# Composition, Repellent, and Insecticidal Activities of Two South American Plants against the Stored Grain PestsTribolium castaneum and Tribolium confusum (Coleoptera: Tenebrionidae)

Authors: Verónica Benzi; Natalia Stefanazzi; Ana Paula Murray; Jorge O. Werdin González; Adriana Ferrero
Journal: ISRN Entomology
 (2014)

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## Abstract

As part of a screening program to evaluate the biological activity of indigenous plants, we report the composition and the bioactivity of essential oils (EOs) extracted from Té de BurroAloysia polystachya [(Griseb.) Moldenke] and Lemon Verbena Aloysia citriodora [Palau] against two of the most widespread secondary pests of stored products, the red flour beetle Tribolium castaneum [Herbst] and the confused flour beetle Tribolium confusum [Jacqueline du Val]. Analysis by gas chromatography-mass spectrometry of the EOs led the identification of their major constituents and their relative proportions. EO of A. citriodora showed the highest repellent activity against both beetles (>70%). On the other hand, both plants showed fumigant toxicity only against T. confusum, without significant differences between them (LC50 values of 5.92 and 5.53 mg/L air for A. polystachya and A. citriodora, resp.). For contact toxicity (topical applications) the EO of A. polystachya was more effective (LD50 = 7.35 μg/insect) than the EO of A. citriodora (LD50 = 13.8 μg/insect) only against T. castaneum. On the other hand, T. confusum was not susceptible by contact to any of these EOs. These results provide important tools for the development of an Integrated Pest Management (IPM) program.


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## Body

## 1. Introduction

The red flour beetleTribolium castaneum (Herbst) and the confused flour beetle Tribolium confusum(Duval) (Coleoptera: Tenebrionidae) are the most widespread and destructive secondary pests of stored grains and grain-derived products. They have been reported as serious pests in Argentina. Particularly, T. castaneum has been found as one of the most prevalent secondary pests in port areas of Buenos Aires province [1]. Control of these insects is primarily dependent upon continuous application of synthetic insecticides, which produce disturbance in the environment, increasing costs of application, pest resurgence, pest resistance, and lethal effects on nontarget organisms, in addition to direct toxicity to users. Thus, the risks associated to the use of these products have led to the growth of environmentally sustainable alternatives.In the last couple of decades, agrochemical companies have promoted the study of natural products for the development of new insecticides. An evidence of that is the number of organic agriculture products that reached the market [2, 3]. Thus, insecticides of natural origin are proposed as rational alternatives to synthetic ones and, among the biopesticides, essential oils (EOs) are growing rapidly on the botanical pesticide markets [4]. The diversity in their composition and mixture of compounds enhance their insecticide efficacy and reduce the evolution of tolerance and resistance to these products. For this purpose, biological activities of diverse plants have been recorded by several authors [5–9]. EOs and their constituents exert insecticidal effects, repel or deter insect food consumption, or reduce and disrupt insect growth. Several species of Verbenaceae have such EOs with biological properties [10–12]. In particular, the genus Aloysiaincludes approximately 200 species of herbs, shrubs, and small trees. Aloysia polystachya (Griseb.) Moldenke, and Aloysia citriodora (Palau) are native from Argentina and are distributed throughout South and Central America and tropical Africa. The EOs and extracts from these species are valued medicinally, and also their aromatic properties in culinary and cosmetic industries, due to the presence of phenolic compounds (flavonoids) [13]. Moreover, terpenes extracted from these plants have been shown to have important ecological roles in plant defense and attract pollinators, and they have been reported as repellents and insecticides against several pests [4, 14].As part of a screening program to evaluate the bioactivity of native plants as insect control agents, we report the composition, repellent activity, and fumigant and contact toxicity of EOs ofA. polystachya and A. citriodora against adults of T. confusumand T. castaneum.


