Fabien Gasc

Sophie Thiebaud-Roux
email: sophie.thiebaudroux@ensiacet.fr

Zephirin Mouloungui

Methods for synthesizing diethyl carbonate from ethanol and supercritical carbon dioxide by one-pot or two-step reactions in the presence of potassium carbonate

Keywords: Diethyl carbonate, diethyl ether, supercritical carbon dioxide, ethanol, potassium carbonate, one-pot process, two-step reaction, hybrid carbonate

Carbon dioxide sequestration was studied by synthesizing diethyl carbonate (DEC) from ethanol and CO 2 under supercritical conditions in the presence of potassium carbonate as a base. The co-reagent was ethyl iodide or a concentrated strong acid. This sequestration reaction occurs in two steps, which were studied separately and in a one-pot reaction. An organic-inorganic carbonate hybrid, potassium ethyl carbonate (PEC) is generated at the end of the first step. This intermediate was characterized and was found to be a target molecule for CO 2 capture. Different co-reactants, such as ethyl iodide and concentrated strong Brönsted acid, were compared in the second step and used to investigate the reactivity of the hybrid. With ethyl iodide as the co-reactant, one-pot DEC synthesis gave higher yields (46%) than two-step production. The supercritical CO 2 acts as a swelling solvent and compatibilizing agent in the reaction medium, favoring interactions between ethanol and CO 2 and between PEC and ethyl iodide. The use of a phase transfer catalyst (PTC) increased DEC production (yield 51%) without increasing the amount of diethyl ether (DEE) produced as a by-product (yield 2%).

Introduction

Carbon dioxide is one of the greenhouse gases (together with CH 4 , NO x and CFCs) responsible for global warming. By 2005, CO 2 concentration in the atmosphere had increased from the 280 ppm level, thought to be present before the Industrial Revolution, to 379 ppm. Over the last 10 years (1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)) it has increased substantially, at a mean rate of 1.9 ppm.year -1 [START_REF] Metz | Carbon dioxide capture and storage[END_REF]. No single technology currently available could, if used alone, decrease worldwide emissions of CO 2 into the atmosphere to the level required by the Kyoto protocol. The most appropriate approach to reducing CO 2 emissions into the atmosphere is currently thought to involve selecting a certain number of technologies, each able to reduce emission by a given amount and to make use of the cumulative effects of these technologies to reach carbon dioxide emission targets [START_REF] Aresta | Utilisation of CO 2 as a chemical feedstock: opportunities and challenges[END_REF]. A number of current technologies for recovering, binding and sequestering carbon dioxide are being developed with a view to increasing their efficiency, reliability and cost [START_REF] Herzog | What future for carbon capture and sequestration?[END_REF]. One of these methods is the geological sequestration of CO 2 by the carbonation of minerals rich in calcium, magnesium, sodium and potassium. In nature, this process is exceedingly slow (100,000 years), so various experiments have been carried out to optimize the conversion of carbon dioxide into its solid stable form: mineral carbonate [START_REF] Kharoune | Use of alkaline materials for sequestering and recycling of carbon dioxide[END_REF].

Comment citer ce document : Gasc, F., Thiebaud-Roux, S., Mouloungui, Z. (2009). Methods for synthesizing diethyl carbonate from ethanol and supercritical carbon dioxide by one-pot or two-step reactions in the presence of potassium carbonate. Journal of Supercritical Fluids, 50 [START_REF] Metz | Carbon dioxide capture and storage[END_REF], 46-53. , DOI : 10.1016/j.supflu.2009.03.008

2

The sequestration of CO 2 into organic carbonates uses up only limited amounts of the total amount of CO 2 produced, but is nonetheless of real interest to the scientific community. CO 2 has been exploited as a substitute for phosgene [START_REF] Heyden | Patent DE 109933[END_REF] or carbon monoxide [START_REF] Romano | Synthesis of dimethyl carbonate from methanol, carbon monoxide, and oxygen catalyzed by copper compounds[END_REF][START_REF] Romano | Dimethyl carbonate[END_REF] in the synthesis of various organic carbonates, and particularly in the carbonation of epoxides [START_REF] Bhanage | Synthesis of dimethyl carbonate and glycols from carbon dioxide, epoxides, and methanol using heterogeneous basic metal oxide catalysts with high activity and selectivity[END_REF][START_REF] Tian | Efficient synthesis of dimethyl carbonate from methanol, propylene oxide and CO 2 catalyzed by recyclable inorganic base/phosphonium halide-functionalized polyethylene glycol[END_REF] or alcohols [START_REF] Ballivet-Tkatchenko | Reactivity of Carbon Dioxide with n-Butyl(phenoxy)-, (alkoxy)-, and (oxo)stannanes: Insight into Dimethyl Carbonate Synthesis[END_REF]. These processes were reviewed by Shaikh et al. [START_REF] Shaikh | Organic Carbonates[END_REF], and more recently, by Sakakura et al. [START_REF] Sakakura | Transformation of Carbon Dioxide[END_REF]. However, from the perspective of green chemistry, the most attractive method [START_REF] Anastas | Origins, current status, and future challenges of green chemistry[END_REF] is the use of an alcohol and carbon dioxide as synthons.

However, several studies on the synthesis of dimethyl carbonate from alcohol and CO 2 have shown the major problem to be the rate of conversion of methanol, due to the thermodynamic limitations of the reaction and deactivation of the homogeneous catalysts (e.g. tin complexes: distannoxanes) [START_REF] Sakai | Reaction of organotin alkoxides with carbon disulfide, carbony sulfide, or carbon dioxide[END_REF][START_REF] Yamazaki | Chemical reactions of carbon dioxide[END_REF][START_REF] Kizlink | Synthesis of dimethyl carbonate from carbon dioxide and methanol in the presence of organotin compounds[END_REF][START_REF] Kizlink | Preparation of dimethyl carbonate from methanol and carbon dioxide in the presence of Sn(IV) and Ti(IV) alkoxides and metal acetates[END_REF][START_REF] Ballivet-Tkatchenko | The role of distannoxanes in the synthesis of dimethyl carbonate from carbon dioxide[END_REF] and heterogeneous catalysts (e.g. zirconium oxide : ZrO 2 ) [START_REF] Tomishige | Catalytic Properties and Structure of Zirconia Catalysts for Direct Synthesis of Dimethyl Carbonate from Methanol and Carbon Dioxide[END_REF][START_REF] Ikeda | Promoting effect of phosphoric acid on zirconia catalysts in selective synthesis of dimethyl carbonate from methanol and carbon dioxide[END_REF][START_REF] Tomishige | A novel method of direct synthesis of dimethyl carbonate from methanol and carbon dioxide catalyzed by zirconia[END_REF][START_REF] Zhao | Novel reaction route for dimethyl carbonate synthesis from CO 2 and methanol[END_REF]. The only means of improving methanol conversion to date has been to use a co-reactant, such as methyl iodide, in the presence of basic catalysts [START_REF] Fang | Direct synthesis of dimethyl carbonate from carbon dioxide and methanol catalyzed by base[END_REF][START_REF] Cao | Catalytic esterification of carbon dioxide and methanol for the preparation of dimethyl carbonate[END_REF][START_REF] Cai | Synthesis of Dimethyl Carbonate from Methanol and Carbon dioxide using Potassium Methoxide as Catalyst under Mild Conditions[END_REF][START_REF] Hou | Synthesis of dimethyl carbonate using CO 2 and methanol: enhancing the conversion by controlling the phase behavior[END_REF]. Potassium carbonate has been reported to be the most effective base tested. In 2001, Fujita et al. [START_REF] S.-I. Fujita | Synthesis of dimethyl carbonate from carbon dioxide and methanol in the presence of methyl iodide and base catalysts under mild conditions: effect of reaction conditions and reaction mechanism[END_REF] reported a study of the reaction conditions required for the synthesis of DMC and proposed a reaction mechanism in which DMC and dimethyl ether (DME) were produced by parallel pathways. Increasing CO 2 pressure to 80 bar results in an increase in DMC yield with the amount of catalyst or methyl iodide added.

Carbon dioxide can also be sequestrated into a hybrid organic-inorganic carbonate. The existence of such hybrids and their synthesis have been known since the 1960s [START_REF] Howe | Carbonic Acid and its Derivatives[END_REF]. Indeed, although alkyl hydrogen carbonates are not stable at room temperature, metal salts (e.g. of Li, Na, K, Mg and Ba) may be obtained by saponifying dialkylcarbonate [START_REF] Adam | Crystal structure of potassium monomethylcarbonate[END_REF] or by bubbling dried CO 2 through a solution containing dry methanol and sodium [START_REF] Kunert | Novel arrangement of methyl carbonate units: x-ray crystal structure of sodium methyl carbonate[END_REF].

In this study, we investigated two carbon dioxide sequestration reactions for DEC synthesis (Figure 1). The first of these reactions can be broken down into two steps: a. Preparation of two potassium salts: potassium ethylcarbonate (PEC) as a hybrid carbonate and potassium bicarbonate (KHCO 3 ), according to an ionic pathway (Figure 1a). b. Alkylation of PEC in the presence of ethyl iodide (EtI) or acidified ethanol (EtOH/H + ) to obtain DEC (Figure 1b). EtI and EtOH/H + are co-reagents in the O-alkylation of PEC giving rise to an electrophilic alkyl group.

The second one reaction involves the direct mixing of ethanol, supercritical CO 2 and ethyl iodide in a one-pot process (Figure 1c). The best yields reported for ethanol conversion to date have not exceeded 0.6% [START_REF] S.-I. Fujita | Synthesis of dimethyl carbonate from carbon dioxide and methanol in the presence of methyl iodide and base catalysts under mild conditions: effect of reaction conditions and reaction mechanism[END_REF].

Comment citer ce document : Gasc, F., Thiebaud-Roux, S., Mouloungui, Z. In this work, we used ethanol rather than methanol, and we assessed its reactivity with supercritical carbon dioxide. Ethanol was preferred over methanol as it is a highly abundant, non toxic biomolecule used in the production of biofuel and because it participates in the advantageous cycle reducing the greenhouse effect. For these reasons, carbon dioxide sequestration with ethanol is in keeping with most green chemistry principles. However, ethanol is less reactive than methanol, as it is less acidic [START_REF] Murto | Nucleophilic reactivity of alkoxide ions towards 2,4-dinitrofluorobenzene and the acidity of alcohols[END_REF].

