Chemical Composition and Acaricidal Properties of Deverra

scoparia Essential Oil (Araliales: Apiaceae) and Blends of

Its Major Constituents Against Tetranychus urticae (Acari:
Tetranychidae)
Author(s): S. Attia , K. L. Grissa , G. Lognay , S. Heuskin , A. C. Mailleux , and T.
Hance
Source: Journal of Economic Entomology, 104(4):1220-1228. 2011.
Published By: Entomological Society of America
DOI: http://dx.doi.org/10.1603/EC10318
URL: http://www.bioone.org/doi/full/10.1603/EC10318

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 ECOTOXICOLOGY

Chemical Composition and Acaricidal Properties of Deverra scoparia

Essential Oil (Araliales: Apiaceae) and Blends of Its Major
Constituents Against Tetranychus urticae (Acari: Tetranychidae)
S. ATTIA,1,2 K. L. GRISSA,3 G. LOGNAY,4 S. HEUSKIN,4 A. C. MAILLEUX,1 AND T. HANCE1

J. Econ. Entomol. 104(4): 1220Ð1228 (2011); DOI: 10.1603/EC10318

ABSTRACT The essential oil of Deverra scoparia Coss. & Durieu was investigated for its acaricidal
activity against the worldwide pest twospotted spider mite, Tetranychus urticae Koch (Acari: Tet-
ranychidae). The essential oil was analyzed by fast gas chromatography (GC) and GC-mass spec-
trometry. The activities of its individual and blended constituents were determined. Our study showed
that female mortality increased with increasing D. scoparia oil concentrations, with LD50 and LD90
values at 1.79 and 3.2 mg liter⫺1, respectively. A reduction in fecundity had already been observed
for concentrations of 0.064, 0.08, and 0.26 mg liter⫺1 D. scoparia essential oil. Ten major components,
comprising 98.52% of the total weight, were identiÞed; ␣-pinene was the most abundant constituent
(31.95%) followed by sabinene (17.24%) and ⌬3-carene (16.85%). The 10 major constituents of D.
scoparia oil were individually tested against T. urticae females. The most potent toxicity was found with
␣-pinene, ⌬3-carene, and terpinen-4-ol. The presence of all constituents together in the artiÞcial
mixture caused a signiÞcant decrease in the number of eggs laid by females, at 0.26 mg liter⫺1 (11 eggs),
compared with the control (50 eggs). The toxicity of blends of selected constituents indicated that
the presence of all constituents was necessary to reproduce the toxicity level of the natural oil.

KEY WORDS essential oil, Deverra scoparia, Tetranychus urticae, toxicity, fecundity

Twospotted spider mite, Tetranychus urticae Koch
(Acari: Tetranychidae), is a well-known mite pest
worldwide, with a wide range of host plants. It causes
serious damage in outdoor crops and greenhouses
(Hazan et al. 1974, Goff 1986, Lee et al. 1994, Ho et al.
1997). Synthetic acaricides have been widely used for
the control of this pest in greenhouses, orchards and
many other cropping systems, a practice that has led
to the development of resistance (Sundaram et al.
1995, Van Leeuwen et al. 2006). Indeed, ⬎550 species
of insects and mites have developed resistance to one
or more classes of synthetic biocides (Van Leeuwen et
al. 2009). Consequently, the number of effective
chemicals for the control of crop pests and disease
vectors is rapidly decreasing (Georghiou 1990). More-
over, some chemical products are known for their
negative effects on human health and the environ-
ment (Sathiyamoorthy et al. 1997, Cavalcanti et al.
2010). These concerns have provided the basis for the
recent interest in bioactive acaricidal compounds
from higher plants. Among other bioactive natural
1 Earth and Life Institute, Biodiversity Research Centre, Université
Catholique de Louvain, 4-5, Place Croix du sud, B-1348 Louvain-la-
Neuve, Belgium.
2 Corresponding author, e-mail: sabine_bio5@yahoo.fr.
3 Laboratoire dÕEntomologie-acarologie, Institut National Agro-
nomique de Tunisie, 1082 Cité Mahrajène, Tunis, Tunisia.
4 Université de Liège Gembloux Agro-Bio Tech Unité de Chimie
analytique. Passage de Déportés, 2, B-5030 Gembloux, Belgium.
compounds, several plant essential oils have been
demonstrated as potential alternative compounds for
mite control (Choi et al. 2004, Floris et al. 2004, Calm-
asur et al. 2006, Pontes et al. 2007, Cavalcanti et al.
2010, Chermenskaya et al. 2010, Han et al. 2010, Neves
et al. 2011). Essential oils have a short residual activity
and are less persistent than conventional pesticides
because they tend to be volatile and are susceptible to
temperature and UV light degradation (Miresmailli et
al. 2006, Cloyd et al. 2009). Consequently, most of
these oils are environmentally nonpersistent and rel-
atively nontoxic to warm-blooded animals and humans
(Crowell et al. 1992, Kubo et al. 1994), with some
exceptions (Roe 1965, Hjorther et al. 1997).
Essential oils are secondary metabolites that are
frequently concentrated in the leaves, bark, or fruit of
aromatic plants. The oils are generally obtained by
steam distillation and are composed of a complex mix-
ture of monoterpenes, phenols, and sesquiterpenes
(Isman 2000). These constituents are also of interest
to industrial markets, especially because of their tox-
icity to the numerous species of insects that have
developed resistance to synthetic biocides (Bekele
and Hassanali 2001). Many plants, including garlic
(Allium sativum L.), rosemary (Rosmarinus officinalis
L.), cinnamon (Cinnamomum verum J.Presl), citrus
(Citrus spp.), and cedar (Cedrus spp.), have been used
to control a variety of insects (Isman 2004). In this
context, Deverra scoparia Coss. & Durieu, an endemic

