Purification and characterization of acetylcholinesterase in Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

Journal Pre-proofs Purification and Characterization of Acetylcholinesterase in Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) Magda A. Mohamed, Shebl Shaalan, Abd-Elhady M. Ghazy, Atef A. Ali, Ahmed M. Abd-Elaziz, Manal M.E. Ghanem, Sarah A. Abd-Elghany PII: DOI: Reference:

S0141-8130(19)36165-3 https://doi.org/10.1016/j.ijbiomac.2019.10.071 BIOMAC 13565

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International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

4 August 2019 5 October 2019 7 October 2019

Please cite this article as: M.A. Mohamed, S. Shaalan, A.M. Ghazy, A.A. Ali, A.M. Abd-Elaziz, M.M.E. Ghanem, S.A. Abd-Elghany, Purification and Characterization of Acetylcholinesterase in Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.071

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Purification and Characterization of Acetylcholinesterase in Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

Magda A. Mohameda, Shebl Shaalanb, Abd-Elhady M. Ghazya, Atef A. Alib, Ahmed M. Abd-Elaziza, Manal M.E. Ghanema, , Sarah A. Abd-Elghanya

aMolecular

Biology Department, National Research Centre,

33-El Bohouth st. Dokki, Giza, Egypt, P.O.12622 bZoology

Department, Faculty of science, Cairo University, Giza, Egypt, P.O.12316

Corresponding to Magda A. Mohamed Molecular Biology Department, National Research Centre, 33- El Bohouth st. Dokki. Giza. Egypt e mail. [email protected]

Abstract Red palm weevil (RPW), Rhynchophorus ferrugineus, is one of the most destructive pests of cultivated palm trees. The application of synthetic insecticides is currently a main strategy for RPW control. In this study we estimated the distribution of acetylcholinesterase (AChE), as a detoxifying enzyme and the target site of inhibition by insecticides, using ASChI as substrate in different organs of the pest including whole gut, cuticle, fat body, head and haemolymph. The activity ranged from 314.9 to 3868 U in individual organs while the specific activity ranged from 99 to 340.8 U/mg proteins; the cuticle had the highest enzyme level. During larval development, the 11th instar larvae had the highest enzyme content with 5630 U in the cuticle, with a specific activity of 140 U/mg protein. The two major AChE isoenzymes were purified by chromatography on gel filtration and ion exchange columns. They had specific activities of 3504.3 and 2979 U/mg protein, molecular weights of 33 and 54 kDa and activation energies of 8.3 and 4.4 kCal/mol, respectively. Both isoenzymes had monomeric forms, optimum activity at pH 8.0 and 40o C, were completely inhibited by Hg2+ and Cu2 and showed similar trends towards the inhibitors eserine, BW284C51 and iso-OMPA. The catalytic properties were compared with those previously recorded for different insect species. This work will pave the way for more studies for improving the understanding of insecticide resistance and developing the field application of synthetic insecticides for controlling R. ferrugineus to ensure successful application.

Key words: biochemical characterization; detoxification; cuticle; larval development; inhibition; kinetics; specificity; Red palm weevil.

1. Introduction The red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) is considered the most serious tissue-boring pest of palms. Severe R. ferrugineus infestations have been reported on Cocos nucifera, Phoenix dactylifera and Phoenix canariensis (Hussain et al., 2013). The most destructive stage of R. ferrugineus is the legless, creamy white larval stage (grubs), which chews the tender, soft tissues of the palms and moves toward the interior of the plant. Larval lasts for about three months (Mahmoud et al.,2015; El-Zoghby and Naglaa 2018). The damage

caused by R. ferrugineus is difficult to detect during the early stages of infestation. Consequently, the damage can be seen only after long infection. This late detection of R. ferrugineus constitutes a serious problem in the fight against the pest and in any effort to guarantee the pest-free status in adult trees (Vatanparast et al., 2014).

The management of red palm weevil presents a tremendous challenge because of its cryptic life cycle. Different strategies are used to combat R. ferrugineus; however the application of synthetic organophosphate (OP) and carbamate insecticides remains the main strategy for R. ferrugineus control (Al-Ayedh et al., 2016; Bamidele et al., 2017). The primary toxicity of these insecticides is due to their inhibition of acetylcholinesterase (AChE, EC 3.1.1.7), the target site of inhibition (Kim et al., 2012; King and Aaron, 2015). Palm farmers have noticed ineffectiveness of currently available insecticides against RPW, and this observation may be due to the development of insecticide resistance in the target species (Al-Ayedh et al., 2016).

Earlier reports documented that high level of AChE is one of the major reasons for the development of resistance by metabolic detoxification of insecticides. The probable role of high AChE levels for conferring resistance has been reported in various insect species (Yu, 2006; Pethuan et al., 2007; Yang et al., 2008; Kim et al., 2012; Mohamed et al., 2016). Recently, the high level of AChE in entomopathogenic nematodes Heterorhabditis bacteriophora has been contributed for conferring resistance against different insecticides (Mohamed et al., 2017).