Experimental procedures

Dimethyl and diethyl carbonates (99%), ethyl iodide (≥ 99%), potassium carbonate (≥ 99%) and Aliquat 336 (trioctylmethylammonium chloride, 88%) were all purchased from Sigma-Aldrich, France. Before use, potassium carbonate was dried for 48 hours at 104 °C. Anhydrous absolute ethanol was purchased from API, France. Compressed carbon dioxide was used in a specific installation purchased from Top-Industrie, France, and described in Figure 2. All experiments in the presence of supercritical CO 2 were carried out in a stainless steel autoclave with an internal volume of 200 ml equipped with a mechanical stirrer and a heating collar cooled by a vortex. The CO 2 is introduced into the reactor via a pneumatic pump, after heating to the required temperature. Pressure and temperature are kept constant with the aid of pressure and temperature regulators. All the parameters (temperature ± 0.1 °C, pressure ± 0.1 bar, stirring speed ca. 700 rpm, volume of CO 2 introduced ± 0.01 ml) were monitored and recorded. After the reaction, the reactor was cooled to ca. 10 °C by a vortex and bled via a manual valve equipped with a fritted disk.

Typical procedures are described below:

For the mixed carbonate synthesis method (procedure A), two different procedures were used for the first step: Procedure A1: Ethanol (340 mmol) and K 2 CO 3 (340 mmol) were loaded into the reactor. The autoclave was then heated to 100 °C and CO 2 was injected to the desired pressure with stirring. The desired pressure (ca. 80 bar) was reached within one minute.

Procedure A2: Ethanol (340 mmol) and K 2 CO 3 (340 mmol) were loaded into the reactor. CO 2 was then injected into the reactor to a pressure of ca. 20 bar at room temperature. The autoclave was heated to 100°C at a rate of 5 °Cmin -1 . The pressure obtained was about 50 bar. Finally, CO 2 was injected to the desired pressure, with stirring.

For assessment of the reactivity of the mixed carbonate with a co-reactant (procedure B), experiments were carried out in a three-necked round-bottomed flask equipped with a mechanical stirrer and a reflux condenser. The solid hybrid carbonate obtained during procedure A (30 g), was introduced in ethanol (50 ml) and the co-reactant solution was then via a dropping funnel once the desired temperature had been reached. The reaction was performed at 110 °C or -80 °C for 3 hours. Finally, once cold, the reaction medium was filtered to obtain a liquid phase and a solid, which was dried for 24 h in a vacuum at 104 °C.

For the one-pot reaction (procedure C), ethanol (856 mmol), C 2 H 5 I (107 mmol) and K 2 CO 3 (107 mmol) were loaded into the reactor, into which CO 2 was then injected at a pressure of ca. 20 bar at room temperature. The autoclave was heated to 100 °C and CO 2 was injected to the desired pressure, with stirring.

The collected liquid phase was analyzed by infrared spectroscopy (JASCO, FT/IR 460 plus) and gas chromatography. Synthesized DEC and ethyl iodide were quantified with DMC as the internal standard. Methanol was used as the internal standard for diethyl ether (DEE) quantification. The solid hybrid carbonate was analyzed by infrared spectroscopy with FT-IR, CPMAS 13 C NMR (Bruker Avance 400WB) and by evaluating solid mass-gain.

The mass-gain MG(%) of the solid at the end of the reaction was determined as follows:

100 (%) 0 0 1 × - = m m m MG (1)
where m 0 =mass of introduced K 2 CO 3 (g), m 1 = mass of dried solid phase (g). The theoretical mass-gain TMG(%) was defined by equation ( 2):

Comment citer ce document : Gasc, F., Thiebaud-Roux, S., Mouloungui, Z. ( 2009). Methods for synthesizing diethyl carbonate from ethanol and supercritical carbon dioxide by one-pot or two-step reactions in the presence of potassium carbonate. Journal of Supercritical Fluids, 50 (1), 46-53. , DOI : 10.1016/j.supflu.2009.03.008

5 100 ) ( (%) 0 ) ( 2 × × = + m M n TMG CO EtOH Et (2)
where m 0 is the mass of introduced solid (g), n E is the number of moles of introduced ethanol (mol), and

) ( 2 CO EtOH M +
is the molar weight of EtOH + the molar weight of CO 2 (gmol -1 ).

The TMG(%) should be 65% if all the ethanol introduced has reacted with K 2 CO 3 and CO 2 according to the reaction described in Figure 1a. We consider that the KHCO 3 formed to be in the solid phase, because it is insoluble in ethanol.

Results and discussion

For validation of the mechanism of this carbon dioxide sequestration reaction, in which PEC is formed as an intermediate (Figure 1), the two reaction steps were studied separately and then in a one-pot reaction.

Two-step reaction method

Carbon dioxide sequestration into a hybrid organic-inorganic carbonate (PEC)

Our research, based on green chemistry principles, led us to perform carbon dioxide sequestration in safer conditions (without the cogeneration of hydrogen gas), using ethanol and potassium carbonate.

The reactivity of carbon dioxide with ethanol and potassium carbonate was demonstrated by determining the mass-gain of the solid obtained after reaction (Table 1). Theoretically, the maximum mass-gain is 65% for a total sequestration reaction. Operating conditions: 100°C and 80 bars with Procedure A1 or Procedure A2 (entry 10*).

Entry

Table 1: Effect of reaction time on the mass-gain of the solid product

An EtOH/K 2 CO 3 molar ratio of 1/1 was required to respect the stoichiometry of the reaction, making it possible to obtain PEC and KHCO 3 (entry 2). During this reaction, the organized structure of the Lewis and Brönsted base (K 2 CO 3 ) is disrupted by an exchange of the hydroxyl group proton from ethanol with the potassium cation.

Comment citer ce document : Gasc, F., Thiebaud-Roux, S., Mouloungui, Z. (2009). Methods for synthesizing diethyl carbonate from ethanol and supercritical carbon dioxide by one-pot or two-step reactions in the presence of potassium carbonate. Journal of Supercritical Fluids, 50 (1), 46-53. , DOI : 10.1016/j.supflu.2009.03.008 6 As shown in Table 1, PEC yield was maximal in the presence of K 2 CO 3 (entries 3 to 9), peaking two hours into the reaction. We accelerated the rate of reaction for this step by modifying the operating conditions for procedure A1 (section 2). With the second procedure, A2, a total reaction time of 30 minutes was required for sequestration of the carbon dioxide into PEC. With this method, the ethanolate generated from the ionic exchange between the base and ethanol is in contact with gaseous CO 2 during heating (ca. 10 minutes) and the reaction can start in subcritical CO 2 conditions. The mass gain in these conditions is slightly smaller than that in supercritical conditions (entry 6), when the reaction occurs with gaseous CO 2 (entry 11). Furthermore, the potassium bicarbonate (KHCO 3 ) produced did not react with ethanol and carbon dioxide due to a loss of the organized structure (monoclinic phase) of potassium carbonate [START_REF] Carbonnel | The water-potassium carbonate system[END_REF]. Indeed, although potassium is complexed with eight oxygens in potassium carbonate and potassium bicarbonate, the additional hydrogen bonding in KHCO 3 results in dimeric bicarbonate units [HCO 3 ] 2 2-capable of bidentate complexation [START_REF] Kunert | Novel arrangement of methyl carbonate units: x-ray crystal structure of sodium methyl carbonate[END_REF]. Thus, hydrogen bonding controls the formation of complexes between the carbonate units and changes the structure established by van der Waals forces in K 2 CO 3 . It leads to a solid mass loss of 3% due to manipulation (entry 1 Table 1): potassium bicarbonate is not an efficient base for the sequestration of CO 2 in PEC synthesis.

The solid product containing PEC was initially characterized by infrared spectroscopy. Its FT-IR spectrum is shown in Figure 3, together with those of commercial potassium carbonate and commercial potassium bicarbonate. The characteristic absorption bands identified for the three products are shown in Table 2. A large O-H band is visible for K 2 CO 3 and KHCO 3 . Potassium carbonate is very hygroscopic and deliquesces rapidly. The hydration form of K 2 CO 3 is known to depend on the synthesis procedure [START_REF] Carbonnel | The water-potassium carbonate system[END_REF] (Figure 4). In the monoclinic structure, K 2 CO 3 consists of Stretching C-H bands (1294 and 874 cm -1 ) and bending bands (2634 and 2971 cm -1 ), characteristic of the presence of alkyl chains, were observed in the analyzed solid. Moreover, the presence of an organic carbonate was revealed by two large bands: one at ca. 1660 cm -1 (composite of 4 bands), corresponding to C=O vibration in the carbonate group, and the other at ca. 1400 cm -1 (a composite of two bands), corresponding to C-O vibration in the carbonate group. The C=O wavelength for the intermediate was lower than that for diethyl carbonate (1760 cm -1 ) because PEC is an organic salt. Gatehouse et al. [START_REF] Gatehouse | The infrared spectra of some simple and complex carbonates[END_REF] showed that the separation of the C=O and C-O bands increases along the following series: basic salt < carbonate complex < organic acid < organic carbonate. This difference between the C=O wavelengths of different compounds results from the structure and coordination of the molecules (Figure 5) [START_REF] Nakamoto | Infrared spectra of inorganic and coordination compounds[END_REF]. The C-O distances and bond angles are between different carbonate molecules may differ considerably, due to the presence of different interactions in each structure [START_REF] Nitta | Crystal structure of potassium bicarbonate[END_REF]. For K 2 CO 3 , the CO 3 group coordinates one of the K atoms in a bidentate manner, and the other in a unidentate manner [START_REF] Ogden | Matrix isolation studies on cesium, rubidium, and potassium carbonates. The shape of molecular potassium carbonate[END_REF], whereas it coordinates K exclusively in a bidendate manner in PEC and KHCO 3 , because of the resonance phenomenon (Figure 5). Comparison of the IR spectra obtained for the commercial potassium carbonate, commercial potassium bicarbonate and the reaction product led to the identification of PEC and KHCO 3 in the solid product. For confirmation of this qualitative composition, we carried out CPMAS 13 C NMR analysis. In our case, cross-polarization to study 13 C functionality (CPMAS 13 C NMR) was not quantitative, because of the poor signal-to-noise ratio. In Figure 6, two singlets can be observed, corresponding to the presence of an ethyl chain: at 15 ppm for the methyl group and 60 ppm for the CH 2 bound to oxygen. The other two signals correspond to the carbonyl group: one signal at 161 ppm is ascribed to the carbonyl group of KHCO 3 and the other, at 158 ppm, is ascribed to PEC. Correlations of the results of these analyses with mass-gain allowed us to draw conclusions concerning the sequestration of CO 2 in PEC and the exchange of protons and potassium cations between ethanol and potassium carbonate to form KHCO 3 .

In this study, CO 2 was sequestrated by reaction with an equimolar amount of ethanol and potassium carbonate to produce a hybrid organic-inorganic carbonate. This hybrid can be used as a nucleophilic reagent for further reactions.