0022-0493/11/1220Ð1228$04.00/0 䉷 2011 Entomological Society of America

August 2011 ATTIA ET AL.: ACARICIDAL PROPERTIES OF D. scoparia AGAINSt T. urticae
plant of North Africa, is locally known for its phar-
maceutical activity (Ben Haj Jilani et al. 2007). Indeed,
Ghrabi-Grammar et al. (2009) investigated the acar-
icidal effect of D. scoparia on the poultry red mite
Dermanyssus gallinae (De Geer), the most economi-
cally deleterious parasite of laying hens in Europe. The
results showed that D. scopria provides good mortality
on D. gallinae (Chauve 1998). So far, no studies of
toxicity have been performed on T. urticae with this
plant, which is very locally distributed throughout the
septentrional ecoregion in the northern region of Al-
geria (Djeridane et al. 2008).
The Þrst purpose of this study was to evaluate the
effect of D. scoparia on the mortality of T. urticae. We
measured the acaricidal activity of the essential oil of
D. scoparia and the concentrations that killed 50 and
90% of individuals (LD50 and LD90, respectively). The
effect of sublethal concentrations on fecundity also
was analyzed by recording the numbers of eggs laid by
the treated females.
For a comprehensive evaluation of the potential of
D. scoparia essential oil as an acaricide, the essential oil
was characterized by gas chromatography (GC) and
GCÐmass spectrometry (GC/MS). Afterward, we
evaluated the effectiveness of the major constituents
of D. scoparia oil by spray applications against T. ur-
ticae. The aim was to compare the toxicity of the
essential oils with that of the major constituents, in-
dividually and in selected blends, with the purpose of
identifying the compounds conferring the biological
activities against T. urticae. This information might
help guide the selection and use of appropriate vari-
eties of such plants in traditional postharvest practices
(Bekele and Hassanali 2001).
Materials and Methods
Collection and Maintenance of T. urticae. T. urticae
were collected from infested plants in citrus orchards
in Tunisia in January 2006 (Tunis, by K.L.-G.). They
were maintained in a climate room at 26⬚C, 50 Ð 60%
RH, and a photoperiod of 16:8 (L:D) h, at the Biodi-
versity Research Center (Ecology of Interactions and
Biological Control team) in the University Catholique
of Louvain (Belgium) for ⬎5 yr without any contact
with acaricides before the experiments. The strain was
reared on bean (Phaseolus vulgaris L.) leaves placed
on moistened cotton in petri dishes (Overmeer 1985).
Only young female adults (⬍24 h old) were chosen for
the bioassays.
Plant and Essential Oil Extraction. D. scoparia in-
dividuals were collected locally in Tunisia (Saddine,
southwest of Kef) in June 2008. This selection was
based on previous work and the use of plant products
in traditional medicine in Tunisia (Ben Haj Jilani et al.
2007). Its essential oil was obtained through steam
distillation of the aerial parts for 4 h by using a Clev-
enger-type apparatus and fresh material. The oil yield
was 0.51% of the dry weight of D. scoparia.
Identification of Essential Oil Constituents. The
essential oil of D. scoparia was analyzed by GC-MS in
the Department of Analytical Chemistry in Gembloux
