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Article

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

by
Marziyeh Oftadeh
1,
Jalal Jalali Sendi
1,2,*,
Asgar Ebadollahi
3,*,
William N. Setzer
4,5 and
Patcharin Krutmuang
6,7,*
1
Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht 416351314, Iran
2
Department of Silk research, Faculty of Agricultural Sciences, University of Guilan, Rasht 416351314, Iran
3
Department of Plant Sciences, Moghan College of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil 5697194781, Iran
4
Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA
5
Aromatic Plant Research Center, 230 N 1200 E, Suite 100, Lehi, UT 84043, USA
6
Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand
7
Innovative Agriculture Research Center, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand
*
Authors to whom correspondence should be addressed.
Foods 2021, 10(2), 210; https://doi.org/10.3390/foods10020210
Submission received: 26 November 2020 / Revised: 18 January 2021 / Accepted: 19 January 2021 / Published: 20 January 2021
(This article belongs to the Special Issue Use of Essential Oils and Volatile Compounds as Biological Control Agents)
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Abstract

:
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.

1. 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 the plant and possess diverse chemical compositions [4]. The effectiveness of essential oils as a more sustainable pest management tool has been noted previously [5,6,7]. It can easily be inferred from their biodegradable nature and safety compared to many of the synthetic insecticides. Since they have multiple target sites in insects, their application is less likely to result in resistance in comparison with synthetic insecticides [8]. It was indicated that plant-derived essential oils may have several effects, including ovicidal, ovipositional deterrents, feeding deterrents, growth retardants, and inhibition in detoxification enzymes [9,10,11].
The annual wormwood, Artemisia annua L. (Asterales: Asteraceae), native to temperate Asia, has been naturalized in many countries [12]. The A. annua has traditionally been used to treat certain diseases of humans, including asthma, fever, malaria, skin diseases, jaundice, circulatory disorders, and hemorrhoids [13]. Although our previous findings of the essential oil or extracts in the vegetative stage of A. annua showed the high potential of this medicinal plant species on insect pest control [14,15,16,17,18], the insecticidal effects of its floral essential oil were evaluated against G. pyloalis in the present study.
The evaluation of lethal (acute) and sublethal (chronic) effects of essential oil extracted from A. annua flowers on G. pyloalis was the main objective of the current study, recommending a biorational and available agent as a possible replacement for synthetic insecticides. Fumigant toxicity is considered to be a non-residual treatment in which no residue will commonly remain for future contaminants. In oral toxicity, the pest is eliminated by swallowing infested food, and it is a suitable method for controlling leaf-eating pests. Therefore, fumigant and oral toxicity and the effect on some key enzymes and biochemical compounds, immunology, digestive system in the larvae, and the ovary of emerged adults of insects, along with the chemical analysis of the essential oil, were evaluated.

2. Materials and Methods

2.1. 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.

2.2. 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.

2.2.2. 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].

2.3. Insecticidal Activity

2.3.1. Oral Toxicity

Initial tests were conducted to assist in selecting the appropriate range of concentrations. Bioassays were carried out on 4th 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.

2.3.2. 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.

2.4. Digestive Enzymes’ Assays

In order to evaluate digestive enzymes activity, the larvae that were treated with LC50, LC30, and LC10 (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.

2.4.1. 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).

2.4.2. 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.

2.4.3. Lipase Estimation

The method of Tsujita et al. (1989) [23] was adopted to estimate lipase. Concisely, 10 μL enzyme, 18 μL p-nitrophenyl butyrate (50 mM), and 172 μL universal buffer (pH 7) were mixed and incubated at 37 °C for 30 min. The absorbance was recorded at 405 nm in the ELISA reader.

2.4.4. The α- and β-Glucosidase Estimation

Here, we used Triton X-100 in order to hydrolyze glucosidases (α- and β-) for 20 h at 40 °C in a ratio of 10 mg of Triton X-100/mg protein. Then, we incubated 75 mL p-nitrophenyl-α-d-glucopyranoside (pNaG, 5 mM), p-nitrophenyl-β-d-glucopyranoside (pNbG, 5 mM), 125 mL universal buffer (made of 2%Mol MES (2-(N-morpholino)ethanesulfonic acid), glycine, and succinate, 100 mM, pH 5.0), and 50 mL enzyme solution. In order to stop the reaction, 2 mL of sodium carbonate (1 M) was used and the absorbance was read at 450 nm [24].

