Available online at www.sciencedirect.

com

Bioresource Technology 99 (2008) 2507–2515

Mosquito repellent activity of essential oils of aromatic plants

growing in Argentina
a,b a,* b
Y.G. Gillij , R.M. Gleiser , J.A. Zygadlo
a
Centro de Relevamiento y Evaluación de Recursos Agrı́colas y Naturales (CREAN), Facultad de Ciencias Agropecuarias,
Universidad Nacional de Córdoba, Av. Valparaı´so s.n, P.O. Box 509, Córdoba 5000, Argentina
b
Cátedra de Quı´mica Orgánica, Facultad de Ciencias Exactas, Fı´sicas y Naturales, Universidad Nacional de Córdoba, Av. Velez Sarsfield 1611,
Córdoba 5016, Argentina

Received 19 October 2006; received in revised form 26 April 2007; accepted 26 April 2007
Available online 20 June 2007

Abstract

Mosquitoes are important vectors of diseases and nuisance pests. Repellents minimize contact with mosquitoes. Repellents based on
essential oils (EO) are being developed as an alternative to DEET (N,N-diethyl-m-methylbenzamide), an effective compound that has
disadvantages including toxic reactions, and damage to plastic and synthetic fabric. This work evaluated the repellency against Aedes
aegypti of EO from aromatic plants that grow in Argentina: Acantholippia seriphioides, Achyrocline satureioides, Aloysia citriodora, Ane-
mia tomentosa, Baccharis spartioides, Chenopodium ambrosioides, Eucalyptus saligna, Hyptis mutabilis, Minthostachys mollis, Rosmarinus
officinalis, Tagetes minuta and Tagetes pusilla. Most EO were effective. Variations depending on geographic origin of the plant were
detected. At a 90% EO concentration, A. satureoides and T. pusilla were the least repellent. At concentrations of 12.5% B. spartioides,
R. officinalis and A. citriodora showed the longest repellency times. Comparisons of the principal components of each EO suggest that
limonene and camphor were the main components responsible for the repellent effects.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Essential oils; Mosquito; Culicidae; Aedes aegypti; Repellent

1. Introduction 2006. Mosquito bites may also cause allergic responses

Mosquitoes are arthropods of public health importance
because many species are vectors of diseases. Argentina is
under risk of dengue, due to the wide distribution of its vec-
tor Aedes aegypti (L.), traffic of people and vehicles from
neighbouring affected regions and adequate climatic condi-
tions (Avilés et al., 2003; Carbajo et al., 2001). Although
arboviral diseases are rare in Argentina, during 2005 an
outbreak of Saint Louis encephalitis with 47 laboratory-
confirmed cases occurred in Cordoba Province (Diaz
et al., 2006). There is concern over the spread of West Nile
virus, first recorded in horses (Morales et al., 2006) and
humans (ProMED-Mail, 2006) in Argentina early in
*
Corresponding author. Tel.: +54 351 4334105x202; fax: +54 351
4334118.
E-mail address: rgleiser@crean.agro.uncor.edu (R.M. Gleiser).
including local skin reactions and systemic reactions such
as urticaria and angioedema (Peng et al., 2004).
Personal protection is one approach to prevent mos-
quito bites (Gupta and Rutledge, 1994). Most commercial
repellents are based on DEET (N,N-diethyl-m-methylben-
zamide) (RED, 1998). This compound is effective against
a wide range of insects; however, it may exert toxic reac-
tions under some circumstances and age groups (Briassou-
lis, 2001; Clem et al., 1993), and may damage plastic,
synthetic fabric and painted surfaces, thus the interest in
exploring alternative new products.
Essential oils (EO) extracted from aromatic plants are a
potential source of pharmacologically active compounds
such as analgesics, anti-inflammatorics, antitumorals, anti-
biotics and digestives (Zygadlo and Juliani, 2000). Bioas-
says on a number of EO show repellence against
mosquitoes (Giménez et al., 1998; Park et al., 2005;

