GOVINDARAJAN 3 Larvicidal efficacy of botanical. 2012

European Review for Medical and Pharmacological Sciences 2012; 16: 386-392 Larvicidal efficacy of botanical extracts against two important vector mosquitoes M. GOVINDARAJAN1, R. SIVAKUMAR1, A. AMSATH2, S. NIRAIMATHI3 1 Division of Vector Biology and Phytochemistry, Department of Zoology, Annamalai University, Annamalainagar (India) 2 Postgraduate & Research Department of Zoology, Khadir Mohideen College, Adirampattinam (India) 3 Postgraduate & Research Department of Biochemistry, RVS College of Arts and Science, Karaikal (India) Abstract. – Objective: To determine the larvicidal efficacy of different solvent leaf extract of Ervatamia coronaria and Caesalpinia pulcherrima against Anopheles subpictus and Culex tritaeniorhynchus. Materials and Methods: Twenty five early third instar larvae of Anopheles subpictus and Culex tritaeniorhynchus were exposed to various concentrations and were assayed in the laboratory. The larval mortality was observed after 24 h of treatment. Results: Among three solvent extracts tested the maximum efficacy was observed in the methanol extract. The LC50 (LC90) values of Ervatamia coronaria and Caesalpinia pulcherrima against early third instar of Anopheles subpictus were 86.47 (159.59) and 113.26 (207.73) ppm and Culex tritaeniorhynchus were 131.53 (245.37) and 165.28 (299.45) ppm, respectively. No mortality was observed in controls. Conclusions: From the results it can be concluded the crude extract of Ervatamia coronaria and Caesalpinia pulcherrima were excellent potential for controlling Anopheles subpictus and Culex tritaeniorhynchus mosquito larvae. Key Words: Larvicidal activity, Anopheles subpictus, Culex tritaeniorhynchus, Ervatamia coronaria, Caesalpinia pulcherrima. Introduction Mosquitoes are the major vector for the transmission of malaria, dengue fever, yellow fever, filariasis, schistosomiasis and Japanese encephalitis (JE), etc. causing millions of deaths 386 every year1. Mosquitoes also cause allergic responses in humans that include local skin and systemic reactions such as angioedema2. Anopheles (An.) subpictus is known to transmit malaria and filariasis, in an isolated study of multiple host-feeding in field populations, and its specific role in transmitting malaria in Sri Lanka revealed that multiple blood feeding within the same gonotrophic cycle was attributed to a local “frequent feeding strategy” in this primarily zoophagic and endophilic malaria vector. On the contrary, in Indonesia, An. subpictus is a potential vector of bancroftian filariasis and fed on microfilaraemia carriers that harbored Wuchereria bancrofti larvae3. An. subpictus breeds profusely in rainwater accumulations and fallow rice fields4, waste water disposal systems, and irrigated sites5, and is also associated with floating and submerged aquatic vegetation in the vicinity of rice plants6. Night time human biting collection in Rajasthan, India, showed two feeding peaks for An. subpictus, one early in the night and the other just before dawn7. Culex tritaeniorhynchus Giles is an important vector of JE in India and South East Asian countries. JE is endemic in few states of India and highly endemic in few districts of Tamil Nadu, Southern India8. Keiser et al9 have reported that approximately 1.9 billion people currently live in rural JE prone areas of the world, the majority of them in China (766 million) and India (646 million). Mosquito control has been become increasing difficult of the indiscriminate uses of synthetic chemical insecticides which has 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. Corresponding Author: M. Govindarajan, Ph.D.; e-mail: drgovind1979@rediffmail.com Larvicidal efficacy of botanical extracts against two important vector mosquitoes 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 generations10. However, one major drawback with the use of chemical insecticides is that they are non-selective and could be harmful to other organisms in the environment. The search for herbal preparations that do not produce any adverse effects in the non-target organisms and are easily biodegradable remains a top research issue for scientists associated with alternative vector control11. 