Chemical composition and bio-pesticidal values of essential oil isolated from the seed of Heracleum persicum Desf . ex Fischer ( Apiaceae )

The present investigation was aimed to analyse the chemical composition of essential oil isolated from Heracleum persicum Desf. ex Fischer and assess its lethal and sub-lethal effects against Tribolium castaneum (Herbst). Essential oil from hydro-distilled seeds of H. persicum was analyzed by gas chromatography-mass spectrometry (GC-MS), and hexyl butyrate (50.58%), octyl acetate (9.80%) and hexyl hexanoate (8.75%) were found as principal constituents. Repellent activity, contact and fumigant toxicity and antifeedant effects of this oil were assessed against the adults of T. castaneum. The essential oil strongly repelled T. castaneum adults even at the lowest concentration (0.035 μL cm). Complete repellency (100%) occurred when the highest concentration (0.212 μL cm) was applied for 8 h. T. castaneum was very susceptible to H. persicum oil at both contact and fumigant bioassays. In the fumigant toxicity, essential oil killed the larvae, pupae and adults and significantly decreased larvae emerged from treated eggs. LC10 to LC40 values of fumigation adult’s bioassay as sub-lethal concentrations were used to evaluate the antifeedant effects. H. persicum essential oil has significant antifeedant effects on T. castaneum adults and decrease of feeding happened when oil concentrations increased. The results of the present study indicate that essential oil of H. persicum, with wide bio-effects on T. castaneum, is a source of biologically active agents which may potentially prove to be efficient insecticides. Additional key words: essential oils; Heracleum persicum; toxicity; repellent; antifeedant. * Corresponding author: asgar.ebadollahi@gmail.com Received: 12-07-14. Accepted: 12-11-14. Abbreviations used: FDI (feeding deterrence index); GC (gas chromatography); LC50 (lethal concentration required to kill 50% of the population); MS (mass spectrometry); PR (percentage of repellency); RH (relative humidity). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2014 12(4): 1166-1174 http://dx.doi.org/10.5424/sjar/2014124-6527 ISSN: 1695-971X eISSN: 2171-9292 RESEARCH ARTICLE OPEN ACCESS Essential oil of Heracleum persicum (Apiaceae) evaluated as insecticide 1167 tion and lethal effects on non-target organisms in addition to direct toxicity to the consumer (Zettler & Arthur, 2000; Khalfi et al., 2008; Nyamador et al., 2010; Wei et al., 2014). The search for natural and environmentally friendly insecticidal substances is ongoing because of the negative effects of conventional chemical insecticides. Essential oils are extracted by distillation and expression, and are popular as ingredients of perfumes, cosmetics and household cleaning products, as well as being used for flavouring food and drink. Recently, as an alternative pest control technology, essential oils have attracted particular attention because of their specificity to pests, biodegradable nature and potential for commercial application (Liu et al., 2006). Essential oils generally have a broad spectrum of bioactivity because of the presence of several active ingredients that work through several modes of action. The toxicity of individual oils or compounds often exerts differential effects depending on both the mode of action and the target pest (Isman, 2006). Investigations in several countries confirm that some plant essential oils not only possess contact and fumigant toxicity against stored product insect pests, but repel insects as well as exhibit feeding inhibition or harmful effects on the reproductive system of insects (Isman, 2006; Rajendran & Srianjini, 2008). Apiaceae (Umbelliferae) is one of the best known families of flowering plants, which comprise 300-450 genera and 3000-3700 species. They are aromatic plants and have a distinctive flavor with diverse volatile compounds present in the fruits and leaves (Pimenov & Leonov, 1993). Heracleum persicum Desf. Ex Fisher (syn. H. pubescens Rech. and H. glabrescens Boiss. & Hohen.) known as Persian Hogweed or “Golpar”, is an annual native plant to the Alborz region, the northern part of Iran with a wide distribution across the country. Its fruits are widely used as a spice and flavoring agent in foods and in the preparation of pickles (Amin, 1991) as well as carminative, antiseptic, digestive, analgesic, antioxidant and anticonvulsant herbal drug in Iranian traditional medicine (Souri et al., 2004; Sayyah et al., 2005; Radjabian et al., 2013). Due to the widespread use of H. persicum as a medicinal plant and flavoring agent, and as part of the search for bio-rational alternatives to synthetic insecticides, this study aims to evaluate the effects of H. persicum essential oil as a contact and fumigant insecticide, repellent, and antifeedant against T. castaneum. In addition, the chemical composition of this essential oil was analyzed by gas chromatography-mass spectrometry (GC-MS). Material and methods Plant material and essential oil analysis The seeds of H. persicum were collected from Meshkin shahr city, Ardabil province, Iran. The seeds were air-dried in the shade at room temperature (26-28°C) for 10 days and the essential oil was isolated by hydrodistillation method using a Clevenger apparatus. Conditions of extraction were: 50 g of air-dried sample, 1:10 in water (w/v), 3 h distillation. Anhydrous sodium sulfate was used to remove water after extraction and extracted essential oil was stored at 4°C. GC-MS analysis was carried out on a HP 7890A GC (Hewlett-Packard, Palo Alto, CA, USA) equipped with a split injector and 5975C mass selective detector system. Chromatographic separation was carried out in a HP-5 capillary column (30 m × 0.25 mm, 0.25 μm in film thickness). The MS was operated in the EI mode (70 eV). The GC-MS interface, ion source, and quadruple temperatures were set at 280°C, 230°C, and 150°C, respectively. The injector temperature was set at 250°C, the column temperature program started at 50°C for 3 min, increased by 10°C min to 110°C and by 10°C min to 180°C, and was maintained for 2 min. Helium (99.999%) was used as the carrier gas with flow rate of 1 mL min. Identification of spectra was carried out by studying their fragmentation and by comparison with standard spectra present in the library of the instrument. Area normalization was used for determination of composition percentage.


