Larvicidal efficacy of Callistemon citrinus Skeels., against Culex quinquefasciatus (Diptera: Culicidae)

Larvicidal efficacy of Callistemon citrinus Skeels., against Culex quinquefasciatus (Diptera: Culicidae) Misvar Ali K 1 and E. M. Aneesh 1 1 Communicable Disease Research Laboratory (CDR, St. Joseph s College, Irinjalakuda, Thrissur, Kerala ABSTRACT The larvicidal activity of hexane, ethyl acetate, acetone and methanol leaf extracts of Callistemon citrinus against the early fourth instar larvae of Culex quinquefasciatus was studied. Insecticidal susceptibility tests were carried out employing WHO standard method and the mortality was observed after 24 h exposure. The methanol leaf extract was found to be the effective larvicide with LC 50 value of 251.7 mg/l after 24 h. The result indicates promising larvicidal activity of C. citrinus against Culex quinquefasciatus. Further investigations are needed to isolate and identify the active principles involved, level of toxicity and their mode of action. Key words: Larvicidal efficacy, Callistemon citrinus, Culex quinquefasciatus Introduction Vector borne diseases are a major source of illness and death worldwide. Mosquitoes are one of the most important vectors that alone transmit diseases to more than 700 million people per annum (Taubes, 1997). They 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 and Misvar, et al., 2014). Mosquitoes belonging to the Genera Culex, Aedes, Anopheles, Mansonia and Armigeres are the major vectors seen in Kerala (Lakshmi et al., 2014). Culex quinquefasciatus, the potential vector of bancroftian filariasis is the most widely distributed mosquito in India (Rahuman et al., 2009). It is responsible for major public health problem in India with around 31 million microfilaraemics, 23 million cases of symptomatic filariasis, and about 473 million individuals potentially at risk of infection (Agrawal et al., 2006). Targeting mosquito larvae is more desirable than controlling adults because the larvae are concentrated in a relatively small area; whatever microbial insecticide adopted needs to be consumed by 99

mosquito larva and must be applied well before the last larval instar stage (Tia et al. 1990). Conventional synthetic pesticides such as DDT, malathion and pyrethroides are used for mosquito management to protect humans from the adverse effects of mosquito borne diseases. However, unsystematic prolonged application of these pesticides can have adverse effects on the environment, as well as cause residual effects and induce the development of resistance to the pesticide by the vector (Mohan and Ramaswamy, 2007). Therefore, it is necessary to develop environmentally safe, biodegradable, economical and indigenous methods for the control of vectors that can be used with minimum care by individuals and communities (Mittal and Subbarao, 2003). Humans have used plant parts, products and metabolites in pest control since early historical times. Plants are the chemical factories of nature, producing many chemicals, some of which have medicinal and pesticidal properties. Larvicides of plant origin are currently receiving considerable attention because of their relatively harmless biodegradable properties. Since 1920s more than 2000 plants have been tested for insecticidal properties. Recently, lot of research work is being carried out in India to search for alternative eco-friendly effective larvicides from botanical origin (Singh et al., 2006). Interest in this field is based on the fact that these substances are least phytotoxic and does not lead to the accumulation of chemical residues in flora, fauna, soil and the entire environment in general. The aim of this study was to find the larvicidal efficacy of C. citrinus of Southern Western Ghats, Kerala against Cx. quinquefasciatus, a potential vector of some arboviral infections as well as filariasis. Materials and Methods Plant material Leaves of C. citrinus were collected from Wayanad, southern part of Western Ghats, in Kerala. It was authenticated by a plant taxonomist from the Department of Botany, St. Joseph s College, Irinjalakuda. A voucher specimen is deposited at the herbarium division, Communicable Disease Research Laboratory, St. Joseph s College, Irinjalakuda. Preparation of extracts Powdered plant materials were extracted successively by using different solvents of increasing polarity (hexane, ethyl acetate, acetone and methanol) in soxhlet apparatus for 8 to 12 h. The extracts were evaporated in a rotary vacuum evaporator at 40 o C to dryness and stored at 4 o C in an air tight bottle for further analysis. Mosquito Larvae Larvae of Culex quinquefasciatus were reared in plastic trays (36x24x7) containing dechlorinated water. Larvae were fed a diet of yeast and powdered dog biscuits in the ratio of 2:1, kept at 26 ± 2º C and 75% - 80% relative humidity, with a photoperiod of 12:12 Light and Dark h for the larval growth. Early fourth instar larvae were used for bioassay. Larval bioassay Larval bioassays were performed according to the standard guidelines of WHO (2005). Stock solutions of the four solvent extracts were prepared and used to make different concentrations. Twenty five larvae were introduced into a 500 ml beaker containing 249 ml of water with one ml of acetone dissolved with desired concentration of the extract. A minimum of three replicates were kept for each concentration along with the control. Mortality was recorded after 24 h at 26 o C.