AChE had been purified from different species of insects including booklice, Liposcelis entomophila (Motschulsky, 1852) (Xiao et al., 2010), oriental fruit fly, Bactrocera dorsalis (Hendel, 1912) (Hsiao et al., 2004), green rice leafhopper, Nephotettix cincticeps (Uhler, 1896) (Kato et al., 2004), western corn rootworm, Diabrotica virgifera (Leconte,1868) (Zhou et al., 2005), oriental migratory locust, Locusta migratoria manilensis (Meyen, 1835) (Ma et al., 2004), green bug, Schizaphis graminum (Rondani, 1852) (Gao and Zhu, 2001), and from adult of R. ferrugineus (Abo-El-Saad and Al-Ajlan, 2006).

Since larvae of R. ferrugineus are the most destructive stage, the aim of this study was to biochemically investigate AChE during larval development. To develop

a strategy for R. ferrugineus management by OP and carbamate insecticide(s), it is important to investigate AChE, the target enzyme, to ensure successful implication of such strategy. To achieve this objective, the present article represents the first report for studying the distribution of AChE content in different organs from a larval stage of R. ferrugineus, in the cuticles during larval development, as well as purification and characterization of the predominant isoenzymes.

2. Materials and Methods

2.1. Insect Different larval instars of R. ferrugineus were obtained from Central Laboratory for Date Palm Research and Development (CLDPRD), Agricultural Research Center (ARC), 9 Cairo university street, Dokki, Giza, Egypt.

2.2. Chemicals Acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), butyrylthiocholine (BuSCh), benzoylthiocholine (BzSCh) iodides, 5,5′-dithiobis (2-nitrobenoic acid) (DTNB), eserine, 1,5-bis (4-allyldimethylammoniumphenyl) pentan 3 one dibromide (BW284C51), tetra (monoisopropyl) pyrophosphortetramide (iso-OMPA), DEAESepharose for chromatography, reagents and resins for polyacrylamide gel electrophoresis (PAGE) were purchased from Sigma-Aldrich, USA. Sephacryl S-200 was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden), metals were purchased from BDH chemicals Ltd Poole England.

2.3. Sample preparation Different organs, including the whole gut, cuticle, fat body, head and haemolymph from the 10th instar larvae of R. ferrugineus were separated. In brief, larvae were placed on ice (about 5 min) for immobilization and dissected under microscope according to Broadway and Duffey (1985) and Alarcon et al. (2002). Haemolymph was collected from the larvae before dissection by puncturing the specimen carefully with a microsurgical knife at the end of the abdomin: the haemolymph was collected from the wound using a capillary then transferred into a microcentrifuge tube by carefully blowing out the capillary (Brakefield et al., 2009).

The whole gut (foregut, midgut and hindgut) was prepared according to Allahyari et al. (2010) with some modifications of Darvishzadeh et al. (2012). The fat tissues were removed by small brush. The entire guts and their contents were taken out. For head preparation, the heads were separated; mouth parts were removed and the brown, hard outer layer of heads was peeled off with the aid of sharp surgical blades according to Al-Rajhy et al. (2005). Finally, the cuticles were separated according to Darvishzadeh et al. (2012). All fractions were stored at -20 oC until used for AChE measurements. The cuticles from 5th to 12th instars larvae were separated weighted and stored at -20oC until used for enzymatic estimation.

2.4. Preparation of AChE crude extracts Crude extracts from different organs of the 10th instar larvae of R. ferrugineus and from cuticles during larval development were prepared. The samples were homogenized in pre-cooled homogenizer (Teflon pestle homogenizer) using Tris-HCl buffer, pH 7.5. The homogenates were centrifuged at 14,000 ×g for 20 min at 4oC. The precipitates were re-extracted as previously described and re-centrifuged. Two supernatants were pooled and frozen at -20oC until used for enzyme estimation.

2.5. Enzyme assay The activity of AChE was estimated using AcSChI as substrate according to Ellman et al. (1961). The reaction mixture (1 ml) consisted in final concentration of 60 mM Tris-HCl buffer, pH 8.0, 1 mM AcSChI, and 1 mM DTNB. The reaction mixtures were incubated at 37oC and the increase in the absorbance was recorded at 412 nm. One unit of AChE activity was defined as the amount of enzyme that catalyses the hydrolysis of 1µmol of substrate per hour under standard assay conditions.

2.6. Protein determination Protein contents were determined according to Bradford (1976) using bovine serum albumin as a standard. The total protein contents of the pooled active fractions were determined spectrophotometrically by measuring the absorbance at 280/260 nm according to Warburg and Christian (1942).