Introduction
Insect pests are a major constraint on crop production, especially in developing countries.The red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), is one of the most widespread and destructive stored-product insect pests throughout the world.Beetles and larvae feed on a very wide variety of dry vegetable substances, such as milled cereal products (Rees, 2004).Infestations not only cause significant losses of grains, they also elevate their temperature and moisture conditions that lead to an accelerated growth of molds, including toxigenic species (Magan et al., 2003).
Currently, the measures to control pest infestation in grain and dry food products rely on the use of liquid insecticides, such as organophosphates, pyrethroids and gaseous insecticides.This can lead to problems such as environmental toxicity, increasing application costs, pest resistance to pesticides, ozone layer deple-tion and lethal effects on non-target organisms in addition to direct toxicity to the consumer (Zettler & Arthur, 2000;Khalfi et al., 2008;Nyamador et al., 2010;Wei et al., 2014).The search for natural and environmentally friendly insecticidal substances is ongoing because of the negative effects of conventional chemical insecticides.
Essential oils are extracted by distillation and expression, and are popular as ingredients of perfumes, cosmetics and household cleaning products, as well as being used for flavouring food and drink.Recently, as an alternative pest control technology, essential oils have attracted particular attention because of their specificity to pests, biodegradable nature and potential for commercial application (Liu et al., 2006).Essential oils generally have a broad spectrum of bioactivity because of the presence of several active ingredients that work through several modes of action.The toxicity of individual oils or compounds often exerts differential effects depending on both the mode of action and the target pest (Isman, 2006).Investigations in several countries confirm that some plant essential oils not only possess contact and fumigant toxicity against stored product insect pests, but repel insects as well as exhibit feeding inhibition or harmful effects on the reproductive system of insects (Isman, 2006;Rajendran & Srianjini, 2008).
Apiaceae (Umbelliferae) is one of the best known families of flowering plants, which comprise 300-450 genera and 3000-3700 species.They are aromatic plants and have a distinctive flavor with diverse volatile compounds present in the fruits and leaves (Pimenov & Leonov, 1993).Heracleum persicum Desf.Ex Fisher (syn.H. pubescens Rech.and H. glabrescens Boiss.& Hohen.)known as Persian Hogweed or "Golpar", is an annual native plant to the Alborz region, the northern part of Iran with a wide distribution across the country.Its fruits are widely used as a spice and flavoring agent in foods and in the preparation of pickles (Amin, 1991) as well as carminative, antiseptic, digestive, analgesic, antioxidant and anticonvulsant herbal drug in Iranian traditional medicine (Souri et al., 2004;Sayyah et al., 2005;Radjabian et al., 2013).
Due to the widespread use of H. persicum as a medicinal plant and flavoring agent, and as part of the search for bio-rational alternatives to synthetic insecticides, this study aims to evaluate the effects of H. persicum essential oil as a contact and fumigant insecticide, repellent, and antifeedant against T. castaneum.In addition, the chemical composition of this essential oil was analyzed by gas chromatography-mass spectrometry (GC-MS).

