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Original Article Ovicidal and Oviposition Deterrent Activities of Medicinal Plant Extracts Against Aedes aegypti L. and Culex quinquefasciatus Say Mosquitoes (Diptera: Culicidae)
Appadurai Daniel Reegan, Munusamy Rajiv Gandhi, Micheal Gabriel Paulraj, Savarimuthu Ignacimuthu
Osong Public Health and Research Perspectives 2014;6(1):64-69.
DOI: https://doi.org/10.1016/j.phrp.2014.08.009
Published online: September 4, 2014
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Division of Vector Control, Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, India

∗Corresponding author. Present address. NVBDCP, ROHFW, Govt. of India, Besant Nagar, Chennai, India. danielreegan85@gmail.com
• Received: July 23, 2014   • Revised: August 18, 2014   • Accepted: August 25, 2014

© 2015 Published by Elsevier B.V. on behalf of Korea Centers for Disease Control and Prevention.

This is an Open Access article distributed under the terms of the CC-BY-NC License (http://creativecommons.org/licenses/by-nc/3.0).

  • Objectives
    To evaluate the ovicidal and oviposition deterrent activities of five medicinal plant extracts namely Aegle marmelos (Linn.), Limonia acidissima (Linn.), Sphaeranthus indicus (Linn.), Sphaeranthus amaranthoides (burm.f), and Chromolaena odorata (Linn.) against Culex quinquefasciatus and Aedes aegypti mosquitoes. Three solvents, namely hexane, ethyl acetate, and methanol, were used for the preparation of extracts from each plant.
  • Methods
    Four different concentrations—62.5 parts per million (ppm), 125 ppm, 250 ppm, and 500 ppm—were prepared using acetone and tested for ovicidal and oviposition deterrent activities. One-way analysis of variance (ANOVA) was used to determine the significance of the treatments and means were separated by Tukey's test of comparison.
  • Results
    Among the different extracts of the five plants screened, the hexane extract of L. acidissima recorded the highest ovicidal activity of 79.2% and 60% at 500 ppm concentration against the eggs of Cx. quinquefasciatus and Ae. aegypti, respectively. Similarly, the same hexane extract of L. acidissima showed 100% oviposition deterrent activity at all the tested concentrations against Cx. quinquefasciatus and Ae. aegypti adult females.
  • Conclusion
    It is concluded that the hexane extract of L. acidissima could be used in an integrated mosquito management program.
Mosquitoes are medically important insects and are considered major public health pests [1]. Mosquitoes transmit many dreadful diseases to humans and other vertebrates; therefore, they have been declared “Public Enemy Number One” [2]. Mosquitoes belonging to the genera Aedes and Culex are transmitting dengue, dengue hemorrhagic fever, yellow fever, chikungunya, Japanese encephalitis, and filariasis [3,4]. Mosquito bites cause allergic responses including local skin reactions and systemic reactions such as angioedema and urticaria [5]. Tropical areas are more vulnerable to mosquito-borne diseases and the risk of contracting arthropod-borne illnesses is increased due to climate change and intensifying globalization [6].
It is imperative to control mosquitoes in order to prevent mosquito-borne diseases and improve public health. Aedes aegypti is the primary vector of dengue, dengue hemorrhagic fever, and chikungunya. Dengue fever is endemic in south-east Asia including India, Bangladesh, and Pakistan [7]. 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 such as dengue hemorrhagic fever and dengue shock syndrome or with unusual symptoms such as central nervous system involvement [8,9]. Culex quinquefasciatus is an important vector of lymphatic filariasis in tropical and subtropical regions. It is a pantropical pest and urban vector of Wuchereria bancrofti [10] and is possibly the most abundant house mosquito in towns and cities of tropical countries. According to [11], about 90 million people worldwide are infected with W. bancrofti, and 10 times more people are at risk of being infected. In India alone, 25 million people harbor microfilaria (mf) and 19 million people suffer from filarial disease manifestations [12].
In recent years, mosquito control programs have suffered a setback because mosquitoes are developing resistance to synthetic chemical insecticides such as organochlorides, organophosphates and carbamates and insect growth regulators such as methoprene, pyriproxyfen, and diflubenzuron [13–16]. Moreover, many organophosphates and organochlorides adversely affect the environment and damage biological systems [17]. These side effects of synthetic chemicals prompted many researchers to find environment-friendly alternatives for mosquito management. Literature reveals sufficient amounts of work on the mosquito control potential of plant extracts and plant essential oils [18–25].
The present study was undertaken to evaluate the ovicidal and oviposition deterrent activities of five medicinal plant extracts namely Aegle marmelos (Linn.), Sphaeranthus indicus (Linn.), Sphaeranthus amaranthoides (burm.f), Limonia acidissima (Linn.), and Chromolaena odorata (Linn.) against Ae. aegypti and Cx. quinquefasciatus mosquitoes.