## 2. Materials and Methods

### 2.1. Insects

T. castaneum and T. confusum are both insecticide-susceptible strains that have been reared in laboratory culture since 1995 and 2005, respectively. Adults of both species were reared in incubators at 28 ± 1°C and 70–80% R.H. in the dark and fed on a mixture of wheat, yeast, and milk in dust (13 : 1 : 1 w : w·w), in glass containers of 500 cm3.


### 2.2. Extraction and Analysis of the Composition of Essential Oils

Young fresh leaves fromA. polystachya were collected from wild plants, during the summer period at Lamarque city, Rio Negro Province, Argentina (39° 24′S. 65° 42′W.). Young fresh leaves from A. citriodora were collected from wild plants in Bahía Blanca, Buenos Aires Province, Argentina (38° 44′S. 62° 16′W.) Leaves from both plants were collected into plastic bags and kept in the freezer until the extraction. EOs were extracted using a Clevenger-type apparatus by hydro-distillation for 4 h. After the extraction, the EOs were dried using anhydrous sodium sulphate and stored in the dark at 4°C. Plant oils yields were 0.54% (mg g−1) for A. polystachya and 0.34% (mg g−1) for A. citriodora.The essential oils composition was determined by gas chromatography-mass spectrometry (GC-MS) using a Hewlett-Packard 5890 Series II gas chromatograph, equipped with a HP-5972 (EI-70 eV) mass selective detector and a 25 m × 0.25 mm i.d., 0.25μm film thickness HP-5MS capillary column with the injection block set 250°C. The GC oven temperature was held at 50°C for 2 min, programmed at 5°C min−1 to 200°C, and then held at this temperature for 10 min. The FID detector temperature was set at 300°C. The carrier gas was He (1 mL min−1, split ratio 1 : 50). Aliquots of the essential oils were dissolved in diethyl ether (injection of 2 μL) for the analysis. Oil components were identified by comparison of their Kobats retention indices with those of known compounds and also by comparison of their mass spectra with those stored in the NBS75K.LMS 86 database.


### 2.3. Bioassays

All bioassays were set up in laboratory under natural light and controlled temperature and humidity (28 ± 1°C and 70–80% R.H). Ten Adults of each species of 3–5 days old were used for each treatment and control. Five independent replicates were conducted, and all the experimental units were placed at 28 ± 1°C and 70–80% R.H., in the dark.


### 2.4. Repellency

Repellency test was conducted according to Talukder and Howse [15]. Filter papers (Whatman N° 1, diameter 9 cm) were divided into two halves. One half was impregnated with 0.5 mL of either EO diluted in n-hexane (treatment) or n-hexane (control). The concentrations evaluated were 90, 120, and 314 μg/cm2. Paper disks were air dried, and then placed inside a Petri dish. Ten adult insects were released in the middle of each disk and covered with plastic tape with some holes to prevent insects from escaping. The number of insects on each half of the paper was counted at hourly intervals for 5 h. The distribution coefficient (DC) was calculated using the formula DC
=
[
(
N
c
-
N
t
)
/
(
N
c
+
N
t
)
]
×
100, where N
c and N
t are the number of insects found on the control and treated zone, respectively [16]. The average values were then categorized according to the scale in Table 1.Table 1
Repellency scale from the less to the most repellent = 0 to V.
Class
DC (%)
0
<0.1
I
0.1 to 20
II
20.1 to 40
III
40.1 to 60
IV
60.1 to 80
V
80.1 to 100
DC: average distribution coefficient.


### 2.5. Fumigant Toxicity

To determine the fumigant toxicity, filter papers (5 cm2 area) were impregnated with EOs diluted in n-hexane and controls with n-hexane alone. Each filter paper was attached to a glass vial, covered with a fine mesh cloth. These vials were then introduced inside a glass flask of 40 mL. Ten adults of each species were placed inside the flask. Concentrations evaluated were from 1 to 12 mg/Lair. Mortality was evaluated 72 h after treatment [17].


