298

Journal of Vector Ecology December 2014

Synergistic repellent and irritant effect of combined essential oils

on Aedes aegypti (L.) mosquitoes
Atirach Noosidum, Theeraphap Chareonviriyaphap, and Angsumarn Chandrapatya*

Department of Entomology, Faculty of Agriculture, Kasetsart University, Chatuchak, Bangkok 10900, Thailand, agramc@ku.ac.th

Received 7 February 2014; Accepted 10 June 2014

ABSTRACT: This study was designed to compare the behavioral responses of Aedes aegypti to a single essential oil and to
a mixture of two or three essential oils using an excito-repellency test chamber. Mixtures were prepared from essential oils
extracted from Litsea cubeba (LC), Litsea salicifolia (LS), and Melaleuca leucadendron (ML). In general, the mixture of essential
oils produced a much stronger escape response by Ae. aegypti, regardless of the test conditions. No significant difference in
escape responses was seen when the mixture of oils was compared with a standard commercial product containing DEET.
Greater contact irritancy was seen from mixed oils of LC and LS than with other mixed oils. Mixtures of LC and LS at 0.075%
showed the highest synergistic action (65.5% escaped) compared to that with unmixed oil alone at the same concentration
(LC=20% and LS=32.2%). In addition, mixtures of LC and LS at 0.075% demonstrated the highest non-contact repellency
(62.7%) and showed a greater effect than the use of LC (20%) or LS (20.3%) alone. We conclude that mixtures of two essential
oils show potential as active ingredients for mosquito repellents. Journal of Vector Ecology 39 (2): 298-305. 2014.

Keyword Index: Excito-repellency, irritancy, essential oils, Melaleuca leucadendron, Litsea cubeba, Litsea salicifolia, Aedes aegypti,
DEET.

INTRODUCTION using an excito-repellency test chamber.

