Mulberry Protection through Flowering-Stage Essential Oil of Artemisia annua against the Lesser Mulberry Pyralid, Glyphodes pyloalis Walker

In the present study, the toxicity and physiological disorders of the essential oil isolated from Artemisia annua flowers were assessed against one of the main insect pests of mulberry, Glyphodes pyloalis Walker, announcing one of the safe and effective alternatives to synthetic pesticides. The LC50 (lethal concentration to kill 50% of tested insects) values of the oral and fumigant bioassays of A. annua essential oil were 1.204 % W/V and 3.343 μL/L air, respectively. The A. annua essential oil, rich in camphor, artemisia ketone, β-selinene, pinocarvone, 1,8-cineole, and α-pinene, caused a significant reduction in digestive and detoxifying enzyme activity of G. pyloalis larvae. The contents of protein, glucose, and triglyceride were also reduced in the treated larvae by oral and fumigant treatments. The immune system in treated larvae was weakened after both oral and fumigation applications compared to the control groups. Histological studies on the midgut and ovaries showed that A. annua essential oil caused an obvious change in the distribution of the principal cells of tissues and reduction in yolk spheres in oocytes. Therefore, it is suggested that the essential oil from A. annua flowers, with wide-range bio-effects on G. pyloalis, be used as an available, safe, effective insecticide in the protection of mulberry.


Introduction
The mulberry (Morus sp. (Rosales: Moraceae)) leaves are used for rearing silkworm (Bombyx mori L. (Lepidoptera: Bombycidae)). The importance of lesser mulberry pyralid Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)) is from the larvae damaging mulberry leaves and the transmission of plant pathogenic agents [1]. The extensive use of synthetic chemical pesticides has led to many concerns about the safety of humans, beneficial insects, and the environment [2,3]. Thus, management of insect pest through eco-friendly and biodegradable agents is critical in sericulture.
The essential oils obtained from several parts of plants, including leaves, flowers, fruits, twigs, bark, seeds, wood, rhizomes, and roots, are made as secondary metabolites in

Insects' Rearing
The larvae of G. pyloalis were handpicked from a mulberry orchard within the University of Guilan campus, Rasht (37.2682 • N, 49.5891 • E), Iran. The larvae were maintained on fresh leaves of 'Shin Ichinoise' mulberry variety in disposable transparent containers (high-density polyethylene plastic containers, 10 × 20 × 5 cm) in a rearing room set at 25 ± 1 • C, 75 ± 5% RH (Relative Humidity), and 16:8 L:D (Light:Dark). The emerging adults were reserved in glass jars (18 × 7 × 5 cm), in which fresh leaves were positioned for egg laying, and 10% honey-soaked cotton wool was provided for feeding.

Essential Oil 2.2.1. Extraction of the Essential Oil
The mature and immature flowers of A. annua (autumn 2018) were collected on the University of Guilan campus. Samples were dried on a table out of direct sunlight for about a week until sufficiently dry to form a powder when ground. The dried flowers were made into a fine powder by a grinder (354, Moulinex, Normandy, France), and a solution was made with distilled water (50 g/750 mL). The solution was let to stand in the dark at laboratory room temperature for 24 h to maximum essential oil extraction. The mixture was distilled to extract the essential oil using a Clevenger apparatus (J3230, Sina glass, Tehran, Iran). The distillation process was run for two hours and the obtained essential oil was dried over anhydrous sodium sulfate. The obtained essential oil was stored in dark glass vials at 4 • C in a refrigerator until used.

Determination of Essential Oil Composition
The essential oil was analyzed through gas chromatography (Agilent Technologies 7890B) coupled with a mass spectrometer (Agilent Technologies 5977A), which was armed with an HP-5MS ((5%-phenyl)-methylpolysiloxane) capillary column with a 30-m length, 0.25-mm width, and an internal thickness of 0.25 µm. Helium gas at a 1 mL/min flow rate was used, while the column temperature started from 50 and reached to 280 • C at a rate of 5 • C/min. A 10% A. annua essential oil solution in methanol (v/v) was prepared, and 1 µL of solution was injected. Spectra were obtained in the electron impact mode with 70 eV of ionization energy. The scan range was between 30-600 m/z. The identification of components was performed by comparing mass spectral fragmentation patterns and retention indices with those described in the databases [19,20].

