Muhammad Khalid Salamat

Mohammed M Moudjou

Jerome Chapuis

Laetitia Herzog

Emilie Jaumain

Vincent Béringue

Human Rezaei

Human Rezaei ‡ Annalisa Pastore

Hubert H Laude

Michel Dron
email: michel.dron@jouy.inra.fr.

Khalid Salamat

Jérôme Chapuis

Integrity of helix 2-helix 3 domain of the PrP protein is not mandatory for prion replication

Keywords: PrP, prion protein, PrP Sc , scrapie-associated PrP, PK, proteinase K, PrP res , PK-resistant PrP Sc, IMAC, immobilized metal affinity chromatography, HSQC, heteronuclear single quantum coherence, PNGase, peptide:N-glycosidase, BisTris,

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mal amino acid substitutions in the PrP protein can hamper prion replication and/or may have a dramatic effect on the susceptibility to infection (28 -31). Deletions within the proteaseresistant domain, whereas inducing neuropathology [START_REF] Shmerling | Expression of amino-terminal truncated PrP in the mouse leading to ataxia and specific cerebellar lesions[END_REF][START_REF] Drisaldi | Genetic mapping of activity determinants within cellular prion proteins. N-terminal modules in PrPC offset pro-apoptotic activity of the Doppel helix B/BЈ region[END_REF][START_REF] Baumann | Lethal recessive myelin toxicity of prion protein lacking its central domain[END_REF][START_REF] Li | Neonatal lethality in transgenic mice expressing prion protein with a deletion of residues 105-125[END_REF], generally do not allow prion replication, except for PrP106, i.e. PrP with two deletions (⌬23-88, ⌬141-176), the so called miniprion [START_REF] Supattapone | Prion protein of 106 residues creates an artifical transmission barrier for prion replication in transgenic mice[END_REF]. However, subsequent studies led these authors to propose the participation of the 141-176 region in the structural changes [START_REF] Govaerts | Evidence for assembly of prions with left-handed ␤-helices into trimers[END_REF]. Recently Myc or tetracysteine tags were introduced by insertion or sequence modification at one each end of the PK-resistant domain (36 -38), and shown to be compatible with prion conversion. However, introduction of such tags farther within this domain had so far prevented prion replication [START_REF] Taguchi | Specific biarsenical labeling of cell surface proteins allows fluorescent-and biotin-tagging of amyloid precursor protein and prion proteins[END_REF][START_REF] Goold | Rapid cell-surface prion protein conversion revealed using a novel cell system[END_REF]. Here we report the conversion into prion of several PrP mutants with an insertion of 8 -16 amino acids in the middle of the H2-H3 region, hence demonstrating an unexpected tolerance to sequence modification in a central part of the PK-resistant core.

EXPERIMENTAL PROCEDURES

Ethic Statement-All animal experiments have been performed in accordance with the European Community Council directive 86/609/EEC.

Generation of PrP Insertion Mutants-Insertions and substitutions were introduced by site-directed mutagenesis (QuikChange II mutagenesis kit, Stratagene) into sheep PrP (allotype Val 136 -Arg 154 -Gln 171 ) cloned into pTRE plasmid (Clontech) using mutagenic primers (supplemental Table S1). The plasmid encoding PrP-ins203, i.e. with an in-frame insertion of 24 bp, was isolated during screening of an experiment originally intended to modify the second site of glycosylation [START_REF] Salamat | Prion propagation in cells expressing PrP glycosylation mutants[END_REF]. The sequences of all the mutant constructs were verified by sequencing. Numbers indicated for each insertion refer to the position of the first amino acid of the insert according to the sheep PrP numbering.

Cell Culture and Isolation of Rov Cells-Rov cells are epithelial RK13 cells that stably express either wild-type (WT) or mutant ovine PrP using a tetracycline-inducible system [START_REF] Salamat | Prion propagation in cells expressing PrP glycosylation mutants[END_REF][START_REF] Vilette | Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein[END_REF]. They were obtained by transfection and puromycin selection and were grown in Opti-MEM medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) and antibiotics, and split at 1:4 after trypsin dissociation once a week. To assess PrP C expression, cells were cultivated in the continuous presence of 1 g/ml of doxycycline (Sigma).

Antibodies-Several mouse anti-PrP monoclonal antibodies (mAb) were used. The 4F2 mAb [START_REF] Féraudet | Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells[END_REF] is an IgG2b directed to the octarepeat domain (54 -92, sheep PrP numbering) and was used to detect PrP C ; Sha31 mAb (epitope 148 -159) [START_REF] Féraudet | Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells[END_REF] was used to detect PrP res on immunoblots. The V14 mAb (IgG2a) recognizes a conformational epitope (191)(192)(193)(194)(195)(196)(197)(198)(199), sensitive to the presence of N-glycan chains [START_REF] Eghiaian | Insight into the PrPC3 PrPSc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants[END_REF][START_REF] Moudjou | Glycan-controlled epitopes of prion protein include a major determinant of susceptibility to sheep scrapie[END_REF]. ICSM33 (IgG2b, epitope 91-110, D-Gen Ltd., London) was used to detect denatured PrP Sc by immunofluorescence [START_REF] Paquet | Efficient dissemination of prions through preferential transmission to nearby cells[END_REF]. 12F10 (IgG2a epitope 142-160, [START_REF] Krasemann | Generation of monoclonal antibodies against human prion proteins in PrP0/0 mice[END_REF] was used for PrP res detection on histoblots. The anti-FLAG M2 mAb was from Sigma. Alexa Fluor (either 468 or 568) conjugated goat anti-mouse IgG, IgG1, IgG2a, and IgG2b were purchased from Molecular Probes and were all used at a 1/500 dilution.

Prion Infection of Cell Cultures-Rov cells were infected with the cloned 127S scrapie strain as previously described (31) using 1% (w/v) brain pool homogenates of terminally ill tg338 mice [START_REF] Vilotte | Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine PrP[END_REF]. Two days postexposure cells were washed, incubated for 2 additional days, trypsinized, and split at 1:10 dilution. Cells were further incubated for 1 week before analysis (passage 1) and split at 1:4 dilution at each following passage. To test the infectivity of cultures propagating mutant PrPr Sc cells were harvested at passage 6 -8 postexposure, pelleted, frozen and thawed, then sonicated, and used as inoculums.

Cell Lysis, Proteinase K Digestion, and PNGase Treatment-Cells were washed twice with cold phosphate-buffered saline and whole cell lysates were prepared at 4 °C in TL1 buffer (50 mM Tris-HCl, pH 7.4, 0.5% sodium deoxycholate, 0.5% Triton X-100). Lysates were clarified by centrifugation for 2 min at 800 ϫ g and protein concentrations were determined by microBCA assay (Pierce). For PrP res , lysates were incubated with 4 g of PK per 1 mg of protein, for 2 h at 37 °C, then centrifuged for 30 min at 22,000 ϫ g. Pellets were dissolved in Laemmli sample buffer and boiled for 5 min at 100 °C. When needed, 500 units of PNGase F (New England Biolabs, MA) and 1% Nonidet P-40 were added to denatured proteins that were further incubated at 37 °C [START_REF] Dron | Endogenous proteolytic cleavage of disease-associated prion protein to produce C2 fragments is strongly celland tissue-dependent[END_REF].

Transgenic Mice, in Vivo Infections, and Tissue Homogenate Preparation-To test for the infectivity of PrP Sc -ins203, 5 ϫ 10 6 Rov-ins203 or RK13 cells were harvested by scraping at passage 8 post-infection. Cell pellets were resuspended in 100 l of medium containing 5% glucose and antibiotics, then frozen and thawed three times before sonication for 1 min. A 20-l aliquot of these cellular lysates was injected intracranially to tg338 mice overexpressing the VRQ allele of ovine PrP [START_REF] Vilotte | Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine PrP[END_REF]. Brains and spleens of infected animals were harvested at the terminal stage of the disease and analyzed for PrP res content and distribution by immunoblots and histoblots, respectively, as described previously [START_REF] Langevin | Marked influence of the route of infection on prion strain apparent phenotype in a scrapie transgenic mouse model[END_REF].

Immunoblotting Detection of PrP C and PrP res -Either 12 or 4 -12% NuPage BisTris polyacrylamide gels (Invitrogen) were used for SDS-PAGE. For PrP C analysis, 50 g of protein were deposited on the gel. For PrP res , samples corresponding to the PK-resistant PrP present in 50 g of protein of the cellular lysate were loaded on the gel, except for PrP res -ins(Gly 6 ) and PrP res -ins(FLAG), which an equivalent of 250 g of protein were loaded as indicated in the figure legends (Figs. 4D and5D). The transfer of proteins, their detection and revelation were described previously [START_REF] Dron | Endogenous proteolytic cleavage of disease-associated prion protein to produce C2 fragments is strongly celland tissue-dependent[END_REF].

Conformational Stability Assay-Cellular lysates were brought to the desired concentration of guanidine HCl, incubated for 1 h at 20 °C under agitation, then diluted to reach the same volume and guanidine HCl concentration (800 l, 0.5 M). 200 l of 5ϫ digestion buffer (750 mM Tris-HCl, pH 7.4, 10% Sarkosyl and 20 g/ml of PK) was added and samples were incubated for 1 h at 37 °C under agitation, before methanol precipitation and analysis by immunoblotting.

Cu 2ϩ -Immobilized Metal Affinity Chromatography (IMAC) Hitrap Chromatography-The AKTA Purifier100 FPLC chromatographic system was used (GE Healthcare) as previously described [START_REF] Dron | Endogenous proteolytic cleavage of disease-associated prion protein to produce C2 fragments is strongly celland tissue-dependent[END_REF][START_REF] Moudjou | Glycan chains modulate prion protein binding to immobilized metal ions[END_REF]. One milliliter of cell lysates (1 mg of protein) made in TNT buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1% Triton X-100) supplemented with protease inhibitors (Roche Applied Science) was injected into the column. Columns were washed and a linear gradient of 3-200 mM imidazole in TNT buffer was applied to elute column-bound proteins at a flow rate of 1 ml/min for 10 min; 0.5-ml fractions were collected. To solubilize aggregates of PrP Sc -ins(His 6 ), all buffers were complemented with 4 M urea. Eluted fractions were either treated with 5 g/ml of PK and precipitated with cold methanol after addition of 20 g of BSA as a protein carrier or methanol precipitated without PK treatment for total PrP. Individual columns were dedicated to each type of lysate.

