Ovicidal and Oviposition Deterrent Activities of Medicinal Plant Extracts Against Aedes aegypti L. and Culex quinquefasciatus Say Mosquitoes (Diptera: Culicidae).

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.

Introduction

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.

Materials and methods

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.

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.

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].

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:

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:where NC is the number of eggs in the control, and NT is the number of eggs in the treatment.

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).

Results

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.

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).

Discussion

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.

Conflicts of interest

The authors do not have any conflicts of interest.

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.