Abdessalem Hammed

Nolan Chatron

Jean Valéry Debaux

Etienne Benoit

Virginie Lattard

Elevated difenacoum metabolism is involved in the difenacoum-resistant phenotype observed in Berkshire rats homozygous for the L120Q mutation in the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene Maylis Boitet

BACKGROUND

Soon after difenacoum began to be used, resistance to this rodenticide was detected in rats in northeast Hampshire and northwest Berkshire in England. Resistance to difenacoum has been reported to be stronger in rats from Berkshire than in rats from Hampshire. Surprisingly, after the discovery of the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene, rats from Berkshire and Hampshire were all shown to be homozygous for the L120Q mutation in Vkorc1.

RESULTS

This study aimed to evaluate the resistance of Berkshire rats to confirm their extreme resistance and determine mechanisms supporting this resistance. For this purpose, we created a quasicongenic rat F7 strain by using a Berkshire rat as a donor to introduce the L120Q mutation in Vkorc1 into the genetic background of an anticoagulant-susceptible recipient strain. The use of F7 rats enabled demonstration of (i) the level of resistance to difenacoum conferred by the L120Q mutation, (ii) co-dominance of the L120 and Q120 alleles, (iii) the extreme resistance of Berkshire rats compared with Q120/Q120 rats as a consequence of additional resistance mechanisms, and (iv) the involvement of cytochrome P 450 (CYP450) enzymes in this extreme resistance.

CONCLUSION

This study demonstrated that elevated CYP450 oxidative metabolism leading to accelerated difenacoum detoxification is involved in the Berkshire phenotype.

INTRODUCTION

Difenacoum, a derivative of 4-hydroxycoumarin, is an anticoagulant molecule used as a rodenticide.1, 2 Like all the molecules in this family, difenacoum is a non-competitive inhibitor of the vitamin K epoxide reductase complex subunit 1 (VKORC1) enzyme involved in the recycling of vitamin K.3 This enzyme catalyzes vitamin K epoxide reductase (VKOR) activity, allowing the reduction of vitamin K epoxide to vitamin K. This activity is crucial to activate clotting factors II, VII, IX and X. Indeed, these clotting factors must be gamma-carboxylated to be active and, during this gamma-carboxylation, one molecule of vitamin K is converted to vitamin K epoxide.4 Dietary vitamin K intake is insufficient, and thus VKOR activity is essential for the maintenance of the pool of vitamin K. By inhibiting the VKORC1 enzyme, difenacoum prevents normal activation of vitamin K-dependent clotting factors and thus impairs the coagulation process.

While the use of 4-hydroxycoumarin derivatives as rodenticides has been reported since the early 1950s, difenacoum was introduced in England in 1974 by UK-based Sorex Ltd (Widnes, UK).1, 2 Its introduction followed a period of intensive use of the first molecules derived from 4-hydroxycoumarin qualified as first-generation anticoagulant rodenticides (FGARs), such as warfarin, coumatetralyl and chlorophacinone.5 This intensive use of FGARs was responsible for the emergence of many rodent strains resistant to FGARs in all European countries and in the rest of the world in the late 1950s.6-8 These cases of resistance, which are still found many years after their detection, have been determined to be predominantly associated with Vkorc1 single nucleotide polymorphisms (SNPs).9 In Europe, these polymorphisms in the Vkorc1 gene have been widely studied in brown rats and three major SNPs were detected, the Y139F, Y139C and L120Q mutations. The Y139F SNP is essentially found in France and Belgium,10, 11 Y139C in Germany, the Netherlands and Denmark12-14and L120Q in England15, [START_REF] Haniza | Large-scale structure of brown rat (Rattus norvegicus) populations in England: effects on rodenticide resistance[END_REF]. In vitro studies suggested a negative effect of these mutations on vitamin K recycling, leading to an increase in the requirement for vitamin K. Moreover, these mutations were shown to lead to resistance to the action of FGARs.17, 18

The emergence of these resistant strains led to the development of second-generation anticoagulant rodenticides (SGARs) in the 1970s effective against the main strains of rodents resistant to FGARs. Difenacoum was the first SGAR available on the market.1, 2 It is now used widely throughout the world. [START_REF] Buckle | Anticoagulant resistance in the United Kingdom and a new guideline for the management of resistant infestations of Norway rats (Rattus norvegicus Berk.)[END_REF][START_REF] Atterby | Whole-carcass residues of the rodenticide difenacoum in anticoagulant-resistant and -susceptible rat strains (Rattus norvegicus)[END_REF][START_REF] Hughes | Monitoring agricultural rodenticide use and secondary exposure of raptors in Scotland[END_REF] Its use is permitted throughout Europe to control populations of rats and mice in and around buildings, in waste sites and in sewers, and, depending on the country, in open areas. For example, difenacoum was reported to be used in approximately 45% of agricultural premises in Great Britain.15, 19 It was initially reported to be efficient to control all resistant strains.21 Nevertheless, some warfarin-resistant rats trapped on a farm in northeast Hampshire in England were rapidly reported also to be resistant to difenacoum.22-25 A derived strain was established and called the homozygous Hampshire strain HH.22 In 1982, rats from a farm in northwest Berkshire, close to the county of Hampshire, were also found to possess resistance to difenacoum. A derived strain was established and called the homozygous Berkshire strain (HB1). [START_REF] Pelz | The genetic basis of resistance to anticoagulants in rodents[END_REF] In contrast to the homozygous Hampshire strain, the homozygous Berkshire strain was shown also to possess moderate resistance to brodifacoum, one of the most efficient SGARs currently available. [START_REF] Gill | Inheritance of Low Grade Brodifacoum Resistance in the Norway Rat[END_REF] The molecular mechanism supporting resistance in the Berkshire strain was thus assumed to be different from that of the Hampshire strain. Surprisingly, after the discovery of the Vkorc1 gene in 2004,9 difenacoum-resistant rats of the Berkshire and Hampshire strains were all shown to be homozygous for the L120Q mutation,13 suggesting the existence of an additional resistance mechanism in Berkshire rats. This study aimed to explore the existence of additional mechanisms of resistance to anticoagulant rodenticides other than pharmacodynamic resistance by characterizing the resistance phenotype of Berkshire rats compared with that of laboratory rats introgressed with the L120Q mutation in the Vkorc1 gene.

MATERIALS AND METHODS

Chemicals

Difenacoum was supplied by Liphatech (Agen, France). Dimethyl sulfoxide (DMSO), acetonitrile, methanol, acetone, diethyl ether and orthophosphoric acid were obtained from VWR International (Fontenay sous bois, France), and Vetflurane® and vitamin K1 were obtained from Alcyon (Miribel, France). High-performance liquid chromatography (HPLC)grade water, prepared using a milli-Q plus system (Millipore, Saint-Quentin en Yvelines, France), was used for preparation of HPLC eluents. All the chemicals used in this study were handled under institutionally accepted safety standards.

Origin and husbandry of animals

HB1 Berkshire rats homozygous for Q120 in Vkorc1 were a generous gift from UK-based Sorex Ltd (Widnes, UK). The founder animals of this strain were initially trapped on an English farm in the Berkshire countryside.13 Since then, the HB1 Berkshire rat strain has been maintained in VetAgro Sup by intercrossing HB1 Berkshire male rats with HB1 Berkshire female rats. F7-introgressed rats homozygous for Q120, F7-introgressed rats homozygous for L120 and heterozygous F7-introgressed rats were derived from backcrossings and intercrossings between Sprague-Dawley rats and rats from the Berkshire wild rat strain (Fig. 1). The Berkshire rat strain with a L>Q mutation at position 120 of the VKORC1 protein was used as a donor.

Recipient strain Sprague-Dawley rats were purchased from Charles River Laboratories (St Germain sur l'Arbresle, France). To generate congenic strains, four L120Q homozygous Berkshire males were crossed with four Sprague-Dawley females to create the F1 hybrid generation. F1 male rats were backcrossed to Sprague-Dawley females to produce the F2 generation. The genotype of young F2 rats was determined by the allele-specific polymerase chain reaction (PCR) method, as described below. Heterozygous females were backcrossed to the recipient Sprague-Dawley strain for five additional generations to yield the F7 generation.

Finally, an F7 intercross of males heterozygous for L120Q with females heterozygous for L120Q was carried out to obtain strains of F7-introgressed rats homozygous for Q120 (Q120/Q120), F7-introgressed rats homozygous for L120 (L120/L120) and F7-introgressed rats heterozygous for L120Q (L120/Q120).

Genetic characterization of animals

Genetic characterization of animals was performed using an allele-specific PCR method, as described in Grandemange et al17 and modified to specifically detect the L120Q mutation in the Vkorc1 gene. Genomic DNA was extracted from tail tissue using the Nucleo-spin Tissue extraction kit (Macherey-Nagel, Hoerdt, France). The allele-specific PCR method employed two pairs of primers purchased from Eurogentec (Angers, France). The forward primer was one of the two primers in which the 3′ nucleotide matched the Vkorc1 sequence of the SD strain (i.e. the rat genome sequence of Vkorc1; L120 primer: 5′-tgtcgctggttctctgtacct-3′) or the 3′ nucleotide matched the mutation in the Vkorc1 sequence of the donor strain (120Q-primer: 5′-tgtcgctggttctctgtacca-3′). The reverse primers matched the Vkorc1 sequence of the rat genome (5′-tcagggctttttgaccttgtg-3′). We amplified 135 base pairs of the Vkorc1 gene using quantitative real-time PCR (qPCR) in a Thermocycler Mx3000P (Stratagene, Massy, France).