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 diseases at the individual as well as at the community level12,13. Anti-feedant and larvicidal activity of acetone, chloroform, ethyl acetate, hexane and methanol peel, leaf and flower extracts of Citrus (C.) sinensis, Ocimum canum, O. sanctum and Rhinacanthus nasutus were studied using fourth instar larvae of Helicoverpa armigera, Sylepta derogata and Anopheles (An.). stephensi14. Rajkumar and Jebanesan15 have reported that the acetone leaves extract of Solanum trilobatum showed maximum oviposition deterrent and skin repellent activity against An. stephensi. The ethanolic extracts of the orange peel (C. sinensis) was tested for the toxicity effect on the larvae of the yellow fever mosquito Aedes (Ae.) aegypti16; The crude chloroform extract of seeds of Millettia dura showed high activity against second-instar larvae of Ae. aegypti17. Larvicidal efficacy of the crude leaf extract of Ficus benghalensis with three different solvents like methanol, benzene and acetone were tested against the early second, third, fourth instar larvae of Culex (Cx.) quinquefasciatus, Ae. aegypti and An. stephensi18. The larvicidal activity of crude carbon tetrachloride, methanol, and petroleum ether extracts of Solanum xanthocarpum fruits was examined against An. stephensi and Cx. quinquefasciatus19; the methanol extracts of leaves of Dysoxylum malabaricum were tested against mature and immature stages of An. stephensi under laboratory conditions20; root bark extracts of Turraea wakefieldii and Turraea floribunda against third-instar larvae21 and extracts of Pelargonium citrosa leaf were tested for their biological, larvicidal, pupicidal, adulticidal, antiovipositional activity, repellency, and biting de- terrency22 against An. stephensi. Mullai et al23 have reported that the leaf extract of Citrullus vulgaris with different solvents viz., benzene, petroleum ether, ethyl acetate, and methanol were tested for larvicidial, ovicidal, repellent, and insect growth regulatory activities against An. stephensi. Elango et al24 have reported that the leaf acetone, chloroform, ethyl acetate, hexane, and methanol extracts of Aegle marmelos, Andrographis lineata, Andrographis paniculata, Cocculus hirsutus, Eclipta prostrate and Tagetes erecta were tested against fourth-instar larvae of An. subpictus and Cx. tritaeniorhynchus. Govindarajan25 reported that the leaf methanol, benzene, and acetone extracts of Cassia fistula were studied for the larvicidal, ovicidal, and repellent activities against Ae. aegypti. The leaf extract of Acalypha indica with different solvents viz., benzene, chloroform, ethyl acetate, and methanol were tested for larvicidal, ovicidal activity, and oviposition attractancy against An. stephensi12. In the present study, we report the mosquitocidal larvicidal properties of the leaves of Ervatamia (E.) coronaria and Caesalpinia pulcherrima against two important vector mosquitoes. Materials and Methods Collection of Plants Fully developed leaves of the E. coronaria and C. pulcherrima were collected from in and around Annamalai University Campus, Annamalainagar, Tamil Nadu, India. It was authenticated by a plant taxonomist from the Department of Botany, Annamalai University. A voucher specimen is deposited at the Herbarium of Plant Phytochemistry Division, Department of Zoology, Annamalai University. Extraction The leaves were washed with tap water, shade dried and finely ground. The finely ground plant leaf powder (3.0 kg/solvent) was loaded in Soxhlet apparatus and was extracted with three different solvents namely benzene, 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 acetone. From this stock solution, different concentrations were prepared and these solutions were used for larvicidal bioassay. 387 M. Govindarajan, R. Sivakumar, A. Amsath, S. Niraimathi Test Organisms The mosquitoes, An. subpictus and Cx. tritaeniorhynchus were 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 one week old chick for blood meal. Mosquitoes were held at 28 ± 20, 70-85% relative humidity (RH), with a photo period of 14 h light, 10 h dark. Larvicidal Bioassay The larvicidal activity of the plants crude extracts was evaluated as per the method recommended by WHO26. Batches of 25 third 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. Six replicate were set up for each concentration and an equal number of control were set up simultaneously using tap water. To this 1 ml of appropriate solvent was added. The LC50 value was calculated after 24 h by probit analysis27. Statistical Analysis The average larval mortality data were subjected to probit analysis for calculating LC50, LC90 and other statistics at 95% confidence limits of upper confidence limit and lower confidence limit, and