Oral Toxicity
Initial tests were conducted to assist in selecting the appropriate range of concentrations. Bioassays were carried out on 4 th instar larvae, which were deprived of nutrition for 4 h before the onset of experiments. The essential oil concentrations of 0.5, 0.7, 1, 1.4 and 2% (W/V) in acetone as solvent (Merck, Darmstadt, Germany) were selected. For bioassays, mulberry leaf disks (8 cm in diameter) were immersed in desired concentrations for 10 s and then air-dried at room temperature for 30 min. Ten 4th instar G. pyloalis were placed on each disk. The mortality was documented after 24 h. Control groups were placed on disks treated with acetone. The control and treated groups were replicated four times.

Fumigant Activity
In order to carry out fumigation bioassays, two transparent polyethylene plastic containers (Pharman polymer company, Rasht, Iran) were used. A 250-mL container was used to place 10 4th instar larvae of mulberry pyralid. They were provided with fresh mulberry leaf disks, and the container top was covered with fine cotton fabric for aeration. The container was then placed inside a 1000-mL container. The desired amount of pure essential oil was poured onto filter papers (Whatman No. 1) cut to 2 cm in diameter using a micro applicator. It was then placed in the corner of the larger container, and its lid tightly sealed using Parafilm. The concentrations of 2, 3, 4, 5 and 6 µL/L air were used for this bioassay based on the initial tests. The controls were treated in the same way without any treatments of the filter papers. All tests were replicated four times.

Digestive Enzymes' Assays
In order to evaluate digestive enzymes activity, the larvae that were treated with LC 50 , LC 30 , and LC 10 (Lethal Concentration to kill 50, 30, and 10% of insects, respectively) dosages of essential oil obtained from oral and fumigant bioassays and the controls were dissected in ringer's solution (9% v/v NaCl and isotonic) 24 h after treatment and their digestive systems (only midguts) were dissected out. Five midguts for each treatment and control were first homogenized in 500 µL of universal buffer (50 mM sodium phosphate-borate at pH 7.1) in a tissue homogenizer (DWK885300-0001-1EA, Merk, Darmstadt, Germany). The supernatant was then kept at −20 • C until analyzed.

The α-Amylase Activity
The reagent dinitrosalicylic acid (DNS, Sigma, St. Louis, MI, USA) in 1% soluble starch was used to estimate α-amylase activity according to the method of Bernfeld (1955) [21]. Briefly, 20 µL of the enzyme was poured into 40 µL of soluble starch and 100 µL of universal buffer (pH 7). The mixture was incubated for 30 min at 35 • C, and DNS (100 µL) was then added to stop the reaction. The absorbance was read at 540 nm in an ELISA reader (Awareness, Temecula, CA, USA).

Protease Assay
The protease activity was assessed by addition of 200 µL of casein solution casein (1%) to 100 µL of enzyme and 100 µL universal buffer (pH 7). Then, the obtained mixture was incubated at 37 • C for 60 min [22]. The mixture was centrifuged at 8000× g within 15 min and the absorbance was read at 440 nm.

Hematological Study
The amount of various circulating blood cells in mm −3 of larval lesser mulberry pyralid treated with sublethal doses of A. annua oil and in controls were assessed. The hemolymph was drawn from one of the larval prolegs, cutting by a fine scissor, using a capillary glass tube (10 µL for each treatment). Then, the blood was diluted five times with a solution of anticoagulant (0.017 M EDTA, 0.186 M NaCl, 0.098 M NaOH, and 0.041 M citric acid at pH 4.5). An improved Neubauer hemocytometer (mlabs, HBG, Giessen, Germany) [30] was used to assess the total cells using the formula of Jones (1962) [31]. A drop of hemolymph was collected from cut proleg of treated and control larvae. A smear was formed and stained with diluted Giemsa (Merck, Darmstadt, Germany) in distilled water (1:9) for 25 min, then just dipped in a saturated solution of lithium carbonate, and, finally, washed with distilled water. Permanent slides were prepared in Canada balsam (Merck Darmstadt, Germany). The percentage profile of different cells was done after identification and counting of 200 cells per slide [32].