Reactions with the hybrid carbonate (PEC)

The second step of the carbon dioxide sequestration reaction to produce DEC from PEC was studied by PEC alkylation with iodoethane in ethanol and by PEC alkylation with ethanol in an acidic medium for ethanol dehydration (Figure 1b). The use of an acid rather than an alkyl halide for the second step of the reaction is advantageous as it is cheaper and less toxic and requires less effluent treatment at the end of the reaction. The number of co-reactant moles was at least as high as that for ethanol in the first step, given that ethanol reacted fully with CO 2 and K 2 CO 3 . The amount of acid used was determined on the basis of requirements for neutralization of the bases (KHCO 3 formed and, possibly, K 2 CO 3 ) present in the solid particles obtained in the first step and ethanol dehydration. All reactions (entry 12-20) took place in a biphasic solid-liquid medium. The best results were obtained when iodoethane was used as the co-reagent (Table 3 DEC was synthesized with a low yield (5%) in the presence of iodoethane. Infrared analysis on the solid phase at the end of the reaction with EtI indicated the persistent presence of PEC. This second step of the reaction, using EtI, is probably the limiting step. The presence of diethyl ether (DEE) at the end of the reaction may result from nucleophilic attacks on the protonated alcohol CH 3 -CH 2 -O + H 2 by ethanol [START_REF] Fridgen | Enthalpy Barriers for Asymmetric SN2 Alkyl Cation Transfer Reactions between Neutral and Protonated Alcohols[END_REF]. A phase transfer catalyst was used to promote the reaction between reactants in the solid (PEC) and liquid (ethyl iodide) phases. As expected, the addition of Aliquat 336 to the reaction medium as a phase transfer catalyst increased yield from 5 to 25% (entry 13). The addition of this catalyst favors contact between the two phases, as shown in the reaction mechanism proposed in Figure 7.

Comment citer ce document : Gasc, F., Thiebaud-Roux, S., Mouloungui, Z. ( 2009). Methods for synthesizing diethyl carbonate from ethanol and supercritical carbon dioxide by one-pot or two-step reactions in the presence of potassium carbonate. Journal of Supercritical Fluids, 50 (1), 46-53. , DOI : 10.1016/j.supflu.2009.03.008 10 We tested several Brönsted acids (entries 14-18), but only concentrated strong acids produced DEC, through attacks on the ethyl carbonate anion of protonated ethanol (CH 3 -CH 2 -O + H 2 ). Thus, although PEC is more basic than ethanol, concentrated strong Brönsted acids protonate a small fraction of ethanol to form CH 3 -CH 2 -O + H 2 , which is transformed by an SN 2 reaction with PEC into DEC and H 2 O. The yields of DEC synthesis were low, probably due to competition with PEC protonation for formation of the monoethyl ester of carbonic acid [CH 3 CH 2 OC(O)OH] [START_REF] Olah | Stable carbonium ions. LXIV. Protonated carbonic acid (trihydroxycarbonium ion) and protonated alkyl (aryl) carbonates and hydrogen carbonates, and their cleavage to protonated carbonic acid and carbonium ions. The possible role of protonated carbonic acid in biological carboxylation processes[END_REF]. This molecule reacts with the hydroxy moiety OH -, as described by Dibenedetto et al. [START_REF] Dibenedetto | On the existence of the elusive monomethyl ester of carbonic acid [CH 3 OC(O)OH] at 300 K: 1 H-and 13 C NMR measurements and DFT calculations[END_REF]. This results in carbon dioxide being inserted into potassium bicarbonate (Figure 8). The monoethyl ester of carbonic acid is not easily decarboxylated to form ethanol and CO 2 as this reaction has a very high dissociation energy [START_REF] Dibenedetto | On the existence of the elusive monomethyl ester of carbonic acid [CH 3 OC(O)OH] at 300 K: 1 H-and 13 C NMR measurements and DFT calculations[END_REF]. The use of concentrated strong acids also leads to the production of larger amounts of diethyl ether, due to the SN 2 reaction between ethanol and protonated ethanol (CH 3 -CH 2 -O + H 2 ). Whatever the temperature, this reaction predominates, preventing the further sequestration of carbon dioxide (entries [START_REF] Yamazaki | Chemical reactions of carbon dioxide[END_REF][START_REF] Kizlink | Synthesis of dimethyl carbonate from carbon dioxide and methanol in the presence of organotin compounds[END_REF][START_REF] Kizlink | Preparation of dimethyl carbonate from methanol and carbon dioxide in the presence of Sn(IV) and Ti(IV) alkoxides and metal acetates[END_REF][START_REF] Ballivet-Tkatchenko | The role of distannoxanes in the synthesis of dimethyl carbonate from carbon dioxide[END_REF][START_REF] Tomishige | Catalytic Properties and Structure of Zirconia Catalysts for Direct Synthesis of Dimethyl Carbonate from Methanol and Carbon Dioxide[END_REF][START_REF] Ikeda | Promoting effect of phosphoric acid on zirconia catalysts in selective synthesis of dimethyl carbonate from methanol and carbon dioxide[END_REF].

Finally, this two-step synthesis method leads to the capture of CO 2 in DEC. The first step involves the production of hybrid carbonate (PEC) from non toxic raw materials (ethanol, K 2 CO 3 , CO 2 ) and the second step consists of the reaction of PEC with ethyl iodide. In the presence of PTC, the global yield of PEC is 25%, based on the initial amount of K 2 CO 3 . Unfortunately, the use of a concentrated acid with the hybrid results in competition between potassium bicarbonate synthesis through the release of ethanol and organic carbonate synthesis. However, carbon dioxide is sequestered in both these reactions.

Carbon dioxide sequestration into an organic carbonate

Comment citer ce document : Gasc, F., Thiebaud-Roux, S., Mouloungui, Z. (2009). Methods for synthesizing diethyl carbonate from ethanol and supercritical carbon dioxide by one-pot or two-step reactions in the presence of potassium carbonate. Journal of Supercritical Fluids, 50 (1), 46-53. , DOI : 10.1016/j.supflu.2009.03.008 11 Carbon dioxide can be sequestered in the form of organic carbonate via the one-pot synthesis of DEC. The synthesis of dimethyl carbonate from an alcohol (methanol) and an alkyl halide (iodomethane) has been studied by several authors. The optimum conditions for the synthesis of DMC are reported in Table 4. Only Fujita et al. [START_REF] S.-I. Fujita | Synthesis of dimethyl carbonate from carbon dioxide and methanol in the presence of methyl iodide and base catalysts under mild conditions: effect of reaction conditions and reaction mechanism[END_REF] used these conditions for DEC production. Given the low yields obtained, we have modified some experimental parameters (entries 5 and 6).

Entry

Reactant The use of equimolar amounts of potassium carbonate and iodoethane increased DEC yield by a factor of six (Table 4), indicating that K 2 CO 3 is a reactant and not a catalyst. Its reaction with ethanol generates a deactivated base: KHCO 3 .K 2 CO 3 can be regenerated by reaction with potassium iodide (figure 10d) or downgraded in the presence of hydrogen iodide. Ethyl iodide is a reactant and not a catalyst, as proposed by Fang et al. [START_REF] Fang | Direct synthesis of dimethyl carbonate from carbon dioxide and methanol catalyzed by base[END_REF] and by Cai et al. [START_REF] Cai | Synthesis of Dimethyl Carbonate from Methanol and Carbon dioxide using Potassium Methoxide as Catalyst under Mild Conditions[END_REF]. Ethyl iodide is soluble in ethanol, which is considered to be the solvent. Ethyl iodide should be less reactive with the organic-inorganic carbonate hybrid than methyl iodide, according to the SN 2 mechanism. There is greater steric hindrance with ethyl iodide and the electrophilic character of the alkyl group of ethyl iodide dissolved in ethanol is less marked than that of methyl iodide dissolved in methanol, due to the lower dissociation power of ethanol than of methanol [START_REF] Murto | Nucleophilic reactivity of alkoxide ions towards 2,4-dinitrofluorobenzene and the acidity of alcohols[END_REF]. However, the mixture of ethanol and ethyl iodide is swollen [START_REF] West | In Situ Formation of Alkylcarbonic Acids with CO 2[END_REF] by the CO 2 , increasing the reactivity of the different species, for example, the reactivity of EtOH with CO 2 and that of PEC with EtI. The reaction medium is triphasic at the start of the reaction. It consists of supercritical CO 2 , a liquid mixture of ethanol and ethyl iodide. As observed by Fujita et al. [START_REF] S.-I. Fujita | Synthesis of dimethyl carbonate from carbon dioxide and methanol in the presence of methyl iodide and base catalysts under mild conditions: effect of reaction conditions and reaction mechanism[END_REF], CO 2 is then solubilized in the liquid phase and swells the volumes of both reactants. This increases reactivity.

Kinetic studies (Figure 9) showed that the production of DEC and DEE increased rapidly with time over the first two hours, reaching a maximum after five hours of reaction. An analysis of the two phases obtained at the end of the reaction provided insight into the reasons for the limited amount of diethyl carbonate produced (less than 50%). These two phases had the following characteristics:

1. The orange color of the liquid phase recovered after 5 hours of reaction was correlated with its UV absorbance, indicating the presence of iodine. Iodine is released when ethyl iodide consumption exceeds the sum of the amounts of DEC and DEE produced (see Table 5).

There may be two reasons for this excess consumption. Ethyl iodide may be broken down by heat to generate hydrogen iodide (HI) and then iodine [START_REF] Maccoll | Heterolysis and the Pyrolysis of Alkyl Halides in the Gas Phase[END_REF], or it may be dehydrohalogenated by EtO -K + as a strong base, according to an E 2 mechanism (Figure 10b).

2. The infrared spectrum of the solid obtained after 5 hours reaction was the same as that obtained at the end of the first step. Thus, the PEC produced did not react completely with ethyl iodide to form DEC.

Other experiments were performed to increase yields and to study the proposed reaction mechanism further (Table 5). 5) show that the carbonyl group introduced during the formation of DEC comes from carbon dioxide and not from potassium carbonate. Without CO 2 , the main product formed was diethyl ether, rather than DEC, as indicated in the proposed mechanism (Figure 10). The very small amount of DEC produced in this case indicates that the carbonate ion of K 2 CO 3 is involved in the reaction, but does not make a significant contribution. Without ethanol, only a very small amount of DEC can be produced by the O-alkylation of potassium carbonate with two equivalents of ethyl iodide in supercritical carbon dioxide (entry 8). The basicity of the catalyst affects the reaction process rather than the carbonate ions [START_REF] Fang | Direct synthesis of dimethyl carbonate from carbon dioxide and methanol catalyzed by base[END_REF]. Potassium carbonate basicity leads to the formation of ethanolate (EtO -), which carries out nucleophilic attack on CO 2 , generating PEC. The presence of the ethanolate at the start of the reaction (entry 9, Table 5) led to the formation of larger amounts of ether, due to competition between the parallel reactions (Figure 10b-d). More diethyl ether was produced because the concentration of ethanolate is higher in the reaction medium when a solution of potassium ethanolate in ethanol is used at the start of the reaction. However, in the experimental conditions used for entry 6 (Table 5), the ethanolate reacted with carbon dioxide as soon as it was produced. As demonstrated in the study of the first step of the reaction, PEC production was complete after 20 minutes of reaction at 100°C.