Plant material and essential oil analysis
The seeds of H. persicum were collected from Meshkin shahr city, Ardabil province, Iran.The seeds were air-dried in the shade at room temperature (26-28°C) for 10 days and the essential oil was isolated by hydrodistillation method using a Clevenger apparatus.Conditions of extraction were: 50 g of air-dried sample, 1:10 in water (w/v), 3 h distillation.Anhydrous sodium sulfate was used to remove water after extraction and extracted essential oil was stored at 4°C.GC-MS analysis was carried out on a HP 7890A GC (Hewlett-Packard, Palo Alto, CA, USA) equipped with a split injector and 5975C mass selective detector system.Chromatographic separation was carried out in a HP-5 capillary column (30 m × 0.25 mm, 0.25 µm in film thickness).The MS was operated in the EI mode (70 eV).The GC-MS interface, ion source, and quadruple temperatures were set at 280°C, 230°C, and 150°C, respectively.The injector temperature was set at 250°C, the column temperature program started at 50°C for 3 min, increased by 10°C min -1 to 110°C and by 10°C min -1 to 180°C, and was maintained for 2 min.Helium (99.999%) was used as the carrier gas with flow rate of 1 mL min -1 .Identification of spectra was carried out by studying their fragmentation and by comparison with standard spectra present in the library of the instrument.Area normalization was used for determination of composition percentage.

Insect rearing
Tribolium castaneum was reared in plastic rectangular containers (20 cm length × 14 cm width × 9 cm height) containing a wheat flour and wheat bran mixture (2:8 w/w).The mouth of the containers was covered with a fine mesh cloth for ventilation and to prevent the beetles from escaping.Cultures were maintained in an incubator at 27 ± 2°C and 60 ± 5% relative humidity (RH) in the dark.Parent adults were obtained from laboratory stock cultures maintained at the Department of Plant Protection, University of Tehran, Iran.All experimental procedures were carried out under the same environmental conditions as the cultures.

Repellent activity
A choice bioassay system was used to study the repellency of H. persicum essential oil.One half of filterpaper disks (6 cm in diameter) was treated with 200 μL of acetonic solution of the essential oil and dried for 5 min.Half of the bottom of a Petri dish was covered with the treated filter paper (Whatman No. 1) with concentration of 0.035, 0.07, 0.106, 0.141 and 0.212 µL cm -2 , while the other half was covered with a filter paper disk impregnated with acetone.Ten unsexed adults were put into each Petri dish and the lid was sealed with parafilm ® .Four replicates were run for each tested concentration, so that 40 adults were assayed per concentration.The number of insects on the two half paper disks was recorded after 2 and 4 h from the beginning of the test.Percentage of repellency (PR) was calculated as follows: PR = [(C -T) / (C + T)] × 100, where C = numbers of insects on the untreated area, and T = numbers of insects on the treated area (Nerio et al., 2009).Positive values express repellent and negative attraction values.

Contact toxicity
The contact toxicity of H. persicum essential oil against adults of T. castaneum was evaluated on filter paper discs of 6 cm in diameter which were treated with the substances diluted in acetone as a solvent.Range-finding studies were done to determine the fair testing concentrations.Concentrations of 1, 1.82, 3.32, 6.04, 10.99 and 20 μL of essential oil diluted in 0.5 mL of acetone were applied to the filter paper discs and the filter papers were placed in Petri's dishes of 6 cm diameter.These concentrations are equivalent to 0.03, 0.06, 0.11, 0.21, 0.38 and 0.71 μL cm -2 .The acetone was allowed to evaporate for 5 min before introduction of 10 unsexed insect adults and these were kept in darkness in the incubator at 27 ± 2°C and 60 ± 5% RH.The lids of Petri dishes were pierced (1 cm diameter) and then covered by a fine mesh cloth to avoid fumigant toxicity.In the control groups only acetone was applied to the filter papers.Each treatment was replicated three times and insect mortality was recorded after 24 h.