The reaction mixture contained 5X HOT BIOAmp EvaGreen (Biofidal, Vaulx-en-Velin, France) qPCR Mix (no ROX). The 15-µL reactions contained 1 µL of genomic DNA, 0.5 pmol µL -1 of each forward and reverse primer, 5 × EvaGreen qPCR buffer including dNTPs, HOT BIOAmp DNA polymerase, MgCl2 at 12.5 mM, and EvaGreen dye. The cycling was initiated by one denaturation step at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s (denaturation), 60°C for 20 s (annealing step) and 72°C for 20 s (elongation). Fluorescence from both the EvaGreen dyes was recorded at the annealing step. The point at which the PCR product was first detected above a fixed threshold, the thermal cycle threshold (Ct), was determined for each sample in duplicate, and the average Ct of duplicate samples was calculated. The three possible genotypes L120/L120, L120/Q120 and Q120/Q120 were deduced from the differences between their characteristic cycle threshold values (ΔCt), as described by Grandemange et al.17); that is, the difference in Ct values between the matched and the mismatched primer extensions for homozygous rats, and the absence of such a difference in Ct values for heterozygous animals.

Phenotypic characterization of animals

Experimental research on the animals was performed according to an experimental protocol following international guidelines (directive 2010/63/EU) and with approval from the ethics committee of the Veterinary School of Lyon. All animals tested were between the age of 10 and 12 weeks. The difenacoum administered was a mix of diastereomers composed of 57% of (1R,3S)(1S,3R) (designed as cis) and 43% of (1R,3R)(1S,3S) (designed as trans). [START_REF] Damin-Pernik | Development of an Ecofriendly Anticoagulant Rodenticide Based on the Stereochemistry of Difenacoum[END_REF] The amount of difenacoum administered per os was expressed as a factor of the acute (i.e. single dose) ED50 values. ED50 is the dose that creates an increase of the prothrombin time, expressed as the international normailzed ratio, higher than 50% compared to the normal population. The ED50 in response to antivitamin Ks (AVKs) in rats differs between females and males. To account for this, as recommended by the technical monograph of the Rodenticide Resistance Action Committee,28 the doses administered were multiples of ED50adjusted for males and females. Difenacoum was dissolved in the vehicle solution 5% DMSO, ethanolamine and sunflower oil and administered by force-feeding. Experiments were performed on female and male rats. Each dosage was assayed in a group of four animals.

Forty-eight hours after difenacoum administration, 3 ml of blood was taken by cardiac puncture into citrate tubes (3.2%; 1:9 v/v) and PT was measured immediately, as previously described by Damin et al. [START_REF] Damin-Pernik | Development of an Ecofriendly Anticoagulant Rodenticide Based on the Stereochemistry of Difenacoum[END_REF]

Determination of difenacoum concentration in liver

Liver tissue (1 g) was extracted with acetone using an Ultra Turrax tissue disperser from IKA Labortechnik® (VWR International, Strasbourg, France) according to the method previously described by Damin-Pernik et al27 and validated according to the guideline on Bioanalytical Method Validation published by the European Medicines Agency. [START_REF]Guideline on bioanalytical method validation[END_REF] The extract was centrifuged at 5000 g for 10 min, and the supernatant was evaporated at 50°C under a gentle nitrogen flow. The dry extract was resuspended in acetonitrile/hexane (50%/50%). The hexane layer was eliminated, and the remainder was dried at 60°C under a gentle nitrogen flow. The final dry extract was dissolved in methanol and concentrations of difenacoum and its metabolites were analyzed by HPLC on a LIChrospher® 100RP18 encapped column (5µm particle size; 250 × 4.6 mm) (VWR International) at detection wavelengths of 258 nM. A gradient elution system was used with a flow rate of 1 mL/min as follows: from 60% acetonitrile/40% water (acidified with 0.2% H3PO4) to 80% acetonitrile/20% water (acidified with 0.2% H3PO4) at 5 min. The recovery rate of difenacoum from tissues was between 75 and 95% in liver with a precision < 15%.

Preparation of liver microsomes

Liver microsomes were prepared from fresh livers by differential centrifugation, as described by Moroni et al. [START_REF] Moroni | The Flavin-Containing Monooxygenase in Rat Liver: Evidence for the Expression of a Second Form Different from FMO1[END_REF] Briefly, livers were resuspended in 50 mM phosphate buffer (pH 7.4) containing 1.15 % (w/v) of KCl. Liver cells were homogenized in buffer using a motor-driven Potter glass homogenizer (VWR International, Fontenay-sous-bois, France and further subjected to differential centrifugation at 4°C. The 100 000 g pellet corresponding to the membrane fraction was resuspended by Potter homogenization in HEPES glycerol buffer (50 mM HEPES and 20% glycerol, pH 7.4). Protein concentrations were evaluated by the method of Bradford31 using bovine serum albumin as a standard. Microsomes were frozen at -80°C and used for kinetic analysis.

CYP450-dependent oxidation assay

Oxidation of difenacoum by liver microsomes was assessed using the amount of hydroxydifenacoum produced in the presence of an Nicotinamide adenine dinucleotide phosphate (NADPH) regenerating system. In a final volume of 1 mL, a typical reaction mixture contained phosphate buffer (100 mM, pH 7.4), 0.3 to 0.5 mg of protein, 12.5 µM of difenacoum as a substrate and a regenerating system containing NADP (1 mM), glucose 6-phosphate (10 mM), glucose 6-phosphate dehydrogenase (1 unit), and MgCl2 (10 mM). Incubations were conducted in glass vials at 37°C for 30 min in air and were performed in duplicate. Reactions were stopped by the addition of 2 ml of ethyl acetate. The ethyl acetate layer was dried under nitrogen. The dry residue was immediately dissolved in 0.2 mL of methanol and reaction products were analyzed by HPLC-UV according to the method as described above.

Statistical analysis

Data are presented as the mean ± standard deviation obtained from four to five individual rats. Comparisons between groups, when more than two groups were present, were performed using the Kruskal-Wallis test or Tukey's multiple comparisons test, with GRAPHPAD PRISM 6 software (GraphPad, San Diego, CA, USA). Comparison between two groups was performed using the Mann-Whitney test. P<0.05 was the accepted level of significance.

RESULTS

Analysis of prothrombin time of rats belonging to the different strains

The PT of our control groups, that is, the basal levels of PT when not exposed to AVK, were 17.9 ± 2.5 and 15.8 ± 1.4 s, respectively, for male and female F7-introgressed rats homozygous for L120; 19.3 ± 0.5 and 16.0 ± 2.1 s, respectively, for male and female F7-introgressed rats homozygous for Q120; 17.0 ± 0.9 and 18.2 ± 2.3 s, respectively, for male and female F7introgressed heterozygous rats, and 17.4 ± 1.4 and 16.5 ± 1.0 s, respectively, for male and female Berkshire rats. Therefore, no significant differences in PT were found between groups when no AVK was administered (Kruskal-Wallis test; P > 0.05).

In vivo determination of the level of resistance to difenacoum associated with the L120Q mutation of the Vkorc1 gene

For the L120/L120 genotype, 48 h after difenacoum administration, PT times increased dramatically with even the smallest dose of difenacoum (i.e. corresponding to 2 ED50 = 1.3 mg/kg for males and 1.58 mg/kg for females) for males (368 ± 42 s) (Fig. 2) and females (213 ± 50 s) (Fig. 2). For the Q120/Q120 and L120/Q120 genotypes, PT increased only slightly at 2.6 mg/kg (i.e. corresponding to 4 ED50) in males. No significant differences in PT were found between Q120/Q120 and L120/Q120 males 48 h after administration of 2.6 mg/kg of difenacoum. In contrast, 48 h after administration of 5.2 mg/kg of difenacoum, Q120/Q120 male rats showed a PT of 101 ± 32 s, higher than that measured for L120/Q120 male rats (181 ± 30 s) (Fig. 2). For the Q120/Q120 and L120/Q120 females, PT increased only slightly (116 ± 25 s) at 6.32 mg/kg (i.e. corresponding to 8 ED50) for heterozygous females, while no increase in PT (21 ± 3 s) was observed for the Q120/Q120 females after the same administration (Fig. 2). Therefore, the PT of the three genotypes L120/L120, L120/Q120 and Q120/Q120 differed significantly 48 h after administration of 8 ED50 of difenacoum (Kruskal-Wallis test; P < 0.01).

Figure 2

Prothrombin times (in seconds) of male and female F7-introgressed rats homozygous for Q120 (Q120/Q120), homozygous for L120 (L120/L120) or heterozygous (L120/Q120) or belonging to the Berkshire strain, 48 h after per os administration of difenacoum. Values are represented as mean + SD. * P<0.05; ** P<0.01; *** P<0.001.

In vivo determination of the level of resistance to difenacoum of rats belonging to the Berkshire wild rat strain

For Q120/Q120 males and females, PT increased dramatically 48 h after administration of 30 ED50 (i.e. 19.5 mg/kg for males and 23.7 mg/kg for females) of difenacoum (i.e. 319 ± 34 s for males and 251 ± 39 s for females) (Fig. 2). Surprisingly, male and female rats belonging to the Berkshire wild rat strain showed a normal PT at 28 ± 2 s for males and 17 ± 2 s for females, 48 h after administration of 30 ED50 of difenacoum. Statistical analysis using Tukey's multiple comparisons test highlighted a significant increase in PT for Q120/Q120 rats compared with rats belonging to the Berkshire wild rat strain (P<0.001) for males and females.

Determination of difenacoum concentration in the liver 48 h after administration of 30 ED 50 of difenacoum

For Berkshire rats or F7-introgressed rats homozygous for Q120 (Q120/Q120), the concentration of difenacoum in the liver was significantly higher in females than in males 48 h after per os administration of 30 ED50 (Fig. 3). Moreover, concentrations of difenacoum in the livers of Berkshire rats were significantly lower than those measured in the livers of Q120/Q120 rats. Difenacoum concentrations in the livers of Berkshire rats were 1.1 ± 0.1 and 1.9 ± 0.4 µg/kg for males and females, respectively, while in the livers of Q120/Q120 rats, concentrations were 3.2 ± 0.2 and 5.4 ± 1.2 µg/kg for males and females, respectively.

Whatever the strain and the sex of the rat were, > 95% of the hepatic difenacoum present in the liver 48 h after administration was cis-isomers (Fig. 3), while the difenacoum administered corresponded to a mixture of 57% cis-isomers and 43% trans-isomers.