### 2.6. Contact Toxicity

For topical applications, aliquots (0.2μL per insect) were applied ventrally to the thorax of adults using a microapplicator. Controls were determined using n-hexane. Both treated and control insects were then transferred to glass vials (10 insects per vial). Ten insects were used for each concentration and control. Concentrations evaluated were from 4 to 18 μg/insect. Five independent replicates were conducted. Mortality was registered after 72 h.


### 2.7. Statistical Analysis

Data from repellency assays (DC) were analyzed using analysis of variance (ANOVA) and minimum significant difference (MSD). Probit analysis was used to estimate LC50 and DL50 values by MicroProbit 3.0. Mortality values were corrected with Abbott’s formula [18] to eliminate natural mortality of control.


## 2.1. Insects

T. castaneum and T. confusum are both insecticide-susceptible strains that have been reared in laboratory culture since 1995 and 2005, respectively. Adults of both species were reared in incubators at 28 ± 1°C and 70–80% R.H. in the dark and fed on a mixture of wheat, yeast, and milk in dust (13 : 1 : 1 w : w·w), in glass containers of 500 cm3.


## 2.2. Extraction and Analysis of the Composition of Essential Oils

Young fresh leaves fromA. polystachya were collected from wild plants, during the summer period at Lamarque city, Rio Negro Province, Argentina (39° 24′S. 65° 42′W.). Young fresh leaves from A. citriodora were collected from wild plants in Bahía Blanca, Buenos Aires Province, Argentina (38° 44′S. 62° 16′W.) Leaves from both plants were collected into plastic bags and kept in the freezer until the extraction. EOs were extracted using a Clevenger-type apparatus by hydro-distillation for 4 h. After the extraction, the EOs were dried using anhydrous sodium sulphate and stored in the dark at 4°C. Plant oils yields were 0.54% (mg g−1) for A. polystachya and 0.34% (mg g−1) for A. citriodora.The essential oils composition was determined by gas chromatography-mass spectrometry (GC-MS) using a Hewlett-Packard 5890 Series II gas chromatograph, equipped with a HP-5972 (EI-70 eV) mass selective detector and a 25 m × 0.25 mm i.d., 0.25μm film thickness HP-5MS capillary column with the injection block set 250°C. The GC oven temperature was held at 50°C for 2 min, programmed at 5°C min−1 to 200°C, and then held at this temperature for 10 min. The FID detector temperature was set at 300°C. The carrier gas was He (1 mL min−1, split ratio 1 : 50). Aliquots of the essential oils were dissolved in diethyl ether (injection of 2 μL) for the analysis. Oil components were identified by comparison of their Kobats retention indices with those of known compounds and also by comparison of their mass spectra with those stored in the NBS75K.LMS 86 database.


## 2.3. Bioassays

All bioassays were set up in laboratory under natural light and controlled temperature and humidity (28 ± 1°C and 70–80% R.H). Ten Adults of each species of 3–5 days old were used for each treatment and control. Five independent replicates were conducted, and all the experimental units were placed at 28 ± 1°C and 70–80% R.H., in the dark.


## 2.4. Repellency

Repellency test was conducted according to Talukder and Howse [15]. Filter papers (Whatman N° 1, diameter 9 cm) were divided into two halves. One half was impregnated with 0.5 mL of either EO diluted in n-hexane (treatment) or n-hexane (control). The concentrations evaluated were 90, 120, and 314 μg/cm2. Paper disks were air dried, and then placed inside a Petri dish. Ten adult insects were released in the middle of each disk and covered with plastic tape with some holes to prevent insects from escaping. The number of insects on each half of the paper was counted at hourly intervals for 5 h. The distribution coefficient (DC) was calculated using the formula DC
=
[
(
N
c
-
N
t
)
/
(
N
c
+
N
t
)
]
×
100, where N
c and N
t are the number of insects found on the control and treated zone, respectively [16]. The average values were then categorized according to the scale in Table 1.Table 1
Repellency scale from the less to the most repellent = 0 to V.
Class
DC (%)
0
<0.1
I
0.1 to 20
II
20.1 to 40
III
40.1 to 60
IV
60.1 to 80
V
80.1 to 100
DC: average distribution coefficient.


