Crop Protection 71 (2015) 95e100

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Crop Protection
journal homepage: www.elsevier.com/locate/cropro

Evaluation of the toxicity of essential oil from Alpinia purpurata

inflorescences to Sitophilus zeamais (maize weevil)
Camila Soledade de Lira a, Emmanuel Viana Pontual b, c, Lidiane Pereira de Albuquerque d,

Vargas de Oliveira f,
Laura Mesquita Paiva e, Patrícia Maria Guedes Paiva c, Jose
c , *
~o , Daniela Maria do Amaral Ferraz Navarro a
Thiago Henrique Napolea
a
Departamento de Química Fundamental-CCEN, Universidade Federal de Pernambuco, Cidade Universita
ria, 50670-901 Recife, Pernambuco, Brazil
b
Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco, 52171-900 Recife, Pernambuco, Brazil
c
Departamento de Bioquímica-CCB, Universidade Federal de Pernambuco, 50670-420 Recife, Pernambuco, Brazil
d
Departamento de Bioquímica e Farmacologia, Universidade Federal do Piauí, 64049-550 Teresina, Piauí, Brazil
e
Departamento de Micologia-CCB, Universidade Federal de Pernambuco, Cidade Universita
ria, 50670-420 Recife, Pernambuco, Brazil
f

Departamento de Agronomia, Area de Fitossanidade, Universidade Federal Rural de Pernambuco, 52171-900 Recife, Pernambuco, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The maize weevil, Sitophilus zeamais Motsch. (Coleoptera: Curculionidae), is a cosmopolitan pest that
Received 26 December 2014 causes extensive and irrecoverable damage to storage grains. This work investigated the chemical
Received in revised form composition, insecticidal activity, feeding-deterrent and repellent effects of an essential oil from Alpinia
4 February 2015
purpurata inflorescences against S. zeamais. Analysis by gas chromatography coupled to mass spec-
Accepted 5 February 2015
Available online
trometry (GCeMS) revealed the presence of 30 volatile constituents (mainly mono- and sesqui-
terpenes). The main compounds found in the oil were b-pinene, a-pinene, trans-caryophyllene,
camphene, and 7-epi-a-selinene. When ingested by S. zeamais adults, the A. purpurata oil did not kill
Keywords:
Red ginger
the insects but disrupted their nutritional status since biomass gain rate and efficiency in conversion of
Botanical insecticide ingested food were both negative or near zero. A weak feeding-deterrent effect was also detected. No
Essential oil toxicity was observed when the oil was applied directly on the cuticle of S. zeamais adults. The fumigant
Stored grain pest toxicity assay revealed that the A. purpurata oil killed S. zeamais adults with LC50 ¼ 41.4 mL/L in air;
however, no repellent property was detected. The essential oil from A. purpurata inflorescences was toxic
to S. zeamais adults by fumigation and due to interferences in digestion of food. Fumigant toxicity is likely
due to the presence of some monoterpene hydrocarbons and oxygenated monoterpenes.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction
Sitophilus zeamais Motsch. (Coleoptera: Curculionidae),
commonly known as the maize weevil, is primarily a cosmopolitan
pest that shows cross-infestation and attacks stored grains such as
wheat (Triticum spp. L.), rice (Oryza sativa L.), barley (Hordeum
vulgare L.), and triticale (a hybrid of Triticum aestivum L. and Secale
cereale L.) (Lorini, 2008). The extensive damage caused by this in-
sect in stored grain is usually irrecoverable, reaching as much as
30% (Miranda et al., 1995; Botton et al., 2005).
Currently, control strategies for S. zeamais are primarily con-
ventional insecticides such as pyrethroids, organophosphates, and
* Corresponding author.
~o).
E-mail address: thiagohn86@yahoo.com.br (T.H. Napolea
phosphine, which are extremely volatile, toxic, and have led to the
evolution of resistance (Perez-Mendoza, 1999). Cleaning and drying
of grains, maintenance of aeration, and temperature regulation can
also minimize maize weevil attack (Ileleji et al., 2007; Noomhorm
et al., 2009; Carvalho et al., 2012).
Strategies of integrated pest management (IPM) aim to maintain
the population density of an organism below the level of economic
injury and intend to include methods with the least possible haz-
ards to people, non-target organisms, and the environment. IPM
practices including plant-derived insecticides may be an inter-
esting alternative since these compounds are usually rapidly
degraded, resulting in low persistence and residual action. Plant
extracts, leaf powders, wood ash, seed oils, lectins, and essential
oils have been investigated as possible integrated management
strategies for S. zeamais (Kehinde and Angela, 2004; Demissie et al.,
~o
2008; Yuya et al., 2009; Chu et al., 2010; Ukeh et al., 2009; Napolea

