Purification of acetylcholinesterase from Halyomorpha halys (Stål) (Heteroptera: Pentatomidae) and management of control of the Pest through inhibition of the enzyme

Halyomorpha halys (Stål) (Heteroptera: Pentatomidae) has brought about agricultural harm throughout the Eastern Black Sea coastline since 2017. It continues to come to a threat because there are no adequate studies on managing this pest. One of the major rein strategies of insecticides is acetylcholinesterase (ACHE) inhibition. Therefore, this study aims to investigate an alternative way to struggle H. halys by inhibiting ACHE. The ACHE was purified from H. halys using edrophonium-Sepharose 6B affinity chromatography and characterized by examining some kinetic properties. The molecular weight of the purified enzyme was determined by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and natural polyacrylamide gel electrophoresis. In addition, the inhibitory effects of tacrine and edrophonium chloride, and water extracts of olive leaf, walnut leaf and alder leaf on this ACHE were investigated. The acetylcholinesterase was purified 403-fold with an 83.3% yield. H. halys ACHE was found to have six subunits and a molecular weight of approximately 350 kDa. The ACHE’s Km, Vmax, and kcat values were assigned to be 0.02 ± 0.006 mM, 3,333.3 ± 481 EU.mg protein− 1, and 1070.2 ± 184 min− 1, respectively. All inhibitors highly inhibited of activity of h.halys ACHE. Especially, the fact that the water extracts of these plants are effective in ACHE inhibition is significant due to being environmentally amicable pesticides that may be benefited in the struggle with the pest.