Fumigant toxicity
To determine the fumigant toxicity, f ilter paper (Whatman No. 1, cut into 2-cm diameter pieces) was impregnated with essential oil at doses calculated to give equivalent fumigant concentrations of 17.86, 24.64, 34, 46.92, 64.71 and 89.28 μL L -1 air.The impregnated filter paper was then attached to the undersurface of the screw cap of a glass vial (280 mL).The caps were screwed tightly on a vial containing 10 insects.Each concentration and the control were replicated tree times.Larvae (10-12 d-old), pupae (8-9 dold) and unsexed adults (1-7 d-old) were tested as above.When no leg or antennal movements were observed, insects were considered dead.Mortality was determined after 24 h from commencement of exposure for larvae and adults, while adults emerging from pupae were noted after 10 days.
For bioassay of eggs, 50 newly emerged beetles (male and female) were placed in 280 mL glass jars containing wheat meal and after 24 h, the insects were removed.Then treated filter papers were placed in each glass jars containing meal and 24 h old eggs.The jars were checked after 14 days to compare the number of emerged larvae between treated and control groups.

Antifeedant activity
The antifeedant effect of sub-lethal doses on 1 to 7day old adults was assessed as described for fumigant toxicity with concentrations ranging from LC 10 to LC 40 for 24 h exposure time.Surviving adults were removed and used immediately for the antifeedant assay.Ten grams of wheat flour and 50 insects previously treated with sub-lethal doses of oil was placed in each Petri dish (φ 6 cm).The lids of Petri dishes were pierced (φ 1 cm) and then covered by a fine mesh cloth for ventilation.Control groups were treated in the same way without oil.Each experiment was replicated three times.Reduction in the flour weight in each Petri dish was calculated after 72 h as follows: Feeding Deterrence Index or FDI (%) = [(Control -Treatment) / Control] × 100, where 'Control' is the weight of meal consumed by the insects without essential oils and 'Treatment' the weight of meal consumed by the insects treated with essential oils.
The experiments were arranged in a completely randomize design and the data were analyzed with ANOVA.The means were separated using the Tukey's test at the 5% level.The LC 50 values with confidence limits were calculated by probit analysis using the SPSS version 16.0 software package.
According to Fig. 1, the essential oil of H. persicum strongly repelled T. castaneum adults even at the lowest concentration, the repellency increased with increasing concentrations of essential oil.Complete repellency (100%) occurred when the highest concentration (0.212 μL cm -2 ) was applied for 8 h.
Fig. 2 shows that the adults of T. castaneum were very susceptible to the contact toxicity of essential oil of H. persicum and even the lowest concentration (0.03 μL cm -2 ) was significantly different from the control according to Tukey's test.The mortality was dose-dependent and increased with increasing concentrations (F = 284.57,p = 0.0001).Results of the probit analysis showed that the 24 h LC 50 with 95% confidence limits was 0.194 (0.118-0.363) μL cm -2 (Table 2).The essential oil of H. persicum demonstrated fumigant toxicity to all stages of T. castaneum.This essential oil killed the larvae, pupae and adults and significantly decreased larvae emerging from treated eggs.The activity depended on essential oil concentrations (F = 47.25, p = 0.0001 for eggs, F = 263.93,p = 0.0001 for larvae, F = 89.82,p = 0.0001 for pupae, F = 1591.57,p = 0.0001 for adults) (Fig. 3).In the fumigant toxicity test, according to Table 2, the LC 50 values with their 95% confidence limits were 41. 779 (29.991-60.831), 62.871 (53.333-79.427) and 46.005 (35.558-63.715)for larvae, pupae and adults, respectively.It is obvious that larvae were more susceptible than adults and pupae.On the other hand, pupae were more tolerant than larvae and adults (Table 2).
Probit analysis for fumigant toxicity on adults showed that sub-lethal concentrations from LC 10 to LC 40   were 16.06 to 37.36 μL L -1 air respectively and these concentrations were used to assess feeding deterrent activity.Fig. 4 shows that the essential oil of H. persicum had significant antifeedant effects on adults of T. castaneum in a dose-dependant manner.The concentrations of 53.89, 59.71, 67.5, 72.79, 77.85, 87.38 and 93.2% were used in FDI bioassay.This effect was also dose-dependent (F = 18.45, p = 0.0001) and concentration of 37.36 μL L -1 air (the LC 40 ) caused the highest antifeedant effect in which food consumption and FDI values were 0.09 g and 93.2% respectively (Fig. 4).