2.5. Detoxifying Enzymes’ Assays

Quantitative analyses of biochemical constituents were carried out on insects remaining after treatments with LC10, LC30, and LC50 and controls. To quantify the whole body protein, the method of Bradford (1976) [25], using the kit (GDA01A, Biochem Co., Tehran, Iran), was incorporated, while glucose and triglyceride were measured by Siegert (1987) [26] method and the triglyceride diagnostic kit, respectively (Pars Azmoon Co., Tehran, Iran). Key enzymes including esterase (general esterases with α- and β-naphthyl acetate substrates), glutathione S-transferase (GST), and phenol oxidase (PO) were assessed by the method described by van Asperen (1962) [27], Habing et al. (1974) [28], and Parkinson and Weaver (1999) [29], respectively.

2.6. 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 × 104 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.

2.7. 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).

2.8. 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).

3. Results

3.1. A. annua Essential Oil Analysis

The chemical composition of extracted A. annua essential oil is presented in Table 1. We identified 55 compounds in flowers of this plant, which represent 93.0% of the total composition. Camphor (13.1%), artemisia ketone (11.8%), β-selinene (10.7%), pinocarvone (7.4%), 1,8-cineole (6.8%), and α-pinene (5.9%) were considered as the major compounds detected, all of which are terpenes. However, other groups such as ester and phenylpropene were also recognized (Table 1).

3.2. 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 LC50 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 LC50, the LC10 and LC30 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).

3.3. 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 (LC10 to LC50) 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.

3.4. 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 LC50 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 LC50 was applied in both oral and fumigation methods compared to the controls (Table 5).

3.5. 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 (Figure 1, Figure 2, Figure 3 and Figure 4). Total hemocyte counts (THC), plasmatocytes and granular cells, nodule formation, and phenol oxidase activity was recorded the lowest in LC50 both in oral and fumigation assays, respectively.

3.6. 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).

4. 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 α- and 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].

5. 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.

Author Contributions

Conceptualization, M.O., J.J.S., and A.E.; methodology, M.O. and J.J.S.; formal analysis, M.O., J.J.S., A.E., and W.N.S.; investigation, M.O.; writing—original draft preparation, M.O., J.J.S., A.E. and P.K.; writing—review and editing, M.O., J.J.S., A.E., P.K. and W.N.S.; supervision, J.J.S. and A.E.; funding acquisition, P.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

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

Acknowledgments

This research was financially supported by the University of Guilan, Rasht, Iran, and was partially supported by Chiang Mai University, Thailand, which is greatly appreciated. W.N.S. participated in this work as part of the activities of the Aromatic Plant Research Center (APRC, https://aromaticplant.org/).

Conflicts of Interest

The authors declare no conflict of interest.