Figure 3 Difenacoum concentration in livers of male and female rats of the Berkshire strain and the F7-introgressed strain homozygous for L120Q (Q120/Q120), 48 h after per os administration of 30 ED50 of commercial difenacoum composed of 57% cis-isomer and 43% trans-isomer, with the relative proportions of cis and trans isomers. Values are represented as mean ± SD. * P<0.05; ** P<0.01; *** P<0.001.

Determination of the metabolites of difenacoum in the liver 48 h after administration of 30 ED 50 of difenacoum

Three different metabolites were detected with retention times of 6.4, 6.9 and 7.4 min, respectively, and a UV spectrum similar to that obtained for difenacoum (data not shown).

These metabolites were detected in the livers of Berkshire rats, but also in the livers of Q120/Q120 rats. The molecular mass of these metabolites, investigated by mass spectrometry, was 459 uma (data not shown). This molecular mass corresponded to the molecular mass of difenacoum added of 16 amu, corresponding to the molecular mass of a hydroxylated metabolite. Quantification of hydroxylated metabolites was performed by UV-HPLC by comparison to a range of difenacoum. The results are presented in Figure 4. The results correspond to the amount of hydroxylated metabolites measured in the liver 48 h after the administration of 30 ED50 of difenacoum. In the livers of female Berkshire rats, the concentration of hydroxylated metabolites (i.e. 9.4 ± 0.5 µg/g of liver) was significantly higher (30%) than that measured in the livers of female Q120/Q120 rats (i.e. 7.0 ± 0.1 µg/g of liver) (Mann-Whitney test; P < 0.01). In the livers of male Berkshire rats, the concentration of hydroxylated metabolites (i.e. 5.5 ± 0.8 µg/g of liver) was not statistically different from that of male Q120/Q120 rats (i.e. 4.3 ± 0.7 µg/g of liver).

Figure 4

Concentration of hydroxylated metabolites of difenacoum, 48 h after per os administration of 30 ED50 of difenacoum in male and female rats of the Berkshire strain and the F7-introgressed strain homozygous for L120Q (Q120/Q120). Values are represented as mean ± SD. * P<0.05; ** P<0.01; *** P<0.001.

Determination of CYP450-dependent oxidative activity in the liver

Incubation of liver microsomes of male Sprague-Dawley rats with difenacoum led to the production of only one detectable hydroxylated metabolite by UV-HPLC at 258 nm with a retention time of 6.9 min, corresponding to the major metabolites detected in the liver after per os administration of difenacoum to rats. In the presence of a saturating concentration of difenacoum (i.e. 12.5 µM of difenacoum), the specific activity obtained to produce this hydroxylated metabolite in the presence of liver microsomes of male Sprague-Dawley rats was 27.9 ± 3.2 nmol/min/mg of total protein. For Berkshire and Q120/Q120 rats, the results obtained are presented in Figure 5. The specific activity obtained to produce hydroxylated metabolites was 1.5-fold higher in Berkshire rats compared with Q120/Q120 rats and this difference was statistically significant (Mann-Whitney test; P < 0.01 for males and P < 0.05 for females).

Figure 5 CYP450-dependent oxidative metabolism of difenacoum by liver microsomes of male

Berkshire rats or male F7-introgressed rats homozygous for L120Q (Q120/Q120). Values are represented as mean ± SD. * P<0.05; ** P<0.01; *** P<0.001.

DISCUSSION

Since the description of wild Norway rats resistant to second-generation AVK rodenticides in the counties of Hampshire and Berkshire, the degree of resistance in Hampshire and Berkshire rats has been widely discussed because of (i) inconsistencies in results between laboratory studies and field trials and (ii) detection of the same mutation in the Vkorc1 gene associated with differences in the degree of resistance. [START_REF] Pelz | The genetic basis of resistance to anticoagulants in rodents[END_REF][START_REF] Gill | Inheritance of Low Grade Brodifacoum Resistance in the Norway Rat[END_REF] To advance the understanding of Berkshire/Hampshire resistance, we tried first to define the degree of resistance induced by the unique L120Q mutation by studying the relationship between genotype and phenotype. For this, we introgressed the L120Q mutation present in Berkshire rats into a laboratory rat strain through a series of seven successive backcrosses in order to remove all additional genetically based mechanisms that may contribute to the physiological response to AVK exposure. Indeed, Greaves and Cullen-Ayres22 postulated that resistance to difenacoum might be multigenic. After these seven repeated backcrosses, the genome of the original wild Berkshire rat is so diluted in the genome of the laboratory rat that its influence on the phenotype is negligible. Indeed, after seven backcrosses, the percentage of the genome of the original wild rat may be < 0.8%. However, as a consequence of our method of selection based on the selection of the L120Q mutation, there is still a fragment of chromosome 1 of the wild Berkshire rat around the introgressed gene, which was in a similar experiment estimated at 20 megabases or about 10% of chromosome 1 (250 megabases).17

The newly generated quasicongenic strain enabled us to observe the physiological response mediated by the L120Q mutation in the Vkorc1 gene. Unsurprisingly, the L120Q mutation induces a severe difenacoum-resistant phenotype. For introgressed rats homozygous for the L120Q mutation, an 8-fold increase of the normal dose of difenacoum necessary to increase the PT in warfarin-susceptible rats was required to impair the coagulation function. These results are consistent with the in vitro results obtained previously with recombinant VKORC1 enzymes expressed in the yeast Pichia pastoris,18 although the resistance factor induced by the L120Q mutation determined in vitro (×30) was higher than that determined in vivo (×8).

This study is thus a supplementary demonstration17, 32 revealing that in vitrocharacterization of recombinant VKORC1 enzymes expressed in yeast by the DTT-driven VKOR assay is a perfect tool to characterize the in vivo phenotype when no mutation other than the mutation in the Vkorc1 gene is involved in the resistance.

Moreover, the use of rats of the same F7 strain, but with different Vkorc1 genotypes generated by intercrosses (i.e. L120/L120, L120/Q120 and Q120/Q120), enabled establishment of the codominance of the Q120 and L120 alleles. This codominant property was also demonstrated for the F139 allele which is also widely distributed among the population of Norway rats in Europe.17 The codominance of the L120 and Q120 alleles is a crucial point for the selection of the L120Q mutation in the field. In practice, during rodent control efforts, the presence of the mutation even in the heterozygous state and with low frequency in the population can lead to resistance even at high dosages of difenacoum.

Because of this resistance, the heterozygotes, which are usually most frequent in the initial phase of the selection, survive better in the control treatment than the susceptible animals.

When the proportion of heterozygotes increases, homozygotes for the L120Q mutation appear and are then selected by difenacoum treatment because they survive better. Furthermore, the use of rats of the F7 introgressed strain enabled us also to demonstrate the extreme resistance of Berkshire rats compared with rats homozygous for the L120Q mutation. Indeed, after administration of almost 20 mg/kg of difenacoum (i.e. 30-fold the dose of difenacoum required to increase PT in warfarin-susceptible rats), PT appeared unchanged in Berkshire rats, while for an 8-fold increased dose, PT was already clearly increased for F7introgressed rats homozygous for the L120Q mutation. The resistance of the Berkshire strain is the most potent resistance described until now very severe. In the field, the use of bait containing currently 50 ppm of difenacoum cannot cause death of such rats and is thus certainly inefficient to manage such populations of rats. However, such baits might be still effective to manage populations homozygous for the L120Q mutation, although repeated consumption of this bait is certainly required. Considering the feeding behavior and the neophobia of rats, repeated consumption of baits by rats is quite possible.

Comparison of these strains has enabled us to confirm without any possible ambiguity that, for the same L120Q mutation of the Vkorc1 gene, two different phenotypes exist. Thus, Berkshire resistance involves an additional mechanism compared with Hampshire resistance.

In addition to the Vkorc1 gene, different additional genetically based mechanisms have been proposed to be able to contribute to the physiological response to AVK exposure. Modified expression of calumenin33 and mutation of epoxyde hydrolase 1,34 both proteins suggested to be components of the VKOR complex, have been proposed to mediate resistance, but the contributions of such mechanisms have never been confirmed. Mutations of CYP4F2, a vitamin K oxidase,35 have been shown to influence the vitamin K1 plasma concentration in humans, and thus the dose of anticoagulant required to maintain an anticoagulation status.36, 37 Nevertheless, the extreme degree of resistance observed in the Berkshire strain seems to exclude such a mechanism. In addition to these various mechanisms, an increase in the ability to eliminate a particular xenobic can result in resistance to this xenobiotic. In insects, this mechanism has been widely described and allows them to adapt to an insecticidecontaminated environment. [START_REF] Scott | Cytochromes P450 and insecticide resistance[END_REF] In humans, it has been demonstrated that CYP3A induction increases the tolerance for higher doses of warfarin in humans.39 Such a mechanism has also been previously suggested for warfarin-resistant wild roof rats in the Tokyo area of Japan.40, 41 It is thus possible that modified expression or activity of the drug metabolism enzymes in Berkshire rats accelerates difenacoum detoxification. To explore this hypothesis, difenacoum residues were quantified in the livers of Berkshire and F7-introgressed rats after oral administration and were shown to be 2.5-fold lower in Berkshire rats compared with F7introgressed rats. Elevated difenacoum metabolism is thus involved in the difenacoumresistant phenotype observed in Berkshire rats. Metabolic pathways of anticoagulant rodenticides have not yet really been explored, except for warfarin. Warfarin has been shown in rats to be principally oxidized by different CYP450 enzymes.42 Therefore, we monitored the production of hydroxylated metabolites. Effectively, three different hydroxylated metabolites were observed after difenacoum administration, suggesting the involvement of phase I metabolism enzymes in difenacoum metabolism. These metabolites were found in Berkshire and F7-introgressed rats and no additional metabolites have been identified in Berkshire rats.