For decades, synthetic insecticidal compounds within MATERIALS AND METHODS

different chemical classes have been used by the private and
public health sectors. Synthetic pyrethroids have become
ubiquitous due to their relatively low mammalian toxicity
and ability to rapidly immobilize invertebrates at low levels
(Jirakanjanakit et al. 2007, Thanispong et al. 2008). Because
of the frequent use of pyrethroids, selection pressure for
insecticide resistance has become a major concern (Roberts
et al. 1997, Jirakanjanakit et al. 2007, Thanispong et al.
2008). In addition to toxicants, effective repellents such as
DEET (N, N-diethyl-meta-toluamide) are readily available
and commonly used (Fradin and Day 2002). DEET is used
extensively to protect against biting mosquitoes, however it
has been shown to have significant adverse or toxic effects
to humans (Brown and Hebert 1997, Odalo et al. 2005).
Consequently, there is interest in the development of insect
repellents from natural plant extracts that might be less toxic
to humans, such as thyme (Park et al. 2005) and citronella
(Yang and Ma 2005).
Recently, several plant species were evaluated as potential
repellents or toxicants against blood-feeding invertebrates
and pestiferous flies in Lao PDR (De Boer et al. 2010).
Although several essential oils have exhibited significant
repellent activity against target insects (Noosidum et al. 2008,
Kim et al. 2012, Suwansirisilp et al. 2013), comparatively little
has been done to identify the degree of behavioral responses
using the mixtures of at least two essential oils. In this study,
we compared the behavioral responses of Aedes aegypti (L.) to
a single essential oil and mixtures of essential oils from Litsea
cubeba (Lour.) Persoon (LC), Litsea salicifolia (Roxburgh ex
Nees) Hook. f. (LS), and Melaleuca leucadendron L. (ML)
Mosquitoes
Aedes aegypti populations were collected from Pu Teuy
Village, Sai Yok District, Kanchanaburi Province, Thailand
(14°17’N, 99°1’E). All female mosquitoes were identified to
species and colonies were established using modified methods
from Kongmee et al. (2004). Two days post-emergence,
mosquitoes were blood-fed on live guinea pigs held for three
days until oviposition occurred on filter paper contained
in dishes with tap water. Egg and larval stages were reared
in plastic pans and pupae were maintained in plastic cups,
which were held at 25 ± 5° C, 12:12 (L:D) photoperiod, and
80 ± 10% relative humidity. Adults were held in 30×30×30
cm screened holding cages and provided with cotton pads
soaked with 10% sugar solution. Only three to five day-old
female Ae. aegypti were used and were sugar-starved (water
only) for 12 h before testing.
Oil extraction
Fresh ML leaves were collected from Chanthaburi
Province, Thailand, in October, 2010 (13°98’N, 48°18’E).
Mature fruits of LC and LS were collected from Doi Ang-
khang, Fang District, Chiang Mai Province in November,
2010 (19°54’N, 99°2’E). Essential oils from the plants were
extracted over 6 h by water distillation using a Clevenger-
type apparatus. The superior phase was collected from the
condenser, dried over anhydrous sodium sulfate, and stored
in amber-colored vials at 0° C until testing (Noosidum et al.
2008).
Vol. 39, no. 2 Journal of Vector Ecology 299
Behavioral tests
This experiment was designed to compare the repellent
(non-contact response) and irritant response (contact
response) from a single oil and oil mixture in various
concentrations using excito-repellency assays described
in Noosidum et al. (2008). Based on our previous study
(Noosidum et al. 2008), LC and LS at 0.025, 0.05, 0.075, 0.1,
0.125, and 0.15 (v/v), and ML at 0.3 and 0.5 (v/v) were diluted
in absolute ethanol and impregnated onto a (15×17.5 cm)
filter paper. All filter papers were treated with 2.75 ml of test
solution per 180 cm2. DEET (15% w/w) and 11 concentrations
of the oil mixture were also evaluated. The control papers were
treated with absolute ethanol only. All tests were performed
during 08:00-16:30 under laboratory-controlled conditions.
Each experimental series consisted of two test chambers
treated with essential oil, one contact and one non-contact, and
two identical untreated control chambers. Female mosquitoes
were sugar starved and held in cups approximately eight to
ten h before testing. Fifteen mosquitoes were placed in each
test chamber (23×23×23 cm stainless steel box) using a mouth
aspirator. Mosquitoes were allowed a 3-min resting period in
the test chamber, after which the escape tunnel was opened to
begin the observation period. A receiving cage (23×23×23 cm
paper box) was connected to the exit portion for collecting
and all escaped mosquitoes were recorded at 1-min intervals
for a period of 30 min. After test completion, the number
of dead or knockdown mosquitoes in each treatment test