http://dx.doi.org/10.1016/j.cropro.2015.02.004
0261-2194/© 2015 Elsevier Ltd. All rights reserved.
96 C.S. de Lira et al. / Crop Protection 71 (2015) 95e100
et al., 2013).
Essential oils have been broadly investigated as insecticides
against other economic pests and disease vectors (Bakkali et al.,
2008; Pandey et al., 2012; Kedia et al., 2014; Navarro et al., 2013).
They are complex mixtures of volatile and lipophilic substances,
mostly liquid and odoriferous, containing terpenes, aldehydes,
ketones, phenols, esters, oxides, peroxides, furans, organic acids,
lactones, and coumarins (Dorman and Deans, 2000; Simo ~ es and
Spitzer, 2004).
Alpinia purpurata (Vieill.) K. Schum. (Zingiberaceae), popularly
known as “red ginger”, has great ornamental value and is also used
for production of fibers, paper, dyes, spices, and perfumes. It is
cultivated on a large scale due to its ability to bloom throughout the
year as well as the profusion and durability of its flowers (Medeiros

rio et al., 2010). Santos et al. (2012) were the first
et al., 2009; Victo
to report the insecticidal action of A. purpurata inflorescence
essential oil; essential oils extracted from inflorescences of the red
and pink variants were active against the dengue vector, Aedes
aegypti L., by killing the fourth instar larvae and exerting an
oviposition-deterrent effect.
In this study, we aimed to evaluate the insecticidal effect of an
essential oil from A. purpurata inflorescences (red variant) on
S. zeamais by contact, fumigation, and ingestion assays.
2. Materials and methods
2.1. Plant material and insects
Inflorescences of A. purpurata (red variant) were collected from
decorative specimens in the Departamento de Química Fundamental
of the Universidade Federal de Pernambuco (08 030 0700 S
34 560 5900 W, tropical climate) at Recife city, State of Pernambuco,
northeastern Brazil. The inflorescences were collected from speci-
mens cultivated without any application of insecticides. The dried
specimen was designated number 53,376 at the herbarium UFP-
Geraldo Mariz from the Centro de Ci^ encias Biolo
gicas at the Uni-
versidade Federal de Pernambuco.
S. zeamais adults were reared in the Laborato
rio de Bioquímica de
Proteínas, Departamento de Bioquímica at the Universidade Federal
de Pernambuco, Recife, Brazil. The insects originated from colonies
maintained at the Departamento de Micologia at the Universidade
Federal de Pernambuco, Recife, Brazil and taxonomic identification
was confirmed by experts from the Departamento de Biologia, Area
Benevides, PA, Brazil) in 5 mL of oil solution (in ethanol) to achieve
de Entomologia at the Universidade Federal Rural de Pernambuco. The
colonies were maintained in glass containers (1 L capacity) covered
with unwoven fabric at 28 ± 2  C, 70% relative humidity, 12:12 L:D
photoperiod. The diet consisted of maize grains (acquired from
local farms and cultivated without insecticides) selected according
to integrity, sanitary condition, size, and absence of insect
contamination. Insects with 30e60 days of age were used in the
assays.
Both plant collection and insect rearing had permission (num-
ber 36301-2) from the Instituto Chico Mendes de Conservaça ~o da
Biodiversidade (ICMBio) of the Brazilian Ministry of the
Environment.
2.2. Essential oil extraction
Oil extraction was performed immediately after collection as
described by Santos et al. (2012). Inflorescences (528.3 g, corre-
sponding to 25 inflorescences collected at the same time) were
homogenized with distilled water using a blender and treated with
a hydrodistillation technique. The homogenized material was
placed in a 5 L round-bottom flask containing 2.5 L of distilled
water. The flask was attached to a heating mantle (160e170  C) and
connected to the Clevenger apparatus and a condenser. Hydro-
distillation lasted 3 h after which the oil was collected and treated
with anhydrous sodium sulfate to remove any remaining water.
2.3. Chemical composition analysis by gas chromatography coupled
to mass spectrometry (GCeMS)
For analysis of chemical composition, 1 mL of the sample
(essential oil diluted 1:50, v/v, with hexane) was injected in split
mode (1:20) in a Thermo Finnigan Voyager Mass Spectrometer
coupled with a Thermo Trace Gas Chromatographer 2000 (Thermo
Fisher Scientific) equipped with a DB-5 column (Agilent J&W;
30 m  0.25 mm  0.25 mm). A standard solution of C9eC34 hy-
drocarbons (SigmaeAldrich, USA) in hexane was also evaluated by
injection in split mode (1:20). In addition, a mixture of essential oil
and hydrocarbon standards (0.8 mL of oil plus 0.2 mL of alkanes) was
injected in the gas chromatographer in splitless mode. The oven
temperature was set at 60  C for 3 min, and increased by 2.5  C/min
until 240  C where it was held at this temperature for 10 min.
Helium carrier gas flow was maintained at 100 kPa. The interface
with MS was at 200  C and mass spectra were taken at 70 eV in
electron impact ionization mode, with a scanning speed of 0.5
scans/s from m/z 20e350.
The retention times of compounds present in the oil, hydro-
carbon standards, and compounds in the mixture containing oil
and hydrocarbon standards were used to calculate retention
indices according to the Kratz equation (Adams, 2001). The eluted
compounds were identified by comparison between the mass
spectra and retention indices of authentic standards available in the
reference libraries MassFinder 4, NIST08, and Wiley Registry™ 9th
edition, all integrated in the software Agilent MSD Productivity
ChemStation (Agilent Technologies, Palo Alto, USA). The areas of
peaks in chromatograms were integrated in order to obtain the
total ionic signal, and the values were used to determine the rela-
tive proportion of each compound.
2.4. Evaluation of the ingestion toxicity of the essential oil
Toxicity by ingestion of the essential oil was evaluated using an
adaptation of the Xie et al. (1996) method described by Napolea ~o
et al. (2013). The artificial diet was prepared from a suspension of
autoclaved wheat flour (2.0 g; Dona Benta®, Bunge Alimentos S.A.,
the final concentrations of 18.75 or 37.5 mL/g (mL of neat oil/g of
wheat flour). Five aliquots (200 mL) of the suspension were
measured with a micropipette fitted with a disposable tip cut at the
narrow end (2 mm of internal diameter) and the flour suspension
was applied in Petri dishes (90 mm  100 mm; with known weight)
for disk formation. The dishes containing the flour disks were left in
incubator (37  C) overnight to dry. After that time, the weight of the
dishes was determined again and the mass of the flour disks were
calculated. Twelve S. zeamais adults with known weight were then
transferred to each dish. Each assay had four replicates. After 7 days
at 28 ± 2  C in darkness, the mortality rate and the weights of
broken flour disks and insects were recorded. The control diet
consisted of wheat flour suspended in ethanol.
The deterrent and nutritional indices were determined using
the results of the toxicity by ingestion assays. The feeding-
deterrence index (FDI) was calculated as: FDI (%) ¼ 100  [(A e
B)/(A)], where A is the mass of food ingested by insects in the
control, and B is the mass of food ingested by insects in the test
group (Isman et al., 1990). Samples were classified according to the
FDI as having promoted (Liu et al., 2007): no feeding deterrence
(FDI < 20%), weak (50% > FDI  20%), moderate (70% > FDI  50%),
or strong (FDI  70%) feeding deterrence.
 C.S. de Lira et al. / Crop Protection 71 (2015) 95e100 97