Data analysis The control mortality was corrected using Abbott s (1925) formula, if mortality was between 5-20%. The corrected mortality data were subjected to regression analysis of probit mortality on log dosage Finney (1971). LC 50 values were considered to be significantly different if the 95% FLs of two LC 50 values did not overlap each other (Yang et al., 2002). Results The results for the toxicity of the Callistemon citrinus leaf extracts by different solvents tested against Cx. quinquefasciatus larvae are presented in Table 1. The leaf extracts of C. citrinus showed larval mortality against the fourth instar larvae of Cx. quinquefasciatus. Methanol extract has exhibited minimum LC 50 and LC 90 of 251.7 ppm and 819.1 ppm respectively. It was followed by the acetone extract by producing 50% mortality with 327.95 ppm. The LC 50 value of ethyl acetate extract was 471.81ppm with LC 90 of 2052.99. The least larvicidal activity was detected in hexane extract of C. citrinus with LC 50 of 511.96 ppm. The log dose probit mortality responses of all the four solvent extracts are shown in Fig. 1. Table 1 Efficacy of different solvent extracts of Callistemon citrinus leaf extract against fourth instar larvae of Cx. quinquefasciatus Extracti on solvent Conc. in ppm %mortality Slop e±se LC 50 (mg/ (95%F LC 90 (mg/ (95%F χ 2 (df) Regressi on equation Hexane 0 14.2 ±2.0 26.5 ±1.1 37.3 ±2.1 52.0 ±0.5 82.0 ±3.6 1.78 ±0.4 0 511.96 275.03-1425.9 5 2673.40 1112.5-0 14.00( 3) 1.7853 X +.1631 Ethyl acetate 0 09.8±3.6 28.6±2.0 42.5±2.0 63.6 ± 1.5 77.3 ± 2.5 2.00 ±0.1 9 471.81 411.00-545.73 2052.99 1551.3 3-3051.5 3 0.90(3 ) 2.0067 X + -.3656 Acetone 0 15.9±0.5 35.6±1.0 53.6±1.5 74.2±2.0 94.3±1.5 2.28 ±0.3 4 327.95 205.00-479.18 1194.31 735.50-3963.0 6 9.62(3 ) 2.2831 X + -.7440 Methan ol 0 19.8±1.1 46.1±1.0 65.1±2.5 83.7±1.6.0±0 2.50 ±0.4 0 251.73 140.59-369.95 819.12 524.19-2420.9 9 11.68( 3) 2.5010 X + - 1.0048 #LC 50 median lethal concentration, FL fiducial limits, LC 90 90% lethal concentration, df degree of freedom. 101