chamber and the escape-receiving cage were recorded. All
knockdown and live specimens were transferred separately to
clean holding cups and a cotton pad with 10% sugar solution
was provided for 24 h. Mortality was recorded at the end of
the period. All experimental treatments were replicated four
times.
Data analysis
Kaplan-Meier survival analysis was used to analyze and
interpret the behavioral response data. Survival analysis was
used to estimate the probability of escape time (ET) and to
compare differences in mosquito response between the three
oils (Kleinbaum 1995) based on mosquito escape patterns
at 1-min intervals. For purposes of analysis, mosquitoes
that escaped were treated as ‘deaths’ and those remaining in
the chambers as ‘survivals’ (Chareonviriyaphap et al.1997).
The escape time (ET) in minutes for 30% (ET30) and 50%
(ET50) was calculated using survival analysis and patterns of
escape response were determined using the log-rank method
(Mantel and Haenzel 1959). For those tests in which the
control mortality exceeded 5%, a Abbott’s correction factor
was applied to the data (Abbott 1925). Statistical significance
for all tests was set at P < 0.05.
RESULTS
Escape responses of Ae. aegypti that were exposed to
a single or a mixture essential oil from LC, LS, or ML were
defined in contact and non-contact trials. A commercial
DEET (15% w/w) was used as the standard repellent and
irritant. Knockdown of escaped and unescaped Ae. aegypti
females were observed during the exposure period (30
min). Overall, no knockdown specimen was recorded from
chambers treated with all treatments, except LC 0.175+LS
0.175 (Tables 1-4). Also, mortalities of Ae. aegypti females
after 24 h post-exposure were obtained.
The percentage and rate of escape response during
the 30 min exposure to contact and non-contact chambers
treated with LC, LS, ML, DEET, and from LC, LS, and ML
combinations in different concentrations are illustrated in
Figures 1-4. The rates represent the probability of escape
from a chamber with a given oil and concentration. Strong
escape patterns were observed with concentrations of 0.125%
LC and 0.15% LC in both contact and non-contact trials, in
comparison to other doses (Figure 1). Similarly, 0.125% LS
and 0.15% LS showed marked escaped responses in contact
and non-contact trials, respectively (Figure 2). With LC+LS,
0.075% LC + 0.075% LS showed strong escape patterns in
both contact and non-contact trials compared to other doses
(Figure 3). Other combination treatments and DEET also
showed strong escape patterns (> 30%) (Figure 4).
Low mortality (0-7.02%) was observed in all contact,
non-contact, and control trials. For contact trials of a single
oil, mortalities ranged between 0 and 5.1% in chambers
treated with 0.1% LS, 0.15% LS, 0.3% ML, 0.5% ML, and
DEET (Table 1). Mortality rates of <7% were observed in
non-contact trials in 0.1% LS, 0.3% ML, and DEET and
other experimental trials did not result in mortality (Table
2). In contact trials of oil mixture treatments, mortality rates
from escape and not escape specimens varied between 0 and
6.7% (Table 3). The percent mortality for non-contact trials
of combined treatments was 6.7% or less in escape and not
escape specimens (Table 4).
The escape patterns from chambers treated with test
compounds were defined as an escape time for 30% (ET30)
and 50% (ET50) of the test population to leave the treated
chambers in 19 min (Table 5). In contact trials, the greatest
ET50 was found at 0.075% LC+0.075% LS at 4 min, followed by
0.05% LC+0.05% LS at 6 min (Table 5). In non-contact trials,
ET30 and ET50 values for all three pure compounds and DEET
were over 3 min (Table 5). The number of escaped specimens
from LC and LS were low, with no ET50 values observed in any
concentration (Table 5). The LC+LS combinations at 0.075%
and 0.1% had ET50 values of 7 and 28 min, respectively. The
mixture of essential oils combined from two plants, LC, LS
and ML, showed ET50 values from 9 to 29 min (Table 5).
However, the results of this study showed that combined
essential oils in each concentration typically had higher
effectiveness than pure essential oils alone, with the greatest
efficacy at 0.075% LC+0.075% LS in contact and non-contact
trials. Most of the mixture oils increased the percentage of
repellent and irritant behaviors more than pure oils in both
contact and non-contact trials.
Multiple log-rank comparisons were made in contact,
non-contact, and control trials for the three compounds by
concentration. Significant differences in escape patterns were
observed in comparison with contact vs non-contact trials (P
< 0.05) for 0.05% LC, 0.05% LS, 0.5% ML, and 0.025% LC+LS.
Statistical differences in escape responses were found in all
300 Journal of Vector Ecology December 2014

