Int J Pharm Pharm Sci, Vol 9, Issue 5, 156-160Original Article


ADULTICIDAL ACTIVITY OF BOTANICAL OILS BY IMPREGNATED PAPER ASSAY AGAINST CULEX QUINQUEFASCIATUS SAY

M. RAMAR1,3*, S. IGNACIMUTHU1, P. MANONMANI2, K. MURUGAN3

1Entomology Research Institute, Loyola College, Chennai-34, Tamil Nadu, India, 2Department of Biotechnology, PRIST University, Vallam, Thanjavur, 3Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar, University, Coimbatore, Tamil Nadu 641046, India
Email: ramareri@gmail.com

Received: 19 Oct 2016 Revised and Accepted: 31 Mar 2017

ABSTRACT

Objective: The present study was undertaken with the aim of finding out the efficacy of essential oils (EOs) as anti-mosquito agents for commercial purposes. Plant source insecticides as an alternative to chemical insecticide, this study were evaluated to assess the knock-down and adulticidal prospective of the essential oils against Culex quinquefasciatus. The plant essential oil is largely cultivated throughout India and in all Tropical countries.

Methods: The selected botanical essential oils were procured from commercial producers of plant essential oils and aromatic substances were used in this study. Knock-down and Adulticidal bioassay was performed according to WHO protocol. A single dose of the essential oils was used in the preliminary screening. 20 adult mosquitoes (3-5 d old glucose fed mosquitoes) were exposed on treated paper for one hour and knocked down and live mosquitoes were counted at 5 minute intervals.

Results: Among the twenty three oils tested, 100% knock-down and adult mortality was recorded at 10%/cm2dose of calamus, camphor, cinnamon, citronella, clove, eucalyptus, lemongrass, pine, thyme and tulsi oils respectively. At 10 % concentration, clove oil (KT50 =1.8 and KT90 = 2.03 min) was found to be the most potential treatment. After 15 min exposure period clove oil registered the lowest knock-down dose which was calculated as (KD50 =1.8 %/cm2and KD90 =11.2 %/cm2). The lower and upper 95 % confidence limits for clove oil were calculated as 0.2 and 4.2 min respectively.

Conclusion: From the results it can be concluded that the adult of the Cx. quinquefasciatus were susceptible to the essential oils. Such findings would be useful in promoting research aiming at the development of new agent for mosquito control on basis of chemical compounds from indigenous plant sources as an alternative to chemicals.

Keywords: Essential oils, Screening, Filaria Mosquito, Culex quinquefasciatus, Knock-down-Adulticidal

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2017v9i5.15764

INTRODUCTION

Mosquito diseases, such as malaria, Japanese encephalitis, filariasis, dengue and yellow fever a major source of illness and death worldwide, particularly in tropical and subtropical area [1]. The Culex quinquefasciatus is a major public health problems in India and filariasis is potentially risk of infection [2] These diseases affect the health and quality of life of millions of people in Worldwide [3]. An estimated 120 million people in tropical and subtropical areas of the world are infected with lymphatic filariasis [4]. Moreover, mosquitoes cause allergic responses in humans that include local skin and systemic reactions [5].

Such serious diseases are becoming increasingly difficult because of the high rate of reproduction and development of resistance to insecticides in mosquitoes [6]. The use of chemical control of mosquitoes is remaining as a main part of integrated vector management [7-8]. According to Word Health Organization pesticide, the major insecticides used against mosquitoes are pyrethroids and organophosphates [7-8]. Although, factors including the development of resistance to insecticides are leading to morbidity and mortality due to malaria and other vector-borne diseases. Insect resistance has been reported to chemical class of insecticide used in vector control programs and insect growth regulators [9].

