Insecticidal and repellent activity of essential oil from Amomum villosum Lour. and its main compounds against two stored-product insects

ABSTRACT The Amomum villosum essential oil was obtained from hydrodistillation and was investigated by GC-MS. The main constituents were bornyl acetate (51.6%), camphor (19.8%), camphene (8.9%) and limonene (6.2%). Insecticidal toxicity of the essential oil was evaluated in this study. It showed that the essential oil possessed contact toxicity against Tribolium castaneum and Lasioderma serricorne (LD50 = 32.4 and 20.4 μg/adult). Three monoterpenoids camphor, camphene and limonene showed strong fumigant toxicity against T. castaneum (LC50 ＜ 2.3, LC50 ＝ 6.2 and 6.2 mg/L air). In addition, repellency of the essential oil was also evaluated. Data showed that the essential oil and all four compounds had repellent activity against T. castaneum and L. serricorne at high concentration (78.63 nL/cm2). But with the decrease of concentration, they showed a different degree of attractant properties.


Introduction
Stored-product insects are serious pests of oilseeds, stored pulses, cereals crops, of many value-added food products and nonfood derivatives of agricultural products. [1,2] These pests are worldwide distributed and cause huge economic losses. [3][4][5][6][7] Tribolium castaneum (Herbst) is among the most economically important stored-product pests. [8] It is one of the common pests infesting many indoor food storage facilities. [9] Infestations not only cause economic losses because of the consumption of grains; they also result in elevated temperature and moisture conditions that lead to an accelerated growth of molds, including toxigenic species. [10] Lasioderma serricorne is widely distributed in tropical and subtropical regions. The cigarette beetle is a strong flyer and highly mobile, and has been scattered from the food warehouse to other habitats in the field. [11] The females lay eggs masses in the crevice of tobacco bales and its eggs can survive during the cigarette production. Its larvae are quick and feed on tobacco leaves. [12,13] Besides being a primary pest of tobacco, the adults of L. serricorne also infests packaged foods and cause problems in food products as dry fruits, crumbs, grains and flours. [11,14] Chemical control using synthetic insecticides have been well applied because of their rapid action and ease of use. [15] However, the widespread extensive use has many negative consequences (i.e., insecticide resistance, toxicity to mammals and other non-target animals, residue problems, environmental pollution). [11,14] Therefore, it is extremely important to develop a safe, low toxicity to nontarget animals and environment friendly natural drug to control pests. Essential oils have received much attention as pest control agents. They are characterized by low toxicity to human and animals, high volatility and toxicity to insect pests of stored products. [16] Some literatures have shown that essential oil has good control over the red beetle and the tobacco beetle. [17,18] There are also numerous reports on insecticidal activity of the essential oils and other products from Zingiber species. [19][20][21][22] Amomum villosum Lour. belongs to the Zingiberaceae family and is widely distributed in the south of China. The fruit of A. villosum can be used for cooking and it also has a variety of medicinal value, for example, A. villosum essential oil and its main component bornyl acetate could inhibit intestinal mucositis. [23] And it can improve gastrointestinal function and start the production of digestive juice, as well as analgesic and antidiarrheal effects. [24] However, few studies have investigated bioactivity of the plant extract obtained from A. villosum against pests. Therefore, we decided to evaluate the insecticidal toxicity of the essential oil for the first time.

Plant material and extraction of essential oil
Fruits (1 kg) of A. villosum were collected in July 2013 from Yunnan Province, China. The species were identified according to the voucher specimen (BNU-CMH-Dushushan-2013-07-11-011) deposited at the Herbarium of College of Resources Science and Technology, Beijing Normal University. Fruits of A. villosum were subjected to hydrodistillation by a modified Clevenger-type apparatus for 6 h. After extraction, the water in the essential oil was removed by anhydrous sodium sulfate. The essential oil was stored in an closed container in 4°C refrigerator.

Insects
Cultures of T. castaneum and L. serricorne were maintained in the laboratory without exposure to any insecticide before use. The two storage insect pests were obtained in incubators about 30°C and 75% r.h in the dark. They were fed on wheat flour mixed with yeast (10:1, w/w) and the moisture content is about 12%. The unsexed insect adults used in the experiments were 1-2 weeks old.