Table 1. Mortality of Ae. aegypti females after a 24-h holding period following exposure in
excito-repellency contact trials of pure compounds.
Number % Knockdown % Mortality3
Treat1 Dose (%)2
Tested Esc (%) Esc Not esc Esc Not esc
LC 0.025 56 3 (5.4) 0 0 0 0
0.05 58 10 (17.2) 0 0 0 0
0.075 60 12(20.0) 0 0 0 0
0.1 59 13 (22.0) 0 0 0 0
0.125 60 30 (50.0) 0 0 0 0
0.15 58 30 (51.7) 0 0 0 0
LS 0.025 58 5 (8.6) 0 0 0 0
0.05 59 17 (28.8) 0 0 0 0
0.075 59 19 (32.2) 0 0 0 0
0.1 58 17 (29.3) 0 0 3.5 5.1
0.125 60 25 (41.7) 0 0 0 0
0.15 60 26 (43.3) 0 0 0 2.3
ML 0.3 60 10 (16.7) 0 0 1.7 0
0.5 60 17 (28.3) 0 0 0 1.7
15% DEET 60 26 (43.3) 0 0 1.7 1.7

1
LC: Litsea cubeba; LS: Litsea salicifolia, ML: Melaleuca leucadendron, DEET: a commercial
DEET 15% w/w. 2Essential oil dosages: 0.025%, 0.05%, 0.075%, 0.1%, 0.125%, 0.15%, 0.3%, 0.5%.
3
Mortalities based on number dead following 24-h holding period after specimens were removed
from exposure chamber or females escaped from exposure chambers. Esc: escape mosquitoes,
Not esc: non-escaped mosquitoes.

Table 2. Mortality of Ae. aegypti females after a 24-h holding period following exposure in excito-
repellency non-contact trials of pure compounds.
Number % Knockdown % Mortality3
Treat 1 Dose (%)2
Tested Esc (%) Esc Not esc Esc Not esc
LC 0.025 60 2 (3.3) 0 0 0 0
0.05 57 3 (5.3) 0 0 0 0
0.075 60 12 (20.0) 0 0 0 0
0.1 60 14 (23.3) 0 0 0 0
0.125 60 29 (48.3) 0 0 0 0
0.15 58 28 (48.3) 0 0 0 0
LS 0.025 60 7 (11.7) 0 0 0 0
0.05 55 7 (12.7) 0 0 0 0
0.075 59 12 (20.3) 0 0 0 0
0.1 57 15 (26.3) 0 0 3.51 7.02
0.125 60 20 (33.5) 0 0 0 0
0.15 58 27 (46.6) 0 0 0 0
ML 0.3 59 8 (13.6) 0 0 0 6.8
0.5 59 6 (10.2) 0 0 0 0

15% DEET 59 27 (45.8) 0 0 0 1.7

1
LC: Litsea cubeba; LS: Litsea salicifolia, ML: Melaleuca leucadendron, DEET: a commercial
DEET 15% w/w. 2Essential oil dosages: 0.025%, 0.05%, 0.075%, 0.1%, 0.125%, 0.15%, 0.3%, 0.5%.
3
Mortalities based on number dead following 24-h holding period after specimens were removed
from exposure chamber or females escaped from exposure chambers. Esc: escape mosquitoes,
Not esc: non-escaped mosquitoes.
Vol. 39, no. 2 Journal of Vector Ecology 301

Figure 1. Escape probability of Ae. aegypti females in contact (top) and non-contact (bottom) trials using six different
concentrations of Litsea cubeba essential oil extract.

Figure 2. Escape probability of Ae. aegypti females in contact (top) and non-contact (bottom) trials using five different
concentrations of Litsea salicifolia essential oil extract.
302 Journal of Vector Ecology December 2014

Figure 3. Escape probability of Ae. aegypti females in contact (top) and non-contact (bottom) trials using five different
concentrations of combination between Litsea cubeba and Litsea salicifolia essential oil extracts.

Figure 4. Escape probability of Ae. aegypti females in contact (top) and non-contact (bottom) trials using different concentrations
of DEET, Melaleuca leucadendron essential oil extract and various combinations.
Vol. 39, no. 2 Journal of Vector Ecology 303