Nevertheless, the production of hydroxylated metabolites has been shown to be higher in male Berkshire rats compared with male F7-introgressed rats, suggesting overexpression or an increase in the activity of CYP450 enzymes, although the difference in production was not statistically significant in females. The involvement of other metabolic pathways such as glucuronidation leading to the elimination of hydroxylated metabolites may explain the absence of hydroxylated metabolite accumulation. To confirm the involvement of CYP450dependent oxidative metabolism in the Berkshire resistance, in vitro assays were performed.

The results obtained confirmed the increase in the CYP450 oxidative metabolism of difenacoum in Berkshire rats. Further study of the metabolic pathway of difenacoum will be required to identify the specific isoform involved.

CONCLUSIONS

This study is the first demonstration of resistance caused by increased metabolism of anticoagulant rodenticides in brown rats. Markussen et al43 had previously suggested that changes in the expression of CYP450 could coexist in a bromadiolone-resistant rat strain carrying the Y139C mutation in the Vkorc1 gene, but without any apparent consequences. In Berkshire rats, the presence of this metabolic component, in addition to the L120Q mutation in the Vkorc1 gene, appears to substantially enhance the resistance phenotype. However, it is unclear whether only these two mechanisms are involved or if another mechanism is required to explain the extreme resistance to difenacoum of Berkshire rats.

In practical terms, it is essential to determine the limits of the resistance of Berkshire rats to optimize management methods. Because CYP450-dependent metabolism pathways are modified in Berkshire rats, metabolism of other AVKs must be characterized in these rats. AVK molecules able to control rats homozygous for the L120Q mutation (i.e. brodifacoum, difethialone and flocoumafen)18 with a different metabolism pathway from difenacoum should be used to control such rats and avoid selection of Berkshire rats.44 Alternation of active substances should also be considered to avoid development of other metabolic resistance. Moreover, if Berkshire resistance is associated with a biological cost, the suspension of the use of AVK rodenticides for a while could be recommended to decrease the frequencies of the alleles that confer resistance to difenacoum. It would be also essential to develop a simple tool to detect metabolic resistance to difenacoum to be able to distinguish in the field the different phenotypes revealed in this study.

Figure 1

 1 Figure 1 Schema of introgression of the L120Q mutation into the Sprague-Dawley Rattus norvegicusstrain. WT, wild type.

ACKNOWLEDGEMENTS

This work was supported by grant ISI n°I1301001W "NEORAMUS" from Bpi France.


Keywords: mobile, internet, body, position, perspective, tilt angle mobile, internet, body, position, perspective, tilt angle, gyro marketing

, we present two experimental studies in which we show (1) that mobile device tilt angle impacts purchase intention of advertised products, (2) that purchase intention is increased when device tilt angle allows viewers to stand in a body posture that is consistent with the image perspective, and (3) that mental simulation process mediates this effect. The results contribute to a better understanding of the conditions of advertising effectiveness on mobile devices.

Mobile advertising: the effect of tablet tilt angle on users' purchase intentions E-commerce growth is primarily being driven by consumers using their mobile devices, phones and tablets, to acquire goods and services (statista, 2018). Retail e-commerce sales reached $2.3 trillion in 2017, a 23.2% increase over the previous year. The mobile share of this stood at 58.9%, or $1.4 trillion. In 2021, mobile e-commerce could rake in some $3.5 trillion and then make up almost three quarters (72.9 %) of e-commerce sales (statista, 2018). Laptop and PC e-commerce drops off, although e-commerce on smartphones and tablets increases dramatically.

Many industries that rely on e-commerce are challenged by the rise of mobile interfaces because they fundamentally change the experience of the online advertisement. The body posture consumers adopt as they stare at their mobile screen (smartphone and tablet) is very different from laptop and desktop users' posture. While using mobile devices, consumers may hold the screen flat (near-horizontal device position) or hold the screen straight (near-vertical device position). Therefore, depending on how the mobile device is tilted, a mobile ad can be seen by viewers bending their head forward and dropping their chin on to their chest (downward head position) or by viewers keeping their head straight and looking forward to the screen (frontward head position).

Surprisingly, the impact of device tilt angle position and viewers' body posture on mobile advertising efficacy has received little attention in literature. Research shows that purchase intention towards advertised product is determined by mental simulation processes provoked by the advertisement (e.g. [START_REF] Elder Ryan | Visual Depiction Effect in Advertising: Facilitating Embodied Mental Simulation through Product Orientation[END_REF]. Product mental simulation is a form of automatic mental imagery process that helps individuals to mentally imagine product interaction. Various features of advertisements have been shown to impact mental simulation and purchase intention: product orientation within the picture [START_REF] Förster | How body feedback influences consumers' evaluation of product[END_REF][START_REF] Elder Ryan | Visual Depiction Effect in Advertising: Facilitating Embodied Mental Simulation through Product Orientation[END_REF], camera viewing angles (Royo-Vela and Black, 2018;[START_REF] Van Rompay | Embodied Product Perception: Effects of Verticality Cues in Advertising and Packaging Design on Consumer Impressions and Price Expectations[END_REF] or wording of messages on the ad [START_REF] Jennifer | Narrative Processing: Building Consumer Connections to Brands[END_REF][START_REF] Jennifer | Self-Referencing and Persuasion: Narrative Transportation versus Analytical Elaboration[END_REF][START_REF] Jeong | Imagine yourself being healthy: the mental simulation effect of advertisements on healthy menu promotion[END_REF]. But literature has not yet examined the potential impact of the media device position on mental simulation and purchase intention towards the advertised product.

Yet, embodied cognition literature highlights that body postures have a strong impact on information processing and decision making [START_REF] Barsalou Lauwrence | Grounded cognition[END_REF]. Several studies demonstrate that approach behaviors towards objects vary depending on individuals' actual body postures [START_REF] Förster | How body feedback influences consumers' evaluation of product[END_REF]. For example, [START_REF] Van Kerckhove Anneleen | The Floor Is Nearer than the Sky: How Looking Up or Down Affects Construal Level[END_REF] show that consumers prefer and choose different products when they look down versus up. Indeed, mental simulation with the product is facilitated, and purchase intentions are increased, when viewers' body postures are aligned with product orientation within the ad [START_REF] Elder Ryan | Visual Depiction Effect in Advertising: Facilitating Embodied Mental Simulation through Product Orientation[END_REF].

Depending on how the mobile device is held (near-horizontal or near-vertical tilt angle position), the product advertised in the mobile ad can be aligned (or unaligned) with actual viewers' body posture. In instance, when the product is photographed with a downward view to the object (so called birds' eyes perspective), viewers holding their mobile device in a near-horizontal position (tilting their head forward and dropping their chin on to their chest in a downward head position) should stand in a body posture that is aligned with product orientation within the ad. To the contrary, when the product is photographed with a frontward view to the object (so called frontal perspective), viewers' body posture should be aligned with the advertised product orientation when they hold their device straight in a near-vertical position (keeping their head straight and looking forward to the screen in a frontward head position). In this paper, we focus on the impact of mobile device tilt angle on mental simulation processes and purchase intention of advertised products. We present two experimental lab studies where we manipulate mobile device position (near-horizontal or nearvertical device tilt angle position) and product image perspective in the ad (vertical view to the object, so-called birds' eyes perspective or frontal view to the object, so-called frontal perspective). We study whether vertical (vs frontal) viewers' head position required by mobile device position (near-horizontal or near-vertical tilt angle position) impacts mental simulation and purchase intention for advertised products on mobile devices. In doing so, we extend the literature on mental simulation with advertised products (e.g. [START_REF] Marcelo | Drone images versus terrain images in advertisements: Images' verticality effects and the mediating role of mental simulation on attitude towards the advertisement[END_REF][START_REF] Yung | How do 3-dimensional images promote products on the internet?[END_REF][START_REF] Elder Ryan | Visual Depiction Effect in Advertising: Facilitating Embodied Mental Simulation through Product Orientation[END_REF][START_REF] Förster | How body feedback influences consumers' evaluation of product[END_REF] by underlining the role of the device tilt angle, mental simulation and purchase intention.

In the first section, we define the conceptual framework that allows us to establish research hypotheses. Next, we present the two experimental studies that we conducted on users of digital tablets. In the first study, we show that purchase intention towards an advertised product varies when the internet user is holding the tablet flat (in a so-called "near horizontal" position) or upright (a so-called "near vertical" position). In the second study, we show that tablet tilt angle impacts purchase intention towards advertised product because it creates a consistency (or inconsistency) with the viewer's body posture, that promotes (or reduces) mental simulation with the advertised product. Recent research highlights that perceptions of online advertised products and purchase intentions are impacted by the way viewers interact with them [START_REF] Adam | Tablets, touchscreens, and touchpads: How varying touch interfaces trigger psychological ownership and endowment[END_REF][START_REF] Hao | Computer Interfaces and the "Direct-Touch" Effect: Can iPads Increase the Choice of Hedonic Food?[END_REF].

One distinctive feature of tablets and smartphones is that they are held in the hand of the viewer. Therefore, unlike a desktop computer which is always positioned vertically (usually against a wall), mobile devices are held in various positions, such as horizontally (when rested on the knees, for example) or vertically (when standing straight).

Therefore, mobile advertisements can be viewed with different head positions (see figure 1 for an example). Indeed, ergonomics research has shown that individuals adapt their entire body posture to the tilt angle of the mobile device. Even though the eye can move in its socket, people tend to adjust their head and neck posture to the position in which the mobile device is held [START_REF] Justin | Touch-screen tablet user configurations and case-supported til taffect head and neck flexion angles[END_REF][START_REF] Yu | Consumers' resistance to using mobile banking: evidence from Thailand and Taiwan[END_REF].

When the mobile device is tilted forward (near-horizontal device position), the chin is dropped on to the chest, the head is tilted forward [START_REF] Hansraj | Assessment of stresses in the cervical spine caused by posture and position of the head[END_REF] and the mobile advertisement is viewed with the head in a downward position [START_REF] Vasavada | Gravitational demand on the neck musculature during tablet computer use[END_REF].

On the other hand, when the device is held straight in front (near-vertical device position), the head remains straight in a frontward position.