1221
Agro-Biotech, University of Liège, Liège, Belgium.
For quantitative analyses (percentage determina-
tion), we used a fast GC, which proved to be powerful
enough to analyze the essential oil constituents (Bic-
chi et al. 2004, Heuskin et al. 2009).
GC-MS Analysis. Conventional GC-MS analyses
were carried out on a thermo trace MS Finnigan mass-
selective detector equipped with an Optima 5 MS
(Macherey-Nagel) capillary column (30 m by 0.25 mm
i.d., 0.25-␮m Þlm thickness) and a split/splitless in-
jector (splitless mode) at 250⬚C. The oven tempera-
ture was programed from 40 to 210⬚C. Helium was the
carrier gas at 1 ml/min. Volatile compounds were
identiÞed by comparing the mass spectra obtained
with those from the Wiley 275 liters spectral library
and with their retention indices. The retention indices
were determined relative to the retention times of a
series of n-alkane standards (C9-C30, 0.025 ␮g/␮l in
n-hexane, Sigma-Aldrich, Bornem, Belgium), mea-
sured under the chromatographic conditions de-
scribed above, and compared with values in the liter-
ature (Adams 2001).
Fast GC Analysis. Fast GC analyses were conducted
on a Thermo Ultra-Fast Trace GC operated with a
split/splitless injector and a Thermo AS 3000 autosam-
pler (Thermo Fisher ScientiÞc, Waltham, MA). The
GC system was equipped with an ultrafast module
(UFM) incorporating a direct resistively heated col-
umn (Thermo Fisher ScientiÞc): UFC-5, 5% phenyl (5
m by 0.1 mm i.d., 0.1-␮m Þlm thickness). The following
chromatographic conditions were used to obtain a
suitable peak resolution. The UFM temperature pro-
gram was as follows: initial temperature at 40⬚C, held
for 0.1 min, ramp 1 at 30⬚C min⫺1 to 95⬚C, ramp 2 at
35⬚C min⫺1 to 155⬚C, ramp 3 at 200⬚C min⫺1 to 280⬚C,
held for 0.5 min. Injection temperature was 240⬚C;
injection volume was 1 ␮l; carrier gas was He, at a
constant ßow rate of 0.5 ml min⫺1; and split ratio was
1:100. The GC unit had a high-frequency fast ßame
ionization detector (300 Hz), at 250⬚C; H2 ßow was 35
ml min⫺1; air ßow was 350 ml min⫺1; and make-up gas
ßow (N2) was 30 ml min⫺1. Data processing was per-
formed using Chromcard software version 2.3.3. (Lou-
vain-la-Neuve, Belgium).
Effect of D. scoparia Essential Oil on Mortality. The
acute toxicity (measured as mortality at 72 h) of the
essential oil was determined via a spray application
method on young females of T. urticae aged 24 h. D.
scoparia essential oil was diluted 1:100 in ethanol. The
acaricidal effect of 12 concentrations of essential oil
was investigated which corresponded to 0.064, 0.08,
0.26, 0.66, 1.32, 2, 2.66, 3.32, 4, 4.66, 5.32, and 6 mg
liter⫺1, respectively.
A group of 25 young females aged 24 h was randomly
selected and transferred to fresh bean leaf discs (di-
ameter, 35 mm) mounted with the adaxial side up-
permost on moistened cotton in petri dishes (90 by 15
mm). A Potter spray tower that produced a deposit
after a settling time of 1 min 30 s was used to spray the
mites on the leaf discs at a pressure 1.4 bar under 20 ⫾
2⬚C. The distance of the sprayer nozzle from the leaves
was 50 cm. The tests were replicated Þve times per
1222 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 104, no. 4