Fig. 1: Dose-mortality response of different solvent extracts of Callistemon citrinus against fourth instar larvae of C. quinquefasciatus Discussion A number of researches on the field of vector control have revealed the efficacy of different phytochemicals obtained from various plants against different species of mosquitoes. Botanical derivatives are considered to be ecofriendly and can be used in mosquito larval control ensuring maximum safety in any ecological condition. The biological activity of plant extracts must be due to diverse compounds including phenolics, terpenoids and alkaloids present in plants (Rafael et al., 2009). The present study was aimed to utilize the leaves of Callistemon citrinus as mosquito control agent. Our investigations revealed that methanol extracts of C. citrinus exhibited 2.03 fold more larvicidal potential than hexane extracts against early fourth instars of Cx. quinquefasciatus. The type of solvents selected affects extract efficacy between different phytochemicals of varying volatility is present in the final extraction (Shaalan et al., 2005). The current study tested the larvicidal efficacy of extracts employing solvents of increasing polarity from hexane (non polar, p=0.1) to water (the most polar one with a polarity index (p) of 10.2). Data on C. citrinus leaf extract showed an equivalent relationship exists between extract efficacy and solvent polarity where efficacy increases with increasing polarity. This is not in line with the observation made by Aivazi and Vijayan(2009) in oak gall extracts and Prathibha and Vijayan(2010) in Euodia ridleyi leaf extract as where they got converse relationship of increasing efficacy with decreasing polarity. Previous investigations have shown that the peel oil extracts of C. aurantium, C. lemon, and C. sinensis possessed excellent larvicidal potential against Cx. quinquefasciatus larvae with lemon peel oil proving as the best larvicidal agent (Mwaiko, 1992; Mwaiko and Savaeli 1994). Mullai and Jebanesan (2007) have reported that ethyl acetate, petroleum ether and methanol leaf extracts of C. colocynthis 102

and Cucurbeta maxima showed LC 50 values of 47.58, 66.92 and 1189.74 ppm and 75.91, 117.73 and 171.64 ppm, respectively, against Cx. quinquefasciatus larvae. The methanol extract of Cassia fistula showed LC 50 values of 17.97 and 20.57 mg/l against A. stephensi and Cx. quinquefasciatus, respectively (Govindarajan et al., 2008). The larvicidal efficacy of the crude leaf extracts of Ficus benghalensis with three different solvents like methanol, benzene and acetone was tested against the early second, third, fourth instar larvae of Cx. quinquefasciatus, Ae. aegypti and An. stephensi. Among the three solvents the maximum efficacy was observed in methanol. The LC 50 values of Ficus benghalensis against early second, third and fourth larvae of Cx quinquefasciatus, Ae. aegypty and An. stephensi were 41.43, 58.21 and 74.32 ppm, 56.54, 70.29 and 80.85 ppm and 60.44, 76.41 and 89.55 ppm, respectively (Govindarajan, 2010). Conclusion The first hand information of the current research clearly shows the potential of C. citrinus as a possible larvicidal agent against Cx. quinquefasciatus. The perusal of literature on phytochemical assays and the present study on the target species reveal that botanical derivatives can be explored as a powerful weapon in preventing deadly transmission of vector borne diseases. The effective larvicide obtained from the current study with LC 50 value of 251.7 mg/l after 24 h indicates promising larvicidal activity of C. citrinus against Culex quinquefasciatus. Further study is required to reveal the active principle involved, level of toxicity and their mode of action. Conflict of interest statement We declare that we have no conflict of interest Acknowledgements The authors are thankful to the Principal, St. Joseph s College for the laboratory facilities provided. We acknowledge University Grants Commission, New Delhi for providing funds under UGC Major Research Project. We also thank the scientists of the VBRL, University of Mysore for their cooperation. References 1. Abbot, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Con. Entomol., 18: 265-267. 2. Agrawal Lt., V.K. Col, Wg. Sashindran, V.K. Cdr. 2006. Lymphatic Filariasis in India: Problems, Challenges and New Initiatives. MJAFI. 62: 359-362. 3. Aivazi, A. A. and V.A. Vijayan, 2009. Larvicidal activity of oak Quecus infectoria oliv. (Fagaceae) gall extracts against Anopheles stephensi, liston. J.Parasitol. Res., 104: 1289-1293. 4. Finney, D.J., 1971. Probit Analysis. 3 rd Edn., Cambridge University Press, Cambridge, London. 103

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