The search for alternative pesticides and control measures that pose no risk or posing minimal risk to human health and the environment is of great interest from the preventive medicine point of view [10]. Pyrethrin-based mosquito liquid formulations are widely used in many countries, especially in the house hold of rural population. Interest in botanical pesticides revived during recent years, because of the deleterious effects of synthetic insecticides, including lack of selectivity, impact on the environment and the emergence and spread of pest resistance. The naturally occurring pesticides appear to have a promising role in the development of future commercial pesticides for safety of the environment and public health [11-12]. Essential oils from plant species, celery (Apiumgraveolens), caraway (Carum carvi), zedoary (Curcuma zedoaria), long pepper (Piper longum) and Chinese star anise (Illicium verum), were studied adulticidal activity against mosquito vectors [13]. Adulticidal activity of five essential oils such as Citrus sinensis, Mentha pipreta, Carvocryl oil, Citronela oil and citral oil against Cx. Quinquefasciatu [14]. The fumigant toxicity of essential oils from 15 species of African plants against Anopheles gambiae in the laboratory conditions [15].

Plant essential oils are volatile substances found in a various plants. This oil isolated from plant sourse, but thus oils are consisting of mixtures of many compounds. Botanical based oils were the first preservatives used by man, their natural state within plant tissues and as oils obtained by water distillation. The oils composed by isoprenoid compounds, mainly mono-and sesquiterpenes are the carriers of the smell found in aromatic plants [16]. The uses of commercial, essential oils are used in four primary ways: pharmaceuticals, flavor enhancers in many food products, odorants in fragrances and insecticides.

At present, evaluation of essential oils against mosquitoes and isolation, identification and development of natural products from them are under the focus of numerous research programmes around the globe. There is a renewed interest in plant essential oils products as sources of new insect controlling agents, because they may be biodegradable to nontoxic compounds, thus minimizing the accumulation of harmful residues, leading them to be more environmentally friendly compared to synthetic compounds [17]. The present study was undertaken to evaluate the bio-efficacy of some essential oils (EOs) against Culex quinquefasciatus adults mosquito and to study effects of the effective oils on adults of the mosquito.

MATERIALS AND METHODS

Materials

Botanical essential oils

For the present study, twenty two botanical essential oils were procured from Tegraj and Co (P) Ltd, India (A commercial producer of plant essential oils and aromatic sources) were used in this study. The essential oils (EOs) were selected primarily on the basis of ethno-botanical information and various biological effects, the following 22 essential oils (EOs) were selected: Aniseed (Pimpinella anisum Linn.), Bergamot (Citrus bergamia Risso.), Calamus (Acorus calamus L.), Camphor (Cinnamomum camphora Linn.), Cedarwood (Cedrus atlantica Endl), Cinnamon (Cinnamomum veerum J. S. Presl), Citronella (Cymbopogon nardus Linn.), Clove (Myrtus caryophyllus Linn.), Eucalyptus (Eucalyptus globulus Labill.), Geranium (Pelargonium graveollens L.), Lemon (Citrus limon Linn.), Lemongrass (Cymbopogon flexuosus J. F. Watson), Lime (Citrus aurantifolia Christm.), Luchi (Gaultheria fragrantissima Cham), Nutmeg (Myritica fragrans Houtt.), Orange (Citrus sinensis Linn.), Palmarosa (Cymbopogon martin Stapf), Pine (Pinus radiate D. Don), Rose mary (Rosmarinus officinalis Linn.), Thyme (Thymus vulgaris Linn.), Tulsi (Ocimum sanctum Linn.) and Vetiver (Vetiveria zizanioides Linn.). The essential oils were stored in the refrigerator until used for bioassay experiments.

Maintenance of test organism

The study organism of Cx. quinquefasciatus, was maintained in several generations at Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, India. Mosquitoes were free of exposure to pathogens and insecticides and maintained at 26±1 °C and 60-80% relative humidity (RH). Larvae were fed on dog biscuits and yeast powder in a ratio 3:2 until moulting to become pupae, pupae was transferred into a mosquito rearing cage. The pupae were transferred from culture trays to glass beakers containing tap water and placed in cages (45 x45 x 45 cm), where adults emerged. The cages was made up of metal and covered with a muslin cloth. The emergent adults were fed with 10% glucose solution dipped in cotton.

