Impact of essential oil from plants on migratory activity of Sitophilus granarius and Tenebrio molitor

Measures against pests should be performed in the context of integrated management of agricultural crops and complex control of pests. Therefore, use of ecologically safe approaches is the best option. Essential oils of plants can make an impact on the main metabolic, biochemical, physiological and behavioural functions of insects. We evaluated the effect of 18 essential oils and 18 dried plants on migratory activity of Sitophilus granarius (Linnaeus, 1758) and Tenebrio molitor Linnaeus, 1758 in conditions of laboratory experiment. Notable repellent activity against S. granarius was exhibited by Citrus sinensis and Picea abies. Repellent action against T. molitor was displayed by dried and cut leaves of Origanum vulgare and Eucalyptus globulus, and also essential oils from Juniperus communis, Р. abies, Pterocarpus santalinus, C. sinensis and C. aurantiifolia. Therefore, out of 18 studied essential oils, only two samples had a notable biological effect on migratory activity of S. granarius and five samples – on T. molitor. These data indicate a possibility of using essential oils or their main components as ecologically safe natural repellents against pests of stored wheat and products of its processing.


Introduction
Measures against pests should be performed in the context of integrated management of agricultural crops and complex control of pests, and also should be ecologically-based. Therefore, use of ecologically safe approaches is the most promising variant (Koul & Walia, 2009). Over the recent 50 years, control of pests in agriculture has been based on use of synthetic chemical insecticides in field agrocenoses and conditions of greenhouse cultivation. However, synthetic insecticides are toxic, they cause a non-favourable impact on the environment, polluting soil, water and air, and also their large scale use leads to development of resistance in the target species and significant damage to populations of non-target species of invertebrates (Benhalima et al., 2004;Pimentel et al., 2009;Boyko & Brygadyrenko, 2017;Martynov & Brygadyrenko, 2017, 2018Martynov et al., 2019). Strict measures of ecological regulation of use of pesticides have led to growth of a number of studies on use of natural plant extracts as an alternative to synthetic preparations (Isman, 2004;Pérez et al., 2010).
There are 17,500 species of aromatic plants and around 300 essential oils, which have commercial importance for cosmetics, pharmaceutics and the food industry (Bakkali et al., 2008;Pushpanathan et al., 2008;Ebadollahi et al., 2015). Over 2,000 species of plants have insecticidal activity (Klocke, 1989). Many commercial essential oils are included in the list Generally Recognized as Safe, fully recognized by the Environmental Protection Agency and Food and Drug Administration of the USA (Burt, 2004).
Essential oils affect the main metabolic, biochemical, physiological and behavioural functions of insects (Mann & Kaufman, 2012), and can also block airways, leading to asphyxiation and death of pests (Kaufmann & Briegel, 2004;Rotimi et al., 2011). They can exert toxic, fumigative, repellent, antifeedant, ovicidal, attractant and other actions (Werdin-González et al., 2008). A number of researchers (Isman, 2000;Gutiérrez et al., 2009) report neurotoxic, cytotoxic, phototoxic and mutagenic effects of essential oils on insects. Botanical insecticides have a number of advantages: they do not persist in the environment, they pose relatively low risk for non-target organisms (useful predators and parasites) and are relatively non-toxic for mammals (Weinzierl, 1998;Scott et al., 2003). They usually quickly decompose in the environment and are easely metabolized by the animals that receive sublethal doses (Grdiša & Gršić, 2013). Reasons for limited commercial use of biological insecticides are their relatively slow action, variable efficiency, absence of stability and non-constant availability compared to synthetic analogues (Isman, 2008). Other obstacles to commercializing botanical insecticides are deficiency of natural resources, difficulties of standardization, control of quality and registration (Isman, 1997).