2.7. Purification of AChEs from R. ferrugineus

Unless otherwise stated all steps were performed at 4-7 oC. 2.7.1. Step 1: Preparation of crude extract Enzyme crude extract was prepared from the cuticles of the 11th instar larvae of R. ferrugineus. Two cuticles were homogenized in 4 ml of 50 mM Tris-HCl buffer, pH 7.5 using a Teflon pestle homogenizer and a small amount of glass beads (<106 µm, Sigma) were added during homogenization with cooling. The homogenate was centrifuged at 14,000 ×g for 20 min at 4°C. The precipitate was re-extracted as previously described and recentrifuged. The two supernatants were pooled, stored at -20°C and designated as enzyme crude extract. 2.7.2. Step 2: DEAE-Sepharose chromatography The crude extract was dialyzed against 20 mM Tris-HCl buffer, pH 8.0 over night then introduced into a DEAE-Sepharose column (15 x1.6 cm i.d.) previously equilibrated with the same buffer. The adsorbed materials were eluted with a stepwise gradient NaCl ranging from 0.0 to 0.4 M prepared in the same buffer at a flow rate of 60 ml/h. Protein fractions exhibiting AChE activities were eluted with zero, 0.05 and 0.3 M NaCl and designated as AChEI, AChEII and AChEIII according to their elution order. 2.7.3. Step 3: Sephacryl S-200 chromatography The pooled active fractions of the most predominant activities, AChEII and AChEIII, were applied separately to a Sephacryl S-200 column (95 x 1.6 cm i.d.) equilibrated with 20 mM Tris-HCl buffer, pH 8.0 and developed with the same buffer at a flow rate of 20 ml/h. The most active fractions were pooled and designated as AChEIIb and AChEIIIb

2.8. Polyacrylamide gel electrophoresis (PAGE) Enzyme purity was examined by electrophoresis under non-denaturing PAGE conditions according to Davis (1964) using 12% (w/v) acrylamide condition. The protein bands were stained by silver staining according to Morrissey (1981).

2.9. Molecular weight determination The native molecular weights of AChEIIb and AChEIIIb were estimated by gel filtration using Sephacryl S-200 column according to Oberg and Philipson (1967) which was calibrated with standard proteins ranged from 12.4 to 200 kDa. Subunit molecular weights were estimated by SDS-PAGE according to Laemmli (1970), under denaturing conditions using 12% (w/v) acrylamide separating gel condition in a

Bio-Rad, (USA) Mini-protean II electrophoresis cell (Bio-Rad, USA). The subunit molecular weights were calculated using standard proteins ranging from 20 to 245 kDa.

2.10. Optimum temperature for activity and stability The effect of temperature on activity and stability of purified AChEIIb and AChEIIIb were estimated over a range of temperatures (20 to 80°C). For optimum temperature for activity, AChE assays were performed at different temperatures. For temperature stability, AChEIIb and AChEIIIb were pre-incubated individually at different temperatures for 1 hr and quickly cooled before estimating the residual activities.

2.11. Effect of metals Effect of different metal cations in chloride form including Co2+, Mg2+, Hg2+, Mn2+, K+, Ni2+, Zn2+, Cu2+ , Ca2+, Ba2+ and Na+on the activities of R. ferrugineus AChEs was estimated at final concentrations of 1 and 10 mM. Each isoenzyme was pre-incubated in Tris-HCl buffer, pH 8.0 for 15 min at room temperature with the different cations prior to substrate addition for estimating the residual activities.

2.12. Substrate specificity and kinetic parameters The relative rate of hydrolysis of four different substrates, AcSCh-, PrSCh-, BuSCh- and BzSCh iodides were measured at a concentration of 1 mM. The kinetic parameters, Michaelis-Menten constant (Km), maximal velocity (Vmax) and catalytic efficiency (Km/Vmax) for purified AChEIIb and AChEIIIb were estimated using computer analysis of Lineweaver-Burk double reciprocal plots of experimental data according to Lineweaver and Burk (1934) using 6 concentrations for each substrate in the range from 0.1 to1.0 mM.

2. 13. Inhibition specificity The inhibition specificity of purified R. ferrugineus AChEs was investigated according to Zhu and Clark (1994) and Mohamed et al. (2016). Each of purified AChEs was pre-incubated with six different concentrations of each inhibitor individually at room temperature for 15 min. The residual activity was estimated as previously described using AcSChI as substrate. The inhibitors were eserine,

BW284C51 and iso-OMPA as non-specific AChE, specific AChE and specific BuChE inhibitors, respectively. The bimolecular rate constants (Ki) and the mechanism of inhibition for eserine and BW284C51 were estimated by the double reciprocal plots of initial velocities of R. ferrugineus AChEIIb and AChEIIIb versus reciprocal concentrations of AcSChI as substrate in absence and presence of three different concentrations of eserine or BW284C51, individually.

2.14. Statistical analysis The enzyme activities for crude extracts from different organs of R. ferrugineus and in the cuticles from different instar larvae were replicated three times. One larva was used for each extraction. The data were analyzed using Statistical Package for the Social Sciences (SPSS) version 22. Data were displayed as means ± standard error. Duncan’s test for homogeneity was applied to study similarities in the studied variables among the different instars and among the different organs.