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.04.066
2508 Y.G. Gillij et al. / Bioresource Technology 99 (2008) 2507–2515
Trongtokit et al., 2005; Yang et al., 2004) usually attrib-
uted to its main compounds. However, synergistic phenom-
ena between the diverse components of the EO may result
in a higher bioactivity (an increased repellent response) of
the oil as a whole compared to its isolated components
(Hori, 2003). Thus, the fact that an EO contains specific
main components may be an indication of its potential
use, but does not warrant its use without confirmation of
activity. Also, composition may vary considerably between
aromatic plant species and varieties and between the same
varieties from different geographic areas (Zygadlo and Juli-
ani, 2003).
Repellent properties of several EO appear to be associ-
ated with the presence of monoterpenoids and sesquiter-
penes (Jaenson et al., 2006; Sukumar et al., 1991).
Monoterpenes such as a-pinene, limonene, terpinolene, cit-
ronellol, citronellal, camphor and thymol are common con-
stituents of a number of EO described in the literature as
presenting mosquito repellent activity (Ibrahim and Zaki,
1998; Jaenson et al., 2006; Moore, 1974; Park et al.,
2005; Yang et al., 2004). Among the sesquiterpenes,
b-caryophyllene is the most cited as a strong repellent
against Ae. aegypti (Jaenson et al., 2006; Trongtokit et al.,
2005). Essential oils of aromatic plants in this study have
been reported to contain a number of mono- or sesquiter-
penes mentioned above (CDC, 2005; Juliani et al., 2004;
Labuckas et al., 1999; Zygadlo and Juliani, 2003). This work
assessed the repellent potential against Ae. aegypti mosqui-
toes of EO extracted from Argentine aromatic plants.
2. Methods
2.1. Plant material
The following native Argentine plant species were stud-
ied (common name indicated in quotation marks): Achyro-
cline satureioides (Lam.) D.C. ‘‘marcela’’, Baccharis
spartioides (H. et al.) J. Rimy ‘‘carqueja’’ Tagetes minuta
L. ‘‘suico’’ or ‘‘Mexican marigold’’, Tagetes pusilla Kunth
‘‘lesser marigold’’ (Asteraceae), Hyptis mutabilis (Rich.)
Briq. ‘‘tropical bushmint’’, Minthostachys mollis (H.B.K.)
Griseb ‘‘peperina’’ (Lamiaceae), Anemia tomentosa (Sav-
igny) Sw. var. anthriscifolia (Schrad.) Mickel ‘‘Hairy Flow-
ering Fern’’ (Schizaeaceae), Acantholippia seriphioides (A.
Gray) Mold ‘‘tomillo’’ and Aloysia citriodora Palau
‘‘cedrón’’ or ‘‘lemon verbena’’ (Verbenaceae). The follow-
ing exotic species that grow extensively in Argentina were
also included: Chenopodium ambrosioides L. ‘‘Paico’’ or
‘‘wormseed’’ (Chenopodiaceae), Rosmarinus officinalis L.
‘‘rosemary’’ (Lamiaceae) and Eucalyptus saligna Sm. ‘‘blue
gum’’ (Myrtaceae). These plants were selected because
some of the components of their EO were either previously
reported as potential insect repellents or were chemically
similar to other components with repellent properties. A
herborized specimen of each plant was deposited in the
Botanical Museum CORD, Universidad Nacional de
Córdoba.
2.2. Essential oil extraction and analysis
Essential oils were extracted by hydro-distillation in a
Clevenger type apparatus for 2 h with a separated extrac-
tion chamber. The resulting EO were dried over anhy-
drous-sodium sulphate to extract the oil, and analyzed
with gas–liquid chromatography and mass spectrometry,
on a Perkin Elmer Q-700 equipment. A polar column of
30 m · 0.25 mm · 0.25 lm (CBwax) and an apolar column
of 30 m · 0.25 mm · 0.25 lm (DB-5) were successively
used. Both columns were held for 3 min at 60 C, then pro-
grammed at 4 C/min to 240 C. The injector was held at
250 C. Helium was used as the carrier gas at a 0.9 ml/
min caudal. The mass spectrum was obtained at a 70 eV
ionization voltage. The identification of components was
done with the use of the NIST (version 3.0) data base, bib-
liography (Adams, 1969) and authentic standards. The