The following nutritional indices were calculated according to
Xie et al. (1996), using data recorded at the conclusion of the
insecticidal assays: (a) Relative consumption rate ¼ C/(D  days),
where C is the mass of ingested food in mg and D corresponds to the
initial insect biomass in mg; (b) Relative biomass gain rate ¼ E/
(D  days), where E corresponds to the biomass gained in mg; (c)
Efficiency in conversion of ingested food (%) ¼ (E/C)  100.
2.5. Evaluation of the contact toxicity of the essential oil
The contact toxicity assay was performed according to Liu and
Ho (1999). The essential oil of A. purpurata inflorescences was
diluted with ethanol to achieve test solutions at 0.75, 1.88, 7.5, 37.5,
and 75 mL/mL. Next, aliquots (0.5 mL) from the test solutions were
topically applied on the dorsal surface of the thorax of insects using
a micropipette. The insects in the negative control were treated
with ethanol, and pyrethrum extract at 2.0e6.0 mg/insect (PESTA-
NAL®, Fluka) was used as a positive control. Following this proce-
dure, the insects were transferred to plastic containers (diameter of
2.5 cm, length of 5.5 cm) and the mortality rate was recorded daily
for 8 days. Each experiment had four replicates.
arcsine square-root transformation, using the Origin 6.0 and Action
2.8.29.357.515 softwares. For fumigation assay, the mortality rates
were calculated using the Abbott's correction. The lethal concen-
tration required to kill 50% of insects (LC50) was then calculated by
probit analysis with a reliability interval of 95% using the software
XLSTAT version 2015.1.01 (Addinsoft, USA).
3. Results
3.1. Extraction and characterization of the oil
Starting from 528.3 g of A. purpurata inflorescences, the
hydrodistillation procedure resulted in a 0.008% essential oil yield.
GCeMS analysis revealed a total of 30 volatile constituents
(Table 1), mainly mono- and sesqui-terpenes, corresponding to
98.8% of the A. purpurata essential oil. The major constituents, in
descending order, were b-pinene, a-pinene, trans-caryophyllene,
camphene, and 7-epi-a-selinene.
3.2. Evaluation of toxicity by ingestion

2.6. Evaluation of the fumigant toxicity of the essential oil
The fumigant toxicity assay was performed according to Chu
et al. (2010). The assay used plastic containers (2.5 cm width and
5.5 cm length, and 24 mL volume) whose lids were covered with
filter paper. The filter paper had previously been soaked in 20 mL of
the oil diluted in ethanol to provide doses of 16.0, 32.0, and 64.0 mL/
L in air. In the control treatment, the filter paper was soaked with
ethanol only. After a 30 s evaporation time, 20 S. zeamais were
placed in each container and the lids were tightly closed in order to
form a sealed chamber. After 24-h exposure, the insects were
transferred to containers with ambient air, and the mortality rate
was recorded after 8 days. Each assay had four replicates.
In the test of toxicity by ingestion, the essential oil did not
significantly (p > 0.05) induce mortality of S. zeamais in treatments
at either concentration (18.75 and 37.5 mL/g), in comparison with
the negative control using ethanol. However, it was observed that
the presence of A. purpurata oil in the diet interfered with insect
nutrition (Table 2). Biomass gain rate (Fig. 1A) and efficiency in
conversion of ingested food (Fig. 1B) were negative or near zero,
revealing that the insects did not digest the food, and instead
metabolized their own reserves leading to a loss in biomass. The
Table 1
Constituents of the essential oil extracted from Alpinia purpurata (red variant)
inflorescences.

