Fumigant toxicity of five essential oils rich in ketones against Sitophilus zeamais (Motschulsky)

Essential oils (EOs) and individual compounds act as fumigants against insects found in stored products. In fumigant assays, Sitophilus zeamais Motschulsky adults were treated with essential oils derived from Aphyllocladus decussatus Hieron, Aloysia polystachya Griseb, Minthostachys verticillata Griseb Epling and Tagetes minuta L , which are rich in ketones and their major components: αthujone, R-carvone, S-carvone, (-) menthone, R (+) pulegone and E-Zocimenone. M. verticillata oil was the most toxic ( LC50: 116.6 μl /L air) characterized by a high percentage of menthone (40.1%) and pulegone (43.7%). All ketones showed insecticidal activity against S. zeamais. However, pulegone (LC50: 11.8 μl/L air), Rcarvone (LC50: 17.5 μl/L air), S-carvone (LC50: 28.1 μl/L air) and E-Z-ocimenone (LC50: 42.3 μl/L air) were the most toxic. These ketones are α,b-unsaturated carbonyl. This feature could play a fundamental role in the increase of insecticidal activity against S. zeamais.


InTrODuCTIOn
The central area of Argentina is the most important in the production of corn.The amount of corn exported to the United States, Europe and China during 2011 -2012 was approximately 75,000 tons (Lezcano, 2012).Worldwide maize grain storage sites are affected by different species of beetle pests.Sitophilus zeamais (Motschulsky) is a serious primary pest of stored maize throughout the world causing quantitative and qualitative losses (Boyer et al., 2012).The control of these pests is dependent upon applications of synthetic insecticides: methyl bromide and phosphine (Benhalima, 2004;Athie & Mills, 2005;Pimentel et al., 2012).These control measures have several limitations, such as the development of resistance (Benhalima, 2004;Pimentel et al., 2009;Pimentel et al., 2012 ) and environmental damage, which cause serious concerns about human health.
In view of the problems with the current fumigants, there is a global interest in generating alternative strategies.There are numerous studies describing essential oils (EOs) and their components as potential insecticides (Pérez et al., 2010;Caballero-Gallardo et al., 2011;Kurdelas et al., 2012).This insecticidal activity has been evaluated against the maize weevil.The results showed that many EOs could control Sitophilus zeamais populations in corn grain stores (Liu et al., 2011;Suthisut et al., 2011;Yang et al., 2011).However, the insecticidal activity of synthetic products is far superior to that of EOs but the pressure generated by the consuming public about the use of synthetic pesticides has raised the option of an integrated management of insects affecting food storage sites (Yigezu et al., 2010).In this framework EOs are shown as an important alternative (Correa et al., 2011).Plant essential oils are promising in that they are easily biodegradable, do not present described resistance and they are environmentally friendly (Pérez et al., 2010).
Therefore, in the present work, we report insecticidal activity of EOs rich in ketones of Aphyllocladus decussatus Hieron, Aloysia polystachya Griseb, Minthostachys verticillata Griseb Epling, Tagetes minuta L. and their major components against S. zeamais.

Insects
Adults of the strain of S. zeamais were obtained from Metán, Salta province, Argentina.The colony was maintained in our laboratory for one year without exposure to insecticides.
S. zeamais were reared on sterilized whole maize grain in sealed containers.Insect rearing was carried under controlled temperature and humidity (28 ºC and 60-70%) and a light/dark regime of 12:12 h (FAO, 1974).Experiments were conducted under complete darkness, at 28 ºC and 60-70% relative humidity.The unsexed adult weevils used were about 2-weeks old.

essential oils and pure compounds
Aphyllocladus decussatus Hieron, Aloysia polystachya Griseb (population 1) were collected from La Rioja province, Argentina.Plants of Aloysia polystachya Griseb (population 2), Minthostachys verticillata Griseb Epling and Tagetes minuta L. were collected from Córdoba province, Argentina.We deposited all voucher plant specimens at the Museo Botánico de Córdoba (CORD).The EOs were obtained by hydrodistillation (Demo et al., 2005) and stored at -20ºC in airtight microtubes prior to analysis by gas chromatography-mass spectrometry (GC-MS).The α-thujone, R-carvone, S-carvone, (-) menthone and R (+) pulegone used for bioassays were purchased from Sigma Aldrich (Steinheim, Germany).The E-Z-ocimenone was isolated from T. minuta EO by supercritical carbon dioxide fractionation.The highest selectivity was obtained at 80 bar and 40 ºC, with a static period of 30 min followed by dynamic extraction at a flow rate of 0.1 g/min until complete removal of monoterpenes, leaving a residue of purified ocimenone (Figure 1).