concentration of each EO component was calculated from
the integration area of the chromatographer.
For repellence assays the whole EO extract diluted in
ethanol was tested.
2.3. Mosquitoes
Ae. aegypti was selected for the repellence assays
because it is an important dengue and yellow fever vector
(Carbajo et al., 2001; Avilés et al., 2003), easy to colonize
in the laboratory and an anthropophilic species that prefers
to bite during the day. Mosquitoes were reared in the lab-
oratory using methods described by Gerber et al. (1994)
and Dominguez et al. (2000). Egg hatching was induced
with dechlorinated water. Larvae were reared in open plas-
tic containers (1000 ml) and fed with lyophilized liver
(0.25 mg/larvae/day). Adult mosquitoes were maintained
in entomological cages (30 · 40 · 30 cm) and fed a sucrose
solution (10% in water).
2.4. Test procedure
Repellence of each EO was tested by means of an arm-
in-cage assay, in which subjects insert their product-treated
arm into a cage with a fixed number of blood unfed mos-
quitoes and the time elapsed to the first bite was recorded
(Fradin and Day, 2002). The forearm and hand of a human
subject were washed with unscented neutral soap, thor-
oughly rinsed and allowed to dry 10 min before product
application. The hand wore a latex glove to protect it from
mosquito bites. The forearm was protected with a paper
sleeve leaving a 30 mm circle of exposed skin, where 60 ll
of the product was applied. One arm was treated with
90% EO (dose of 7.64 ll/mm2 of skin) and the other
with ethanol as a control. Batches of 25 female mosquitoes,
3–10 days old, fed on sucrose solution, were housed in
30 · 22 · 22 cm cages. Each cage had a cotton stockinet
access sleeve on the front, clear glass top and back (to facil-
itate viewing), an aluminium sheet bottom and screen on
 Y.G. Gillij et al. / Bioresource Technology 99 (2008) 2507–2515
remaining sides. The testing environment was maintained
at 27 C and 60% RH. The authors were the test subjects.
Tests were conducted by placing the repellent-treated
forearm into a test cage for 5 min, 10 min after EO applica-
tion, and then every 30 min until the test subject received a
mosquito bite. Protection time was recorded as the time
elapsed between repellent application and the observation
period immediately preceding that in which a confirmed
bite was obtained. If no bites were confirmed at 90 min,
tests were discontinued and protection time was recorded
as 90 min. An attempt of the mosquito to insert its stylets
was considered a bite. A control arm was placed in the cage
randomly before or after the treated arm to assess predis-
position of mosquitoes to bite. In the rare case when no
mosquito attempted to bite the control arm during the
observation period, that trial was discarded and the test
was repeated with a new batch of mosquitoes, to ensure
that lack of bites was due to repellence and not to mosqui-
toes not being predisposed to get a blood meal at the time.
Each product was tested once on three subjects, using a
completely randomized design. Each subject tested one
treatment a day.
Those products showing higher repellence were tested
again at decreasing concentrations of 50%, 25% and
12.5% (doses of 4.25, 2.12 and 1.06 ll/mm2 of skin, respec-
tively) using 50 mosquitoes per cage. Again, each concen-
tration was tested once on three subjects.
2.5. Statistical analysis
One-way analysis of variance followed by Fisher LSD
mean comparison a posteriori test was used to compare
the mean repellence time (time to first bite) for the 14
EOs at 90% concentration. Data on repellence times at
lower concentrations of the EO were analyzed by two-
way ANOVA that assessed the effects of EO (A. seriphio-
ides, A. citriodora, B. spartioides, E. saligna, M. mollis,
R. officinalis and T. minuta), the concentration of each