Table 3. Mortality of Ae. aegypti females after a 24-h holding period following exposure
in excito-repellency contact trials of combined compounds.
Number % Knockdown % Mortality 2
Treat # Dose (% v/v) 1
Tested Esc (%) Esc Not esc Esc Not esc
LC 0.025 + LS 0.025 59 18 (30.5) 0 0 0 0
LC 0.05 + LS 0.05 58 33 (56.9) 0 0 0 0
LC 0.075 + LS 0.075 58 38 (65.5) 0 0 5.2 5.2
LC 0.1 + LS 0.1 60 32 (53.3) 0 0 3.3 1.7
LC 0.125 + LS 0.125 60 31 (51.7) 0 0 1.7 0
LC 0.15 + LS 0.15 59 30 (50.9) 0 0 0 0
LC 0.175 + LS 0.175 59 15 (25.4) 0 67.8 1.7 15.3
LC 0.1 + ML 0.3 59 29 (49.2) 0 0 0 2.3
LS 0.1 + ML 0.3 60 33 (55.0) 0 0 0 0
LC 0.1 + ML 0.5 60 26 (43.3) 0 0 5.0 2.2
LS 0.1 + ML 0.5 60 29 (48.3) 0 0 3.3 6.7
LC 0.05 + LS 0.05 + ML 0.3 60 23 (38.3) 0 0 5.0 5.0

1
LC: Litsea cubeba; LS: Litsea salicifolia, ML: Melaleuca leucadendron. 2Mortalities based on
number dead following 24-h holding period after specimens were removed from exposure
chamber or females escaped from exposure chambers. Esc: escape mosquitoes, Not esc:
non-escaped mosquitoes.

Table 4. Mortality of Ae. aegypti females after a 24-h holding period following exposure
in excito-repellency non-contact trials of combined compounds.

Number % Knockdown % Mortality2

Treat # Dose (% v/v)1
Tested Esc (%) Esc Not esc Esc Not esc

LC 0.025 + LS 0.025 58 7 (12.1) 0 0 0 0

LC 0.05 + LS 0.05 60 27 (45.0) 0 0 0 0

LC 0.075 + LS 0.075 59 37 (62.7) 0 0 3.4 3.4

LC 0.1 + LS 0.1 60 31 (51.7) 0 0 6.7 0

LC 0.125 + LS 0.125 59 25 (42.4) 0 0 0 0

LC 0.15 + LS 0.15 58 23 (39.7) 0 0 0 0

LC 0.175 + LS 0.175* 59 19 (32.2) 0 39.0 1.7 1.7

LC 0.1 + ML 0.3 58 29 (50.0) 0 0 0 4.44

LS 0.1 + ML 0.3 60 30 (50.0) 0 0 0 0

LC 0.1 + ML 0.5 59 31 (52.5) 0 0 5.1 1.7

LS 0.1 + ML 0.5 60 32 (53.3) 0 0 5.0 5.0

LC 0.05 + LS 0.05 + ML 0.3 60 21 (35.0) 0 0 5.0 3.3

1
LC: Litsea cubeba; LS: Litsea salicifolia, ML: Melaleuca leucadendron. 2Mortalities based on
number dead following 24-h holding period after specimens were removed from exposure
chamber or females escaped from exposure chambers. Esc: escape mosquitoes, Not esc: non-
escaped mosquitoes.
304 Journal of Vector Ecology December 2014