2.1 Collection of plant material
The matured leaves of each plant were collected from Chennai, Tirunelveli and surrounding areas in Tamil Nadu, India and the plant species were authenticated by a Botanist at Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, India. The voucher specimens (ERI-LA-MOS-210-214) of each plant species were deposited in the herbarium of the institute. The collected leaves were shade-dried for 5 days and coarsely powdered using an electric blender.
2.2 Preparation of solvent extracts
Crude extracts were prepared from the powdered leaves of each plant by a sequential extraction method using hexane, ethyl acetate, and methanol solvents (Fisher Scientific and Himeddia, Chennai, India). Leaf powder (1 kg) of each plant was soaked in 3 L of hexane for 48 hours with intermittent shaking. The extract was filtered through Whatman No. 1 filter paper, concentrated in a rotary evaporator (Medica instruments Mgf.Co. Sl.No:EV11.JF.012), and finally dried in vacuum. The residue was soaked in other solvents consecutively and extracted. All the crude extracts were stored at 4°C in air-tight glass vials in the dark until used.
2.3 Test mosquitoes
The mosquito life stages used in this study were obtained from the Entomology Research Institute, and they were free of exposure to pathogens, insecticides, or repellents. The rearing conditions were: 28 ± 1°C; 70–75% relative humidity (RH); and 11 ± 0.5-hour photoperiod [26].
2.4 Ovicidal assay
Ovicidal activity was studied following the method of Elango et al [27]. Twenty five freshly laid eggs of Ae. aegypti and Cx. quinquefasciatus were separately exposed to four different concentrations, namely 62.5 parts per million (ppm), 125 ppm, 250 ppm, and 500 ppm, prepared using acetone. Each concentration was replicated five times. Control (acetone in water) was maintained separately and egg mortality was observed under the microscope. Azadirachtin (10 ppm) and temephos (10 ppm) were used as positive controls for comparison with five replications each. The percent ovicidal activity was assessed at 120 hours post-treatment using the following formula:
Percentovicidalactivity:NumberofunhatchedeggsTotalnumberofeggsintroduced×100
2.5 Oviposition deterrent assay
The oviposition deterrent activity was assessed using earlier reported methods [27,28] with slight modifications. Ten blood-fed females of Ae. aegypti and Cx. quinquefasciatus (10 days old, 2 days after blood feeding) were transferred to separate cages (45 cm × 45 cm × 45 cm) made of mosquito net with a muslin socket on the front side for access. In each cage, four plastic bowls holding 200 mL of tap water were placed in opposite corners of each cage; one bowl was treated with the test material (extract), two bowls were used for positive control (temephos and azadirachtin), and the other one served as control. The concentrations used were 62.5 ppm, 125 ppm, 250 ppm, and 500 ppm. Each concentration was replicated five times. Sucrose solution (10%) was provided to the adult as feed throughout the study period. Experiments were carried out at room temperature (28 ± 1°C; RH: 70–75%) for a period of 72 hours. After 72 hours, the number of eggs laid in each bowl was counted and recorded. The percent effective repellency (ER) for each concentration was calculated using the following formula:
Effectiverepellency(ER)(%)=NCNTNC×100(%)
where NC is the number of eggs in the control, and NT is the number of eggs in the treatment.
2.6 Statistical analysis
The mean values and standard deviations were calculated from replication data. One-way analysis of variance (ANOVA) was used to determine the significance of the treatments and means were separated by Tukey's test of multiple comparisons using SPSS software (version 11.5; SPSS Inc., Chicago, IL, USA).
3.1 Ovicidal activity results
Among the different extracts of the five plants screened, the hexane extract of L. acidissima recorded the highest ovicidal activity of 79.2% and 60% at 500 ppm concentration against the eggs of Cx. quinquefasciatus and Ae. aegypti, respectively (Tables 1 and 2). The hexane extract of A. marmelos recorded moderate ovicidal activity of 53.6% and 48.8% at 500 ppm concentration against the eggs of Cx. quinquefasciatus and Ae. aegypti, respectively (Tables 1 and 2). The ethyl acetate extract of C. odorata recorded 42.4% and 13.6% at 500 ppm concentration against the eggs of Cx. quinquefasciatus and Ae. aegypti, respectively. The other two plant extracts showed much less ovicidal activity. The positive control azadirachtin recorded ovicidal activity of 95.2% and 92.8% at 10 ppm concentration against the eggs of Cx. quinquefasciatus and Ae. aegypti, respectively. Temephos recorded 46.4% and 44% at 10 ppm concentration against the eggs of Cx. quinquefasciatus and Ae. aegypti, respectively (Tables 1 and 2). Overall, the ovicidal activity was higher against Cx. quinquefasciatus eggs than Ae. aegypti eggs.