Immunofluorescence-Cells were fixed with 4% paraformaldehyde and PrP C was detected as previously described [START_REF] Salamat | Prion propagation in cells expressing PrP glycosylation mutants[END_REF][START_REF] Dron | Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells[END_REF]. For PrP Sc immunodetection, fixed and permeabilized cells were treated for 5 min with 3.5 M guanidium thiocyanate and washed three times before exposure to ICSM33 mAb as previously described [START_REF] Paquet | Efficient dissemination of prions through preferential transmission to nearby cells[END_REF][START_REF] Dron | Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells[END_REF], or to both ICSM33 and M2 anti-FLAG mAbs and then to fluorescent anti-IgG2b and anti-IgG1. Images were acquired with an Axio observer Z1 microscope (Zeiss) and a coolsnap HQ 2 camera (Photometrics) driven by the Axiovision imaging system software.

NMR Measurements-Recombinant proteins (WT PrP, PrP-ins191, and PrP-ins193) were produced and purified from Escherichia coli as published previously [START_REF] Adrover | Prion fibrillization is mediated by a native structural element that comprises helices H2 and H3[END_REF]. HSQC experiments were run at 25 °C on 0.5-0.7 mM 15 N-labeled samples of wildtype PrP and mutants using a VARIAN INOVA 600 MHz. The samples were in 5 mM MOPS buffer, pH 7.0. Water suppression was achieved with a standard Watergate pulse sequence.

Circular Dichroism-CD measurements were carried out on a Jasco-715 spectropolarimeter equipped with a thermostated cell holder controlled by a Jasco Peltier element. Far-UV CD spectra were recorded from 260 to 195 nm at 25 °C in a 1-mm path-length quartz cuvette at a protein concentration of 12 M in 5 mM MOPS buffer, pH 7.0. Each CD spectrum was obtained by averaging 15 scans collected at a scan rate of 200 nm/min. Baseline spectra obtained with buffer were subtracted for all spectra. The thermal denaturation scans were obtained by heating a 12 M sample from 20 to 97 °C at 2 °C/min and by monitoring the ellipticity at 220 nm with a 4-s averaging time and a 0.2 °C data pitch. The thermogram was analyzed assuming a two-state model.

RESULTS

PrP C with Extended H2-H3 Loop Can Convert into Prion-In a previous study aimed at producing PrP glycosylation mutants [START_REF] Salamat | Prion propagation in cells expressing PrP glycosylation mutants[END_REF], we identified a mutant that contained an insertion of 8 amino acids downstream from the second glycosylation site, within the unstructured loop joining helices H2 and H3 of sheep PrP (Fig. 1, A andB). This mutant, denoted PrP-ins203, arose fortuitously probably due to a misalignment of the mutagenic primers used. To examine the effects of this mutation on PrP glycosylation and permissiveness to prion infection, PrP-ins203 was stably expressed in RK13 cells. Immunoblotting analysis revealed the presence of mono-and diglycosylated PrP-ins203 but the apparent lack of the unglycosylated form (Fig. 1C). By quantification on overexposed blots, this latter form was found to be 15-20-fold less abundant than WT PrP (Fig. 1D). PrP-ins203 polypeptide showed a slightly reduced mobility compared with WT PrP, consistent with the presence of additional residues (Fig. 1E). The anti-PrP mAb V14, which recognizes a conformational epitope (amino acids 191-199) encompassing a region located nearby upstream of the site of insertion of the octapeptide ins-203 [START_REF] Eghiaian | Insight into the PrPC3 PrPSc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants[END_REF][START_REF] Moudjou | Glycan-controlled epitopes of prion protein include a major determinant of susceptibility to sheep scrapie[END_REF], was unable to recognize PrP-ins203 (Fig. 1E and supplemental Fig. S1). This suggested that the presence of additional amino acids just downstream of the V14 epitope somehow modifies the local conformation of the PrP. Yet the insertion mutant was correctly routed to the cell surface, as ascertained by PrP immunofluorescence labeling of intact cells (Fig. 1F) and presence in lipid rafts (supplemental Fig. S2).

Cells expressing either WT or mutant PrP were infected with the 127S strain of ovine prion and analyzed for their PrP res content at subsequent passages of cultures. The immunoblot in Fig. 2 shows that PrP-ins203 was converted into a self-propagating, protease-resistant species with amounts of PrP res comparable with those in cells expressing WT PrP (Fig. 2). After PNGase treatment, the PrP res fragment of the insertion mutant was shown slightly bigger in size than WT PrP, thus indicating that the inserted amino acid stretch was protected from protease digestion in PrP Sc . To further characterize the PrP-ins203 mutant we subjected it to a conformational stability assay using guanidine hydrochloride as a denaturing agent [START_REF] Peretz | Strain-specified relative conformational stability of the scrapie prion protein[END_REF]. The insertion did not appreciably modify the resistance to denaturation (Fig. 3A), because half-denaturation of the mutant and WT PrP Sc occurred at 1.4 Ϯ 0.2 and 1.3 Ϯ 0.4 M, respectively.

PrP-ins203 conferred cells similar susceptibility to prion infection as WT PrP in cell cultures exposed to serial dilutions of 127S-infected mouse brain homogenate. Both Rov-wt and Rov-ins203 produced large amounts of PrP res after exposure to inoculum at the 10 Ϫ3 dilution, whereas no PrP res was detected beyond the 10 Ϫ4 dilution (Fig. 3B). To confirm that PrP-ins203 led to the generation of infectious prion, the cell homogenate from 8 passages on Rov-ins203 cultures was inoculated to naive Rov-ins203 or Rov-wt cells, and to tg338 mice. Both homotypic and allotypic cell cultures were successfully infected based on PrP res accumulation (Fig. 3C). Mice inoculated with the PrP-ins203-derived homogenate died at 61 Ϯ 2 days with typical TSE signs (Fig. 3D) and showed the presence of PrP res with a glycoform pattern and regional deposition in the brain typical of the 127S strain (supplemental Fig. S3). Altogether these results demonstrate that a mutant PrP with an 8-amino acid insertion in the PK-resistant core is efficiently convertible into bona fide prion.

Insertion of Glycine Residues, or Extension of the H2-H3 Loop Up to 16 Amino Acids Do Not Prevent Prion Replication-We

modified the original insertion already containing 3 glycine residues to create a segment containing 6 glycines (Fig. 4A). Because a feature of the polyglycine stretch is to be incompatible with the formation of secondary, either ␣-helix or ␤-sheet structures, it was assumed that the insert ins203(Gly 6 ) would neither participate to elongation of H3 nor convert into a ␤-sheet segment if PrP Sc were to be produced. When expressed in cells, PrP-ins203(Gly 6 ) showed a glycoform pattern with a low level of the unglycosylated form as for the original PrP-ins203, and some decrease in the proportion of the diglycosylated form (Fig. 4B). PrP-ins203(Gly 6 ) was efficiently routed to the cell surface and found to be associated with lipid raft fractions (supplemental Fig. S2) and was converted into PK-resistant species upon infection with 127S prion (Fig. 4, C andD). PrP Sc -ins203(Gly 6 ) was readily propagated along with five subpassages, although the amount of PrP res was reduced by at least Infected cell cultures were split once a weak and samples of PK-digested cellular lysates were analyzed by immunoblotting, the passage number is indicated at the top of the upper panel. Note that the unglycosylated form of PrP Sc -ins203 was not detected to this level of exposure and that, as expected, mock-infected cells (m.i.) did not produce protease-resistant PrP. In the lower two panels, PrP res was treated with PNGase F before immunoblotting. Sha31 anti-PrP antibody recognizes an epitope located in the helix H1, whereas V14 antibody recognizes a conformational epitope previously localized nearby and upstream of the insert. 10-fold compared with that in WT PrP Rov cells (Fig. 4D). Thus, extension of the loop between H2 and H3 by a polyglycine stretch remained compatible with PrP Sc formation, suggesting that this part of the protein is unstructured in misfolded conformers.

We also introduced an 8-amino acid FLAG sequence into the original ins203 octapeptide, enabling us to recognize the presence of the insert using a relevant antibody (Fig. 5A). As for other ins203 insertions, the unglycosylated forms of PrP-ins-(FLAG) were produced in low amounts in RK13 cells (Fig. 5B), and FLAG-PrP was efficiently expressed at the cell surface (Fig. 5C). Rov-ins203(FLAG) were susceptible to prion infection and generated self-propagating PrP Sc , however, the PrP res levels were markedly reduced compared with WT PrP (Fig. 5D). Cells in which PrP Sc had accumulated efficiently exhibited numerous aggregates labeled, after guanidium thiocyanate denaturation, by anti-PrP and anti-FLAG antibodies (Fig. 5E). We further showed that PrP Sc -ins(FLAG) autopropagated in cell culture was also infectious for Rov-wt cells (Fig. 5F). Altogether these results established that a PrP C with a 4-fold increase, i.e. from 5 to 21 amino acids, of the size of the sequence between H2 and H3 can be converted into bona fide prions.

Production of His-tagged PrP Sc -As a means to facilitate PrP purification, the first 6 amino acids of ins203 were substituted by histidine residues and the resulting PrP-ins203(His 6 ) was expressed in RK13 cells (Fig. 6A). The functionality of the tag was tested by loading cellular lysates onto Cu 2ϩ -IMAC columns. Because PrP already contains an N-terminal octarepeat sequence, a strong binding site for divalent ions, both WT and the His-tag PrP C were retained on such columns (Fig. 6B). However, PrP-ins(His 6 ) was eluted at a much higher concentration of the Cu 2ϩ competitor imidazole (150 mM) than WT PrP (90 mM), indicating that the His tag actually increased the avidity for immobilized copper ions of PrP (Fig. 6C), including diglycosylated molecules where complex N-glycan chains are attached close to the tag. Interestingly, the fractions in which His tag PrP was eluted contained at least 10-fold less contaminant proteins than the fractions containing WT PrP (not shown). To determine whether the H2-H3 segment was accessible to interaction into PrP Sc molecules as well, Rov-ins(His 6 ) cells were subjected to prion infection. Infected cells produced PrP res from the first passage on, the amount of which increased gradually on subpassaging to reach a level comparable with that produced by Rov-wt after 7-8 passages. His-tag PrP Sc was able to infect cultures expressing homotypic as well as WT PrP sequence (Fig. 6, C and D). Anti-His antibodies recognized the PK-resistant fragment (Fig. 6C, right panel). The half-denaturation value of PrP Sc -ins(His 6 ) in conformational stability assay was 1.3 Ϯ 0.25 M, similar to WT and ins203 PrP res (see above).