GC-FID and GC-MS analysis
GC-MS analysis was performed on a Thermo Finnigan Trace DSQ instrument equipped with a flame ionization detector and an HP-5MS (30 m × 0.25 mm × 0.25 μm) capillary column. The column temperature was programmed at 50°C for 2 min, then increased at 2°C/min to the temperature of 150°C and held for 2 min, and then increased at 10°C/min until the final temperature of 250°C was reached, where it was held for 5 min. The injector temperature was maintained at 250°C and the volume injected was 1 μL of 1% solution (diluted in n-hexane). The carrier gas was helium at a flow rate of 1.0 mL/min. Spectra were scanned from 50 to 550 m/z. The retention indices were determined in relation to a homologous series of n-alkanes (C 5 -C 36 ) under the same operating conditions. GC retention time and their mass spectra that stored in NIST 05 and Wiley 275 libraries or from literature were used for identifying the essential oil compounds. [25] Relative percentages of the individual compounds were obtained by averaging the GC peak area % reports.

Contact toxicity
The essential oil and individual components against the two storage insect pests were measured as described by Liu and Ho. [9] The four compounds (L-bornyl acetate, D-camphor, camphene and D-limonene) were obtained from Beijing Inno Chem Science & Technology Co., Ltd., Beijing, China. Range-finding studies were run to determine the appropriate testing concentrations. A series of testing samples (five concentrations) were prepared in n-hexane for the essential oil and individual components. Aliquots of 0.5 µL of the dilutions were applied topically to the dorsal thorax of the insects. Pyrethrins (pyrethrin I and II, 37%) were used as positive controls and n-hexane were used as negative controls. Ten insects were used for each concentration and control, and five parallel experiments were carried out. Both treated and control insects were then transferred to glass vials and preserved in the incubators. Mortality was recorded after 24 h.

Fumigant toxicity
The appropriate testing concentrations were determined by range-finding studies. A serial dilution of the essential oil and compounds with five concentrations were prepared in n-hexane. The essential oils and above samples were tested with the method described by Liu and Ho. [9] Ten insects were put into a glass vial (diameter 2.5 cm, height 5.5 cm, volume 25 ml). A filter paper (diameter 2 cm) was placed at the bottom of cap and treated with 10 µL sample solution. The solvent was allowed to evaporate for 20 s before the cap was placed tightly on the glass vial to form a sealed chamber. n-Hexane was used as a control. Five repeated experiments were carried out for all treatments and controls, and they were incubated under the same conditions as rearing. The mortality was measured after 24 h.

Repellency tests
The repellent effects of the essential oil and its main compounds against T. castaneum and L. serricorne were assessed by using assays on Petri dishes. [26] Petri dishes 9 cm in diameter were used to confine insects during the experiment. The essential oil of A. villosum and its main compounds were prepared in n-hexane (78.63, 15.73, 3.15, 0.63, and 0.13 nL/cm 2 ). Filter paper 9 cm in diameter was cut in half and 500 μL of each concentration was applied separately to half of the filter paper as uniformly as possible. The other half (control) was treated with 500 μL of absolute n-hexane. 20 insects were released at the center of each filter disk, and a lid was covered on the Petri dish. Five parallel experiments were done, and each experiment was repeated three times. A commercial repellent, DEET (N,N-diethyl-3-methylbenzamide), was brought from the National Center of Pesticide Standards (8 Shenliao West Road, Tiexi District, Shenyang 110021, China) and used as a positive control. Counts of insects presented on each paper were made at 2 and 4 h after exposure. The percent repellency (PR) of the essential oil and individual compounds was then calculated using the formula: where Nc is the number of insects present in the negative control half and Nt is the number of insects present in the treated half. The averages were then assigned to different classes (0 to V) using the following scale (percentage repellency). [9] Class, % repellency: 0, > 0.

Data analysis
The data were corrected for control mortality using Abbott's formula. The LD 50 (Contact toxicity) and LC 50 (Fumigant toxicity) values were calculated by using Probit analysis (IBM SPSS V 20.0). [26,27] In repellency tests, means and standard errors were conducted by Microsoft Excel 2007 for Windows 7.