## 2.5. Fumigant Toxicity

To determine the fumigant toxicity, filter papers (5 cm2 area) were impregnated with EOs diluted in n-hexane and controls with n-hexane alone. Each filter paper was attached to a glass vial, covered with a fine mesh cloth. These vials were then introduced inside a glass flask of 40 mL. Ten adults of each species were placed inside the flask. Concentrations evaluated were from 1 to 12 mg/Lair. Mortality was evaluated 72 h after treatment [17].


## 2.6. Contact Toxicity

For topical applications, aliquots (0.2μL per insect) were applied ventrally to the thorax of adults using a microapplicator. Controls were determined using n-hexane. Both treated and control insects were then transferred to glass vials (10 insects per vial). Ten insects were used for each concentration and control. Concentrations evaluated were from 4 to 18 μg/insect. Five independent replicates were conducted. Mortality was registered after 72 h.


## 2.7. Statistical Analysis

Data from repellency assays (DC) were analyzed using analysis of variance (ANOVA) and minimum significant difference (MSD). Probit analysis was used to estimate LC50 and DL50 values by MicroProbit 3.0. Mortality values were corrected with Abbott’s formula [18] to eliminate natural mortality of control.


## 3. Results and Discussion

Analysis by gas chromatography-mass spectrometry of the steam distilled oils showed that the major components ofA. polystachya were carvone (83.5%) and limonene (16.5%), while A. citriodorawas mainly made up of citronellal (51.29%) and sabinene (22.93%) (Table 2).Table 2
Major identified constituents ofAloysia polystachya and Aloysia citriodoraessential oils and their relative proportion in the oils.
A. citriodora
A. polystachya
Constituents
corr.%
Constituents
corr.%
Citronellal
51.29
Carvone
83.5
Sabinene
22.93
Limonene
16.5
α-curcumene
9.57
Limonene
7.44
Caryophyllene
2.37
α-pinene
2.28
γ-cedrene
2.27
p-cymene
1.82Both EOs had repellent effects againstT. castaneum and T. confusum (Table 3). However, based on the repellency scale, the activity was stronger with A. citriodora, and it was higher than 70% to all concentrations evaluated (Table 3). The repellent effect of A. citriodora is possibly due to the presence of citronellal, which is one of the main constituents (>50%) and is a botanical compound use in commercial insect repellents [19]. Similar results were found by Olivero-Verbel et al. [20] when tested the repellent effect of Eucalyptus citriodora against T. castaneum, which main component is citronellal (40%). Indeed, the repellent effect of these plants has been also tested against other insects; for instance, Gillij et al. [11] found A. citriodora as one of the most promising EO of the fourteen plants test against Aedes aegypti. Moreover, Gleiser et al. [21] showed a dose-dependent activity of the EO of A. polystachyaagainst the same mosquito.Considering that the EO of A. citriodora contains also other compounds such as limonene (7.4%) with potential repellent effects, synergistic phenomena should not be discarded [22].Table 3
Average repellency of EOs ofA. polystachya and A.  citriodora against T. castaneumand T. confusum.
Average distribution coefficient (DC%)1
RCb
Plant
Insect
C
a
Hours after treatment
MRc
1
2
3
4
5
A. polystachya
T. castaneum
90
−13a
20a
13a
40a
20a
16
I
120
−6a
−46a
−60a
−66a
−80a
−51.6
0
314
46a
33a
66a
53a
46a
48.8
III
T.  confusum
90
93a
86a
73a
93a
93a
87.6
V
120
86a
40a
40a
20ab
33a
43.8
III
314
80a
60a
53a
46b
46b
57
III
A. citriodora
T. castaneum
90
80a
93a
93a
80a
100a
89.2
V
120
93a
86a
73a
100a
80a
74.4
IV
314
80a
80a
66a
93a
93a
82.4
V
T.  confusum
90
93a
100a
100a
100a
86a
95.8
V
120
80a
100a
93b
100a
100a
