Sriswaroop Dasari

Abhinav Jagetia

Y.-J Chang

Vishal Soni

Bharat Gwalani

S Gorsse

A.-C Yeh

Rajarshi Banerjee
email: raj.banerjee@unt.edu

5 high entropy alloy

Keywords: High entropy alloys, Complex concentrated alloys, Heterogenous microstructure, Intermetallic precipitates, Mechanical properties

Engineering multi-scale B2 precipitation in a heterogeneous FCC based microstructure to enhance the mechanical properties of a Al0.5Co1.5CrFeNi1.

Introduction

Conventional alloy design has been largely based on one principal element alloyed with small additions of multiple other elements, to obtain desired properties like corrosion resistance, toughness, strength, ductility and thermal properties. However, this strategy limits alloy design to the corners of phase diagrams, leaving unexplored the vast central regions of multi-component phase space. About fifteen years ago, a new strategy emerged, shifting the alloy design paradigm to include equi-atomic or concentrated mixtures of multiple principal alloying elements, leading to the concept of high entropy alloys (HEAs) or multicomponent alloys [START_REF] Yeh | Nanostructured highentropy alloys with multiple principal elements: novel alloy design concepts and outcomes[END_REF][START_REF] Cantor | Multicomponent and high entropy alloys[END_REF]; another widely accepted name is complex concentrated alloys (CCAs) [START_REF] Miracle | A critical review of high entropy alloys and related concepts[END_REF]. Four core effects have been attributed to these alloys; sluggish diffusion effect, lattice distortion, high configurational entropy and the cocktail effect [START_REF] Yeh | Nanostructured highentropy alloys with multiple principal elements: novel alloy design concepts and outcomes[END_REF][START_REF] Ranganathan | Alloyed pleasures: Multimetallic cocktails[END_REF]. Using the HEA/CCA design strategy has expanded the scope of alloy design to include the central unexplored regions of complex multi-component phase diagrams.

The effects of Al addition to CoCrFeNi based alloys has been extensively studied in the past decade [START_REF] Kao | Microstructure and mechanical property of ascast, -homogenized, and -deformed AlxCoCrFeNi (0 ≤ x ≤ 2) high-entropy alloys[END_REF][START_REF] Yang | Effects of AL addition on microstructure and mechanical properties of AlxCoCrFeNi High-entropy alloy[END_REF][START_REF] Chou | Microstructure, thermophysical and electrical properties in AlxCoCrFeNi (0 ≤ x ≤2) high-entropy alloys[END_REF][START_REF] Wang | Phases, microstructure and mechanical properties of AlxCoCrFeNi high-entropy alloys at elevated temperatures[END_REF][START_REF] Cao | The influence of Al elements on the structure and the creep behavior of AlxCoCrFeNi high entropy alloys[END_REF]. With increasing Al content, it was shown that the phase composition of the alloy changes from FCC/L12 to FCC/L12+BCC/B2 to BCC/B2. In addition to Al, minor Ti additions has also proven beneficial in stabilizing ' precipitates, increasing both their phase fraction and solvus temperature, as well demonstrated in previous work on Ni-base superalloys [START_REF] Reed | The Superalloys fundamentals and applications[END_REF]. Similarly, in case of HEAs/CCAs, the addition of Ti results in higher ' phase fractions. This has led to the recent development of a number of HEAs exhibiting an excellent combination of strength and ductility.

He et.al. developed a CoCrFeNi base alloy containing 6 at.% Al+Ti that produced a microstructure containing fine L12 coherent nano-precipitates in an FCC matrix [START_REF] He | A precipitation-hardened high-entropy alloy with outstanding tensile properties[END_REF]. The tensile yield strength and ductility of this microstructure were reported to be over 1 GPa and 17% respectively. Yang et.al. [START_REF] Yang | Multicomponent intermetallic nanoparticles and superb mechanical behaviors of complex alloys[END_REF] designed a CoFeNi base alloy with Al and Ti constituting 14 at.%. Themomechanical processing of this alloy led to a high phase fraction of L12 nanoparticles in FCC matrix that exhibited a yield strength of 1.03 GPa and ~50% ductility.