3. Results 3.1. Distribution of AChEs in different organs of R. ferrugineus The total and specific activities of AChE were estimated in different organs isolated from the 10th instar larvae of R. ferrugineus (Fig. 1 A, B). One-way ANOVA revealed significant effect of the organ type on the total activity (F4,10=1808.19, p<0.001) and specific activity (F4,10=691.54, p<0.001). The enzyme activity in terms of total U/organ and specific activity (U/mg protein) ranged from 314.9 - 3868 and from 99 - 340.8, respectively. Among the organs examined, cuticle displayed the highest AChE activity 3868 U/cuticle. However, the highest specific activity (340.8 U/mg protein) was recorded in the whole gut.

3.2. AChEs during larval development The activities of AChE in the cuticles of R. ferrugineus were estimated, during larval development (Fig. 2 A, B). According to one-way ANOVA, the larval development significantly affected both the total activity (F7,16=177.73, p<0.001) and specific activity (F7,16=68.84, p<0.001) activities of AChE. The enzyme activity in terms of total activity/cuticle and specific activity (U/mg protein) ranged from 948 5630 and from 51.6 - 140, respectively. No significant differences in total enzymatic

activity could be observed between the 5th - 8th instar larvae (P<0.05). On contrary, from the 9th instar larvae, the enzyme activity sharply increased and maximally reached in the 11th instar larvae with enzyme activity of 5630 U/cuticle with specific activity 140 U/mg protein. Thereafter, the enzyme activity declined in the 12th instar larvae with 4178 U/cuticle with specific activity 112 U/mg proteins. Therefore, purification and characterization of AChE were restricted from the cuticle of 11th instar larvae of R. ferrugineus.

3.3. Purification of R. ferrugineus AChEs Two major isoenzymes AChEIIb and AChEIIIb were purified to homogeneity from two cuticles of the 11th instar larvae of R. ferrugineus (Table 1). Unbound isoenzyme AChEI and two bound isoenzymes, AChEII and AChEIII, were resolved upon chromatography of crude extract on a DEAE-Sepharose column (Fig. 3). The isoenzymes were eluted with 0.0, 0.05 and 0.3 M NaCl, respectively. R. ferrugineus AChEII and AChEIII have 2.4- and 1.78-folds higher enzyme activities than the unbound isoenzyme AChEI. The two predominant isoenzymes, AChEII and AChEIII, were further purified by applying separately on a Sephacryl S-200 column. Each isoenzyme resolved into a minor and a major isoenzyme (Fig. 4 A and B). The two major R. ferrugineus AChEIIb and AChEIIIb isoenzymes were purified to homogeneity with final specific activities of 3504.3 and 2979 U/mg protein which represented 24.9 and 21.2-fold purification over crude extract, respectively.

3.4. Homogeneity The presence of one single protein band for each R. ferrugineus AChEIIb and AChEIIIb indicates the purity of the enzyme (Fig. 5).

3.5. Estimation of molecular weights. R. ferrugineus AChEIIb and AChEIIIb had molecular weights of 32 and 57 kDa, respectively by chromatography. They migrated as a single protein band on SDSPAGE, with molecular weights of 33 and 54 kDa, respectively confirming the existence of the two isoenzymes in monomeric forms (Fig. 6).

3.6. Characterization of R. ferrugineus AChEs

3.6.1 Effect of pH and temperature R. ferrugineus AChEIIb and AChEIIIb had optimum activity at pH 8.0 using Tris-HCl buffer and at temperature 40oC with activation energies 8.3 and 4.4 kcal/mol, respectively (Fig. 7 A, B). The two isoenzymes showed high stability where 29 and 15 % of activities were lost upon incubation for 1hr at 70oC. 3.6.2. Effect of metal cations No stimulatory effects could be recorded upon incubation of R. ferrugineus AChEs with the metal cations examined. Most of metal cations had potent inhibitory effect ranging from 20 to 100 % inhibition upon incubation with 10 mM concentration (Fig. 8 A, B). R. ferrugineus AChEIIb and AChEIIIb activities were completely inhibited upon incubation separately with 10 mM of each Hg2+, Mn2+ and Cu2+. Neither Mg2+ nor Na+ at concentrations of 1 and 10 mM had inhibitory effect on the activities of R. ferrugineus AChEs. 3.6.3. Substrate specificity The purified R. ferrugineus AChEs showed broad substrate specificities towards commercially available pseudosubstrates, AcSChI, PrSChI, BuSChI and sparingly towards BzSChI (Table 2). The relative activity arranged in a descending order: AcSChI > PrSChI > BuSChI > BzSChI, indicating the preferability towards AcSChI. They showed Michaelis-Menten behavior in the concentration range 0.1- 1.0 mM for the three former substrates (Fig. 9A, B). By computer analysis

of lineweaver-Burk double-reciprocal plots

of

experimental data for the three former substrates (Table 2) Km, Vmax, Vmax/Km were calculated. R. ferrugineus AChEIIb and AChEIIIb exhibited higher catalytic efficiency (Vmax/Km) toward AcSChI than BuSChI and the relative efficiency of BuSChI versus AcSChI hydrolysis (REH) as determined by the ratio Vmax (BuSCh)/Vmax (AcSCh) were 0.71 and 0.67, respectively.

