Archives of Phytopathology and Plant Protection

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Toxicity of Artemisia annua (Asteraceae) essential oil on the tea mealy bug, viburni Sigornet (: Pseudococcidae)

Samar Ramzi, Ali Seraji, Reza Azadi Gonbad, Seyyedeh Kimia Mirhaghparast, Zahra Mojib Haghghadam & Shiva Haghighat

To cite this article: Samar Ramzi, Ali Seraji, Reza Azadi Gonbad, Seyyedeh Kimia Mirhaghparast, Zahra Mojib Haghghadam & Shiva Haghighat (2018): Toxicity of Artemisia annua (Asteraceae) essential oil on the tea mealy bug, Pseudococcus viburni Sigornet (Hemiptera: Pseudococcidae), Archives of Phytopathology and Plant Protection, DOI: 10.1080/03235408.2017.1352223 To link to this article: https://doi.org/10.1080/03235408.2017.1352223

Published online: 08 Jan 2018.

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Download by: [EPFL Bibliothèque] Date: 08 January 2018, At: 07:49 ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION, 2018 https://doi.org/10.1080/03235408.2017.1352223

Toxicity of Artemisia annua (Asteraceae) essential oil on the tea mealy bug, Pseudococcus viburni Sigornet (Hemiptera: Pseudococcidae)

Samar Ramzia, Ali Serajia, Reza Azadi Gonbada, Seyyedeh Kimia Mirhaghparastb, Zahra Mojib Haghghadamc and Shiva Haghighata

aTea Research Center, Horticulture Science Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Lahijan, Iran; bDepartment of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran; cPlant protection Department, Research Center for Agriculture and Natural Resources, Agricultural Research, Education and Extension Organization (AREEO), Rasht, Iran

ABSTRACT ARTICLE HISTORY A combination of bioassay and biochemical approaches Received 1 April 2017 were used to determine toxicity of Artemisia annua essential Accepted 28 June 2017 oil (AaEO) Pseudococcus viburni. AaEO via leaf dipping KEYWORDS bioassay showed LC50 values of 0.693 and 0.419% after two Artemisia annua; essential time exposures. Different concentrations of AaEO caused oil; Pseudococcus viburni; deterrence index between 28.58 to 86.26% by the calculated toxicity; physiology; ED50 of 0.4%. Although, α-esterase activity using α-naphtyl deterrence acetate increased in the treated nymphs by AaEO after 24 hours but it showed the lower activity in the treated nymphs using β-naphtyl acetate. Glutathione S-transferase assayed by CDNB showed the higher activity in the treated nymphs than control after 24 hours while the adverse results gained not only after 48 hours but also after 24 hours by using DCNB. No significant differences were found in the activity of alanine aminotransferase versus control, but aspartate aminotransferase and γ-glutamyl transferase showed the statistically higher activities in the treated nymphs in comparison with control. Activities of aldolase and lactate dehydrogenase were significantly lower than those of control.

Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 Only acid phosphatase showed the significantly altered activity in the treated nymphs in comparison with control after 24 hours. Results of our study indicated significant toxicity, deterrence and physiological effects of AaEO on P. viburni.

Introduction

Tea (Camelia sinensis L.) is one of the most popular beverages which are cultivated commercially in Asia, Africa and (FAO 2012). Although Iran is

CONTACT Samar Ramzi [email protected] © 2018 Informa UK Limited, trading as Taylor & Francis Group 2 S. RAMZI ET AL.