Number Compound RI Relative

2.7. Evaluation of the repellent effect of the essential oil area (%)

1 a-Thujene 926 0.05

The repellence assay was performed using arenas similarly to 2 a-Pinene 932 20.57
that used by Gonz
alez et al. (2011), but had only two options 3 Camphene 946 7.57
(control and test) per assay. The arena consisted of two plastic 4 gValerolactone 952 0.18
5 Sabinene 972 0.04
containers, connected symmetrically to a central container by two
6 b-pinene 975 35.76
plastic tubes. A piece of filter paper soaked with 20 mL of ethanol 7 Myrcene 991 0.48
(control) was placed in one container and another piece soaked 8 aeTerpinene 1016 0.07
with 20 mL of A. purpurata essential oil was place in the other one. 9 orto-Cymene 1024 0.21
Ten S. zeamais adults were released in the central container. 10 Limonene 1028 3.99
11 g-Terpinene 1058 0.22
Different oil concentrations (18.75, 37.5, and 75 mL/mL) were tested 12 Terpinolene 1088 0.70
separately in a randomized design. After seven days, the number of 13 Linalool 1100 0.92
insects present in each container was counted. Repellence index 14 Nonanal 1105 0.50
(RI) was calculated according to Mazzonetto and Vendramim 15 Fencholeendo 1113 0.06
16 aeTerpineol 1190 0.21
(2003) using the formula: RI ¼ 2T/(T þ C), where T is the percent-
17 Fenchyl acetateeendo 1221 0.19
age of insects in the oil container and C is the percentage of insects 18 Bornyl acetate 1287 1.49
in the control container. Standard deviations (SD) were calculated 19 trans-Caryophyllene 1422 13.23
and the results were classified as: RI ± SD > 1, attractive effect; 20 a-Humulene 1456 0.93
RI ± SD < 1, repellent effect. Each assay had four replicates. 21 b-Selinene 1486 1.64
22 Viridiflorene 1489 1.42
23 Drim-8-(12)ene 1492 0.22
2.8. Statistical analysis 24 Valencene 1496 2.28
25 Selinene(7)-epi-a 1521 4.21
The data were expressed as a mean of replicates ± standard 26 trans-Nerolidol 1565 0.08
27 Caryophyllene oxide 1586 1.09
errors, which were calculated using GraphPad Prism version 4.0 for
28 Humulene epoxide II 1613 0.06
Windows (GraphPad Software, San Diego, California, USA). One way 29 a,becaryophylla-4(12),8(13)-dien-5-ol 1640 0.07
fixed-effects ANOVA (significance at p < 0.05) was conducted using 30 Eudesmol-7-epi-alfa 1662 0.36
the Action 2.8.29.357.515 software. Significant differences between The constituents are listed according to elution order in a DB-5 column. RI: retention
treatment groups from flour disk bioassays and repellence assay indices calculated in regard to standards mixture of hydrocarbons (C9eC34) for
were analyzed using Tukey's test (significance at p < 0.05), after 30 min.
98 C.S. de Lira et al. / Crop Protection 71 (2015) 95e100

Table 2
Data from one-way ANOVA of the effects of A. purpurata essential oil by ingestion
and fumigation on S. zeamais.

Parameter Degrees of freedom F-statistics p value

Numerator Denominator

Ingestion assay
Biomass gain rate 2 9 49.72 0.002
Relative consumption rate 2 9 4.24 0.071
Efficiency in conversion 2 9 158.65 0.000
of ingested food
Fumigation assay
Mortality 3 12 17.95 0.0001

In ingestion assay, three treatments were used: negative control and tests with oil at
18.75 and 37.5 mL/g. In fumigation assay, four treatments were used: negative
control and tests with oil at 16.0, 32.0, and 64.0 mL/L.

relative consumption rates (Fig. 1C) were not significantly different

than the control (p > 0.05) and a weak feeding-deterrent effect
(FDI: 23%) was observed for the oil only at the 18.75 mL/g concen-
tration. There was no deterrent effect observed at the 37.5 mL/g
concentration.

3.3. Evaluation of toxicity by contact

A. purpurata essential oil did not show toxicity by contact since

there was no mortality at any tested concentration. The pyrethrum
extract (positive control) promoted mortality of insects, with LC50
of 4.77 (95% confidence interval: 4.59e4.92) mg/insect (goodness-
of-fit c2: 19.305; degrees of freedom: 18; p ¼ 0.373).

3.4. Evaluation of fumigant toxicity and repellent effect

The fumigant toxicity assay showed that A. purpurata essential

oil led to increased insect mortality rates. Statistical analysis
showed that the data from control and treatments at different
concentrations were significantly different (Table 2). The LC50
determined was 41.4 (95% confidence interval: 33.4e53.9) mL/L in
air (goodness-of-fit c2: 12.484; degrees of freedom: 10; p ¼ 0.254).
The repellent activity of A. purpurata oil was also evaluated, and the
results showed that the oil did not repel or attract the insects at any
of the tested concentrations (F-statistics: 0.71; p ¼ 0.517; numer-
ator and denominator degrees of freedom: 3 and 12, respectively).