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Foods 10 00210 g001 550
Figure 1. The effect of Artemisia annua floral essential oil on total hemocyte counts (THC) of Glyphodes pyloalis larvae treated with oral (A) and fumigant (B) assays. Bars with different letters above them indicate significant differences between means at p < 0.05, Tukey’s test. Number of hemocytes ×104.
Figure 1. The effect of Artemisia annua floral essential oil on total hemocyte counts (THC) of Glyphodes pyloalis larvae treated with oral (A) and fumigant (B) assays. Bars with different letters above them indicate significant differences between means at p < 0.05, Tukey’s test. Number of hemocytes ×104.
Foods 10 00210 g001
Foods 10 00210 g002 550
Figure 2. The effect of Artemisia annua floral essential oil on the plasmatocytes and granular cells of Glyphodes pyloalis larvae treated with oral (A) and fumigant (B) assays. Bars with different letters indicate significant differences among means of each hemocyte at p < 0.05, Tukey’s test. The number of hemocytes ×104.
Figure 2. The effect of Artemisia annua floral essential oil on the plasmatocytes and granular cells of Glyphodes pyloalis larvae treated with oral (A) and fumigant (B) assays. Bars with different letters indicate significant differences among means of each hemocyte at p < 0.05, Tukey’s test. The number of hemocytes ×104.
Foods 10 00210 g002
Foods 10 00210 g003 550
Figure 3. Effects of Artemisia annua floral essential oil on the nodule formation of Glyphodes pyloalis larvae treated with oral (A) and fumigant assays (B) and inoculated with Beauveria bassiana spores or latex beads. Bars with different letters indicate significant differences between means at p < 0.05. Tukey’s test. The number of hemocytes ×104.
Figure 3. Effects of Artemisia annua floral essential oil on the nodule formation of Glyphodes pyloalis larvae treated with oral (A) and fumigant assays (B) and inoculated with Beauveria bassiana spores or latex beads. Bars with different letters indicate significant differences between means at p < 0.05. Tukey’s test. The number of hemocytes ×104.
Foods 10 00210 g003
Foods 10 00210 g004 550
Figure 4. The effect of Artemisia annua floral essential oil on phenol oxidase (PO) activity of Glyphodes pyloalis larvae treated with oral (A) and fumigant (B) assays. Bars with different letters above them indicate significant differences between means at p < 0.05, Tukey’s test. The number of hemocytes ×104.
Figure 4. The effect of Artemisia annua floral essential oil on phenol oxidase (PO) activity of Glyphodes pyloalis larvae treated with oral (A) and fumigant (B) assays. Bars with different letters above them indicate significant differences between means at p < 0.05, Tukey’s test. The number of hemocytes ×104.
Foods 10 00210 g004
Foods 10 00210 g005 550
Figure 5. Light microscopy of the larval midgut of Glyphodes pyloalis in control (a) and after oral treatment with Artemisia annua floral essential oil (b). Normal texture of all cell types (a) was contrasted to changes in size and texture in treated larvae (b). In the midgut of insects treated with essential oil from A. annua the cohesion of the columnar epithelial layer was damaged. (BM) basement membrane, (CC) columnar cell, (GC) goblet cell, and (PM) peritrophic membrane.
Figure 5. Light microscopy of the larval midgut of Glyphodes pyloalis in control (a) and after oral treatment with Artemisia annua floral essential oil (b). Normal texture of all cell types (a) was contrasted to changes in size and texture in treated larvae (b). In the midgut of insects treated with essential oil from A. annua the cohesion of the columnar epithelial layer was damaged. (BM) basement membrane, (CC) columnar cell, (GC) goblet cell, and (PM) peritrophic membrane.
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Foods 10 00210 g006 550
Figure 6. Histology of ovaries in adults of Glyphodes pyloalis emerging from untreated (a) and treated larvae by Artemisia annua floral essential oil (b). In treatments of the ovarian sheath significant changes and yolk granules were reduced under the influence of vacuolization in cytoplasm compared to the control. (FE) follicular epithelium, (V) vacuole, and (Y) yolk granules.