the twelfth country in tea production, it accounts for more than four per cent of the world’s tea consumption (FAO 2012). Regions of tea cultivation have been restricted to around ofCaspian Sea but it suffers incident of some serious pests like Pseudococcus viburni Sigornet (Hemiptera: Pseudococcidae) and Brevipalpus obovatus Donnadieu (Prostigmata: Tenuipalpidae) (Mafi 1997). Nowadays, P. viburni has emerged as the key constraint on tea production of Iran since both adults and nymphs intensively feed on foliages leading to wilt leaves and stems and to cover them by production of (Mafi1997 ). Since leaves of tea are directly processed and consumed by human, chemical spraying has been banned due to biohazardous cautions so biological control using Cryptolaemus montrouz- ieri Mulsant (Coleoptera: Coccinellidae) is recommended although it sometimes failed because of environmental extremes or quality of produced biocontrol agent. Plant extracts and essential oils have been suggested as the reliable alternatives for chemical not only for their direct toxicity, repellency and physiolog- ical turbulences on pests, but also for low toxicity on non-target organisms besides negligible potential of environmental pollution (Isman 2006). In details, plant secondary metabolites lead to toxicity on insect pests in low concentrations in addition to ovicidal, larvicidal, anti-feedant and sterilising properties (Isman 2006). As the toxic compounds, plant metabolites increase permeability and ionic leak of cells due to destruction of their membranes. Hence, mitochondria, mem- brane proteins and cytoplasm components are degenerated and cell death occurs in the target tissue or whole body (Bhakuni et al. 2001). Artemisia annua L. (Asteraceae), called wormwood, is an annual short-day plant native of Asia which grows as a wild weed around paddy field of northern Iran (Shekari et al. 2008). It is a hairless plant with 30–100 cm of length, the leaves have an intensive aromatic scent which are divided by deep cuts into two or three small leaflets (Simonnet et al. 2006). Although Artemisin is a well-known compound of A. annua, researches on extracts and essential oils revealed several medicinal characteristics as anti-malarial, anti-bacterial, anti-inflamatory, plant growth regulatory and cytotoxicity activities (Bhakuni et al. 2001). Extract or

Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 essential oil of A.annua have shown considerable toxicity on several pests along with different physiological disorders on nutrition, reproduction, immune responses, intermediary metabolism etc. (Shekari et al. 2008; Zibaee and Bandani 2010a, 2010b; Hasheminia et al. 2011; Zibaee 2011; Mojarab-Mahboubkar et al. 2015). Since P. v ibur ni is one of the main pests reducing tea yield in northern Iran, control techniques through safe procedures, such as plant compounds, seems to be an efficient way with the lowest impact on non-target organisms, environment and agricultural products. Moreover, there are a few studies on the toxicity of essential oils mainly A. annua on and there is no study on their physiological alterations due to essential oil treatment. Hence, the current study was carried out determining the effect of A. annua essential oil on toxicity, deterrence, detoxifying and intermediary-involved enzymes in the third nymphal of P. v ibur ni . ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 3

Material and methods Insect rearing P. v ibur ni was obtained from a laboratory stock held in Department of Plant Protection, Tea Research Institute of Iran. The insects were fed on squash at 25 ± 2 °C, 70% of relative humidity and 16L:8D of photoperiod to get stock population. Containers were cleaned twice a week and squashes were replaced every three weeks.

Preparation of A. annua essential oil

The leaves of A. annua collected from Lahijan in northern Iran (37°12′26″N 50°00′14″E) were dried in the shade and were ground to be powder. Then, 50 g was poured into 750 ml of distilled water and left in 4 °C for 24 h. The mixture was transferred to a Clevenger-type apparatus as recommended by the British Pharmacopoeia (Yazdani et al. 2014). Distillation took about 2 h to obtain the essential oil while the process was repeated several times to gain the desirable amounts. Finally, the essential oil was dehydrated by sodium sulphate and kept at 4 °C for less than a month to onset of bioassay (Yazdani et al. 2014).

Bioassay of A. annua essential oil on P. viburni

The leaves of C. sinensis (tea) were cut in pieces of 3 × 3 cm and put in different solution of AaEO as 0.1, 0.2, 0.5, 1, 1.5 and 2% (based on a preliminary test) for 30 s. The concentrations were prepared in a Triton X-100 (0.01%) solution and the control leaves were only treated by Triton X-100. The treated leaves were dried on filter paper (Whatman No.1) for 60 min and the third nymphal instars (24 h age) were transferred to leaf discs. Each concentration including control contained forty nymphs in five replicates of eight individuals in each (N = 280).

After 24 and 48 h, the mortality of nymphs were recorded and LC50 values were

Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 calculated by POLO-PC software (LeOra Software 1987).

Deterrence tests

The method of Xie and Isman (1992) were followed to find deterrence index on the third nymphal instars of P. v ibur ni versus AaEO. The five concentrations were prepared as 0.4, 0.8, 1.6, 3.2 and 6.5% and the experiment was repeated three times by 20 nymphs in each. The two leaves of tea in the same size were selected, dipped in the desired concentration for 30 s and dried in air for 60 min. Control leaves were dipped in distilled water containing Triton X-100 (0.01%). The control and treated leaf were placed in a container (10 × 20 cm), then 20 nymphs were released at the centre of container. The explained procedure was done for each concentration versus control. The numbers of insects attracted to the control 4 S. RAMZI ET AL.

or treated leaf were recorded after 24 h. Deterrence index was calculated by the following equation: C − T DI = × 100 C + T

where C is the number of insects on the control leaf and T is the number of insects on the treated leaf.