4. Discussion

Santos et al. (2012) showed the presence of 42 mono- and
sesqui-terpenes in an essential oil extracted from A. purpurata in-
florescences, and b-pinene (13.93%), a-pinene (6.28%) and 7-epi-a-
selinene (8.26%) were also among the major constituents. However,
there are remarkable quantitative differences between the oils in
our study and Santos et al. (2012); the concentrations of a-pinene
and camphene were 3.2 and 2.7 fold higher, respectively, in the oil
obtained in the present study, whereas the concentrations of
linalool and a-terpineol were 10.4 and 34 fold higher, respectively,
in the oil obtained in the previous study. These differences are
probably due to seasonal factors, which usually lead to differences
in the composition of oils extracted from conspecific individuals
collected at the same location or even from the same specimen at
different time periods (Celiktas et al., 2007; Hussain et al., 2008;
Soliman et al., 2009).
A. purpurata oil exerted anti-nutritional effects but was a weak
deterrent agent indicating that the insects detected the oil, but this
did not lead to rejection of food. Thus, the anti-nutritional effects
observed for the oil were probably linked to post-ingestion effects,
such as intoxication, and not pre-ingestion effects. It is not a
Fig. 1. Nutritional parameters of S. zeamais adults reared on artificial diets containing
ethanol (control) or ethanolic solution of A. purpurata essential oil (18.75 or 37.5 mL of
oil per g of wheat flour). (A) The relative biomass gain rate indicates the biomass (mg)
gained every day per mg of initial body weight. (B) The efficiency in conversion of
ingested food (%) indicates the amount of ingested food converted in biomass by the
insects. (C) The relative consumption rate indicates the amount of food consumed (mg)
per mg of body weight per day. Each bar corresponds to the mean ± SD of four rep-
licates. Different letters indicate significant (p < 0.05) differences between treatments
by Tukey's test.
requirement that insecticides have feeding-deterrent effects. In
fact, these effects may have limited importance because of the
behavioral plasticity of insects, which can be initially deterred, but
rapidly desensitized in months, days, or even hours (Isman, 2006).
The anti-nutritional effects found in this study could potentially
be useful in attempts to control S. zeamais, which is a very
destructive pest due to the speed with which it consumes grain
(Huang et al., 1997). Thus, even though A. purpurata oil did not lead
to high mortality rates, the anti-nutritional effects could minimize
reproductive fitness by reducing assimilation of ingested food.
 C.S. de Lira et al. / Crop Protection 71 (2015) 95e100
Similar to the results found in this study, essential oil from Myristica
fragrans Houtt. (nutmeg) seeds also affected biomass gain and
reduced food consumption of S. zeamais adults in the flour disk
bioassay (Huang et al., 1997).
The compound a-pinene, one of the major components of
A. purpurata oil, was previously assessed for effects on feeding and
growth of S. zeamais using the flour disk bioassay; this compound
showed feeding deterrent effects, but did not exert anti-nutritional
effects (Huang et al., 1998). Camphene and b-pinene were also
among the major components of the essential oils from other
Zingiberaceae species and interfered with the nutritional status of
S. zeamais adults, but when tested individually, these compounds
did not show any effect. It is possible that the anti-nutritional ef-
fects of A. purpurata oil are due to a synergistic effect of its com-
ponents or are the result of specific action of one or more of the
minor components.
Many oils have been reported to exert contact toxicity effects on
the maize weevil and other stored grain pests, and the degree of
toxicity has been attributed to characteristics such as low to
moderate volatility and ease with which it penetrates the cuticle of
insects (Sampson et al., 2005; Ogendo et al., 2011; Suthisut et al.,
2011). It is possible that the A. purpurata oil did not have suffi-
cient time to penetrate the S. zeamais highly chitinized elytra or
may have interacted with cuticle lipids, which did not allow it to be
absorbed into the insect's body. Suthisut et al. (2011) investigated
the toxicity of the compounds camphene, a-pinene, and b-pinene
to S. zeamais by topical application; and found that indeed, these
compounds, which correspond to 63.9% of the A. purpurata essen-
tial oil, were ineffective as contact insecticides.
Fumigation is the most commonly used method for pest control
in stored products because it is usually inexpensive, fast, effective
against insects at all life stages, and direct application to insects is
possible (Graver, 2004; Nenaah, 2014). The A. purpurata oil was less
effective than methyl bromide, which is able to kill S. zeamais adults
with LC50 of 0.67 mg/L (Liu and Ho, 1999). Nevertheless, methyl
bromide and phosphine, the two fumigants currently used, are
strongly toxic to humans (Yang et al., 1995; Nath et al., 2011). Thus,
finding a natural fumigant agent from A. purpurata inflorescences
for control of S. zeamais is attractive.
Yildirim et al. (2013) evaluated the fumigant toxicity of several
monoterpenes against S. zeamais and, interestingly, their results are
in agreement with ours as they detected insecticidal activity for
some of the compounds found in A. purpurata oil, including the
major components. The monoterpene hydrocarbons a-pinene, b-
pinene, and camphene caused a reduction in survival of approxi-
mately 50% in S. zeamais after 96 h, while limonene was more
active, inducing mortality of 79% in insects in this same time period