We also asked whether a His tag inserted in the PK-resistant core would still bind to copper-immobilized ions. Although we did show binding of the His tag PrP Sc to IMAC Cu 2ϩ columns (Fig. 7), the bulk of PrP Sc did not bind and was either excluded in the flow-through (Fig. 7, top panel) or retained on the column even after application of imidazole at high concentrations (not shown). To improve the purification of His-tagged PrP Sc , cellular lysates were incubated with 4 M urea prior to application on the columns. Proteins recovered by elution with increasing imidazole concentrations were PK-digested and analyzed by immunoblotting. WT PrP Sc , present in Rov cellular lysates essentially as N-terminal truncated species [START_REF] Dron | Endogenous proteolytic cleavage of disease-associated prion protein to produce C2 fragments is strongly celland tissue-dependent[END_REF], bound to Cu 2ϩ columns with low avidity and started to elute at 40 mM imidazole (Fig. 7, lower panel). His-tagged PrP res was eluted at higher concentrations of imidazole, starting at 88 mM (Fig. 7, middle panel), indicating that the tag could be functional for the PrP res species under adequate conditions of solubility.

Integrity of C-terminal Part of Helix-2 Is Not Essential for

Prion Conversion-To determine how variable the exact location of the 8-amino acid insertion could be, we moved it upstream and downstream of the original position (Fig. 8A). The insert was introduced at the other end of the inter helix loop (position 198), or in helix H2 (195 to 187) or helix 3 (207 and 210). These mutants were expressed and efficiently reached the cell surface (supplemental Fig. S4A), except PrP-ins210, which was not detectably expressed at the membrane but showed intracellular accumulation, essentially at a juxtanuclear position (supplemental Fig. S4B). PrP-ins198 did not confer Rov cells susceptibility to prion infection, indicating that even in the unstructured segment considered, the site of insertion might be critical. Cells expressing H3 mutants, Rov-ins207 and Rov-ins210, were not permissive to the infection, yet the convertibility of PrP-ins210 could not be adequately tested due to impaired trafficking.

In contrast, PrP with insertion into H2, 3 amino acids before the end of the helix (position 195) and then further upstream with 1 amino acid increment up to 193 conferred permissiveness to the cells and led to reproducible and robust infection as attested by the formation of self-propagating PrP res (Fig. 8B). Demonstration of the infectivity of each of the three mutant PrP Sc was ascertained by using cellular homogenates to challenge naive Rov-wt and homotypic cells (supplemental Fig. S5). Insertion further upstream markedly affected (PrP-ins192) or even hampered (PrP-ins191 and PrP-ins187) prion replication (Fig. 8B). These results showed that insertion of extra amino acids in the part of the protein corresponding to last two turns of H2 (Fig. 9) did not prevent prion formation, whereas introduction of the same insertion further upstream in the helix impaired conversion.

Insertions in H2 C Terminus Do Not Interfere with PrP-fold-To understand whether the inserted sequences affect the fold of PrP, we recorded NMR 15 N HSQC experiments, which are sensitive to protein tertiary fold. As insertions in H2 could be more destabilizing for mutants than insertion in the loop, we specifically analyzed PrP-ins193 and PrP-ins191. The former is competent for prion conversion but not the latter, although they are both expressed at the cell surface and found associated with lipid rafts (supplemental Figs. S2 andS4). Moreover the insertion is moved only 2 amino acids toward the center of H2 in the PrP-ins191 with respect to the PrP-ins193. The spectra of the wild-type and mutant proteins were similar (Fig. 10A). They all showed a good resonance spreading that is typical of folded proteins indicating that the insertions do not significantly affect the tertiary structure, which is thus retained. Minor differences can reasonably be attributed to a different packing of the secondary structural elements in the three proteins that leads to different local chemical environments. These results are consistent with previous observations that had proven that the presence of the disulfide bridge in the middle of H2 and H3 is essential for retaining the fold of the core element H2-H3 (25): we do not observe a major destabilization because the insertions introduced do not affect the disulfide bridge.

To understanding further the role of the insertions, which is whether they introduce additional secondary structures by elongating the helices or merely substitute the native structure, we recorded far-UV CD spectra of PrP-ins191 and PrP-ins193 and compared them with that of the wild-type protein (Fig. 10B). They were identical within experimental error suggesting that the additional residues do not promote formation of an additional secondary structure. Finally, we questioned whether the insertions would have an effect on the fold stability. We recorded temperature scans followed by CD. We observed that, whereas having very similar highly cooperative behavior, the wildtype seemed to reproducibly be 2-3 °C more stable than the two mutants suggesting that, if at all, the insertions have a mild destabilizing effect (Fig. 10B). Taken together these results tell us that these insertions do not perturb the overall fold of PrP C .

Conversion into Prion of Mutant with Two Insertions-We have also constructed a double mutant by inserting the octapeptide concomitantly at two places previously found to be tolerant to insertion, i.e. at position 195, into H2, and at position 203, in the loop between H2 and H3. Upon infection, PrP-ins(195 ϩ 203) was convertible into a prion that was readily and reproducibly (3 times out of 3 trials) propagated in cell culture (Fig. 8). Thus again, mutant PrP with 16 additional amino acids in a central part of the PK-resistant core proved to be apt to form prion.

DISCUSSION

In this study, we expressed a number of sheep PrP mutants displaying a peptide insertion at various places and we show that nine mutants with exogenous sequences introduced in the central part of the H2-H3 region remain convertible into prions, thus questioning how critical is the integrity of this region for the conversion into PrP Sc . This issue is important particularly in view of several recent reports proposing the crucial involvement of the H2-H3 region in misfolding events leading to prion formation [START_REF] Smirnovas | Structural organization of brain-derived mammalian prions examined by hydrogen-deuterium exchange[END_REF][START_REF] Cobb | Molecular architecture of human prion protein amyloid. A parallel, inregister ␤-structure[END_REF][START_REF] Tycko | The ␣-helical C-terminal domain of full-length recombinant PrP converts to an in-register parallel ␤-sheet structure in PrP fibrils. Evidence from solid state nuclear magnetic resonance[END_REF][START_REF] Kunes | Left-handed ␤-helix models for mammalian prion fibrils[END_REF][START_REF] Chakroun | The oligomerization properties of prion protein are restricted to the H2H3 domain[END_REF][START_REF] Prigent | PrP assemblies. Spotting the responsible regions in prion propagation[END_REF].

PrP Proteins with Extended H2-H3 Interhelix Segment Remain Convertible into Prions-Four PrP mutants were built with insertions at position 203 that increased about 2-4-fold the size of the loop joining H2 and H3. The resulting proteins were glycosylated and properly routed to the cell surface, whereas sharing a notable reduction of unglycosylated PrP species. The latter feature might result from an increased glycosylation efficiency [START_REF] Ben-Dor | Biases and complex patterns in the residues flanking protein N-glycosylation sites[END_REF] or a reduced stability of this form, or both, as observed for the F198S mutation in human [START_REF] Dlouhy | Linkage of the Indiana kindred of Gerstmann-Sträussler-Scheinker disease to the prion protein gene[END_REF][START_REF] Zaidi | Characterization of the F198S prion protein mutation. Enhanced glycosylation and defective refolding[END_REF][START_REF] Meli | Dynamic diagnosis of familial prion diseases supports the ␤2-␣2 loop as a universal interference target[END_REF] and for its equivalent in the sheep sequence, F201S [START_REF] Salamat | Prion propagation in cells expressing PrP glycosylation mutants[END_REF], both of which are close to the insertion site. Upon prion infection ins203 mutants produced self-propagating PrP Sc albeit at different levels. The original PrP-ins203 generated as much PrP res as WT PrP, whereas cells expressing the His-tagged PrP had to undergo several passages before reaching similar levels, suggesting an adaptation of the prion strain to this particular substrate or selection of most performing components [START_REF] Li | Darwinian evolution of prions in cell culture[END_REF]. The other two PrP mutants produced lower quantities of PrP res , which may reflect reduced replication efficiency, or a lower stability of the misfolded protein in the cells. Nevertheless, these insertion mutants of PrP Sc were de novo infectious for naive cells or for transgenic mice expressing the sheep PrP. Altogether these results proved that bona fide prions could be generated despite a substantial increase in size of the segment separating H2 and H3, the heart of structured PrP Sc . The magnitude of acceptable changes in this region was impressive because not only was the size of the H2-H3 connecting segment increased, but the physicochemical characteristics of the loop were also drastically altered. In PrP-ins(His 6 ) and PrP-ins-(FLAG), in particular, the calculated isoelectric point and charge at pH 7 of the loop segment were markedly modified.

PrP C and PrP Sc Conformations of Insertion Mutants in Position 203-The loss of the conformational V14 epitope in PrP-ins203 might suggest some local dynamic change of the conformation of the region immediately upstream of the insert (56), as the epitope was reported to cover the domain between the two glycosylation sites, from the middle of H2 to the beginning of the H2-H3 loop [START_REF] Eghiaian | Insight into the PrPC3 PrPSc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants[END_REF][START_REF] Moudjou | Glycan-controlled epitopes of prion protein include a major determinant of susceptibility to sheep scrapie[END_REF]. However, another possibility is that the size of the segment encompassing the epitope would have been previously underestimated. PrP Sc -ins203 had a similar resistance to guanidium denaturation as WT PrP Sc , suggesting no major structural variation between the two misfolded proteins, despite dramatic under-representation of aglycosylated species in PrP Sc -ins203. Moreover, the strain features in tg338 mice of PrP Sc -ins203 were typical of the 127S strain propagated on WT PrP cultures. Importantly, the introduction of six consecutive glycines in the insert did not preclude prion conversion. This finding strongly suggests that the corresponding segment was not changed into the ␤-sheet in PrP Sc , and comes in support of the structurally based prediction that the loop between H2 and H3 remains unstructured in PrP Sc [START_REF] Govaerts | Evidence for assembly of prions with left-handed ␤-helices into trimers[END_REF][START_REF] Cobb | Molecular architecture of human prion protein amyloid. A parallel, inregister ␤-structure[END_REF].

Prions Can Be Tagged inside the PK-resistant Core-The insertion of tags might be useful to study the biology of prion infection. Previous observations tended to indicate that the ends of the PK-resistant fragment could be modified, whereas the inside part could not (36 -38). In fact, as shown here, tags can be introduced within the protease-resistant segment without blocking prion replication. Despite their location between two heavy N-glycan chains, the His tag and FLAG tag were easily accessible in PrP C and proved to be useful for purification and immunodetection experiments. Yet, purification or detection of His-tagged PrP Sc could not be achieved without partial solubilization of aggregates by urea. Also, PrP Sc -ins(FLAG) was not recognized by anti-FLAG antibodies without prior guanidine treatment of the cells. Both results are consistent with previous observations on other tagged PrP Sc and the inaccessibility of all these tags to proteolytic enzymes in misfolded PrP (36 -38).