Results and discussion
Chemical composition of the essential oil A. villosum essential oil was light blue with a yield of 1.9% (v/w) and density of 1.01 g/mL. The chemical component of the essential oil was summarized in Table 1. A total of 17 components of the essential oil were identified. The quantity of each constituent was determined by GC-FID. The main constituents of the essential oil were bornyl acetate (51.6%), camphor (19.8%), camphene (8.9%) and limonene (6.2%). According to the previous paper about the essential oil from different origins of fruits of A. villosum, the results showed that their main components were similar but the contents were different. For example, A. villosum oil from Guangxi contains more bornyl acetate (69.3%) and camphor (20.9%) than from Guangdong (50.6% and 10.3%) and Vietnam (48.2% and 14.8%). [28] Chemical analysis indicated that bornyl acetate and camphor were the main components of A. villosum essential oil. The result was in agreement with ours. But our analysis revealed that there are more limonene (6.2%) than in Guangdong (1.0%), Vietnam (1.2%) and Guangxi (0.3%). In addition, camphene was not detected in the essential oil from other origins. These differences in chemical composition and content among the essential oils could be due to different effects of environmental factors (such as sunlight, water and soil) or may result from different metabolic pathways in the plant. Thus, further studies on plant cultivation and essential oil standardization are necessary.

Contact toxicity
The consequence of contact toxicity of A. villosum essential oil and its main constituents against the two stored-product insects were showen in Table 2. The essential oil of A. villosum showed contact toxicity against T. castaneum and L. serricorne adults with LD 50 values of 32.4 and 20.4 µg/adult, respectively ( Table 2). Compared with the positive control pyrethrins, the essential oil demonstrated 125 times less toxicity against the red flour beetle and 102 times less toxicity against the tobacco beetle. In this context, compared with other essential oils mentioned in the literature, the essential oil of A. villosum showed stronger contact toxicity against T. castaneum, e.g. essential oils of Ajania fruticulosa (LD 50 = 106 µg/adult), [29] Platycladus orientalis (LD 50 = 48.59 µg/adult) [30] and Acorus calamus (LD 50 = 32.55 µg/adult). [31] Moreover, compared with another essential oil from the ginger family in the previous studies, the essential oil of A. villosum exhibited stronger contact toxicity than Zingiber purpureum (LD 50 = 39.0 µg/adult). [32] In addition, the toxicity of limonene (LD 50 = 15.0 μg/adult) [33] and camphene (LD 50 = 21.6 μg/adult) were stronger than camphor (LD 50 > 50.0 μg/adult) [34] and bornyl acetate (LD 50 = 66.0 μg/adult). At the same time, our research showed that L. serricorne was more susceptible than T. castaneum to the contact toxicity of the essential oil of A. villosum. Moreover, there were many literatures showed that the A. Villosum essential oil exhibited stronger contact toxicity against L. serricorne than other essential oils mentioned in the literature, e.g. essential oils of Artemisia anethoides (LD 50 = 24.03 μg/adult), [35] Vitex negundo L. var. cannabifolia (Sieb. et Zucc.) Hand.-Mazz. (LD 50 = 25.30 μg/adult), [36] Forsythia suspensa (LD 50 = 23.66 μg/adult), [37] Laggera pterodonta (LD 50 = 32.97 μg/adult) [38] and Platycladus orientalis (LD 50 = 50.45 µg/adult). [30] In addition, compared with the other essential oils from the ginger family in the previous studies, the essential oil of A. villosum exhibited stronger contact toxicity against L. serricorne, e.g. essential oil of Alpinia kwangsiensis (LD 50 = 24.59 μg/adult) [39] and Zingiber zerumbet (L.) Smith (LD 50 = 48.3 μg/ adult). [40] The four monoterpenoids (bornyl acetate, camphor, camphene and limonene) exhibited fair contact toxicity against T. castaneum and L. serricorne respectively. e.g., limonene showed 50 and 68.5 times less toxicity to T. castaneum and L. serricorne when compared to pyrethrins. Monoterpenoids from plants are considered to have anticholinesterasic properties which cause high levels of mortality of insects when higher concentrations were used. [41] For instance, limonene exhibited contact toxicity with LD 50 values of 0.187 mg/adult and 0.283 mg/adult against S. zeamais and T. castaneum, respectively. [42] And from the data, we found that L. serricorne were more sensitive to the contact toxicity of monoterpenes than T. castaneum. The experimental results of linalool, β-pinene and 3carene also drew the same conclusion. [43] However, all the four compounds possessed stronger activity against L. serricorne adult than the essential oil, suggesting that there may be some other weaker active compounds in small amounts in the essential oil or may be some antagonistic action between the various compounds.

