Chemical composition and insecticidal properties of essential oil from aerial parts of Mosla soochowensis against two grain storage insects

Purpose: To determine the insecticidal properties of essential oil from Mosla soochowensis aerial parts against two insect pests, Sitophilus zeamais and Tribolium castaneum. Methods: Hydro-distillation of M. soochowensis was used to extract the essential oil. Gas chromatography/mass spectrometry (GC/MS) analysis was performed, and the contact (topical application) and fumigant toxicity (sealed space) of the essential oil were evaluated. Results: Thirty-nine chemical compounds were identified by GC-MS analysis of M. soochowensis essential oil. The major components are β-caryophyllene (12.82 %), spatulenol (6.34 %), β-eudesmol (6.26 %), carvone (6.12 %), α-thujone (5.12 %), γ-eudesmol (4.86 %), α-cedrol (4.23 %), and αcaryophyllene (4.04 %). The plant essential oil exerted contact toxicity against adults of S. zeamais and T. castaneum (median lethal concentration (LC50), 25.45 and 10.23 μg/adult, respectively). Moreover, the essential oil exhibited pronounced fumigant toxicity towards adults of both species (LC50 12.19 and 10.26 mg/L air, respectively). Conclusion: These results show that M. soochowensis essential oil can be used in development of safer and more natural and effective fumigants/insecticides for stored products.


INTRODUCTION
Contamination and destruction of stored food products by insects is a major problem for manufacturers and consumers, as the cost of such events runs to millions of dollars annually.In China, Sitophilus zeamais (Motsch.),commonly known as the maize weevil, and Tribolium castaneum (Herbst), commonly called the red flour beetle, have destructive effects on stored grain products [1], and infestations are typically controlled by fumigation techniques [2].However, excessive use of synthetic fumigants and insecticides has several detrimental effects, including but are not limited to insecticide resistance, environmental contamination, and damage to human health and other non-target organisms [2,3].Research into alternative treatments has shown that essential oils and their constituents possess insecticidal properties against many stored-product insect pests [4][5][6]; therefore, these may be alternatives to currently used fumigants and insecticides.

Mosla soochowensis
Matsuda (Family: Lamiaceae) is an annual plant distributed predominantly in Anhui, Jiangsu, Jiangxi, and Zhejiang Provinces in China [7].The aerial parts  [8,9] and the chemical composition of its essential oil [10,11].Nevertheless, a literature search revealed that the insecticidal effects of M. soochowensis essential oil on grain insects have not been reported.Therefore, this study aimed to determine the chemical composition of M. soochowensis and the effects of its essential oil on two insect species.

EXPERIMENTAL Plant material and extraction of essential oil
First, in August 2013, we gathered 5 kg of fresh M. soochowensis aerial parts at the flowering stage from Lishui (27.54 N, 119.20 E), located in Zhejiang Province, China.Dr. Wang from Lishui Academy of Forestry, Zhejiang, assisted with identification, A voucher specimen (category no.lsxy2013008) was kept at the herbarium of College of Ecology, Lishui University, Zhejiang, China.Samples were cut into pieces with pruning shears, and 900 g was transferred to three Lround-bottom flasks, each containing 1500 mL of tap water.The mixtures were then boiled for 6-8 hours, and the steam distilled.Because the essential oil is highly volatile, it was collected in a flask, separated from the aqueous layer using nhexane in a separation funnel, and stored at 4 C.Na 2 SO 4 was used to dry the extract, and the solvent was removed using a vacuum rotary evaporator.

Analysis of the essential oil
M. soochowensis essential oil was analyzed by gas chromatography-mass spectrometry (GC-MS) using a mass selective detector (Agilent 5973 N) and a non-polar HP-5ms capillary column of 30 m × 0.25 mm (film thickness, 0.25 µm).The column temperature program was as follows: hold at 60 C for 1 min; increase to 180 C at 10 C per min; hold at 180 C for 1 min; increase to 280 C at 20 C per min; and hold at 280 C for 15 min.The injector temperature was 270 C.Samples (1 μL were diluted 1:100 portion with acetone) were instilled with 1:10 split ratio.Helium at a flow rate of 1.0 mL per min was used as the carrier gas.Spectra were scanned from 20 to 550 m/z at 2 scans per second.Constituents were identified based on retention indices in the literature or data from our laboratory.The retention indices were estimated under identical operating conditions using a homologous series of n-alkanes (C 8 -C 24 ).Further identification was performed by comparing the mass spectra with those in the NIST 05 and Wiley 275 libraries or in the literature [12].Relative percentages were calculated based on GC peak areas, and no correction factor was applied.