Entry

The mechanism shown in Figure 10 was confirmed by the production of a mixed organic carbonate (ethyl methyl carbonate, EMC) with methanol and ethyl iodide (containing two different alkyl chains).

As expected from the results of the study of the second step, the use of a phase transfer catalyst resulted in a small increase in DEC production, with no change in the amount of DEE produced (entry 11 and 6). Aliquat 336 was less effective in the one-pot reaction than in the two-step reaction (Figure 7), because its role in the one-pot reaction is secondary, with interaction phenomena due to the swelling of the reactants playing a much more important role. Similar quantities of DEE were produced in the presence and absence of Aliquat 336. This CTP did not affect the reaction of ethanolate with ethanol, because this reaction occurred in a homogeneous liquid medium.

Comparison of the yields of DEC produced when EtI was used as a co-reactant in the one-pot and two-step reactions showed that the one-pot synthesis reaction gave the best results. Thus, performing

Comment citer ce document : Gasc, F., Thiebaud-Roux, S., Mouloungui, Z. (2009). Methods for synthesizing diethyl carbonate from ethanol and supercritical carbon dioxide by one-pot or two-step reactions in the presence of potassium carbonate. Journal of Supercritical Fluids, 50 (1), 46-53. , DOI : 10.1016/j.supflu.2009.03.008 14 the entire reaction in a supercritical CO 2 medium accelerates the rate of the DEC formation. The supercritical CO 2 solubilized in the liquid phase containing ethanol and ethyl iodide favors interactions between EtOH and CO 2 and between PEC and ethyl iodide by swelling the reactants. It therefore acts as a compatibilizing agent and a swelling solvent.

Conclusion

We studied the synthesis of diethyl carbonate (DEC) from ethanol and supercritical CO 2 in the presence of potassium carbonate and a co-reagent (ethyl iodide or concentrated acidic alcoholic solution) by two processes: a direct "one-pot" procedure and an indirect two-step procedure. The twostep procedure led to the isolation of a reaction intermediate, potassium ethyl carbonate (PEC), which was characterized by infrared spectrometry and 13 C NMR. This hybrid carbonate was then used as a nucleophilic agent for the second step of the reaction, the O-alkylation of PEC to generate DEC.

Studies of the synthesis of DEC with EtI showed that the second step was limiting for the synthesis of DEC. We also identified other reactions competing with the O-alkylation reaction of PEC (SN 2 ), such as the etherification of ethanolate (produced by the reaction of ethanol with the base K 2 CO 3 ) and the EtI dehydrohalogenation reaction, according to an E 2 elimination mechanism.

The reaction of PEC with a concentrated alcoholic solution of a strong acid as a co-reactant made it possible to prepare DEC for the first time. Admittedly, the yields obtained with this method were low, but this method is more compatible with the principles of green chemistry. It will be used in future studies based on superacids and polyoxygenated reagents such as orthoesters or ketals.

The study of the effect of the operating conditions for each of the procedures on the yield and selectivity of the reaction (diethyl carbonate versus diethyl ether) showed that the one-pot procedure favored the synthesis of DEC. Indeed, the O-alkylation of PEC is favored when the reaction occurs in supercritical carbon dioxide, because the reactants are in the swelling solvent, CO 2 . The use of a PTC increased the production yield of DEC without increasing the amount of DEE produced. However, the one-pot synthesis procedure is too inefficient for industrial use. Further work, involving an exhaustive study of the reaction with a PTC, is required to increase reaction yields.

In this study, carbon dioxide was captured in two forms: an organic carbonate and an organicinorganic hybrid that could be reused without purification for the synthesis of organic carbonates.

3 Figure 1 :

 31 Figure 1: DEC synthesis from ethanol, carbon dioxide, potassium carbonate and a co-reactant

Figure 2 :

 2 Figure 2: Experimental setup for reactions in supercritical CO 2

Figure 3 :

 3 Figure 3: FTIR spectra of othe btained intermediate solid, KHCO 3 and K 2 CO 3 .

7 Figure 4 :

 74 Figure 4: Potassium carbonate hydration form [22]

Figure 5 :

 5 Figure 5: Resonance in potassium ethyl carbonate

Figure 6 :

 6 Figure 6: CPMAS 13 C NMR spectra of K 2 CO 3 (a), KHCO 3 (b) and the solid phase (c)

Figure 7 :

 7 Figure 7: Mechanism of phase transfer catalysis at the solid/liquid interface

Figure 8 :

 8 Figure 8: Reaction of PEC in an acidic medium

  Comment citer ce document : Gasc, F., Thiebaud-Roux, S., Mouloungui, Z. (2009). Methods for synthesizing diethyl carbonate from ethanol and supercritical carbon dioxide by one-pot or two-step reactions in the presence of potassium carbonate. Journal of Supercritical Fluids, 50 (1), 46-53. , DOI : 10.1016/j.supflu.2009.03.008 12 DEE: diethyl ether, DEC: diethyl carbonate

Figure 9 :

 9 Figure 9: Concentration-time profiles for DEC (straight line) and DEE (dotted line)

Figure 10 :

 10 Figure 10: Proposed reaction mechanism: competition between carbonate and ether formation

Table 2 : Main IR bands of the intermediate solid obtained, commercial KHCO 3 and commercial K 2 CO 3

 2

			Wavenumber (cm -1 )
	Vibration	K 2 CO 3	KHCO 3	Intermediate solid product
	ν O-H	3218	3377	3417
	ν as C-H			3073
	ν as C-H			2971
	ν O-H + ν s C-H		2726	2730
	ν O-H + ν s C-H		2627	2634
	ν as C=O		1855	1853
	ν as C=O		1693	1698
	ν as C=O			1681
	ν as C=O	1655	1649	1665
	ν as C=O	1638	1631	1632
	ν s C-O	1446	1402	1406
	ν

s C(-O) 2 -+ δ C-H 1378 1369 1371 Comment citer ce document : Gasc, F., Thiebaud-Roux, S., Mouloungui, Z. (2009). Methods for synthesizing diethyl carbonate from ethanol and supercritical carbon dioxide by one-pot or two-step reactions in the presence of potassium carbonate. Journal of Supercritical Fluids, 50 (1), 46-53. , DOI : 10.1016/j.supflu.2009.03.008

Table 3 : Effect of the co-reactant on the formation of DEC from the PEC synthesized (Table 1, entry 8)

 3 ).

	Entry	Reactant	n EtOH (mmol)	n meq H+ or n EtI (mmol)	T (°C)	t (h)	n DEC (mmol)	n DEE (mmol)	% Yield DEC (K 2 CO 3 basis)
	12	EtI / EtOH	1800	206	110	4	7.1	3.5	5
	13	EtI / EtOH / PTC a 1800	206	110	4	22.8	3.2	25
	14	PTSA b	1800	45	110	3	0.4	0.3	0.3
	15 16 17 18	c c d H 2 SO 4 H 2 SO 4 H 3 PO 4 HCl e	1800 1800 1800 1800	400 3600 4000 3300	110 110 110 110	3 3 3 3	0.1 3.6 0.2 2.8	0.2 42.0 0 3.9	0.1 2.7 0.1 2
	19 20	H 2 SO 4 HCl e	c	1800 1800	3600 3300	-80 -80	3 3	3.1 0	17.4 2.5	2.3 0

DEE: diethyl ether, DEC: diethyl carbonate, a PTC: 14 mmol Aliquat 336 (trioctylmethyl chloride), b PTSA: p-toluene sulfonic acid (≥ 98.5%), c H 2 SO 4 (≥ 98%), d H 3 PO 4 (≥ 85%), e HCl (≥ 37%).

Table 4 : Effect of the experimental conditions on the yield of DAC

 4

			n ROH (mmol)	n RX (mmol)	Molar ratio (ROH/RX/K 2 CO 3 )	T (°C) P (bar) t (h)	% Yield DAC (RX basis)
	1 [23] MeOH/MeI	192	24	64/8/1	100	>> 50 (n.a. a )	2	50
	2 [27] MeOH/MeI	200	24	67/8/1	70	80	4	17
	3 [27]	EtOH/EtI	200	24	67/8/1	70	80	4	0.6
	4 [24] MeOH/MeI	3730	160	64/3/1	90	70	n.a. a	53
	5	EtOH/EtI	846	108	64/8/1	110	80	5	8
	6	EtOH/EtI	846	108	8/1/1	110	80	5	46

ROH: alcohol, RX: alkyl halide, DAC: dialkyl carbonate, a n.a.: not available

  : diethyl ether, DEC: diethyl carbonate, a without CO 2 , b 24% w/w EtOK in EtOH, c Aliquat 336, d not detected, e relative % of ethyl methyl carbonate (EMC).

		Reactant	Molar ratio	T (°C)	P (bar)	t (h)	n DEE (mmol)	n DEC (mmol)	Conversion EtI (%)	% Yield DEC (EtI basis)
	6 7 a 8 9 b	EtOH / EtI / K 2 CO 3 EtOH / EtI / K 2 CO 3 EtI / K 2 CO 3 EtOH / EtOK / EtI	8/1/1 8/1/1 2/1 6/1/1	110 110 110 110	80 0 80 80	5 5 5 5	3.0 15 n.d. d 8.9	50 0.1 0.4 32	97.6 86.7 82.5 95.5	46 0.1 0.2 30
	10 11 c EtOH / EtI / K 2 CO 3 / PTC 8/1/1/0.2 110 8/1/1 110 MeOH / EtI / K 2 CO 3	80 80	5 5	n.d. d 2.6	0.1 55	98.7 99.5	0.1 (ca. 14) e 51

DEE

Table 5 : Effect of operating conditions on yield and selectivity of the reaction

 5

Comment citer ce document : Gasc, F., Thiebaud-Roux, S., Mouloungui, Z. (2009). Methods for synthesizing diethyl carbonate from ethanol and supercritical carbon dioxide by one-pot or two-step reactions in the presence of potassium carbonate. Journal of Supercritical Fluids, 50 (1), 46-53. , DOI : 10.1016/j.supflu.2009.03.008 13 Entries 7-8 (Table


Jahangir Khajehali

Thomas Van Leeuwen
email: thomas.vanleeuwen@ugent.be

Maria Grispou

Evangelia Morou

Haoues Alout

Mylene Weill

Luc Tirry

John Vontas

Anastasia Tsagkarakou
email: tsagkarakou@nagref.gr

Haou És Alout

Myl Ène Weill

Acetylcholinesterase point mutations in European strains of Tetranychus urticae

Keywords: Tetranychus urticae, spider mite, organophosphate resistance, acetylcholinesterase mutations, ace-1, resistance mechanisms