Methods

Knock-down and adulticidal activity

The Knock-down and killing bioassay was performed according to WHO protocol [18]. The selected essential oil was prepared in 2 ml of acetone. A single dose of the oil (10%) was used in the preliminary screening. Each essential oil were prepared in 2 ml of acetone and applied on Whitman no. 1 filter papers (size 12 x15 cm2), control papers were treated with 2 ml of acetone alone and placed in exposure tubes. 3-5 d old sugar fed mosquitoes (In each tube, 20 adult mosquitoes) were exposed on treated paper for one hour and knocked down and live mosquitoes were recorded at every 5 minute intervals. After one-hour exposure mosquitoes was transferred into recovery test tubes for 24 hour mortality observation. Five replicates were run at a time with control. The reference insecticide was used with 0.05 % deltamethrin impregnated paper as a comparison. The median knock-down time (KT50 and KT90) and knock-down dose (KD50 and KD90) were calculated by Probit analysis.

Data analysis

Statistical analysis was performed using SPSS software package, version 15. The values were analyzed by one way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT)[19]. The median Knock down time (KT50 and KT90) and lethal dose (KD50 and KD90) was carried out by Profit analysis [20]. p value of<0.05 was considered to represent Significant differences. The corrected percent mortality was evaluated by using Abbott’s formula [21].

RESULTS

Percentage of knock-down and killing effect

The screening efficacy of Knock-down and killing effect of the plant oil on Culex quinquefasciatus was studied and is illustrated in the table 1. In the present preliminary screening study a single dose (10%) of EOs was used and acetone served as control and reference insecticide (0.05 % deltamethrin) was also used as comparison.

Table 1: Preliminary screening (mean±SE, No. of five replicates) of essential oils (EOs) for knock-down and killing effect at 10%/cm2 by impregnated paper method against adult Culex quinquefasciatus mosquito

Plant essential oils % knock-down (cm2) at 1 h Mortality (%) after 24 h
Aniseed 73.0±0.61f 54.3±1.23f
Bergamot 60.0±0.52cde 36.3±1.21bc
Calamus 100±0.00h 100±0.00i
Camphor 100±0.00h 100±0.00i
Cedarwood 63.3±0.31def 39.0±0.51de
Cinnamon 100±0.00h 96.6±0.82i
Citronella 100±0.00h 100±0.00i
Clove 100±0.00h 100±0.00i
Eucalyptus 100±0.00h 100±0.00i
Geranium 86.6±0.31g 58.3±0.82f
Lemon 36.6±0.32b 16.6±0.34b
Lemongrass 100±0.00h 91.3±2.34h
Lime 66.6±0.32ef 38.6±0.81de
Luchi 62.0±0.31def 38.0±0.52de
Nutmeg 53.0±0.32cd 27.3±1.22b
Orange 53.3±0.34cd 35.3±0.31cd
Palmarosa 50.0±1.00c 36.6±0.82cd
Pine 100±0.00h 100±0.00i
Rose mary 73.3±0.32f 62.0±0.53g
Thyme 100±0.00h 97.6±0.81i
Tulsi 100±0.00h 96.6±0.82i
Vetiver 60.0±0.51cde 38.6±1.33de
Deltamethrin (0.05%) 100±0.00h 100±0.00i
Solvent (Acetone) 0.0±0.00a 3.12±1.24a

Values with the same letter were not significantly different (P<0.05) by one way ANOVA with DMRT.

The results clearly indicated that 100% knock-down and adult mortality was recorded at 10% dose of calamus, camphor, cinnamon, citronella, clove, eucalyptus, lemongrass, pine, thyme and tulsi oils respectively. The lowest knock-down and adulticidal activity of 36.6% and 15.4% were recorded in lemon oil. Reference insecticide (0.05% detramethrin) showed 100% knock-down and adult mortality against Cx. quinquefasciatus.