Sitophilus granarius (Linnaeus, 1758) is one the most harmful and common pests of grain. When feeding, an adult beetle damages different grains and products of its processing. Larvae can develop in grain of wheat, rye, barley, oat, rice, maize, buckwheat, panicgrass, and sometimes live in macaroni products and congealed flour. Another common pest of grain storages is Tenebrio molitor Linnaeus, 1758, which can damage different fractured grains of maize, wheat, soybean and other crops (Punzo & Mutchmor, 1980;Fazolin et al., 2007;Cosimi et al., 2009). Presence of T. molitor in stored grain leads to its contamination with enzymes of the body and products of vital activity of the insect, and also contributes to development of saprophytic microflora, reducing the quality of the product (Loudon, 1988;Schroeckenstein et al., 1990;Barnes & Siva-Jothy, 2000). This insect causes loss of up to 15% of grain and flour products throughout the world (Dunkel, 1992;Flinn et al., 2003;Neethirajan et al., 2007).
The objective of this article was to evaluate the impact of different essential oils on migratory activity of S. granarius and T. molitor in the conditions of laboratory experiment.

Materials and methods
The study on the impact of plant essential oils was conducted in a series of three experiments on two species of insects: S. granarius and T. molitor. In the first experiment, we used imagoes of S. granarius. Before the beginning of the experiment, the animals were kept in a common container with grain of wheat. Insects for the experiment were selected randomly. The experiment was undertaken in polyethylene tubes of 105 cm length and 2.5 cm diameter with marks each 10 cm. The tubes were filled with grain of wheat. At one end of the tube, 40 individuals of S. granarius and a cotton disk of 0.4 cm in diameter, saturated with 0.06 mL of essential oil -0.48 mL/cm 2 concentration were placed (Table 1). The tubes were closed with a plug at both ends and put randomly on the tables of the laboratory with same illuminance and temperature, out of reach of direct sunlight. After two days, each 10 cm of the tube with grain were sieved through a laboratory sieve with diameter of cell of 2 mm for count of weevils in each part of the tube. Each variant of the experiment was performed in five replications. In all variants of the experiment, 1,800 individuals of S. granarius were used. The results were statistically analyzed in Statistica 8.0 (Statsoft Inc., USA) software pack using χ 2 criterion. In the second and the third experiments, we used third age larvae of T. molitor. For one month before the beginning of the experiments, they were kept in a general container and fed with a single component diet (dry rolled oats). Larvae for experiments were selected randomly. The second experiment was undertaken in polyethylene tubes of 4 cm diameter and 105 cm length with marks each 10 cm. In one part of the tube, 400 g of wheat grain with cut up plants was placed (Table 2), and 400 g of grain with no additions was put in the other part. At 10 cm intervals along the tube, 5 larvae of T. molitor were placed. The tubes were put randomly on the tables in the laboratory with same illuminance and temperature, out of reach of direct sunlight. After two days, from each 10 cm of the tube, grain was extracted and sieved through a laboratory sieve with cells of 2 mm in diameter for detecting and counting larvae of T. molitor in each section of the tube. Each variant of the experiment was performed in ten replications. In all variants of the experiment, 9,500 individuals of T. molitor (9,000 in 18 variants of the experiment and 500 in the control) were used.   The third experiment was performed in a container (50 х 33 х 19 cm) in which wheat flour of highest sort was put (400 g) in a 1 cm layer. Then, 17 plastic cups with removed bottom (100 mL capacity) were put in the container at a distance of 0.5 cm one from another with 80 g of flour and 5 cups with 5 larvae of T. molitor in each. In 15 cups, into flour, a cotton disk of 0.4 cm diameter, saturated with 0.06 mL of essential oil of one of the plants (0.48 mL/cm 2 ), was placed at a 3 cm depth. For each of the 20 studied types of essential oils (Table 3) we used one cup. The other two cups were the control ( a 4 cm diameter cotton disk not processed with any of the essential oils was placed in them). Each cup was covered with a separate plastic cover to prevent mixing of the odours of the essential oils. The experiment was performed in five replications (n = 5). Duration of each experiment was 48 hours. After this period, the flour from the cups was sieved for counting live and dead insects. The results of the second and the third experiments were statistically analyzed in Statistica 8.0 (Statsoft Inc., USA) software pack. The differences between the selections were considered reliable at P < 0.05 (one-way ANOVA). All the variants of the experiment were performed in the same conditions. Fluctuations in temperature over the day did not exceed 3 °С (+18…+21 °С), length of daylight in October-November of 2018 equalled 8.30-11.00 h and was prolonged by artificial illumination to 14 hours; relative air moisture was 60-70%.

