ISSN: MOSQUITO LARVICIDAL, OVICIDAL AND PUPICIDAL ACTIVITIES OF ABRUS PRECATORIUS LINN

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1 OPEN ACCESS at journalijcir.com Research Article ISSN: MOSQUITO LARVICIDAL, OVICIDAL AND PUPICIDAL ACTIVITIES OF ABRUS PRECATORIUS LINN. (FABACEAE) AGAINST DENGUE VECTOR, AEDES AEGYPTI (LINN) MALARIAL VECTOR, ANOPHELES STEPHENSI (LISTON) AND FILARIAL VECTOR, CULEX QUINQUEFASCIATUS (SAY) (DIPTERA : CULICIDAE) Mathivanan Thangarasu, Krishnappa Kaliyamoorthy, *Elumalai Kuppusamy Abstract The present investigation was aimed to investigate the mosquito larvicidal, ovicidal and pupicidal activities of different extracts of Abrus precatorius Linn. (Fabaceae) against selected mosquito species of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus (Diptera: Culicidae). Abrus precatorius hexane, diethyl ether, dichloromethane, ethyl acetate and methanol extracts were tested against 4 th instar larvae of Ae. aegypti, An. stephensi and C. quinquefasciatus for 24 h and mortality were recorded at various concentrations. The LC 50 and LC 90 values were determined following probit analysis. Similarly, plant extracts were tested against pupae of selected mosquitoes at various concentrations and mortality of each pupa was recorded after 24 hrs post treatments of exposure. Furthermore, selected extracts were tested against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus eggs for 48 hrs post treatment and percentage of egg mortality recorded at various concentrations. The significant larvicidal activity was recorded from the highest concentration of methanol extract at 500 ppm and the least larvicidal activity was recorded from the 100ppm concentration of hexane extract. Lethal concentration, LC 50 value of hexane, diethyl ether, dichloromethane, ethyl acetate and methanol extract of Abrus precatorius againt Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus were , , , , , , , , , , , , , and ppm respectively. The chi-square values are significant at p 0.05 level. Among five solvent extracts, the methanol extract was responsible for strong lethal activity observed against selected mosquitoes and pest species. Moreover, among the five solvent extracts tested for pupicidal activity against selected mosquitoes at 100ppm concentrations, the methanol was found to be most effective for pupicidal activity provided (58.00%), (64.00%) and (72.00%) against Ae. aegypti, An. stephensi and C. quinquefasciatus, respectively. It has been noticed that the higher concentrations of Abrus precatorius methanol extracts possesses strong ovicidal activity at 300, 400 and 500ppm concentration against Ae. aegypti, An. stephensi and C. quinquefasciatus, no egg hatchability was recorded. In the same way, methanol extracts showed maximum ovicidal activity followed by ethyl acetate, dichloromethane, diethyl ether and hexane against selected vector mosquitoes. Key Words: Aedes aegypti, Anopheles stephensi, Culex quinquefasciatus, Abrus precatorius, Larvicidal activity, Ovicidal activity, Pupicidal activity. INTRODUCTION Mosquitoes are the major vector for the transmission of several communicable diseases like malaria, dengue fever, yellow fever, filariasis, schistosomiasis, Japanese encephalitis, etc., causing millions of deaths every and also cause allergic responses in humans that include local skin and systemic reactions such as angioedema [1,2]. Aedes aegypti (L.) is generally known as a vector for an arbovirus responsible for dengue fever, which is endemic to Southeast Asia, the Pacific island area, Africa, and the Americas. This mosquito is also the vector of yellow fever in Central and South America and West Africa. Dengue fever has become an important public health problem as the number of reported cases continues to increase, especially with more severe forms of the disease, Unit of Entomotoxicity, Department of Advanced Zoology & Biotechnology, Govt. Arts College (Autonomous), Nandanam, Chennai , Tamilnadu, India. Correspondence and Reprint Requests: Kuppusamy Elumalai Received: A p r i l 2 5, 2015 Accepted: May 5, 2015 Published Online: May 28, 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (creativecommons.org/licenses/by/3.0) Conflict of interest: None declared Source of funding: Nil

