For many years, there has been a long and persistence fight against insects attacking the food and food products. Many of the earlier pesticides were of plant origin and were used locally and traditionally before, during and after food production [1]. Synthetic or fumigant pesticides used for plant protection and pests controlling in stores usually bring about resistance in these pests [2,3]

The persistent use of synthetic pesticides in agriculture, silviculture and even animal husbandry has created several difficulties on public health. However, even today the pesticides constitute one of the major parts in IPM for controlling insect pests. Plant origin pesticides are trustworthy, economical and safe for ecosystems and can control various ranges of pests. These insecticides- due to their little side effect on natural enemies, little toxicity on vertebrates and fast degradation in environment - are of special importance [4,5]. Hence, it seems that plant origin insecticides are safe alternatives to traditional insect control measures [6,7]. Among plant products used for pest control, essential oils are used as fumigant [8,9], contact [10-12], deterrents [13- 15] and antifeedants [16]. They may also affect some life parameters like development, longevity, reproduction and as well as sex finding behaviors [2] and finally growth regulatory effect [17]. The insecticidal effect of plant origin insecticides is determined by insect species, the chemical compounds of the plants and duration of exposure of the insect pest [18]. P. interpunctella has been the subject of some studies on the effect of essential oils [19-22] but its mode of action has not been elucidated. A. annua is an indigenous plant growing wild in north of Iran around paddy fields and has shown considerable effect on some insect species of agriculture and forestry importance [23-26]; moreover it has the potential of screening on other important pests. There are also reports on its fumigants activity on some stored product insects [19]. The essential oils used for stored product insects should have the potential of killing all life stages of insects [2]. Due to this reason the study was undertaken against eggs, larvae and adults of Indian meal moth. We also studied the sub lethal doses of this essential oil to examine fecundity, fertility, longevity and some important biochemical compounds essential for insect development and survival (Lipids, Proteins and carbohydrates) to have insight into its probable mode of action.

Insect

Plodia interpunctella (Huebner) was collected from infested rice in stores. The insects were reared in plastic jars (7.5 ×145×18.5 cm), holes (2×2cm) were provided with meshing cloth for aeration. The insects were reared in a controlled room (26±1°C, 14:10 LD and RH% 65±5). They were provided with artificial diet made of 800gms wheat, 160gm yeast, 200cc glycerol and 200cc natural honey [24]. The 17 day old larvae (unsexed) and adults (sexed) of cohort age were used for this study.

Plant essential oil

Artemisia annua L. was collected from field; leaves were separated, then washed with water and dried in shade and finally made into powder. Essential oil was extracted by a modified clevenger type apparatus. The oil was subsequently dehydrated by anhydrous sodium sulphate and kept in refrigerator (4°C) until used for experiments.

Bioassay and treatment

The bioassay for 17 day old larvae were carried out on filter paper [25]. The essential oil was diluted in acetone and used in 15%, 11.8%, 5.5% and 4% concentrations after initial bracketing test. The control received pure acetone (Merck, Germany). This test was carried out in four replications for each concentration and one control including 10 larvae (unsexed) of cohort age. Filter papers were impregnated with 1CC of essential oil in acetone and after evaporation of acetone, 10 larvae were released into each Petri dish. The Petri dishes were closed and tightly covered by Para films. After 24h, the number of dead or live insects were counted. The data were analyzed by Polo-Pc software and LC₂₅, LC₅₀ and LC₇₅ were estimated. Live larvae were separated and were reared separately in plastic jars and food material was provided to them. The parameters like longevity and percent adult emergence of male and female insects were calculated.

The bioassay on adults were performed on male and female based on the presence of claspers in male and after initial bracketing tests which included 4 concentration ranges of 9%, 8%, 6% and 5% along with acetone as a control. This experiment was carried out in 3 replications and by 32 insects for each replication [25]. For this purpose adults of cohort age were separated and 2 adults were transferred to vials (50 mm high and 29.5 mm width). Filter paper of 15mm width was located on the underside of the cap and was impregnated with essential oil or acetone alone in case of controls. A cloth mesh was also placed between the filter paper and vial cap in order to prevent adults from direct contact with filter paper. After 3 minutes of acetone evaporation the insects were released in the vials and the mortality was recorded after 24h. The mortality was separately recorded for male and female adults and with the help of Polo-Pc software LC₂₅, LC₅₀ and LC₇₅ was estimated. The insects remaining alive after 24h were transferred to plastic jars 76.3mm width × 187mm height whose lids were partly cut and replaced by meshing cloth for aeration. The daily mortality was recorded and was compared with the controls.

Biochemical analyses

Some important biochemical analyses (proteins, carbohydrates and lipids) were investigated on 17 day old larvae. 24 larvae, treated with LC₂₅, LC₅₀ and LC₇₅ were considered in 3 replicates for male and female insects and one control treated with acetone alone. After 24 h from every replicate 3 larvae were randomly selected from each sex (based on the presence of testis visible under stereomicrocope), homogenized in 2CC double distilled water and then centrifuged for 10 min at 4°C in 10000rpm and the supernatant was preserved at -20°C until the onset of experiments. The Bradford method [26] was adopted for measuring total protein and the absorbance was read at 630nm in an Eliza reader (Awareness Technology INC USA). The method of Van Handel [27] was adopted for analysis of lipid content and the absorbance was read at 630 nm. The method of Van Handel et al. and Yuval et al. [27,28] was adopted for analyzing carbohydrates and the absorbance was read at 450 nm in the same Eliza reader.