Results
The impact of essential oils on movement of S. granaries in conditions of a 48 h laboratory experiment is demonstrated in Figures 2-8. Essential oils from C. sinensis and P. abies stimulated migratory activity of S. granarius and are promising for use as repellents.  Table 5. The strongest effect on distribution of larvae of T. molitor in the fodder substrate was exerted by essential oils from J. communis, Р. abies, P. santalinus, C. sinensis and C. aurantiifolia (P < 0.01).

Discussion
The obtained data indicate that the essential oils from C. sinensis and P. abies exert notable repellent action towards S. granarius at concentration of 0.48 mL/cm 2 . Repellent effect on T. molitor was displayed by dried and cut leaves of O. vulgare and E. globulus, and also essential oils from J. communis, Р. abies, P. santalinus, C. sinensis and C. aurantiifolia (P < 0.01). Absence of notable effects among the rest of the tested samples can be related to insufficient concentration of essential oils, or resistance of the pest species. Resistance of insects to the vapours of essential oils can be associated with the activity of cytochromes of P 450 -dependent monooxygenase, carboxyl esterase, superoxide dismutase and catalase (Ryan & Byrne, 1988;Boyer et al., 2012).
Today, the effect of essential oils and their constituents on S. granarius and T. molitor and other economically harmful species is described in a number a scientific works. Yildrim et al. (2005) studied efficiency of eight essential oils: Hypericum scabrum Linnaeus, 1753, Hyssopus officinalis Linnaeus, (1753), Micromeria fruticosa Druce, 1914, Origa-num acutidens (Handel-Mazzetti) Ietswaart, Satureja hortensis Linnaeus, 1753, Salvia limbata C. A. Meyer, 1831 and S. nemorosa Linnaeus, 1762 against S. granarius. Mortality of insects at concentration of essential oils equaling 10 µL was 74%, 66%, 73%, 4%, 12%, 10% and 14%, respectively. Level of mortality rose with increase in the concentration of essential oils and duration of their action. Note: * -x is share of initial number of larvae distributed in particular section of the tube (%). Note: * -x is share of the initial number of larvae placed in the cup before the beginning of the experiment (%). Rozman et al. (2006) studied the efficacy of essential oils from L. angustifolia, L. nobilis, R. officinalis and Thymus vulgaris Linnaeus, 1753, and also their components: 1,8-cineole, camphor, eugenol, linalool, carvacrol, thymol, borneol, bornylacetate and lynalylacetate against S. granarius. The studied substances in concentration of 0.1 µL/720 mL demonstrated high efficiency with average mortality of 96.5-100.0% after 24 h exposure.
Ebadollah & Mahboubi (2011) studied the efficacy of essential oil from Azilia eryngioides (Pau) Hedge & Lamond, 1987 against S. granarius. The studied oil had high insecticidal activity, 100% death rate was achieved at impact of concentration of 37.0 µL/L over 28 h. Values of LC 50 of essential oil from A. eryngioides for S. granarius equaled 20.1 µL/L after 24 h of impact.
In a study on toxicity of different essential oils against S. granarius, Lamiri et al. (2001) determined that the highest toxic effect on the insect was caused by essential oils from Mentha pulegium Linnaeus, 1753, M. spicata Linnaeus, 1753, E. globulus and Origanum compactum Bentham, which exerted killing effect on the insect in concentration of 10 and 5 µL/mg over 24 and 48 h respectively. Mahmoudvand et al. (2011) studied toxicity of essential oil from C. sinensis for S. granarius, Tribolium castaneum (Herbst, 1797) and Callosobruchus maculatus (Fabricius, 1775). Values of LC 50 of the tested substance equaled 367.8, 391.3 and 223.5 µL/L of air after 24 h and 320.5, 362.4 and 207.2 after 48 h of exposure for S. granarius, T. castaneum and C. maculatus, respectively. Mortality of the insects depended on concentration and duration of impact of the tested essential oil.