3.6.4. Effect of high substrate concentration R. ferrugineus AChEs were inhibited by AcSChI, PrSChI and BuSChI at concentrations above 1mM, resulting in bell-shaped curves (Fig. 10A, B). R. ferrugineus AChEIIb and AChEIIIb activities were completely inhibited at 5 and 4 mM, respectively.

3.6.5. Inhibition specificity For confirming the status of R. ferrugineus AChEs, three inhibitors were examined. The inhibitors were eserine, BW284C51 and iso-OMPA. R. ferrugineus AChEIIb and AChEIIIb activities were suppressed by 50% upon incubation with 2 and 0.75 mM eserine, respectively. In contrast, the two isoenzymes were not affected by iso-OMPA at high concentration up to 50 mM. The inhibition kinetic parameters IC50 and Ki values by eserine and BW284C51 are indicated in table (4). R. ferrugineus AChEs competitively inhibited by such inhibitors (Fig. 11, 12 A and B).

Figures

a

A

3500 3000 2500 2000 1500 1000

b 500

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Head

Haemolymph

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Specific activity (U/mg protein)

Total activity (U in individual organs)

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e

0

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Cuticle

Fat body

Gut

Cuticle

th

Different organs of 10 Instar Larval

Fat body

Head

Haemolymph Gut Different organs of 10 Instar Larval th

Fig. 1. The activity of AChE in different organs of 10th instars larvae of R. ferrugineus. Each value represents the mean ±standard error of the mean (SEM). Bars marked with the same letters are similar (P>0.05) whereas those with different one are significantly different (P<0.05).

Total activity (U in the cuticle)

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Larval instar

Fig. 2. The activity of AChE in the cuticles of R. ferrugineus during larval development. Each value represents the mean ±standard error of the mean (SEM). Bars marked with the same letters are similar (P>0.05) whereas those with different one are significantly different (P<0.05).

1200

5

0.05 M NaCl

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0.3 M NaCl 0.4 M NaCl

Absorbance at 280 nm

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AChEII

800

3

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AChEI 1

200

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Fraction No.

Fig. 3. A typical elution profile for the chromatography of R. ferrugineus AChE crude extract from two cuticles on DEAE-Sepharose column. Absorbance at 280 nm (o—o) and enzyme activity (•—•).

AChE activity (units/fraction)

0.0 M NaCl

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A

AChEIIb

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ACHEIII activity (units/fraction)

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Elution volume (ml)

Fig. 4. A typical elution profiles for the chromatography of DEAE-Sepharose active fractions of R. ferrugineus AChEII (A) and AChEIII (B) on a Sephacryl S-200 column. Absorbance at 280 nm (o—o) and enzyme activity (•— •).

AChEIIIb

AChEIIb

a

b

c

Fig. 5. Electrophoretic analysis of R. ferrugineus AChEs (a) AChEIIb, (b) AChEIIIb and (c) Crude extract.

Fig. 6. Electrophoretic analysis of R. ferrugineus AChEs on 12% SDS-PAGE for estimating the subunit molecular weights. (a) Molecular weight marker proteins (b) denatured purified AChEIIb and (c) denatured purified AChEIIIb.

Fig. 7. Effect of temperature on the activities (x——x) and stabilities (•——•) of AChEIIb (A) and AChEIIIb (B) of R. ferrugineus. Activation energies were calculated from the replot of log activity versus 1/T (Arrhenius plot) shown in the inset.

140

A

1 mM 10 mM

Relative activity (%)

120

a a*

a a*

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a

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Metal cations

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1 mM 10 mM

Relative activity (%)

120 100

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h f*

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None Co2+ Mg2+ Hg2+ Mn2+ K+ Ni2+ Zn2+ Cu2+ Ca2+ Ba2+ Na+

Metal cations

Fig.8. Effect of metal cations on the activities of R. ferrugineus AChEIIb (A) and AChEIIIb (B). *letters referrers to statistical analysis for metal concentration

10mM

-3

26

1/ µmol substrate hydrolyzed/ml/h X 10

A

Km AcSChI (•―•) = 1 mM Km PrSChI (x―x) = 1.3 mM Km BuSChI (o―o) = 1.7 mM

24 22 20 18 16 14 12 10 8 6 4 2 0

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Km AcSChI (•―•) = 0.67 mM Km PrSChI (x―x) = 0.95 mM Km BuSChI (o―o) = 1.15 mM

40

30

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Fig. 9. Estimation of Km, Vmax using AcSChI, PrSChI and BuSChI as substrates for R. ferrugineus AChEIIb (A) and AChEIIIb (B) by Lineweaver–Burk plots.

800

A

AcSChI PrSChI BuSChI

AChEIIIb (Units/ml/h)

700 600 500 400 300 200 100 0 -1.0

-0.5

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AChEIIIb (Units/ml/h)

600 500 400 300 200 100 0 -1.0

-0.5

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Substrate concentration (Log mM)

Fig. 10. Effect of high substrate concentrations on the activities of AChEIIb (A) and AChEIIIb (B) of R. ferrugineus in 60 mM Tris-HCl buffer, pH 8.0

16

Zero eserine 1.5 mM eserine 2 mM eserine 3 mM eserine

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Ki=0.52 mM

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Zero eserine 0.5 mM eserine 0.75 mM eserine 1 mM eserine

18 16 14 12 10 8 61.25 4 2 0

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6

-1

Fig.11. Plots of reciprocal of initial velocities of AChEIIb (A) and AChEIIIB (B) versus reciprocal concentrations of AcSChI in absence and presence of different concentrations of eserine. Inhibition constant (Ki) of eserine as the competitor of AcSChI was calculated from the replot of S/V against serine concentrations.

