# Relative Toxicity of Leaf Extracts ofEucalyptus globulus and Centella asiatica against Mosquito Vectors Aedes aegypti and Anopheles stephensi

Authors: Savitha Sekhar Nair; Vinaya Shetty; Nadikere Jaya Shetty
Journal: Journal of Insects
 (2014)

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## Abstract

The larvicidal activity of different solvent leaf extracts (hexane, diethyl ether, dichloromethane, and methanol) ofEucalyptus globulus and Centella asiatica against two geographically different strains of Aedes aegypti and Anopheles stephensi was investigated. The extracts were tested against the late third instar larvae of Aedes aegypti and Anopheles stephensi, and larval mortality was observed after 24 hours of treatment. LC50 and LC90 were calculated. The LC50 values of hexane extract of Eucalyptus globulus against the late third instar larvae of the BSN and JPN strains of Aedes aegypti and the DLC and KNG strains of Anopheles stephensi were 225.2, 167.7, 118.8, and 192.8 ppm, while those of the hexane extract of Centella asiatica were 246.5, 268.7, 50.6, and 243.5 ppm, respectively. The LC50 values of diethyl ether extract of Centella asiatica were 339.6, 134.5, 241, and 14.7 ppm. The hexane extracts of both plants and the diethyl ether extract of C. asiatica presented the highest potential for the control of Aedes aegypti and Anopheles stephensi. The present findings also reveal the necessity of assaying multiple strains of a species to fully comprehend the larvicidal efficacy of a compound.


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## Body

## 1. Introduction

Apart from being a social nuisance, mosquitoes pose serious health threats to both men and animals considering that they are the principal vectors for many vector borne diseases including malaria, dengue, yellow fever, and Chikungunya [1, 2] in men and equine encephalitis, haemorrhagic septicaemia of buffaloes, and enzootic hepatitis in animals [3]. In some individuals, mosquito bites also result in acute systemic allergic reactions defined by the presence of one or more of the following: urticaria, angioedema, wheezing, dyspnea, hypotension, and decrease or loss of consciousness [4]. The mosquito,Aedes(Stegomyia)aegypti (Linn.), (Diptera: Culicidae) is the primary vector of dengue, yellow fever, and Chikungunya [2]. According to WHO, over 40% of the world’s population is now at risk of dengue and there are 200 000 estimated cases of yellow fever, causing 30 000 deaths, worldwide each year. Malaria, on the other hand, a life threatening disease which caused an estimated 627 000 deaths in 2012 is  transmitted exclusively through the bites ofAnopheles mosquitoes [5].Anopheles stephensiListon (Diptera: Culicidae) is a major vector in India as well as in some of the West Asian countries and has been shown to be directly responsible for about 40–50% of the annual malarial incidence [6, 7].Mosquito control is an extensively researched topic considering that presently we do not have vaccines for malaria and dengue [8, 9] and, therefore it becomes necessary to check the spread of these diseases. Vector control methods, thus are of utmost prominence. The incessant use of chemical insecticides has often led to the disruption of natural biological control system and outbreak of insect species [10]. The selective pressure of conventional insecticides is enhancing the resistance of mosquito strains at an alarming rate [11], increasing the demand for new products that are eco-friendly, target-specific, and biodegradable. Plant-derived insecticides provide an alternative to synthetic pesticides because of their generally low environmental pollution, low toxicity to men, and other advantages [12].Earlier studies have shown the efficacy ofEucalyptusoils as a larvicide against mosquitoes [13–16]. In the year 2004, Yang  et al. reported thatEucalyptus leaf oil, particularly some of its components such as 1,8-cineole, 1-α-terpineol, and (E)-pinocaveol, merits further study as lead compounds for the control of the human hair louse,Pediculus humanus capitis De Geer (Pediculidae) [17]. Constituents of the essential oil ofEucalyptus camaldulensis Dehnh andEucalyptus urophyllaS. T. Blake (Myrtaceae) a-phellandrene, limonene, p-cymene, c-terpinene, terpinolene, and a-terpinene have been shown to possess strong larvicidal effects againstAedes aegypti andAedes albopictus[15]. The various components ofEucalyptus essential oil have been shown to act synergistically (and not additively) to bring the overall bactericidal activity [18].Centella asiaticais mostly studied for its medicinal properties. Very few studies have documented its potential as a biopesticide. A study has suggested that the leaf extract ofC. asiatica is promising as a larvicide and adult emergence inhibitor againstCulex quinquefasciatus Say (Culicidae) [19]. Shukla et al. [20] isolated and studied the growth inhibitory effect of 2 α, 3 β, 6 β, 23-α tetrahydroxyurs-12-ene-28-oic acid, a triterpenoid glycoside against larvae ofSpilarctia obliquaWalker (Arctiidae).Numerous studies have examined the variations in larvicidal efficacies of plant extracts tested against different mosquito species; however, we were not able to find any reports of the larvicidal efficacies of plant extracts tested against different strains of mosquitoes of the same species. Susceptibility studies involving strains from different locations could be beneficial to gauge the efficacy of the drug being studied and as a prelude to investigations involving the inherent resistance mechanisms of the vectors to the drugs.Different solvent types can significantly affect the potency of the extracted plant compounds and there is a difference in the chemoprofile of the plant species [21]. This is because polar solvents extract polar constituents of the plant, while nonpolar solvents extract the nonpolar constituents of the plant. Hence, the best results will be observed using the plant extract of the solvent whose polarity matches that of the molecules in the plant responsible for insecticidal activity (if any). Additionally, extracts or pure compounds derived from specific solvents can influence the bioactivity, probably because of the active components which are present in large quantities [22].In the present study, we have investigated the larvicidal activity of the hexane, diethyl ether, dichloromethane, and methanol extracts of the leaves ofEucalyptus globulusLabill (Myrtaceae) andCentella asiatica(Linn.) Urban (Apiaceae) against two geographically different strains ofAedes(Stegomyia)aegypti (Linn.) (Diptera: Culicidae) andAnopheles stephensiListon (Diptera: Culicidae).