analysis of essential Oils
The EOs were analyzed by Perkin Elmer Clarus 500 chromatograph equipped with a detector FID and a capillary column DB-5 (60 m x 0.25 mm i.d. and 0.25 µm coating thickness).The temperature of the column was programmed from 60 o C to 240 o C at 4 o C/min.The detector and injector temperatures were 250 o C. Helium was used as carrier gas with a flow rate of 0.9 ml/min.The quantitative composition was obtained by peak area normalization, and the response factor for each component was considered to equal 1.For the determination of the composition, EOs samples were diluted with n-hexane.The injection volume was 1 μl.The identification of the EOs' components was carried out by GC-MS.A Perkin-Elmer Clarus 600 GC-MS coupled with an ion trap mass detector which was employed for the identification.A capillary column DB-5 (60 m x 0.25 mm i.d. and 0.25 µm coating thickness) was used for the separation of the components.Helium was used as carrier gas with a flow rate of 0.9 ml/min.The temperature program for the oven and injector was the same as that for the GC-FID.Ionization was realized by electron impact at 70 eV.Mass spectral data were acquired in the scan mode in the m/z range 35-250.Retention indices (RI) of the sample components were determined on the basis of homologous n-alkane hydrocarbons under the same conditions.The compounds were identified by comparing their retention indices and mass spectra with published data (Adams, 2007) and libraries NIST and Adams.The main components were further identified by coinjection of authentic standards (SIGMA, USA).

Fumigant Toxicity assay
The insecticidal activity against S. zeamais was evaluated using fumigant toxicity assay described by Huang et al. (2000), with some modifications.Briefly, glass vials (30 ml) were used as fumigation chambers.Different amounts of EOs and pure compounds were applied to Whatman filter paper disks (2 cm diameter) placed on the underside of the screwcap of a glass vials at the doses corresponding to 15-600 µl/L air.A series of concentrations of each EOs and pure compound were prepared in nhexane.Solvent was allowed to evaporate for 2 min prior to introduction of insects.Ten adults S. zeamais were placed into each vial (5 replicas / dose).Control insects were kept under same conditions without EOs and pure compounds.Insect mortality was checked after 24 h.The mortality percentages and LC 50 values were calculated according to Finney (1971).

statistical analysis
The concentration-mortality data were subjected to Probit analysis to obtain the LC 50 values.The lethal concentrations LC 50 and LC 95 were calculated using SPSS Statistics program version 17.0 (SPPSS Inc).The values of LC 50 were considered to be significantly different, if 95% confidence limits did not overlap.Treatment means were compared and separated by Duncan's test at p = 0.05 using the InfoStat software Professional 2010 (Di Rienzo et al., 2010).

Toxicity of essential oils
Comparison of LC 50 values for the five EOs against S. zeamais showed that M. verticillata oil was the most toxic ( LC 50 = 116.6 µl/L air), toxicity being twice lower at 24 h after exposure than in the rest of the EOs studied.LC 50 of EO of T. minuta could not be calculated because it did not show dose dependent linear behavior (Table 2).
The EOs and the individual compounds act as fumigants against insects found in stored products.In the study, EO of M. verticillata was the most bioactive, which can be attributed to its content in pulegone (43.6 %) (Table 1).However, S. zeamais showed greater sensitivity to pure compounds than EOs.
Pulegone was more toxic than carvone and E-Zocimenone (Table 3).In addition, carvone isomers presented differences in toxicity.Carvone R was more active than S isomer.Controversial results of insecticidal activity of isomers of carvone were shown by Lee et al. (2003) and Tripathi et al. (2003).Numerous studies have shown ketones with highest toxicity against Sitophilus in fumigant and contact assays (Lee et al., 2003;Tripathi et al., 2003;Liu et al., 2011;Germinara et al., 2012).These results suggest that the presence of carbonyl groups augments toxicity.On the other hand, ketones belonging to Group 1 were α,b-unsaturated.This feature could be playing a fundamental role in the increase of insecticidal activity.Thus, ketones of Group 1 were more toxic than those of Group 2 (Table 3).Xavier & Rauter (2008) revealed that compounds containing the α,b-unsaturated act as Michael acceptors for the addition of protein nucleophilic groups.Also, α,b-unsaturated carbonyl compounds were described as potent inhibitors of the enzymes Glutathione-S-transferases that contribute to the phase II metabolism of xenobiotics (Yu & Abo-Elghar, 2000).
In conclusion, the present results indicate that α,b-unsaturated carbonyl ketones act as potential fumigants against S. zeamais.Based on these findings, they could serve as viable alternative to synthetic insecticides.

aCKnOWLeDGemenTs
This research was supported by grants from Secretaría de Ciencia y Técnica de la Universidad Nacional de Córdoba and the Agencia Nacional de Promoción Científica y Técnica, FONCYT, PICT 2012-2146.The authors thanks Dra.Gloria E. Barboza for her valuable help.We thank Iliana A. Martinez, MA for revising the English language.

Fig. 1 .
Fig. 1.Chemical structures of natural ketones compounds studied in the present work.

Table 3 .
Fumigant toxicity of pure compounds against S. zeamais adults at 24 h after exposure.