Immunity Responses
Initially the treated or control larvae were made immobile by keeping them on ice cubes for five minutes. Then, they were surface sterilized and injected with 1 × 10 4 spores/mL in 0.01% Tween-80 of Beauveria bassiana (IRAN403C isolate) or latex beads (1:10 dilution for each suspension and Tween-80, respectively) on the second abdominal sternum using a 10-µL Hamilton syringe. The treated larvae were then transferred to glass jars and were given fresh leaves of mulberry. The control larvae were injected with 1 µL of distilled water comprising 0.01% of Tween-80 only. The hemolymph was collected 24 h post-injection from each larva, and the number of nodules formed was scored in a hemocytometer [33]. The counting was repeated four times for each group.

Histological Studies of Larvae Midgut and Adults' Ovary
The larvae midguts were separated from the whole dissected gut in insect ringer and were immediately fixed in aqueous Buine solution for 24 h [10]. Also, the ovary of adults (2 days old), emerging from either treated or control larvae, were separated and fixed. The tissues were processed for embedding in paraffin after being dehydrated in grades of ethanol alcohol and also cleaned by xylene. The fixed tissues were then cut by 5-µM thickness through a rotary microtome (Model 2030; Leica, Wetzlar, Germany). The hematoxylin and eosin were used for staining and then permanent slides were thus prepared, observed, and photographed under a light microscope (M1000 light microscope; Leica, Wetzlar, Germany) armed with an EOS 600D digital camera (Canon, Tokyo, Japan).

Statistical Analysis
LC values were determined using the Polo-Plus software (2002) [34]. All the data were analyzed by ANOVA (SAS Institute, Cary, Cary, NC, USA, 1997) [35], and the comparison of means was performed using Tukey's multiple comparison test (p < 0.05).

Insecticidal Activity
Based on oral and fumigant bioassays, A. annua essential oil was toxic to 4th instar larva of G. pyloalis 24 h post treatments. Probit analysis revealed that the LC 50 values were 1.204 % W/V and 3.343 µL/L air for oral and fumigant toxicity, respectively. The mortality of tested larvae was augmented with increasing concentration (Table 2). Besides LC 50 , the LC 10 and LC 30 values were used to evaluate sublethal bio-activities, including effects on energy reserves, digestive and detoxifying enzymes activity, and hematological and immunity responses and histological study of midgut and ovary of larvae (Table 2).

Energy Reserves
The essential oil of A. annua flowers on the energy reserves of G. pyloalis larvae is shown in Table 3. As can be seen, for all macromolecules, increasing dose of essential oil decreased the concentrations of protein, glucose, and triglycerides. For example, doubling the essential oil concentration (LC 10 to LC 50 ) reduced glucose by 29% in oral tests, while a 1.7-fold increase in fumigant concentration resulted in a 32% drop in glucose levels. The protein was also affected but the decrease in protein with increasing essential oil levels was insufficient to detect given background variability.

Digestive and Detoxifying Enzymes
The effects of A. annua floral essential oil on digestive enzymes' activity of G. pyloalis larvae was manifested by a decrease in protease, α-glucosidase, β-glucosidase, α-amylase, and lipase contents. The difference was significant between the LC 50 versus the control in both oral and fumigant applications while other concentrations of the essential oil produced intermediate responses (Table 4). The effect of essential oil of A. annua flowers on the activity of esterase and glutathione S-transferase (GST) of G. pyloalis larvae is shown in the Table 5. Glutathione S-transferase and esterase contents were reduced significantly when LC 50 was applied in both oral and fumigation methods compared to the controls (Table 5).

Hematological Study and Immunity Responses
The essential oil affected the immune system, which included cellular quantity and quality, phenol oxidase activity, and the immune responses after B. bassiana and latex beads' injection (Figures 1-4). Total hemocyte counts (THC), plasmatocytes and granular cells, nodule formation, and phenol oxidase activity was recorded the lowest in LC 50 both in oral and fumigation assays, respectively.