Effect of A. annua essential oil on detoxifying and intermediary-involved enzymes

The leaf discs were prepared as above and divided into two groups. One group was

dipped into control solution but another one was dipped into LC50 concentration of AaEO and dried as explained earlier. Thirty nymphs were transferred on the two groups of leaves, respectively, and kept for 24 and 48 h. Then, the nymphs were divided into three groups (Control and treatment separately), transferred to eppendorff tubes and homogenated in 500 μl of distilled water. The samples were centrifuged at 20,000 g for 20 min at 4 °C and the supernatant was used for biochemical experiments.

Determination of general esterase activity

The activities of α- and β-esterases in the control and treated nymphs of P. v ibur ni were determined using α-naphtyl acetate and β-naphtyl acetate as substrates. Based on Han et al. (1998), 20 microlitre of substrates (10 mM) was separately mixed by 50 μl of fast blue RR salt (1 mM). Then, 10 μl of enzyme solution was added to each tube containing different substrates and optical density (OD) was recorded at 450 nm using microplate reader after 5 min during the linear phase of the reaction.

Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 Determination of glutathione S-transferase (GST) activity (2.5.1.18)

Based on the method of Oppenorth (Oppenoorth et al. 1979), 20 μl of CDNB (1-chloro-2,4-dinitrobenzene, 20 mM) and DCNB (1,2-dichloro-4-nitro-benzene, 40 mM) were separately mixed by the reduced glutathione in the microplate wells, then 10 μl of enzyme solution was added and OD value was recorded at 340 nm after 5 min of incubation during the linear phase of the reaction.

Assay of alanine (EC 2.6.1.1) and aspartate (EC 2.6.1.1) aminotranferases

The activities of Alanine- and aspartate aminotrasferase (ALT, AST) were deter- mined based on the procedure of Thomas (1998). The assay mixture contains ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 5

2,4-dinitrophenyl hydrazine in which produced pyruvate is combined with 2,4-dintitrophenyl puruvate and synthesised pyruvate hydrazine. Briefly, reagent A (for AST) and reagent B (for ALT) were incubated separately with reagent D for 5 min. Then, 10 μl of the enzyme solution was added and incubation prolonged for 60 min. Finally, reagent C was added and absorbance was read at 340 nm (Biochem Co., Iran).

Assay of γ-glutamyl transferase (γ-GT) (EC 2.3.2.2)

Based on the method of Szasz (1976), 50 μl of buffer reagent and 20 μl of substrate reagent (L-ɤ-glutamyl-3-carboxy-4-nitrianilide) were mixed gently, then 10 μl of enzyme solution was added prior to read the absorbance at 405 nm after 3 min (ZiestChem Diagnostic Co., Tehran-Iran).

Assay of aldolase (EC 4.1.2.13)

As instruction of the manufacturer (ZiestChem Diagnostics Co., Tehran-Iran), 50 μl of buffer reagent, 25 μl of substrate reagent (Fructose-1,6 di-phosphate), 10 μl of cofactor reagent (NADH) were incubated for 5 min with 20 μl of sample. Then, the absorbance was read at 340 nm (Pinto et al. 1969).

Assay of lactate dehydrogenase (EC 1.1.1.27)

King (1965) method was used to evaluate activity of lactate dehydrogenase (LDH). To standardise volumes, 0.2 ml NAD+ solution was added to the test tubes and 0.2 ml of water was added to control test tubes, each containing 1 ml of the buff- ered substrate and 0.01 ml of the sample was also added to the test tubes. Test tube samples were incubated for exactly 15 min at 37 °C and then arrested by adding 1 ml of colour reagent (2,4-dinitrophenyl hydrazine) to each tube and the incubation continued for an additional 15 min. Then, the contents were cooled

Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 at room temperature, 10 ml of 0.4 N NaOH was added to each tube to make the solutions strongly alkaline. At exactly 60 s after the addition of alkali to each tube, the intensity of colour was measured at 340 nm.