2 Kuppusamy Elumalai et al., dengue hemorrhagic fever, and dengue shock syndrome, or with unusual manifestations such as central nervous system involvement [3]. The disease remains endemic in more than 100 developing tropical countries, and its control is a major goal for improved worldwide health. Anopheles stephensi Liston is the common vector of malaria in India and other West Asian countries. Malaria remains one of the most prevalent diseases in the tropical world. With 200 million to 450 million infections annually worldwide, it causes upto2.7 million deaths [4]. The disease remains endemic in more than 100 developing tropical countries, and its control is a major goal for improved worldwide health. Lymphatic filariasis is a major vector borne disease making about 120 million peoples in 83 countries physically disabled [5] which is transmitted by C. quinquefasciatus mosquito having cosmopolitan distribution. WHO, (1992) [6] have suggested various controlling strategies to control vector transmission at different levels. Among the available vector control methods, chemical control is decisively superior over environmental and biological control strategies that have limited applicability in mitigating sporadic unpredictable outbreaks of vector borne disease. However, C. quinquefasciatus has also shown resistance to different insecticides used in mosquito control such as organochlorines, organophosphorous, pyrethroids and microbial insecticides throughout the world [7]. Mosquito control has been becoming increasingly difficult because of the indiscriminate uses of synthetic chemical insecticides which have an adverse impact on the environment and disturb ecological balance. Majority of the chemical pesticides are harmful to man and animals, some of which are not easily degradable and spreading toxic effects. The increased use of these insecticides may enter into the food chain, and thereby, the liver, kidney, etc., may be irreversibly damaged. They even result in mutation of genes and these changes become prominent only after a few generations [8]. Mosquito control is very costly. In larval mosquito control, application of insecticides in ponds, wells, and other water bodies may cause health hazards to human and larvivorus fishes. Nowadays, mosquito coils containing synthetic pyrethroids and other organophosphorus compounds causes so many side effects, such as breathing problem, eye irritation, headache, asthma, itching, and sneezing to the users. With the use mosquito repellent, people complained of ill health effect and sometimes required medical treatment. In addition, pests were becoming resistant to chemical treatments. Indoor residual spraying of insecticides stains the walls and leaves a long lasting unpleasant odour. These problems have highlighted the need for the development of new strategies for selective mosquito control. Phytochemicals are advantageous due to their eco-safety, targetspecificity, non development of resistance, reduced number of applications, higher acceptability, and suitability for rural areas. Botanicals can be used as alternative to synthetic insecticides or along with other insecticides under integrated vector control programs. The plant product of phytochemical, which is used as insecticides for killing larvae or adult mosquitoes or as repellents for protection against mosquito bites. Phytochemicals obtained from the whole plant or specific part of the plant by the extraction with different types of solvent such as aqueous, methanol, chloroform, benzene, acetone, etc., depending on the polarity of the phytochemical. Some phytochemicals act as toxicant (insecticide) both against adult as well as larval stages of mosquitoes, while others interfere with growth and growth inhibitor or with reproduction or produce an olfactory stimulus, thus acting as repellent or attractant [9-14]. Plants may be a source of alternative agents for control of mosquitoes because they are rich in bioactive chemicals, are active against a limited number of species including specific target insects, and are biodegradable. They are potentially suitable for use in integrated pest management programs [15-22]. 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 ovicidal and pupicidal potential of the hexane, diethyl ether, dichloromethane, ethyl acetate and methanol extracts of Abrus precatorius against the medically important vector mosquitoes, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus (Diptera : Culicidae). MATERIAL AND METHODS Plants collection and solvent extraction Matured leaves of Abrus precatorius (Fabaceae) were collected during the flowering season (December 2013-January 2014) in and around Yercaud hill station ( N, E) Salem District of the Tamilnadu India. The collected leaves brought to the laboratory where, they were washed thoroughly with tap water and kept in sunlight for 45 minutes for the complete evaporation of water and then shade dried on blotting paper spread at room temperature (28 ± 2 C). The dried plant material was powdered using electric blender and extracted with selected solvent using Soxhlet apparatus. The solvent from the crude extract was evaporated using 74