EO (50%, 25% and 12.5%) and their interaction. Previous
to this analysis, data had to be transformed to ranks as
described by Shirley (1987), to better reach the ANOVA
assumptions. When significant effects of the factors evalu-
ated were detected, data were analyzed a posteriori by
Fisher LSD mean comparison test. A value of p 6 0.05
was considered statistically significant.
3. Results
Essential oils were extracted from 12 aromatic plant spe-
cies that grow in Argentina. The main components of each
EO are shown in Table 1. As expected, in most plants com-
ponents that had previously indicated repellent activity
were detected, such as limonene, geranial and p-cymene.
Populations of A. citriodora and C. ambrosioides from dif-
ferent geographic regions were analyzed. A. citriodora vari-
ations were mainly quantitative, with higher levels of
limonene in Traslasierra and higher levels of neral in Cór-
2509
doba. The EO composition of C. ambrosioides differed
qualitatively between the two regions. Over 99% of the
EO of C. ambrosioides from Córdoba was ascaridol, while
the major components of the EO from Tucumán included
trans-pinocarveol, trans-pinocarvyl acetate, and cis-
carveol.
Assays at 90% concentration indicated that more than
70% of the EOs repelled mosquitoes for 60 min or longer
after application (Table 2). A one-way ANOVA showed
a significant effect of the factor EO (F(13, 28) = 6.44;
p < 0.01). Mean comparisons a posteriori showed that
repellence times of A. satureioides (3.33 min) and T. pusilla
(6.67 min) were significantly shorter than the other EO,
except H. mutabilis. H. mutabilis and A. citriodora from
Córdoba presented intermediate repellence times (20 and
50 min, respectively) and significantly shorter times than
the EO of A. seriphioides, A. citriodora (from Traslasierra),
B. spartioides, E. saligna, M. mollis, R. officinalis and
T. minuta. In every case the positive predisposition of mos-
quitoes to bite was verified in a control arm (treated with
ethanol).
Essential oils of A. seriphioides, A. citriodora (from
Traslasierra), B. spartioides, E. saligna, M. mollis, R. offici-
nalis and T. minuta were selected to test repellence at lower
concentrations, because they presented maximum times to
first bite (90 min) in the assays at 90% concentration (Table
2). Table 3 illustrates the mean repellence time of EO at
decreasing concentrations (50%, 25% and 12.5%). The
two-way ANOVA showed a significant effect of the EO
(F(6, 42) = 4.08; p < 0.003) and the concentration of each
EO (F(2, 42) = 4.14, p = 0.02), but not of their interaction
(p = 0.07). A Fisher LSD test indicated that E. saligna
repellence time was significantly shorter than A. citriodora
(Traslasierra), B. spartioides and R. officinalis. Mean com-
parison analysis a posteriori evaluating the effect of EO
concentration showed that lower concentrations of the
EO resulted in shorter repellence times.
Considering that the interaction between the EO factor
and the concentration factor showed a probability value
(p = 0.07) very close to the statistical significance criteria
(p < 0.05), a mean comparison analysis a posteriori of the
interaction was carried out (Table 3). This analysis indi-
cated that repellence times of E. saligna EO at 12.5% and
25% were significantly shorter than repellence times of
A. citriodora (Traslasierra), B. spartioides and R. officinalis
at 12.5%. No statistically significant differences were
detected between EO at 50% concentrations.
4. Discussion
This study evaluated repellence of 14 EO against Ae.
aegypti mosquitoes. Repellence tests with EO at 90% con-
centration indicated that most were effectively repelling
mosquitoes for 90 min. The EO of A. seriphioides was
repellent even at the lowest concentration tested (12.5%).
Repellence by this EO was expected because its main
components are p-cymene (53%) and thymol (47%). Both
Table 1