concentration. Each replicate consisted of 25 females Table 1. Major constituents of D. scoparia essential oil and
of T. urticae, with Þve mites on each leaf disc. Control their relative proportions in the pure oil
tests were performed using water and ethanol. After
Retention
spraying, the bean leaf discs were maintained at room No. Component
Retention
index %
temperature (26 ⫾ 1⬚C) 60 ⫾ 10% RH, and a photo- time (min)
(measured)
period of 16:8 (L:D) h. 1 ␣-Thujene 5.62 930 13.71
Mites were included in the mortality count 72 h 2 ␣-Pinene 5.85 939 31.95
posttreatment if their appendages did not move when 3 Sabinene 6.91 975 17.24
prodded with a Þne pencil. For each concentration, 4 Myrcene 7.43 991 3.46
5 ⌬-3-Carene 8.75 1031 16.85
after using AbbottÕs correction (Abbott 1925), we cal- 6 Ocimene 13.20 1144 9.75
culated the mortality rate according to (mean number 7 Terpinene-4-ol 14.66 1177 2.84
of deaths after the treatment ⫺ mean number of 8 Pulegone 17.27 1237 0.16
deaths after the control)/the total number of females 9 Eugenol 22.70 1359 1.80
10 ␤-Eudesmol 34.79 1651 0.76
at the beginning of the tests. The data obtained in this
experiment also were submitted to probit analysis IdentiÞcation with retention index was obtained by GC-MS. Per-
(Finney and Stevens 1948). centage were obtained by Fast-GC-FID.
Effect of D. scoparia Essential Oil on Fertility. Dur-
ing the mortality experiments (see above), a few dead
mites were found after being sprayed with low con- carene, and terpinene-4-ol, three constituents that
centrations (0.064, 0.08, and 0.26 mg liter⫺1) of the oil. were found to be highly toxic to T. urticae (i.e., that
The experimental method was similar to that used were lethal to at least 65.6% of the individuals). TM2
to test the lethal toxicity described previously. A was composed of eugenol, pulegone, ␣-thujene,
group of 25 young females aged 24 h were randomly ␤-eudesmol, sabinene, and ocimene, six moderately
selected and transferred to fresh bean leaf discs on active constituents with mortality rates between 23
moistened cotton in petri dishes. These females and 65.6%. TM3 was composed of myrcene, an inactive
were sprayed with a Potter spray tower. The tests constituent with a mortality rate ⬍23%. This classiÞ-
were replicated Þve times per concentration. Each cation of toxicity was similar to that used by Mires-
replicate consisted of 25 females of T. urticae, with mailli et al. (2006).
Þve mites in each leaf disc. Control tests were per- Finally, we tested the effects of different blends of
formed using water and ethanol. After spraying, the constituents on fecundity: PM1 was composed of
each female was transferred to bean leaf discs (di- myrcene, eugenol, and ⌬3-carene, three constituents
ameter, 15 mm), which had been completely that were found to be highly active on the fecundity
cleaned, to check fecundity. These bean leaf discs of T. urticae (the number of eggs laid never overpasses
were maintained at room temperature (26 ⫾ 1⬚C), 30 eggs per female. PM2 was composed of terpinene-
60 ⫾ 10% RH, and a photoperiod of 16:8 (L:D) h. 4-ol, ocimene, sabinene, and ␣-thujene, four moder-
To test the effect of the sublethal concentrations of ately active constituents with laying rates between 30
the essential oil on fecundity, the numbers of eggs laid and 34 eggs. PM3 was composed of ␣-pinene, ␤-eudes-
by the treated females was recorded daily for 12 d, the mol, and pulegone, three inactive constituents with a
point when maximum fecundity was expressed laying rate ⬎34 eggs. The results were compared with
(Hance and Van Impe 1999), and the eggs were de- the results obtained from the complete essential oil.
stroyed after counting. The fecundity of each female Statistical Analysis. All the data were corrected by
treated with the three solutions (0.064, 0.08, and 0.26 using AbbottÕs formula (Abbott 1925). Probit anal-
mg liter⫺1) and with the control solution (water and ysis was used to determine the LD50 and LD90 values
ethanol) was compared. using the StatPlus program (AnalystSoft Inc., www.
Effect of Constituents of D. scoparia Essential Oil analystsoft.com).
on Mortality and Fecundity. The toxicity experiments Regarding mortality, the data also were analyzed
were repeated with commercially available constitu- using Prism version 5.01 for Windows (GraphPad Soft-
ents of the essential oil and with blends of these at ware, San Diego, CA) for analysis of variance
their natural proportions (GC-MS; Table 1) in the oil (ANOVA). All tests were applied under two-tailed
and in amounts present in the minimum 100% lethal hypotheses and the signiÞcance level P was set at 0.05.
dose of the oil (3.42 mg liter⫺1). TukeyÕs test was used to compare means.
Pure compounds were purchased from Sigma-Al- In the fecundity experiments, the cumulative
drich: ␣-thujene, ␣-pinene, sabinene, myrcene, ⌬3- number of eggs was best-Þtted to a sigmoidal curve
carene, ocimene, terpinene-4-ol, pulegone, eugenol, (Prism) using the formula Y ⫽ B ⫹ (A ⫺ B)/(1 ⫹