## 2. Materials and Methods

### 2.1. Mosquito Culture

Four strains which consisted of laboratory colonies ofAedes aegypticollected from J. P. Nagar (JPN) (12.9120°N; 77.5930°E) and Basaveshwaranagar (BSV) (12.9867°N; 77.5386°E) andAnopheles stephensi collected from Dollars Colony (DLC) (13°2′30′′N; 77°34′3′′E) and Kengeri (KNG) (12.9100°N; 77.4800°E) were used for this study. Larvae collected from the said locations of BBMP region, Karnataka, India, during the months March-April 2012, had been reared continuously in the insectary at the Centre for Applied Genetics, Bangalore University, following the method of Shetty [23].


### 2.2. Collections of Plant Materials

The leaves ofEucalyptus globulus andCentella asiatica were collected from Bangalore city (11°1′48′′N; 77°2′36′′E; elevation: 399 m), Karnataka, India, in July 2013, and were authenticated at the Department of Botany, Bangalore University, Bangalore, India.


### 2.3. Preparation of Plant Extracts

The leaves (2 kg each) were air dried in shade for 15–30 days. The dried leaves were then powdered mechanically using a commercial electrical stainless steel blender. One kg of powdered leaves was extracted successively by maceration using nonpolar to polar solvents, namely, hexane, diethyl ether, dichloromethane, and methanol. In each solvent, the plant material was soaked for 48 h at 35°C and filtered twice using Whatman number 1 filter paper to obtain the extract, and to the residue the same solvent was added again. The procedure was repeated twice to obtain maximum extract. The extracts were concentrated at reduced temperature using a rotary vacuum evaporator and stored at a temperature of 4°C. One gram of the concentrated plant extract was dissolved in 100 mL of 1 : 1 acetone : dimethyl sulfoxide (DMSO) and considered as 1% stock solution. From this stock solution, varying concentrations of each extract was prepared and these solutions were used for larvicidal bioassay. All chemicals used in this study were of extra pure grade and were obtained from Sisco Research Laboratories PVT. Ltd., India.