Assay of acid (EC 3.1.3.2) and alkaline (EC 3.1.3.1) phosphatase (ACP, ALP)

Based on the method of Bessey et al. (1946), the buffered substrate (Tris-HCl, 20 mM, pH 8 for ALP and pH 5 for ACP) phosphate buffer, 0.02 m, pH 7.2) was incubated with samples for 30 min. Then, NaOH (1 M) was added to stop the reaction and the absorbance was read at 405 nm. 6 S. RAMZI ET AL.

Table 1. Toxicity of Artemisia annua essential oil on the 3rd nymphal instars of Pseudococcus viburni.

2 Time of treatment (h) LC50 (%) Confidence limit (95%) Slope ± SE X df 24 0.693 0.286–1.642 0.703 ± 0.231 2.6535 3 48 0.419 0.095–0.864 1.208 ± 0.243 6.1051 3

Table 2. Repellency effect of Artemisia annua essential oil on the 3rd nymphal instars of Pseudoc- cocus viburni after 24 h.

Treatments (%) DI (%) ± SE ED50 Confidence limit (95%) Slope ± SE X2 df 0.4 28.58 ± 5.06 0.4 0.09774 ± 0.70776 1.069 ± 0.28 0.79 3 0.8 38.11 ± 4.81 1.6 54.21 ± 8.73 3.2 60.41 ± 6.89 6.5 86.26 ± 5.65

Protein determination

Protein concentrations in the samples were assayed according to the method described by Lowry et al. (1951) by bovine serum albumin as standard (Recommended by Ziest Chem. Co., Tehran-Iran).

Statistical analysis

The experiments were done in a complete randomised design. The obtained data were compared by one-way analysis of variance (ANOVA) followed by T-Test at a probability less than 5% and marked by asterisks in figures and tables.

Results Effect of A. annua essential oil on mortality and deterrence of P. viburni A. annua essential oil caused significant mortality on the third nymphal instars of

Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 P. v ibur ni in the two time intervals. The LC50 concentrations of AaEO were found to be 0.693 and 0.419% after 24 and 48 h post-treatment with confidential limit of 0.286–1.642 and 0.095–0.864, respectively (Table 1). The concentrations of AaEO used for deterrence of nymphs showed different values in a dose-dependent manner so that the concentration of 0.4% led to 28.58% deterrence index as the lowest value and the concentration of 6.5% led to the highest deterrence index as 86.26% (Table 2). Moreover, ED50 was calculated to be 0.4% on the third nymphal instars of P. v ibur ni (Table 2).

Effect of A. annua essential oil on detoxifying enzymes of P. viburni

The assay of general esterase was done using two substrates in the two time inter- vals. The activity of assayed α-esterase by α-naphtyl acetate statistically increased in the treated nymphs in comparison with control after 24 h of treatment. Although ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 7

-esterase (-naphtyl acetate) 0.06

* 0.05 in) 0.04

0.03 Control 0.02 LC50

EST activity (OD/m 0.01

0 24 48 Time (hour)

-esterase (-naphtyl acetate) 0.1 * 0.09 * 0.08 in) 0.07 0.06 0.05 Control 0.04 0.03 LC50

EST activity (OD/m 0.02 0.01 0 24 48 Time (hour)

Figure 1. Effect of Artemisia annua essential oil on the activity of α- and β-esterases using α-naphtyl acetate and β-naphtyl acetate as substrates in the 3rd nymphal instars of Pseudococcus viburni. Statistical differences have been done within each time interval and marked by asterisks (t-test, p ≤ 0.05).

Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 the enhanced enzymatic activity was also observed after 48, it showed no statistical difference (Figure1 ). Activity of β-esterase in the treated nymphs was significantly lower than those of control for both time intervals (Figure 1). By using CDNB as the reagent, activity of GST significantly increased in the treated nymphs after 24 h but it sharply decreased after 48 h. Such an alleviation was also observed in the enzymatic activity by using DCNB after 24 h but it showed no statistical differences after 48 h (Figure 2).

Effect of A. annua essential oil on the activity of intermediary-involved enzymes

Activities of the three transaminases including alanine aminotransferase, aspartate aminotransferase and γ-glutamyl transferase were assayed in the third nymphal 8 S. RAMZI ET AL.