and ␤-eudesmol. Purities of the compounds varied 10ˆ((LT50 ⫺ X))). In this formula, Y represents the
from 95 to 99%. value of the cumulative number of eggs, B is the
To identify the relative contribution of each con- minimum number of eggs (constrained with a min-
stituent to the toxicity of the oil, we tested each of imal value of 0), A is the maximum number of eggs
these constituents individually. We then tested a (plateau value), and LT50 is the period of time
blend of all major constituents, called a full mixture (days) that provided a response halfway between
(TFM). Finally, we tested the toxicity of three se- zero and the maximum of eggs (plateau), and X is
lected blends: TM1 was composed of ␣-pinene, ⌬3- the period of time of the experiment (days).
August 2011 ATTIA ET AL.: ACARICIDAL PROPERTIES OF D. scoparia AGAINSt T. urticae
Fig. 1. Mortality caused by the oil, full mixture, and individual constituents to T. urticae when applied at levels equivalent
to those found in the 100% lethal concentration of the pure oil (LD100 ⫽ 3.42 mg liter⫺1). Values are mean ⫾ SE of Þve
replicates with 25 adult female mites per replicate. Treatments with different letters are signiÞcantly different from each other
(TukeyÕs test).
Results
Identification of Essential Oil Constituents. Table 1
showed the composition of D. scoparia essential oil.
Ten components were identiÞed (electron impact-
mass spectra and retention index comparison) by
GC-MS comprising 98.52% of the total weight;
␣-pinene was the most abundant compound (31.95%)
followed by sabinene (17.24%) and ⌬3-carene
(16.85%). The essential oil was then analyzed by fast
GC on a UFM column of the same polarity as in
GC-MS (apolar stationary phase). The retention times
of the components of interest from the essential oil
were compared with those of the reference com-
pounds.
Effect of D. scoparia Essential Oil and Its Constit-
uents and Blends on Mortality. Toxicity of Essential
Oil. All mites (females) in the control group survived
72 h. A low mortality rate (⬍20%) was observed at
0.064, 0.08, and 0.26 mg liter⫺1. The LD50 of D. scoparia
was 1.79 mg liter⫺1 for the adult female spider mites
(with a SE of 0.123 and a ␤ value of 0.84). The LD90
and LD100 values for the mites were obtained at 3.16
and 3.42 mg liter⫺1, respectively.
Toxicity of Single Constituents. A signiÞcant differ-
ence was found in the lethal toxicity of the single
constituents of D. scoparia when all treatments were
compared (including the essential oil and the control
treatments, one-way ANOVA; F ⫽ 88.91; df ⫽ 12, 104;
P ⬍ 0.001) (Fig. 1). Indeed, post hoc TukeyÕs tests
comparing the toxicity of single constituents revealed
that three constituents (␣-pinene, ⌬3-carene, and ter-
pinene-4-ol) were similarly and highly toxic, but they
were not as toxic individually as the essential oil (Fig.
1). Six constituents (eugenol, pulegone, ␣-thujene,
␤-eudesmol, sabinene, and ocimene) were slightly
toxic, whereas the remaining constituent (myrcene)
was not toxic to T. urticae and did not differ signiÞ-
cantly (post hoc TukeyÕs tests) from the mortality rate
found in the control test (Fig. 1).
1223
Toxicity of Recomposed Oil. Based on the 100%
lethal concentration LD100 ⫽ 3.42 mg liter⫺1 and
following the natural composition of the oil as in-
dicated by GC-MS (Table 1), bioassays with the
recomposed oil (␣-thujene, ␣-pinene, sabinene,
myrcene, ⌬3-carene, ocimene, terpinene-4-ol, pule-
gone, eugenol, and ␤-eudesmol) showed that the
greatest mortality rate was obtained when all 10
constituents were present (full mixture). This was
signiÞcantly higher than the mortality rates for the
three most toxic constituents (␣-pinene, ⌬3-carene,
and terpinene-4-ol) when tested individually (Fig.
1). The mortality rate caused by the full mixture (a
blend of all 10 major constituents) was signiÞcantly
lower than the mortality caused by pure D. scoparia
oil (post hoc TukeyÕs tests) (Fig. 1).
Bioassay of Selected Blends. The mortality rates
caused by the selected blends differed with the blend
(one-way ANOVA; F ⫽ 248,2; df ⫽ 5, 54; P ⬍ 0.001)
(Fig. 2). The mortality rate caused by all of the se-
lected blends (TM1, TM2, TM3, TM1 ⫹ 2 ⫽
TM1⫹TM2) was signiÞcantly lower than the mortality
rate caused by pure D. scoparia oil (Figs. 1 and 2).
The mortality rates of TM1, TM2, and TM3 were
lower than the mortality rate of the full mixture of the
10 major components (Fig. 2), the mortality rate of
TM1 ⫹ 2 was not signiÞcantly different from that of
the full mixture (Fig. 2), and TM3 did not cause any
signiÞcant mortality in T. urticae.
Effect of D. scoparia Essential Oil and Its Constit-
uents and Blends on Fecundity. Effect of Essential Oil.
Fecundity was affected by the concentration of the
essential oil (Fig. 3). The experimental data were
correlated with the theoretical data and followed a
sigmoid model. The maximum values of the cumula-
tive numbers of eggs were signiÞcantly different be-
tween the control experiments and the treatments
(Fig. 3).
1224 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 104, no. 4