Di Stefano (2016) studied insecticidal activity of essential oil from L. angustifolia against S. granarius. Mortality of insects at concentration of essential oil equaling 449.1 µg/mg equaled 91.7% and 100.0% after 24 and 48 h of impact, respectively. Values of LD 50 and LD 90 equaled 83.8 and 379.7 µg/mg after 24 h and reduced to 58.3 and 208.3 µg/mg after 48 h, respectively. Fumigant toxicity of essential oil from L. angustifolia for S. granarius reached its maximum at doses of 11.9 and 47.5 mg/L of air. Values of LC 50 and LC 90 equaled 1.6 and 4.1 mg/L at absence of substrate (grains of wheat) and 10.9 and 47.6 mg/L at presence of grains respectively. The studied essential oil had notable repellent activity against imagoes of S. granarius.
Study on toxicity of essential oils from Artemisia absinthium, A. santonicum Linnaeus, 1753 and A. spicigera K. Koch, 1851, and also their constituents against S. granarius were undertaken by Kordali et al. (2006). Essential oils of all three species killed 80-90% of the insects at concentration of 9 L/L of air during 48 h of exposure. Mortality increased with increase of doses of essential oils and duration of exposure. The main components of essential oils: borneol, bornyl acetate, camphor, α-terpineol killed 100% of S. granarius at concentration equaling 1.0 L/L of air, and 1,8-cineole and terpinen-4-ol -at concentration of 0.5 L/L of air, respectively, at over 12 h exposure. Mohamed & Abdelgaleil (2008) studied toxicity of different essential oils against Sitophilus oryzae (Linnaeus, 1763) and T. castaneum. Highest toxicity for S. oryzae was exhibited by essential oil from Citrus limon (Linnaeus) Osbeck, 1765, C. sinensis and C. paradisi, LC 50 for which equaled 9.9, 19.7 and 24.1 mg/L of air, respectively. Highest toxicity against T. castaneum was exerted by C. sinensis and C. paradisi with parameters of LC 50 equaling 24.6 and 25.5 mg/L of air, respectively. Abdelgaleil et al. (2015) studied insecticidal activity of plant essential oils against S. oryzae. At fumigation, the most toxic for the insect were essential oils from O. vulgare, C. limon, Callistemon viminalis (Solander ex Gaertner) G. Done x Loudon (1830), Cupressus sempervirens Linnaeus, 1753 and C. sinensis, values of LC 50 of which equaled 1. 64, 9.89, 16.17, 17.16 and 19.65 mg/L of air, respectively. The highest contact toxicity towards S. oryzae was exhibited by essential oils from Artemisia judaica Linnaeus, 1759, C. viminals and O. vulgare with parameters of LC 50 equaling 0.08, 0.09 and 0.11 mg/cm 2 , respectively. Essential oil from A. judaica in concentration of 16.1 mg/L exerted inhibiting effect on the activity of acetylcholinesterase, whereas oils from C. viminals and O. vulgare were strong inhibitors of ATRs at concentration of 4.69 and 6.07 mg/L, respectively. In a similar study, Lee et al. (2001) determined that most toxic essential oils for S. oryzae were those from E. globulus and R. officinalis, LD 50 of which equaled 28.9 and 30.5 L/L of air. Essential oils from L. angustifolia, T. vulgaris, Cananga odorata (Lamarck) Hooker & Thomson, 1855 and C. paradisi were less toxic: LD 50 -54.0, 63.9, 73.1 and 87.0 L/L of air, respectively. Most toxic for S. oryzae were the following components of essential oils: 1,8cineole, p-cymene, α-pinene and limonene, LD 50 of which equaled 23.5, 25.0, 54.9 and 61.5 L/L of air, respectively.