94.6
V
314
93a
86a
80b
80a
67a
81.2
V
1DC% = [
(
N
c
-
N
t
)
/
(
N
c
+
N
t
)
]
×
100; a
C: concentration (μg cm−2); bRC: repellency class; cMR: mean rate.
Numbers in the same column of each plant followed by the same letters do not differ significantly in ANOVA test.For fumigant assay, both EOs were toxic againstT. confusum but there were no significant differences (P
>
0.05) between them. However, there were not lethal effects against T. castaneum(Table 4). More studies must be undertaken to elucidate the differences found between the susceptibility of both beetles, but a possible explanation can be based on the differences in the respiration rates of insects. T. castaneum has a lower rate of air exchange and consequently a smaller diffusion of toxic compounds into the insect [23, 24].Table 4
Fumigant activity of EOs ofA. polystachyaand A. citriodoraagainst T.  confusum.
Essential oil
LC
50
a
95% CIc
LC
95
b
95% CIc
Slope ± SEd
X
2
A. polystachya
5.92a
(5.2–6.5)
11.9
(10.08–15.9)
5.37 ± 0.75
2.75
A. citriodora
5.53a
(2.6–6.8)
13.2
(10.5–30.6)
4.35 ± 1.39
0.12
a
LC
50: lethal concentration 50 (mg litre−1 air); b
LC
95: lethal concentration 95 (mg liter−1 air); cCI 95%: confidence interval of 95%; dSE: standard error. Numbers in the same column followed by the same letters do not differ significantly.For contact toxicity, no lethal effects were found against adults ofT. confusum. However, significant differences were found between EOs, against T. castaneum(P
<
0.05). A. polystachyashowed the highest toxicity (Table 5). Carvone, the main compound of A. polystachya, has been found to be toxic against diverse insects, for example, Fang et al. [25] found strong contact toxicity of carvone and limonene, against Sitophilus zeamais and T. castaneum. Moreover, carvone caused the highest mortality against larvae of T. castaneum among the several monoterpenes evaluated (LC50 = 19.8 μg/cm2) [26]. On the other hand, the concentration of these products has been found as an important factor that determines their toxicity, and it is directly proportional to the rate of penetration of a substance [27–29]. Carvone found in A. polystachya is denser than citronellal found in A. citriodora, thus this can be related with the differences found in the contact activity of both EOs. Similar results were found earlier with the EOs of A. polystachya against R. dominica (Fabricius) and Nezara viridula (L.) [12, 30].Table 5
Contact activity of EOs ofAloysia polystachya, and Aloysia citriodoradetermined by topical application to T.  castaneum.
Essential oil
LD
50
a
95% CIc
LD
95
b
95% CIc
Slope ± SEd
X
2
A. polystachya
7.35a
(6.38–8.5)
21.95
(16.6–34.5)
3.46
±
0.47
7.37
A. citriodora
13.8b
(9.5–43.7)
124
(41–158.2)
1.71
±
0.59
0.4
a
LD
50: lethal dose 50 (μg insect−1); b
LD
95: lethal dose 95 (μg insect−1); cCI 95%: confidence interval of 95%; dSE: standard error. Numbers in the same column followed by the same letters do not differ significantly.


## 4. Conclusions

This is the first report on the repellent and insecticidal activities of EOs ofA. citriodora and A. polystachya (Verbenaceae) against T. castaneum and T. confusum, two of the most widespread secondary pests in the world. The results present herein indicate that EOs extracted from these indigenous plants showed repellent and bioinsecticides properties. The EO of A. polystachya had the greatest contact toxicity against T. castaneum. Both EOs had the same fumigant activity against T. confusum.In summary, these EOs are good options for the control of stored grain pests but they need to be studied under commercial storage conditions.



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*Source: 175827-2014-02-20.xml*