Embodied cognition literature highlights that body postures have a strong impact on information processing and decision making [START_REF] Barsalou Lauwrence | Grounded cognition[END_REF]. Several studies demonstrate that approach behaviors towards objects vary depending on individuals' actual body postures [START_REF] Förster | How body feedback influences consumers' evaluation of product[END_REF]. For example, [START_REF] Van Kerckhove Anneleen | The Floor Is Nearer than the Sky: How Looking Up or Down Affects Construal Level[END_REF] show that consumers prefer and choose different products when they look down versus up. Various theories are explored to explain how body postures can influence advertising perception and behavioral reactions (for a review see [START_REF] Förster | How body feedback influences consumers' evaluation of product[END_REF]. Many empirical studies show that human individuals tend to get ready to implement the appropriate body movement when they are exposed to a visual image of a product [START_REF] Schlosser | Experiencing Products in the Virtual World: The Role of Goal and Imagery in Influencing Attitudes versus Purchase Intentions[END_REF]. Therefore, consistency between product orientation and viewers' body postures results in increased approach behaviors. This effect is called the principle of perceptualmotor compatibility [START_REF] Förster | How body feedback influences consumers' evaluation of product[END_REF]. It shows that approach behavior towards advertised products tends to be activated more easily and faster by the viewer when his actual body posture is in line with the gesture that he would have to perform to grasp the product in is hand. For instance, Elder and Krishna (2012) demonstrate that purchase intention for a product orientated to the left (vs right) within the advertisement is higher when viewers stand with their left (vs right) hands ready for usage, and their right (vs left) hands occupied with a physical task that prevents their usage. As far as mobile advertising is concerned, with viewers' body postures depending on device tilt angle position (frontward when device is held in a near-vertical position and downward when device is held in a near-horizontal position), purchase intention towards advertised products should vary depending on how the viewers' position is in line with the way the product is depicted within the advertisement.

In advertising, there are several ways of depicting an object. In front-views perspective, the camera is positioned in front of the product although in high-angle views, the camera is positioned above the product. Following the perceptual-motor compatibility principle [START_REF] Förster | How body feedback influences consumers' evaluation of product[END_REF], approach behavior towards products on frontal perspective advertisements should be easier to activate when the mobile device is held in a nearvertical position that allow the viewers to keep their heads straight and to look at the picture in front of them. Approach behaviors should thus be higher because the Indeed, high-angle pictures show the products from the top, and approach behaviors should thus be increased when viewers hold their device flat so as to bend their head downward, because the birds' eye view of the product is consistent with the downward position of the viewers' head.

Building on this literature, we hypothesize: H1: Purchase intention of advertised product is higher when the mobile device is held in a position that is consistent with the advertised product orientation, in such a way that viewers with a frontward head position watch front-view product advertisement (H1a) and viewers with a downward head position watch high-angle view product advertisements (H1b) -Figure 1-

Mental simulation towards advertised products in advertisement

Advertising research shows that purchase intention towards advertised product is determined by mental simulation processes provoked by the product picture on the advertisement [START_REF] Yung | How do 3-dimensional images promote products on the internet?[END_REF][START_REF] Elder Ryan | Visual Depiction Effect in Advertising: Facilitating Embodied Mental Simulation through Product Orientation[END_REF]. Mental simulation, or the reenactment of perceptual experiences, is an automatic form of mental imagery that is initiated by exposure to visual representations of objects. Product mental simulation is an automatic operative process that helps individuals to imagine product interaction.

Seeing a visual depiction of a product automatically activates a quick cerebral motor response that consists in imagining how to use the product. The link between mental simulation and motor responses has been demonstrated several times with neuroscientific studies (e.g. [START_REF] Linda | Representation of Manipulable Man-Made Objects in the Dorsal Stream[END_REF]. Several authors (e.g. Choi and Taylor, 2014) establish a causal link between body postures, mental simulation with the product, and purchase intention. Indeed, advertised products cannot be physically tested, manipulated, or touched, and viewers tend to automatically perform a mental simulation of the advertised product to determine whether they like the product and want to buy it [START_REF] Elder Ryan | Visual Depiction Effect in Advertising: Facilitating Embodied Mental Simulation through Product Orientation[END_REF]. Many studies have shown that the ease with which a product is mentally visualized does not change attitudes [START_REF] Schlosser | Experiencing Products in the Virtual World: The Role of Goal and Imagery in Influencing Attitudes versus Purchase Intentions[END_REF][START_REF] Elder Ryan | Visual Depiction Effect in Advertising: Facilitating Embodied Mental Simulation through Product Orientation[END_REF][START_REF] Yung | How do 3-dimensional images promote products on the internet?[END_REF] and has a direct positive effect on the intent to buy the object (Gregory, Cialdini, Carpenter, 1982;[START_REF] Schlosser | Experiencing Products in the Virtual World: The Role of Goal and Imagery in Influencing Attitudes versus Purchase Intentions[END_REF][START_REF] Yung | How do 3-dimensional images promote products on the internet?[END_REF]. Indeed, approach behaviors toward products in an advertisement are the consequence of a mental process in which the approach movements are first mentally prepared as part of mental imagery or mental simulation activities (Stanford University's Encyclopedia of Motor Cognition). Mentally imagining a product in an advertisement acts directly on behavior without going through an affective route during which the product would be assessed further [START_REF] Yung | How do 3-dimensional images promote products on the internet?[END_REF].

According to embodied cognition theory [START_REF] Barsalou Lauwrence | Grounded cognition[END_REF], body postures affect advertising information processing, and behavioral reactions for advertised products, because they affect how easily viewers can mentally simulate the movement needed to mentally grasp or use the product in the advertisement [START_REF] Hao | Computer Interfaces and the "Direct-Touch" Effect: Can iPads Increase the Choice of Hedonic Food?[END_REF][START_REF] Elder Ryan | Visual Depiction Effect in Advertising: Facilitating Embodied Mental Simulation through Product Orientation[END_REF]. Various features of the ad have been shown to impact the mental simulation process, which in turn impacts purchase intentions : product orientation within the picture [START_REF] Förster | How body feedback influences consumers' evaluation of product[END_REF][START_REF] Elder Ryan | Visual Depiction Effect in Advertising: Facilitating Embodied Mental Simulation through Product Orientation[END_REF] Black, 2018) or wording of messages on the ad [START_REF] Jennifer | Narrative Processing: Building Consumer Connections to Brands[END_REF][START_REF] Jennifer | Self-Referencing and Persuasion: Narrative Transportation versus Analytical Elaboration[END_REF][START_REF] Jeong | Imagine yourself being healthy: the mental simulation effect of advertisements on healthy menu promotion[END_REF]. Empirical studies show that the visual perspective on a product (e.g. vertical pictures taken from a drone or grounded perspective) may result in modified information processing of advertising, such as modified mental simulation processes [START_REF] Marcelo | Drone images versus terrain images in advertisements: Images' verticality effects and the mediating role of mental simulation on attitude towards the advertisement[END_REF]. In particular, research shows that mental simulation with a product is stronger when there is consistency between the viewers' body posture and the product perspective in the advertisement. Some studies examine the impact of product orientation within the picture (e.g. the handle of a mug is orientated to the left or the right within the picture of the advertisement) (e.g. [START_REF] Elder Ryan | Visual Depiction Effect in Advertising: Facilitating Embodied Mental Simulation through Product Orientation[END_REF].

They demonstrate that consistency between product orientation (to the left or to the right) and viewer's body posture leads to increased mental simulation and purchase intention. Other studies focus on image perspective and demonstrate that alignment of the viewer's gaze direction with image perspective facilitates mental simulation with advertised product and increases purchase intention [START_REF] Schlosser | Experiencing Products in the Virtual World: The Role of Goal and Imagery in Influencing Attitudes versus Purchase Intentions[END_REF][START_REF] Yuwei | Imagining Yourself in the Scene: The Interactive Effects of Goal-Driven Self Imagery and Visual Perspectives on Consumer Behavior[END_REF]. Indeed, mental simulation is facilitated when viewer's body posture is consistent with the mental movement that is needed to mentally grasp the advertised product.

As far as mobile advertising is concerned, building on these previous studies, mental simulation should be easier, and purchase intentions higher, when the device is held in a tilt angle position that allows the viewer to align his body posture with the product image perspective within the advertisement. Indeed, when device position allows the viewers to stand in a body posture that is consistent with the picture perspective, viewers can easily perform a mental interaction with the advertised product and declare higher purchase intention for the advertised product.

When product is displayed in a high-angle perspective (birds' eye view), the camera positioned above the product suggests that the mental movement needed to grasp the product is a downward movement. Therefore, the mental simulation with the product should be easier when the device is held flat because it allows the viewers to stand with a downward head posture that is consistent with the image perspective. Indeed, in this tilt angle position, viewers have to bend their head downward, ready to perform a downward mental movement of approach toward the advertised product. Thus, holding the device flat provokes a consistency between image perspective and viewer's body posture when the advertisement is a high-angle (birds' eye) product image perspective.

To the contrary, when the product is shot with a front-view perspective, mental movement needed to mentally interact with the product is a frontal movement. Mental simulation with the product is easier, and purchase intention is higher, when the device is held near-vertically because it allows the viewers to keep their head straight, ready to activate a frontal mental approach movement towards the advertised product. To do so, we conducted a study of usage patterns on digital tablets. This study was conducted by e-mail via the software wysuforms. It was sent to 150 undergraduate students in a French management university and resulted in the collection 130 responses from tablet users (43% of men, 57% of women). Various questions were asked about their tablet usage (preferred tablet brand, tablet use frequency), and especially what type of body posture they adopted while using their tablet. Various body postures were proposed and respondents were asked to evaluate how often they adopted the specific body posture for tablet and laptop usage (never, very rarely, rarely, often, very often, nearly always, always). For example, they were asked how often they sat on couch or armchair when they use their tablet, and how often they sat down in a working position to use their device. The results reveal that 84% of respondents use always or nearly always their tablet on a couch or an armchair (compared to only 29.4% for laptops).