3 Mosquito larvicidal, ovicidal and pupicidal activities of abrus precatorius linn. (fabaceae) against dengue vector, aedes aegypti (linn) malarial vector, anopheles stephensi (liston) and filarial vector, culex quinquefasciatus (say) (diptera : culicidae) Table 1 Larvicidal activity of Abrus precatorius against the freshly moulted 4 th instar larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Larvicidal activity (%), Vectors mosquitoes Concentration (ppm) A. aegypti An. stephensi C. quinquefasciatus Hexane Control 1.8±0.8 a 1.4±1.2 a 1.6±1.2 a ±1.9 b 18.6±1.4 b 19.2±1.4 b ±1.8 c 36.7±1.4 c 35.6±2.8 c ±2.4 d 57.6±2.8 d 58.2±2.9 d ±2.7 e 78.5±1.4 e 79.3±2.5 e ±2.3 f 94.8±2.7 f 95.6±2.3 f Diethyl ether Control 1.2±0.8 a 1.4±1.2 a 1.6±1.2 a ±1.6 b 20.4±1.4 b 18.8±1.3 b ±1.7 c 31.6±1.4 c 34.6±1.5 c ±2.4 d 56.7±1.2 d 56.8±1.6 d ±2.5 e 78.8±2.7 e 78.3±2.7 e ±2.7 f 95.5±2.5 f 96.4±2.9 f Dichloromethane Control 1.2±0.6 a 1.4±1.2 a 1.2±1.2 a ±1.8 b 17.6±1.9 b 16.2±1.4 b ±1.2 c 31.7±1.6 c 30.7±1.5 c ±2.9 d 56.8±1.2 d 57.2±2.8 d ±2.6 e 78.5±2.8 e 78.8±2.6 e ±2.8 f 95.6±2.4 f 95.4±2.4 f Ethyl acetate Control 1.2±0.8 a 1.4±0.6 a 1.6±0.8 a ±1.9 b 19.4±1.4 b 21.7±1.4 b ±1.5 c 33.7±1.8 c 33.8±1.3 c ±1.4 d 59.6±1.2 d 61.5±1.5 d ±2.7 e 78.5±2.3 e 81.3±2.8 e ±2.4 f 96.5±2.9 f 97.9±2.7 f Methanol Control 1.2±0.8 a 1.4±0.6 a 1.6±0.2 a ±1.6 b 27.8±1.9 b 25.2±1.5 b ±2.4 c 33.4±1.5 c 36.6±1.9 c ±2.7 d 68.6±2.8 d 64.2±2.4 d ±2.9 e 84.2±2.6 e 85.3±2.7 e ±0.0 f 100.0±0.0 f 100.0±0.0 f Values represent mean ± S.D of five replications. Values with different alphabet in the column differs statistically, p<0.05% level. (LSD, DMRT) rotary evaporator at 45ºC until the complete evaporation of solvent, the crude extract was weighed and stored in an aseptic amber bottle vials at 4ºC in the refrigerator. Extraction The leaves of Abrus precatorius were washed with 75 Table 3. Ovicidal activity of different extracts of Abrus precatorius against the freshly laid eggs of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Mosquito species and their Percentage of egg hatch ability Extracts tested Concentrations Aedes Anopheles Culex (ppm) aegypti stephensi quinquefasciatus ± ± ± ± ± ±1.71 Hexane ± ± ± ± ± ± ± ± ±2.92 Diethyl ether ± ± ±2.42 Dichloromethane ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.84 Ethyl acetate ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.59 Methanol ± ± ± ± ± ± ± ± ±0.0 Control ± ± ±0.00 Values represent mean ± S.D of five replications. Mortality of the eggs observed after 48hrs of exposure period (Su and Mulla,1998). Values in the column with a different superscript alphabet are significantly different at P < 0.05 (MANOVA; LSD -Tukey s Test) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.18 tap water, shade-dried, and finely ground with the help of electrical blender. The finely ground plant leaf powder (1.0kg) was loaded in Soxhlet apparatus and was extracted with hexane, Table 2 Leathal concentration of different extracts of Abrus precatorius against the 4 th instar larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Extracts tested Mosquitoes LC50 95% Confidence Limit 95% Confidence Limit LC90 LCL UCL LCL UCL Slope 2 value Hexane Aedes aegypti Anopheles stephensi Culex quinquefasciatus Aedes aegypti Diethyl ether Anopheles stephensi Culex quinquefasciatus Aedes aegypti Dichloromethane Anopheles stephensi Culex quinquefasciatus Aedes aegypti Ethyl acetate Anopheles stephensi Culex quinquefasciatus Aedes aegypti Methanol Anopheles stephensi Culex quinquefasciatus LC50=Lethal Concentration 50%; LC90=Lethal Concentration 90%; LCL=Lower Confidence Limit; UCL= Upper Confidence Limit; 2 = Chi square. diethyl ether, dichloromethane, ethyl acetate and methanol by adapting a standard protocol [23]. The solvents from the extracts were removed using a rotary vacuum evaporator to collect the crude extract. Standard stock solutions were prepared to 100, 200, 300,400 and 500ppm by dissolving the residues in their respective solvent.

