Effect of Artemisia Annua L. (Asterales: Asteraceae) Essential Oil on Mortality, Development, Reproduction and Energy Reserves of Plodia Interpunctella (Hübner)

lizers & rti P e e F s f t o i c l i Zamani et al., J Biofertil Biopestici 2010, 2:1

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s DOI: 10.4172/2155-6202.1000105 u

o J Biofertilizers & Biopesticides ISSN: 2471-2728

Research Article Open Access Effect of Artemisia Annua L. (Asterales: Asteraceae) Essential Oil on Mortality, Development, Reproduction and Energy Reserves of Plodia Interpunctella (Hübner). (Lepidoptera: Pyralidae) Sheyvan Zamani, Jalal Jalali Sendi* and Mohammad Ghadamyari Department of Plant Protection, Faculty of Agriculture, University of Guilan, Rasht, Iran

Abstract The study was conducted to determine the insecticidal and physiological activity of essential oil from Artemisia annua L. on Indial meal moth Plodia interpunctella (Hübner) in laboratory condition. The yellowish oil of wormwood Artemisia annua L. was diluted in acetone and different concentrations (15%, 11%, 8%, 5.5% and 4%) were assayed on

17 days old larvae. LC25, LC50 and LC75 were estimated 5.96%, 8.4% and 11.3% respectively after 24 h. The sub lethal doses showed that essential oil reduced adult emergence, longevity of male and female insects, fecundity and fertility

of females. Evaluation of toxic vapors on adult insect was also considered and LC25, LC50 and LC75 were estimated to be 6.35%, 8.13% and 10.45%. There was no difference in mortality on either sex. The protein, carbohydrate and lipid contents of treated larvae were significantly reduced compared with the controls.

Keywords: Worm-wood; Indian meal moth; Reproduction; Protein; Material and Methods Carbohydrate; Lipid Insect Introduction Plodia interpunctella (Huebner) was collected from infested rice For many years, there has been a long and persistence fight against in stores. The insects were reared in plastic jars (7.5 ×145×18.5 cm), insects attacking the food and food products. Many of the earlier holes (2×2cm) were provided with meshing cloth for aeration. The pesticides were of plant origin and were used locally and traditionally insects were reared in a controlled room (26±1°C, 14:10 LD and RH% before, during and after food production1 [ ]. Synthetic or fumigant 65±5). They were provided with artificial diet made of 800gms wheat, pesticides used for plant protection and pests controlling in stores 160gm yeast, 200cc glycerol and 200cc natural honey [24]. The 17 day usually bring about resistance in these pests [2,3] old larvae (unsexed) and adults (sexed) of cohort age were used for this study. The persistent use of synthetic pesticides in agriculture, silviculture and even animal husbandry has created several difficulties on public Plant essential oil health. However, even today the pesticides constitute one of the major parts in IPM for controlling insect pests. Plant origin pesticides are Artemisia annua L. was collected from field; leaves were separated, trustworthy, economical and safe for ecosystems and can control then washed with water and dried in shade and finally made into various ranges of pests. These insecticides- due to their little side effect powder. Essential oil was extracted by a modified clevenger type on natural enemies, little toxicity on vertebrates and fast degradation apparatus. The oil was subsequently dehydrated by anhydrous sodium in environment - are of special importance [4,5]. Hence, it seems sulphate and kept in refrigerator (4°C) until used for experiments. that plant origin insecticides are safe alternatives to traditional insect Bioassay and treatment control measures [6,7]. Among plant products used for pest control, essential oils are used as fumigant [8,9], contact [10-12], deterrents [13- The bioassay for 17 day old larvae were carried out on filter paper 15] and antifeedants [16]. They may also affect some life parameters [25]. The essential oil was diluted in acetone and used in 15%, 11.8%, like development, longevity, reproduction and as well as sex finding 5.5% and 4% concentrations after initial bracketing test. The control behaviors [2] and finally growth regulatory effect17 [ ]. The insecticidal received pure acetone (Merck, Germany). This test was carried out in effect of plant origin insecticides is determined by insect species, the four replications for each concentration and one control including 10 chemical compounds of the plants and duration of exposure of the larvae (unsexed) of cohort age. Filter papers were impregnated with insect pest [18]. P. interpunctella has been the subject of some studies 1CC of essential oil in acetone and after evaporation of acetone, 10 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]; *Corresponding author: Jalal Jalali Sendi, Department of Plant Protection, Faculty of Agriculture, University of Guilan, Rasht, Iran, Fax: +98 131 669028; moreover it has the potential of screening on other important pests. E-mail: [email protected] There are also reports on its fumigants activity on some stored product insects [19]. The essential oils used for stored product insects should Received October 11, 2010; Accepted June 15, 2011; Published July 05, 2011 have the potential of killing all life stages of insects [2]. Due to this Citation: Zamani S, Sendi JJ, Ghadamyari M (2011) Effect of Artemisia Annua reason the study was undertaken against eggs, larvae and adults of L. (Asterales: Asteraceae) Essential Oil on Mortality, Development, Reproduction and Energy Reserves of Plodia Interpunctella (Hübner). (Lepidoptera: Pyralidae). J Indian meal moth. We also studied the sub lethal doses of this essential Biofertil Biopestici 2:105. doi:10.4172/2155-6202.1000105 oil to examine fecundity, fertility, longevity and some important biochemical compounds essential for insect development and survival Copyright: © 2011 Zamani S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted (Lipids, Proteins and carbohydrates) to have insight into its probable use, distribution, and reproduction in any medium, provided the original author and mode of action. source are credited.

