Attractiveness of essential oils of three Cymbopogon species to Tribolium castaneum (Herbst) adults

Nikola Đukić1, Anđa Radonjić1, Goran Andrić2*, Petar Kljajić2, Milica Drobac3, Eihab Omar4 and Nada Kovačević3 1 University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia 2 Institute of Pesticides and Environmental Protection, Banatska 31b, 11080 Belgrade, Serbia 3 University of Belgrade, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia 4 Omdurman Islamic University Faculty of Pharmacy, P.O. Box #382, Omdurman, Sudan *Corresponding author: goran.andric@pesting.org.rs Received: 28 October, 2016 Accepted: 7 December, 2016


INTRODUCTION
Red flour beetle, Tribolium castaneum (Herbst) is one of the most important species of storage insects in economic terms, especially in mills and other facilities for processing and storage of plant products, where it causes direct losses. Besides direct damage, their presence and excrements contaminate food which affects product trade name reputation (Rees, 2004;Mahroof & Hagstrum, 2012). Due to the specificity of food processing facilities and processed commodities only a small number of synthetic insecticides (fumigants and contact insecticides) can be used to control this and other stored-product insect pests (Fields & White 2002;Arthur, 2008;Arthur & Subramanyam, 2012). However, the use of synthetic insecticides is limited due to the resistance of certain populations of T. castaneum and other storage insects to insecticides (Kljajić & Perić, 2005;Boyer et al., 2012;Andrić et al., 2010) and increasingly restrictive standards relating to food and environment safety (Arthur, 1996;Arthur & Subramanyam, 2012). As a result, alternative methods of protection of stored products are being tested and introduced (Kljajić, 2008;Phillips & Throne, 2010).
One of potential alternative solutions is to use plant extracts and essential oils (EOs). Although a large number of plant species have shown insecticidal activity (Phillips & Throne, 2010;Pugazhvendan et al., 2012;Regnault-Roger et al., 2012;Sung-Wong et al., 2013;Nenaah, 2014), only a few are currently used on a commercial scale. Pyrethrum, a commercial mixture of compounds derived from Chrysanthemum cinerariifolium and various extracts from the neem tree, Azadirachta indica, with azadirachtin as the active compound, are the most common options (Phillips & Throne, 2010). Products based on azadirachtin have been proved to have lethal effects on insects by inhibiting their growth and development, and interfering with their mating, feeding and oviposition (Gahukar, 2012;Surajwo et al., 2016). However, as EOs are assumed to usually require high doses for achieving lethal effects on storage insects, the influence of EOs on insect behavior has been further examined in recent years. The focus has been on whether EOs attract or repel insects at certain concentrations, and whether their attractant or repellent potentials can be used for improving the existing pest management programs for storage insects (Koul, 2004;Adarkwash et al., 2010;Caballero-Gallardo et al., 2012;Laznik et al., 2012;Regnault-Roger et al., 2012;Licciardello et al., 2013).
Various plant species and their products are traditionally used in many countries as stored-product protection agents, such as essential oils from the genus Cymbopogon. These are perennial plants belonging to the grass family (Poaceae), distributed throughout the warm and tropical regions of the Old World and Oceania (Bertea & Maffei, 2010). Earlier research has shown that Cymbopogon essential oils can be effective against different insect pests important in agriculture (Rajendran & Sriranjini, 2008;Nerio et al., 2010;Hernandez-Lambrano et al., 2015;Wang et al., 2016), but significant differences have also been revealed regarding the strength of effect, depending on plant species and applied concentration (Bossou et al., 2015).
The aim of this study was to investigate the effects of essential oils of three Cymbopogon species: C. nervatus (Hochst.) Chiov., C. proximus Stapf. and C. shoenanthus (L.) Spreng, on T. castaneum adults. The olfactometer was used in tests with selected concentrations (0.0001, 0.001 and 0.01%) of essential oils and the effects were compared with those of azadirachtin in commercial bioinsecticide.