4 Test organisms The larvae of mosquitoes, Ae. aegypti, Anopheles stephensi and C. quinquefasciatus were collected from the agricultural gardens and field and continuously reared in the laboratory. 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 27 ± 2 C, 75±5% RH, with a photo period of 12L: 12D. Larvicidal bioassay The larvicidal activity of the plants crude extracts was evaluated as per the method recommended by World Health Organization (2005) [24]. Batches of Kuppusamy Elumalai et al., Table 4 Pupicidal activity of different extracts of Abrus precatorius against the pupae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. 2days post treatment Extracts tested Concentration Agricultural pests Mortality Adult emergence Pupal mortality % Mortality Adult % Emergence Aedes aegypti 9.93± ± ppm Anopheles stephensi 7.28± ± Culex quinquefasciatus 9.62± ± Aedes aegypti 16.39± ± Hexane 300ppm Anopheles stephensi 16.97± ± Culex quinquefasciatus 17.91± ± Aedes aegypti 25.82± ± ppm Anopheles stephensi 24.71± ± Culex quinquefasciatus 25.55± ± Aedes aegypti 8.36± ± ppm Anopheles stephensi 10.81± ± Culex quinquefasciatus 11.15± ± Aedes aegypti 14.82± ± Diethyl ether 300ppm Anopheles stephensi 18.26± ± Culex quinquefasciatus 21.73± ± Aedes aegypti 24.87± ± ppm Anopheles stephensi 25.29± ± Culex quinquefasciatus 25.96± ± Aedes aegypti 10.46± ± ppm Anopheles stephensi 10.80± ± Culex quinquefasciatus 10.20± ± Aedes aegypti 19.58± ± Dichloromethane 300ppm Anopheles stephensi 18.93± ± Culex quinquefasciatus 20.07± ± Aedes aegypti 26.27± ± ppm Anopheles stephensi 26.58± ± Culex quinquefasciatus 26.32± ± Aedes aegypti 12.64± ± ppm Anopheles stephensi 11.87± ± Culex quinquefasciatus 12.72± ± Aedes aegypti 21.55± ± Ethyl acetate 300ppm Anopheles stephensi 22.94± ± Culex quinquefasciatus 21.56± ± Aedes aegypti 30.00± ± ppm Anopheles stephensi 28.67± ± Culex quinquefasciatus 30.00± ± Aedes aegypti 17.35± ± ppm Anopheles stephensi 19.31± ± Culex quinquefasciatus 21.49± ± Aedes aegypti 27.93± ± Methanol 300ppm Anopheles stephensi 28.16± ± Culex quinquefasciatus 30.00± ± Aedes aegypti 30.00± ± ppm Anopheles stephensi 30.00± ± Culex quinquefasciatus 30.00± ± Control 0.00ppm Selected mosquitoes 0.00± ± Value represents mean ±S.D. of five replications. Mortality of the pupae observed after 7 days of exposure period (WHO, 2005). Values in the column with a different superscript alphabet are significantly different at P < 0.05 (MANOVA; LSD -Tukey s Test). 25 fourth instar larvae were transferred to a small disposable test cups, each containing 200 ml of water. The appropriate volume of dilution was added to 200 ml water in the cups to obtain the desired target dosage, starting with the lowest concentration. Five replicates were set up for each concentration, and an equal number of controls were set up simultaneously using tap water. To this, 1 ml of appropriate solvent was added. The larval mortality was calculated by using the formula of Abbott (1925) [25] and LC 50 value was calculated after 24 h by probit analysis [26]. Ovicidal activity For ovicidal activity, slightly modified method of Su and Mulla (1998) [27] was performed. The 76

5 Mosquito larvicidal, ovicidal and pupicidal activities of abrus precatorius linn. (fabaceae) against dengue vector, aedes aegypti (linn) malarial vector, anopheles stephensi (liston) and filarial vector, culex quinquefasciatus (say) (diptera : culicidae) eggs/egg rafts of Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus were collected from vector control laboratory, Department of Zoology, Govt. Arts College, Nandanam. The different leaf extracts diluted in the appropriate solvent to achieve various concentrations ranging from 100 to 500 ppm. Eggs of these mosquito species (100) were exposed to each concentration of leaf 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 five times along with appropriate control. Pupicidal activity: Batches of thirty number of pupae were introduced into 500 ml of the test medium containing 100 and 500ppm concentration of the crude extract in a plastic cups in five replications. In