chi-square values were calculated using the SPSS12.0 (Statistical Package for Social Sciences: Chicago, IL, USA) software. Results with p < 0.05 were considered to be statistically significant. Results The efficacy of methanol, benzene and ethyl acetate solvent extract of leaf of E. coronaria and C. pulcherrima were tested against the early third larvae of An. subpictus and Cx. tritaeniorhynchus. The data were recorded and statistical data regarding the LC50, LC90, Chi-square and 95% confidence limits were calculated (Tables I-IV). Among three solvents tested the methanolic extract of E. coronaria and C. pulcherrima showed highest larvicidal activity against An. subpictus and Cx. tritaeniorhynchus. The LC50 values were 86.47 (159.59) and 113.26 (207.73) ppm for An. subpictus and 131.53 (245.37) and 165.28 (299.45) ppm for Cx. tritaeniorhynchus respectively. No mortality was observed in control. The chi-square values were significant at p < 0.05 level. Table I. Larvicidal activity of different solvent leaf extracts of Ervatamia coronaria against Anopheles subpictus. Solvents Methanol Benzene Ethyl acetate Concentration (ppm) % of mortality ± SD LC50 (LCL-UCL) (ppm) LC90 (LCL-UCL) (ppm) Control 40 80 120 160 200 Control 40 80 120 160 200 Control 40 80 120 160 200 0.0 ± 0.0 30.4 ± 0.8 49.1 ± 1.2 70.6 ± 1.8 84.2 ± 1.4 99.8 ± 1.6 0.0 ± 0.0 21.6 ± 1.2 42.5 ± 1.8 67.4 ± 1.4 78.6 ± 0.8 97.6 ± 0.6 0.0 ± 0.0 17.5 ± 1.8 35.6 ± 1.6 64.8 ±1.2 72.1 ± 1.4 95.2 ± 0.8 86.47 (61.00-109.99) 159.59 (131.72-216.09) 19.158* 97.53 (77.85-116.76) 172.44 (147.91-215.56) 12.734* 106.76 (88.97-126.86) 185.04 (159.00-231.53) 12.604* χ2 *Significant at p < 0.05 level. LC50: Lethal Concentration; LCL: Lower Confidence Limit; UCL: Upper Confidence Limit. 388 Larvicidal efficacy of botanical extracts against two important vector mosquitoes Table II. Larvicidal activity of different solvent leaf extracts of Ervatamia coronaria against Culex tritaeniorhynchus. Solvents Methanol Benzene Ethyl acetate Concentration (ppm) % of mortality ± SD LC50 (LCL-UCL) (ppm) LC90 (LCL-UCL) (ppm) Control 60 120 180 240 300 Control 60 120 180 240 300 Control 60 120 180 240 300 0.0 ± 0.0 31.4 ± 1.8 48.3±1.2 69.4 ± 1.6 81.8 ± 1.4 99.7 ± 0.8 0.0 ± 0.0 24.3 ± 0.8 44.9 ± 1.6 66.3 ± 2.0 78.7 ± 1.8 98.1 ± 1.4 0.0 ± 0.0 20.9 ± 0.8 41.6 ± 1.6 61.8 ± 1.4 76.3 ± 1.2 96.6 ± 0.8 131.53 (88.81-170.72) 245.37 (199.77-344.59) 21.956* 143.14 (109.39-175.74) 258.18 (217.12-334.82) 15.472* 152.45 (122.80-182.00) 269.89 (231.51-337.89) 12.397* χ2 *Significant at p < 0.05 level. LC50: Lethal Concentration; LCL: Lower Confidence Limit; UCL: Upper Confidence Limit. Discussion Today, the environmental safety of an insecticide is considered to be of paramount importance. An insecticide does not have to cause high mortality on target organisms in order to be ac- ceptable28. Phytochemicals may serve as suitable alternatives to synthetic insecticides in future as they are relatively safe, inexpensive, and are readily available in many areas of the world. According to Bowers et al29 the screening of locally available medicinal plants for mosquito control Table III. Larvicidal activity of different solvent leaf extracts of Ervatamia coronaria against Anopheles subpictus. Solvents Methanol Benzene Ethyl acetate Concentration (ppm) % of mortality ± SD LC50 (LCL-UCL) (ppm) LC90 (LCL-UCL) (ppm) Control 50 100 150 200 250 Control 50 100 150 200 250 Control 50 100 150 200 250 0.0 ± 0.0 28.6 ± 1.8 48.3 ± 1.2 65.6 ± 1.4 81.2 ± 2.2 99.9 ± 0.8 0.0 ± 0.0 21.5 ± 1.2 42.6 ± 1.6 62.9 ± 1.2 76.3 ± 1.4 97.8 ± 1.8 0.0 ± 0.0 18.2 ± 1.4 33.6 ± 1.8 59.9 ± 0.8 72.8 ± 1.4 96.1 ± 1.2 113.26 (79.09-145.32) 207.73 (170.16-288.23) 21.147* 124.96 (97.88-151.74) 221.41 (187.7-284.39) 14.721* 135.42 (112.41-159.08) 232.28 (201.15-286.15) 11.319* χ2 *Significant at p < 0.05 level. LC50: Lethal Concentration; LCL: Lower Confidence Limit; UCL: Upper Confidence Limit. 