J Biofertil Biopestici ISSN:2155-6202 JBFBP, an open access journal Volume 2 • Issue 1 • 1000105 Citation: Zamani S, Sendi JJ, Ghadamyari M (2011) Effect of Artemisia Annua L. (Asterales: Asteraceae) Essential Oil on Mortality, Development, Reproduction and Energy Reserves of Plodia Interpunctella (Hübner). (Lepidoptera: Pyralidae). J Biofertil Biopestici 2:105. doi:10.4172/2155- 6202.1000105

Page 2 of 6 larvae were released into each Petri dish. The Petri dishes were closed LC LC LC No 25 50 75 Slope±SE X2 (df) and tightly covered by Para films. After 24h, the number of dead or (90% CI) (90% CI) (90% CI) 5.96 8.14 11.13 live insects were counted. The data were analyzed by Polo-Pc software A. annua 24 4.97±0.63 2.05 (3) (5.2-6.6) (7.43-8.89) (10.12-12.53) and LC25, LC50 and LC75 were estimated. Live larvae were separated and were reared separately in plastic jars and food material was provided CI: confidence limits to them. The parameters like longevity and percent adult emergence of Table 1: The LC25, LC50, LC75 values. Confidence limits (%90) and regression slop male and female insects were calculated. after 24 h exposure to A. annua essential oil in larva of P. interpunctella (Hubner).

The bioassay on adults were performed on male and female LC LC LC Chi- No 25 50 75 Slope±SE X2 (df) based on the presence of claspers in male and after initial bracketing (90% CI) (90% CI) (90% CI) square tests which included 4 concentration ranges of 9%, 8%, 6% and 5% 6.35 8.13 10.45 A. annua 284 1.32±1.14 (2) 0.072 along with acetone as a control. This experiment was carried out in 3 (5.44-6.93) (7.51-8.91) (9.42-12.65) replications and by 32 insects for each replication [25]. For this purpose 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).

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 Figure 1: Regression of dose-response for Artemisia annua L. essential oil on cut and replaced by meshing cloth for aeration. The daily mortality was 17 day old larva of Plodia interpunctella. 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] Figure 2: Mean (±SE) percentage adult emergence of P. interpunctella was adopted for analyzing carbohydrates and the absorbance was read (Huebner) after larval treatment by A. annua L essential oil. Different letters at 450 nm in the same Eliza reader. indicate significance at p<0.01. 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. Results The toxicity and physiological activity of essential oilon larvae

Figure 3: Mean (±SE) percentage adult male and female emergence of P. Dose-response of essential oil on 17 day old larvae of Plodia interpunctella (Huebner) after larval treatment by A. annua L. essential oil. interpunctella is presented in Figure 1. The LC25, LC50 and LC75 values Different letters indicate significance at p<0.01. after 24 h exposure are shown inTable 1.

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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 significantFigure ( 3) (F= 3.32, df= 1, p>0.05). 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

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.

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

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

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.

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

Figure 6: Mean (± SE) of eggs oviposition rate of females after larval treatment interpunctella by A. annua L. essential oil. Different letters indicate significance at p<0.01. Dose-response of essential oil on adults is depicted in Figure 8 and

Page 4 of 6 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).

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.

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

The effect of different concentrations25 (LC , LC50 and LC75) on protein content of treated larvae is shown in Figure 11. The result Figure 10: Mean (± SE) percentage mortality of male and female P. showed that the mean for protein content of control was 2.9128±0.096 interpunctella fumigated by A. annua L. essential oil. 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).

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, Figure 11: Mean (±SE) effects of A. annua L. essential oil on protein content df= 1, P> 0.05). of P. interpunctella17-day old larva 24 h after. Different letters indicate significance at p<0.01. The effect of different concentrations25 LC , 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). Discussion 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[ ].

Figure12: Mean (± SE) Effects of A. annua L. essential oil on carbohydrate The results of this investigation clearly indicate that by increasing content of P. interpunctella 17-day old larva 24 h after treatment. Different concentrations, the rate of mortality increases; it is similar to results letters indicate significance at p<0.01. reported on P. interpunctella adults by various essential oils [18].

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The results of [22] also showed significant mortality of Tribolium 3. White NDG (1995) Insects, mites and insecticides in stored grain ecosystems. castaneum, when A. annua extract at 0.5 % concentration was used. Stored grain ecosystem. Marcel Dekker, New York. 4. Isman MB (1994) Botanical insecticides and antifeedant: new sources and The species of Artemisia have shown a good effects on mortality Perpectives. Pesticide Research Journal 6: 11-19. of different species; for example the studies of [15] on Xanthogaleruca 5. Negahban M, Moharamipour S, Sefidkon F (2007) Fumigant toxicity of essential luteola Mull have shown LC50 values of 48% and 43% after 24 and oil from Artemisia sieberi Besser against three stored-product insect. Journal of 48 h of exposure. Another study by [5] on A. sieberi showed 100 % Stored Products Research 43: 123-128. mortality by 37μl / air for Callosobruchus maculates (F), Triboliun 6. 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J Biofertil Biopestici ISSN:2155-6202 JBFBP, an open access journal Volume 2 • Issue 1 • 1000105