### 2.4. Larvicidal Bioassay

Larvicidal activity of each extract derived from the leaves ofEucalyptus globulusandCentella asiatica against the two strains ofAe. aegyptiandAn. stephensiwas assessed by using a slightly modified version of the WHO standard method [24].Initially, the mosquito larvae were exposed to a broad range of test concentrations to determine the activity range of each extract. Based on the results of preliminary screening, batches of 25 late third instar larvae were added to 300 mL wide mouth disposable bowls containing serial concentrations (6–9 concentrations, yielding between 10% and 95% mortality in 24 hrs) of each plant extract made up to 250 mL by volume using tap water. The test was carried at a temperature of25
±
2°C and relative humidity of 75
±
5%. The numbers of dead larvae were counted after 24 hours of exposure and percentage mortality was calculated for each test as follows:
(1)
Percentage
Mortality
=
Number
of
dead
larvae
/
pupae
Number
of
larvae
introduced
×
100
.
The final percentage mortality was calculated from the average of three replicates. Solutions containing tap water and 1 : 1 acetone : dimethyl sulfoxide, but without the plant sample, served as controls. The control mortalities were corrected by using Abbott’s formula [25].


### 2.5. Statistical Analysis

The average larval mortality data were subjected to probit analysis for calculating LC50 and LC90 and other statistics at 95% fiducial limits of upper confidence limit (UCL) and lower confidence limit (LCL), and Chi-square values were calculated using the SPSS 12.0 (Statistical Package of Social Sciences) software (Finney, 1971). The Chi-square values were considered significant at P
<
0.05 level.


## 2.1. Mosquito Culture

Four strains which consisted of laboratory colonies ofAedes aegypticollected from J. P. Nagar (JPN) (12.9120°N; 77.5930°E) and Basaveshwaranagar (BSV) (12.9867°N; 77.5386°E) andAnopheles stephensi collected from Dollars Colony (DLC) (13°2′30′′N; 77°34′3′′E) and Kengeri (KNG) (12.9100°N; 77.4800°E) were used for this study. Larvae collected from the said locations of BBMP region, Karnataka, India, during the months March-April 2012, had been reared continuously in the insectary at the Centre for Applied Genetics, Bangalore University, following the method of Shetty [23].


## 2.2. Collections of Plant Materials

The leaves ofEucalyptus globulus andCentella asiatica were collected from Bangalore city (11°1′48′′N; 77°2′36′′E; elevation: 399 m), Karnataka, India, in July 2013, and were authenticated at the Department of Botany, Bangalore University, Bangalore, India.


## 2.3. Preparation of Plant Extracts

The leaves (2 kg each) were air dried in shade for 15–30 days. The dried leaves were then powdered mechanically using a commercial electrical stainless steel blender. One kg of powdered leaves was extracted successively by maceration using nonpolar to polar solvents, namely, hexane, diethyl ether, dichloromethane, and methanol. In each solvent, the plant material was soaked for 48 h at 35°C and filtered twice using Whatman number 1 filter paper to obtain the extract, and to the residue the same solvent was added again. The procedure was repeated twice to obtain maximum extract. The extracts were concentrated at reduced temperature using a rotary vacuum evaporator and stored at a temperature of 4°C. One gram of the concentrated plant extract was dissolved in 100 mL of 1 : 1 acetone : dimethyl sulfoxide (DMSO) and considered as 1% stock solution. From this stock solution, varying concentrations of each extract was prepared and these solutions were used for larvicidal bioassay. All chemicals used in this study were of extra pure grade and were obtained from Sisco Research Laboratories PVT. Ltd., India.


## 2.4. Larvicidal Bioassay

Larvicidal activity of each extract derived from the leaves ofEucalyptus globulusandCentella asiatica against the two strains ofAe. aegyptiandAn. stephensiwas assessed by using a slightly modified version of the WHO standard method [24].Initially, the mosquito larvae were exposed to a broad range of test concentrations to determine the activity range of each extract. Based on the results of preliminary screening, batches of 25 late third instar larvae were added to 300 mL wide mouth disposable bowls containing serial concentrations (6–9 concentrations, yielding between 10% and 95% mortality in 24 hrs) of each plant extract made up to 250 mL by volume using tap water. The test was carried at a temperature of25
±
2°C and relative humidity of 75
±
5%. The numbers of dead larvae were counted after 24 hours of exposure and percentage mortality was calculated for each test as follows:
(1)
Percentage
Mortality
=
Number
of
dead
larvae
/
pupae
Number
of
larvae
introduced
×
100
.
The final percentage mortality was calculated from the average of three replicates. Solutions containing tap water and 1 : 1 acetone : dimethyl sulfoxide, but without the plant sample, served as controls. The control mortalities were corrected by using Abbott’s formula [25].