Figure 6. Histology of ovaries in adults of Glyphodes pyloalis emerging from untreated (a) and treated larvae by Artemisia annua floral essential oil (b). In treatments of the ovarian sheath significant changes and yolk granules were reduced under the influence of vacuolization in cytoplasm compared to the control. (FE) follicular epithelium, (V) vacuole, and (Y) yolk granules.
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Table
Table 1. Chemical composition of the of Artemisia annua floral essential oil.
Table 1. Chemical composition of the of Artemisia annua floral essential oil.
RIcalcRIdbCompound%RIcalcRIdbCompound%
923926Tricyclene MH0.212581259Lepalone OM0.1
938939α-Pinene MH5.912811278Lepalol OM0.3
978975Sabinene MH0.312991290p-Cymen-7-ol OM0.2
982979β-Pinene MH0.113371327p-Mentha-1,4-dien-7-ol OM0.2
992990Myrcene MH0.413611359Eugenol PP0.6
1013999Yomogi alcohol OM1.213741376α-Copaene SH1.0
10211024p-Cymene MH0.813911392Benzyl 2-methylbutanoate E0.3
10261026o-Cymene MH0.814021392(Z)-Jasmone OC0.1
103010311,8-Cineole OM6.814201419(E)-β-Caryophyllene SH3.1
10611062Artemisia ketone OM11.814261432β-Copaene SH0.2
10741070cis-Sabinene hydrate OM0.514481454α-Humulene SH0.3
10821083Artemisia alcohol OM1.414551456(E)-β-Farnesene SH 1.0
110411143-Methyl-3-butenyl 3-methylbutanoate E0.814711477β-Chamigrene SH0.2
11191126α-Campholenal OM0.714781485Germacrene D SH0.7
11311144trans-Pinocarveol OM0.414891490β-Selinene SH10.7
11441146Camphor OM13.115101516Isobornyl isovalerate OM0.1
11611164Pinocarvone OM 7.415171523δ-Cadinene SH0.1
11691169Borneol OM 1.515471555iso-Caryophyllene oxide OS0.3
11791177Terpinene-4-ol OM2.215851583Caryophyllene oxide OS5.4
11921188α-Terpineol OM0.915881590β-Copaene-4α-ol OS0.2
11991195Myrtenol OM2.615941594Salvial-4(14)-en-1-one OS0.2
12111205Verbenone OM 0.316431640Caryophylla-4(12),8(13)-dien-5β-ol OS1.3
12191216trans-Carveol OM0.617001695Germacra-4(15),5,10(14)-trien-1β-ol OS1.7
12271230cis-p-Mentha-1(7),8-dien-2-ol OM0.217651767β-Costol OS1.3
12291235(3Z)-Hexenyl 3-methylbutanoate E0.218541847Phytone OC0.4
12341236n-Hexyl 2-methylbutanoate E0.119841960Palmitic acid OC1.2
12401241Cuminaldehyde OM0.220872106Phytol DT0.3
12441243Carvone OM0.1Total identified93.0
RIcalc = retention index determined with respect to a homologous series of n-alkanes on a HP-5 ms column; RIdb = retention index from the databases [19,20]; MH = monoterpene hydrocarbone; OM = oxygenated monoterpene; SH = sesquiterpene hydrocarbone; OS = oxygenated sesquiterpene; DT = diterpene; PP = phenylpropene; E = ester; OC = other components.
Table
Table 2. Probit analysis of the oral and fumigant toxicity of Artemisia annua floral essential oil on 4th instar larva of Glyphodes pyloalis.
Table 2. Probit analysis of the oral and fumigant toxicity of Artemisia annua floral essential oil on 4th instar larva of Glyphodes pyloalis.
BioassayLC10
(95% CL)		LC30
(95% CL)		LC50
(95% CL)		LC90
(95% CL)		Slope ± SEX2
(df = 3)
Oral toxicity0.593
(0.395–0.735)		0.901
(0.725–1.058)		1.204
(1.024–1.466)		2.445
(1.882–4.128)		4.165 ± 0.6313.2567
Fumigant toxicity1.945
(1.568–2.240)		2.678
(2.347–2.948)		3.343
(3.048–3.632)		5.745
(5.112–6.825)		5.449 ± 0.7882.976
LC: lethal concentration (% W/V for oral toxicity and µL/L for fumigant toxicity), CL: confidence limits, X2: Chi-square value, and df: degrees of freedom. According to Chi-square values, no heterogeneity factor was used in the calculation of confidence limits. Concentration rates were 0.5–2% (W/V) and 2–6 µL/L air for oral and fumigant toxicity, respectively.
Table
Table 3. Effect of Artemisia annua flowers’ essential oil on macromolecules in 4th instar larvae of Glyphodes pyloalis.
Table 3. Effect of Artemisia annua flowers’ essential oil on macromolecules in 4th instar larvae of Glyphodes pyloalis.
Bio-assayConcentrations Protein (mg/dL)Glucose (mg/dL)Triglyceride (mg/dL)
Oral toxicity (% W/V)Control1.0200 ± 0.0360 a1.7733 ± 0.0247 a1.8800 ± 0.0145 a
LC100.9833 ± 0.0088 a1.6666 ± 0.0033 a1.8033 ± 0.0617 a
LC300.9700 ± 0.0057 a1.6533 ± 0.0290 a1.6557 ± 0.0531 a
LC500.9533 ± 0.0088 a1.1733 ± 0.0783 b1.1700 ± 0.0577 b
F-Value2.1629.5119.65