Median knock-down time (KT50) and KT90

The KT50 and KT90 of effective essential oils are shown in table 2. At 10 % concentration, clove oil (KT50 =1.8 and KT90 = 2.03 min) was found to be the most potential treatment. It recorded 1.8 min as KT50 after 1 h exposure period for citronella (KT50 = 2.5 and KT90 = 28.0 min) and eucalyptus (KT50 = 3.6 and KT90 = 44.3 min) oils were also recorded as potentially effective adulticides against Cx. quinquefasciatus. In 0.05% detamethrin (KT50 = 2.3 and KT90 = 27.13 min) was registered after 1 hour exposure treatment period.

Median knock-down dose (KD50) and KD90

Ten essential oils were identified as potential treatments in screening bioassay of knock-down and killing activity. Tables 3a and 3b present the median knock-down dose (KD50 and KD90) of the essential oils against Cx. quinquefasciatus adults at 10 % concentration.

After 15 min exposure period clove oil registered the lowest knock-down dose which was calculated as (KD50 =1.8 %/cm2and KD90 =11.2 %/cm2). Next to clove oil citronella (KD50 = 3.0% and KD90 = 11.5%/cm2) and eucalyptus (KD50=3.4 and KD90 = 11.6%/cm2) oils were also effective since they recorded lower median knock-down dose.

Tulsi (KD50 =7.4 and KD90 =16.4%/cm2) and calamus (KD50 =8.7 and KD90 =22.4%/cm2) oils were the least effective knock-down treatment. The lower and upper 95 % confidence limits for clove oil were calculated as 0.2 and 4.2 min respectively.

Table 2: Median knock-down time (KT50) and KT90 of effective essential oils against Culex quinquefasciatus at 10%/cm2 after 1 hour exposure period

Plant ssential oils KT50 (min) 95% CLb KT90(min) 95% CLb Chi-square
LCL UCL LCL UCL
Calamus 22.4 19.35 34.17 48.1 35.98 124.4 6.1
Camphor 11.0 8.324 54.58 32.7 27.35 58.23 7.2
Cinnamon 7.6 4.322 24.45 47.6 36.56 65.56 11.8
Citronella 2.5 1.123 5.842 28.0 15.34 36.54 9.6
Clove 1.8 0.214 4.231 2.03 12.33 30.12 8.1
Eucalyptus 3.6 1.243 15.44 44.3 28.45 67.84 9.6
Lemongrass 20.3 16.36 23.50 44.6 40.86 49.86 3.5
Pine 15.4 12.44 26.34 26.3 43.07 32.10 4.2
Thyme 19.7 15.67 27.56 43.6 35.56 78.54 3.1
Tulsi 24.4 13.24 25.23 52.3 35.43 67.54 4.2
Deltamethrin (0.05%) 12.0 9.310 64.59 42.6 37.37 68.24 7.5

Chi-square value significant at P<0.05 level, bCL: confidence limits; LCL: lower confidence limits; UCL: upper confidence limits, KT50 = Knock down time required to kill 50% of the population exposed, KT90 = Knock down time required to kill 90% of the population exposed

Table 3a: Median knock-down dose (KD50) and KD90 of effective essential oils against female adult Cx. quinquefasciatus at 10%/cm2 after 1 h exposure period

Plant ssential oils Exposureperiod (min) KD50 (%) 95% CLb KD90 (%) 95% CLb Chi-square
LCL UCL LCL UCL
Calamus 15304560 8.75.01.90.3 *3.790.87-2.25 *9.152.750.59 22.417.112.46.1 *12.19.084.80 *40.121.99.13 5.83.52.42.5
Camphor 15304560 1.81.0-1.8-2.4 -10.80.23-6.58-8.20 4.1611.30.60-0.67 18.16.29.65.7 10.418.26.614.22 337.9428.222.710.3 1.51.72.62.7
Cinnamon 15304560 4.62.80.2-1.8 ***-5.39 ***-0.48 11.410.16.04.6 ***3.61 ***7.21 4.412.58.50.8
Citronella 15304560 3.00.8-0.4-0.6 2.22-0.90-2.25-2.07 4.341.660.380.00 11.58.95.12.5 8.446.644.112.06 20.715.37.283.31 0.80.60.22.9
Clove 15304560 1.8-0.2-0.5-0.8 0.54-2.02-1.83-2.82 2.820.580.06-0.46 11.25.72.31.4 7.994.541.891.14 22.18.283.022.02 0.010.11.70.1