## 2.5. Statistical Analysis

The average larval mortality data were subjected to probit analysis for calculating LC50 and LC90 and other statistics at 95% fiducial limits of upper confidence limit (UCL) and lower confidence limit (LCL), and Chi-square values were calculated using the SPSS 12.0 (Statistical Package of Social Sciences) software (Finney, 1971). The Chi-square values were considered significant at P
<
0.05 level.


## 3. Results

The larvicidal efficacies of hexane, diethyl ether, dichloromethane, and methanol extracts of bothEucalyptus globulus andCentella asiaticawere established for two different mosquito strains of each of the above said species from the Bangalore, Karnataka. The data were recorded and statistical data including regression equation, LC50, LC90, LCL, UCL, and Chi-square values were calculated and presented (Table 1). No mortality was observed in the control.Table 1
Effects of relative toxicity of solvent extracts (hexane, diethyl ether, dichloromethane, and methanol) of the leaves ofEucalyptus globulus and Centella asiatica against the late third larval stage of  BSV and JPN strains of Aedes aegypti and DLC and KNG strains of Anopheles stephensi.
Botanical name
Solvents
Species
Strain
LC50 (ppm) (UCL-LCL)
LC90 (ppm) (UCL-LCL)
Regression equation
r
χ
2
df
Eucalyptus  globulus
Hexane
Ae. aegypti
BSV
225.2  (210.9–263.5)
423.6  (400.4–598.9)
y
=
4.6638
x
-
5.9716
0.9364
0.1902
7
JPN
167.7  (152.1–186.7)
304  (266.3–400.9)
y
=
4.9559
x
-
6.0248
0.984
0.0238
6
An. stephensi
DLC
118.8  (88.1–151.2)
450.4  (373.8–953.3)
y
=
2.2119
x
+
0.4106
0.9137
0.1123
7
KNG
192.8  (154.7–255.1)
827  (655.2–1634)
y
=
2.0242
x
+
0.3744
0.9378
0.1134
7
Diethyl ether
Ae. aegypti
BSV
686.8  (630.8–756.4)
1216.7  (1073.7–1582.5)
y
=
5.1546
x
-
9.6228
0.9594
0.0244
7
JPN
318.8  (146.7–641.2)
2069.9  (1085.1–34888.5)
y
=
1.5756
x
+
1.0554
0.9114
0.1811
6
An. stephensi
DLC
464.1  (428.8–524.2)
800.1  (726.1–1050.7)
y
=
5.4107
x
-
9.428
0.9382
0.0767
6
KNG
318  (270.6–391.7)
951.9  (737.4–1618.7)
y
=
2.688
x
-
1.7265
0.9279
0.0605
6
Dichloromethane
Ae. aegypti
BSV
314.6  (276.1–385)
829.9  (677.1–1290.7)
y
=
3.038
x
-
2.588
0.9846
0.0832
6
JPN
362.2  (171.6–620.1)
2904.6  (1068.6–13925.1)
y
=
1.4157
x
+
1.3773
0.889
0.1362
6
An. stephensi
DLC
300.3  (256.3–394.2)
1235.4  (872.5–2283.9)
y
=
2.0839
x
-
0.163
0.9747
0.1345
7
KNG
162  (112.5–199.4)
582.4  (452.6–1323.4)
y
=
2.3036
x
-
0.0899