Introduction
Acetylcholinesterase (EC3.1.1.7)plays a role in the termination of impulse conduction by swift hydrolysis of the neurotransmitter acetylcholine in multiple cholinergic pathways in the central and peripheral nervous systems (Xu et al. 2023;Dinçer and Kızıl 2022;Cavdar et al. 2019;Colovic et al. 2013).
Vertebrates have both acetylcholinesterase and butyrylcholinesterase though insects only have acetylcholinesterase; therefore, it is the target of numerous insecticides (Li and Han 2002;Fournier and Mutero 1994).The research about ACHE has aroused caution due to the significance of (BSMB), is domestic to East Asia (Japan, China, and Korea).The highly polyphagous invasive pest has rapidly extended throughout the Northern Hemisphere since its initial detection in the USA in the 1990s and in Europe in 2004.It has also been statemented from diverse zones in Italy since 2012 (Ak et al. 2019;Cesari et al. 2015;Maistrello et al. 2014;Wermelinger et al. 2008).It proceeds to diffuse in France, Switzerland, Greece, Hungary, Germany, Austria, Liechtenstein, Serbia, Romania, Spain, Russia, Georgia and Turkey (Özdemir and Tuncer 2021;Lupi et al. 2017;Gapon 2016;Haye et al. 2015;Vetek et al. 2014).
The BSMB was first recorded in Turkey by Istanbul (Çerçi and Koçak 2017) and Artvin, located in the Eastern Black Sea Region close to the Georgian border, in 2017 (Güncan and Gümüş 2019).Considering the regions of Turkey at potential risk for this invasive species (Kriticos et al. 2017), it is understood that hazelnut is one of the most important export products under threat in the Black Sea Region.It has been reported that the pest causes serious yield and quality losses due to feeding directly on the fruit (Bosco et al. 2018;Haye and Weber 2017;Hedstrom et al. 2014).In addition, this pest has considered a significant potential threat to many agricultural products in Turkey as in other countries, considering the broad host range, as well as agricultural products such as kiwi, persimmon, corn, citrus beans, and tomatoes, are grown in the region and are essential hosts for the pest.
The brown skunk spends the winter among plant residues that have fallen to the ground, under the bark of trees, in holes, and in various settlements, and prefers dry areas in particular (Lee et al. 2013).Although it is stated that it can give 4-6 generations per year in South Asia (Lee et al. 2013), it has been determined that it gives 1 or 2 generations in its spreading areas to date (Rice et al. 2014).It has been determined that the brown skunk gives 2 offspring per year in Italy and its reproductive power is high (Costi et al. 2017).Although the pest is mainly stated as a pest that produces 2 offspring in most sources, it has also been found in regions where it gives only one generation (Lee et al. 2013).The lower and upper growth thresholds of the brown skunk are 12 and 35 °C, respectively (Kiritani 2012;Cira et al. 2016).In addition, adults can survive at temperatures as low as -17 °C (Cira et al. 2016).
Adults and nymphs of the brown skunk are harmful by injecting digestive enzymes directly into the fruit and absorbing the plant juice, thanks to their stinging-sucking mouth structure (Rice et al. 2014).Damaged products lose a great deal of their market value (Bariselli et al. 2016;Rice et al. 2014;Hedstrom et al. 2014).Moreover, it creates a major urban problem because of the entry of this pest into the houses during overwintering, their gathering in huge groups in such structures, and the negative effects of the stench it emits on human health (Inkley 2012).It is also known to be a vector of Paulownia tomentosain, a phytoplasma disease in Asia (Hoebeke and Carter 2003) and is suspected to be a vector of other phytoplasmas (Jones and Lambdin 2009).
Synthetic pesticides are mostly used to control pests.The use of synthetic pesticides has caused a number of negative side effects, including teratogenic, carcinogenic, and acutely toxic residues in food, groundwater, soil, and air.(Dinçer and Kızıl 2022;Bughio and Wilkins 2004;Lingk 1991).In order to accomplish these problems, it is necessary to search for alternative pest control methods that are secure, practical, environmentally friendly and cost-effective.Among the diverse alternatives from the methods, many natural herbal products which are biodegradable, non-phytotoxic, and eco-friendly engage the interest of researchers worldwide (Dinçer and Kızıl 2022;Mishra 1990).They submit a relative nadir threat to nontarget organisms (beneficial predators and parasites), and can generally be swiftly degraded in the surroundings and metabolized readily by animals ingesting unlethal doses (Dinçer and Kızıl 2022;Scott et al. 2003).Moreover, different from classic insecticides grounded on one only active component, plant-derived insecticides are less likely for pests to evolve resistance to such matters because of containing a set of chemical compounds that work in harmony with behavioral and physiological procedures (Hassan Adeyemi 2010).The products have been researched for insect control, such as acute toxicity, anti-nutrition or repellent, attractant, fumigant effects, and inhibiting the reproduction of many pest species (Cox 2004;Hamouda et al. 2015a).Numerous studies focused on the olive tree (Olea europaea sativa L.), particularly its leaf displaying powerful biological activities (Hamouda et al. 2015b;Zari et al. 2011;Jemai et al. 2009).These influences come from the many phenolic compounds, especially oleuropein, in the olive leaf.In fact, it has been previously reported that polyphenols adversely affect the development of insects due to their capacity to inhibit many enzymes of the hydrolase class (Dinçer and Kızıl 2022;Céspedes et al. 2004).
This study contains purification, inhibition, and characterization of the ACHE from Halyomorpha halys by affinity chromatography edrophonium-Sepharose, and investigating some of its kinetic features.Also, it was studied the effect of tacrine and edrophonium, which are competitive ACHE inhibitors, and some plant leaf water extracts on the activity of the ACHE.Thus, it is aimed to propose a natural pesticide that can be used to combat this pest through the inhibition of acetylcholinesterase with the findings to be obtained in this study.

Chemicals and materials
Whole chemicals used in this study were bought from Sigma Chemical Co.The adults of Halyomorpha halys were collected by pheromone trap from Rize in Turkey in July and stored at -20 o C. Olive leaves (Olea europaea sativa L.) were gathered from Bursa, and walnut leaves (Juglans regia L.) and alder leaves (Alnus glutinosa subsp.barbata) were collected from Trabzon, Turkey.They were dried up at 40 °C and powdered by a grinder and then sieved into a 60-mesh-screen.