Test insects
A laboratory population of T. castaneum, reared in the insectary of the Institute of Pesticides and Environmental Protection, Belgrade, Serbia, was used in the tests, and procedures described by Harein and Soderstrom (1966), and Davis and Bry (1985) were applied. Tribolium castaneum were reared in 2.5 L glass jars containing wheat flour with 5% yeast. Air temperature in the insectary was 25 ± 1ºC, and relative humidity 60 ± 5%. Unsexed 3-5 week old adults were used in all trials. Before using them in the experiment the adults were starved for 24 h (Wakefield et al., 2005).
The commercial product NeemAzal-T/S (manufactured by Trifolio-M GmbH, Germany) is a formulation of NeemAzal, a high content oil-free extract of A. indica seeds containing 34% of azadirachtin-A and around 20% of other isomers. The content of azadirachtin-A in the product is standardized to 1% (10gL -1 ).

Bioassays
Olfactory responses of T. castaneum adults were measured using a two-way airflow olfactometer consisting of two stimulus zones (arms) directly opposite each other, with a central neutral zone separating them (Ninkovic et al., 2013). Air was drawn from the centre of the olfactometer using a vacuum pump, establishing discrete air currents in the side arms.
Insect olfactory response was tested by adding the EOs or biopesticide at a volume of 10 μL to small pieces of filter paper placed into plastic tubes (4 mm diameter) connected to the holes on the sides of the olfactometer arms. Filter papers with the tested EOs or biopesticide were attached to one side of the olfactometer and control to the opposite side. The experiment was conducted using serial dilutions of EOs and biopesticide in n-Hexane (0.0001, 0.001 and 0.01%), and n-Hexane was used as the control.
Insects were randomly chosen, using a fine paintbrush. A single insect was introduced into the olfactometer through a hole in the top. After an adaptation period of three minutes, insects staying in olfactometer arms were recorded over a 10 minute period.
The accumulated time (in seconds) of stay of a single insect in the arms with different odour sources was regarded as one replicate. Pseudo replication was avoided by using a single insect in each replicate, testing an insect only at a time, and by using a clean olfactometer for each replicate. The number of replications was 20.
The experiment was conducted at 23 ± 1ºC and 50 ± 5% r.h. in a dark room with a light above the olfactometer.

Data analysis
Prior to statistical data analysis, we calculated the indices obtained by substracting total time which insects spent in the control arm from total time that insects spent in olfactometer arms with scents of the essential oils or azadirachtin during the same experiment.
Thus we obtained the numbers, indices, which were either positive or negative -positive when the substance acted as an attractant, and negative when it acted as a repellent. The absolute value of the index indicates a potential for attraction or repellence of that substance for insects.
The indices were further analyzed using the analysis of variance for repeated measurements and the mean values were compared using the t and LSD post hoc tests. The data were run on StatSoft (2005)

RESULTS
The essential oils of all three Cymbopogon species and azadirachtin at all concentrations showed statistically significant (p <0.05) repellent effects on adults of T. castaneum, with the exception of C. proximus oil which did showed no statistically significant (p = 0.321) repellent effect at the lowest concentration on this insect species (Table 1). Comparing the effects between the tested concentrations of each EO and azadirachtin, no statistically significant difference was found between the concentration of C. schoenanthus oil and azadirachtin ( Table 2). The highest concentration of C. nervatus oil repelled insects significantly more than the lowest concentration. Insects spent 15.8% and 8.4% of total time in the olfactometer arms with concentrations of 0.01% and 0.0001%, respectively (Figure 1). The essential oil of C. proximus at the highest concentration (13.9% of total time) had a significantly stronger repellent effect than at the lowest concentration (28.4% of total time), while the mean concentration did not differ from the other two (Table 2, Figure 1) By comparing the effects of Cymbopogon EOs and the commercial product based on azadirachtin at the same concentrations, it was revealed that all tested oils at the concentrations of 0.0001% and 0.001% had the same repellent effect on T. castaneum adults as the commercial product (Table 3). At the highest concentration, the oil of C. nervatus showed the strongest repellent effect, followed by C. proximus oil and azadirachtin with the same level of repellency, while the C. schoenanthus oil showed the lowest repellent effect ( Table 3).
The differences in index values at the highest concentration are shown in Figure 2.