Fig. 2. Mortality caused by selected blends of active and inactive constituents of D. scoparia oil when applied at levels
equivalent to those found in the 100% lethal concentration of the pure oil (LD100 ⫽ 3.42 mg liter⫺1) Values are mean ⫾
SE of Þve replicates with 25 adult female mites per replicate. Treatments with different letters are signiÞcantly different
from each other (post hoc TukeyÕs tests). TM1 (active), ␣-pinene ⫹ ⌬3-carene ⫹ terpinene-4-ol; TM2 (moderately
active), eugenol ⫹ pulegone ⫹ ␣-thujene ⫹ ␤-eudesmol ⫹ sabinene ⫹ ocimene; and TM3 (inactive), myrcene.

Effect of Single Constituents. The maximum values
of the cumulative numbers of eggs obtained for
␣-pinene, ␤-eudesmol, and pulegone did not differ
signiÞcantly from that obtained for the control (Table
2). The maximum values of the cumulative numbers of
eggs obtained for the remaining constituents (ter-
pinene-4-ol, ocimene, sabinene, ␣-thujene, myrcene,
eugenol, and ⌬3-carene) were signiÞcantly lower than
those obtained by the control and the other constit-
uents.
Effect of Selected Blends. For PM2 ⫹ PM3, the max-
imum value of the cumulative number of eggs was 32,
which was signiÞcantly different to the 17 eggs ob-
tained with the full mixture PFM (Fig. 4). Removal of
the mixture containing the moderately active constit-
uents (PM2) increased the maximum value of the
cumulative number of eggs to 24 compared with the
full mixture, and removal of the inactive constituents
(PM3) increased the maximum value of the cumula-
Fig. 3. Mean ⫹ SE cumulative number of eggs laid by
female T. urticae after treatment with a 0.064, 0.08, or 0.26 mg
liter⫺1 Deverra oil, or after treatment with a control (water
and ethanol) solution.
tive number of eggs to 19 compared with the full
mixture (17 eggs).
Effect of Recomposed Oil. The combination of the
active constituents with either the moderately active
(PM1 ⫹ PM2) or inactive constituents (PM1 ⫹ PM3)
restored a level of toxicity that was consistent with the
full mixture of 10 compounds (Fig. 4). The absence of
PM1 constituents from the mixture (PM2 ⫹ PM3)
caused a signiÞcant increase in egg production (P ⬍
0.05).
In our study, the lowest concentrations did not
affect female survival but they did affect female fe-
cundity in comparison with the control group. Our
results showed that females laid eggs during similar
periods of time but that the laying rate was decreased
by treatment with D. scoparia essential oil. The total
number of eggs was lower (38, 34, and 11 eggs per
female for 0.064, 0.08, and 0.26 mg liter⫺1; 14 compared
with 50 eggs in the control group) at the end of the
laying period.
Although it only represented 16.85% of D. scoparia
oil, ⌬3-carene was the major contributor to the de-
crease in the maximum cumulative number of eggs
obtained after 12 d. Similarly to the toxicity experi-
ments, individual constituents in the fecundity inhi-
bition assays showed that the absence of certain con-
stituents (myrcene, eugenol, and ⌬3-carene) in the
artiÞcial mixture caused a signiÞcant increase in the
cumulative number of eggs laid by the females.
Here, we showed that the artiÞcial blend TM1
(myrcene, eugenol, and ⌬3-carene) was responsible
for decreasing the cumulative number of eggs to 25
eggs per female compared with 50 eggs per female in
the control, whereas myrcene, eugenol, and ⌬3-
carene decreased the cumulative number of eggs to 30,
30, and 27 eggs per female, respectively, allowing us to
conclude that the active constituents had synergistic
August 2011 ATTIA ET AL.: ACARICIDAL PROPERTIES OF D. scoparia AGAINSt T. urticae 1225

Table 2. Cumulative number of eggs laid by females treated with constituents of the essential oil