Insects
The insects used in this study were from laboratory-reared colonies maintained for ≥10 years.T. castaneum adults were kept on wheat flour blended with yeast (10:1, w/w) at 29 -30 °C and 70 -80 % relative humidity, while S. zeamais adults were reared on whole hard wheat (12 -13 % moisture) under the same conditions.Adults insects used in this study had been growing for 2 weeks.All containers and petri dishes used were designed to prevent escape by the insects, and were coated with polytetrafluoroethylene (Fluon, Blades Biological, UK).

Test of contact toxicity
Liu and Ho [13] proposed a method for determining the contact toxicity of M. soochowensis essential oil.First, essential oil concentrations were evaluated.n-Hexane was used to serially dilute M. soochowensis essential oil to 3.5 -10.0 % v/w in six steps.A Burkard hand micro-applicator (Burkard Scientific, Uxbridge, UK) was used to apply 0.5 μL aliquots to the dorsal thorax of the insects.Ten treated insects were transferred to a glass bottle (25 mL) in five replicates.n-Hexane was used as a negative control, and pyrethrum extract (25 % pyrethrin I and pyrethrin II, Fluka Chemie AG, Switzerland) as a positive control.The treated and control insects were kept in incubators (29 -30 °C, 70 -80 % relative humidity) for 24 h without a food supply, and mortality was monitored.Insects were considered dead if they did not react to a gentle touch with a dissecting needle or did not walk during a 3 min examination.

Fumigant toxicity bioassay
Essential oil fumigant toxicity was confirmed using the method of Liu and Ho [13], with minor modifications.To determine suitable test concentrations of the essential oil, several rangefinding studies were conducted.n-Hexane was used to prepare six M. soochowensis essential oil solutions (3.0 -15.0 % v/v) using Whatman filter paper (2 cm diameter) and a screw-capped glass vial (2.5 cm diameter, 5.5 cm height, 24 mL volume).An aliquot of essential oil solution (10 μL) was dropped onto filter paper in the cap of a glass vial.The cap was then replaced on the glass vial, which contained 10 unsexed insects, and closed to form an airtight chamber.n-Hexane was used as a control.The treated and control insects were kept in incubators (29 -30 °C, 70 -80 % relative humidity) for 24 h without a food supply, and mortality was monitored.Insects were considered dead if they did not react to a gentle touch with a dissecting needle or did not walk during a 3 min examination.

Statistical analysis
Abbott's formula was applied to estimate corrected percent mortality.The PriProbit software ver.1.6.3 was used to determine LD 50 and LC 50 values and their 95 % confidence intervals [14].Samples with non-overlapping 95 % fiducial limits were regarded as significantly different.
The M. soochowensis essential oil showed significant contact toxicity against both insect species.However, the toxic effect was weaker than that for the positive control (Table 2).

CONCLUSION
The findings suggest that M. soochowensis essential oil exerts contact and fumigant toxic effects on two species of grain insect, suggesting its potential as a natural fumigant for stored products.Development of viable alternatives to commercially used, synthetic compounds is important because of the spread of resistance to these pesticides and their environmental toxicity.
To best of our knowledge no study has assessed the effect of M. soochowensis essential oil on human health.However, because aerial parts of this plant are consumed in China as a traditional medicine, the threat to human health is likely low.Further work should include evaluation of its nonspecific toxic effects.
of M. soochowensis are applied in Chinese folk medicine to cure the common cold, tonsillitis, abdominal pain, and heartburn, and are used in ointments to treat insect and centipede bites[7].

Table 1 :
Major compounds of Mosla soochowensis essential oil ** Identification by co-injection of authentic compounds

Table 2 :
Contact and fumigant toxicity of Mosla soochowensis essential oil against adults of S. zeamais (SZ) and T. castaneum (TC) a p < 0.05