Histological Studies
The histological texture of larval midgut upon treatment with A. annua essential oil revealed significant differences with the controls, the most significant of which was the elongation and separation of epithelial cells losing the compactness ( Figure 5). The most significant changes in ovarian structure was thinning of epithelial cells around each follicle compared with that of control. Also, the significant reduction in cytoplasm was seen

Histological Studies
The histological texture of larval midgut upon treatment with A. annua essential oil revealed significant differences with the controls, the most significant of which was the elongation and separation of epithelial cells losing the compactness ( Figure 5). The most significant changes in ovarian structure was thinning of epithelial cells around each follicle compared with that of control. Also, the significant reduction in cytoplasm was seen after vacuolization in yolk spheres of the oocytes (Figure 6).

Histological Studies
The histological texture of larval midgut upon treatment with A. annua essential oil revealed significant differences with the controls, the most significant of which was the elongation and separation of epithelial cells losing the compactness ( Figure 5). The most significant changes in ovarian structure was thinning of epithelial cells around each follicle compared with that of control. Also, the significant reduction in cytoplasm was seen after vacuolization in yolk spheres of the oocytes (Figure 6).

Discussion
The chemical composition of A. annua essential oil in the vegetative stage was investigated in the previous studies [15,[36][37][38][39], in which terpenes such as 1,8-cineole, camphor, and artemisia ketone were introduced as major constituents. Although 1,8-cineole (6.8%), camphor (13.1%), and artemisia ketone (11.8%) were also identified as main compounds in the essential oil extracted from A. annua flowers, some other terpenes such as β-selinene (10.7%), pinocarvone (7.4%), and α-pinene (5.9%) had high amounts. However, a range of minor constituents, including compounds from ester and phenylpropene groups, were also recognized. Such differences can be caused by exogenous and endogenous factors, including geographic location, harvesting time, and the growth stage of plants [40]. The chemical composition of each essential oil has a significant impact on its insecticidal activity. For example, the promising insecticidal effects of terpenes like camphor and 1,8cineole identified and extracted from essential oils were reported [41,42].
Our study clearly showed decreased enzymatic activity in G. pyloalis larvae related to ingestion of A. annua essential oil-treated mulberry leaves. Our findings support earlier