Slope (S/V)

1.0 0.8 0.6 0.4 0.2 0.0 -1

0

1

2

3

BW284C51 concentration (mM)

Ki= 0.78 mM

A

Zero BW284C51 1 mM BW284C51 2 mM BW284C51 3 mM BW284C51

-3 1.2

1/µmol Substrate hydrolyzed/ml/hx10

1.4

60

50

40

30

20

10

0 -10

-8

-6

-4

-2

0

2

(1/Substrate) mM

0.2

0.0 -0.2

0.0

0.2

0.4

BW284C51 concentration (mM)

Ki= 0.3 mM

6

8

Zero BW284C51 0.15 mM BW284C51 0.3 mM BW284C51 0.45 mM BW284C51

-3 0.4

1/µmol Substrate hydrolyzed/ml/hx10

Slope (S/V)

0.6

4

40

10

-1

B

30

20

10

0 -10

-8

-6

-4

-2

0

2

(1/Substrate) mM

4

6

8

10

-1

Fig. 12. Plots of reciprocal of initial velocities of a) AChEIIb and b) AChEIIIb versus reciprocal concentrations of AcSChI in absence and presence of different concentrations of BW284C51. Inhibition constant (Ki) of BW284C51 as the competitor of AcSChI was calculated from the replot of S/V against BW284C51 concentrations.

Tables Table 1. A typical purification scheme for AChE from R. ferrugineus. Protein (mg) 80

Activity (U) 11260

Specific activity (U/mg protein) 140.7

Fold purification 1

Recovery (%) 100

AChE I

4.2

769.2

183

1.18

8.4

AChE II

12

6155

512.9

3.6

54.7

AChE III

10

3942

394.2

2.1

35

AChEIIa

1.9

431

226.8

1.6

3.8

AChEIIb

1.4

4906

3504.3

24.9

43.6

AChEIIIa

1.7

233

137

0.97

2.07

AChEIIIb

1

2979

2979

21.2

26.5

Purification step Crude extract DEAE-Sepharose

Sephacryl S-200

Table 2. Substrate specificity and the kinetic characteristics of R. ferrugineus AChEs Relative activity (%) Substrate AChEIIb

Km (mM)

Vmax (U/ml/h)

Vmax /Km

AChEIIIb

AChEIIb

AChEIIIb

AChEIIb

AChEIIIb

AChEIIb

AChEIIIb

AcSChI

100

100

1

0.67

800

400

800

597

PrSChI

70.1

68

1.3

0.95

666

334

512.3

351.6

BuSChI

52.2

49

1.7

1.1

571

266.7

336

242.5

BzSChI

4.5

4

N.D.

N.D.

N.D.

N.D.

N.D

N.D

N.D.---- not detected.

Table 3. Substrate affinity (Km) towards AcSChI for R. ferrugineus AChEs and for different insect species. Species

Km (mM)

References

AChEIIb

1.0

Present study

AChEIIIb

0.67

R. ferrugineus

B. dorsalis

0.088

Hsiao et al. (2004)

S. graminum

0.059

Gao and Zhu (2001)

D. virgifera

0.02

Gao et al. (1998)

T. confusum

0.57

Chaudhary et al. (1966)

A. citricola

0.17

Manulis et al. (1981)

Manduca sexta

0.27

Lester and Gilbert (1987)

Table 4. Inhibition specificity and parameters (IC50 and Ki) for R. ferrugineus AChEs and different insect species.

AChEIIb

Eserine IC50 Ki (mM) (mM) 2 2*

AChEIIIb

0.75

S

11.1x10-8 6.4x10-7

1.26x10-7

Guedes et al.,

5.44x10-6

1997

0.011 0.05

0.82

7x10-7 9.8x10-7

0.82

1.2x10-8 4.48x10-6

10.4x10-4 9.75x10-4

4.75x10-4

1.45x10-4

Species

R. ferrugineus R. dominica

R S

L. entomophila

R

L.

S

bostrychophila

R S

B. dorsalis

R

S. graminum S S. frugiperda

R

0.52*

BW284C51 IC50 Ki (mM) (mM) 2 0.78* 0.3

1.3

1.3

0.8x10-3 1.6x10-3

* Competitive inhibition, R: resistant and S: susceptible.