Integrity of the Last Two Turns of H2 Is Not Mandatory for

Prion Replication-Insertion of the octapeptide (AQQ-GGGFT) either at the beginning of the H2-H3 loop (position 198) and in the N-terminal part of H3 (position 207) impaired the formation of prion. Nevertheless, the integrity of the extremity of H3 or of the first residue of the H2-H3 loop is unlikely to be essential for prion conversion because insertion of another sequence at close proximity appeared to be compatible with PrP res formation [START_REF] Geissen | Inhibition of prion amplification by expression of dominant inhibitory mutants. A systematic insertion mutagenesis study[END_REF]. In contrast, introduction of the octapeptide in the C-terminal part of H2 allowed the generation of four PrP mutants that all remained prion-compatible as attested by self-propagation of PrP res and de novo infectivity of the mutant PrP Sc . Of note, the levels of newly formed PrP res decreased while moving the insertion from position 195 to 192, with no conversion occurring beyond this position. As the thread of helix comprises a little more than 3 amino acids, it means that insertion in the last two C-terminal turns of H2 allowed the formation of bona fide prions.

Furthermore, a double mutant with an insert in the last turn of H2 and another in the H2-H3 loop was competent for prion conversion and propagation, showing that the simultaneous modification of both regions was tolerated. Like the PrP-ins-(FLAG) mutant mentioned above, PrP-ins(195 ϩ 203) harbors 16 additional amino acids, strengthening the view that neither the size of the middle part of the H2-H3 region nor the amino acid sequence are critical for 127S prion replication. Altogether these findings were unexpected as the H2-H3 region is proposed to be essential for prion conversion according to several, recently published models [START_REF] Cobb | Molecular architecture of human prion protein amyloid. A parallel, inregister ␤-structure[END_REF][START_REF] Kunes | Left-handed ␤-helix models for mammalian prion fibrils[END_REF][START_REF] Chakroun | The oligomerization properties of prion protein are restricted to the H2H3 domain[END_REF], in which these two helices are proposed to undergo drastic conformational change and to adopt a ␤-sheet conformation in the prion state [START_REF] Smirnovas | Structural organization of brain-derived mammalian prions examined by hydrogen-deuterium exchange[END_REF][START_REF] Lu | ␤-Sheet core of human prion protein amyloid fibrils as determined by hydrogen/deuterium exchange[END_REF][START_REF] Prigent | PrP assemblies. Spotting the responsible regions in prion propagation[END_REF]. Other models, however, consider that it is mainly the N-terminal moiety of the PK-resistant fragment that supports structural modification [START_REF] Govaerts | Evidence for assembly of prions with left-handed ␤-helices into trimers[END_REF][START_REF] Wille | Natural and synthetic prion structure from x-ray fiber diffraction[END_REF][START_REF] Demarco | From conversion to aggregation. Protofibril formation of the prion protein[END_REF]. Also, a recent paper has reported productive infection in cell cultures expressing a PrP molecule with two point mutations substituting original amino acids by cysteines, one in the H2 end and the other in the loop [START_REF] Hafner-Bratkovic | Globular domain of the prion protein needs to be unlocked by domain swapping to support prion protein conversion[END_REF].

Insertions in H2 End Preserve Overall Fold of the H2-H3 Domain-We wonder whether the insertions could have destabilizing effects on the structure of PrP C and particularly on the H2-H3 domain. This question concerned both the PrP construct competent for prion conversion and those that are not. We thus turned to structural analysis of the recombinant insertion mutant PrP assuming that their structures would reflect characteristics of the corresponding glycosylated PrP anchored at the membrane of mammalian cells. The conclusions from these works that insertions ins193 are compatible with prion conversion and ins191, are not compatible, do not induce significant alteration of the overall fold of PrP.

As a whole, if we assume the increasingly supported hypothesis of a conformational change of the H2-H3 domain in prions [START_REF] Cobb | Molecular architecture of human prion protein amyloid. A parallel, inregister ␤-structure[END_REF][START_REF] Lu | ␤-Sheet core of human prion protein amyloid fibrils as determined by hydrogen/deuterium exchange[END_REF][START_REF] Tycko | The ␣-helical C-terminal domain of full-length recombinant PrP converts to an in-register parallel ␤-sheet structure in PrP fibrils. Evidence from solid state nuclear magnetic resonance[END_REF][START_REF] Adrover | Prion fibrillization is mediated by a native structural element that comprises helices H2 and H3[END_REF][START_REF] Kunes | Left-handed ␤-helix models for mammalian prion fibrils[END_REF], the comparison of infection data with structural analysis of PrP-ins193 and PrP-ins191 may suggest that when approaching the disulfide bridge, which could induce specific constraints to surrounding areas, modifications of H2 are less important for the overall fold of PrP C than for its conversion into PrP Sc .

In conclusion we showed that it is possible to generate bona fide prions with 8 -16 additional amino acids inserted in the middle of the PK-resistant domain, revealing an unexpected tolerance to PrP sequence change in the prion core. More specifically we showed that octapeptide insertions in either the H2-H3 loop or the last two C-proximal turns of H2 were not critical for prion conversion. These findings indicate that the integrity and sequence specificity of the loop and the two adjacent turns of H2 are not determinant for the structure of PrP Sc and will help to validate and refine structural models.

FIGURE 1 .

 1 FIGURE 1. Characteristics of a PrP with 8 amino acids inserted in front of helix-3. A, the sequences of mammalian PrP included between the 2 cysteines are highly conserved. Identical amino acids are boxed and those corresponding to the H2-H3 loop are underlined. B, nucleotide (top line) and primary sequences of PrP-ins203. In this and subsequent figures, the insert is in red and helices H2 and H3 are highlighted in green and blue, respectively. The asparagine of the second site of glycosylation is marked. C, glycoforms profiles of WT PrP and PrP-ins203 as shown by immunoblotting (mAb 4F2). Molecular masses markers (in kDa) are indicated. D, ratios of the unglycosylated form as quantified from four immunoblots. E, PNGase treatment of samples showing the lower mobility of deglycosylated PrP-ins203 compared with WT PrP on a resolutive gel (left, 4F2 mAb). Analysis of a similar blot with the V14 mAb reveals only the WT PrP (right). F, immunofluorescence with anti-PrP antibody shows a similar cell surface expression of the WT and mutant protein (4F2 mAb).

FIGURE 2 .

 2 FIGURE 2. 127S-infected Rov-ins203 cells produce self-propagatingPrP res . Infected cell cultures were split once a weak and samples of PK-digested cellular lysates were analyzed by immunoblotting, the passage number is indicated at the top of the upper panel. Note that the unglycosylated form of PrP Sc -ins203 was not detected to this level of exposure and that, as expected, mock-infected cells (m.i.) did not produce protease-resistant PrP. In the lower two panels, PrP res was treated with PNGase F before immunoblotting. Sha31 anti-PrP antibody recognizes an epitope located in the helix H1, whereas V14 antibody recognizes a conformational epitope previously localized nearby and upstream of the insert.

FIGURE 3 .

 3 FIGURE 3. PrP-ins203 is as susceptible as WT PrP to prion infection and PrP Sc -ins203 is infectious. A, WT and ins203 PrP res have a similar sensitivity to guanidine denaturation. A representative experiment of the conformational stability assay shows that resistance to PK digestion of PrP res after denaturation by increasing guanidium chloride concentrations was not appreciably modified for PrP res -ins203 (•) compared with WT PrP res (ࡗ). B, PrP-ins203 has the same susceptibility to prion infection as WT PrP. A 10% (w/v) brain homogenate from 127S-infected mice was diluted and used to infect cells expressing either WT or ins203 PrP as indicated at the top of the panel. The PrP res content was determined at the third passage of the cultures post-infection. C, de novo infection of Rov (wt), RK13 (no PrP), and Rov-in203 cells by PrP Sc -ins203. A cellular homogenate of Rov-in203 cells at passage number six post-infection was used as inoculum for de novo infections. D, PrP Sc -ins203 inoculum killed mice in 2 months. The curves show the survival rate of tg338 mice challenged by intracranial inoculation of homogenates from either 127S-infected Rov-ins203 cells at passage eight (•) or brains of terminally ill mice (ࡗ). All mice (5/5) inoculated with control homogenate from 127S-infected RK13 cells, which do not express PrP, grown in parallel with Rov-ins203, stayed healthy (f).

FIGURE 4 .

 4 FIGURE 4. PrP-ins203(Gly 6 ) is convertible into prion. A, amino acid sequence of the mutated region of PrP-ins203(Gly 6 ). B, immunoblot showing that the unglycosylated species are markedly under-represented for the insertion mutant compared with WT PrP. (4F2 mAb). C, immunofluorescence with anti-PrP antibodies showing that the insertion mutant is properly routed to the cell surface (4F2mAb). D, Western blot analysis of PK-treated samples. The gel was loaded with 250 or 50 g of PK-digested protein for the mutant PrP and WT PrP, respectively. PrP containing the polyglycine stretch was successfully converted into a self-propagating PK-resistant protein but levels of PrP res accumulated in cells were lower than for the WT PrP (Sha31 mAb).

FIGURE 5 .

 5 FIGURE 5. PrP-ins203(FLAG) with a H2-H3 loop extended by 16 additional amino acids is convertible into a true prion. A, primary sequence of the FLAG-tagged mutant PrP. The FLAG sequence (blue) was inserted in the middle of the original ins203 sequence (red). B, analysis of Rov-ins203(FLAG) expression by immunoblotting (4F2 mAb). C, cell surface expression of PrP-ins203(FLAG) on nonpermeabilized cells. D, PrP res detection by immunoblot (left panel, Sha31 mAb) for infected and mock-infected (m) cells. The gel was loaded with 250 or 50 g of PK-digested protein for the mutant PrP and the WT PrP, respectively. Presence of the FLAG was verified by a specific antibody (right panel). E, detection of PrP Sc by immunofluorescence. Prion-infected Rov-ins203(FLAG) cells were immunolabeled by anti-PrP and anti-FLAG antibodies. Merge images with nuclei labeled by DAPI (blue) showed colocalization of PrP Sc and FLAG signals in infected cells (yellow). F, de novo infection of WT Rov cells challenged with homogenate from Rov-ins203(FLAG) cells propagating a PrP Sc for five passages. The third passage post-infection is shown (left panel). RK13 cells infected by 127S brain homogenate at the same time and maintained in the same conditions as Rov-ins203(FLAG) were used to control the absence of residual infectious brain material in the culture (right panel).

FIGURE 6 .