Discussion
There are many studies related to sensitivity of Tribolium castaneum to plant essential oils that support the results of the present study.For example, Zapata & Smagghe (2010) reported the repellent activity and the contact and fumigant toxicity of four essential oils extracted from the leaves and bark of Laurelia sempervirens Tul.(Monimiaceae) and Drimys winteri J.R. Forster and G. Forster (Winteraceae) against T. castaneum.After 4 h of exposure, >90% repellency was achieved with L. sempervirens oils at low concentrations of 0.032 μL cm -2 , while for D. winteri oils concentrations 3-10 times higher were needed to achieve it.LC 50 values by topical application of L. sempervirens oils were from 39 to 44 μg mg -1 insect; for D. winteri oils these were from 75 to 85 μg mg -1 insect.By fumigation, LC 50 values for L. sempervirens oils were 1.6-1.7 μL L -1 air, while these were 9.0-10.5 μL L -1 air for D. winteri oils.In the study of Abbasipour et al. (2011), the insecticidal activity of the essential oil from Elettaria cardamomum L. (Zingiberaceae) on the adults of Ephestia kuehniella Zeller (Pyralidae), T. castaneum and Callosobruchus maculatus (Fabricius) was investigated.Results indicated that essential oil of E. cardamomum was toxic to all insects and E. kuehniella adults were more sensitive than the Coleoptera and T. castaneum was more tolerant compared to C. maculatus.Also, the highest mortality of these insects was seen after 12 hours.Caballero-Gallardo et al. (2011) tested the repellent activity of essential oils from Tagetes lucida Cav., Lepechinia betonicifolia (Lam.),Lippia alba (Mill.),Cananga odorata (Lam.),Rosmarinus officinalis L. and several of their constituents against T. castaneum.All essential oils were repellents, followed a dose-response relationship, and had bioactivity similar to or better than that of commercial compound IR3535.Essential oils from C. odorata and L. alba were the most active.Compounds from essential oils, such benzyl benzoate, β-myrcene, and carvone, showed good repellent properties.All mentioned studies emphasized the results of the present study for susceptibly of T. castaneum to plant essential oils from lethal to sub-lethal bio-effects.
Lethal and sub-lethal insecticidal effects of H. persicum essential oil have been reported in many studies.For example, the fumigant toxicity of this essential oil on C. maculatus was reported by Manzoomi et al. (2010).LC 50 value in this study was 337.38 μL L -1 that was much more than our LC 50 values.In the study of Izakmehri et al. (2013), the lethal and sublethal effects of essential oils from H. persicum were evaluated on C. maculatus adults.The LC 50 value of H. persicum was 219.4 μL L -1 air after 12 h and 136.4 μL L -1 air after 24 h of exposure, respectively.The results showed that low lethal concentration (LC 20 ) of essential oils negatively affected the longevity, fecundity, and fertility of female adults.In another study, Sedaghat et al. (2011) found that H. persicum essential oil exerted significant larvicidal potential with LC 50 value of 26.30 ppm against four instar larvae of Anopheles stephensi Liston after 24 h and a positive correlation was observed between the essential oil concentrations and the mortality percentage.In the study of Faraji et al. (2012), the effect of H. persicum essential oil was tested on egg-laying of Phthorimaea operculella (Zeller) with fresh potato leaves and without them; the essential oil concentration in both series of experiments affected to the female egg-laying rate, the number of eggs laid was significantly higher in the control than in the treated plants.The egg-laying inhibition observed in both series of experiments was 56.8% and 48% respectively.Furthermore, Amizadeh et al. (2013) studied the acaricidal activity of this oil against eggs and adults of Tetranychus urticae Koch (Acari: Tetranychidae).Their f indings are in accordance with our results for lethal and sub-lethal effects of H. persicum essential oil.
The mechanisms of essential oil toxicity to insects have not been fully identified.Treating the insects with essential oils or pure compounds may cause symptoms due to neurotoxic activity.These symptoms include hyperactivity, seizures, and tremors followed by knocking down.Such symptoms are very similar to those produced by pyrethroid insecticides (Isman, 2006).Enan (2001) suggested that toxicity of the essential oil constituents is related to the octopaminergic nervous system of insects.Octopamine is a neurotransmitter, neurohormone, and circulating neurohormone-neuromodulator.Disruption of octopamine results in total breakdown of the nervous system in insects.The lack of octopamine receptors in vertebrates may provide the mammalian selectivity of essential oils as insecticides.Several reports illustrated that essential oils cause insect mortality by inhibiting acetylcholinesterase enzyme (AChE) activity (Kostyukovsky et al., 2002;Houghton et al., 2006;Abd El-Galeil et al., 2009).However, some activity on the hormone and pheromone system and on the cytochrome P450 monooxygenase enzyme has also been detected (Tsao & Coats, 1995;De-Oliveira et al., 1997).In our previous study (Ebadollahi et al., 2013), the essential oil isolated from Agastache foeniculum (Pursh) Kuntze (Lamiaceae) not only showed high toxicity on T. castaneum larvae, but activity of esterase and glutathione S transferase enzymes were also decreased besides reducing total carbohydrate, lipid and protein contents.These studies showthat the insecticidal activity of essential oils is due to several mechanisms that affect multiple targets.Sefidkon et al. (2004) assessed the chemical composition of essential oils of the stems (before and at full flowering stage), unripe and ripe seeds of H. persicum by a combination of GC and GC/MS.Twenty-four components were characterized from the stem oil before flowering with (E)-anethole (47.0%), terpinolene (20.0%), γ-terpinene (11.6%) and limonene (11.5%) as the main constituents.At full flowering stage, 33 compounds were identified in the stem oil with (E)anethole (60.2%), terpinolene (11.3%) and γ-terpinene (7.1%) as the major components.Among the 30 compounds identified in the seed oil of H. persicum, the major constituents were hexyl butyrate (22.5% and 35.5%), octyl acetate (19% and 27%) and hexyl isobutyrate (9.1% and 3.2%) in unripe and ripe seeds, respectively.In the study of Radjabian et al. (2013), the essential oils of flat-oval shaped fruits of 17 wild populations of H. persicum collected from different locations in Iran were obtained by hydro-distillation and analyzed by GC-MS.The oil yields varied greatly among populations and ranged from 1.6% to 4.9% based on dried plant material.Thirty-six compounds, which accounted for 89.7-99.0.% of the total oil, were identif ied in the essential oils.Octyl acetate (7.5-40.8%),hexyl butyrate (13.3-43.0%),hexyl isobutyrate (2.9-7.2%) and hexyl 2-methyl butyrate (4.8-11.9%)were the major components.Aliphatic esters were the most abundant class of compounds identified in the essential oils and of them, both octyl acetate and hexyl butyrate were the characteristic constituents of fruit essential oils.In the present study, hexyl butyrate, octyl acetate and hexyl hexanoate were found as main constituents of essential oil from H. persicum seed and it confirm the results of previous mentioned studies.Generally, the chemical composition of essential oil varies with species, season, climate, soil type, age of the leaves, soil fertility regimen, the method used for drying the plant material and the method of oil extraction (Batish et al., 2008).
The bioactivity of the essential oils depends on the type and nature of the constituents and their concentration.The toxicity, repellent, antifeedant and other biological activities of plant essential oils are mainly related to their constituents (Koul & Walia, 2009) and it may be concluded that the biological effects of H. persicum essential oil are related to its main components, such as hexyl butyrate, octyl acetate and hexyl hexanoate.The strong repellency, fumigant toxicity and the safety of essential oil suggest that H. persicum is a promising candidate to be used in insect pests' management.Furthermore, this oil is used as flavoring and medicinal agents, and is considered to reducing the harmful effect of conventional insecticides on humans and the environment.For the practical application of the essential oil as insecticide, further studies on formulation development are necessary to improve efficacy and stability, and cost reduction.