AbdEl-Salam (2010) studied toxic action of essential oils for S. oryzae. Percentage of mortality heightened with increase in concentration of different essential oils and period of exposure. Essential oils from C. verum and M. alternifolia killed 90.0% of insects at concentrations of 8.0 and 16.0 µL/50 mL of air, respectively at impact over 24 hours. Values of LC 95 of essential oils from C. verum, S. aromaticum and E. globulus equaled 3.67, 4.07 and 8.73 µL/50 mL of air for S. oryzae at period of impact of 72 hours.
Insecticidal activity of essential oils against S. oryzae was studied by Yazdgerdian et al. (2015). The most active fumigants of the insect were Gaultheria procumbens Linnaeus, 1753, Thuja plicata Donn ex D. Don, 1824 and Bursera graveolens (Kunth) Triana & Planchon, 1872, values of LC 50 of which equaled 6.8, 19.8 and 21.4 µL/L of air, respectively. Rajkumar et al. (2019) studied insecticidal activity of essential oil from M. piperita and its constituents against S. oryzae and T. castaneum. Values of LC 50 of essential oil, menthone and menthol for S. oryzae equaled 43.2, 46.7 and 49.4 µL/L of air, and for T. castaneum -48.7, 51.9 and 54.5 µL/L of air, respectively. Both insect species were observed to have dose-dependant inhibition of activity of acetylcholinesterase when exposed to essential oil from M. piperita, menthone and menthol concentration, LC 50 equaled 29.7%, 18.8% and 14.3% for S. oryzae and 20.7%, 13.7% and 9.2% for T. castaneum, respectively at 24 hours exposure. Also, under the impact of the studied substances in dose equal to LC 50 , an increase was observed in the activity of superoxide dismutase by 17.4, 15.2 and 13.3 units/mg of protein and 31.9, 28.3 and 25.3 units/mg of protein for S. oryzae and T. castaneum, respectively. Bertoli et al. (2012) studied biological activity of essential oils against Sitophilus zeamais (Motschulsky), 1855. Toxic effect of essential oils from A. millefolium and Foeniculum vulgare Miller, 1768 was displayed by minimum concentration of 0.05 µL, similar to the effect of L. angustifolia -at concentration of 0.10 µL. Level of mortality of insects was never higher than 76.0% (essential oil from L. angustifolia in dose of 0.75 µL during more than 96 hours of exposure). Buneri et al. (2019) studied efficiency of essential oil from Cedrus deodara (Roxburghe x D. Don) G. Don, 1830 in concentrations of 0.8%, 1.5%, 3.0%, 6.0% and 12.0% as insecticide against larvae of Т. molitor. Death rate of insects after 48 h equaled 17%, 33%, 49%, 64% and 87%, respectively. LC 50 of oil from C. deodara equaled 3.1%. After the exposure, the insects were observed to have increase in the level of total protein from 2.12 to 4.14 mg/mL. Essential oil from C. deodara has a notable repellent action against larvae of Т. molitor. Fazolin et al. (2007) studied toxicity of essential oils from Piper aduncum Linnaeus, 1753, P. hispidinervum C. de Candolle, 1914 and Tanaecium nocturnum (Barbosa Rodrigues) Bureau & K. Schumann, 1896 against larvae of Т. molitor. Values of LC 50 for these essential oils were 0.045, 0.033 and 1.515 mL/cm 2 , LD 50 -0.000025, 0,009 and 0.000015 mL/g, respectively.