Reference

Present study

0.3*

Xiao et al., 2010

Chai et al., 2007 Hsu et al., 2008

Gao and Zhu, 2001 Yu, 2006

4. Discussion Red palm weevil (RPW) R. ferrugineus is the most destructive pest of many palm trees in vast areas of the world (Al-Rajhy et al., 2005). The intensive use of synthetic insecticides for controlling RPW resulted in the emergence of insecticide(s)resistance strains. Resistance that arises from increase AChE level due to overexpression and target site insensitivity in different insect species had been investigated (Yu, 2006; Yang et al., 2008; Polsinelli et al., 2010; Vohra et al., 2014). The present study estimated the distribution of AChE level in different organs of R. ferrugineus, in the cuticle during larval development as well as purification and biochemical characterization of two major AChEs isoenzymes.

The current study investigated AChE activity levels in different organs of R. ferrugineus. The enzyme activity in terms of total U/organ in the cuticle of the 10th instar larvae was 18.2- 16.5- 12.3- and 6.0- times higher than that recorded in head, haemolymph, gut and fat body, respectively. The highest specific activity of AChE in the whole gut could be attributed to the low protein content due to the presence of high proteolytic activities in the larval midgut of R. ferrugineus as recorded by (Alarcon et al., 2002). The high AChE activity in the cuticle suggested that such organ could play the most important role in the detoxification for scavenging the insecticide(s). In addition, such organ represents the first line for protecting the whole larval systems by degrading the detrimental chemicals before entering the entire body for surviving R. ferrugineus in a chemically unfriendly environment. In addition, the various levels of AChE in different organs deduced that the individual organ has a role for scavenging the toxicity of an insecticide and might handle the toxic chemical assault differently.

While all insects probably possess capacity to detoxify the synthetic insecticide(s) chemicals, AChE levels as insecticide(s) scavenger can be expected to vary among insect species, with the nature of insect's recent environment and with developmental stage (Bamidele et al., 2017). The present study demonstrated changes in AChE contents in the cuticles of R. ferrugineus during larval development. The cuticle of the 11th instar larvae had the highest AChE level with 5630 U/cuticle which represents 5.9- and 1.2-fold higher than that estimated in the cuticle of the 5th instar larvae and the late larval stage, 12th respectively. Therefore, purification and

characterization of AChE isoenzymes were carried out from 11th instar larvae. The decline in AChE activity in the cuticle of the 12th instar larvae may be due to low synthesized enzyme by the larvae when they are near to the next stage (pupal stage).

Two predominant isoenzymes AChEIIb and AChEIIIb were purified from the R. ferrugineus cuticle of 11th instar larvae. While, one major isoenzyme was partially purified from adults (Abo-El-Saad and Al-Ajlan, 2006). R. ferrugineus AChEs had specific activities of 3504 and 2979 U/mg protein with fold purification 24.9 and 21.2, respectively. These values were congruent with those estimated for western corn rootworm Diabrotica virgifera virgifera (20.4) (Gao et al., 1998). The purification folds for R. ferrugineus AChEs were ranged from 2.7- to 120-fold lower than those recorded for different insect species as Spodoptera frugiperda (Yu, 2006), Galleria mellonella (Keane and Ryan, 1999) and insecticide-resistant strains of L. entomophila (Xiao et al., 2010), B. dorsalis (Hsu et al., 2008), Rhyzopertha dominica (Guedes et al., 1997), S. graminum (Gao and Zhu, 2001). This could be attributed to the high and low contents of activity and protein, respectively, in the crude homogenates of R. ferrugineus cuticle compared to those estimated for different other insect species.

The native molecular weights of R. ferrugineus AChEIIb and AChEIIIb were 32 and 57 kDa, respectively, by gel filtration on Sephacryl S-200 column. These values were confirmed by SDS-PAGE indicating monomeric proteins. The molecular weight of R. ferrugineus AChEIIIb was comparable to those recorded for S. frugiperda (66 kDa) (Yu, 2006), head and appendage of Pardosa astrigera (66 kDa) (Ma et al., 2011) and for partially purified AChE (70 kDa) from adult R. ferrugineus (Abo-ElSaad and Al-Ajlan, 2006).

The optimum activities for R. ferrugineus AChEs were observed at Tris-HCl, pH 8.0 and at temperature 40oC. These results agree with those reported for green rice leafhopper, N. cincticeps (Kato et al., 2004) and adult stage of Dictyocaulus viviparus (Mckeand et al., 1994). While, oriental fruit fly B. dorsalis (Hsiao et al, 2004), cotton aphid, Aphis gossypii Glover (Li and Han, 2002) and field populations of L. entomophila (Xiao et al., 2010) showed optimum for activities at pH 7.5 and at temperature range 35-37oC.

The most examined metal cations inhibited R. ferrugineus AChEs and the inhibitory effect ranged from 20-100%. They were completely inhibited by 10 mM of Mn2+, Hg2+ and Cu2+. Abo-El-saad and Al-Ajlan (2006) reported that the partially purified AChE from adults of R. ferrugineus were stimulated up to 1.2 and 1.5- fold increase upon incubation with 10 mM Mg2+ and Ca2+, respectively. We have no indication about the physiological role of Mn2+, Hg2+ and Cu2+ on R. ferrugineus AChEs. However, it can be hypothesized that the inhibition of AChE by such metals resulted in an increase in ACh accumulation in R. ferrugineus as recorded by (Lester and Gilbert, 1987) and may open a way for development of a new strategy for controlling R. ferrugineus.