H2: Mental simulation of advertised product is increased when the mobile device is held in a position that is consistent with the advertised product orientation, in such a way that viewers with a frontward head position watch front-view product advertisements (H2a) and viewers with a downward head position watch high-angle view product advertisements (H2b) H3: Mental simulation of advertised product mediates the impact of the consistency between mobile device position and advertised product orientation on purchase intention towards advertised product

Only 6% of the individuals queried say they always or nearly always adopt a working position (sitting at a desk or table) when using their tablet (versus 64% of respondents using laptops). This first study suggests that internet users are usually sitting on a couch when they are using their tablet, rather than at a table. The second pilot study was an observational study. Ten users of digital tablets were summoned to the home of the researcher. These users were selected from the social circle of the researcher to represent a range of ages and different body types. Each participant came to the home of the researcher with his/her digital tablet. He/she was shown into the living room/dining room, a room with a living area (couch, coffee table )   and a dining area (table and chairs). The respondent received the instruction to sit wherever he/she wished to answer a survey on his/her digital tablet. All 10 respondents spontaneously went to the couch. They adopted two types of positions : 6 of them held the tablet flat (this position will be described as near-horizontal in the rest of the study), resting on the knees, hands, or even sometimes the respondent's bag, and 4 of them held the tablet slightly inclined (this position will be described as near-vertical in the rest of the study), with the tablet resting on the raised legs, the hands, or the tablet easel (figure 3).

-Figure 3 -After these two pilot studies, we selected two positions on which to test the research hypotheses: the so-called "near-horizontal" position (the screen forms an angle with the ground of less than 45°, it is nearly parallel with the ground, and the internet user's gaze is looking downward) and the so-called "near-vertical" position (the screen forms an angle with the ground of greater than 45°, it is nearly perpendicular to the ground and the gaze is toward the front) (figure 3). In these two positions, the users are seated on a sofa. These positions have the advantages of maintaining the same observation distance from the screen (approximately 35 cm). This distance corresponds to the ideal observation distance (also called orthoscopic distance) for a tablet with a ten-inch screen 1 (17 x 24 cm). These two positions also have the advantage of placing the screen against a static and neutral background (i.e. the user's pants), which does not distract attention from the screen. The literature on visual saturation has emphasized the extent to which the visual elements located near the target of the eyes can create interference and disturb perception [START_REF] Wim | Online advertisements and congruency effects: it depends on how you look at it[END_REF][START_REF] Adam | The Role of Visual Attention In Internet Advertising[END_REF][START_REF] David | Visual Crowding: A Fundamental Limit on Conscious Perception and Object Recognition[END_REF].

Study 1

Design of study 1.

The purpose of the first experimental study was to select an advertisement using a photograph with a particular perspective and to study whether the position in which the internet user holds the tablet (so-called "near-vertical position", so-called "nearhorizontal position" -see figure 3) has an impact on mental simulation of the product and on advertised product purchase intention. Two experimental conditions were selected based on the position in which the internet user was placed and each respondent was assigned randomly to one of the two conditions (near-vertical position or nearhorizontal). A between-subject design was selected, and each respondent adopted only one position.

Selection of the advertisement for study 1.

For this study, we selected a photograph of a product using a front-view perspective on the product (the camera is placed in front of the product).

In the frontal perspective, the plane of the photograph is vertical (figure 3). With a photograph in a front-view perspective, the position of the tablet allows the viewer to 1 The respondents all used full-size tablets of approximately ten inches. We decided to use an advertisement on a new kitchen utensil (a kit used to store liquids, such as yogurt drinks) that evokes grasping representation relatively automatically (appendix 1). Indeed, mental simulation processes are more easily activated when the advertisement depicts a product for which usage is easy to understand (Zhao et al,

2011).

The photograph was displayed in 11cm x 15cm on the tablet. We took care to position the photograph in the center of the screen because unusual positions on the screen can enhance (or decrease) the attention of internet users and consequently change their reactions to the advertisement (Janssens et al, 2012). The image was static so as not to distract the internet users [START_REF] Jaana | The impact of salient advertisements on reading and attention on web pages[END_REF] Sample of study 1.

The study was administered to 76 users of digital tablets recruited through an announcement posted in the hallway of a French university. The respondents were not compensated. 32 respondents were randomly assigned to a "near-vertical tablet" condition and 44 respondents were assigned to a "near-horizontal tablet" condition.

The respondents were between 14 and 63 years of age and the ages were fairly well distributed between the two experimental conditions (Mhoriz. = 35 years old; Mvert. = 40 years old). The data sheet administered at the end of the survey indicates that the respondents use their digital tablet frequently and that there is no major difference in usage between the two groups (Mhoriz. = 5.3; Mvert. =4.8; p=0.162).

Study 1 procedure. The survey was sent by e-mail. In the e-mail invitation, respondents were informed that they had to spend five minutes seated on a couch without being disrupted, moving, or changing position. Depending on the position to which they were assigned, they were asked to hold their tablet flat in a "near horizontal position" or in a "near-vertical position". The device positon and the body posture that they had to adopt during the study was illustrated by a picture (figure 3). They were given the instruction to assume the same position as the person in the picture.

During the survey, reminders were regularly displayed: "we remind you that it is essential that you remain in the same position when answering this survey." At the beginning of the survey, the respondent agreed to remain in this same position for the entire survey by checking the item "I agree to maintain this position for the entire duration of the survey."

Selection of measurements for study 1.

To measure how much mental simulation process was activated in viewers' mind, we built on previous literature on mental simulation and mental imagery. Indeed, mental simulation measures are inspired by mental imagery scales (e.g. [START_REF] Schlosser | Experiencing Products in the Virtual World: The Role of Goal and Imagery in Influencing Attitudes versus Purchase Intentions[END_REF][START_REF] Yung | How do 3-dimensional images promote products on the internet?[END_REF].

There are various correlated indicators of the degree of mental simulation elicited by the message. Mental simulation process can be measured by asking questions that refer to the quantity of mental images that are activated by the stimuli (study 3, Elder and Krishna, 2012) or by questions that refer to the ease of the mental simulation process (study 4, Elder and Krishna, 2012). Indeed, various studies on mental imagery demonstrate that ease and quantity of mental imagery are strongly correlated (for a [START_REF] Ellen | The generation and consequences of communication-evoked imagery[END_REF] empirically shows that the quantity and ease items load together.

In this paper, the items to measure mental simulation process focus on ease of the mental simulation process. Indeed, it may be more difficult for respondents to evaluate the quantity of mental images process than the easiness of the mental simulation process. It seems easy for respondents to evaluate if mental simulation with advertised product came easily to mind, or if they felt that mental interaction with the advertised product was very demanding. To the contrary, as the mental simulation process is a narrative and continuous process [START_REF] Jennifer | Self-Referencing and Persuasion: Narrative Transportation versus Analytical Elaboration[END_REF], it may be more difficult for respondents to give an evaluation of the number of mental images processed when they were exposed to the advertisement. Therefore, we chose in this paper, to measure mental simulation process by focusing on ease of the mental simulation process.

We therefore asked the respondents to note on a 7-point scale, To what extent it was difficult to imagine using the product (from extremely difficult to extremely easy), and How much time did it take you to imagine using the product (from a very long time to very quickly). The respondents also evaluated whether they were in agreement with the statement It took effort of thought to imagine myself using the product (from entirely agree to entirely disagree). These three elements were combined to form a measurement of mental simulation (α = 0.818).

To measure behavioral intentions with regard to the object shown in the advertisement, we asked the respondents to rate "the probability that they would buy this product".

Results of study 1

As an initial step, we also tested whether intent to buy and mental simulation varied according to screen position. We performed a difference of means test between the two F(1,74)=5.71; p=0.019). The respondents more easily imagined themselves using the product when holding their tablet in a position that allowed them to align their body posture with the perspective of the advertisement (in this study, a frontal view/frontal perspective).

With regard to intent to buy, the test also indicates that a near-vertical tablet position leads internet users to form a more favorable purchase intention than a near-horizontal tablet position (Mhoriz. = 1.45; Mvert. = 2.21; F(1;74)=8.30; p=0.005). Therefore, online advertising creates greater purchase intention when viewed on a tablet held in a position that allows the user to align his/her body posture with the perspective of the image. H1 is validated for an advertisement with a frontal perspective.

-Table 1 -

To test the link between mental simulation and purchase intention, we performed a linear regression between the two variables. The analysis indicates that the regression of mental simulation on purchase intention is significant (b=0.31; SE=3.61; p<0.001).

Thus, the more the respondents could easily imagine themselves using the product, the more they reported that they wanted to buy the product.

To test the mediating effect of mental simulation, we followed the method recommended by Hayes and Preacher (2013) and performed a simple mediation test using a bootstrapped procedure (Model 4, n=1000), in which we set purchase intention as the dependent variable, tablet position as the independent variable, and mental simulation as the mediating variable. Results indicated that tablet position was a significant predictor of mental simulation and that mental simulation was a significant predictor of purchase intention. These results support the mediational hypothesis. Tablet position was no longer a significant predictor of purchase intention after controlling for mental simulation, consistent with full mediation. The indirect effect was tested using a bootstrap estimation approach with 1000 samples. These results indicated the indirect coefficient was significant, b = -0.191, 95% CI = -0.5610, -0.0187. The effect of mediation of mental simulation is significant with mental simulation averages that exclude zero and a confidence interval of 95% [-0.5610; -0.0187]. H3 is validated for an advertisement with a frontal perspective.

-Table 2 -Discussion of study 1.

This first experimental study confirms that the position in which the tablet is held can affect behavioral intentions toward online advertising. However, in this stage of the research, the role of consistency between image perspective and tablet position has not been demonstrated. The hypothesis was made that a tablet position enabling alignment of the body posture with the perspective of the photograph would increase mental simulation and purchase intention. But at this stage of the research, some may argue that the orientation of the head downward could have provoked more negative reactions than an upward head. In fact, following the work of Van Kerckhove, Geuens, and Vermeir Therefore, in order to demonstrate the role of consistency of viewers' body posture and image perspective, we conducted a second experimental study in which we simultaneously manipulated tablet position (near-horizontal, near-vertical) and image perspective (front-view, birds' eye view).