(Yildirim et al., 2013). These four compounds accounted for 67.89%
of A. purpurata oil composition, and thus, are probably linked to the
fumigant toxicity detected in our study. In regard to the oxygenated
monoterpenes found in A. purpurata oil, Yildirim et al. (2013) re-
ported that linalool was highly successful against S. zeamais,
causing 100% mortality in 72 h, and aeterpineol was also able to
increase mortality of all tested insects in 96 h. In A. purpurata oil,
these compounds account for a small fraction (1.13%), but since they
were reported as highly effective against S. zeamais adults, it is
plausible that they contributed greatly to the fumigant toxicity of
the oil.
When applied by fumigation, essential oils can be inhaled,
ingested, and/or absorbed through the cuticle by the insect
(Nenaah, 2014). Ogendo et al. (2011) suggested that toxicity by
fumigation of essential oil terpenoids on Tribolium castaneum
Herbst may be linked to induction of cells changes and blocking of
octopamine receptors, a neuromodulator/neurohormone present
in all invertebrates. Coitinho et al. (2011) also detected a fumigant
99
effect of essential oils from several plants against S. zeamais.
A. purpurata oil did not act as a repellent to maize weevil adults.
It is probable that if the S. zeamais adults detected the presence of
the oil, it was not interpreted by them as a cue to avoid exploitation
of the site containing the oil. In addition, the absence of repellent
effects of the A. purpurata oil may be linked to a relatively high
volatility similar to that reported for the essential oil from Evodia
rutaecarpa Benth., which loses its repellent activity when evapo-
rated for 5 min or longer (Liu and Ho, 1999). On the other hand, in
the fumigant assay employed here, the evaporation time before the
oil was trapped in the sealed chambers was only 30 s, which
probably allowed the oil to cause insect mortality.
Studies have focused on nanoformulations of essential oils
aiming at minimize loss by evaporation or degradation, facilitate
handling, and establish conditions for controlled release (Gonz
alez
et al., 2014). The fumigant toxicity of A. purpurata essential oil
should lead to future studies evaluating possible formulations to be
used for S. zeamais control.
In conclusion, the essential oil from A. purpurata inflorescences
has the potential to be used in control of S. zeamais since it was toxic
to adult insects by fumigation, and exerted anti-nutritional effects
when ingested. The insecticidal effect is probably due to the activity
of some monoterpene hydrocarbons and oxygenated mono-
terpenes, which were previously reported to act as insecticides
against the maize weevil. A. purpurata essential oil can act as an
alternative, without the need to isolate these compounds. The
seasonal variation in the composition of A. purpurata oil, in com-
parison with previous work, highlights the importance of chemical
standardization strategies in the use of essential oils as insecticides.
Acknowledgments
The authors express their gratitude to the Conselho Nacional de
Desenvolvimento Científico e Tecnolo
gico (CNPq; 472546/2012-0),
the Fundaça ~o de Amparo a Ci^
encia e Tecnologia do Estado de Per-
nambuco (FACEPE; PRONEX/2008-FACEPE) and the Brazilian Min-
istry of Science, Technology and Innovation (MCTI; 01200.003711/
2011-11) for financial support. E.V. Pontual would like to thank
FACEPE and CAPES for post-doctoral scholarship (APQ-0137-2.08/
12; BCT-0421-2.08/12). P.M.G. Paiva and J.V. Oliveira would like to
thank CNPq for research grants and fellowship.
References
Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatog-
raphy Quadupole Mass Spectroscopy. Allures Publishing Coorporation, Illinois.
Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of
essential oils e a review. Food Chem. Toxicol. 46, 446e475.
Botton, M., Lorini, I., Afonso, A.P.S., 2005. Ocorre ^ncia de Sitophilus zeamais Mots.
(Coleoptera: Curculionidae) danificando a cultura da videira no Rio Grande do
Sul. Neotropical Entomol. 34, 355e356.
Carvalho, M.O., Pires, I., Barbosa, A., Barros, G., Riudavets, J., Garcia, A.C., Brites, C.,
Navarro, S., 2012. The use of modified atmospheres to control Sitophilus zeamais
and Sitophilus oryzae on stored rice in Portugal. J. Stored Prod. Res. 50, 49e56.
Celiktas, O.Y., Kocabas, E.E.H., Bedir, E., Sukan, F.V., Ozek, T., Baser, K.H.C., 2007.
Antimicrobial activities of methanol extracts and essential oils of Rosmarinus
officinalis, depending on location and seasonal variations. Food Chem. 100,
553e559.
Chu, S.S., Liu, Q.R., Liu, Z.L., 2010. Insecticidal activity and chemical composition of
the essential oil of Artemisia vestita from China against Sitophilus zeamais.
Biochem. Syst. Ecol. 38, 489e492.
Coitinho, R.L.B.C., Oliveira, J.V., Gondim Junior, M.G.C., Ca ^mara, C.A.G., 2011. Tox-
icidade por fumigaça ~o, contato e ingest~ ao de o
leos essenciais para Sitophilus
zeamais Motschulsky, 1885 (Coleoptera: Curculionidae). Cie ^ncia Agrotecnologia
35, 172e178.
Demissie, G., Tefera, T., Tadesse, A., 2008. Efficacy of Silicosec, filter cake and wood
ash against the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera:
Curculionidae) on three maize genotypes. J. Stored Prod. Res. 44, 227e231.
Dorman, H.J.D., Deans, S.G., 2000. Antimicrobial agents from plants: antibacterial
activity of plant volatile oils. J. Appl. Microbiol. 88, 308e316.
Gonza
lez, J.O.W., Gutie
rrez, M.M., Ferrero, A.A., Band, B.F., 2014. Essential oils
100 C.S. de Lira et al. / Crop Protection 71 (2015) 95e100
nanoformulations for stored-product pest control e characterization and bio-
logical properties. Chemosphere 100, 130e138.
Gonza