control, the same number of pupae was maintained in 500 ml of dechlorinated water containing appropriate volume of DMSO. All containers were maintained at room temperature (28±2 C) with naturally prevailing photoperiod (12: 12h/L: D) in the laboratory. Any pupa was considered to be dead if did not move when prodded repeatedly with a soft brush. Mortality of each pupa was recorded after 24 of exposure to the extract [25]. RESULTS Results of the present study reflected spectrum of activity with Abrus precatorius different solvent crude extracts tested against the selected mosquitoes larvae, pupae and eggs. Generally, as the concentration increases the rate of larval mortality are also increases. The significant larvicidal activity was recorded from the highest concentration of methanol extract at 500 ppm and the least larvicidal activity was recorded from the 100ppm concentration of hexane extract (table -1). Lethal concentration, LC 50 value of hexane, diethyl ether, dichloromethane, ethyl acetate and methanol extract of Abrus precatorius againt Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus were , , , , , , , , , , , , , and ppm respectively. The chisquare values are significant at p 0.05 level. Among five solvent extracts, the methanol extract was responsible for strong lethal activity observed against selected mosquitoes species (table 2). It has been noticed that the higher concentrations of Abrus precatorius methanol extracts possesses strong 77 ovicidal activity at 300, 400 and 500ppm concentration against Ae. aegypti, An. stephensi and C. quinquefasciatus, no egg hatchability was recorded (table 3). In the same way, methanol extracts showed maximum ovicidal activity followed by ethyl acetate, dichloromethane, diethyl ether and hexane against selected vector mosquitoes. Moreover, among the five solvent extracts tested for pupicidal activity against selected mosquitoes at 100ppm concentrations, the methanol was found to be most effective for pupicidal activity provided (58.00%), (64.00%) and21.49 (72.00%) against Ae. aegypti, An. stephensi and C. quinquefasciatus, respectively (table 4). Results of this study show that the Abrus precatorius selected solvent crude extracts may be a potent source of natural larvicidal, ovicidal and pupicidal activities against selected important vector mosquitoes. DISCUSSIONS Due to indiscriminate use of synthetic chemicals to control the mosquitoes in the natural habitats, they have developed strong resistance to almost all the chemicals that are available today. Moreover, chemical pesticides gradually altered the behaviour of non-target organisms. Thus, in this context, the world scientific community intensively searching for the alternative mosquitocidal agent preferably from plants available in nature. Today, the environmental safety of an insecticide is considered to be of important milestone in the field of pest control in general and vector control programme in particular. An insecticide must not cause high mortality in target organisms in order to be acceptable [28]. The extract treated eggs exhibited an allayed hatchability and this may be due to the action of phytochemicals present in the extract. The extract may inhibit the hatchability of the eggs by interfering with their chorion. It is evident from the present study that exposure of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus eggs to the leaf extracts of Abrus precatorius various solvents not only elicited egg mortality but also delayed hatchability to larval stages. Similar kind of observation was also noted earlier by several workers [29, 30]. The ovicidal activity indicated an important finding that the larvae which hatched out of the treated eggs were succumbed to death within an hour or two. In the present study, our aim was to determine whether Abrus precatorius could be used for mosquito control. We observed a functional response of the ovicidal activity exhibited by the methanol extract. In the case of ovicidal activity, exposure to the freshly laid eggs was more effective than that to the older eggs.