0.9623
0.0536
7
Methanol
Ae. aegypti
BSV
1839.3  (1695.8–2049.7)
3235.9  (2930.2–4051.1)
y
=
5.2171
x
-
12.032
0.9805
0.0504
7
JPN
1467.9  (1281–1786.8)
3530.2  (2911.7–5612.3)
y
=
3.3587
x
-
5.636
0.9644
0.0634
5
An. stephensi
DLC
1263.3  (798.1–1817.3)
3614.3  (2988.3–12736.6)
y
=
2.8038
x
-
3.696
0.8769
0.2807
7
KNG
889.6  (764.7–1124.3)
2655.1  (2177.0–4907.4)
y
=
2.6955
x
-
2.9496
0.9204
0.1292
6
Centella  asiatica
Hexane
Ae. aegypti
BSV
246.5  (213.1–307.1)
593.1  (527.3–1013.9)
y
=
3.3561
x
-
3.027
0.9284
0.1366
5
JPN
268.7  (174.4–374.7)
1606.8  (1082.1–5866.2)
y
=
1.6479
x
+
0.9969
0.929
0.0583
5
An. stephensi
DLC
50.6  (18.3–105)
328.8  (198.5–8730)
y
=
1.5752
x
+
2.3154
0.8489
0.2479
6
KNG
243.5  (221.1–274.2)
428.6  (362.7–584.5)
y
=
5.2115
x
-
7.4372
0.9963
0.0055
5
Diethyl Ether
Ae. aegypti
BSV
339.6  (258.1–472.3)
1599.3  (1173.6–3840.5)
y
=
1.9022
x
+
0.1855
0.8489
0.0844
5
JPN
134.5  (20.6–204.1)
770.8  (576.7–3191.7)
y
=
1.6885
x
+
1.4054
0.9362
0.0463
5
An. stephensi
DLC
241  (203.4–301.5)
630.7  (540.4–1116.2)
y
=
3.063
x
-
2.2959
0.8489
0.1046
5
KNG
14.7  (3–35)
118  (63.7–17122)
y
=
1.4175
x
+
3.3433
0.8862
0.1491
5
Dichloromethane
Ae. aegypti
BSV
339.7  (28.1–746.3)
2127.7  (1241.9–403246)
y
=
1.6065
x
+
0.9337
0.9384
0.1913
5
JPN
837.6  (777.9–1030.2)
1627.2  (1542.3–2558.8)
y
=
4.4382
x
-
7.973
0.9
0.1722
5
An. stephensi
DLC
236.8  (145.5–393.3)
3347.1  (1576.7–27927.2)
y
=
1.1127
x
+
2.3581
0.9338
0.0533
5
KNG
155.5  (20.9–295.5)
3022.9  (1732.5–33559.1)
y
=
0.9933
x
+
2.8229
0.8881
0.0528
5
Methanol
Ae. aegypti
BSV
776.5  (590.99–1090.78)
3853.4  (2728.01–10164.21)
y
=
2.1701
x
-
1.2027
0.8905
0.0966
5
JPN
721.5  (369.54–1358.04)
2806  (1866.07–32489.71)
y
=
2.1701
x
-
1.2027
0.8905
0.1928
5
An. stephensi
DLC
476.8  (461.94–493.49)
587.9  (567.22–643.99)
y
=
14.064
x
-
32.668
0.9832
0.0333
8
KNG
177  (119.28–225.09)
559.9  (483.97–1260.17)
y
=
2.5595
x
-
0.7538
0.9002
0.1107
5
LC50: lethal concentration that kills 50% of the exposed larvae, LC90: lethal concentration that kills 90% of the exposed larvae, UCL: upper confidence limit, LCL: lower confidence limit, r: correlation coefficient, χ
2: Chi-square significant at P < 0.05 level, and df: degree of freedom.All plant extracts showed varied level of toxic effects with species after 24 hours of exposure, while the methanol extract demonstrated very low larvicidal efficacy in the case of both of the plants.