R. ferrugineus AChEs were most active against AcSChI and showed less activity towards PrSChI, BuSChI and BzSChI. R. ferrugineus AChEIIb and AChEIIIb had high catalytic activity 800 and 400 U/ml/h with Km values of 1.0 and 0.67 mM, respectively for the former substrate, AcSChI. The substrate affinity (Km) for R. ferrugineus AChEs were compared with those previously reported for different insect species (Table 3). It can be concluded that R. ferrugineus AChEIIIb had an affinity for AcSChI comparable to that recorded for beetle Tribolium confusum (Chaudhary et al., 1966). While the affinities of R. ferrugineus AChEs represent 3.9- to 50-fold lower than those recorded for different insect species (Manulis et al., 1981; Lester and Gilbert, 1987; Gao et al., 1998; Gao and zhu, 2001; Hsiao et al., 2004). Recently, AChEs from EPN, Heterorhabditis bacteriophora, as a biocontrol agent against R. ferrugineus, had Km values of 1.3 and 0.9 mM using AcSChI also as a substrate (Mohamed et al., 2016). The low affinity towards substrate and a high catalytic activity had been documented in insecticide-resistant populations of B. dorsalis (Hsu et al., 2008), S. frugiperda (Yu, 2006), Liposcelis bastrychophila (Chai et al., 2007), L. manilensis (Yang et al., 2008) and L. entomophila (Xiao et al., 2010). Consequently, it can be hypothesized that R. ferrugineus larvae is an insecticideresistant pest and it can be solve the complained problem by the farmers in the ineffectiveness of insecticides for controlling R. ferrugineus. The catalytic efficiency for R. ferrugineus AchEs as determined by the (Vmax/Km) toward AcSChI, 2.39- and 2.46-fold higher than BuSChI confirming the typical substrate specificity of AChEs (Hsiao et al., 2004; Mohamed et al., 2007; Kim et al., 2012). The relative efficiency of hydrolysis (REH) of BuSChI versus AcSChI

by R. ferrugineus AChEs as determined by the Vmax (BuSChI)/Vmax (AcSChI) ratio according to Hsiao et al. (2004) were 0.71 and 0.67. The REH values were in the same range of AChEs from B. dorsalis (0.66) (Hsu et al., 2008), Dendrobaena veneta (0.6) (Talesa et al., 1996) and H. bacteriophora AChEs (0.8 and 0.71) (Mohamed et al., 2016), indicating the substrate spectrums of AChEIIb and AChEIIIb were similar.

High concentrations of the substrates, AcSChI, PrSChI and BuSChI, (>1mM) were inhibited R. ferrugineus, showing a bell-shaped curves. This substrate inhibition phenomenon could be due to the binding of excess substrate to the peripheral regulatory site leading to inactive enzyme- substrate- substrate complex (Kato et al., 2004). It is a phenomenon of typical true AChEs capable of hydrolyzing BuSChI. Similar finding had been well documented for AChEs from different insect species (Marcel et al., 2000; Kato et al., 2004; Hsu et al., 2008; Xiao et al., 2010; Mohamed et al., 2016).

The inhibition specificity of R. ferrugineus AchEs towards eserine, BW284C51, as non specific and specific inhibitors for AchEs, respectively and iso-OMPA as specific inhibitor for human BuChEs had been investigated. R. ferrugineus AchEs had similar trends towards there inhibitors, where they had higher sensitivity to inhibition by BW284C51 than eserine, competitively inhibited by these inhibitors, and are no inhibited by iso-OMPA. The inhibition parameters and kinitics of R. ferrugineus AchEs were compared with those previously reported for different insect species table (4). It can be concluded that R. ferrugineus AchEs had sensitivity to inhibition by eserine, as a carbamate inhibitor and the IC50 and Ki values were several folds higher than those recovered for resisitant and susceptible strains of different insect species (Guedes et al., 1997; Gao and Zhu, 2001; Yu, 2006; Chai et al., 2007; Hsu et al., 2008; Xiao et al., 2010)

Conclusively, high activity of AChE, particularly in cuticle strongly suggested that cuticle might play the most important role for detoxifying insecticide(s). During larval development, the 11th instar larvae had the highest AChE level. AChEIIb had higher efficiency for detoxifying eserine, as a carbamate insecticide, than AChEIIIb. The in vitro studies indeed reflect on in vivo sensitivity of RPW to insecticides. Therefore, the sensitivity of purified R. ferrugineus AChEs and the toxicological

characteristics towards a wide range of insecticides in vitro and in vivo, respectively are in progress to overcome the failure of synthetic insecticides field application for controlling R. ferrugineus.

5. Acknowledgement This research was supported by the National Research Centre (11/3/10), the authors would like to thank Dr. Ibrahim M. Shams, Central Laboratory for Date Palm Research and Development (CLDPRD), Agricultural Research Center, Dokki, Giza, Egypt.

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Leptinotarsa