Chi-square value significant at P<0.05 level, bCL: confidence limits LCL: lower confidence limits UCL: upper confidence limits, KD50= Knock-down dose required to kill 50% of the population exposed, KD90 = Knock-down dose required to kill 90% of the population exposed, * = Knock-down dose could not be determined.

Table 3b: The KD50 and KD90 of effective essential oils against female adult Cx. quinquefasciatus

Plant ssential oils Exposure period (min) KD50 (%) 95% CLb KD90 (%) 95% CLb Chi-square
LCL UCL LCL UCL
Eucalyptus 15304560 3.41.90.30.2 *0.80-1.54* *2.760.83* 11.610.36.12.7 *7.614.88* *18.48.92* 4.10.62.24.0
Lemongrass 15304560 5.34.21.90.7 ***-9.79 ***2.00 11.22.27.33.9 ***2.57 ***25.3 4.96.64.05.1
Pine 15304560 5.74.31.2-1.5 4.49*-2.390.23 6.719.07-2.534.32 13.816.214.06.50 10.0*8.958.31 24.6*47.319.2 2.15.51.34.3
Thyme 15304560 7.54.61.91.2 **0.870.56 **2.752.54 16.513.010.19.8 **7.496.81 **17.416.5 5.14.92.42.1
Tulsi 15304560 5.34.02.20.2 ***-1.62 ***1.25 14.412.512.97.6 ***4.82 ***13.6 6.35.24.33.4
Tulsi 15304560 7.44.12.50.2 ***-1.66 ***1.15 16.411.511.97.7 ***5.89 ***12.6 6.65.24.53.3
Deltamethrin (0.05%) 15304560 1.20.8-0.4-0.6 -2.39-0.90-2.25-2.07 -2.531.660.380.00 14.08.95.12.6 8.956.644.112.06 47.315.37.283.32 1.40.60.22.8

Chi-square value significant at P<0.05 level, bCL: confidence limits; LCL: lower confidence limits; UCL: upper confidence limits, KD50= Knock-down dose required to kill 50% of the population exposed, KD90 = Knock-down dose required to kill 90% of the population exposed, * = Knock-down dose could not be determined

DISCUSSION

In the present study 22 essential oils registered knock-down and adulticidal effects. Plenty of literature is available with regard to bio efficacy of volatile oils against vector mosquitoes. These findings are comparable to those of Vartak and Sharma [22] have reported the knock-down effect of terpenoids of volatile oils against A. aegypti adult females.

Prajapati et al. [23] have studied the essential oils viz., Cinnamomum zeylanicum, Cuminum cyminum, Cyperus scariosus, Curcuma longa, Juniperus macropoda, Ocimum basilicum, Rosmarinus officicinalis, Nigella sativa, Pimpinella anisum, and Zingiber officinale for adulticidalt activity against three mosquito species; Anopheles stephensi, Aedes aegypti and Culex uinquefasciatus. Omoloa et al. [24] have reported the fumigant toxicity of essential oils from 15 species of African plants against Anopheles gambiae in the laboratory. They reported that oils of 6 plant species viz., Tarchonanthus camphoratus, Lippia javanica, Plectranthus marruboides, Tetradenia riparia, Lippia ukambensis and Conyza newii were found to be relatively more toxic, with C. newii and P. marruboides showing the highest potency.