Parameters of the sigmoid Comparison with the control

Constituent
Bottom Top TL50 R2 F (DFn, DFd) Bottom Top TL50
␣-Pinene 7.11 38.07 5.08 0.90 1.35 (1, 9) 0.28 0.9143 0.0642
␤-Eudesmol 6.37 38.18 3.81 0.91 0.63 (1, 9) 0.45 0.9392 0.9038
Pulegone 6.34 38.32 3.75 0.91 0.53 (1, 9) 0.49 0.9988 0.7945
Terpine-4-ol 6.68 33.57 4.22 0.89 0.98 (1, 9) 0.35 0.0474 0.5145
Ocimene 5.18 33.73 4.01 0.92 0.43 (1, 9) 0.53 0.0188 0.7208
Sabinene 5.53 31.67 4.95 0.90 0.68 (1, 9) 0.43 0.009 0.084
␣-Thujene 5.87 30.98 5.47 0.90 0.77 (1, 9) 0.40 0.0092 0.0278
Myrcene 4.98 30.35 4.75 0.92 0.40 (1, 9) 0.54 0.0011 0.1332
Eugenol 4.81 30.04 3.77 0.92 0.26 (1, 9) 0.62 0.0003 0.8233
⌬3-Carene 4.35 27.58 4.16 0.90 0.16 (1, 9) 0.70 0.0002 0.5262
Full mixture 2.80 19.74 4.44 0.94 0.09 (1, 9) 0.77 ⬍0.0001 0.1663
Essential oil 1.87 13.69 5.14 0.93 2.30 (1, 9) 0.16 ⬍0.0001 0.0093

Comparison between the maximal numbers of eggs laid by the females treated with the pure oil, full mixture, individuals constituents of D.
scoparia and with the control solution when applied at levels equivalent to those found in 3.42 mg liter⫺1. LT50 is the period of time (days)
that provided a response halfway between zero and the maxiumum of eggs (plateau), and X is the period of time of the experiment (days).
Values are mean of n ⫽ 5 replicates with 25 adult female mites per replicate (df ⫽ 9 for all).

effects because the number of eggs laid was signiÞ-
cantly higher when the constituents were tested in-
dividually rather than when they were in a mixture
with other active constituents.
Discussion
This study is the Þrst to qualitatively show how the
concentration of D. scoparia essential oil varies in its
efÞcacy against the twospotted spider mite.
Mortality. Our results clearly indicated that the
essential oil of D. scoparia is toxic to T. urticae females
with LC90 and LC100 values of 3.2 and 3.42 mg liter⫺1,
respectively. We identiÞed 10 components in the es-
sential oil that had differing levels of toxicity:
␣-pinene, the major constituent (31.95%) of D. sco-
paria oil, was the major contributor to the toxicity of
the artiÞcial blend, followed by ⌬3-carene (16.85%)
and terpinene-4-ol (2.84%). Myrcene was inactive on
its own. However, the toxicity of our artiÞcial mixture
only reached 83% mortality when the blends of the
active constituents were mixed with myrcene (inac-
tive). The highest mortality rate was obtained when all
of the constituents were present in the mixture, but
the mortality caused by the artiÞcial mixture contain-
ing all of the constituents remained signiÞcantly lower
than that caused by the natural essential oil. This
suggests that unknown constituents comprising 1.5%