6 Kuppusamy Elumalai et al., Similarly, ovicidal and gravid mortality effects of ethanolic extract of Andrographis paniculata was assessed by Kuppusamy et al., [31] against An. stephensi. Larvicidal and oviposition activity of Cassia obtusifolia leaf extract against An. stephensi Liston was also evaluated by Rajkumar and Jebanesan [32]. Similarly, the aqueous and hydro-alcoholic extracts of Melia azedarach leaves and seeds were tested to explore the in vitro ovicidal and larvicidal activity against Haemonchus contortus Kamaraj et al., [33] and the results were comparable with our results. Additionally, through screening several plants for their larvicidal activity, Sharma et al., [34] found that Artimisia annua was the most toxic against anopheles with an LC 50 of ppm and ppm after 24 and 48 h of exposure, respectively. In addition, the larvicidal effects of Momordica charantia fruit on An. stephensi (LC 50 of ppm) were also investigated by Singh et al., [35]. The biological activity of the plant extract might be due to a variety of compounds in Abrus precatorius a may jointly or independently contribute to cause larvicidal, ovicidal and pupicidal activity against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. The main chemical compounds present in the E. pedunculatum might responsible for the activities recorded in the present experiments. It would have been suggested that the direct and indirect contributions of such compounds to treatment efficacy while on the use of botanical insecticides for the control of An. stephensi. These and other naturally occurring insecticides may play a crucial role in vector control programs in the near future [36]. Since An. stephensi breeds in drinking water tank, many of the plant extracts are subject to risk factors in mosquito control [37]. In the present study, Abrus precatorius extract showed promising effect on Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus and it has no deleterious effects against human beings. CONCLUSION The methanol extract of Abrus precatorius at higher concentration showed maximum larvicidal, ovicidal and pupicidal activity against selected vector mosquitoes. Hence it is inferred that the methanol extract of Abrus precatorius can be used further for the isolation of active molecules and to develop a new botanical formulation for the management of dipteran mosquitoes. This may serve as an effective phytopesticides to control the vectors in the near future. We are continuing to develop phytopesticides as a biocide for medically important vector mosquitoes. Conflict of interests We declare that we have no conflict of interests. Acknowledgements Authors are gratefully acknowledged to Professor N. Kalaichelvi, Head, Department of Zoology The Principal, Govt. Arts College (Autonomous) Nandanam for their support and laboratory facilities provided. The authors are also thankful to University Grants Commission (UGC, New Delhi; Ref. No /2013 (SR) for their financial assistance. References 1. A.I. Vogel, "Text book of practical organic chemistry". The English Language Book Society and Longman, London, pp 1368, Balu selvakumar J. Gokulakrishnan K. Elumalai, S. Dhanasekaran, A. Anandan and K. Krishnappa. "Mosquito ovicidal and repellent activity of Melothria maderaspatana plant leaf extracts against Aedes aegypti (Diptera: Culicidae)". International Journal of Recent Scientific Research, vol 3(5), pp , 2012b. 3. Balu selvakumar, J. Gokulakrishnan, K. Elumalai, S. Dhanasekaran, A. Anandan and K. Krishnappa. "Mosquito larvicidal activity of Oxystelma esculentum plant extracts against Anopheles stephensi (Diptera: Culicidae) ". International Journal of Recent Scientific Research, vol 3(5), pp , 2012a. 4. C. Kamaraj, A.A. Rahuman, A. Bagavan, J.M. Mohamed, G. Elango, G. Rajakumar, "Ovicidal and larvicidal activity of crude extracts of Melia azedarach against Haemonchus contortus (Strongylida) ". Parasitology Research, vol 106, pp , C. Kuppusamy, K. Murugan, "Oviposition deterrent, ovicidal and gravid mortality effects of ethanolic extract of Andrographis paniculata Nees against the malarial vector Anopheles stephensi Liston (Diptera:Culicidae) ". Entomological Research, vol 38, pp , C. Pancharoen, W. Kulwichit, T. Tantawichien, U. Thisyakorn, C. Thisyakorn. "Dengue infection: a global concern". Journal of Medical Association, Thailand. vol.85, pp 25 33, C.B. Wandscheer, J.E. Duque, M.A.N. da Silva, Y. Fukuyama, J.L. Wohlke, J. Adelmann, J.D. Fontana, "Larvicidal action of ethanolic extracts from fruit endocarps of Melia azedarach and Azadirachta indica against the 78

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