3.2 Oviposition deterrent activity results
Among the five plant extracts screened, the hexane extract of L. acidissima showed 100% oviposition deterrent activity at all the tested concentrations against Cx. quinquefasciatus and Ae. aegypti adult females (Tables 3 and 4). At 500 ppm concentration, the hexane extract of A. marmelos recorded 76.74% and 71.79% oviposition deterrent activity against Cx. quinquefasciatus and Ae. aegypti, respectively (Tables 3 and 4). The ethyl acetate extract of S. amaranthoides recorded 22.31% and 20.48% oviposition deterrent activity at 500 ppm concentration against Cx. quinquefasciatus and Ae. aegypti, respectively. The extracts of S. indicus and C. odorata showed the least oviposition deterrent activity at all the tested concentrations against two mosquito species (Tables 3 and 4).
Over the past 5 decades, synthetic pesticides have been indiscriminately used against vector mosquitoes. As a result, side effects such as environmental pollution and toxic hazards to humans and other nontarget organisms were created. These side effects of synthetic chemicals created awareness of the need for ecofriendly and target-specific pesticides for mosquito control [29,30]. It is clearly proven that plant extracts and plant compounds are ecofriendly, target-specific, less expensive, and highly efficacious pesticides for the control of vector mosquitoes [31,32].
In the present study, the hexane extract of L. acidissima recorded the highest ovicidal activity of 79.2% and 60% at 500 ppm concentration against the eggs of Cx. quinquefasciatus and Ae. aegypti, respectively. Previously, some investigators studied the ovicidal activity of plant extracts against mosquito eggs. Elango et al [27] reported that Cocculus hirsutus methanol extract caused 86% and 100% ovicidal activity at 500 ppm and 1000 ppm, respectively against An. subpictus. In another study, 100% ovicidal activity was recorded by a methanol extract of Andrographis paniculata at 150 ppm concentration in An. stephensi eggs [33].
Furthermore, the same hexane extract of L. acidissima showed 100% oviposition deterrent activity at all the tested concentrations (62.5–500 ppm) against Cx. quinquefasciatus and Ae. aegypti adult females. Previously, some investigators reported the oviposition deterrent effect of plant extracts against vector mosquitoes. Coria et al [34] reported 100% oviposition deterrent effect obtained with Melia azedarach L. leaf extract at 1 g/L concentration against Ae. aegypti. Autran et al [35] recorded the oviposition deterrent effect of essential oil obtained from leaves, inflorescence, and stem of Piper marginatum Jacq. Their results showed that essential oil of leaves and stems of P. marginatum exhibited oviposition deterrent effect on Ae. aegypti females at 50 ppm and 100 ppm concentration and that the number of eggs laid was significantly lower (<50%) compared to control. Similarly, Prajapati et al [36] reported that the bark oil of Cinnamomum zeylanicum reduced the oviposition of Ae. aegypti to 50% at 33.5 ppm concentration.
In conclusion, the hexane extract of L. acidissima was the most potent treatment against the two tested mosquito vectors. Based on these results, the hexane extract of L. acidissima could be used in vector mosquito control and may be further probed to isolate the active constituent responsible for the bioactivities.
The authors do not have any conflicts of interest.
The authors are thankful to the Entomology Research Institute for financial assistance. The authors would like to thank Mr. S. Mutheeswaran, Entomology Research Institute, Loyola College, Chennai, India for his help in identifying plant materials.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Table 1
Percent ovicidal activity of crude extracts against Culex quinquefasciatus eggs.
Mosquito species Plant Treatment Concentration (ppm)
62.5 125 250 500
Culex quinquefasciatus Aegle marmelos Hexane 7.1 ± 2.08b 14.4 ± 6.06b 22.4 ± 2.19b 53.6 ± 2.19b
Ethyl acetate 6.4 ± 2.19bc 9.6 ± 2.19bc 20.0 ± 2.82bc 43.2 ± 1.78c
Methanol 3.2 ± 1.78bcd 7.2 ± 1.78c 14.4 ± 2.19cd 28 ± 2.82ef
Limonia acidissima Hexane 17.6 ± 2.19a 36 ± 2.82a 56.8 ± 3.34a 79.2 ± 3.34a
Ethyl acetate 0cd 0.8 ± 1.78d 1.6 ± 3.57f 4.0 ± 2.82hi
Methanol 4 ± 2.82bcd 8.8 ± 3.34c 18.4 ± 2.19bcd 39.2 ± 1.38cd
Sphaeranthus indicus Hexane 3.2 ± 1.78bcd 7.2 ± 3.34c 13.6 ± 2.19cd 25.6 ± 2.19f
Ethyl acetate 2.4 ± 2.19bcd 7.2 ± 1.78c 14.4 ± 2.19cd 29.6 ± 2.19ef
Methanol 1.6 ± 2.19cd 4.8 ± 1.78cd 6.4 ± 2.19ef 15.2 ± 3.34g
Sphaeranthus amaranthaides Hexane 3 ± 2.73bcd 7.8 ± 3.03c 16 ± 4.48bcd 33 ± 2.73de
Ethyl acetate 4 ± 2.82bcd 8 ± 2.82c 15.2 ± 1.78cd 30.4 ± 2.19ef
Methanol 4 ± 2.82bcd 9.6 ± 2.19bc 16 ± 2.82bcd 32.8 ± 3.34de
Chromolaena odorata Hexane 3.2 ± 3.34bcd 6.4 ± 2.19c 12.8 ± 4.38de 24.8 ± 3.34f
Ethyl acetate 4 ± 2.82bcd 9.6 ± 2.19bc 19.2 ± 3.34bcd 42.4 ± 2.19c
Methanol 0cd 0d 4 ± 2.82f 9.6 ± 2.19gh
Control 1.6 ± 2.19cd 0.8 ± 1.78d 0.8 ± 1.78f 0i
Azadirachtin (10 ppm) 95.2 ± 1.78
Temephos (10 ppm) 46.4 ± 3.57