Study on repellent activity of essential oils from L. nobilis, С. bergamia, F. vulgare, Lavandula x intermedia Emeric ex Loiseleur-Deslongchamps in 0.01% and 0.10% concentrations against larvae of Т. molitor was performed by Cosimi et al. (2009). Essential oil from F. vulgare had a repellent effect in concentrations of 0.10% after 1 h of exposure, whereas the rest of the oils showed low effectiveness even after 5 h of exposure. Wang et al. (2016) studied the effect of essential oils and their components on larvae of Т. molitor. Significant repellent effect on the insect was presented by geraniol, cis-3-hexenol and ionone in concentration of 1 mol/L, and also essential oil of eucalypt in concentration of 1%. Attractant effect on Т. molitor was caused by isoeugenol and α-pinene in concentrations of 1.0 and 0.1 mol/L, and also peppermint oil and turpentine oil in concentrations of 1.0% and 0.1%. Plata-Rueda et al. (2017) studied insecticidal activity of essential oil from Allium sativum Linnaeus, 1753 for different stages of Т. molitor. The insect was more sensitive at the larva stage compared to the pupa and imago stages. Values of LC 50 for the different stages equaled 0.771, 2.371, 2.032 µL/mL, respectively at impact over 48 h. Essential oil from A. sativum was noted for repellent action against Т. molitor at all stages of the development, and also caused reduction of frequency of breathing of the insect after 1 h of impact. George et al. (2009) studied toxicity of different essential oils for Т. molitor. Most toxic for the insects were essential oils from Carum carvi Linnaeus, 1753, Mentha spicata Linnaeus, 1753 and J. communis, which killed 95%, 87% and 85%, respectively at concentration of substances of 0.14 mg/cm 3 after 24 h of impact. Less effective were oils from T. vulgaris and Piper nigrum Linnaeus, 1753, the killing power of which equaled 50% and 40%, respectively at the same concentration and duration of exposure. Kuusik et al. (1995) studied toxicity of extract of R. tomentosum for pupaes of Т. molitor. Exposure of the pupae with extract of R. tomentosum led to different morphological effects depending on duration of exposure. The affected pupae transformed into immature imagoes. Furthermore, changes were observed in the movement activity of the pupaeweak bending activity turned into energetic and more prolonged.
Effectiveness of plant essential oils against Т. molitor was studied by Wang et al. (2015). Values of LC 50 of essential oils from Litsea cubeba (Loureiro) Persoon, 1807 and C. limon equaled 19.6 and 42.2 mg/cm 2 respectively at 24 h exposure and 13.9 and 21.2 mg/cm 2 at 48 h exposure. The studied essential oils exhibited notable repellent activity against larvae of Т. molitor, and also caused increase in duration of the development of larva stage. Martínez et al. (2017) studied insecticidal activity of essential oils from C. verum and S. aromaticum, and also some of their components against Т. molitor. Values of LC 50 of essential oil from C. verum for larvae, pupae, and imagoes of Т. molitor equaled 30.4, 10.7 and 29.8 µg/mL, and LC 50 of S. aromaticum were 35.1, 6.5 and 21.6 µg/mL, respectively. LC 50 of eugenol, caryophyllene oxide, α-pinene α-humulene and α-phellandrene for larvae of Т. molitor were 9.2, 9.2, 14.0, 15.2 and 17.1 µg/mL, respectively. Caryophyllene oxide showed a notable repellent action towards larvae of Т. molitor, while eugenol, α-humulene and essential oil from C. verum showed an attractant effect.
Despite the relevance of developing methods of using essential oils and their constituents as insecticidal preparations, their introduction into technologies of integrated control of pests remains impossible due to insufficient amount of data on this problem, difficulties in standardization and control of plant production.

Conclusions
Notable repellent activity against S. granarius was exhibited by essential oils from C. sinensis and P. abies at concentration of 0.48 mL/cm 2 . Repellent effect on T. molitor was displayed by dried and cut leaves of O. vulgare and E. globulus, and also essential oils from J. communis, Р. abies, P. santalinus, C. sinensis and C. aurantiifolia (P < 0.01). Therefore, out of 18 studied essential oils, only two samples had notable biological action on migratory ability of S. granarius and only five samples -on T. molitor. These data are confirmed by many studies on insecticidal activity of essential oils and the possibility of using them as ecologically safe natural pesticides. Studies on biological activity of essential oils against economically harmful species of insects is a relevant issue necessary for the development of ecologically-based control of agricultural pests.