Statistical analysis

The analysis of data was performed using SAS software (1997). For comparing different effects of essential oil in used concentrations, equalized complete randomized design and sometimes unequalized complete randomized were used and also for comparing the effects of essential oil in sex and concentrations, factorial design was used. For biochemical analyses the complete randomized design was adopted. The Tukey test was used for comparing the means. Wherever necessary the percentage data were Arc-Sin transferred.

The toxicity and physiological activity of essential oil on larvae

Dose-response of essential oil on 17 day old larvae of Plodia interpunctella is presented in Figure 1. The LC₂₅, LC₅₀ and LC₇₅ values after 24 h exposure are shown in Table 1.

Table 1: The LC₂₅, LC₅₀, LC₇₅ values. Confidence limits (%90) and regression slop after 24 h exposure to A. annua essential oil in larva of P. interpunctella (Hubner).

Figure 1: Regression of dose-response for Artemisia annua L. essential oil on 17 day old larva of Plodia interpunctella.

The result showed that there were significant differences between percent emergence of adult in control (89.75 ±1.57) and the treatments. The least emergence was recorded for LC₇₅ (F= 19.44, df= 3, p<0.01). The percent adult emergence for male was less than female, but this difference was not statistically significant (Figure 3) (F= 3.32, df= 1, p>0.05).

Figure 2: Mean (±SE) percentage adult emergence of P. interpunctella (Huebner) after larval treatment by A. annua L essential oil. Different letters indicate significance at p<0.01.

Figure 3: Mean (±SE) percentage adult male and female emergence of P. interpunctella (Huebner) after larval treatment by A. annua L. essential oil. Different letters indicate significance at p<0.01.

The comparison of means of adult longevity of treated larvae in 3 concentrations (LC₂₅, LC₅₀ and LC₇₅) showed that the control having 8.08 ±0.17 days had the maximum mean and stayed first in ranking (Figure 4). Even though the mean for male longevity was less than female insects but the differences were not significant (Figure 5). The analysis variance of the effect of concentration × sex between 3 concentrations was statistically significant (F= 87.94, df= 3, P<0.01).

Figure 4: Mean (± SE) adult longevity (days) after larval treatment by A. annua L essential oil. Different letters indicate significance at p<0.01.

Figure 5: Male and female adult longevity (days) after larval treatment by A. annua L. essential oil.

The evaluation of the effect of essential oil of A.annua on reproduction showed that both fecundity and fertility were significantly reduced (Figure 6 and Figure 7).

Figure 6: Mean (± SE) of eggs oviposition rate of females after larval treatment by A. annua L. essential oil. Different letters indicate significance at p<0.01.

Figure 7: Mean (± SE) egg hatchability deposition by females after larval treatment by A. annua L. essential oil Different letters indicate significance at p<0.01.

The toxicity effect of essential oil on adults of Plodia interpunctella

Dose-response of essential oil on adults is depicted in Figure 8 and in Table 2. The results of toxicity on adults showed the lowest mortality in controls (3.6±0.014) and the highest mortality in 9% concentration (33.36±0.014). Therefore the higher is the dose, the higher will mortality be (Figure 9) (F=37.14, df=4, p<0.01). Analysis of variance in adults does not show any significant difference in male and female (F= 0.28, df= 1, p> 0.05). Similarly, concentration into sex does not show any differences (Figure 10) (F= 2.28, df=4, p>0.05).

Table 2: The LC₂₅, LC₅₀, LC₇₅ values. Confidence limits (%90) and regression slop after 24 h exposure to A. annua essential oil in adult of P. interpunctella (Hubner).

Figure 8: Regression of dose-response for Artemisia annua L. essential oil vapor on adult Plodia interpunctella (Hubner).

Figure 9: Mean (± SE) percentage of adult mortality P. interpunctella fumigated by A. annua L. essential oil. Different letters indicate significance at p<0.01.

Figure 10: Mean (± SE) percentage mortality of male and female P. interpunctella fumigated by A. annua L. essential oil.

The effect of A.annua essential oil on energy reserves of Plodia interpunctella

The effect of different concentrations (LC₂₅, LC₅₀ and LC₇₅) on protein content of treated larvae is shown in Figure 11. The result showed that the mean for protein content of control was 2.9128±0.096 mg/larvae and in concentrations LC₂₅, LC₅₀ and LC₇₅ were 2.6472, 2.6471 and 2.4113 mg/larvae respectively; it also showed significant reduction compared with the control 24 h post treatment (F7.94, df= 3, P<0.01). The mean for protein content of female (2.635 mg / larvae) was more than male (2.479 m g/ larva) but this difference was not statistically significant (F= 2.38, df= 1, P>0.05).