Pr0.01700.00010.0005
Fumigant toxicity
(μL/L)		Control1.0400 ± 0.0208 a1.8100 ± 0.0655 a1.9200 ± 0.0964 a
LC100.9900 ± 0.0057 ab1.7266 ± 0.0384 a1.7533 ± 0.0635 ab
LC300.9700 ± 0.0032 b1.6900 ± 0.0208 a1.433 ± 0.2185 ab
LC500.9366 ± 0.0088 b1.1633 ± 0.0317 b1.3000 ± 0.0765 b
F-Value12.9447.805.04
Pr0.00190.00010.0300
In each separate column, means followed by different letters designate significant differences at p < 0.05 according to Tukey’s test.
Table
Table 4. Effect of Artemisia annua floral essential oil on digestive enzyme activities in 4th instar larvae of Glyphodes pyloalis.
Table 4. Effect of Artemisia annua floral essential oil on digestive enzyme activities in 4th instar larvae of Glyphodes pyloalis.
Bio-assayDigestive Enzymes
(U/mg Protein)		ControlLC10LC30LC50F-ValuePr
Oral toxicity
(% W/V)		Protease1.9467 ± 0.3525 a1.7833 ± 0.1201 ab1.5433 ± 0.0876 ab1.0667 ± 0.0437 b3.960.0531
α-glucosidase1.374 ± 0.192 a1.046 ± 0.0825 ab0.7119 ± 0.0333 b0.5640 ± 0.0360 b9.310.0055
β-glucosidase1.4451 ± 0.1165 a1.1635 ± 0.0955 a0.8757 ± 0.05365 b0.6873 ± 0.0515 b15.610.0010
α-amylase0.3066 ± 1.732 a0.2633 ± 0.0202 ab0.2333 ± 0.01763 b0.0833 ± 0.0120 c41.320.0001
Lipase0.0571 ± 0.032 a0.0387 ± 0.064 ab0.03806 ± 0.089 b0.03700 ± 0.059 b22.750.0003
Fumigant toxicity
(μL/L)		Protease1.8333 ± 0.1244 a0.8967 ± 0.1197 b 0.7167 ± 0.1591 b0.4067 ± 0.1591 b15.830.0010
α-glucosidase1.2034 ± 0.039 a1.1083 ± 0.266 a0.8870 ± 0.064 b0.6921 ± 0.038 b20.800.0004
β-glucosidase1.3451 ± 0.0330 a1.3183 ± 0.1830 a0.9537 ± 0.0282 ab0.7591 ± 0.0717 b8.070.0084
α-amylase0.2800 ± 0.0057 a0.2700 ± 0.01731 ab0.2300 ± 0.11541 ab0.1333 ± 0.0145 b37.490.0001
Lipase0.0559 ± 0.0010 a0.0436 ± 0.0012 b0.0378 ± 0.0027 b0.02620 ± 0.0025 c37.680.0001
In each separate row, means followed by different letters designate significant differences at p < 0.05 according to Tukey’s test.
Table
Table 5. Effect of the different concentrations of Artemisia annua flowers’ essential oil on the activity of glutathione S-transferase (GST) and esterase in 4th instar larvae of Glyphodes pyloalis.
Table 5. Effect of the different concentrations of Artemisia annua flowers’ essential oil on the activity of glutathione S-transferase (GST) and esterase in 4th instar larvae of Glyphodes pyloalis.
Bio-assayConcentrationsGST (U/mg Protein) Esterase (U/mg Protein)
Oral toxicity
(% W/V)		Control0.02300 ± 0.001 a 0.0953 ± 0.004 a
LC 100.01733 ± 0.0032 a 0.08266 ± 0.007 ab
LC 300.0065 ± 0.0025 b 0.07366 ± 0.002 ab
LC 500.0001 ± 0.00001 b 0.06700 ± 0.001 b
F-Value23.46 14.13
Pr0.0003 0.0483
Fumigant toxicity
(μL/L)		Control0.02266 ± 0.0008 a 0.09566 ± 0.004 a
LC 100.01533 ± 0.0006 a 0.07966 ± 0.0005 ab
LC 300.0010 ± 0.0001 b 0.06066 ± 0.0063 ab
LC 500.0001 ± 0.0000 b 0.04600 ± 0.0024 b
F-Value30.13 22.27
Pr0.0001 0.0003
In each separate column, means followed by different letters indicate significant differences at p < 0.05 according to Tukey’s test.
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Oftadeh, M.; Sendi, J.J.; Ebadollahi, A.; Setzer, W.N.; Krutmuang, P. Mulberry Protection through Flowering-Stage Essential Oil of Artemisia annua against the Lesser Mulberry Pyralid, Glyphodes pyloalis Walker. Foods 2021, 10, 210. https://doi.org/10.3390/foods10020210

AMA Style

Oftadeh M, Sendi JJ, Ebadollahi A, Setzer WN, Krutmuang P. Mulberry Protection through Flowering-Stage Essential Oil of Artemisia annua against the Lesser Mulberry Pyralid, Glyphodes pyloalis Walker. Foods. 2021; 10(2):210. https://doi.org/10.3390/foods10020210

Chicago/Turabian Style

Oftadeh, Marziyeh, Jalal Jalali Sendi, Asgar Ebadollahi, William N. Setzer, and Patcharin Krutmuang. 2021. "Mulberry Protection through Flowering-Stage Essential Oil of Artemisia annua against the Lesser Mulberry Pyralid, Glyphodes pyloalis Walker" Foods 10, no. 2: 210. https://doi.org/10.3390/foods10020210

APA Style

Oftadeh, M., Sendi, J. J., Ebadollahi, A., Setzer, W. N., & Krutmuang, P. (2021). Mulberry Protection through Flowering-Stage Essential Oil of Artemisia annua against the Lesser Mulberry Pyralid, Glyphodes pyloalis Walker. Foods, 10(2), 210. https://doi.org/10.3390/foods10020210

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