 6 FIGURE 6. Insertion of six histidines increased the copper affinity of PrP C and PrP-ins(His 6 ) was converted into a His-tagged prion. A, the glycoform pattern of PrP-ins(His 6 ) shows a reduction of the unglycosylated form similar to the original ins203 PrP (4F2 mAb). B, WT PrP was eluted from the Cu 2ϩ IMAC column at a lower concentration of imidazole than PrP-ins(His 6 ). Note that the monoglycosylated forms were eluted after the diglycosylated ones for both WT and ins203(His 6 ) PrP. Immunoblots were revealed with the 4F2 mAb. C, immunoblot showing that PK-resistant PrP was generated upon infection. The rate of PrP res increased notably after five to six passages of the cell culture (left panel, Sha31 mAb). The presence of the His tag on the PK-resistant fragment was ascertained using an anti-His 6 mAb (right panel). D, Rov cells expressing WT PrP and Rov-ins(His 6 ) were susceptible to de novo infection by cellular homogenate of 127S-infected Rov-ins(His 6 ) harvested at passage six (PK-treated samples and Sha31 mAb).

FIGURE 7 .

 7 FIGURE 7. Retention of PrP-ins(His 6 ) prion by chromatography affinity. A, part of the PK-resistant PrP-ins(His 6 ) produced by infected cells was retained with high affinity to the column. B, PrP Sc -ins203(His 6 ) solubilized with 4 M urea was more efficiently retained on IMAC copper columns than the WT PrP res . At this particular concentration urea allowed complete PK digestion of PrP C but preserved the protease resistance of both the His tag and WT PrP Sc . Eluted samples were treated by PK and analyzed by immunoblotting (Sha31 antibody).

FIGURE 8 .

 8 FIGURE 8. The C-terminal part of helix-2 is not critical for prion conversion. A, sequence of different mutant PrP generated with an insertion located upstream or downstream of the 203 position. The insert is in red and amino acids that belong to, or are originally from helix-2 are in green. Note that the bottom sequence contains two inserts, each at a different position. The name of the mutant is on the left and its capacity to convert into prion is indicated on the right. B, assay for PrP res content of the insertion mutant PrP modifying the loop, the helices, or both. The same amount of protein digested by PK was used throughout. The passage number is indicated above each panel and the asterisk indicates that a longer exposure is shown.

FIGURE 9 .

 9 FIGURE 9. Schematic structure of WT PrP with the indication of regions tolerant to insertion. ␣-Helices are shown in red and the two short ␤-sheets in yellow. H2 and H3 are linked by a disulfide bond that maintains them close to each other, and the unstructured inter-helix segment forms a loop. The position where the four different insertions (ins203, ins203(Gly 6 ), ins203(His 6 ), and ins203(FLAG)) were introduced, just before H3, is indicated. The C-terminal part of H2 in which octapeptides can be introduced without preventing prion conversion is colored in dark blue.

FIGURE 10 .

 10 FIGURE 10. Structural analyses of PrP with insertions in H2 conferring permissiveness to 127S or not. A, comparison of the NMR HSQC spectra at 25 °C of ins191 (top), ins193 (middle), and wild-type PrP (bottom) recorded at 600 MHz. B, comparison of the far-UV CD (top) and the temperature scans followed at 220 nm (bottom) of ins191 (red), ins193 (blue), and wild-type (black) PrP recorded at 25 °C. The spectra were normalized for the concentration.

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Acknowledgments-We acknowledge Stéphanie Prigent and Sylvie

Noinville (INRA Jouy-en Josas, France), Kris Pauwels and Stephen Martin (NIMR London, UK) for help with recombinant PrP and structural analysis. We also acknowledge Jacques Grassi and Stéphanie Simon (CEA, Saclay, France), Jean-Philippe Deslys (CEA, Fontenay aux Roses, France), Jan P. Langeveld (Central Institute for Animal Disease Control, Lelystad, Netherlands), and John Collinge (MRC Prion Unit, UCL Institute of Neurology, UK), for supply of antibodies. We thank Calum MacKichan for English style editing.

by the fact that mini-* This work was supported by the Institut National de la Recherche Agronomique (INRA). □ S This article contains supplemental Table S1 and Figs. S1-S5. 1 Supported by the Higher Education Commission of Pakistan and the INRA Animal Health division.


Orianne Rollin
email: orianne.rollin@avignon.inra.fr

Vincent Bretagnolle

Axel Decourtye

Jean Aptel

Nadia Michel

Bernard E Vaissière

Mickaël Henry

Differences of floral resource use between honey bees and wild bees in an intensive farming system

Keywords:

Bees provide an essential pollination service for crops and wild plants. However, substantial declines in bee populations and diversity have been observed in Europe and North America for the past 50 years, partly due to the loss of natural habitats and reduction of plant diversity resulting from agricultural intensification. To mitigate the negative effects of agricultural intensification, agri-environmental schemes (AES) have been proposed to sustain bees and others pollinators in agrosystems. AES include the preservation of semi-natural habitats such as grasslands, fallows, woodlots, hedgerows or set-aside field margins. However, empirical evidence suggest that the use of those semi-natural habitats by bees may vary greatly among bee functional groups and may further be influenced by the presence of alternative foraging habitats such as mass-flowering crops. The present study sets out to investigate whether the three bee groups typically targeted by AES (honey bees, bumble bees and other wild bees) differ in the way they use those semi-natural habitats relative to common mass-flowering crops (oilseed rape, sunflower, alfalfa) in an intensive agricultural farming system. A clear segregation pattern in the use of floral resources appeared between honey bees and wild bees, with the former being tightly associated with mass-flowering crops and the latter with semi-natural habitats. Bumble bees had an intermediate strategy and behaved as habitat generalists. Therefore, it would be sensible to treat the three bee groups with distinct AES management strategies, and to further consider potential effects on AES efficiency of alternative foraging habitats in the surrounding. This study also stresses the importance of native floral resources, particularly in semi-natural herbaceous habitats, for sustaining wild bee populations.

Introduction

Flower-visiting animals provide substantial pollination services for a wide range of wild plants and crops worldwide [START_REF] Klein | Importance of pollinators in changing landscapes for world crops[END_REF][START_REF] Ollerton | How many flowering plants are pollinated by animals?[END_REF]. The economic value of insect-mediated pollination for agriculture at the global scale was estimated to be D 153 billion in 2005, considering only the crops that are used by humans for food [START_REF] Gallai | Economic valuation of the vulnerability of world agriculture confronted with pollinator decline[END_REF]. Bees (Hymenoptera, Apoidea) are a keystone group in this pollination process. However, the last 50 years have witnessed a global decline in both honey bees and wild bees in European and North American agricultural landscapes the local scale by sowing attractive flower covers like scorpion weed Phacelia tanacetifolia [START_REF] Decourtye | Landscape enhancement of floral resources for honey bees in agro-ecosystems[END_REF], clover Trifolium spp., alfalfa Medicago sativa or mustard Sinapsis spp. [START_REF] Dicks | Bee Conservation: Evidence for the Effects of Interventions, Synopses of Conservation Evidence[END_REF]. However the chosen "pollen and nectar mixes" are usually foraged by a limited subset of bee species, due to morphological or phenological incompatibilities [START_REF] Kirk | Plants for Bees: A Guide to the Plants That Benefit the Bees of the British Isles[END_REF]. Alternatively, the habitat-oriented approach consists in protecting and restoring (semi-) natural habitats with the aim to promote wild floral resources co-evolved with native bees. Natural and semi-natural habitats are fields or landscape areas with reduced management, including grasslands, fallows, woodlots, hedgerows or set-aside field margins [START_REF] Gathmann | Trap-nesting bees and wasps colonizing set-aside fields: succession and body size, management by cutting and sowing[END_REF][START_REF] Steffan-Dewenter | Succession of bee communities on fallows[END_REF][START_REF] Ricketts | Landscape effects on crop pollination services: are there general patterns[END_REF].

However, both theoretical and empirical evidence suggest that the use of those semi-natural habitats in intensive farming systems may vary greatly among bee functional groups and also depending on the presence of attractive mass-flowering crops. Mass-flowering crops may be intensively used by bees in agrosystems and are liable to influence the way bees use other habitats in the environment [START_REF] Holzschuh | Expansion of mass-flowering crops leads to transient pollinator dilution and reduced wild plant pollination[END_REF][START_REF] Tscharntke | Landscape moderation of biodiversity patterns and processes -eight hypotheses[END_REF]. For instance, flowered field margins tend to be less used by honey bees when alternative floral resources increase in the vicinity [START_REF] Henry | Spatial autocorrelation in honeybee foraging activity reveals optimal focus scale for predicting agro-environmental scheme efficiency[END_REF]. Furthermore, all bee groups may not be influenced in the same way by the presence of mass flowering crops in the environment. Honey bees may show a strong preference for mass-flowering crops because they need to store large amounts of food to sustain colony development, and have evolved social and behavioural adaptations to optimize foraging efficiency in that respect [START_REF] Dyer | The biology of the dance language[END_REF][START_REF] Dornhaus | Why do honey bees dance?[END_REF]. In particular, mass-flowering crops can be the major pollen suppliers for honey bees throughout the year in an intensive farming system (62% of the year-round pollen harvest, compared to 32% and 4.5% for trees and grasslands plants, respectively; [START_REF] Odoux | Territorial biodiversity and consequences on physico-chemical characteristics of pollen collected by honey bee colonies[END_REF]. Conversely, wild bees need to provision each cell in their nest with enough food for one larva to complete its growth [START_REF] Michener | The Bees of the World, 2nd Revised Edition[END_REF], and therefore might perform well with less abundant but more diversified native floral resources. Bumble bees, which are eusocial species but with annual and much smaller colonies than honey bees, might display an intermediate resource use pattern.

To test these predictions, we compared five different foraging habitats among the three broad bee groups targeted by AES. Studied foraging habitats were either semi-natural areas or mass flowering crops. Within the former, we distinguished semi-natural herbaceous habitats (grasslands and road and field margins) and semi-natural woody habitats (hedgerows and forest edges). Massflowering crops include oilseed rape Brassica napus, sunflower Helianthus annuus and alfalfa Medicago sativa. These crops offer big-bang floral resources, i.e. locally very abundant monofloral resources of short duration [START_REF] Herrmann | Genetic diversity and mass resources promote colony size and forager densities of a social bee (Bombus pascuorum) in agricultural landscapes[END_REF][START_REF] Westphal | Mass flowering oilseed rape improves early colony growth but not sexual reproduction of bumblebees[END_REF]. The use of those different foraging habitats by bees was assessed by means of flower-visit surveys in order to test whether the three bee groups differed in the way they used mass-flowering crops vs. semi-natural habitats. The implication of those results for the landscape scale planning of agri-environmental schemes will also be discussed.