Crude Extract Preparation
Approximately 5 g of Halyomorpha halys adults (about three months old) were homogenized in 20 mL 50 mM pH 7.4 sodium phosphate buffer containing 0.5% Triton X-100 and 1 mM EDTA in an ice bath.The supernatant was filtered by a syringe filter unit having a pore size of 0.45 μm after the homogenate was centrifuged at 20,000xg at 4 °C for 45 min.The supernatant was utilized as a raw extract (Dinçer and Kızıl 2022;Son et al. 2002).Hodgson and Chubb's (1983) procedure was used to prepare the edrophonium sepharose 6B affinity gel.After washing the epoxy-activated Sepharose 6B with distilled water, it was quoted onto 20 mL 12.0 mM borate buffer (pH 11.0) containing 20.0 mM edrophonium chloride.The pH of the gel slurry was adjusted to 12.0 by adding 1.0 M NaOH.It was incubated at 50 °C for 48 h on a shaking incubator.The affinity gel was washed in turn with 10 volumes each of 0.1 M sodium acetate buffer (pH 4.5), 12.0 mM sodium borate buffer (pH 10.0), and distilled water (Son et al. 2002).This washing process continued until the absorbance was 0.001 at 280 nm.

Acetylcholinesterase purification
The extract was performed to a column of edrophonium-Sepharose 6B (1.5 × 7 cm, Øxh) previously equalized by sodium phosphate buffer (0.05 M, pH 7.4).The affinity column was washed with the 50 mM phosphate buffer (pH 7.4) until the change of absorbance caused by the protein material was under 0.01 at 280 nm, then it continued that the column was washed with the same buffer embodying 0.5 M NaCl.Specific elution of the ACHE was realized with 50.0 mM sodium phosphate buffer (pH 7.4) containing 0.5 M NaCl and 12.0 mM edrophonium chloride.Each fraction was collected as 3 mL.The fraction showing the ACHE activity was used as a source of the pure enzyme (Dinçer and Kızıl 2022;Askar et al. 2011;Son et al. 2002).

Sodium dodecyl sulfate and natural polyacrylamide gel electrophoresis
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and natural polyacrylamide gel electrophoresis (natural-PAGE) were performed according to the method of Laemmli (1970).As a standard protein marker, a standard having a molecular weight range of 14.4-98.0kDa (BioBasic trademark) in SDS-PAGE and a natural molecular weight standard containing xanthine oxidase (283 kDa) and urease (544.6 kDa) in natural PAGE, were used.Natural electrophoresis gel was stained with silver staining method and SDS-PAGE was stained with Coomassie Brilliant Blue R-250.

Enzyme assay and protein determination
The ACHE activity was specified spectrophotometrically using the ATC substrate (Ellman et al. 1961). 1 mL of the reaction mixture in 0.1 M phosphate buffer (pH 8.0) contained 1.5 mM acetylthiocholine iodide (ATC), 0.2 mM 5,5'-dithiobis (2-nitrobenzoic acid) and the enzyme solution.The increase in absorbance was recorded for 3 min at 412 nm at 25 °C caused by the rise in the amount of the colored compound formed by the product formed as a result of the enzymatic reaction with the chromophore ligand.The amount that catalyzed the hydrolysis of 1.0 µM of ATC per minute at room temperature was described as a unit of the enzyme (EU) (Cavdar et al. 2019;Son et al. 2002;Ellman et al. 1961).The average of three measurements was used to calculate the value of each activity.
The kinetic parameters of the ACHE, Michaelis-Menten constant (Km), maximal enzyme velocity (Vmax), and catalytic constant (kcat), were estimated by Lineweaver and Burk plots (1934) using seven substrate doses ranging from 0.001 to 0.1 mM.The protein material was determined according to the Lowry method (Lowry et al. 1951), using bovine serum albumin as a standard protein.

ACHE inhibition
The inhibition effects of tacrine and edrophonium chloride which are specific inhibitors of acetylcholinesterase, and the water extracts of olive leaf, walnut leaf, and alder leaf on ACHE activity were performed in the availability of ATC substrate at pH 8.0.By plotting the % inhibition graph against the inhibitory concentration, the IC 50 value, defined of these extracts on the ACHE isolated from H. halys in the 5-150 g dry matter/mL concentration range was assigned.