### 3.1.Eucalyptus globulus

Among theAn. stephensi strains, the DLC strain displayed the highest susceptibility to the hexane extract (LC50 118.8 and LC90 450.4 ppm) followed by the diethyl ether extract (LC50 464.1 and LC90 800.1 ppm). The KNG strain ofAnopheles stephensi, on the other hand, showed the highest larval mortality in the dichloromethane extract (LC50 162.0 and LC90 582.4 ppm), followed by the hexane extract (LC50 192.8 and LC90 827 ppm). The BSN and JPN strains ofAe. aegypti were most susceptible to the hexane extract (LC50 225.2, 167.7 and LC90 423.6, 304.0 ppm, resp.). The methanol extract was the least effective in all cases. A higher level of tolerance was recorded in the BSN strain ofAe. aegyptito the methanol extract (LC50 1839.0 and LC90 3235.9 ppm).


### 3.2.Centella asiatica

The hexane extract produced the highest mortality in the case of the BSN and JPN strains ofAe. aegypti(LC50 246.5, 268.7 and LC90 593.1, 1606.8 ppm, resp.), and also the DLC strain ofAn. stephensi (LC50 50.6 and LC90 328.8 ppm), the KNG strain ofAn. stephensihowever was most susceptible to the diethyl ether extract (LC50 14.7 and LC90 118.0 ppm). Again, the methanol extract was the least potent in all cases. The BSN strain ofAe. aegypti displayed a higher level of tolerance (LC50 776.5 and LC90 3853.4 ppm) upon treatment with the methanol extract.


## 3.1.Eucalyptus globulus

Among theAn. stephensi strains, the DLC strain displayed the highest susceptibility to the hexane extract (LC50 118.8 and LC90 450.4 ppm) followed by the diethyl ether extract (LC50 464.1 and LC90 800.1 ppm). The KNG strain ofAnopheles stephensi, on the other hand, showed the highest larval mortality in the dichloromethane extract (LC50 162.0 and LC90 582.4 ppm), followed by the hexane extract (LC50 192.8 and LC90 827 ppm). The BSN and JPN strains ofAe. aegypti were most susceptible to the hexane extract (LC50 225.2, 167.7 and LC90 423.6, 304.0 ppm, resp.). The methanol extract was the least effective in all cases. A higher level of tolerance was recorded in the BSN strain ofAe. aegyptito the methanol extract (LC50 1839.0 and LC90 3235.9 ppm).


## 3.2.Centella asiatica

The hexane extract produced the highest mortality in the case of the BSN and JPN strains ofAe. aegypti(LC50 246.5, 268.7 and LC90 593.1, 1606.8 ppm, resp.), and also the DLC strain ofAn. stephensi (LC50 50.6 and LC90 328.8 ppm), the KNG strain ofAn. stephensihowever was most susceptible to the diethyl ether extract (LC50 14.7 and LC90 118.0 ppm). Again, the methanol extract was the least potent in all cases. The BSN strain ofAe. aegypti displayed a higher level of tolerance (LC50 776.5 and LC90 3853.4 ppm) upon treatment with the methanol extract.