Fumigant toxicity
The essential oil of A. villosum fruits showed fumigant toxicity against T. castaneum adults with LC 50 values of 28.3 mg/L air (Table 3). Compared with the positive control MeBr (LC 50 = 1.8 mg/L air), essential oil was almost 16 times less toxic. In this context, compared with essential oil mentioned in the literature, the essential oil of A. villosum showed stronger fumigant toxicity against T. castaneum than Platycladus orientalis (LC 50 = 39.25 mg/L air). [30] And we had done fumigation tests with camphor [34] and limonene [33] under the same conditions. In order to further evaluate the relationship between the essential oil and its main compounds in the toxicity activity, we had direct references to the past data. Analysis of the data showed that the compounds camphor, camphene and limonene had strong contact toxicity against T. castaneum adults (LC 50 < 2.3 mg/L air, LC 50 = 6.2 and 6.2 mg/L air, respectively). There were many literatures also showed that the three compounds had good fumigant toxicity for other storage pests such as Sitophilus oryzae, [44,45]  * data from Wang et al. [33] , ** data from Guo et al. [34] Sitophilus zeamais, [46,47] Rhyzopertha dominica [45,48] and Cryptolestes pusillus. [48] In addition, the three compounds with significant fumigation effect (camphor, camphene and limonene) all belong to monoterpenoids. We suspected T. castaneum may be very susceptible to the fumigation toxicity of some monoterpenoids. To prove this point, we had consulted many literatures. These results showed that other monoterpenoids, such as 1,8-cineole, menthone, pulegone, l-fenchone and linalool, also had significant fumigation activity to T. castaneum. [49,50] So the existence of the three monoterpenoids comprounds might be one of the key elements which affected the fumigant toxicity of A. villosum essential oil against T. castaneum. However, in our preliminary experiments, fumigating activity of essential oil on L. serricorne was less effective for 24 h, the differences in mortality between T. castaneum and L. serricorne maybe because the susceptibility of different insects to essential oils constituents can vary depending on endogenous and exogenous factors.

Repellent activity
The repellent activities of the essential oil from A. villosum and its major constituents to T. castaneum adults were tested using the area preference method 2 h and 4 h after treatment ( Figure 1). Data showed that at 78.63 nL/cm 2 , the essential oil of A. villosum, camphor and limonene showed strong repellent activity against T. castaneum with the percentage repellency (PR) over 70% after 2 h. The repellent activities of the essential oil of A. villosum and isolated constituents to L. serricorne adults were tested using the area preference method 2 h and 4 h after treatment (Figure 2). Data showed that at 78.63 nL/cm 2 , the essential oil of A. villosum, camphor, bornyl acetate, limonene showed repellent activity against L. serricorne with the PR over 70% after 2 h. Compared with the positive control DEET and camphor showed the same level (Class V) of repellency after 2 h treatment. However, at the dose of 15.73 nL/cm 2 and 0.13 nL/cm 2 , the essential oil of A. villosum showed strong attractant properties (PR = −56% and −58%) and at the dose of 15.73 nL/cm 2 , 3.15 nL/cm 2 and 0.63 nL/cm 2 , bornyl acetate showed strong attractant properties (PR = −42%, −62%  [34] , ** data from Wang et al. [33] , *** data from Liu et al. [9] and −46%). Consulting the past literature, borneol acetate also showed similar attraction effect to L. serricorne. [51] At almost all tested concentration, the essential oil and the constituents show the weak repellency at 4 h after exposure. And at 3.15 nL/cm 2 , camphor and bornyl acetate showed insect attractant properties (PR = −42% and −52%). The results showed that the essential oil had repellent activity against T. castaneum and L. serricorne at high concentration (78.63 nL/cm 2 ). However, with the decreasing of concentration, the repellent effect of the essential oil was also reduced, even attractant properties appeared. On T. castaneum larvae, Tagetes terniflora, Cymbopogon citratus and Elyonorus muticus essential oils also showed repellent action at high concentrations while attractive at low concentrations. [52] The same substance can behave as repellent or attractant, depending on the conditions used in the bioassay. [53] And the attractant effects of isolated constituents maybe had made a negative effect on the essential oil. But change an angle to consider this problem, the strong attraction of borneol acetate at 0.13 nL/cm 2 can provide useful information for developing attractive chemicals for T. castaneum.

Conclusion
The chemical composition of the essential oil from A. villosum fruits was analyzed. And the contact, fumigant and repellent activities of the essential oil against two stored-product insects were reported for the first time. This work investigated the insecticidal bioactivity of the essential oil of A. villosum fruits against T. castaneum and L. serricorne. However, further studies are needed to focus on the safety of the essential oil to humans and to improve the potency and stability of these crop protection products for practical use.