GST (CDNB) 0.2 * 0.18 * 0.16 in) 0.14 0.12 0.1 0.08 Control 0.06 LC50

GST activity (OD/m 0.04 0.02 0 24 48 Time (hour)

GST (DCNB) 0.3 * 0.25 in) 0.2

0.15 Control 0.1 LC50

GST activity (OD/m 0.05

0 24 48 Time (hour)

Figure 2. Effect ofArtemisia annua essential oil on the activity of glutathione S-transferase using CDNB and DCNB as reagents in the 3rd nymphal instars of Pseudococcus viburni. Statistical differences have been done within each time intervals and marked by asterisks (t-test, p ≤ 0.05). Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 of P. v ibur ni treated by AaOE. No statistical differences were obtained in the activities of ALT between treated and control nymphs in both time intervals (Figure 3). The activity of AST showed no statistical differences between treated and control nymphs after 48 h but it significantly increased in the treated nymphs by AaEO (Figure 3). The activity of γ-GT significantly increased in the treated nymphs by AaEO after 24 h but it showed no significant difference after 48 h (Figure 3). Activities of both aldolase and LDH in the treated nymphs by AaEO were statistically lower than those of control after 24 h while no significant differences were obtained after 48 h (Figure 4). Although similar results were found in activity of ACP for control and treated nymphs, the activity of ALP showed no statistical differences between nymphs in both time intervals (Figure 5). ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 9

ALT 0.12

0.1

0.08 g protein)

0.06 Control 0.04 LC50

0.02 ALT activity (U/m

0 24 48 Time (hour)

AST 0.2 0.18 * 0.16 0.14 g protein) 0.12 0.1 0.08 Control 0.06 LC50 0.04

AST activity (U/m 0.02 0 24 48 Time (hour)

-GT 0.25 *

0.2 g protein)

Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 0.15

0.1 Control LC50 0.05 -GT activity (U/m

0 24 48 Time (hour)

Figure 3. Effect of Artemisia annua essential oil on the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (γ-GT) in the 3rd nymphal instars of Pseudococcus viburni. Statistical differences have been done within each time intervals and marked by asterisks (t-test, p ≤ 0.05). 10 S. RAMZI ET AL.

Aldolase 0.25

* 0.2 g protein)

0.15

0.1 Control activity (U/m LC50 0.05 Aldolase 0 24 48 Time (hour)

LDH 0.14

0.12 *

0.1 g protein)

0.08

0.06 Control LC50 0.04

0.02 LDH activity (U/m

0 24 48 Time (hour)

Figure 4. Effect of Artemisia annua essential oil on the activities of aldolase and lactate dehydrogenase (LDH) in the 3rd nymphal instars of Pseudococcus viburni. Statistical differences have been done within each time intervals and marked by asterisks (t-test, p ≤ 0.05).

Discussion

Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 Although there are some well-established examples of biocontrol programmes against sucking insects, spraying by chemical insecticides seems to be an on-going way to suppress their population outbreaks. Such a control procedure has its own costs as toxicity on wildlife and human, environmental pollutions and some ineffi- ciencies like insecticide resistance and resurgence of secondary pests. In case, plant secondary metabolites in the two prepared compounds, essential oils and extracts, would be of interest because of their insecticidal potential, low toxicity on non-tar- get organisms and low persistence in environment (Isman 2006; Zibaee 2011). Our study showed that AaEO had the significant effects on mortality and deterrence

on the third nymphal stage of P. v ibur ni in low concentrations. Moreover, the LC50 value after 48 h post-treatment was lower than that of 24 h indicating prolonged mortality of the nymphs by AaEO. Rizvi et al. (2015) reported that extracts of Neem and Tobacco led to significant mortalities on cotton , Phenacoccus ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 11

ACP 0.08 * 0.07

0.06

g protein) 0.05

0.04 Control 0.03 LC50 0.02

ACP activity (U/m 0.01

0 24 48 Time (hour)

ALP 0.1 0.09 0.08 0.07 g protein) 0.06 0.05 0.04 Control 0.03 LC50 0.02

ALP activity (U/m 0.01 0 24 48 Time (hour)