Study 2

Design and procedure for study 2.

We implemented a study in which the same advertised product is photographed from two different perspectives: a front-view perspective and a high-angle perspective. We selected an advertisement showing a cup of coffee. In the front-view perspective, the cup of coffee is photographed from the front. In the high-angle perspective, the cup of coffee is photographed from the top. We verified that, in all four conditions, the image was easy to understand and that information in the advertisement was easy to process (1.4<M<1.6; ps>0.495). To do so, we used two 7 points Likert scale items and calculated an average score of the two items: (understanding this image requires effort of thought and to understand this image, you have to concentrate).

The experimental design includes four different conditions (near-vertical, nearhorizontal) x (front-view, birds' eye view). A between-subject design was selected, and each respondent viewed only one image in a single position. The survey was sent by e-mail, and the procedure was exactly the same as for study 1.

-Table 3 -Measurements of study 2.

We adapted the measurements from the first study to the context of study 2. The respondents had to indicate on a 7-point scale, To what extent they imagined drinking the coffee (from not at all to a lot), How much time it took them to imagine drinking coffee (from a very long time to very quickly). To what extent it was easy to imagine the taste of coffee and To what extent they were immersed in the image of the coffee (from not at all to a lot). These four items were combined to form a mental simulation measurement (α = 0.693).

To measure the behavioral intentions with regard to the object shown in the advertisement, we used three items in combination to create a measurement of approach behavior. The respondents were asked to note the probability that they would take a coffee break?, that they would take a moment to have a coffee? They also had to respond using the following Likert scale: I feel like having a cup of coffee (α = 0.805).

Sample of study 2.

We recruited users of digital tablets who agreed to participate in an online survey by posting an announcement in the hallways of a French university. The participants were assigned randomly to the four conditions. This procedure resulted in the collection of 74 completed surveys, of which 37 respondents were exposed to the image in a frontal perspective and 49 in a high-angle perspective. Forty respondents were women and thirty-four respondents were men. The average age of the respondents is 32.

There was no difference in terms of familiarity with digital tablets and general attitudes towards the experimental stimuli. On average, in all four conditions, the respondents used their digital tablets fairly often (1. 7<M<2.5; ps>0.115). In all four conditions, the respondents moderately enjoy drinking coffee (2.20<M<3.20; ps>0.070).

-Table 4 -Results of study 2.

In order to study whether the consistency between the perspective of the advertising image (frontal, high-angle) and the screen position (vertical, horizontal) facilitated mental simulation and increased purchase intention, we conducted two tests of analysis of variance with interaction. The purchase intention and mental simulation were introduced in the model as dependent variable. The device position (near-horizontal, near-vertical position) and the ad perspective (frontal perspective, high-angle perspective) were set as the independent interacting variables.

First, the analysis indicates that there is no main direct effect of device position and image perspective on purchase intention and on mental simulation (p>0.05). Second, there is a significant interaction between the position of the tablet and the perspective of the advertising image (F(3;70)=5.857, p=0.001 for purchase intention and F(3;70)=3.560, p=0.018 for mental simulation).

- The contrast analysis indicates that the advertisements shot from a front-view perspective reduces mental simulation of the product when viewed on a screen in the horizontal position (versus vertical) (Mhoriz. = 2.91;Mvert. = 4.20;p=0.04).

Furthermore, it triggers less purchase intention when viewed on a screen in the horizontal position (versus vertical) (Mhoriz. = 2.43; Mvert. = 3.93; p=0.06). Thus, when the advertisement is from a front-view perspective, the perspective consistency between the advertisement and the viewer's body posture increases mental simulation and creates stronger purchase intention.

However, the results are more nuanced for advertisements with a high-angle perspective. They trigger less intent to buy when viewed on a screen in the vertical position (versus horizontal) (Mhoriz. = 3.02; Mvert. = 1.96; p=0.019). However, the mental simulation is not significantly impacted by the position of the screen (Mhoriz. = 3.17; Mvert. = 3.72; p=0.186). Therefore, holding the screen in a position that allows viewers to align their body posture with the perspective of the image increases purchase intention for the two tested perspectives but only increases mental simulation for the front-view perspective. H2 is validated, but H1 is only partially validated for images with a frontal perspective.

-Figure 4 -We conducted a regression analysis using the Hayes and Preacher (2013) macro in order to examine whether mental simulation mediates the positive effect of perspective consistency on purchase intention. To do so, we created a perspective consistency variable (yes, no) in which the frontal (high-angle) image viewed on a position in near- -Table 6 -Discussion of study 2.

Study 2 shows that purchase intention for an online advertised product is higher when the advertisement is seen on a mobile device that is held in a position that allows the viewers to stand in a body posture that is consistent with the product image perspective within the advertisement. Therefore, purchase intention for front-view advertisement is increased when the mobile device is held in a near-vertical position. Conversely, purchase intention for high-angle perspective (birds' eye view) is higher when the mobile device is held in a near-horizontal position.

Study 2 also demonstrates that this positive consistency effect is mediated by mental simulation processes. Indeed, consistency between viewers' body posture and image perspective results in increase mental simulation, which in turn results in higher purchase intention. Results also suggest that inconsistency between body postures and image perspective is less impactful for high-angles perspectives (birds' eye view). Indeed, although front-view advertisements lead to lower mental simulation process when it is seen on a device held in a near-vertical position, mental simulation is not significantly and negatively impacted by the mobile device position when the advertisement depicts a high-angle product perspective. For high-angle perspectives, consistency between the position and the perspective of the image does not have any significant impact on mental simulation. This is consistent with previous studies on mental simulation processes triggered by the verticality of images. Indeed, Royo-Vela and Black (2018) show that images pictured from high-angle perspective have a greater effect on the viewer than images produced at ground level. This explains why, in study 2, mental simulation was not disrupted by tablet position when the perspective of the image is high angle.

CONCLUSION

Both studies that we conducted confirm that the impact of commercial photographs on internet users depends on the position of the mobile screen on which the photographs are viewed. Although we might expect that individuals would gradually become accustomed to processing the images on their mobile device regardless of the position and their actual body posture, this is not the case. Advertisements showing products from a high-angle perspective trigger stronger purchase intention if they are shown on a screen held near-horizontally. To the contrary, advertisements showing products with a The results thus contribute to mobile marketing research by showing that the effectiveness of a visual advertisement is improved when the image is viewed on a device held in a position that allows viewers' body postures (downward position, frontward position) to be consistent with the perspective of the advertising image (downward position for vertical perspective ad, frontward position for frontal perspective ad).

These results extend the perceptual-motor compatibility principle [START_REF] Förster | How body feedback influences consumers' evaluation of product[END_REF] by showing that approach behavior toward advertised products are easier to activate when the mobile device is held in a position that is consistent with the image perspective.

Perspective consistency helps the viewers to generate mental simulation of the product, which consequently has a positive effect on purchase intention. In frontal views advertisements, where the products are shot with the cameras are positioned in front of them, the mental movement needed to mentally grasp the products are frontward movements; they are easier to activate when the mobile device is held vertically because the viewers hold their heads straight, looking in front of them.

Although mental simulation mediates, for both types of perspective (front-views and high-angle views), the impact of body posture and image perspective on purchase intention, we find in this study that the device position does not have a significant impact on mental simulation for high-angle perspective advertisements. Previous research has shown that an advertisement with a high-angle perspective can engender surprise, entertainment, and potentially positive emotions (Royo-Vela and Black, 2018).

The literature on non-canonical perspectives (Kosslyn, Thompson and Alpert, 1999) points to the roles of surprise, attention, and commitment. Yet, when attention is higher, interferences have lower negative effects on individuals [START_REF] Catherine | Attentional Effects of Hand Proximity Occur Later in Older Adults: Evidence From Event-Related Potentials[END_REF]. Therefore, inconsistency between body posture and image perspective could be less impactful for high-angle images than for front-views images because viewers benefit from higher available cognitive resources to control the negative effect of inconsistency between their body posture (frontward head position) and image perspective (high-angle perspective).

This research extends the literature on the impact of digital interfaces on advertising efficacy [START_REF] Adam | Tablets, touchscreens, and touchpads: How varying touch interfaces trigger psychological ownership and endowment[END_REF][START_REF] Hao | Computer Interfaces and the "Direct-Touch" Effect: Can iPads Increase the Choice of Hedonic Food?[END_REF]. This paper contributes to a better understanding of mental simulation processes on images displayed on a mobile screen.

Very few research demonstrate empirically that mental simulation with advertised products on mobile happens spontaneously and drives products evaluation and intention to buy (Zhao et al, 2011). Usually mental simulation is considered to happen when a specific priming message such as "Imagine yourself interacting with the product" is proposed to consumers. Yet, we demonstrate in this paper that mobile advertising leads to spontaneous generation of mental simulation process, even if there is no priming, and that mobile device inclination modifies purchase intention toward advertised products.

Based on the literature on embodied cognition and advertisement effectiveness (e.g. [START_REF] Elder Ryan | Visual Depiction Effect in Advertising: Facilitating Embodied Mental Simulation through Product Orientation[END_REF], this research establishes a link between viewers' body postures and purchase intention, without mobilizing attitudes. An avenue for future research will be to examine what attitudinal mechanisms are at work when the advertisement image is shot from a high-angle perspective. A link between perspective consistency and the emotional reactions of internet users will have to be explored. This study also have several limits that are also potential future avenues of research.

Limits and future avenues of research

First, viewers' familiarity with the advertisements have not been controlled. Yet, the two advertisements present objects that evoke grasping representations relatively automatically. But there is still work to be done to understand the role of product previous knowledge on mental simulation processes and their impact on purchase intentions (Zhao et al, 2011).