Table 5. Estimated time in min for 30% (ET30) and 50% (ET50) friendly have been become more popular. Essential oils from
of Ae. aegypti females to escape from exposure chambers in numerous plant species have been extensively tested to assess
excito-repellency of contact and non-contact trials of the oil their repellent properties (Noosidum et al. 2008, De Boer et
compounds. al. 2010, Sritabutra et al. 2011, Suwansirisilp et al. 2013).
In a previous study, essential oils extracted from ML,
Contact Non-contact LC, and LS had shown high irritant and repellent properties
Treat # Dose (% v/v)1
ET 30
2
ET50 ET302 ET50 against Ae. aegypti females (Noosidum et al. 2008). In this
study, the combinations of mixture oils demonstrated the
15% DEET 19 3
capability to repel Ae. aegypti, which is similar to Suwonkerd
0.125% LC 1 20 3 and Tantrarongroj (1994). They reported that repellent cream
0.15% LC 1 14 4 made from 2.5% citronella oil, 5% galingale oil, and 0.5%
vanillin prevented Anopheles minimus Theobald biting for
0.075% LS 26
6 h, while a cream containing 10% citronella oil prevented
0.125% LS 11 21 mosquito biting for only 2 h. Sritabutra et al. (2011) reveal that
0.15% LS 12 5
the mixtures of essential oils derived from 5% citronella oil +
5% eucalyptus oil, and 5% eucalyptus oil + 5% sweet basil oil
0.05% LC + 0.05% LS 2 6 5 also showed greater than 98 h of protection time against Ae.
0.075% LC + 0.075% LS 1 4 1 7 aegypti, compaired to 65-81 h of protection with 10% of pure
oils alone. Both formulations had an additive effect, however,
0.1% LC + 0.1% LS 1 16 1 28
some combinations had a reduction (antagonistic effect)
0.125% LC + 0.125% LS 1 9 1 in protection time when compared with pure compound.
0.15% LC + 0.15% LS 1 20 7
Similar to this study, the synergistic effect was also found in a
combination of 5% eucalyptus oil + 5% sweet basil oil which
0.1% LC + 0.3% ML 1 3 27 could provide 210 h of protection time for Anopheles dirus
0.1% LS + 0.3% ML 1 21 4 29 Peyton & Harrison, while pure compounds only had 95 and
45 h of protection time, respectively (Sritabutra et al. 2011).
0.1% LC + 0.5% ML 3 1 8
In summary, combine-extracted oils produced vigorous
0.1% LS + 0.5% ML 1 1 9 contact irritancy and non-contact repellency at low
0.05% LC + 0.05% LS + 0.3% ML 3 5 concentrations, suggesting that combinations of two essential
oils may serve as a more effective mosquito repellent than
1
LC: Litsea cubeba; LS: Litsea salicifolia, ML: Melaleuca each separate essential oil. However, combinations of different
leucadendron: DEET: a commercial DEET 15% w/w. oils will decrease or increase their efficacy depending upon
2
Survival analysis estimate time in min for 30 and 50% of test plant-oil species and concentration and will also give us more
populations to escape from exposure chambers. (-) indicates knowledge of alternative chemicals for mosquito control.
less than 30% of test specimens escaped from exposure Plant extracts and essential oils can be an effective alternative
chambers, therefore ET30 and ET50 estimates could not be to synthetic insecticides for vector control. Additional studies
calculated for 30 min exposure period. are recommended to determine the secondary metabolites
of effective plant extracts against mosquitoes to further
develop new and safe insecticides. Development of better
treatments when contact was compared to the controls (P < formulations, such as using a fixative component, could
0.05), except 0.025% LC, 0.075% LC, 0.025%-0.075% LS, 0.5% improve efficacy (Tawatsin et al. 2001, Tuetun et al. 2005, Kim
ML, and 0.025% LC+0.025% LS. Likewise, marked differences et al. 2012).
in escape responses were observed in paired non-contact and
control trials (P < 0.05) for several treatments, except 0.025% Acknowledgments
LC, 0.075% LC, and 0.025% LS.
This project was financially supported by the Thailand
DISCUSSION Research Fund (TRF Senior Research Scholar #RTA 4880006).
We are indebted to Associate Professor Atchariya Prabaripai,
In this study, the essential oils which demonstrated Department of Computer and Biostatistics, Kasetsart
repellent activities were tested against Ae. aegypti, a vector of University, for her valuable suggestions on the statistical
the dengue pathogen. The importance of the synergistic effects analysis. We also thank Miss Nutchanart Vareesomboon,
between essential oils from different plants (LC, LS, and ML) Mrs. Wilailak Tassanaphakdee, and Mrs. Prachup Ingsantae
was the main focus of this study. Although synthetic chemicals for their helpful advice and valuable assistance.
repellents are more frequently used than essential oils, natural
products have been shown to provide effective and relatively
safer repellency against insect vectors (Fradin and Day 2002,
Sritabutra et al. 2011). In general, the use of natural products
that possess reliable efficacy, safety, and are environmentally
Vol. 39, no. 2 Journal of Vector Ecology 305
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