389 M. Govindarajan, R. Sivakumar, A. Amsath, S. Niraimathi Table IV. Larvicidal activity of different solvent leaf extracts of Caesalpinia pulcherrima against Culex tritaeniorhynchus. Solvents Methanol Benzene Ethyl acetate Concentration (ppm) % of mortality ± SD LC50 (LCL-UCL) (ppm) LC90 (LCL-UCL) (ppm) Control 75 150 225 300 375 Control 75 150 225 300 375 Control 75 150 225 300 375 0.0 ± 0.0 27.8 ± 1.4 48.4 ± 1.8 69.5 ± 1.4 84.3 ± 1.2 100.0 ± 0.8 0.0 ± 0.0 23.8 ± 1.2 44.4 ± 1.8 67.2 ± 1.4 80.7 ± 1.2 98.6 ± 1.6 0.0 ± 0.0 19.5 ±1.2 40.9 ± 1.8 63.4 ± 1.6 77.8 ± 1.4 97.1 ± 0.8 165.28 (121.10-206.77) 299.45 (249.13-396.41) 17.546* 177.05 (138.09-214.71) 315.86 (268.67-401.29) 14.050* 189.41 (155.75-222.85) 330.69 (286.89-404.56) 10.903* χ2 *Significant at p < 0.05 level. LC50: Lethal Concentration; LCL: Lower Confidence Limit; UCL: Upper Confidence Limit. would generate local employment, reduce dependence on expensive imported products and stimulate local efforts to enhance public health. Different parts of plants contain a complex of chemicals with unique biological activity30 which is thought to be due to toxins and secondary metabolites, which act as mosquitocidal agent31. The crude extracts may be more effective compared to the individual active compounds, due to natural synergism that discourages the development of resistance in the vectors32. The findings of this study showed that crude ethyl acetate, benzene, and methanol extracts of the leaf of the plants, C. pulcherrima, and E. coronaria have a significant larvicidal activity. Rahuman and Venkatesan33 reported that the petroleum ether extract of Citrullus colocynthis, methanol extracts of Cannabis indica, Cannabis sativus, Momordica charantia and acetone extract of Trichosanthes anguina were against the larvae of Ae. aegypti (LC50=74.57, 309.46, 492.73, 199.14, and 554.20 ppm) and against Cx. quinquefasciatus (LC50=88.24, 377.69, 623.80, 207.61, and 842.34 ppm), respectively. Larvicidal activity of acetone extracts of Murraya koenigii, Coriandrum sativum, Ferula asafoetida, and Trigonella foenum graceum reported maximum activity ranging 25-900 ppm against Ae. aegypti34. Mullai and Jebanesan35 have reported that ethyl acetate, petroleum ether and methanol leaf extracts of Citrullis colocynthis and Cucurbita maxima 390 showed LC50 values of 47.58, 66.92 and 118.74 ppm and 75.91, 117.73 and 171.64 ppm, respectively, against Cx. quinquefasciatus larvae. The methanol extract of C. fistula exhibited LC50 values of 17.97 and 20.57 mg/L, An. stephensi and Cx. quinquefasciatus, respectively13. Larvicidal activity of crude extract of Sida acuta against Cx. quinquefasciatus, Ae. aegypti and An. stephensi with LC50 values ranging between 38 to 48 mg/l36. Sosan et al37 reported larvicidal activities of essential oils of Ocimum gratissimum, Cymbopogon citrus, and Ageratum conyzoides against Ae. aegypti and achieved 100% mortality at 120, 200, and 300 ppm concentrations, respectively. Thirteen oils from 41 plants (camphor, thyme, amyris, lemon, cedarwood, frankincense, dill, myrtle, juniper, black pepper, verbena, helichrysum, and sandalwood) were found to induce 100% mortality in third instar larvae of Ae. aegypti, An. stephensi, and Cx. quinquefasciatus after 24 h, or even after shorter periods38. Similarly, it was reported that the essential oil of Ipomoea cairica Linn. possesses remarkable larvicidal properties as it could produce 100% mortality in the larvae of C. tritaeniorhynchus, Ae. aegypti, An. stephensi, and Cx. quinquefasciatus mosquitoes at concentrations ranging from 100 to 170 ppm39. Tiwary et al40 observed the larvicidal activity of linalool rich essential of Zanthoxylum armatum against different mosquito species viz., Cx. quinquefasciatus Larvicidal efficacy of botanical extracts against two important vector mosquitoes (LC50=49 ppm), Ae. aegypti (LC50=54 ppm) and An. stephensi (LC50=58 ppm). Singh et al41 reported the larvicidal activity of Ocimum canum oil against vector mosquitoes, namely, Ae. aegypti and Cx. quinquefasciatus (LC50=301 ppm) and An. stephensi (LC50=234 ppm). Plants could be an alternative source for mosquito larvicides because they constitute a potential source of bioactive chemicals and generally free from harmful effects. The findings of the present investigation revealed that the leaf extract of E. coronaria, and C. pulcherrima possess remarkable larvicidal activity against An. subpictus and Cx. tritaeniorhynchus. Further investigations are needed to elucidate this activity against a wide range of mosquito species and also the active ingredient(s) of the extract responsible for larvicidal activity in An. subpictus and Cx. tritaeniorhynchus should be identified and utilized, if possible, in preparing a commercial product/formulation to be used as a mosquitocidal. –––––––––––––––––––– Acknowledgements The Authors are 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. References 1) JAMES AA. Mosquito molecular genetics: the hands that feed bite back. Science 1992; 257: 37-38. 2) PENG Z, YANG J, WANG H. 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