Materials and methods

This study was carried out in the springs and summers of 2010, 2011 and 2012 in the Zone Atelier Plaine et Val de Sèvre, a 500 km 2 intensive agricultural area managed as a long-term ecological research (LTER) site by the Centre d'Etudes Biologiques de Chizé (CEBC-CNRS) within the Poitou-Charentes Region in Western France. The study site comprises over 16,000 fields, mostly used for cereal production. During 2010-2012, annual crops accounted on average for 80% of total land cover (40% for cereals, 9% for oilseed rape, 12% for sunflower and 9% for maize). Forest patches and permanent grassland covered only 3% and 7% of the total land of the study area, respectively. The information on land use is updated twice a year since 1995, for every single field, and stored into a geographic information system (ArcGIS, ESRI).

Due to phenological differences between floral resources present in the different habitat types [START_REF] Odoux | Territorial biodiversity and consequences on physico-chemical characteristics of pollen collected by honey bee colonies[END_REF], it was not possible to perform all pairwise comparisons of resource use. Instead, surveys were grouped by crop flowering period. During oilseed rape full flowering, in April-May, other available foraging habitats considered for comparison were semi-natural herbaceous and woody habitats. During sunflower full flowering, in July-August, other available foraging habitats for comparison with sunflower, were flowering alfalfa and semi-natural herbaceous habitats. Flowering woody species were too scarce for meaningful comparisons during the latter period (see Section 3).

Sampling design

The relative use of foraging habitats by the different bee groups was investigated using surveys of flower-visiting bees. In order to cover a wide variety of ecological contexts across the whole study area, we favoured an extensive sampling strategy, which consisted of short bee surveys in many sites. Over the three years, a total of 812 sites were surveyed, located in 30 grid cells (10 cells/year) randomly drawn without replacement from a 3.3 × 3.3 km spacing grid covering the whole study area (Appendix A). The grid cell size (about 10 km 2 ) was set to encompass the approximate foraging range of honey bee colonies [START_REF] Steffan-Dewenter | Honeybee foraging in differentially structured landscapes[END_REF]. Sampled habitats were either (i) a mass-flowering crop field (oilseed rape, sunflower or alfalfa), (ii) a flowering tree, or a patch of flowering shrubs or epiphytic plants in semi-natural woody habitats (hedgerows, forest edges or stand-alone trees), or (iii) a conspicuous patch of spontaneous wild flowers in semi-natural herbaceous habitats (permanent grasslands, external field margins, spontaneous weed plants in crops).

Sites were surveyed once each by capturing flower-visiting bees along walking transects (crops and herbaceous habitats) or at observation plots (woody habitats) during 15 min sampling sessions. At flowering trees, shrubs or epiphyte observation plots, field workers captured bees on accessible flowers. Walking transects were 50 m long and 2 m wide. The 50 m length was estimated to be the distance required to cover the whole floral species richness in patches of herbaceous wild flowers typically sampled in our study area.

Transects in flowering crops were established from the median part of the most accessible field margin and towards the field centre. A buffer distance of 10 m from the field margin was used to reduce edge effect. Bees were captured with a net, exclusively when foraging on a flower. The visited flowers were identified in situ to the finest taxonomical level possible, or collected for subsequent identification in the laboratory. Whenever necessary, ladders and insect nets mounted on a 2 m long perch were used to permit captures in higher vegetation strata.

Unlike flowering crops and grasslands, whose location were exhaustively recorded prior to each sampling period, the occurrence of wild flower patches could not be determined a priori, which precluded purely random sampling site selection. Instead, we favoured a pseudo-random sampling strategy whereby wild flower patches were chosen along predetermined prospection itineraries in a way that reproduced a random-like selection. Within each grid cell, we had randomly selected a transversal itinerary along trails and secondary road networks prior to each sampling session. In a first step, all mass-flowering crops and grasslands (with wild flowers patches) located within a 500 m array from the chosen itinerary were systematically visited and sampled. While travelling among those fields, all accessible wild flower patches were sampled in semi-natural habitats other than grasslands (e.g. field margins, hedgerows), respecting a minimal 50 m distance among samples. In a second step, field workers further sampled as many additional flowering crops and wild flowers patches in grasslands and other semi-natural habitats as possible within the grid cell. Exhaustiveness was often reached within a grid cell for the least common field types (alfalfa and grasslands).

Wild herbaceous flower patches (e.g. in grasslands or field margins) were not sampled if the flower cover was too scarce, or if they were shorter than the 50 m length requirement. As an exception to this 50 m rule, two smaller contiguous flower patches were occasionally assimilated to a single one, provided that they were located <10 m apart.

Overall, this strategy led to the survey of a large number of flowering crops and wild flower patches, located at various ranges of distances from each other, and with a preserved homogeneity between habitat types in terms of spatial and temporal distribution. The 10 grid cells to be investigated during a given year and crop period could be processed within about 12-15 days.

Sites were sampled between 10:00 and 19:00, and only under good weather, that is with temperatures ranging from 16 • C to 35 • C and wind speed less than 15 km/h (see also [START_REF] Hoehn | Relative contribution of agroforestry, rainforest and openland to local and regional bee diversity[END_REF][START_REF] Westphal | Measuring bee diversity in different european habitats and biogeographical regions[END_REF]. Temperature and wind speed were systematically checked prior to each site sampling. Bees were sampled during full bloom periods of oilseed rape and sunflower. Oilseed rape sampling periods were from April 16th to May 1st in 2010, from April 1st to 13th in 2011, and from April 6th to May 5th in 2012. Sunflower sampling periods were from July 16th to 23rd in 2010, from June 28th to July 12th in 2011, and from July 17th to August 7th in 2012.

Characterization of resource use by bees

Foraging bees were classified into three groups, namely honey bees, bumble bees and all other wild bees. This classification matches the usual broad AES target bee groups [START_REF] Dicks | Bee Conservation: Evidence for the Effects of Interventions, Synopses of Conservation Evidence[END_REF], and is further justified by major differences in their biological traits and status. One bumble bee species, Bombus terrestris, is reared as well, but these colonies are used mainly for the pollination of greenhouse crops [START_REF] Velthuis | A century of advances in bumblebee domestication and the economic and environmental aspects of its commercialization for pollination[END_REF], which are absent or uncommon in the study area. Non-managed bees were also split into bumble bees, usually of greater body size and eusocial, and other wild bees.

Resource use was defined as the relative foraging intensity recorded for each bee group (honey bees, bumble bees, wild bees) in the different floral habitat types, as revealed by the number of foraging bees found in sampling sites. Bee counts in sampling sites were zero-inflated, i.e. displayed much more null values than expected from a usual frequency distribution (e.g. Poisson distribution). This indicates that additional mechanisms may further limit the use of resource patches by bees. To avoid possible biases, bee occurrence frequency, i.e. presence-absence data at sampling sites, were first computed and analysed. In a second step, bee abundance (counted bees per 50 m transect or 15 min observation plot) among habitat types was compared on presence-only sampling sites.

Data analysis

Analyses consisted of comparing among habitat types and bee groups the bee occurrence frequency and bee abundance. Therefore, in the subsequent analyses, generalized linear modelling approaches were used with occurrence (0-1 binary data) and abundance (counts) as dependent variables, while habitat type, bee group, and their two-way interaction, were the main explanatory variables. A binomial error distribution family was specified in occurrence models, and a Poisson family, which is well suited for count data, in abundance models.

Direct abundance comparisons between transect data (crops and herbaceous wild habitats) and observation plot data (woody habitats) were not fully informative due to the methodological differences. However, what is at issue here is whether habitat use differed among bee groups, which statistically would result as a significant interaction between habitat type and bee group, regardless of habitat-specific sampling method.

Prior to the statistical comparison of resource use descriptors among habitat types and bee groups, possible confounding factors or other sources of significant statistical noise were investigated in our dataset. The possible biases were (i) spatial autocorrelation at small scales, (ii) distribution gradients at the study area scale (i.e. non-stationarity), (iii) inter-annual variations and (iv) temperature-dependent variations in bee foraging activity at the daily scale.

Auto-correlation in honey bee foraging activity may occur over a range of about 80-400 m [START_REF] Henry | Spatial autocorrelation in honeybee foraging activity reveals optimal focus scale for predicting agro-environmental scheme efficiency[END_REF]. To assess whether our dataset was subject to autocorrelative biases, we proceeded in three steps. First, we sought possible spatial dependence among residuals of null models, i.e. simple statistical models with no explanatory variable specified. Generalized linear models (GLMs) where performed separately by habitat type and by bee group to figure out where in the dataset autocorrelation might occur. Second, the autocorrelation analysis was repeated a posteriori on the residuals of the final models used for hypothesis testing (see below). This was to check whether the autocorrelation potentially detected at the first step persisted in the analysis or was removed by our model parameterization. Third, would the assumption of independence of residuals still be unsatisfied, an additional spatial grouping variable was introduced in models to fix the non-independency among neighbouring samples. To do so, within each grid cell, sampling sites were clustered based on Euclidian distances. Clusters of neighbouring sites were determined by cutting the cluster tree at the level of the autocorrelation upper distance range. The cluster identity was introduced as an additional random grouping level in the mixed model structure described thereafter. In all cases, autocorrelation was analysed using spline correlograms on model residuals [START_REF] Rhodes | GLMM applied on the spatial distribution of Koalas in a fragmented landscape[END_REF]. Spline correlogram uncertainty was assessed by Monte Carlo simulations [START_REF] Bjornstad | Nonparametric spatial covariance functions: estimation and testing[END_REF] with 1000 simulated random data sets, using the nfc package [START_REF] Bjornstad | ncf: spatial nonparametric covariance functions[END_REF] in R version 2.12.0 (R Development Core Team, 2010).

Possible bias due to large-scale distribution gradient was systematically accounted for by including the grid cell identity as a random factor within the frame of a mixed model structure, i.e. generalized linear mixed models (GLMMs). Likewise, interannual variations were accounted for by including the year as a higher-level random variable, within which grid cells were nested. Finally, temperature-dependant variations could be accounted for by introducing temperature ( • C) as a fixed variable in the models. Bee foraging activity is affected by temperature [START_REF] Corbet | Temperature and the pollinating activity of social bees[END_REF][START_REF] Kelber | Light intensity limits foraging activity in nocturnal and crepuscular bees[END_REF][START_REF] Kwon | Effect of temperature on the foraging activity of Bombus terrestris L. (Hymenoptera: Apidae) on greenhouse hot pepper (Capsicum annuum L.)[END_REF], but its effect on bee occurrence frequency or bee abundance might not be linear. To model a quadratic shape, raw and squared temperature data where implemented jointly, after raw data were standardized to avoid collinearity [START_REF] Saveliev | Incorporating temporal correlation in seal abundance data with MCMC[END_REF]. An interaction term between standardized temperature and bee group was also specified because all bee groups are not equally affected by temperature [START_REF] Corbet | Temperature and the pollinating activity of social bees[END_REF].