2510
Components of the essential oils assayed, expressed as percent of total volume
Component of the essential oils Plant species
A. se. A. sa. A. ci. (C) A. ci. (TS) A. to. B. sp. C. am. (C) C. am. (T) E. sa. H. mu. M. mo. R. of. T. mi. T. pu.
Hydrocarbonated monoterpenes
Camphene 0.4 0.5 0.1 0.3 1.0 tr
D-3-Carene 0.2
2-Carene 0.7
p-Cymene 52.8 0.2 tr 0.1 0.2 0.6 <0.1
Limonene 0.6 7.0 19.7 4.3 10.1 tr 66.3
Myrcene tr 0.3 1.9 0.1 1.1 1.1 0.5 tr
(Z)b-Ocimene 0.4 tr 0.5
(E)b-Ocimene 0.2
a-Pinene 0.5 1.1 tr 0.5 5.2 1.0 tr 11.8
b-Pinene 0.1 0.6 2.1 1.5 tr

Y.G. Gillij et al. / Bioresource Technology 99 (2008) 2507–2515

Sabinene 0.3 0.1 0.6 1.1 0.6
c-Terpinene 0.1 0.4 tr 0.8 0.9 0.1
a-Thujene 0.7 0.1 5.5 tr
Oxygenated monoterpenes
1,8-Cineole 0.6 0.2 93.2 5.7 0.1
Anethol 74.5
Ascaridol 99.4
Borneol 0.2 1.2 0.1
Bornyl acetate 0.2
Camphenilone 0.6 tr
Camphor tr 5.2 50.5 1.4 33.6
Carvacrol tr 1.2
cis-Carveol 0.2 0.1 10.6
Carvone 0.8 0.2 5.3 0.1
Estragol 23.7
Geranial 22.7 21.0 6.9
Linalool 0.5 0.2 0.1 <0.1 4.1
Menthone tr tr 30.7
Myrtenol 0.3
Neral 19.4 14.0 4.9 0.8 0.2
(Z)-Ocimenone 2.7
(E)-Ocimenone 0.5 0.3 19.1
trans-Pinocarveol 0.5 42.4
Pinocarvone 0.4 0.3
trans-Pinocarvyl acetate 22.4
Pulegone 0.8 51.2
cis-Sabinol 4.6
trans-Sabinene hydrate 0.6
Terpinen-4ol 0.1 1.3 0.4 tr 8.4
a-Terpineol 0.8 0.4 tr 1.3 8.2
a-Thujone 14.2 0.3
Thymol 46.8 1.0
Verbenone 24.9
cis-Verbenyl acetate 0.1 9.1
Hydrocarbonated sesquiterpenes
Aromadendrene 1.6 0.4
Allo-aromadendrene 0.3
Bicyclogermacrene 4.1 8.0 2.3
b-Bisabolene 1.5
b-Bourbonene 0.8 0.8 0.4 2.7 66.3
c-Cadinee 2.9 2.5
d-Cadinene 9.4 0.2 tr 0.4 2.5
a-Calacorene 2.1
Calamenene 1.5 11.8
(E)-Caryophyllene tr 39.7 0.4 0.3 0.1 3.5 14.7 0.1 14.8
a-Cedrene 3.0 2.8
a-Copaene 14.2 0.7 0.7
a-Cubebene 0.3 0.4
b-Cubebene 0.3 0.1 0.2

Y.G. Gillij et al. / Bioresource Technology 99 (2008) 2507–2515

b-Curcumene tr 0.9 0.5 0.1
b-Elemene 0.1
c-Elemene 0.9
D-Elemene 5.0
Farnesyl acetate 1.1
Germacrene B 9.5
Germacrene D 3.3 6.9 0.8 11.1 0.4
(Z)-b-Guaiene 0.4
a-Gurjunene 4.0
b-Gurjunene 2.6 2.0
a-Humulene 2.4 0.6 0.7 0.2 1.5 0.2 2.7 1.4
a-Muurolene 0.7 3.3
c-Muurolene 0.9
b-Selinene 1.1
Viridiflorene 1.1
a-Ylangene 2.9
a-Zingiberene 0.4 0.4
Oxygenated sesquiterpenes
T-Muurolol 2.6
a-Muurulol 3.2
14-Hydroxy-9-epi-(E)-caryophyllene 1.4
a-Bisabolol 51.4
b-Bisabolol 1.1
a-Bisabol oxido 1.1
Epi-a-Cadinol 0.8
T-Cadinol 1.4
a-Cadinol 1.5
Caryophyllene oxide 2.1 0.9 7.7 3.2 1.6
Cubebol tr 1.4
a-Eudesmol 1.2
b-Eudesmol 0.1 0.6 0.4
Germacrene D-4-ol 0.7
Globulol 0.8 0.3
Guaiol 0.3 0.2
Ledol 0.9

2511
(continued on next page)
2512 Y.G. Gillij et al. / Bioresource Technology 99 (2008) 2507–2515