lez, S., Pino, O., Herrera, R.S., Valenciaga, N., Fortes, D., Sa
nchez, Y., 2011.
Potencials of the powders of Lonchocarpus punctatus in the control of Sito-
philus zeamais. Cuban J. Agric. Sci. 45, 89e94.
Graver, J.E., van, S., 2004. Guide to Fumigation under Gas-Proof Sheets. Food and
Agricultural Organization of the United Nations, Rome.
Huang, Y., Tan, J.M.W.L., Kini, R.M., Ho, S.H., 1997. Toxic and antifeedant action of
nutmeg oil against Tribolium castaneum (Herbst) and Sitophilus zeamais Motsch.
J. Stored Prod. Res. 33, 289e298.
Huang, Y., Hee, S.K., Ho, S.H., 1998. Antifeedant and growth inhibitory effects of a-
pinene on the stored-product insects, Tribolium castaneum (Herbst) and Sito-
philus zeamais Motsch. Int. Pest Cont. 40, 18e20.
Hussain, A.I., Anwar, F., Sherazi, S.T.H., Przybylski, R., 2008. Chemical composition,
antioxidant and antimicrobial activities of basil (Ocimum basilicum) essential
oils depends on seasonal variations. Food Chem. 108, 986e995.
Ileleji, K.E., Maier, D.E., Woloshuk, C.P., 2007. Evaluation of different temperature
management strategies for suppression of Sitophilus zeamais (Motschulsky) in
stored maize. J. Stored Prod. Res. 43, 480e488.
Isman, M.B., 2006. Botanical insecticides, deterrents, and repellents in modern
agriculture and an increasingly regulated world. Annu. Rev. Entomology 51,
45e66.
Isman, M.B., Koul, O., Luczynski, A., Kaminski, J., 1990. Insecticidal and antifeedant
bioactivities of neem oils and their relationship to azadirachtin content. J. Agric.
Food Chem. 38, 1406e1411.
Kedia, A., Prakash, B., Mishra, P.K., Chanotiya, C.S., Dubey, N.K., 2014. Antifungal,
antiaflatoxigenic, and insecticidal efficacy of spearmint (Mentha spicata L.)
essential oil. Int. Biodeterior. Biodegrad. 89, 29e36.
Kehinde, K., Angela, O., 2004. Comparative biological activity of Synzygium aro-
matieum (L.) and Xylopia efhiopiea on Rhizopetha dominica F. (Coleoptera: Bos-
trychidae) and Sitophilus zeamais Motsch (Coleoptera: Curculionidae) in maize
grains. J. Asia-Pacific Entomology 7, 339e342.
Liu, Z.L., Ho, S.H., 1999. Bioactivity of the essential oil extracted from Evodia
rutaecarpa Hook f. et Thomas against the grain storage insects, Sitophilus zea-
mais Motsch. and Tribolium castaneum (Herbst). J. Stored Prod. Res. 35,
317e328.
Liu, Z.L., Goh, S.H., Ho, S.H., 2007. Screening of Chinese medicinal herbs for bioac-
tivity against Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst).
J. Stored Prod. Res. 43, 290e296.
Lorini, I., 2008. Manejo Integrado de Pragas de Gra ~os de Cereais Armazenados
(Embrapa Trigo, Passo Fundo).
Mazzonetto, F., Vendramim, J.D., 2003. Efeito de po
s de origem vegetal sobre
Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae) em feija ~o armazenado.
Neotrop. Entomol. 32, 145e149.
Medeiros, S.R.R., Moura, G.B.A., Giongo, P.R., Silva, A.P.N., 2009. Potencial agro-
clim
atico para a Alpinia purpurata, no Estado de Pernambuco. Rev. Bras. Eng.
Agrícola Ambient. 13, 165e169.
Miranda, M.M.M., Araújo, J.M., Picanço, M., Faleiro, F.G., Machado, A.T., 1995.
Detecça ~o de na ~o-prefere
^ncia a Sitophilus zeamais Mots. em espigas e gr~ aos de
49 populaço ~es de milho. Rev. Bras. Armazenamento 20, 21e25.
Napole~ ao, T.H., Belmonte, B.R., Pontual, E.V., Albuquerque, L.P., S
a, R.A., Paiva, L.M.,
Coelho, L.C.B.B., Paiva, P.M.G., 2013. Deleterious effects of Myracrodruon urun-
deuva leaf extract and lectin on the maize weevil, Sitophilus zeamais (Coleop-
tera, Curculionidae). J. Stored Prod. Res. 54, 26e33.
Nath, N.S., Bhattacharya, I., Tuck, A.G., Schlipalius, D.I., Ebert, P.R., 2011. Mechanisms
of phosphine toxicity. J. Toxicol. 2011, 494168.
Navarro, D.M.A.F., Silva, P.C.B., Silva, M.R., Napolea ~o, T.H., Paiva, P.M.G., 2013. Lar-
vicidal activity of plant and algae extracts, essential oils and isolated chemical
constituents against Aedes aegypti. Nat. Prod. J. 3, 268e291.
Nenaah, G.E., 2014. Chemical composition, toxicity and growth inhibitory activities
of essential oils of three Achillea species and their nano-emulsions against
Tribolium castaneum (Herbst). Industrial Crops Prod. 53, 252e260.
Noomhorm, A., Sirisoontaralak, P., Uraichuen, P., Ahmad, I., 2009. Effects of pres-
surized carbon dioxide on controlling Sitophilus zeamais (Coleoptera: Curcu-
lionidae) and the quality of milled rice. J. Stored Prod. Res. 45, 201e205.
Ogendo, J.O., Deng, A.L., Kostyukovsky, M., Ravid, U., Matasyoh, J.C., Omolo, E.O.,
Kariuki, S.T., Kamau, A.W., Bett, P.K., Shaaya, E., 2011. Biocontrol potential of
selected plant essential oil constituents as fumigants of insect pests attacking
stored food commodities. Baraton Interdisciplinary Research Journal 1, 13e19.
Pandey, A., Chattopadhyay, P., Banerjee, S., Pakshirajan, K., Singh, L., 2012. Anti-
termitic activity of plant essential oils and their major constituents against
termite Odontotermes assamensis Holmgren (Isoptera: Termitidae) of North East
India. Int. Biodeterior. Biodegrad. 75, 63e67.
Perez-Mendonza, J., 1999. Survey of insecticide resistance in Mexican populations of
maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae).
J. Stored Prod. Res. 35, 107e115.
Sampson, B.J., Tabanca, N., Kirimer, N., Demirci, B., Baser, K.H.C., Khan, I.A.,
Spiers, J.M., Wedge, D.E., 2005. Insecticidal activity of 23 essential oils and their
major compounds against adult Lipaphis pseudobrassicae (Davis) (Aphididae:
Homoptera). Pest Management Science 61, 1122e1128.
Santos, G.K.N., Dutra, K.A., Barros, R.A., Ca ^mara, C.A.G., Lira, D.D., Gusma ~o, N.B.,
Navarro, D.M.A.F., 2012. Essential oils from Alpinia purpurata (Zingiberaceae):
chemical composition, oviposition deterrence, larvicidal and antibacterial ac-
tivity. Industrial Crops Prod. 40, 254e260.
Simo