Figure 5. Effect of Artemisia annua essential oil on the activities of acid and alkaline phosphatases in the 3rd nymphal instars of Pseudococcus viburni. Statistical differences have been done within each time intervals and marked by asterisks (t-test, p ≤ 0.05). Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 solenopsis Tinsley (: Pseudococcidae) by low concentrations in comparison with some synthetic insecticides. Prishanthini and Vinobaba (2014)

demonstrated that Neem, Tobacco, Calotropis and Garlic extracts had the LC50 of 0.82, 0.89, 0.95 and 1.15% on the cotton mealybug, Phanaccocus solenopis (Tinsley) (Hemiptera: Pseudoccocidae), respectively. Piragalathan et al. (2014) compared toxicities of some plant extracts including Neem, Pavetta leaf extract and garlic bulb on Papaya Mealybug, Paracoccus marginatus Williams y Granara de Willink (Hemiptera: Pseudococidae) in the different times of exposure. The mortality increased along with time exposure to 72 h in which garlic extract showed the highest mortality on the mealybug. El-Hefny et al. (2011) also reported higher mortality on citrus mealybug, Planococcus citri (Risso) (Pseudococcidae: Homoptera) treated by different plant extracts by increase of exposure time. 12 S. RAMZI ET AL.

Hollingsworth (2005) determined toxicity of Limonene, a cyclic terpene, of Citrus on longtail mealybugs. He reported that 1% concentration of limonene solution killed 43.9 to 98.2% of third and fourth instars based on type and percentage of emulsifier. Roonjho et al. (2013) determined repellency of some plant extracted from Prunus persica L. (Rosaceae), Eucalyptus globulus L. (Myrtaceae), Polyalthia longifolia Sonnerat (Annonaceae), Silybum marianum L. (Asteraceae) and Sonchus oleraceus L. (Asteraceae) on the cotton mealybug in the concentrations of 250, 500 and 1000 ppm. The authors found that the extracts caused different detterence on the cotton mealybug depending on type of extraction (Ether, Acetone and Etanol) and time of exposure (1–3 weeks) by the highest value caused by P. longifolia extract. Moreover, deterrence indices of plant extracts increased in a dose-de- pendent manner so that the highest values were obtained at the concentration of 1000 ppm. Singh et al. (2012) reported that different plant extracts of Azadirachtin indica Juss (Lamiaceae), E. globules and Ocimun basilicum L. (Meliaceae) caused repellency of 88–99% against cotton and cotton mealybug. Results of our study emboss significant effects of botanical compounds on mortality and repel- lency of mealybugs. The differences observed in detailed findings may be attrib- uted to the composition nature of materials in plant species or the place where the medicinal plant and mealybug exist or interact by environmental factors i.e. insecticides. P. v ibur ni treatment by AaOE showed changes in activities of the two detoxi- fying enzymes, α- and β-esterase and glutathione S-transferase. Although activity of esterase using β-naphtyl acetate in the treated nymphs were lower than control but the enzyme showed the enhanced activity by using α-naphtyl acetate. These results indicated that an isozyme of esterase which its activity can be detected by α-naphtyl acetate involves in detoxification of AaEO in the first time-interval after treatment. No statistical differences in esterase activity after 48 h may show efficacy in detoxifying of essential oil at the first 24 h of exposure. Moreover, lower activity of the enzyme using β-naphtyl acetate may demonstrate no engagement in AaEO detoxification. Accordingly, Mojarab-Mahboubkar et al. (2015) reported

Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 lower activity of general estarese assayed by both substrates in the treated larvae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) by AaEO. In con- trast, Zibaee and Bandani (2010a) reported the higher activity of the enzyme in the treated Eurygaster integriceps Puton (Hemiptera: Scutelleridae) by A. annua extract. Since there are no similar findings on other mealybugs, it could be con- cluded that involvement of esterases in detoxifying of plant materials depends on previous exposure of the insect to xenobiotics like insecticides in the fields. On the other hand, no spraying is made against P. v ibur ni so lower activity of esterase can be expectable. Another detoxifying enzyme used in our study was glutathione S-transferase which its activity increased after 24 h of exposure when CDNB was used as reagent. Another time interval by CDNB and the activity using DCNB in the treated nymphs by AaEO were lower than control nymphs. Similar to esterase, we believe an isozyme of GST detected by CDNB may involve in detoxification ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 13

