration of 2 hours. Resemblances of the results help us to presume that the ethyl acetate extract of the fruits of D. roxburghii was twice as much efficient as the crude extract of the same as a repellent agent of mosquitoes. In addition both the extracts had no adverse effects like allergic manifestations or other kind of visible reactions during the exposure period or beyond that.

The plant based products are generally not so expensive, easily accessible, relatively less toxic to environment besides being effective on mosquitoes. Entire study herein clearly demonstrated a high potential for using crude and ethyl acetate extracts of D. roxburghii fruit as adulticide and repellent against mosquitoe species like Cx. quinquefasciatus and An. stephensi without causing any allergic reaction which may lead to new and more effective strategies to prevent and control mosquitoes.

4 Materials and Methods
4.1 Collection of plant material of D. roxburghii
Mature fruits of D. roxburghii were collected from trees grown within the University campus during November to January, 20013-2014. It was authenticated and voucher specimens were deposited in the Mosquito, Microbiology and Nanotechnology Research Units, Department of Zoology, The University of Burdwan.

4.2 Preparation of crude extract
Fruits were washed thoroughly with distilled water. Crude extract was prepared by grinding the plant material in a mixer grinder. Then the ground material was passed through cheesecloth. Required concentrations of crude extract were prepared by mixing the liquid extract with suitable amount of sterilized distilled water.

4.3 Preparation of solvent extracts
Cleaned mature fruits were dried in shade at room temperature and milled into fine powder with mixer grinder. For solvent extraction procedure 200 gm of finely ground powdered fruit were extracted with ethyl acetate for 72 hours, in a grease free Soxhlet apparatus. After collection and filtration, the extract was subjected to evaporation in a vacuum rotary evaporator below 40°C, and then the extract was stored in a refrigerator within air tight glass container until further use.

4.4 Test mosquitoes
Adult Cx. quinquefasciatus and An. stephensi mosquitoes were collected from the mosquito colonies maintained in pathogen free and hygienic condition in the laboratory of Mosquito, Microbiology and Nanotechnology Research Units, Department of Zoology, The University of Burdwan. Adult mosquitoes were reared in humidified cages and fed with 10% aqueous glucose solution. Female mosquitoes were periodically blood-fed on restrained albino rat for egg production.

4.5 Adulticidal bioassay with the fruit extracts
This bioassay was performed according to WHO (1981) protocol with minor modifications. Appropriate amount of residue of ethyl acetate extract was dissolved in 2.5 ml acetone to achieve solutions of desired concentrations (50, 100, 150, 200, 250 ppm) and applied on Whatman no. 1 filter papers (size 12 × 15 cm²) (Dua et al., 2008). For experimentation with crude extract 2.5 ml juice extracted from fresh mature fruits was applied on the filter papers. Control papers were treated only with acetone. After drying the filter papers were kept inside plastic holding tube covering the inner side of the tube. Separate batches of 25 An. stephensi and 25 Cx. quinquefasciatus adult mosquitoes (2-5 days old blood starved, glucose fed) were cautiously transferred into a plastic holding tube and they were allowed there for acclimatization till 1 hour. Then the mosquitoes were exposed to the treated papers containing holding tube for 1 hour. They were transferred back to the previous holding tube and kept there for 24 hour for recovery. Mosquitoes were provided with 10% glucose solution soaked in cotton ball. Control experiments were performed similarly. The test was repeated four times for each mosquito species. Adulticidal efficacy of the plant extracts were expressed in terms of percent mortality of the mosquitoes after 24 hour of recovery period.
Percent mortality was corrected by using Abbott’s formula (1925).


4.6 Repellency potentiality test with the fruit extracts
Repellent activity test of plant extract was executed through percentage of protection in relation to dose method. One hundred (3-4 days old) blood starved female mosquitoes were kept inside two net cages (45 x 30 x 45 cm³) for concomitant test and control experiments. A stock solution of 50 ml of 1000ppm concentration was made by dissolving the ethyl acetate extract in ethanol. A number of ranges of test solutions (1%, 25%, 50%, 75% and 99%) of the ethyl acetate extract were prepared by dissolving the stock solution in ethanol. The forehands of the volunteer (self volunteer) were cleaned with ethanol. Different test solutions were applied thoroughly from the elbow to the tip of the fingers on one hand followed by drying (test for each concentration was done separately). The other forehand was treated with ethanol only to serve as control. After gently tapping the sides of the bioassay cage to stimulate the mosquitoes, the treated and control forehands were put into the cages first and kept there for 5 min. If at least two mosquitoes landed on the test areas, the hands were shaken off before imbibing any blood and withdrawn from the cages.  The number of bites was counted over 5 min/hour from 6.00 p.m to 6.00 a.m. Care was taken between the testing periods to minimize contact of the test sites with any other influence. All the experiments were conducted four times with control. Repellency of the crude extract was also assessed by applying it directly on the forearm without preparing any graded concentration. In that case the other forearm was washed with distilled water to serve as control. The percentage protection was calculated by using the following formula (Venkatachalam and Jebanesan, 2001)


Conflict of Interest
We declare that we have no conflict of interest.

Acknowledgements
The authors gratefully acknowledge the financial support provided by UGC [No.F.17-8/08 (SA-I)] and UGC-DRS. They are indebted to Professor Dr. A. Mukhopadhyay, Botany Department, The University of Burdwan, for botanical identification of the plant species.

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