Data are the mean ± standard deviation (SD) of five replicates; Means were separated by Tukey's test of multiple comparisons, one-way analysis of variance (ANOVA).

ppm = parts per million.

p ≤ 0.5, level of significance.

Results with same letters in the column are not significantly different.

Table 2
Percent ovicidal activity of crude extracts against Aedes aegypti eggs.
Mosquito species Plant Treatment Concentration (ppm)
62.5 125 250 500
Aedes aegypti Aegle marmelos Hexane 6.4 ± 1.78ab 13.6 ± 2.19b 26.4 ± 5.21a 48.8 ± 4.38b
Ethyl acetate 1.6 ± 2.19cd 5.6 ± 3.57c 10.4 ± 5.36b 24.8 ± 4.38c
Methanol 4 ± 2.82bc 7.2 ± 1.78c 11.2 ± 4.38b 24.8 ± 1.34c
Limonia acidissima Hexane 8 ± 2.82a 17.6 ± 2.19a 29.6 ± 2.19a 60 ± 2.82a
Ethyl acetate 2.4 ± 2.19cd 5.6 ± 1.19c 11.2 ± 1.78b 19.2 ± 3.34cd
Methanol 0d 0.8 ± 1.78e 4 ± 2.82cd 6.4 ± 1.34fg
Sphaeranthus indicus Hexane 0d 1.6 ± 2.19de 4.0 ± 2.82cd 8.8 ± 3.34ef
Ethyl acetate 0d 0e 0d 3.2 ± 1.78fg
Methanol 0d 0e 0.8 ± 1.78cd 2.4 ± 3.57fg
Sphaeranthus amaranthaides Hexane 0d 0e 2.4 ± 3.57cd 4.8 ± 4.38fg
Ethyl acetate 0d 0e 0d 0g
Methanol 0d 0.8 ± 1.78e 1.6 ± 2.19cd 3.2 ± 1.78fg
Chromolaena odorata Hexane 0d 0e 0.8 ± 2.19cd 3.2 ± 4.38fg
Ethyl acetate 1.6 ± 3.57cd 4.8 ± 3.34cd 6.4 ± 2.19bc 13.6 ± 2.19de
Methanol 0d 0e 0d 1.6 ± 2.19g
Control 0d 00.8 ± 1.78e 00.8 ± 1.78cd 1.6 ± 2.19g
Azadirachtin (10 ppm) 92.8 ± 3.34
Temephos (10 ppm) 44 ± 2.82

Data are mean ± standard deviation (SD) of five replicates. Means are separated by Tukey's test of multiple comparisons, one-way analysis of variance (ANOVA).

ppm = parts per million.

p ≤ 0.5, level of significance.