## 4. Discussion

The leaf extracts ofEucalyptus globulus andCentella asiatica showed an overall moderate larvicidal effect when tested against different strains ofAe. aegypti andAn. stephensi. Among the four extracts studied, the hexane extracts of both plants and the diethyl ether extract ofC. asiatica demonstrated higher potential for the control of strains of mosquito vectors, followed by dichloromethane. The methanol extract produced a comparatively less promising result. The effect of the extracts on larval mortality was dose-dependent, with increasing rates of larval mortality observed at increasing concentrations. The highest level of tolerance was observed in the BSN strain ofAe. aegypti towards the methanol extract ofE. globulus.Although the larvicidal efficacy of the extracts is not as promising as that of synthetic insecticides commonly in use today [26], the present results are comparable to those of earlier authors who worked on various plant extracts as larvicides against different mosquito species. The methanol leaf extract ofClitoria ternateaL. (Leguminosae) showed a dose-dependent larvicidal activity againstAn. stephensi with an LC50 value of 555.6 ppm [27]. In the case ofMorinda citrifoliaL. (Rubiaceae) the highest larvicidal activity was exhibited by the methanol extract of the leaf when compared to the hexane, chloroform, acetone, and water extracts; the LC50 in case ofAn. stephensi andAe. aegypti was observed to be 261.96 and 277.92 ppm, respectively. The chloroform leaf extracts ofNyctanthes arbortristis L. (Oleaceae) demonstrated an LC50 value of 780.6 ppm, while the LC50 value of the methanol flower extract of the same plant was 679.4 ppm when tested againstAn. stephensi. Studies have also been carried out on the larvicidal potential of the essential oil extracted from theEucalyptus species [13–15] andE. globulus in particular [28]. However, this is the first time report of the larvicidal efficacy of serial extracts ofEucalyptus globulus andCentella asiatica leaves using multiple solvents against different geographical strains ofAe. aegypti andAn. stephensi. In the present study, strains of both species of mosquitoes were found to be highly susceptible to hexane leaf extracts when compared to the other solvent extracts.Numerous authors have observed a converse relationship between extract effectiveness and solvent polarity where the efficacy increases with decreasing polarity [29–32]. Singh et al. [33] reported the LC50 values of the hexane extract of Eucalyptus citriodoraHook (Myrtaceae) against the IVth instar larvae ofAn. stephensi (69.86 ppm) andAe. aegypti (91.76 ppm). Hexane extracts of other plants have also shown similar satisfactory larvicidal potency. LC50 of hexane leaf extract ofCitrus sinensis (L.) Osbeck (Rutaceae) against early fourth instars ofAe. aegypti was found to be 446.84 ppm [34]. The LC50 of hexane leaf extract ofMurraya koenigii (L.) Spreng (Rutaceae) against third instarsCulex quinquefasciatus was found to be 963.53 ppm [35]. In the case ofCitrullus colocynthis (L.) Schrad (Cucurbitaceae), however, the LC50 of the hexane extract was found to be 1451.29 ppm while that of the diethyl ether extract was found to be 503.39 ppm. This result is similar to that of the KNG strain ofAn. stephensi in our studies, where the diethyl ether extract ofC. asiatica produced a better result than the hexane extract. Contrarily, the otherAn. stephensistrain that we worked with (DLC) exhibited higher susceptibility for the hexane extract against the diethyl ether extract.Even though our studies show methanol extracts to produce the least mortality, other plants such asMorinda citrifolia L. (Rubiaceae) andErythrina indica Lam (Leguminosae) have been shown to produce greater susceptibility for their methanol extract when compared to their hexane extract [36, 37]. Therefore, the present study suggests that, in the case ofE. globulus andC. asiatica, the compounds majorly responsible for the insecticidal activity are most probably in the nonpolar and intermediate range and are not polar.There is probably also the result of the synergistic activity of the active compounds that influences the efficacy of the extract, against the physiological characteristics of each strain tested. For instance, dichloromethane and diethyl ether are solvents with very similar polarity and should typically elute compounds of the same class, and the results show that in some instances dichloromethane extracts prove that they have higher efficacy, while diethyl ether extracts show greater potency. Further studies involving larger number of strains and purified isolated constituents should reveal a clearer picture of interaction between the bioactive molecules and target species.In the present investigation, it was also interesting to note that the variation in the response of different strains of mosquito of the same species to a specific plant extract is almost the same as the variation in the response of different strains of mosquito of different species to the same extract. For instance, the LC50 values for diethyl ether extract ofC. asiatica against theAn. stephensi strains KGR and DLC are 14.7 ppm and 241 ppm, while the LC50 values for the same extract against theAe. aegypti strains JPN and BSV are 134.5 and 339.6 ppm. From this study, it is revealed that it is imperative to assay the extract against different geographical strains of the same species to ascertain the efficacy of a compound.Earlier studies on different geographical strains ofAn. stephensi andAe. Aegypti have reported varied levels of susceptibility to various insecticides commonly used in mosquito vector control [26, 38–43]. Unlike conventional insecticides which are based on a single active ingredient, plant derived insecticides comprise botanical blends of chemical compounds which act concertedly on both behavioral and physiological processes. Thus, there is very little chance of pests developing resistance to such substances. Identifying bioinsecticides that are efficient, as well as being suitable and adaptive to ecological conditions, is imperative for continued effective vector control management [21]. In order to develop cost effective pesticides from the plant leaves used in the present study, it will be necessary to carry out further studies to determine the active compounds in these plants, their larvicidal efficacies, their individual and synergistic modes of action, the feasibility of large scale use, and stability of these active compounds under field conditions. Most studies using biological extracts have been tested against a single strain of a species. This is probably because one would not expect to observe large variations in the susceptibility of one strain to another. Unlike the varied resistance status observed in the case of synthetic insecticides, the biological extracts used in this study have not been used by municipal bodies as insecticides. Therefore, the observed variation in response between different strains does not appear to have been caused by such a selection event, but rather by the intervention of biological and genetic factors, resulting in the change in the susceptibility to different plant extracts.


## 5. Conclusion

In the present study, the hexane extracts of both plants and the diethyl ether extract ofC. asiatica demonstrated the highest potential for the control of strains of mosquito vectors. The results indicate that the compounds responsible for the insecticidal activity of both plants are most probably in the nonpolar and intermediate range. Also, the present findings show that it is necessary to assay multiple strains of a species to fully comprehend the larvicidal efficacy of a compound.



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*Source: 985463-2014-10-22.xml*