Second, potential hand proximity effects have not been explored. Research shows that attentional resources are more focused and have a finer grain near the hands [START_REF] Catherine | Attentional Effects of Hand Proximity Occur Later in Older Adults: Evidence From Event-Related Potentials[END_REF]. In particular, because of this hand proximity effects, where perception and attention is higher, interference effects are shown to be lower proximal to the hands [START_REF] Catherine | Attentional Effects of Hand Proximity Occur Later in Older Adults: Evidence From Event-Related Potentials[END_REF]). Yet, we did not control how the viewers' put their hands on the mobile device to hold it. Negative impact of inconsistency between perspective and body postures could have been lower for viewers that held their screens in their hands, in comparison to those who let their screen on their knees. This hand proximity effect should be investigate in the future so as to better understand the role of viewers' body postures on mobile advertising effectiveness.

Managerial implications

This research encourages marketing to take into account the position in which the screen is viewed. Tablets and smartphones are already equipped with a gyroscope to measure the orientation of the tablet. The question of image perspective arises and certain websites have recently decided to systematically offer several views of the product 2 in order to give internet users multiple perspectives on the product. These initiatives currently do not take into consideration the position of the mobile screen, but 2 Like the jeweler James Allen, who provides the opportunity to view the product from all angles the first signs of questioning concerning online image perspective suggests that this research could quickly resonate with publishers of websites. In particular, pre-existing "web responsive design" practices could be supplemented by introducing the option to adapt product photographs to the orientation of the viewing screen (e.g. horizontal, vertical). Automatically adjusting the photograph of the product to the angle of the screen could maximize the effectiveness of online advertising.

In order to increase the external validity of this research, this work should be replicated on mobile phones. Although marketing resistance of mobile phones is increasing, with the popularity of programs blocking advertisements (such as Adblock Plus), mobile marketing on smartphones affects more than 7 billion people around the world. It would also be interesting to replicate this work using video, a format that is growing rapidly on mobile devices. In conclusion, this initial research is an invitation to further investigation into the interaction between content displayed on the internet and the position in which mobile screens are viewed. In this last version of the manuscript, we added some precisions to the presentation of the results and we edited and proof-read the manuscript so as to be sure there were no mistakes left.

We also carefully examined each of you remark. We addressed each of them in the table below. The reviews are in general favourable and suggest that, subject to minor revisions, your paper could be suitable for publication. Please consider these suggestions, and I look forward to receiving your revision.

We thank you for your encouraging remark. We addressed each comment and suggestion of the reviewers.

Reviewer: 1

Comments to the Author I believe the manuscript has progressed quite well in addressing the comments provided by the review panel. My major concerns raised earlier are about the conceptual development and theoretical contribution of the manuscript. In this round, the authors have addressed my concerns on these issues. To further improve the manuscript, I hope the authors find the following comments helpful:

We thank you for your positive feedback. In this last revised version, we carefully took into consideration each of your suggestions so as to continue improving the manuscript.

1. The statistics in §1(on p. 1) might be updated.

We updated the statistics presented in the introduction : "E-commerce growth is primarily being driven by consumers using their mobile devices, phones and tablets, to acquire goods and services (statista, 2018). Retail e-commerce sales reached $2.3 trillion in 2017, a 23.2% increase over the previous year. The mobile share of this stood at 58.9%, or $1.4 trillion. In 2021, mobile e-commerce could rake in some $3.5 trillion and then make up almost three quarters (72.9 %) of e-commerce sales (statista, 2018). Laptop and PC e-commerce drops off, although ecommerce on smartphones and tablets increases dramatically.

2. The reporting of the results of data analysis should be more professional. For example: (a) Please clarify the measurement scales of the confounding checks in Study 2: "We verified that, in all four conditions, the image was easy to understand and that information in the advertisement was easy to process (1.4<M<1.6; ps > 0.495)". Are the items for the confounding check measured on a 7-pint Likert scale? If so, you might want to report "the image was easy to understand and that information in the advertisement was easy to process (Ms = 1.??/7 vs/ 1.??/7, p > 0.1)" (p. 19).

We clarified the results presentation by indicating what type of scales had been used to measure the images. Then we added : "we used two 7 points Likert scale items and calculated an average score of the two items: "understanding this image requires effort of thought" and "to understand this image, you have to concentrate" We checked that both images were easy to understand in the two tablet positions. Therefore the analysis gave and compare 4 scores. To be simpler, we chose to indicate only the two extreme scores in the manuscript. Please find the four different scores, showing no significant differences between the four conditions : Mverticalfrontview = 1.4333 Mverticalbird'seyeview = 1.4091 Mhorizontal bird'seyeview = 1.5938 Mverticalfrontview= 1.6250 (b) When checking the demographic information of the subjects, it's better to report there is no difference in terms of familiarity of digital tablets and general attitudes towards the experimental Following your suggestion, we explicitly added in the sentence : "There was no difference in terms of familiarity with digital tablets and general attitudes towards the experimental stimuli". (c) Results of Study 2: It would be clearer to report the two ANOVA tests one by one. First, purchase intention was submitted to two-way ANOVA. Does the analysis reveal any main effect of device position and/or ad perspective (in addition to the interaction effect)? If so, please report them accordingly, before proceeding with the contrast analysis. Second, mental simulation was submitted to two-way ANOVA. Similarly, reports any main effects, interaction effect, and the results of contrast analysis.

Following your suggestion, we indicated the results of the main effect analysis before reporting the result of the interaction effect analysis. Indeed, we indicated that there were no direct of device position and image perspective on purchase intention and on mental simulation (p>0.05) 3. Please edit and proof-read the manuscript again to correct the grammar mistakes. For example, "In doing so by underlining the role of the device tilt angle mental simulation and purchase intention" (p. 2) might be changed to " the role of the device tilt angle, mental simulation, and purchase intention" or " the role of the device tilt angle and mental simulation in stimulating consumers' purchase intention".

We thank you for your suggestion. We edited and proof-read the entire manuscript again.

Reviewer: 2

Comments to the Author Please do a proof reading and make sure there is no mistake in data analysis before final submission.

Finally

  , the article reaches a conclusion on the contributions and limitations of the research and suggests several avenues for future research.

  their head is consistent with the frontward perspective of the picture. Conversely, advertisements showing products from a high-angle perspective should trigger stronger purchase intention if they are seen on a device held horizontally.

  pilot studies in order to determine in what positions internet users usually hold their tablet. The objective is to test the hypotheses on positions that are often used by internet users and thereby maximize the external validity of the results.

  posture with the perspective of the picture when he holds the device in a near-vertical position.

  [START_REF] Laurie | A Modified Scale for the Measurement of Communication-Evoked Mental Imagery[END_REF]. For example, mental imagery research of Bone and

  the mental simulation and intent to purchase variables. The test indicates that mental simulation is easier when the tablet is in the near-vertical position, in other words, when it is aligned with the frontal perspective of the image (versus a tablet in a near-horizontal position) (Mhoriz. = 3.15; Mvert. = 4.05;

(

  2015) on gaze orientation and level of thought, some might say that looking downward leads one to adopt more concrete thinking, which encourages the individual to focus on the negative elements of the product[START_REF] White Katherine | It's the Mind-Set that Matters: the role of construal level and Message Framing in influencing consumer efficacy and conservation Behaviors[END_REF]. Indeed, different points of views could convey various associations of ideas. A study conducted in 2012 by and Schwarz demonstrated that the viewing angle of photographs of cars impacted the assessment of the product and characteristics related to power. As a result of this work, holding the device horizontally and adopting a high-angle view could increase the impression of dominating one's environment and thus decrease preferences for low-value products (such as the kitchen aid product depicted in the study 1 advertisement).

  -horizontal) position was consistent in terms of perspective. Results indicated that perspective consistency was a significant predictor of mental simulation, b = -0.31, SE = 0.13, p < .05, and that mental simulation was a significant predictor of purchase intention, b = 0.80, SE = 0.11 p <.05. These results support the mediational hypothesis. Perspective consistency was no longer a significant predictor of purchase intention after controlling for the mediator, mental simulation, b = 0.0008, SE = 0.13, ns, consistent with full mediation. The indirect effect was tested using a bootstrap estimation approach with 5000 samples. These results indicated the indirect coefficient was significant, b = -0.25, SE = 0.13, 95% CI = -0.5437, -0.0355. In fact, zero is not included in the confidence interval [-0.5437; -0.0355]. The results indicate that mental simulation mediates the effect of perspective consistency on purchase intention. H3 is validated.

  perspective result in higher purchase intention when the device is held in a near-vertical position.

  Hoeffler Steve, ZauberMan Gal (2011), "Mental Simulation and Product evaluation: the affective and Cognitive Dimensions of Process versus outcome Simulation", Journal of Marketing Research, 48 (October), 827 -839. Zhou Tao (2013), "The effect of flow experience on user adoption of mobile TV", Behaviour & InformationTechnology, 32 (January), 263-272.

  FIGURES AND TABLES

Figure 4 :

 4 Figure 4: Effect of tilt angle tablet position (study 2)

Table 5 -

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Table 1 : Position of the tablet, mental simulation, and purchase intention (study 1)

 1

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	Tablet in near-	Tablet in near-
	vertical position	horizontal position
	(frontal view <45°)	(high-angle view >45°)
	40°	80°

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Table 2 : Mediation of mental simulation on purchase intention (study 1)

 2

	4 conditions	Position	Position
		near-vertical	near-horizontal
	High-angle perspective
		Inconsistency of perspective	Consistency of perspective
	Frontal perspective
		Consistency of perspective	Inconsistency of
			perspective

Table 3 : Experimental design of study 2

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Table 4 : Sample of study 2

 4

	Frontal	High-angle
	perspective	perspective

Table 5 : Tablet position, mental simulation, and behavior (study 2)

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We thank you for your suggestion. We edited and proof-read the entire manuscript again. We also carefully checked that there were not any mistake in the data analysis before final submission.

APPENDIX Appendix 1: Image used for study 1

Appendix 2: Images used for study 2

Frontal perspective

High-angle perspective