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Organophosphates (OPs) are among the first chemical groups used to control the two-spotted spider mite Tetranychus urticae Koch, an economically important agricultural pest with a wide range of host plants and geographic distribution. OP insecticides act by inhibiting acetylcholinesterase (EC 3.1.1.7, AChE), a key enzyme in the nervous system of both vertebrates and invertebrates. Since the first reported cases of OP resistance in the late 1940s, [START_REF] Helle | Genetics of resistance to organophosphorus compounds and its relation to diapause in Tetranychus urticae Koch (Acari)[END_REF] T. urticae has developed resistance towards almost every acaricide used for its control. Within the OP family, cross-resistance is a general rule in T. urticae. 2 -8 This can result from enhanced detoxification [START_REF] Van Leeuwen | Comparative acaricide susceptibility and detoxifying enzyme activities in field-collected resistant and susceptible strains of Tetranychus urticae[END_REF][START_REF] Voss | Resistance to organophosphorus compounds in the two-spotted spider mite: two different mechanisms of resistance[END_REF] and/or target-site insensitivity. The insensitivity of T. urticae AChE towards OPs was first reported in 1964, 9 suggesting the presence of some modifications at the active site of the enzyme. [START_REF] Voss | Resistance to organophosphorus compounds in the two-spotted spider mite: two different mechanisms of resistance[END_REF] Since then, AChE insensitivity to OPs has been demonstrated in various strains of T. urticae from the Netherlands, Germany, the USA, New Zealand, Israel, Egypt, Greece and Japan. 5,10 -15 Many insects and ticks have at least two genes, termed ace-1 and ace-2, encoding for AChE1 and AChE2 respectively. These genes correspond to the Drosophila paralogous and orthologous genes. [START_REF] Weill | A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila[END_REF] In the dipteran order of the Cyclorrhapha (true flies), only the ace-2 gene is present. [START_REF] Huchard | Acetylcholinesterase genes within the diptera: takeover and loss in true flies[END_REF] In insects that have both genes, the main synaptic function is encoded by ace-1. [START_REF] Weill | A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila[END_REF][START_REF] Weill | Insecticide resistance in mosquito vectors[END_REF] In this gene, seven point mutations associated with resistance to OPs and CARBs have been reported, resulting in the following amino acid substitutions: G119S, A201S, F290V, G227A, S331F and F331W/C; 15,18 -25 numbering based on Torpedo californica AChE nomenclature. [START_REF] Massoulié | Recommendations for Nomenclature in Cholinesterases, Multidisciplinary Approaches to Cholinesterase Functions[END_REF] In true flies, one or a combination of mutations in the ace-2 gene, including substitutions F78S, I129V/T, V150L, G227A/V, F290Y, G328A and G396S, conferred in vitro insensitivity to OP insecticides. 27 -31 Although biochemical evidence of AChE insensitivity in T. urticae was already revealed in 1964 by Smissaert,9 the exploration of the molecular basis of AChE1 insensitivity in Tetranychidae started only quite recently. Among phytophagous mites, ace-1 gene sequences encoding for the enzyme AChE1 have been determined in two tetranychid species, T. urticae and T. kanzawai. [START_REF] Anazawa | Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant twospotted spider mite, Tetranychus urticae[END_REF][START_REF] Aiki | Amino acid substitution in Ace paralogous acetylcholinesterase accompanied by organophosphate resistance in the spider mite Tetranychus kanzawai[END_REF] The comparison of the AChE1 amino acid sequence from susceptible and OP-resistant T. urticae from Japan revealed the presence of an F331C substitution. [START_REF] Anazawa | Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant twospotted spider mite, Tetranychus urticae[END_REF] In OP-resistant T. kanzawai Kishida, a tryptophane was detected at position 331 (W331). [START_REF] Aiki | Amino acid substitution in Ace paralogous acetylcholinesterase accompanied by organophosphate resistance in the spider mite Tetranychus kanzawai[END_REF] In the present study, a more thorough examination was made of the presence of mutations in the AChE1 of several OP-and CARB-resistant T. urticae strains by combining toxicological, biochemical and molecular data.

MATERIALS AND METHODS

Strain characteristics

This study characterised three laboratory T. urticae strains commonly used as reference strains (reference strains SAMB, GSS and LS-VL) and three resistant strains maintained in the laboratory under continuous OP selection (OP strains WI, MR-VL and ATHRos-Pm). SAMB and GSS had both been maintained in the laboratory without treatment since 1965. SAMB was initially collected on Sambucus nigra (L.) in the Netherlands and obtained from the Centre de Biologie et de Gestion des Populations, Montpellier, France. GSS was obtained from Bayer CropScience (Monheim, Germany) and was originally collected from an unknown host in Germany. LS-VL was collected in 2000 from roses in a garden near Ghent, Belgium, [START_REF] Van Leeuwen | Genetic analysis and crossresistance spectrum of a laboratory-selected chlorfenapyr-resistant strain of two-spotted spider mite (Acari: Tetranychidae)[END_REF] and since then had been maintained in the laboratory without treatment.

The historical German resistant strain WI has a well-studied target-site resistance to OPs and CARBs. [START_REF] Stumpf | Resistance to organophosphates and biochemical genotyping of acetylcholinesterases in Tetranychus urticae (Acari: Tetranychidae)[END_REF] It was obtained from Bayer CropScience where it had been maintained in the laboratory under biannual selection with the OP oxydemeton-methyl since 1954. [START_REF] Nauen | Acaricide toxicity and resistance in larvae of different strains of Tetranychus urticae and Panonychus ulmi (Acari: Tetranychidae)[END_REF] Biochemical characterisation had shown that the main resistance mechanism was an altered acetylcholinesterase with a 110-fold and 340-fold lower sensitivity to inhibition by chlorpyrifos-oxon and ethyl paraoxon respectively. [START_REF] Stumpf | Resistance to organophosphates and biochemical genotyping of acetylcholinesterases in Tetranychus urticae (Acari: Tetranychidae)[END_REF] The OP strains MR-VL (from Belgium) and ATHRos-Pm (from Greece) were recently collected from fields on which failure of all insecticide treatments, including OPs, had been claimed by the farmers. MR-VL was collected in 2003 from a greenhouse nursery where poplar cuttings, beans and ornamentals were grown, while ATHRos-Pm was collected in 2007 on roses in a greenhouse near Athens. Both strains had developed resistance to many acaricides currently used to control spider mites. [START_REF] Van Leeuwen | Comparative acaricide susceptibility and detoxifying enzyme activities in field-collected resistant and susceptible strains of Tetranychus urticae[END_REF][START_REF] Tsagkarakou | Identification of pyrethroid resistance mutations in the para sodium channel of the two-spotted spider mite Tetranychus urticae (Acari : Tetranychidae)[END_REF] ATHRos-Pm was maintained in the laboratory under pirimiphosmethyl selection every two generations with concentrations causing ∼80% mortality before its characterisation after eight selections.

All mites were reared on detached bean leaves that were deposited on moist cotton wool to prevent contamination and escape. Rearing conditions were 25 ± 2 • C, 70 ± 5% RH and a 16 : 8 h light : dark photoperiod. Mites were transferred to new bean leaves weekly. Under these conditions, a new generation was obtained every 2 weeks.

Bioassays

Commercial formulations of pirimiphos-methyl 500 g L -1 EC (Actellic; Syngenta, UK), dimethoate 400 g L -1 EC (Dimethoate; Lapafarm, Greece), chlorpyrifos 480 g L -1 EC (Dursban; Dow Agrosciences, USA) and methomyl 200 g L -1 SL (Alpha, Greece) were used. The treatments were conducted on adult females placed on detached bean leaves with a precision Potter spray tower [START_REF] Potter | An improved apparatus for applying direct sprays and surface films with data on the electrostatic charge on atomized spray fluids[END_REF] (Burkard, Rickmansworth, Hertfordshire, UK), as described by Tsagkarakou et al. [START_REF] Tsagkarakou | Identification of pyrethroid resistance mutations in the para sodium channel of the two-spotted spider mite Tetranychus urticae (Acari : Tetranychidae)[END_REF] LC 50 values and 95% confidence limits were calculated by the log-probit program 37 based on Finney. [START_REF] Finney | Probit Analysis[END_REF] Resistance ratios at the LC 50 (RR 50 ) were considered to be significantly different from 1 when their 95% confidence limits did not include this value.

Acetylcholinesterase activity and inhibition studies

For determining AChE1 activity, 1000 adult females were mass homogenised in 1 mL extraction buffer containing 10 mM Tris, pH 7.0, 1 M NaCl and 4 g L -1 Triton X-100. The supernatant obtained after centrifugation (10000 × g, 4 • C, 10 min) was used as the enzyme source. The reaction was conducted in 1 mL substrate-reagent solution containing 30-50 µg of protein and 5,5 -dithio-bis(2-nitrobenzoic acid) (DTNB) and acetylthiocholine (AcSCh) each in a final concentration of 0.5 mM. The mean activity values were compared between strains by one-way analysis of variance (ANOVA) using SPSS statistical software version 13.0 (LSTM, UK). Residual AChE1 activities were measured with increasing concentrations of the analytical-grade inhibitors chlorpyrifos-oxon (varying between 10 -9 and 10 -3 M), paraoxon (varying between 10 -9 and 10 -3 M), carbaryl (varying between 10 -10 and 10 -3 M), pirimiphos-methyl (varying between 10 -7 and 10 -3 M) and omethoate (varying between 10 -7 and 10 -3 M). Briefly, the enzyme source was incubated for 10 min with the inhibitor solution at a given concentration before adding the substrate-reagent solution. Residual activity was estimated by kinetically measuring the variation in optical density at 412 nm with a spectrophotometer (M2e; Molecular Devices, UK). Blanks without homogenate or substrate were used to correct for nonenzymatic activity. Enzyme inhibition was expressed as the mean percentage of activity remaining at different inhibitor concentrations, and the inhibitor concentration inducing 50% inhibition (IC 50 ) was determined using the Microcal Origin 6.0 data analysis program.

Extraction of DNA and RNA, cDNA synthesis, cloning and sequencing

Total DNA (DNA) was extracted from mass homogenates as described by Van Leeuwen et al. [START_REF] Van Leeuwen | Mitochondrial heteroplasmy and the evolution of insecticide resistance: non-Mendelian inheritance in action[END_REF] Total RNA was extracted from mass homogenates of T. urticae mites using TRI reagent (Sigma-Aldrich, Belgium) according to the manufacturers' instructions. The RNA was treated with DNAse RQ1 (Promega, Madison, WI) to remove genomic DNA. A quantity of 2 µg total RNA for each sample was reverse transcribed into cDNA by using the High Fidelity cDNA Synthesis Kit (Roche, Belgium) and random hexamer primers. Primers (Table 1) were designed on the basis of the published sequence of T. urticae AChE1 (AY188448). For cDNA amplification, a long PCR was performed using Acetrur F and Acetrur R primers (Expand Long Range Kit; Roche, Belgium), according to the manufacturer's instructions. Fragments of 2.5 kb were purified with QIAEX II (Qiagen), cloned into the pGEM-T vector (Promega) and sequenced with primer walking. To ensure allele representations, three clones were sequenced for each strain. Plasmids were sequenced by Agowa Sequencing Service (Berlin). Sequence data were analysed using BioEdit 7.0.1. 40

Three-dimensional modelling

The three-dimensional (3D) structure of T. urticae AChE1 was created by automated homology modelling, as described by Weill et al. [START_REF] Weill | The unique mutation in ace-1 giving high insecticide resistance is easily detectable in mosquito vectors[END_REF] The structural templates used were AChE from Torpedo californica (PDB: 1EA5) [START_REF] Sussman | Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein[END_REF] and Drosophila melanogaster (PDB: 1DX4). [START_REF] Harel | Three-dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors[END_REF] The alpha-carbon skeleton of the modelled 3D structure of AChE1 was superimposed on that of the AChE of T. californica. RMS deviation is 1.1 Å on 532 carbon atoms (from Dali pairwise comparison webserver http://ekhidna.biocenter.helsinki.fi/dali lite/start).