Fig. 4. Mean ⫹ SE cumulative number of eggs laid by the female T. urticae treated with the full mixture, and blends of
constituents of D. scoparia essential oil when applied at levels equivalent to those found in 0.26 mg liter⫺1. Value are mean
of n ⫽ 5 replicates with 25 adult female mites per replicate. PM1 (active), ⌬3-carene ⫹ eugenol ⫹ myrcene; PM2 (moderately
active), terpinene-4-ol ⫹ ocimene ⫹ sabinene ⫹ ␣-thujene; PM3 (inactive), ␣Ðpinene ⫹ ␤-eudesmol ⫹ pulegone; and PFM,
PM1 ⫹ PM2 ⫹ PM3.
1226 JOURNAL OF ECONOMIC ENTOMOLOGY
of the oil made a signiÞcant contribution to its overall
toxicity, for example, through synergy.
One interesting aspect of our study was the differ-
ence found in the role of the major constituents in a
mixture as opposed to their individual toxicities. The
artiÞcial blend TM1 (␣-pinene, ⌬3-carene, and ter-
pinene-4-ol) was responsible for 65% mortality,
whereas ␣-pinene, ⌬3-carene, and terpinene-4-ol
caused 68, 65.2, and 63.2% mortality, respectively,
when tested individually. Consequently, our results
strongly suggest that the active constituents had an
antagonistic effect (or at least no additional effect) on
each other, because their toxicity level was signiÞ-
cantly higher when tested individually than when they
were in a mixture with the other active constituents.
Fecundity. We found that the presence of all of the
constituents together in the artiÞcial mixture caused a
signiÞcant decrease in the number of eggs laid by
females, at 0.26 mg liter⫺1 (11 eggs), compared with
the control (50 eggs). Similarly, the highest mortality
rates was obtained when all of the constituents were
present in the mixture at 3.42 mg liter⫺1 (83.6%).
Another interesting aspect of our study was that
our results strongly suggested that D. scoparia oil
might have more than one mode of action (fecun-
dity and mortality). Indeed, three major constitu-
ents (myrcene, eugenol, and ⌬3-carene) strongly
decreased fecundity but only ⌬3-carene had a
strong effect on mortality. Moreover, the most toxic
constituent was ␣-pinene, but this had no effect on
fecundity. Our results conÞrm those of a previous
report (Miresmailli et al. 2006) where the absence
of ␣-pinene from the artiÞcial mixture caused a
signiÞcant decrease in toxicity (80%), which
tempted us to conclude that this constituent is the
major contributor to the toxicity of the essential oil.
We found that the absence of some blends from the
artiÞcial mixture caused a signiÞcant decrease in tox-
icity and an increase in the number of eggs laid by the
females. This indicates that the presence of all of the
constituents was necessary to achieve full toxicity.
Our study shows that terpinen-4-ol can be consid-
ered as being an important contributor to the toxicity
against T. urticae, with a 63.2% mortality rate. Our
results conÞrm a previous report (Kouninki et al.
2007) that found that terpinen-4-ol is responsible for
50% of the mortality against Sitophilus zeamais
Motschulsky adults.
Another previous report showed that 1,8-cineole is
responsible for the major toxicity of rosemary oil and
Listea pungens Hemsl. against T. urticae and Tricho-
pulsia ni (Hübner), respectively (Miresmailli et al.
2006, Jiang et al. 2009).
A few plant-derived essential products are effective
at controlling arthropods (Cloyd et al. 2009). Among
these, some are prohibited because of their potential
phytotoxicity (Cloyd et al. 2009). Previous studies
have reported that the acaricidal effects of plant es-
sential oils are related to their chemical compositions
(Isman et al. 2001, Singh et al. 2001). Mansour et al.
(1986) pointed out that besides toxicity, the residues
of essential oils of some Labiatae species are repellent
Vol. 104, no. 4
and strongly reduce the fecundity of Tetranychus cin-
nabarinus (Boisduval) females.
The octopaminergic nervous system is considered
to be the site of action of essential oils in the American
cockroach (Enan 2001), but this may not be the case
for the twospotted spider mite, and there is also the
possibility that essential oils have more than one site
of action, because they are complex mixtures (Mires-
mailli et al. 2006).
A further step after this study might be to test the
phytotoxicity of D. scoparia essential oil on vegetables
and herbaceous and foliar plant material (Cloyd et al.
2009) However, the extent of plant injury can be
dependent on numerous factors, including the precise
concentration a plant was exposed to during applica-
tions. Consequently, it is necessary to know the pre-
cise effective concentration of an essential oil; this
underlines the importance of efÞcient analyses over a
wide range of concentrations. The present results
strongly suggest that D. scoparia might add to the
arsenal of methods for controlling mites in green-
houses and orchards. However, further studies are
needed to evaluate the cost, efÞcacy, and safety of the
oil. Knowing the role of each constituent in the tox-
icity and effects on fecundity of this oil would provide
an opportunity to create an artiÞcial blend of different
constituents on the basis of their activities against
different pests. Indeed, several constituents are com-
mercially available at a reasonable purity (95%), and
essential oil producers and suppliers can often provide
chemical speciÞcations for even the most complex oils
(Isman 2000). Current information indicates that they
are safe to the user and the environment, with few
qualiÞcations. The exemption of some essential oils
from registration in the United States has stimulated
their development for use as commercial insecticides
(Isman 2000).
Consequently, despite the fact that this oil provides
a good level of T. urticae mortality and inhibition of
female fecundity, if it was to be developed for use in
pest management it might be necessary to look toward
more efÞcient means of oil extraction.
Acknowledgments
We are very grateful to Vincent Hote for useful discussions
about chemical compositions (Université de Liège Gem-
bloux Agro-Bio Tech Unité de Chimie analytique) and to
Ghrabi Gammar Zeineb for providing the D. scoparia sam-
ples. We also indebted to Wallonies-Bruxelles International.
This work was presented at the Journée scientiÞque annuelle
de la société royale de chimie-Chimie verte., held 14 October
2010 in Belgium. This work was supported by funding from
the Institut dÕencouragement de la Recherche ScientiÞque et
de lÕInnovation de Bruxelles (to A.C.M.) and the National
Fund for ScientiÞc Research (Belgium) (to T.H.). This is
publication BRC 190 of the Biodiversity Research Centre at
Université Catholique de Louvain.
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Received 26 August 2010; accepted 12 May 2011.