Results
The ACHE was purified from Halyomorpha halys in a 403fold and yield of 83.3% with an affinity column of edrophonium-Sepharose 6B.The ACHE showed specific activities of 2450.0 ± 62 EU/mg protein (Table 1; Fig. 1).Unbound proteins were eliminated first from the affinity column matrix by treating them with 50.0 mM sodium phosphate buffer (pH 7.4).Then, the negatively charged proteins bound to the positively charged edrophonium were eliminated with the solution of the same buffer containing 0.5 M NaCl.Finally, the ACHE proteins bound to the column matrix-bound edrophonium were isolated from the medium by holding on to free edrophonium in the buffer including 0.5 M NaCl.The ACHE protein came in the 122nd fraction (Fig. 1).
12% SDS-polyacrylamide gel electrophoresis was performed to determine the purity of the enzymes (Fig. 2 ).Six protein bands H.halys ACHE were observed in the obtained SDS-PAGE chromatogram (Fig. 2-C).The molecular weights of these six subunits observed for purified as the inhibitory concentration that inhibits enzyme activity by 50%, was calculated nhibitors concentration ranges were 0.01-10.0µM for tacrine and 0.1-20 µM for edrophonium chloride.Every inhibitor was studied in at least seven concentrations (Dinçer and Kızıl 2022;Mohamed et al. 2020).About 3 g of the dry plant leaf powders were extracted overnight in 30 mL distilled water and they were centrifuged at 10,000xg for 30 min.The final supernatants were diluted 1:1 with distilled water and utilized.The inhibition effect  to be 3333.3± 481 EU/mg protein, 0.02 ± 0.006 mM, and 1070.2 ± 184 min − 1 , respectively (Table 2; Fig. 3).The optimal ACHE activity was assigned at 40.0 o C and pH 8.0 (Figs. 4 and 5; Table 2).IC 50 values were appointed to be respectively 0.08 ± 0.003 and 15.0 ± 1.0 µM for ACHE of H. halys in inhibition studies with edrophonium chloride and tacrine (Table 3).Both inhibitors inhibited this ACHE.Tacrine was more effective than edrophonium chloride on the ACHE activity.The fact that these two inhibitors, which are known inhibitors of ACHEs, are effective on this enzyme is evidence that this enzyme is an ACHE.
In additional inhibition study, IC 50 values were established to be 20.3 ± 1.2 µg dry matter/mL with olive leaf aqueous extract, 108.0 ± 40 µg dry matter/mL with walnut leaf aqueous extract, and 19.0 ± 1.7 µg dry matter/mL with alder leaf aqueous extract (Table 3).The natural extracts inhibited the ACHE very effectively.