Figure 1 .
Figure 1.Repellency of essential oil of Heracleum persicum against the adults of Tribolium castaneum recorded 4 and 8 h after treatment.Repellency % = (C -T) × 100 / (C + T); C = number of insects on the control; T = number of insects on the treated.Ten adults were used per replicate.

Figure 2 .
Figure 2. Contact toxicity of essential oil of Heracleum persicum against the adults of Tribolium castaneum after 24 h of exposition.Means with the same letter on standard error bars are not significantly different (p = 0.05, Tukey's test).Twenty adults were used per replicate.

Figure 4 .
Figure 4. Feeding deterrence effect of sub-lethal concentrations of the essential oil of Heracleum persicum on adults of Tribolium castaneum.FDI was calculated as explained in M&M.Means with the same letter on standard error bars are not significantly different (p = 0.05, Tukey's test).Fifty adults were used per replicate.

Table 1 .
Result of GC-MS analysis of essential oil from Heracleum persicum seed

Table 2 .
Results of probit analysis for contact and fumigant toxicity of the essential oil of Heracleum persicum against Tribolium castaneum Fumigant toxicity of the essential oil of Heracleum persicum against the eggs, larvae, pupae and adults of Tribolium castaneum after 24 h.The mean emerged larvae from treated eggs after 14 days indicate the susceptibility of eggs.For each parameter studied means with the same letter on standard error bars are not signif icantly different (p = 0.05, Tukey's test).Twenty adults were used per replicate.