Figure 11: Mean (±SE) effects of A. annua L. essential oil on protein content of P. interpunctella 17-day old larva 24 h after. Different letters indicate significance at p<0.01.

The effect of different concentrations (LC₂₅, LC₅₀ and LC₇₅) on carbohydrate content of larvae 24 h post treatment with A.annua essential oil is illustrated in Figure 12. The results indicated that the carbohydrate level in control (0.926 ±0.017 mg / larva) was significantly reduced by LC₂₅, LC₅₀ and LC₇₅ treated larvae by 0.2952, 0.1652 and 0.1068 mg/larvae respectively. Therefore, by increasing concentration the carbohydrate level decreased sharply (F=467.34, df= 3, p<0.01). The level of carbohydrates in female larvae (0.3893 mg / larvae) was more than male (0.3570 mg / larvae) but not statistically significant (F= 3.49, df= 1, P> 0.05).

Figure12: Mean (± SE) Effects of A. annua L. essential oil on carbohydrate content of P. interpunctella 17-day old larva 24 h after treatment. Different letters indicate significance at p<0.01.

The effect of different concentrations LC₂₅, LC₅₀ and LC₇₅ of A. annua essential oil and the control on lipid level of larvae is idicated in Figure 13. While the amount of lipid in control is 22.7881±0.63 mg / larva and for LC₂₅, LC₅₀ and LC₇₅ treated larvae are 22.4800, 16.3728 and 12.2643 mg / larva respectively and therefore show significant reduction after treatment(F= 63.72, df= 3, P<0.01). The amount of lipid in female (16.96 mg/larva) was more than male (17.98.mg / larva) but not statistically significant (F= 2.42, df= 1, P>0.05).

Figure 13: Mean (± SE) Effects of A. annua L. essential oil on lipid content of P. interpunctella 17-day old larva 24 h after treatment. Different letters indicate significance at p<0.01.

Plants are great sources of secondary plant metabolites which compete herbivores and pathogens [22]. Iran has a particular geographic condition and thus varied floras which have not been taken into consideration regarding the issue of plant protection. Therefore it seems reasonable to have a plan for further investigation on insecticidal effects of plants in future [5].

The results of this investigation clearly indicate that by increasing concentrations, the rate of mortality increases; it is similar to results reported on P. interpunctella adults by various essential oils [18]. The results of [22] also showed significant mortality of Tribolium castaneum, when A. annua extract at 0.5 % concentration was used.

The species of Artemisia have shown a good effects on mortality of different species; for example the studies of [15] on Xanthogaleruca luteola Mull have shown LC₅₀ values of 48% and 43% after 24 and 48 h of exposure. Another study by [5] on A. sieberi showed 100 % mortality by 37µl / air for Callosobruchus maculates (F), Triboliun castaneum (Herbst), Sitophylus oryzae (L). Similar studies on different species of Artemisia showed fair mortality on different species [30-32]. Camphore, a monoterpinoid in some plants, has been shown to have mortality effect as a fumigant in essential oils [5]. The camphore has been reported in A.annua [33].

The study also showed that different LC values were used to find out whether the adult emergence led to a significant result compared to the controls. This result is indicative of the effect of the essential oil on some morphological characteristics which unable the insect to emerge from pupal shell. In Plutella xylostella L. the Chinnaberry fruit extract showed similar results where, by increasing dosages, the emergence reduced [34]. This effect is definitely due to the metabolites present in either essential oil or in extract [35].

In the present study fecundity and fertility of adults emerging from treated larvae reduced significantly. The tentative answer for such a result may be the reduction in biochemical parameters (Protein and lipid) which has been considerably decreased after larval treatment. [36,37] found similar results in their experiments and concluded that changes in fecundity and fertility may be the result of changes in Protein.

Total protein content of larvae treated with LC₂₅, LC₅₀ and LC₇₅ after 24 h was significantly reduced compared with the controls. The reduction in protein reserves may be due to physiological adoption of the insect to compensate the stress caused by insecticides [38] or due to the production mechanism of lipoprotein for making the cells and tissues. Another possible reason may relatively be the lack of ferric or other essential molecules due to insecticidal or other stresses to some extend [39]. Lipids conservation occurs in larval period since they release high energy necessary for metamorphosis. Any disruption of important biochemical compounds has a direct and indirect effect in resultant adults [39].

Carbohydrate reserves are also affected by plant essential oils [40]. As for lipid we can say that hemolymph and fat body lipids could degrade due to the a hormone controlling lipid metabolism might have been affected by exogenous chemicals. Adipokinetic hormone [41] and other hormones controlling the metabolism of lipid or carbohydrates might be affected [42] and therefore the hormone sites may be the site of effect of exogenous chemicals.

The toxicity and sub lethal action of Artemisia annua L. and other species of this genus reveal that this plant may be a good and safe natural toxicant in stored products which deserves further investigation.

JJS wishes to thank University of Guilan for financial support. The authors also wish to thank the anonymous reviewer for helpful suggestion and we also thank Miss Nasra Roozbeh (M.A. in English) for editing the language of the manuscript.