Discussion
The acetylcholinesterase was purified from Halyomorpha halys adults in one step at a high rate and yield by edrophonium-Sepharose 6B affinity chromatography (Table 1; Fig. 1).The purification of ACHEs from nymphs and adults of Ricania simulans (Walker, 1851) (Hemiptera: Ricaniidae) ACHE were determined to be approximately 71, 69, 65, 60, 45 and 40 kDa (Fig. 2-C).In addition, a single protein band was detected in the gel obtained from the native electrophoresis (Fig. 2).The molecular weight of this identified protein band was calculated as approximately 350 kDa (Fig. 2-E).The only protein band observed in the native electrophoresis gel versus 6 protein bands in the SDS-PAGE gel is evidence that the ACHE of H.halys has 6 subunits.It was determined that the sum of the approximate molecular weights of the calculated subunit protein bands was equal to the molecular weight of the protein band determined in natural electrophoresis.
Some kinetic parameters of the ACHE of H. halys in the attendance of ATC substrate were defined by the Lineweaver-Burk method, and are given a demonstration in Table 2.The V max , K m , and k cat values were established   Mohamed et al. 2020;Meng et al. 2016).These values of pH and temperature match very well with other works in the literature.
The IC 50 values for the H. halys ACHE determined with tacrine and edrophonium chloride, which are among the known inhibitors of ACHEs, are demonstrated in Table 3.These two inhibitors were found to be highly effective against the ACHE.It was reported that IC 50 values of the R. simulans ACHEs were calculated to be 2.4 and 18.0 µM for adults and 0.6 and 1.2 µM for nymphs, respectively for edrophonium chloride and tacrine (Dinçer and Kızıl 2022).Tacrine and edrophonium were found to inhibit the ACHE of Electrophorus electricus at concentrations of 9.16 µM and 0.68 µM, respectively (Martín-Blosca vd., 2009).In another inhibition study, the IC 50 value for tacrine was found to be 0.068 µM for the ACHE of German cockroach (Blattella germanica (Linnaeus, 1767) (Blattodea: Blattellidae), (Mutunga et al. 2009).The IC 50 values for the ACHE of H. halys are fairly nearby the information found in the literature.
Inhibitory effects of water extracts of olive leaves, walnut leaves and alder leaves on H. halys ACHE were determined (Table 3).The water extracts of the leaves used in this study showed a highly effective inhibition effect on the ACHE.Plant-derived insecticides give a relatively low threat to nontarget organisms (beneficial predators and parasites), are generally rapidly degraded in the environment, and are readily metabolized by animals exposed to nonlethal doses.Moreover, unlike conventional insecticides based on a single active ingredient, plant-derived insecticides contain a set of chemical compounds that act harmoniously on both behavioral and physiological processes.Therefore, pests have less opportunity of developing counteraction to such matters (Abdellaoui et al. 2019;Adeyemi Hassan, 20102010;Scott vd., 2003).It is essential for the environment that the extracts used in this study are prepared with water and are effective at very low concentrations.In particular, the pesticides from such natural products may be comfortably used to struggle with such pests, in organic agriculture regions.There are many similar plant-based studies in the literature on the inhibition of ACHE.In a study, the inhibition influences of the aqueous extract of olive leaf on the ACHEs of the nymphs and adults of Rsimulans were investigated.As a result, the IC 50 values were found to be 16.2 µg dry matter/mL for the nymphs and, 20.3 µg dry matter/mL for the adults, respectively (Dinçer and Kızıl 2022).Another study looked at the effect of olive leaf extract on the larvae of the migratory locust, Locusta migratoria (Linnaeus, 1758).The Olive leaf extracts were found to significantly reduce L. migratoria larvae's ACHE activity by 62.9% (Abdellaoui by the same affinity chromatography was carried out with a 65.5-fold purification in a 2.2% yield for nymphs and a 251.6-fold purification in a 34.2% yield for adults (Dinçer and Kızıl 2022).It was declared that the ACHE was isolated from Mytilus galloprovincialis (Lamarck, 1819) in a yield of 3% and a 13-fold by the Edrophonium Sepharose 6B affinity chromatography (Duranay et al. 2019), and from the brain of Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae) with a 283-fold in a yield of 32% by an affinity chromatography (Keane and Ryan 1999).It is obvious that the purification rate and yield of H. halys ACHE are higher than other similar studies in the literature.
Although Halyomorpha halys ACHE was observed to have six protein bands as a result of SDS-polyacrylamide gel electrophoresis, a single band was observed in native electrophoresis (Fig. 2).The sum of the subunit molecular weights of these six protein bands was calculated to be equal to the molecular weight of the protein in native electrophoresis.It was reported that although ACHE purified by affinity chromatography from Galleria mellonella had five subunits (61.6 kDa; 30.9 kDa; 27.8 kDa; 21.2 kDa; and 16.6 kDa) in SDS-PAGE, a single protein band (240 kDa) was observed in native electrophoresis (Keane and Ryan, 1999 The ACHEs molecular weights were similar to those obtained from different sources, for example, 59 kDa for the nymphs and 52 kDa for the adults of Ricania simulans (Dinçer and Kızıl 2022), 51 kDa for Mytilus galloprovincialis (Duranay et al. 2019), 63.5 kDa for the cotton aphid (Aphis gossypii(Glover, 1877) (Hemiptera: Aphididae) (Li and Han 2002) and 25-59 kDa for the electric organ of the electric eel (Electrophorus electricus) (Linnaeus, 1766) (Dudai and Silman 1974).
Table 2 was shown the V max , K m , and k cat values for the ACHE of Halyomorpha halys.The ACHE showed a high affinity for the ATC.It was specified that the K m value (0.02 mM) was similar to the K m values of the nymphs' ACHE of R. simulans (0.02 mM) (Dinçer and Kızıl 2022), and under the K m values of the adults' ACHE of R. simulans (0.04 mM) (Dinçer and Kızıl 2022), Mytilus galloprovincialis (1.3 mM) (Duranay et al. 2019) and Heterorhabditis bacteriophora (Poinar, 1976 ) (0.27 mM) (Mohamed et al. 2007).The V max value (3,333.3± 481 EU/mg protein) in this study was higher than that previously papered for the ACHEs from R. simulans (500 EU/mg protein for nymphs and 2,000 EU/ mg protein for adults) (Dinçer and Kızıl 2022), Mytilus galloprovincialis (183 EU/mg protein) (Duranay et al. 2019), the Nebia albiflora muscle (100 EU) (Shi and Zhang 1981), and the Pseudosciaena crocea (Richardson, 1846) muscle (125 EU) (Dong 1995).The K m , V max values of this enzyme were found to be compatible with other ACHE data.
The optimal conditions were stated at pH 8.0 and 40.0 o C for the ACHE (Figs. 4 and 5; Table 2).In the literature, the et al. 2019).Also, another study indicated that the ACHE activity of the black bean aphid Aphis fabae (Scopoli, 1763) was inhibited with the raw ethanolic extract of Artemisia judaica (Linnaeus) (Acheuk et al. 2017).In a study to determine eeACHE inhibition by extracts of Mentha pulegium L. plant prepared with various solvents, it was determined that the IC50 values were as 40.76 µg/mL for methanol extract (Gülçin et al., 2020), 534 µg/mL for ethanol extract (Mata et al., 2007), 3.13 µg/mL for ethyl acetate extract, 144.59 ± 0.49 µg/mL for diethyl ether extract, 32.28 µg/mL for n-butanol extract (Abbou et al., 2022).