Diagnostic PCR-RFLP for W331

Two primers were designed (AcheDISCF2, AcheDISCR2) (Table 1) around position 331 that amplified a 137 bp fragment using genomic DNA as template. Amplifications from approximately 40 ng gDNA were performed in 1 × Taq reaction buffer (Invitrogen) with a final concentration of 1.5 mM MgCl 2 and 0.5 µM of each primer, with the following cycling conditions: 94 • C for 3 min, 35 cycles of 94 • C for 15 s, 55 • C for 30 s, 72 • C for 40 s, followed by 72 • C for 2 min. A restriction digest using 10 µL PCR product was performed in a total volume of 30 µL using Bgl I according to the manufacturer's instructions (Fermentas). Approximately 20 µL was loaded onto a 3% agarose gel, and after 1.5 h at 50 V the separated restriction fragments were visualised by ethidium bromide staining.

RESULTS

Resistance characteristics of the Tetranychus urticae strains

Among the reference strains, variable levels of sensitivity were found (Table 2). LS-VL proved to be the most susceptible to all insecticides tested: LC 50 of pirimiphos-methyl, chlorpyrifos, methomyl and dimethoate was 16, 11, 28 and 8 mg L -1 respectively. Therefore, this strain was used as a susceptible reference to calculate the RR 50 of the other strains. SAMB exhibited a 31-and 29-fold resistance to chlorpyriphos and dimethoate respectively. GSS was 21-and 1411-fold more resistant to pirimiphos-methyl and dimethoate respectively. The OP strains ATHRos-Pm, MR-VL and WI were resistant to all insecticides tested, with RR 50 of at least 8.4 (Table 2). Among the three OP strains, the highest levels of resistance to pirimiphosmethyl (RR 50 = 43), dimethoate (RR 50 = 4164) and methomyl (RR 50 = 333) were exhibited by ATHRos-Pm, and the weaker levels by WI (RR 50 = 8.9, 137 and 8.4 to pirimiphos-methyl, dimethoate and methomyl respectively). For chlorpyrifos, the resistance ratio was higher in the WI and MR-VL strains (RR 50 = 421 and 586 respectively).

Biochemical characterisation of the AChE1 enzyme

AChE1 activity was highest in the SAMB strain (63 ± 11 mOD min -1 mg -1 protein), followed by the MR-VL strain (37 ± 1 mOD min -1 mg -1 protein) (P < 0.01) (Table 3). The four other strains showed lower activities that did not differ significantly from each other.

As shown by the IC 50 values (Table 3), AChE1 from the OP strains WI, MR-VL and ATHRos-Pm were much less sensitive to inhibition by pirimiphos-methyl, chlorpyrifos-oxon, paraoxon and carbaryl than the AChE1 from the three reference strains, and WI and MR-VL were less sensitive to inhibition by omethoate than LS-VL and SAMB (omethoate was not tested for AChE1 from GSS and ATHRos-Pm strains, and no comparison is possible). AChE1 from SAMB and LS-VL were more sensitive to inhibition by chlorpyrifosoxon but less sensitive to inhibition by carbaryl than the AChE1 from GSS. For paraoxon, IC 50 values for SAMB and GSS were very similar and lower than that of LS-VL. Among the OP strains, IC 50 values were similar for the OPs and the CARBs tested.

Comparison of the AChE1 sequence of the reference laboratory and the OP selected strains

Using specific primers and long-PCR strategy, the cDNA sequence of a 2.5 kb fragment was determined in the six strains (GenBank accessions GQ461336 to GQ461353). This fragment contains an open reading frame encoding for an AChE1 precursor of 687 amino acid residues. Three cDNA clones were sequenced from each strain, resulting in a total of 18 clones, which revealed 88 variable nucleotide sites including inter-and intrastrain polymorphisms. Forty-four of these resulted in amino acid substitutions. By comparing these substitutions in the AChE1 primary sequence from the different strains, six mutations were identified in the OP strains that were not present in the reference strains. Only four of them were present in the mature protein (numbering based on Torpedo californica AChE nomenclature) [START_REF] Massoulié | Recommendations for Nomenclature in Cholinesterases, Multidisciplinary Approaches to Cholinesterase Functions[END_REF] : (1) at position 201, an alanine residue (GCT) is replaced by a serine (TCT) (A201S) in the three clones of the OP strain ATHRos-Pm; (2) at position 280, a threonine residue (ACA) is replaced by an alanine (GCA) (T280A) in all three OP strains, except for one clone of the ATHRos-Pm strain;

(3) at position 328, a glycine (GGA) is replaced by an alanine (GCA) (G328A) in the ATHRos-Pm and MR-VL strains; (4) at position 331, a phenylalanine (TTT) is replaced by a tryptophan (TGG) (F331W) in all clones of the three OP strains. Interestingly, the F331 residue is replaced by a tyrosine (TAT) (F331Y) in two clones of reference strains (one in the SAMB and one in the GSS strain). Moreover, two amino acid substitutions, a valine (GTT) to isoleucine (ATT) at position 7 (V7I) and a glycine (GGC) to serine (AGC) at position 119 (G119S), were found specifically in the reference strain SAMB (Table 4).

Three-dimensional modelling

The positions of the mutations in the structural model of T. urticae AChE1 are shown in Fig. 1. The active site of cholinesterases is subdivided into functional groups important for the hydrolysing process: the catalytic triad (S200, E327 and H440), the peripheral anionic site (D72, Y121, W279 and Y334), the choline-binding site (W84, Y130, Y330 and F331), the acyl-binding pocket (F288 and F290) and the oxyanion hole (G118, G119 and A201). 42,44 -47 Among the two substitutions specifically found in the susceptible strain SAMB, the first one, V7I, is located in the extremity of the N-terminal of the enzyme structure and may not influence the catalytic properties of AChE1. The second substitution, G119S, is located in the active site that belongs to the oxyanion hole and affects an important residue close to the catalytic serine residue (S200); thus, it may modify the catalytic properties of the enzyme.

The T280A mutation was found in all OP strains, and the structural model reveals its position on the surface of the enzyme near the gorge entrance. However, the T280 residue is not involved in the catalytic process and was never shown to confer AChE insensitivity (neither AChE1 nor AChE2). Hence, this mutation might be neutral. In contrast, the F331W mutation that is also found in all OP strains is at a residue that is conserved among AChE1 from both vertebrates and invertebrates, and is a component of the choline-binding site involved in the catalytic process. Thus, it is probably responsible, at least partially, for the modification of the catalytic properties of T. urticae AChE1 in the three OP strains. In addition to the F331W, a G328A substitution was found in ATHRos-Pm and in MR-VL strains. This position is not located in the active site but is in the same α-helix as the 331 position. Therefore, this substitution may indirectly influence the catalytic process. In the ATHRos-Pm strain only, besides the above three substitutions (T280A, G328A and F331W), the A201S substitution was also detected. This substitution is close to the catalytic serine (S200) and therefore may modify some of the biochemical properties of the enzyme.

Diagnostic PCR-RFLP for W331

The authors have developed a PCR-RFLP assay, based on primerinduced mutagenesis, that directly revealed the presence of W at codon 331. Three (CAA to GGC in MR-VL and ATHRos-Pm) or two (GAA to GGC in LS-VL, GSS, SAMB and WI) mutations were 439)

LS-VL-1 (GQ461336) V (GTT) G (GGC) A (GCT) T (ACA) G (GGA) F (TTT) LS-VL-2 (GQ461337) V G A T G F LS-VL-3 (GQ461338) V G A T G F SAMB-1 (GQ461342) I (ATT) S (AGC) A T G Y (TAT) SAMB-2 (GQ461343) I S A T G F SAMB-3 (GQ461344) I S A T G F GSS-1 (GQ461348) V G A T G F GSS-2 (GQ461349) V G A T G Y GSS-3 (GQ461350) V G A T G F WI-1 (GQ4613451) V G A A(GCA) G W (TGG) WI-2 (GQ461352) V G A A G W WI-3 (GQ461353) V G A A G W ATHRos-Pm-1 (GQ461345) V G S (TCT) A A (GCA) W ATHRos-Pm-2 (GQ461346) V G S A A W ATHRos-Pm-3 (GQ461347) V G S T A W MR-VL-1 (GQ461339) V G A A A W MR-VL-2 (GQ461340) V G A A A W MR-VL-3 (GQ461341) V G A A A W
introduced by the forward primer by site-directed mutagenesis, resulting in the generation of a Bgl I restriction site (5 -GCCNNNNNGGC) specifically in the PCR product of the resistant allele carrying W331. Digestion of the PCR products (137 bp) with Bgl I consequently allowed for discrimination between the resistance-associated W331 and the F or Y 331 alleles (Fig. 2). The PCR-RFLP assay with male (haploid) T. urticae displays two different patterns: one with two fragments 112 bp and 25 bp in 331 W males and one with one fragment (137 bp), the original PCR product, in F 331 or Y 331 males (Fig. 2). When pooled DNA from F331 and W331 males was used as template, a third pattern with three fragments (137 bp, 112 bp and 25 bp) was obtained, suggesting that the assay can be used to detect heterozygous females (Fig. 2). The fragment of 25 bp is not detected, as it is too small to be visualised by electrophoresis in a routine agarose gel. Note that sequence conservation and the presence of other resistant alleles should be confirmed by sequencing in any future studies of T. urticae from other geographic origins before this diagnostic test is used, as it will not identify the C331 allele previously suggested to be associated with OP resistance in a Japanese T. urticae strain. [START_REF] Anazawa | Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant twospotted spider mite, Tetranychus urticae[END_REF]