~ es, C.M.O., Spitzer, V., 2004. Oleos vola
teis. In: Simo~es, C.M.O., Schenkel, E.P.,
Gosmann, G., Mello, J.C.P., Mentz, L.A., Petrovick, P.R. (Eds.), Farmacognosia: da
planta ao medicamento. Editora da UFRGS/Editora da UFSC, pp. 468e495. Porto
Alegre/Floriano
polis.
Soliman, F.M., Yousif, M.F., Zaghloul, S.S., Okba, M.M., 2009. Seasonal variation in the
essential oil composition of Origanum majorana L. cultivated in Egypt. Z. für
Naturforsch. C 64, 611e614.
Suthisut, D., Fields, P.G., Chandrapatya, A., 2011. Fumigant toxicity of essential oils
from three Thai plants (Zingiberaceae) and their major compounds against
Sitophilus zeamais, Tribolium castaneum and two parasitoids. J. Stored Prod. Res.
47, 222e230.
Ukeh, D.A., Birkett, M.A., Pickett, J.A., Bowmana, A.S., Luntz, J.M., 2009. Repellent
activity of alligator pepper, Aframomum melegueta, and ginger, Zingiber offici-
nale, against the maize weevil, Sitophilus zeamais. Phytochemistry 70, 751e758.

rio, C.P., Leita
Victo ~o, S.G., Lage, C.L.S., 2010. Chemical composition of the leaf oils of
Alpinia zerumbet (Pers.) Burtt et Smith and A. purpurata (Vieill) K. Schum. from
Rio de Janeiro, Brazil. J. Essent. Oil Res. 22, 52e54.
Xie, Y.S., Bodnaryk, R.P., Fields, P.G., 1996. A rapid and simple flour-disk bioassay for
testing substances active against stored-product insects. Can. Entomol. 128,
865e875.
Yang, R.S., Witt, K.L., Alden, C.J., Cockerham, L.G., 1995. Toxicology of methyl bro-
mide. Rev. Environ. Contamin. Toxicol. 142, 65e85.
Yildirim, E., Emsen, B., Kordali, S., 2013. Insecticidal effects of monoterpenes on
Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). J. Appl. Bot. Food
Qual. 86, 198e204.
Yuya, A.I., Tadesse, A., Azerefegne, F., Tefera, T., 2009. Efficacy of combining Niger
seed oil with malathion 5% dust formulation on maize against the maize weevil,
Sitophilus zeamais (Coleoptera: Curculionidae). J. Stored Prod. Res. 45, 67e70.