Conclusions
In conclusion, ACHE was purified from Halyomorpha halys, which poses a threat to agricultural areas in the Eastern Black Sea Region of Turkey.The enzyme was characterized by examining some kinetic data.It has been determined that the obtained data are compatible with other ACHE data in the literature.The hypothesis of the study was the management of this pest control through the inhibition of ACHE.In this context, success was achieved in the inhibition study and alternative environmentally friendly new inhibitors were determined.
One of the noticeable results of the study is that natural products such as water extracts from olive leaf, walnut leaf, and alder leaf are effective on this enzyme.Another major subject that stands out is that little doses of them, whose can be tolerated by the environment, are influential in ACHE inhibition.Thus, such pests may be combated even in organic farming areas with these plant-derived extracts.It may be proposed to periodically implement these extracts in areas invaded by the pest, commencing in May, during the time that the nymphs of H. halys begin to seem.As a result of the periodic applications, it will be ensured that the extracts are concentrated in the plants that harbor the pest, so that the substances in this extract which they take both owing to nutrition and respiration may be accumulated in the cells (especially in nerve cells) of the pest, which will be able to lead to the death of the pest with the paralysis of nerve conduction via the inhibition of ACHE.
Thanks to this application, the pest population may be controlled in a short time without the requirement for chemical pesticides and its rapid spread may be forestalled.

Fig. 1
Fig. 1 The graphic of demonstrating the ACHE purification stage with the affinity chromatography

Fig. 5 Fig. 4
Fig. 5 The graph of change of the ACHE activity of H. halys with pH

Table 1
Overview of the purification ACHE from H.halys aThe purification fold was calculated from the ratio of specific activities bThe recovery yield (%) of activity was determined from the ratio of the ACHE activity