Table 2

T. pu.

satureioides; A. ci., Aloysia citriodora; A. to., Anemia tomentosa; B. sp., Baccharis spartioides; C. am., Chenopodium ambrosioides; E. sa., Eucalyptus saligna; H. mu., Hyptis mutabilis; M. mo.,
The principal components (>10%) of each essential oil are highlighted in bold font. tr, trace; (C), Córdoba; (T), Tucumán; (TS), Traslasierra; A. se., Acantholippia seriphioides; A. sa., Achyrocline
Mean repellence times (min) against Aedes aegypti of essential oils
extracted from Argentine aromatic plants at 90% concentration
Essential oil Repellence times*
T. mi.
a
Achyrocline satureioides 3.3 ± 3
Tagetes pusillaa 6.7 ± 3
Hyptis mutabilisa,b 20.0 ± 10
R. of.

Aloysia citriodora (C)b,c 50.0 ± 10

Anemia tomentosac,d 60.0 ± 30
Chenopodium ambrosioides (C)c,d 60.0 ± 17
Chenopodium ambrosioides (T)c,d 60.0 ± 30
M. mo.

Acantholippia seriphioidesd 90.0 ± 0

1.1

Aloysia citriodora (TS)d 90.0 ± 0

Baccharis spartioidesd 90.0 ± 0
Eucalyptus salignad 90.0 ± 0
H. mu.

Minthostachys mollisd 90.0 ± 0

14.2

Rosmarinus officinalisd 90.0 ± 0

Tagetes minutad 90.0 ± 0
E. sa.

* Average ± standard error time to first bite. C: Córdoba; T: Tucumán;

TS: Traslasierra. Different super-indexes indicate statistical differences in

repellence (average times to first bite) between the essential oils (P < 0.05
ANOVA and Fisher).
C. am. (T)

Table 3
Mean repellence times (min) against Aedes aegypti of decreasing concen-
trations of essential oils extracted from Argentine aromatic plants
C. am. (C)

Essential oil Essential oil concentration*

50% 25% 12.50%
Aloysia citriodora (TS)A 70 ± 20a 90 ± 0a 90 ± 0a
Baccharis spartioidesA 90 ± 0a 90 ± 0a 90 ± 0a
B. sp.

Rosmarinus officinalisA,B,C 90 ± 0a 80 ± 10a 60 ± 17a,b

0.9

2.1

Mintostachys mollis; R. of., Rosmarinus officinalis; T. mi., Tagetes minuta; T. pu., Tagetes pusilla.

Acantholippia seriphioidesB,D 70 ± 10a 43 ± 24a,b,c 53 ± 23a,b,c

Mintostachys mollisB,D 60 ± 17a,b 50 ± 20a,b,c 40 ± 10a,b,c
A. to.

Tagetes minutaC,D 63 ± 27a,b 90 ± 0a 30 ± 30b,c

0.9

0.4
1.0

0.5

Eucalyptus salignaD 90 ± 0a 17 ± 7c 20 ± 20c

* Average ± standard error time to first bite (repellence) at decreasing
A. ci. (TS)

concentrations of the EO. Different super-indexes indicate statistical dif-

ferences in repellence between the essential oils (Fisher LSD test). TS:
Traslasierra.
0.5
1.0

9.9
1.1

tr
A. ci. (C)

components showed repellent activity for approximately

1 h against another mosquito species, Culex pipiens pallens
0.4

0.4
0.5

0.9

L. (Choi et al., 2002). Also, the essential oil of A. seriphio-

ides from Andino Cuyana and Andino Patagónica regions
A. sa.

containing thymol (29.2%) were toxic against Varroa

Plant species

destructor Anderson and Trueman (Acari: Varroidae) and

Apis mellifera L. (Hymenoptera: Apidae) (Ruffinengo
A. se.

et al., 2005). Thymol was highly toxic against Musca

domestica L. (Diptera: Muscidae) (Lee et al., 1997).
Essential oils extracted from multiple species of Tagetes,
Component of the essential oils

such as T. patula L., T. erecta L. and T. minuta, have

2,2-Dimethyl-3,4-octadienal

shown nematicidal, fungicidal and insecticidal activity

6-Methyl-5-hepten-2-one

(Vasudevan et al., 1997). In this study we evaluated the

(E)-Sesquilavandulol

repellent properties of EO extracted from two species,

Table 1 (continued)