Discussion
The chemical composition of A. annua essential oil in the vegetative stage was investigated in the previous studies [15,[36][37][38][39], in which terpenes such as 1,8-cineole, camphor, and artemisia ketone were introduced as major constituents. Although 1,8-cineole (6.8%), camphor (13.1%), and artemisia ketone (11.8%) were also identified as main compounds in the essential oil extracted from A. annua flowers, some other terpenes such as β-selinene (10.7%), pinocarvone (7.4%), and α-pinene (5.9%) had high amounts. However, a range of minor constituents, including compounds from ester and phenylpropene groups, were also recognized. Such differences can be caused by exogenous and endogenous factors, including geographic location, harvesting time, and the growth stage of plants [40]. The chemical composition of each essential oil has a significant impact on its insecticidal activity. For example, the promising insecticidal effects of terpenes like camphor and 1,8-cineole identified and extracted from essential oils were reported [41,42].
Our study clearly showed decreased enzymatic activity in G. pyloalis larvae related to ingestion of A. annua essential oil-treated mulberry leaves. Our findings support earlier findings where disruption in insects' physiology and their inability to digest food was reported [43,44]. Reduction in α-amylase, protease, and αand β-glucosidase, and disruptions on immunology and digestive system in the larvae and the ovary of emerged adults of G. pyloalis were described in our results. Such activities are common for botanical insecticides against several insect pests [45][46][47]. Also, there were further supports for the interference or even deformation of midgut cells, which were responsible for the production of key enzymes in insects [15,48].
Protein plays a key role in digestion, metabolism, and also energy conversion. Klowden (2007) [49] believes that reduction in the insect's protein content after applying biopesticides may stem from the reduction of growth hormone level. We observed a reduction in protein content and also retardation in growth; however, growth hormone level was not worked out. Lipids are other important macromolecules that help the insect reserve energy from feeding. They play a key role in insects' intermediary metabolism and, therefore, they are essential in insect physiology [49]. Significant reduction in the triglyceride content of G. pyloalis larvae treated with A. annua essential oil was observed in the present study. There are several reasons for reducing insect lipid content after treatments by toxins, alteration in lipid synthesis patterns, and hormonal dysfunction to control its metabolism [49]. Glucose as a key carbohydrate (monosaccharide) was also decreased following treatment with A. annua essential oil. This reduction could be related to reduced feeding following treatment, since the essential oil acts as a deterrent [2]. Any disruption causing reducing resources at larval stages could affect insects' survival and reproduction in their later generations. A reduction in protein, lipid, and glucose contents may have adverse effects on the reproductive parameters such as egg production, fertility, and fecundity [50].
Detoxifying enzymes, including esterases and glutathione S-transferases, are involved in reducing the impacts of exogenous compounds [51]. In the current study, the activity of detoxifying enzymes, including esterases and glutathione S-transferases, was reduced by essential oil of A. annua flowers. Certainly, the reduced activity of these enzymes is related to their production halt somewhere in the process of production [15].
Insect cellular immunity is considered as the main system challenging natural enemies entering the insect body [52]. The immunocytes provide the insect ability to combat invading organisms by several means including phagocytosis, nodulation, and encapsulation [53]. So, the reduced immunocytes, as shown for G. pyloalis larvae treated with A. annua essential oil in the present study, could cause larvae to become susceptible to any invasion [54,55]. The reduced number of hemocytes is mostly due to cytotoxic effect of the botanicals used [56]. We do believe this toxic effect of botanicals to be more reliable as a reasoning for the reduction of immunocytes [57][58][59].
Phenol oxidase system is considered as the key component in the immune system of insect and a bridge in the gap between cellular and humeral insect immunity. Its action is critically required in the last stage of cellular defense in order to form melanization, a process that terminates the action and kills the pathogenic agent. Phenol oxidase inhibition, documented for G. pyloalis larvae treated with A. annua essential oil in the present study, probably helps to make the insects susceptible to pathogenic agents if they have not received the toxic concentration [45,58,60].
The insect midgut principal cells are the main cells taking the role of producing the enzymes needed for digestion and then absorbing the nutrients. Therefore, any damages to these cells will lower the activities in digestive enzymes already reported by other researchers [15,31,61]. The elongation and separation of midgut epithelial cells of G. pyloalis larvae treated by A. annua essential oil were observed in the present study.
Inhibiting insect reproduction has long been the subject of many studies. In lepidopterans, obtaining all nutrients at larval stages is necessary for reproductive development [62]. So, if larval nutrition is disrupted by any means, it will be reflected in adult reproductive function. Our previous findings and the current study display the changes in morphology and histology of emerging adults [15,31]. Our study showed the essential oil of A. annua brought about subtle changes in ovarian tissue, such as disruption of follicular cells. As the insect tries to compromise to reduce nutrients in detoxification processes, follicles' cells deplete its content into the oocytes, which then disrupts the cell texture [63].

Conclusions
Plant-derived allelochemicals are beneficial agents in controlling pests. As we know, the plant kingdom mainly depends on secondary metabolites to defend against herbivores. With this knowledge in mind, scientists exploit the use of secondary plant chemicals for pest control. One of the main reasons for this increased demand is that the plant-originated chemicals are comparatively safer for humans and the environment. Our study's results clearly document that the essential oil of A. annua flowers is toxic to larval mulberry pyralid and disrupt its various physiological systems in a way that the insect can hardly get resistance to it. Consequently, this wild-growing plant in Iran can be considered an efficient natural source capable of controlling insect pests. To apply the research results, it is recommended to evaluate the possible side effects of essential oil on mulberry and the biological control agents in future research. Regarding the insect pest's resistance, identifying specific modes of action of essential oil active components and their overlapping with other insecticides should also be assessed.

Data Availability Statement:
The data that support the findings of this study are available upon request from the authors.