Complete models included six fixed effect variables, namely habitat type (oilseed rape, semi-natural herbaceous habitat and semi-natural woody habitat during oilseed rape flowering; sunflower, semi-natural herbaceous habitat and alfalfa field during sunflower flowering), bee group (honey bees, bumble bees, wild bees), interaction between habitat type and bee group, standardized and squared temperature for quadratic modelling, and interaction between standardized temperature and bee group. Parameter estimates were based on the restricted maximum likelihood estimation (REML) method because this method reduces the biases of maximum likelihood estimates [START_REF] Zuur | Mixed effects modelling for nested data[END_REF]. For the sake of statistical parsimony, complete models were simplified into minimum adequate models (MAMs) by sequentially dropping nonsignificant terms following model comparisons based on likelihood ratio tests. Interactions were assessed first. If they were significant, their individual terms were also kept in the model.

Whenever an interaction between habitat type and bee group was kept in the MAM, the model was repeated on subsets of the initial dataset to test a posteriori all pairwise interactions among habitat types and bee groups. The p-values were adjusted accordingly using the sequential Bonferroni correction for multiple tests (Holm's sequential Bonferroni).

Analyses were performed separately for the two mass flowering periods, i.e. oilseed rape and sunflower. Therefore, four models were produced, one for each resource use descriptor (bee occurrence and abundance) and for each mass flowering period (oilseed rape and sunflower). GLMMs were computed using the lme4 package [START_REF] Bates | lme4: linear mixed-effects models using S4 classes[END_REF] in R version 2.12.0 (R Development Core Team, 2010).

Implications for agri-environmental schemes

To investigate the possible implications of our results for AES planning, occurrence and abundance model predictions were combined to assess how effective -in terms of resource use -would be the conversion of a given land surface into a habitat-oriented AES, compared to a mass-flowering crop. To do so, we evaluated pairwise scenario where bees were offered landscape elements (e.g. field margins) converted into semi-natural herbaceous habitats on the one hand, and equal amounts of unconverted mass-flowering crop surfaces on the other hand. The relative AES effectiveness was measured as:

E = n snh -n mfc n snh + n mfc (1)
where n snh and n mfc are the total number of bees predicted to forage at one particular moment in the semi-natural herbaceous landscape units and in the mass-flowering landscape units, respectively. Effectiveness E varies from -1 (all bees in the mass-flowering crop, minimum AES efficiency) to 1 (all bees in the semi-natural herbaceous habitat, maximal AES efficiency), and tends towards 0 when the two habitats attract equal numbers of foraging bees. Eq. ( 2) gives an estimate of n snh :

n snh = S × P × f snh × a snh ( 2
)
where S is the total surface converted into semi-natural herbaceous habitat, P is the percentage surface S occupied by patches of wild floral resources, and f snh and a snh are the MAM predictions for the bee occurrence frequency and abundance in flower patches. P was set to vary from 1% to 30%. Likewise, n mfc was calculated following Eq. ( 2), whilst P was by definition set to 100%. Herein, S is arbitrary and can be removed from equations whenever scenarios are based on equal surfaces between semi-natural and cropped habitats. Simulations were calculated using bee occurrence frequency and abundance model predictions at minimal and maximal temperatures.

Results

A total of 29,720 foraging bees were recorded (21,852 honey bees, 1356 bumble bees and 6512 wild bees; Table 1) in the 812 sampling sites. Most of the honey bees were found during the sunflower period. Wild bees belonged to 6 families and 22 genera. Samples covered a total of 127 flowering plant species, of which 74 were effectively visited by bees.

Little evidence of autocorrelative bias was found in either occurrence and abundance datasets. Exceptions were the abundance of honey bees in both oilseed rape and sunflower fields, with a positive autocorrelation detected over 2 km, and both the occurrence and abundance of bumble bees in sunflower over 3 km. The spatial dependency of residuals was removed at the MAM stage (see below), indicating that it was unlikely to affect our analysis. Bumble bee abundance in sunflower still displayed a minor autocorrelation at short distance (<1600 m) in the MAM residuals. This was fixed by further grouping neighbouring samples located <1600 m apart from each other into an additional nesting level within the GLMM random effects.

Few model simplifications were allowed to alleviate complete models. In occurrence models, the interaction between temperature and bee group was not significant, both for oilseed rape and sunflower periods (Table 2), and was therefore removed to compute the occurrence MAMs. All other variables had a significant effect or were involved in significant interactions. In abundance models, all variables had a highly significant effect. Therefore complete models were regarded as the abundance MAMs.

Temperature appeared to be an important correlate of the occurrence and abundance of bees during both the oilseed rape and sunflower periods (Table 2). Its overall effect was negative, with fewer bees foraging at high temperatures.

Relative use of flowering crops and semi-natural habitats by bees

Significant differences in resource use were observed for each bee group. The most striking illustration of these differences in resource use is provided during the sunflower period. Honey bees occurred in all of the 177 sampled sunflower fields at an average abundance >40 times greater than that of wild bees (Fig. 1B andD). Conversely, wild bees were found 2.5 times more often than honey bees in semi-natural herbaceous habitats and their abundance was also 2.5 times greater than honey bees. The pattern was not so clear-cut between oilseed rape and woody habitats for honey bees because they were found equally often in these two habitats. No significant difference in the occurrence and abundance of wild bees was found between flowering alfalfa fields relative to semi-natural herbaceous resources. Finally, in agreement with our expectations, bumble bees had an intermediate strategy: like honey bees, they were found significantly less frequently in semi-natural herbaceous habitats compared to other habitats (Fig. 1A andB). However, like wild bees, presence-only data revealed that bumble bees were found in significantly greater abundance in semi-natural habitats than in oilseed rape or sunflower (Fig. 1C andD). The complete pairwise comparison results among habitat types are provided in Appendix B.

The interaction effects between habitat type and bee group merely reveals how the different bee groups differ in the way they use foraging habitats (Fig. 2). The complete pairwise interaction results between bee groups and habitat types are provided in the Appendix C. The most marked and consistent differences in resource use were observed between honey bees, whose occurrence and abundance were strongly biased towards flowering crops compared to all other habitats, and wild bees, whose occurrence and abundance where strongly biased towards semi-natural habitats. Indeed, oilseed rape and sunflower were significantly more used by honey bees than by bumble bees and wild bees, whereas wild bees preferred semi-natural habitats over oilseed rape and sunflower.

The high attractiveness of flowering alfalfa compared to semi-natural herbaceous habitats for the three bee groups was marked. All bee groups were found more frequently in flowering alfalfa fields than in semi-natural herbaceous habitats, and there was no significant difference among honey bees, bumble bees and wild bees regarding their occurrence frequency in alfalfa (Fig. 2B). However, honey bees were more abundant in alfalfa than in semi-natural herbaceous habitats, compared to bumble bees and wild bees (Fig. 2D). There was little or no difference between the three bee groups in the use of semi-natural woody habitats compared to seminatural herbaceous habitats (Fig. 2A andC). However, the difference in abundance of bumble bees between the two types of semi-natural habitat was less than that observed for honey bees and wild bees (Fig. 2C). This propensity of bumble bees to use resources in a balanced way was conformed to our initial predictions of intermediate foraging strategy.

Implications for agri-environmental schemes

The AES efficiency simulations preserved the overall clear-cut segregation pattern between honey bees and wild bees in terms of resource use, as well as the rather intermediate strategy displayed by bumble bees (Fig. 3). Temperature had an overall limited effect on predicted AES efficiency, though more marked on wild bees. In all pairwise scenarii (AES semi-natural herbaceous habitat on one hand vs. oilseed rape, sunflower or alfalfa on the other hand), the semi-natural habitats were overall expected to attract much fewer honey bees, regardless the percentage surface occupied by wild flower patches. Conversely, semi-natural herbaceous habitats would clearly favour wild bees over bumble bees. However, during the oilseed rape period, a wild flower cover threshold appeared (ca. 4-8% of total herbaceous surface) below which a semi-natural herbaceous habitat would hold less foraging wild bees than an equivalent oilseed rape surface (Fig. 3A). Such a threshold was not detected during the sunflower period due to the fairly low sunflower attractiveness for wild bees compared to semi-natural herbaceous habitats (Fig. 3B). Alfalfa, on the contrary, performed better for all bee groups compared to semi-natural herbaceous habitats during this period (Fig. 3C), regardless the percentage surface occupied by native flower patches.

Discussion

Overall, strong evidence of differential habitat use among the three bee groups was found both during oilseed rape and sunflower flowering periods. Honey bees tended to be more frequently observed and more abundant in mass-flowering crops (oilseed rape and sunflower) than in semi-natural habitats, which conformed to our initial predictions. This observation is also congruent with the biannual honey flow from oilseed rape in spring and sunflower in summer, as reported by professional beekeepers in the region, confirming that honey bees make an intensive use of those mass flowering crops.

Conversely, wild bees used more frequently and in greater abundance the native floral resources in semi-natural habitats, particularly woody habitats in spring, and herbaceous habitats in summer. Wild bees were also much rarer than honey bees in massflowering crops. The differential pattern of habitat use between wild bees and honey bees appeared particularly contrasted during the sunflower period, with wild bee occurrence frequency and abundance values being nearly 50 times lower than those of honey bees in sunflower fields, against three or four times lower only in oilseed rape fields. Wild bees may preferentially forage in seminatural habitats because those habitats harbour more diversified food resources combined with more nesting opportunities [START_REF] Potts | Linking bees and flowers: how do floral communities structure pollinator communities?[END_REF][START_REF] Steffan-Dewenter | Succession of bee communities on fallows[END_REF].

However, wild bee abundance in herbaceous semi-natural resource patches was highly variable. A single 50 m transect may return capture numbers ranging from 0 to 95 and from 0 to 225 wild bees during oilseed rape and sunflower periods, respectively. No wild bee was found in nearly one-third (32%) of all semi-natural herbaceous samples. This heterogeneity in wild bee abundances may be largely accounted for by the floral diversity and species composition in sampling sites. Flower species do not attract equally the different wild bee species. Some bee species, also referred to as monolectic or oligolectic, have a very narrow dietary spectrum, while polylectic species have a generalist foraging behaviour [START_REF] Michener | The Bees of the World, 2nd Revised Edition[END_REF]. Dietary preferences are partly driven by morphological characteristics or other life history traits [START_REF] Kirk | Plants for Bees: A Guide to the Plants That Benefit the Bees of the British Isles[END_REF][START_REF] Michener | The Bees of the World, 2nd Revised Edition[END_REF]. For instance, long-tongued bees (such as the families Megachilidae and Apidae) can forage flowers with both deep and short corollas, whereas short-tongued bees (such as the families Halictidae and Andrenidae) can only forage flowers with short corolla, like flowers of Asteraceae family.