of AaEO in P. v ibur ni . Many studies on the effects of plant compounds on insects showed enhanced activities of GSTs (Vanhaelen et al. 2001; Dugravot et al. 2004; Khosravi et al. 2010; Zibaee and Bandani 2010b; Mojarab-Mahboubkar et al. 2015). Intermediary metabolism in insects consists of several catalytic processes by enzymatic and non-enzymatic components which provide required energies for biological activities and keep homeostatic status of insects for efficient physiologi- cal mechanisms (Klowden 2012). Here, the activities of several enzymes including transaminases, aldolase, lactate dehydrogenase and phosphatases were determined to find the effect of AaOE on the intermediary metabolism ofP. v ibur ni nymphs. Transamination is a multiple process in which required amino acids are pro- vided for proper function or maintenance of tissues. The activities of the three known transaminases, alanine aminotransferase, aspartate aminotransferase and γ-glutamyl transferase were compared in the control and treated nymphs of P. v ibur ni by AeEO. ALT showed no significant activities among nymphs in both time intervals while AST had the significant higher activity in the treated nymphs by AaEO after 24 h. Although the activity of γ-glutamyl transferase significantly increased in the treated nymphs after 24 h but no significant difference was found after 48 h of treatment. In transamination process, ALT produces pyruvate and L-glutamate by transferring amino groups of L-alanine to α-ketoglutarate while AST concerts aspartate and α-ketoglutarate to oxaloacetate to be used in Krebs cycle (Klowden 2012). γ-GT leads to glutamate production via mobilisation of a γ-glutamyl moiety of glutathione to receptor producing glutamate. It engages in synthesis or degradation of glutathione or chemical compounds entered into body via γ-glutamyl cycle (Tate and Meister 1985). No significant activity of ALT between the control and treated nymphs may indicate non-reliance of the mealy- bug to provide energy via glycolysis a phenomenon which can be inferred by the activities of aldolase and LDH (see below). The higher activity of AST indicates the imposed energy demands in the treated nymphs which is provided via krebs cycle. Since no activity was observed after 48 h, it could be concluded that the

Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 treated nymphs were supported during the first time interval. Finally, the higher activity of γ-glutamyl transferase indicates its possible role in detoxification of AaEO which corresponds with the higher activity of esterase and GST during the first time interval. Aldolase and LDH are the two important enzymes in the initial and last steps of glycolysis, respectively (Klowden 2012). Aldolase is an isomerase which provide proper form of sugars to continue glycolysis while LDH catalyses the conver- sion of pyruvate to lactate synchronising the conversion of NADH to NAD+. The lower activities of these enzymes in the treated P. v ibur ni nymphs by AaEO versus control could highlight decreased performance of glycolysis to provide required energy hence the initial input for krebs cycle may not provide by pyruvate and it is initiated by amino acids from transamination. 14 S. RAMZI ET AL.

Acid- (ACP) and alkaline phosphatase (ALP) dephosphorylate several biolog- ical molecules such as nucleotides, proteins and alkaloids in alkaline and acidic conditions (Zibaee and Bandani 2010b). Activities of these enzymes in insects depending on the pH of haemocoel may demonstrate digestion efficiency and positive transportation of nutrients among midgut, haemolymph and fat bodies (Senthil-Nathan et al. 2006). No significant difference of ALP between control and treated nymphs of P. v ibur ni treated by AaEO may be attributed to non-involve- ment of the enzyme as regulating factor of homeostasis due to essential oil treat- ment. The lowered activity of ACP in the treated after 24 h may indicate digestive disturbance due to essential oil treatment which can be related to the deterrence effects found earlier. It has been elucidated that plant materials affect chemical sensilla located on mouthparts and inhibits stimulation of glucose, sucrose, ino- sitol and proteins on chemoreceptors (Zapata et al. 2009). Since insects depends on their neural function to find and continue feeding, such a block decreases feeding performance affecting intermediary metabolism which can be detected by the activities of ALP and ACP (Senthil-Nathan et al. 2006). Results of the current study point out the toxicity, deterrence and physiological effects of A.annua essential oil on the third nymphal stage of P. v ibur ni . Due to these results and abundance of A. annua as a wild plant in north of Iran, it may be an appropriate candidate as a biorational insecticide to suppress population outbreaks of the tea mealybug. Our further research would be concentrated to provide a proper formulation mainly nanocapsules which definitely increase effi- ciency of AaEO.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This research was supported by Agricultural Research Education and Extension Organisation Downloaded by [EPFL Bibliothèque] at 07:49 08 January 2018 [grant number 2–21-21–94103].

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