T. minuta and T. pusilla. T. pusilla at 90% was not repellent

(E)-Nerolidol

for Ae. aegypti. This EO is composed mainly of cis-ane-

Spathulenol
Viridiflorol

thole (75%) and estragole (24%). Even though anethole is

Aldehyde
Santalol

Cetone

sometimes an ingredient of commercial repellents, it is used

to improve the aroma of the products and not for its repel-
 Y.G. Gillij et al. / Bioresource Technology 99 (2008) 2507–2515
lent properties. On the other hand, application of T. minuta
at 90% concentration deterred Ae. aegypti from biting, con-
sistently with previous reports indicating a moderate repel-
lence of T. minuta against mosquitoes (Moore and Lenglet,
2004). The composition of the EO of T. minuta is variable
depending on strain and geographic origin (Chisowa et al.,
1998; Craveiros et al., 1988; Gil et al., 2000; Hethelyi et al.,
1987), and its properties may change depending on the
chemotype. Extracts of this plant from Andino Cuyana
and Andino Patagónica regions, whose main component
was b-ocimene (62.8%), were toxic against V. destructor
(Ruffinengo et al., 2005). The samples from Córdoba pre-
sented limonene (66%) and (E) ocimenone (19%). T. min-
uta, as well as other plants assayed that contained
limonene in their EO, A. citriodora (Traslasierra), M. mollis
and B. spartioides (20%, 10% and 4%, respectively),
resulted in maximum repellency times (90 min) (Table 2).
However, considering that these EO also included other
components with potential repellent effects, synergistic phe-
nomena cannot be discarded. Moreover, limonene may
explain the differences in repellence time between A. citrio-
dora from Córdoba and Traslasierra, because its concen-
tration was higher in the EO from Traslasierra, with a
significantly longer repellency time (90 min) than the EO
from Córdoba (50 min), reinforcing the proposal that lim-
onene is an efficient repellent against Ae. aegypti.
The main components of the M. mollis EO samples were
pulegone (51.2%), mentone (30.7%) and limonene (10.1%)
(Table 1). Our results indicate repellence against Ae. aegypti
even at the lowest concentrations tested (Table 3). Coinci-
dentally, hydro-destillation extracts of M. mollis from
Andino Cuyana and Andino Patagónica regions, with a sim-
ilar EO composition (pulegone 52.6%, mentone 35.8% and
limonene 2.6%), were repellent against V. destructor (Ruffin-
engo et al., 2005). However, its repellence was low against
Pediculus humanus capitis L. (Anoplura: Pediculidae), sug-
gesting that the potential of this plant as a repellent varies
in different hematophagous groups (Toloza et al., 2006).
R. officinalis was effective in terms of repellence time
against Ae. aegypti in concentrations as low as 12.5%.
These results are consistent with reports of insect repellent
properties of rosemary, including Cx. pipiens pallens (Choi
et al., 2002) and Myzus persicae (Homoptera: Aphididae)
(Hori, 1998, 1999). Niticidal activity against P. humanus
capitis (Lahlou and Berrenda, 2003) and insecticidal activ-
ity against Ceratitis capitata (Wiedemann) (Diptera: Teph-
ritidae) (Moretti et al., 1998; Passino et al., 1999) was also
reported. The main components of the EO of R. officinalis
included camphor (34%), verbenone (25%) and (E)-caryo-
phyllene (15%). Camphor in R. officinalis (33.6%) and in
B. spartioides (50.5%) may be responsible for repellence
of these plants. This component is commonly used to repel
storage products pests such as moths and weevils (Spurr
and McGregor, 2003). As a principal component of EO
of aromatic plants it has shown repellence against Anophe-
les culicifacies Giles, Cx. quinquefasciatus Say (Ansari and
Razdan, 1995), Anopheles gambiae Giles s.l. and Anopheles
2513
funestus Giles (Seyoum et al., 2002) and by itself against
An. funestus (Seyoum et al., 2003).
Some eucalyptus species have been evaluated for their
potential as mosquito repellents, principally Corymbia
citriodora Hook (also known as Eucalyptus maculata citri-
odora) (Seyoum et al., 2002; Trigg and Hill, 1996). The
repellent properties of E. saligna may be attributed to
1,8-cineole, because it represented 93.2% of its EO. Of
the EO evaluated at concentrations below 90% (Table 3),
it was the only one to significantly decrease in terms of
repellence time at concentrations below 50%. Accordingly,