Bumble bees displayed an intermediate pattern of habitat use. Like honey bees, they occurred less frequently in semi-natural herbaceous habitats compared to mass-flowering crops (oilseed rape, sunflower, alfalfa) and semi-natural woody habitats. But unlike honey bees, they were found at their lowest abundance levels in oilseed rape and sunflower. They had a rather ubiquitous, but low-abundance, distribution pattern, which somehow contrasted with that of honey bees and wild bees. Different processes could explain this intermediate pattern. First, most bumble bees are medium or long-tongued species. They can forage from a wide variety of flowers, with deep or short corolla. However, long-tongued Bombus species are more efficient on flowers with deep corollas than on flowers with short corollas (Ranta and Lundberg, 1980). Thus, oilseed rape and sunflower, both displaying short corollas, might not be viewed as optimal resources compared to e.g. alfalfa deep flowers. Second, sociality and foraging behaviour can largely determine resource use. Honey bees are eusocial and need to store large amounts of food to sustain the development of their large colonies year-round. To do so, they have evolved social and behavioural adaptations to optimize foraging efficiency (the waggle dance to communicate food resource location; [START_REF] Dyer | The biology of the dance language[END_REF][START_REF] Dornhaus | Why do honey bees dance?[END_REF]. Bumble bees, on the other hand, form much smaller, annual colonies [START_REF] Michener | The Bees of the World, 2nd Revised Edition[END_REF] and do not provision food for winter as the queen alone will overwinter. Beside the above-described patterns of ecological segregation, one might suspect that ecological complementary effects occur among habitats. Habitat complementarity means that organisms need two or more habitats in their range to maintain sustainable populations [START_REF] Dunning | Ecological processes that affect populations in complex landscapes[END_REF]. In our study area, honey bees occurred as often in semi-natural woody habitats as in oilseed rape sampling sites and at rather high abundances, indicating that they use both resources in a possibly non-substitutable way. This is further supported by palynological data from our study region [START_REF] Odoux | Territorial biodiversity and consequences on physico-chemical characteristics of pollen collected by honey bee colonies[END_REF] showing that apart from mass-flowering crops, trees are the greatest providers of pollen for honey bees, while pollen from grassland accounts for a marginal part of total harvest. Likewise, possible complementary habitat use might occur (i) between oilseed rape and woody floral resources during the first study period for bumble bees, (ii) between sunflower and alfalfa during the second study period for bumble bees and (iii) between alfalfa fields and semi-natural herbaceous habitats during the second study period for wild bees. However, the habitat complementary hypothesis remains to be documented with deeper details on pollen vs. nectar use, inter-habitat distance effects and species-specific estimates of fitness.

The differential habitat use by the three bee groups might result in high variability in AES effectiveness, as has been observed for a wide range of taxa [START_REF] Kleijn | How effective are European agri-environment schemes in conserving and promoting biodiversity[END_REF][START_REF] Kleijn | Agri-environment schemes do not effectively protect biodiversity in Dutch agricultural landscapes[END_REF][START_REF] Knop | Effectiveness of the Swiss agrienvironment scheme in promoting biodiversity[END_REF]. Therefore, it appears sensible to treat the three bee groups with distinct AES management strategies, and to take into account possible mutual influences among foraging habitats. In an attempt to do so, a series of general recommendations regarding different AES management options is proposed below.

Resource-oriented AES through the promotion of mass-flowering crops. Alfalfa was the most evenly used flowering crop among the three bee groups, considering both occurrence frequency and abundance. However, floral morphology of alfalfa flowers is probably not well suited for all bee species. Only 30% of all wild bee genera reported in this study were observed in alfalfa (Andrena, Halictus, Hoplosmia, Lasioglossum, Megachile, Melitta, Xylocopa). Moreover, honey bees were much more abundant in flowering alfalfa, in August-September, i.e. after the sunflower crop (personal observations, OR). In that respect, alfalfa-oriented AES should be combined with the restoration of semi-natural habitats. Beside alfalfa, promoting the mass-flowering oilseed rape and sunflower will disproportionately benefit honey bees, and to a lesser extent bumble bees. However, given the pervasiveness of those two massflowering crops in modern farming systems (9% and 12% of total land cover, respectively, in our study area), it is unlikely that further increasing their quantity in the environment will make a substantial difference in terms of population fitness. On the contrary, some studies have questioned the effect of excessive oilseed rape cover on bumble bee foraging activity. [START_REF] Diekötter | Oilseed rape crops distort plant-pollinator interactions[END_REF] found that high amounts of oilseed rape in the landscape can have a negative effect on interactions between wild plants and bumble bees. Similarly, [START_REF] Holzschuh | Expansion of mass-flowering crops leads to transient pollinator dilution and reduced wild plant pollination[END_REF] have shown that wild plant pollination by bumble bees decreases with increasing oilseed rape proportion in the landscape. Seed set of Primula veris, whose blooming overlaps with that of oilseed rape, was reduced by 20% when the amount of oilseed rape within 1 km radius raised from 0% to 15%.

Promoting semi-natural woody habitats. Actions intended to favour the recovery of woody vegetation like hedgerows or forest edges, especially including trees from the Rosaceae family, such as Prunus spp., Malus spp., Crataegus spp., will benefit both honey bees and wild bees. Most importantly, honey bees will rely on the pollen of those trees [START_REF] Odoux | Territorial biodiversity and consequences on physico-chemical characteristics of pollen collected by honey bee colonies[END_REF] complementarily with the rather nectar-dominated use of oilseed rape in spring. At this period of the year, brood production rapidly expands in the hives, and the income of pollen protein is critical to support that process.

Promoting semi-natural herbaceous habitats. Habitat-oriented AES dedicated to promote natural herbaceous regeneration in grasslands, field margins or fallows will be the most effective way to favour wild bees in general. However, depending on the context, there might be a minimum flower cover threshold needed for herbaceous habitat-oriented AES to be more profitable to wild bees than resource-oriented AES. There might be an optimum that returns the best compromise among the different wild bee families.

Current AES practices in the study area include especially two types of AES contracts on alfalfa crops, designed to promote little bustard populations, a bird species red-listed in France. The first type involves the conversion of annual crops into perennial grassland and fodder crops (mainly alfalfa) for 5 years to increase the extent of perennial crops; the second type prevents the mowing of alfalfa in June, which may be used by females little bustard during the incubation period [START_REF] Bretagnolle | Rapid recovery of a depleted population of Little Bustards Tetrax tetrax following provision of alfalfa through an agri-environment scheme[END_REF]. This mass-flowering crop can provide alternative floral resources in the landscape during and after the sunflower flowering period. Moreover, this crop was the most evenly used flowering crop by wild bees during sunflower flowering and by all bee groups after the sunflower period. As such, it presents advantages as a candidate resource-oriented environmental scheme for bees, in addition to be a habitat-oriented agri-environmental scheme for birds.

Fig. 2 .

 2 Fig. 2.Representation of statistical interactions between bee groups and habitat types on the use of floral resources. Positive or negative values denote the level of preference for the right hand and the left hand habitats, respectively. Preferences between two habitats differ between two bee groups if there is a statistically significant interaction (NS: p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, see Supplementary data -Appendix C for detailed statistics). Alf = flowering alfalfa; OR = oilseed rape; SNh = semi-natural herbaceous habitats; SNw = semi-natural woody habitats; Sunf = sunflower.

Fig. 3 .

 3 Fig. 3. Effectiveness simulations of habitat-oriented AES relatively to (A) oilseed rape, (B) sunflower, or (C) alfalfa for each bee group, considering field-realistic proportions of semi-natural herbaceous habitats occupied by patches of wild floral resources, at minimal and maximal sampling temperatures (upper and lower lines, respectively).

Table 1

 1 Total number of recorded bees, and sampling sites.

	Sampling period

Table 2

 2 Factors associated with the occurrence frequency and abundance of bee groups, based on GLMMs and log-likelihood ratio tests. The "×" denote statistical interactions.

	Model	Variables	LogLik	2	df	p
	Occurrence frequency during oilseed rape period	Bee group	-759.45	53.608	2	<0.001
		Type of habitat	-759.45	89.487	2	<0.001
		Temperature (quadratic function)	-759.45	21.738	2	<0.001
		Bee group × type of habitat	-651.08	214.66	4	<0.001
		Temperature × bee group	-651.08	1.382	2	0.501
	Occurrence frequency during sunflower period	Bee group	-781.5	4.6814	2	0.096
		Type of habitat	-781.5	13.212	2	0.002
		Temperature (quadratic function)	-781.5	7.4206	2	0.024
		Bee group × type of habitat	-572.21	416.29	4	<0.001
		Temperature × bee group	-572.21	2.0117	2	0.366
	Abundance during oilseed rape period	Bee group	-4821.1	2622.2	2	<0.001
		Type of habitat	-4821.1	1214.7	2	<0.001
		Temperature (quadratic function)	-4821.1	59.023	2	<0.001
		Bee group × type of habitat	-3340.5	2942.3	4	<0.001
		Temperature × bee group	-3340.5	11.769	2	0.003
	Abundance during sunflower period	Bee group	-12,979	25,287	2	<0.001
		Type of habitat	-12,979	6333.4	2	<0.001
		Temperature (quadratic function)	-12,979	121.98	2	<0.001
		Bee group × type of habitat	-5099.2	15,745	4	<0.001
		Temperature × bee group	-5099.2	174.02	2	<0.001

O.Rollin et al. / Agriculture, Ecosystems and Environment 179 (2013) [78][79][80][81][82][83][84][85][86]

Acknowledgements

We would like to thank C. Maffre and A. Haefflinger for field assistance and L. Guilbaud for bee identification to genus level. J.F. Odoux, M. Chabirand and C. Toulet provided valuable help and suggestions on the field. We also thank the farmers of the study sites for allowing us to perform surveys on their fields. We are grateful to the editor and an anonymous reviewer for their constructive corrections. This work was funded by the French Ministry of Agriculture (CASDAR programme no. 9035) and an ANRT CIFRE Ph.D. grant allocated to OR.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.agee.2013.07.007.