DISCUSSION
Overall, our results showed that all three Cymbopogon (C. nervatus, C. proximus and C. schoenanthus) EOs had repellent effects on T. castaneum adults. This is in agreement with many previous studies which reported EOs repellency to stored-product insects (Nerio et al., 2010;Ukeh & Umoetok, 2011;Kedia et al, 2015). Earlier studies also showed repellent effects of oils from different species of the genus Cymbopogon that we used in our research (Licciardello et al., 2013;Caballero-Gallardo et al., 2012)   Also, the results of these studies showed that azadirachtin from A. indica commercial extract of A. indica had a strong repellent effect on T. castaneum adults. This is consistent with previous research in which it was found that A. indica extracts, apart from causing mortality in insect pests, also acted as a repellent (Boeke et al, 2004;Showler et al., 2004;Adarkwash et al., 2010) In our research, all tested EOs showed a repellent effect at the intermediate concentration (0.001%) and the highest concentration (0.01%). It was found that T. castaneum adults spent about 2.5-fold less time in the arm of the olfactometer with the lowest concentration (0.0001%) of C. schoenanthus and C. nervatus EOs and about 2-fold less time in the arm with azadirachtin, compared to the arm containing n-hexane. The C. proximus oil showed no statistically significant effect on T. castaneum at the lowest concentration, suggesting a different repellent potential from EOs obtained from various species of the genus Cymbopogon.
The experiment conducted by Caballero-Gallardo et al., (2012) revealed greater differences in repellent effect between different Cymbopogon species at low concentrations. In those tests it was found that the species C. flexuosus and C. martini had equally high repellency for T. castaneum (> 90%) at the higher EO dose of 0.2 mL cm -2 filter paper. At a significantly lower dose of 0.000022 mL cm -2 filter paper, C. martini oil showed a significantly greater repellency (46%) than the related species C. flexuosus (20%).
Taking into consideration all the EOs and azadirachtin at the highest concentration, T. castaneum adults spent 2.5-7.5-fold more time in the olfactometer arm containing n-hexane than in the arm with the tested oils. The highest tested concentrations of C. nervatus and C. proximus oils caused a double strong repellent effect compared to the lowest concentration tested. A dependence of repellency strength on essential oil concentrations was detected in a research conducted by Licciardello et al. (2013), who found that an EO of C. nardus had a statistically significant repellent effect on T. castaneum applied at concentrations of 0.005-0.02 mL cm -2 of filter paper, while a lower concentration of 0.001 mL cm -2 had no significant repellent effect. In an experiment in which EO was applied to whole oat flour, Olivero-Verbel et al. (2013) reported that increasing concentrations of C. citratus oil increased the repellent effect on T. castaneum. The highest repellency was recorded at the amount of 5 mL cm -2 of flour, and minimum effect was observed at the lowest tested dose of 0.0005 mL cm -2 of flour.
If we compare the repellent effects of the tested EOs, it may be inferred that differences exist only at the highest concentration, where the oil of C. nervatus showed the highest repellent effect, significantly higher than C. schoenanthus oil. Our experiment also showed that the highest tested concentration of C. schoenanthus oil had a weaker repellent effect on T. castaneum adults than the other two tested Cymbopogon oils. Azadirachtin in the commercial biopesticide used in our research as a standard did not show a stronger repellent effect than the tested EOs. In an experiment with the species Aphis gossypii, Ramakrishna (2008) found high repellency (40-60%) of azadirachtin for that species. In our experiment, azadirachtin at the minimum and maximum concentrations had a slightly lower repellent effect than the oil of C. nervatus. Research in which the repellent effect of the commercial synthetic repellent IR3535 was compared to repellent effects of EOs from plants of the genus Cymbopogon also revealed that the oils of C. citratus (Olivero-Verbel et al., 2013), C. martinii and C. flexuosus (Caballero-Gallardo et al., 2012) and C. citratus, C. flexuosus, C. martinii (Hernandez-Lambran et al., 2015) had stronger repellency than the commercial repellent.
The results obtained in this study show that the essential oils of C. nervatus, C. proximus and C. schoenanthus demonstrated repellent effects on T. castaneum adults. Also, taking into consideration the tested concentrations it can be inferred that the tested essential oils showed a pronounced repellent effect at very low concentrations (0.0001%), which suggests that an analysis of their cost-effective use in practice could be positive. However, a more thorough verification of the potentials of these oils as repellents for T. castaneum adults requires detailed studies on treated wheat grain, in the laboratory and/or semi-field conditions.