a study using 1,8-cineole showed moderate effects as a feed-
ing and oviposition deterrent of Ae. aegypti (Wantanabe
et al., 1993).
To our best knowledge, this is the first report of the
potential of C. ambrosioides as a repellent against Ae.
aegypti. The composition of the EO of this plant varied
regionally, as reflected by the composition of the samples
from Córdoba and Tucumán (Table 1). In the sample from
Córdoba the principal component was ascaridole (99.4%),
absent from the EO from Tucumán. The later contained
trans-pinocarveol (42%), trans-pinocarvyl acetate (22%),
and cis-carveol (11%). However, repellence times for both
EO were similar (60 min). These results suggest, on one
hand, that ascaridole may be repellent against mosquitoes.
On the other hand, repellence of the Tucumán sample may
be due to a combined action of its components. A likely
candidate is cis-carveol, because repellence of this compo-
nent against An. gambiae has been reported (Omolo
et al., 2004).
Among ferns there are several species with medicinal
properties, however, repellence of this group of plants
against haematophagous insects has been scarcely
explored. Our results suggest that A. tomentosa may have
potential as a mosquito repellent because biting was pre-
vented for 60 min. Even though its EO contains some com-
ponents for which repellence has been reported, such as
geranial and caryophyllene oxide (Omolo et al., 2004), their
percentages are very low. Also, A. tomentosa has a low con-
tent of neral that has interestingly been described both as
an attractant and a repellent against acari, depending on
the dose (Nishimura et al., 2002). The main component
of this oil is a-bisabolol, for which effects on insects are
not known, and consequently it would be interesting to fur-
ther explore its potential as a repellent or insecticide
against mosquitoes.
Repellence time of H. mutabilis at 90% was significantly
low (20 min). A bibliographic search indicated that compo-
nents showing some repellence against mosquitoes (Yang
et al., 2004) had been isolated from this plant, such as myr-
cene, cis-dehydrocarvone, trans-dehydrocarvone and cam-
phor (Velasco-Negueruela et al., 1995). However, the EO
in our study did not contain these components and it had
a very low percentage of camphor (<2%). Its main compo-
nents were (E)-caryophyllene (14.7%), spathulenol (14.2%)
and D-germacrene (11%). Spathulenol isolated from
Callicarpa presented repellence against Ae. aegypti and
2514 Y.G. Gillij et al. / Bioresource Technology 99 (2008) 2507–2515
Anopheles stephensi (Cantrell et al., 2005). (E)-caryophyl-
lene was also one of the principal components of the EO
of A. satureioides (39.7%) (Table 1). Interestingly, both
the EO of A. satureioides and H. mutabilis showed no or
a low repellence (Table 2). Even though caryophyllene
has been reported as a repellent of mosquitoes and other
insects (Jaenson et al., 2006), it was not effective against
Ae. aegypti, at least at the concentrations applied in the
current work. On the other hand, R. officinalis, with an
(E)-caryophyllene content similar to H. mutabilis, was
repellent, although this property could be attributed to
other EO components such as camphor.
In conclusion, 70% of the aromatic plants that were
tested showed potential as Ae. aegypti repellents because
their application deterred biting for at least 60 min. EOs
from B. spartioides, R. officinalis and A. citriodora (Trasl-
asierra) were the most promising considering their repel-
lence times at low concentrations. Results suggest that
limonene and camphor are the main components responsi-
ble of the repellent effect, because they were important
components of the EO that resulted in longer repellence
times. It would be interesting to determine the lowest con-
centration of these EO at which they are still effective as
repellents, and to study the effects of combining compo-
nents to identify potential synergisms and antagonisms.
Acknowledgements
We thank partial funding from Fundación Mundo
Sano. The study was reviewed and approved by a scientific
advisory board of Universidad Nacional de Córdoba.
RMG and JAZ are Career Members of Consejo Nacional
de Investigaciones Cientı́ficas y Técnicas (CONICET).
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