DISCUSSION

Since the development of OP resistance in T. urticae in the late 1960s, the use of these compounds to control spider mites has largely been abandoned in many parts of the world. However, OPs and CARBs, which account for more than 35% of the total global insecticide sales, [START_REF] Mccaffery | The insecticide resistance action committee (IRAC): public responsibility and enlightened industrial self-interest[END_REF] are still among the most widely used insecticides to control a broad range of arthropod pests of agricultural importance. This keeps spider mites like T.urticae under continuous OP selection pressure in many open environment and greenhouse crops, and OP-resistant spider mite populations can resurge after OP use. This is why OP resistance in T. urticae remains a not to be neglected economic factor. In addition, it was not long ago shown how OPs can antagonise the activity of the recently developed acaricide bifenazate, especially in strains resistant to organophosphates. This is due to an inhibition of general esterase activity by OPs, which is needed to metabolise bifenazate to its active component. [START_REF] Van Leeuwen | Organophosphate insecticides and acaricides antagonise bifenazate toxicity through esterase inhibition in Tetranychus urticae[END_REF][START_REF] Khajehali | Suceptibility of an organophosphate-resistant strain of the two-spotted spider mite (Tetranychus urticae) to mixtures of bifenazate with organophosphate and carbamate insecticides[END_REF] In an attempt to investigate the molecular basis of OP resistance and its relationship to the observed phenotype, a study was made of the characteristics of resistance in three reference laboratory strains (LS-VL, SAMB and GSS) and three strains of T. urticae that were continuously kept under OP selection: a strain that historically developed OP resistance in the 1960s (WI) and two recently collected field strains (ATHRos-Pm from Greece and MR-VL from Belgium) that exhibited OP resistance. Toxicity bioassays on the OP strains revealed varying but high levels of resistance to most of the insecticides tested (Table 2). The WI strain proved to be more resistant to chlorpyrifos, whereas the recently collected strains MR-VL and ATHRos-Pm were particularly resistant to dimethoate. Compared to the reference strains, the biochemical experiments confirmed the presence of a more insensitive AChE1 in all OP strains (Table 3). The high variation found between LC 50 s of OP strains was not reflected in a high variation between IC 50 s. Thus, an insensitive AChE1 is probably not the only mechanism involved in the observed resistant phenotype, and other mechanisms such as metabolic mediated resistance (carboxylesterase, gluthathione-Stransferase or P450 monooxygenase activity) are likely also to be involved.

Target-site insensitivity as a mechanism of OP resistance in T. urticae was the first case ever reported, and provided a proof of principle for resistance development in arthropods in the early 1960s. [START_REF] Voss | Resistance to organophosphorus compounds in the two-spotted spider mite: two different mechanisms of resistance[END_REF]9 Here, the amino acid sequence of AChE1 was determined in three reference strains SAMB, GSS and LS-VL and three OPresistant strains WI, MR-VL and ATHRos-Pm, and four amino acid substitutions were identified in the mature AChE1 that are present in one or more resistant strains and absent in the susceptible ones.

Two substitutions were consistently present in all three OP strains (F331W and T280A). The position of the T280A substitution in the AChE1 structural model indicates that this mutation does not seem to be responsible for the modification of the biochemical properties of AChE1 (Fig. 1). This is further supported by the fact that this residue is not conserved among species and that both resistant and susceptible strains of T. urticae from Japan have a threonine at position 280. [START_REF] Anazawa | Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant twospotted spider mite, Tetranychus urticae[END_REF] In contrast, the phenylalanine 331 residue is involved in substrate guidance and in the hydrolysing process by interacting with the catalytic histidine (H440). [START_REF] Millard | Reaction products of acetylcholinesterase and VX reveal a mobile histidine in the catalytic triad[END_REF] A substitution at this position could lead to the modification of the catalytic properties of AChE1 (i.e. catalytic efficiency and/or inhibitor sensitivity). In T. urticae from Japan, an F331C substitution was found to be associated with OP resistance. [START_REF] Weill | A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila[END_REF] In T. kanzawai, a species closely related to T. urticae, a tryptophane at position 331 has been detected in resistant strains. [START_REF] Maga Ña | Mechanisms of resistance to malathion in the medfly Ceratitis capitata[END_REF] The F331W substitution has also been shown to be responsible for AChE1 insensitivity to OP insecticides in the mosquito Culex tritaeniorhynchus Giles by functional characterisation after site-directed mutagenesis and in vitro expression. [START_REF] Alout | Different amino-acid substitutions confer insecticide resistance through acetylcholinesterase 1 insensitivity in Culex vishnui and Culex tritaeniorhynchus (Diptera: Culicidae) mosquitoes from China[END_REF][START_REF] Oh | Expression of ace paralogous acetylcholinesterase of Culex tritaeniorhynchus with an amino acid substitution conferring insecticide insensitivity in baculovirus-insect cell system[END_REF] In addition, in the aphid species Aphis gossypii (Glov.) and Myzus persicae (Sulzer), a S331F was shown to confer insensitivity to AChE1, especially to the CARB pirimicarb. [START_REF] Andrews | Identification of mutations conferring insecticide-insensitive AChE in the cotton-melon aphid, Aphis gossypii Glover[END_REF][START_REF] Benting | Biochemical evidence that an S431F mutation in acetylcholinesterase-1 of Aphis gossypii mediates resistance to pirimicarb and omethoate[END_REF] More recently, the same substitution was associated with OP resistance in the B-and Q-biotypes of Bemisia tabaci Genn. from Israel and Greece. [START_REF] Alon | Organophosphate resistance in the B-biotype of Bemisia tabaci (Hemiptera: Aleyrodidae) is associated with a point mutation in an ace1-type acetylcholinesterase and overexpression of carboxylesterase[END_REF][START_REF] Tsagkarakou | Molecular diagnostics for detecting pyrethroid and organophosphate resistance mutations in the Q-biotype of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF] In conclusion, the 331 position in AChE1 has been associated with OP resistance in many species, and the functional expression has clearly illustrated the effect of the substitutions on AChE1 sensitivity. Hence, the F331W substitution is most probably the major factor in many OP-resistant spider mite strains. This is further emphasised by the fact that the WI strain, which developed resistance in the 1960s, and the recently collected strains from geographically distant regions (MR-VL and ATHPros-Pm) carry this same F331W substitution.

An allele with an F331Y substitution was sequenced in one clone of reference strains (SAMB and GSS strains). The F331Y substitution may play a role in carbaryl sensitivity, as AChE1 of the GSS strain is more sensitive to carbaryl than AChE1 of the SAMB strain. Genotyping of single mites revealed that in GSS the F331Y is predominant, whereas in the SAMB strain the F331 is predominant (Tsagkarakou A and Van Leeuwen T, unpublished data). However, it is not known whether or not this substitution influences the catalytic parameters of AChE1 in these two strains.

In the recently collected ATHRos-Pm and MR-VL strains, the G328A substitution was found in addition to F331W and T280A. The position 328 in the T. urticae AChE1 model suggests that this substitution may indirectly influence the catalytic process by affecting position 331 (located in the same α-helix). However, when AChE1 sensitivity of the MR-VL and ATHRos-Pm strains is compared with that of the WI strain, the additional G328A substitution does not seem to alter the sensitivity to the tested insecticides, except for carbaryl. This substitution has also been found in the AChE2 of Ceratitis capitata Wied., [START_REF] Maga Ña | Mechanisms of resistance to malathion in the medfly Ceratitis capitata[END_REF] Drosophila melanogaster Meig. [START_REF] Menozzi | Mutations of acetylcholinesterase which confer insecticide resistance in Drosophila melanogaster populations[END_REF] and Muscadomestica L. [START_REF] Walsh | Identification and characterisation of mutations in housefly (Musca domestica) acetylcholinesterase involved in insecticide resistance[END_REF] Site-directed mutagenesis followed by in vitro expression of D. melanogaster and M. domestica AChE2 showed that this mutation conferred insensitivity to chlorpyrifos-oxon, omethoate, malaoxon, methamidophos, paraoxon, paraoxon-methyl and pirimicarb, but increased the sensitivity to coumaphos and carbaryl. [START_REF] Walsh | Identification and characterisation of mutations in housefly (Musca domestica) acetylcholinesterase involved in insecticide resistance[END_REF][START_REF] Menozzi | Mutations of acetylcholinesterase which confer insecticide resistance in Drosophila melanogaster populations[END_REF] In the ATHRos-Pm strain, a fourth substitution (A201S) was found in addition to T280A, G328A and F331W. The proximity of this position to the catalytic serine (S200) may lead to some modifications in AChE1 biochemical properties. However, when IC 50 values were compared between MR-VL and ATHRos-Pm strains (differing only in the presence of the A201S), no effect on AChE1 sensitivity towards the tested inhibitors was detected. This mutation was also found in other arthropod species, such as a resistant clone of A. gossypii [START_REF] Li | Mutations in acetylcholinesterase associated with insecticide resistance in the cotton aphid, Aphis gossypii Glover[END_REF][START_REF] Andrews | Identification of mutations conferring insecticide-insensitive AChE in the cotton-melon aphid, Aphis gossypii Glover[END_REF] and a resistant strain of P. xylostella. [START_REF] Baek | Identification and characterization of ace1-type acetylcholinesterase likely associated with organophosphate resistance in Plutella xylostella[END_REF][START_REF] Lee | Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L)[END_REF] In A. gossypii the A201S substitution was always found in tandem with S331F, and in P. xylostella in tandem with G227A. In both cases it was suggested that its presence increases resistance levels, playing a supplementary role to the main mutations. [START_REF] Baek | Identification and characterization of ace1-type acetylcholinesterase likely associated with organophosphate resistance in Plutella xylostella[END_REF][START_REF] Andrews | Identification of mutations conferring insecticide-insensitive AChE in the cotton-melon aphid, Aphis gossypii Glover[END_REF][START_REF] Lee | Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L)[END_REF] In A. gossypii it was shown that AChE1 from insects carrying both mutations possess a better substrate affinity than when carrying the S331F mutation alone. [START_REF] Andrews | Identification of mutations conferring insecticide-insensitive AChE in the cotton-melon aphid, Aphis gossypii Glover[END_REF] The role of A201S mutation in resistance was characterised by sitedirected mutagenesis in AChE1 of C. tritaeniorhynchus, [START_REF] Oh | Biochemical properties of recombinant acetylcholinesterases with amino acid substitutions in the active site[END_REF] but no functional expression of recombinant A201S AChE1 of A. gossypii or P. xylostella has been performed until now.