Results with same letters in the column are not significantly different.

Table 3
Percent oviposition deterrent activity of crude extracts against Culex quinquefasciatus adult females.
Mosquito species Plant Treatment Concentration (ppm)
62.5 125 250 500
Culex quinquefasciatus Aegle marmelos Hexane 23.09 ± 2.22b 46.79 ± 1.30b 56.67 ± 0.95b 76.74 ± 1.02b
Ethyl acetate 8.73 ± 2.15c 20.25 ± 2.13c 32.87 ± 1.30c 47.56 ± 1.48c
Methanol 0e 4.24 ± 2.30f 11.91 ± 2.22ef 20.91 ± 1.65fg
Limonia acidissima Hexane 100a 100a 100a 100a
Ethyl acetate 2.81 ± 1.79d 8.50 ± 2.39de 20.68 ± 2.06d 30.01 ± 1.75e
Methanol 0e 5.11 ± 2.74f 11.80 ± 2.14f 17.74 ± 1.25g
Sphaeranthus indicus Hexane 0e 0g 0i 0i
Ethyl acetate 0e 0g 7.64 ± 1.55g 11.47 ± 1.75h
Methanol 0e 0g 0i 0i
Sphaeranthus amaranthaides Hexane 2.06 ± 1.21de 5.45 ± 1.62ef 9.93 ± 2.64fg 12.03 ± 1.63h
Ethyl acetate 0.32 ± 0.29de 9.29 ± 2.30d 15.10 ± 1.61e 22.31 ± 2.59f
Methanol 0e 0g 4.03 ± 1.42h 13.34 ± 2.07h
Chromolaena odorata Hexane 0e 0g 0i 0i
Ethyl acetate 0e 0g 0i 0i
Methanol 2.48 ± 2.01de 9.17 ± 1.75d 22.55 ± 1.54d 33.73 ± 1.88d
Azadirachtin (10 ppm) 86.29 ± 1.09
Temephos (10 ppm) 10.27 ± 1.75

Data are mean ± standard deviation (SD). Means are separated by Tukey's test of multiple comparisons, one-way analysis of variance (ANOVA).

p ≤ 0.5, level of significance.

ppm = parts per million.

Results with same letters in the column are not significantly different.

Table 4
Percent oviposition deterrent activity of crude extracts against Aedes aegypti adult females.
Mosquito species Plant Treatment Concentration (ppm)
62.5 125 250 500
Aedes aegypti Aegle marmelos Hexane 21.02 ± 2.11b 45.14 ± 1.69b 52.60 ± 1.77b 71.79 ± 1.57b
Ethyl acetate 3.04 ± 1.54d 7.20 ± 1.63d 13.56 ± 2.21e 31.22 ± 1.63d
Methanol 0e 0g 3.15 ± 1.62hi 13.01 ± 1.56g
Limonia acidissima Hexane 100a 100a 100a 100a
Ethyl acetate 1.20 ± 0.47de 6.44 ± 1.47de 18.06 ± 1.23d 27.71 ± 1.48e
Methanol 0e 1.84 ± 0.88fg 5.87 ± 2.46gh 10.88 ± 2.63gh
Sphaeranthus indicus Hexane 0e 0g 0i 0k
Ethyl acetate 0e 0g 2.59 ± 1.85i 4.34 ± 2.59i
Methanol 0e 0g 0i 0k
Sphaeranthus amaranthaides Hexane 2.27 ± 1.59de 3.90 ± 2.29ef 6.65 ± 1.79g 8.74 ± 1.68h
Ethyl acetate 2.07 ± 1.69de 4.14 ± 0.83ef 10.27 ± 1.75f 20.48 ± 0.83f
Methanol 0e 0g 0i 2.61 ± 1.13ij
Chromolaena odorata Hexane 0e 0g 0i 0k
Ethyl acetate 0e 0g 0i 0k
Methanol 9.93 ± 2.15c 15.41 ± 2.39c 25.53 ± 0.86c 38.23 ± 1.73c
Azadirachtin (10 ppm) 75.21 ± 0.86
Temephos (10 ppm) 4.05 ± 1.36

Data are the mean ± standard deviation (SD). Means are separated by Tukey's test of multiple comparisons, one-way analysis of variance (ANOVA). p ≤ 0.5, level of significance.

ppm = parts per million.

Results with same letters in the column are not significantly different.

Figure & Data

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