Rajkumar and Jabanesan [25] reported the knocking down and killing effects of Solanum aerianthum D. Don. leaf extracts against the mosquito Culex quinquefasciatus Say. Jeyabalan et al. [26] have reported the adulticidal effect of Pelargonium citrosa on Anopheles stephensi, with LC50 and LC90 values as 1.56% and 5.22% respectively. Nathan et al. [27], who reported the adulticidal activity of methanol extract of Dysoxylum malabaricum leaves against Anopheles stephensi. Adulticidal activity of five essential oils (Citrus sinensis, Mentha pipreta, Carvocryl oil, Citronela oil and citral oil) at different concentrations and time intervals was determined. The Rutaceae oil (C. sinensis) was found as the most toxic against Cx. Quinquefasciatus [28]. A large number of plants are reported to have adulticidal activity [28, 29-30]. Kovendan et al. [31] found the adulticidal activity of methanol extract of Acalypha alinifolia leaves against three mosquito species, Ae. aegypti, An. stephensi and Culex quinquefasciatus. Dua et al. [30] reported the adulticidal activity of essential oil of Valeriana jatamansi root against An. stephensi, An. culicifacies, Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus, with LD50 and LD90 values were 0.14, 0.16, 0.09, 0.08, 0.17 mg/cm2 and 0.24, 0.34, 0.25, 0.21, 0.28 mg/cm2, respectively; Whereas KDT50 and KDT90 values were 13, 13, 12, 13, 18 min and 24, 25, 21, 20, 42 min against An. stephensi, An. culicifacies, Ae. aegypti, An. albopictus and Cx. quinquefasciatus, respectively, using 0.28 mg/cm2 impregnated papers.

Yang et al. [32] have studied the adulticidal activity of five essential oils against Culex pipines. They found that the Rutaceae oil obtained from Citrus sinesis was the most effective adulticidal treatment. Other plant species that are reported to possess adulticidal activity includes: Curcuma aromatic against Ae. aegypti [17]. The adulticidal activity of ethanol extract of Apium graveolens seeds against Aedes aegypti has been reported [33]. Dua et al. [29] reported the adulticidal activity of essential oil of leaves of Lantana camara against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluvialitis and An. stephensi, LD50 values were 0.06, 0.05, 0.05, 0.05 and 0.06 mg/cm2 while LD90 values were 0.10, 0.10, 0.09, 0.09 and 0.10 mg/cm2 respectively. Whereas KDT50 values were 20, 18, 15, 12, 14 min and KDT90 values were 35, 28, 25, 18, and 23 min against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluviatilis and An. stephensi, respectively on 0.208 mg/cm2 impregnated paper. The target of many insecticides is the nervous system and in particular, acetylcholine esterase (AChE). However little information is available on mode of adulticidal activity of essential oil. Ware [34] (1994) has reported that the only way volatile insecticides can enter the pest’s body is through the respiratory system.

In the present study the volatile oils showed variation in Knock-down and adulticidal activity when tested by impregnated filter paper method. Calamus, camphor, cinnamon, citronella, clove, eucalyptus, lemongrass, pine, thyme and tulsi oils recorded 100 per cent Knock-down and adult mortality at 10%/cm2 dose only in WHO test tube method. Therefore, the observed Knock-down and adulticidal activity of the EOs may be attributed to these compounds.

CONCLUSION

The obtained results indicate that the essential oil (EOs) has potential to be developed as an eco-friendly insecticide against mosquito. The results of the present study could be useful in promoting research aimed at the development of new insecticidal agents for adult mosquito control with filter paper method. The impregnated paper assay finding may be utilized for the development of botanical based insecticides as alternative to synthetic insecticides for adult mosquito control.

ACKNOWLEDGEMENT

The senior author profoundly indebted to thank the Rv. Fr, Director, Entomology Research Institute, Loyola College, Chennai and former Vice-Chancellor of Bharathiar University, Coimbatore and University of Madras, for the monetary support and for his constructive suggestions.

CONFLICT OF INTERESTS

Declared none

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