Among the two substitutions specifically found in the reference SAMB strain (V7I and G119S), only G119S is located in the active site. This substitution is known to be responsible for AChE1 insensitivity [START_REF] Weill | Insecticide resistance in mosquito vectors[END_REF] or reduced AChE1 activity in mosquitoes. [START_REF] Bourguet | Determination of Ace.1 genotypes in single mosquitoes: toward an ecumenical biochemical test[END_REF][START_REF] Alout | Comparison of Anopheles gambiae and Culex pipiens acetycholinesterase 1 biochemical properties[END_REF] The 119 position is close to the catalytic serine (S200) and the G-to-S substitution would reduce the accessibility to inhibitors and substrates by steric hindrance. Recombinant Culex pipiens G119S AChE1 was similarly or more resistant than the recombinant F331W AChE1 to most of the OPs and CARBs tested, including chlorpyrifos-oxon and paraoxon. [START_REF] Alout | A new amino-acid substitution in acetylcholinesterase 1 confers insecticide resistance to Culex pipiens mosquitoes from Cyprus[END_REF] SAMB exhibited moderate levels of resistance towards chlorpyrifos and dimethoate, and the involvement of an AChE mutation in resistance cannot be ruled out. However, the biochemical data do not support the involvement of G119S mutation in resistance towards the insecticides tested in this study, which is in line with previous reports on Tetranychidae. [START_REF] Anazawa | Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant twospotted spider mite, Tetranychus urticae[END_REF][START_REF] Aiki | Amino acid substitution in Ace paralogous acetylcholinesterase accompanied by organophosphate resistance in the spider mite Tetranychus kanzawai[END_REF] Moreover, the AChE specific activity of the SAMB strain is higher than that of all other strains. However, before any sound conclusions can be made about the role of this mutation in T. urticae OP resistance, the frequency of this mutation should be determined by genotyping single mites of different strains, combined with a functional analysis of this mutation.

Although it is presumed that the F331W substitution reported in this study is essential in determining the insensitivity of AChE1 towards OP and CARB, the A201S and G328A mutations might have been selected by the application of other unknown OPs in the population's history. These substitutions may confer an advantage to other inhibitors. Different mutated AChE1 could cause a strong insensitivity to specific insecticides, as a good relationship between the highest insensitivities detected and the insecticide(s) used locally in mosquito control programs has been found. [START_REF] Alout | Amino-acid substitutions in acetylcholinesterase 1 involved in insecticide resistance in mosquitoes[END_REF] Thus, it is possible that, in T. urticae also, different mutations are selected and maintained in the populations, each one conferring advantage against a particular OP or CARB insecticide. Alternatively, substitutions A201S and G328A might be selected to reduce a possible fitness cost associated with the F331W substitution in T. urticae. Valuable information on the importance of the different substitutions in developing and maintaining an OP-resistant phenotype could be obtained by following their dynamics with molecular diagnostic tests under different OP spraying protocols aimed at controlling other arthropod pests in the field. The development of such a diagnostic test was illustrated for F331W mutation, as it was consistently present in recently resistant spider mite strains from different geographical regions, as well as in a strain that developed resistance in the late 1960s.

Different mutations and their combinations can result in varying levels of resistance to many OP and CARB insecticides. [START_REF] Mutéro | Resistance associated point mutations in insecticide-insensitive acetylcholinesterase[END_REF] Some of them have essentially an additive effect, both in terms of reducing the sensitivity of the enzyme to inhibition by the insecticide and in terms of increasing the stability of the enzyme and its ability to turn over acetylcholine. [START_REF] Shi | Acetylcholinesterase alterations reveal the fitness cost of mutations conferring insecticide resistance[END_REF] Functional analysis should be performed to test the influence of each mutation (alone or in combination) in OP and CARB insensitivity and in the specific activity of the recombinant mutated AChE1 in T. urticae.

Figure 1 .

 1 Figure 1. Three-dimensional structure model of Tetranychus urticae AChE1. The backbone of the enzyme structure is rendered as green ribbon with secondary structure. The catalytic triad (S200, E327 and H440) appears as Van der Waals red spheres. The different amino acid substitutions are shown as Van der Waals coloured spheres. The view points to the entrance of the catalytic gorge.

Figure 2 .

 2 Figure 2. Diagnostic PCR-RFLP for 331 W after primer-induced mutagenesis. Lane 1: uncut PCR product. Lane 2: PCR performed on plasmid DNA containing GSS 331 Y clone. Lane 3: PCR performed on plasmid DNA containing LS-VL 331F clone. Lane 4: PCR performed on plasmid DNA containing WI 331 W clone. Lane 5: PCR performed on DNA extracted from a single male of strain MR-VL (containing 331 W). Lane 6, PCR performed on pooled plasmid DNA containing GSS and WI clones. Lane 7: PCR performed on pooled gDNA from two males from GSS and MR-VL strains. Lane 8: PCR product on pooled mites from GSS. Lane 9: PCR products on pooled mites from LS-VL. Lane 10: MR DNA ladder (Invitrogen, Belgium). The PCR-RFLP assay on heterozygote females (F, Y331/W331) is expected to display the pattern of lanes 6 and 7, whereas the patterns of lanes 1, 2, 3, 8 and 9 are expected from homozygote (F or Y331) females, and the patterns of lanes 4 and 5 from homozygote (W331) females.

Table 1 .

 1 Primers used for the amplification of the Tetranychus urticae

	AChE1
	Primers	Sequence (5 -3 )
	Acetrur F	AAAGGGAGAAGGCAAAAGTGT
	Acetrur R	TGCTCATGTTCAGTTGATCG
	AcETuSeqF1	AATGCCACCTCATTTTCAGG
	AcETuSeqF2	TATCACGCAACGTTTTCAGC
	AcETuSeqF3	ATGAAGATCCCGAGGTTTCC
	AcETuSeqF4	TGGGAGTTATTCATGGTGAGG
	AcETuSeqR1	CATGAATCGATGGGCTTAGG
	AcETuSeqR2	AGCTGATTCACCGAAAATGG
	AcETuSeqR3	AACTGCTTCTTGGGCTAAAGG
	AcETuSeqR4	CAGTGTACTCAGGCCACACG
	AchEDISCF2	AAGCAATCGTGATGAGGGGCCCTATT
	AchEDISCR2	CTAAAGGACTCAGATGGGGATAAAT

Table 2 .

 2 Toxicity data for the OP selected and the reference Tetranychus urticae strains treated with several OP insecticides and one carbamate insecticide

	Treatment	N a	LC 50 (95% CI) (mg L -1 )	S l o p e ( ± SE)	χ 2b	df	RR 50	c (95% CI)
	LS-VL
	Pirimiphos-methyl	522	16 (12-21)	2.66 (± 0.39)	11 *	4
	Chlorpyrifos	435	11 (4.8-24)	0.98 (± 0.27)	14 * * *	3
	Methomyl	435	28 (14-57)	1.20 (±0.30)	24 * * *	4
	Dimethoate	214	8 (2-29)	0.88 (± 0.27)	12 * *	3
	SAMB
	Pirimiphos-methyl	254	18 (16-21)	4.50 (±0.14)	0.4	1	1.1 (0.8-1.7)
	Chlorpyrifos	315	333 (173-646)	2.75 (±1.17)	16 * * *	2	3 1 ( 1 5 -6 4 )
	Methomyl	378	40 (9.34-167)	2.48 (± 1.36)	52 * * *	2	1 . 4 ( 0 . 4 -5 . 4 )
	Dimethoate	226	226 (182-282)	4.97 (± 0.14)	5	2	29 (17-47)
	GSS
	Pirimiphos-methyl	577	332 (166-665)	6.56 (± 6.85)	135 * * *	2	2 1 ( 3 . 9 -1 1 3 )
	Chlorpyrifos	328	63 (19-215)	5.67 (± 7.05)	136 * * *	2	5 . 8 ( 0 . 4 -7 9 )
	Methomyl	279	50 (26-94)	2.11 (± 0.50)	6.7 *	2	1 . 8 ( 1 . 0 -3 . 2 )
	Dimethoate	281	11 164 (7418-16 836)	3.80 (± 1.43)	8.8 *	2	1 4 1 1 ( 6 8 6 -2 9 0 4 )
	WI
	Pirimiphos-methyl	463	142 (96-209)	4.70 (± 1.81)	37 * * *	3	8 . 9 ( 4 . 4 -1 8 )
	Chlorpyrifos	424	4584 (2786-7530)	3.93 (± 1.23)	28 * * *	3	4 2 1 ( 1 9 6 -9 0 7 )
	Methomyl	373	236 (151-373)	3.86 (± 1.26)	17 * * *	3	8 . 4 ( 4 . 2 -1 7 )
	Dimethoate	394	1502 (915-2467)	2.24 (± 0.64)	18 * * *	3	1 3 7 ( 8 4 -2 2 4 )
	MR-VL
	Pirimiphos-methyl	329	314 (266-374)	3.75 (± 0.41)	3.9	3	20 (14-28)
	Chlorpyrifos	392	6386 (4261-9567)	4.05 (± 1.08)	21 * * *	3	5 8 6 ( 3 0 2 -1 1 3 7 )
	Methomyl	419	3759 (3178-4370)	3.08 (± 0.32)	4.2	3	134 (95-189)
	Dimethoate	334	5878 (2218-15 579)	3.16 (± 2.02)	59 * * *	3	7 4 3 ( 2 2 3 -2 4 6 5 )
	ATHRos-Pm
	Pirimiphos-methyl	337	685 (497-944)	2.81 (± 0.73)	12 * *	3	4 3 ( 2 8 -6 6 )
	Chlorpyriphos	366	895 (624-1289)	2.62 (±0.57)	9.4 *	3	7 8 ( 3 9 -1 5 6 )
	Methomyl	301	9338 (4421-19 649)	3.11 (± 0.95)	16 * * *	2	3 3 3 ( 1 3 6 -8 1 8 )
	Dimethoate	319	32 950 (23 977-45 249)	6.09 (± 3.28)	8.5 * * *	1	4 1 6 4 ( 1 7 2 8 -1 0 0 3 6 )

a N, number of mites tested. b χ 2 , chi-square testing linearity: * P < 0.05, * * P < 0.01, * * * P < 0.001. c RR, resistance ratio = LC/LC LS-VL .

Table 3 .

 3 Specific activities and IC 50 of various OP and CX inhibitors on AChE1 extracted from OP selected and reference Tetranychus urticae strains

	IC 50 (M)

Table 4 .

 4 Amino acid polymorphism between reference laboratory and OP selected strains of Tetranychus urticae. Numbers refer to the position in mature AChE1 of Torpedo californica.[START_REF] Massoulié | Recommendations for Nomenclature in Cholinesterases, Multidisciplinary Approaches to Cholinesterase Functions[END_REF] Numbers in brackets refer to the position in T. urticae AChE1 precursor

	Position

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c 2009 Society of Chemical Industry Pest Manag Sci 2010; 66: 220-228

www.interscience.wiley.com/journal/ps c 2009 Society of Chemical Industry Pest Manag Sci 2010; 66: 220-228

www.soci.org J Khajehali et al.

ACKNOWLEDGEMENTS

The authors thank M Navajas (CBGP, Montpellier, France) for providing the SAMB strain and R Nauen (Bayer CropScience) for providing the GSS strain. This work was in part funded by a bilateral Greece-Cyprus cooperation program [0308(BE)/05]. Thomas Van Leeuwen is a post-doctoral fellow of the Research Foundation Flanders (FWO).