In addition to the well-known +' microstructures, HEA/CCAs also offer a variety of phase transformation pathways that can lead to novel microstructures containing intermetallic phases such as L12, B2, , laves and  phases [START_REF] Gwalani | Tensile yield strength of a single bulk Al 0.3 CoCrFeNi high entropy alloy can be tuned from 160 MPa to 1800 MPa[END_REF][START_REF] Gwalani | Microstructure and wear resistance of an intermetallic-based Al0.25Ti0.75CoCrFeNi high entropy alloy[END_REF][START_REF] Tsai | Criterion for sigma phase formation in Cr-and V-Containing high-entropy alloys[END_REF][START_REF] Feng | Phase stability and transformation in a light-weight highentropy alloy[END_REF]. Al0.3CoCrFeNi, in particular, received much attention owing to its tunability of microstructure and mechanical properties [START_REF] Gwalani | Modifying transformation pathways in high entropy alloys or complex concentrated alloys via thermo-mechanical processing[END_REF]. The traditional thermomechanical processing technique namely, cold working, solution treatment followed by precipitation annealing on Al0.3CoCrFeNi results in a homogenous distribution of L12 precipitates in the FCC matrix and grain boundary B2 precipitates. In contrast, when the same alloy is rolled and directly precipitation annealed, concurrent recrystallization of FCC grains and precipitation of B2 and  phases takes place. When compared to single phase FCC condition, the traditional processing technique results in an improvement in tensile yield stress by 250 MPa and the direct annealing improves the yield stress by ~600 MPa. Using Al0.3CoCrFeNi as a prototype, the importance of B2 precipitation in FCC based HEA/CCAs was studied by Choudhuri et.al. [START_REF] Choudhuri | Enhancing strength and strain hardenability via deformation twinning in fcc-based high entropy alloys reinforced with intermetallic compounds[END_REF] Their study reveals that enhanced local stress levels around B2 precipitates trigger deformation twinning thereby improving strain hardenability of the material. Nonetheless, the investigation and fundamental understanding of FCC+B2 microstructures in these alloy systems has been relatively low [START_REF] Yasuda | Formation of ultrafine-grained microstructure in Al0.3CoCrFeNi high entropy alloys with grain boundary precipitates[END_REF][START_REF] Stepanov | Mechanical properties of a new high entropy alloy with a duplex ultra-fine grained structure[END_REF][START_REF] Stepanov | Effect of Al on structure and mechanical properties of Fe-Mn-Cr-Ni-Al nonequiatomic high entropy alloys with high Fe content[END_REF][START_REF] Li | B2 structure of high-entropy alloys with addition of Al[END_REF], especially since they are rarely observed in more conventional alloys based on a single principal element. HEA/CCAs offer the opportunity of exploring such novel phase combinations and microstructures.

Interestingly, FCC+B2 microstructure has also been used to design heterogeneous grain structures [START_REF] Shukla | Hierarchical features infused heterogeneous grain structure for extraordinary strength-ductility synergy[END_REF]. Over the past decade, heterogenous materials have become a fast-emerging area after multiple reports showing strengthductility synergy as opposed to trade-off [START_REF] Fang | Revealing extraordinary intrinsic tensile plasticity in gradient nano-grained copper[END_REF][START_REF] Ma | Towards strength-ductility synergy through the design of heterogeneous nanostructures in metals[END_REF][START_REF] Yang | Dynamically reinforced heterogeneous grain structure prolongs ductility in a medium-entropy alloy with gigapascal yield strength[END_REF]. This is achieved by having both soft and hard domains in the material. The constriction of plasticity in soft domains by relatively harder regions imparts additional strength and the back-stresses produced at interfaces help in enhancing work-hardening and prevent necking of the material.

Previous studies on heterogeneous microstructures have largely focused on bimodal or multimodal distributions of grain size within a single-phase material. Specifically, in the context of precipitation strengthened HEAs/CCAs, these heterogeneous microstructures consist of a complex mixture of fine-grained recrystallized FCC + B2 regions co-existing with coarser nonrecrystallized FCC grains with refined B2 precipitates decorating the deformation bands/twins [START_REF] Shukla | Hierarchical features infused heterogeneous grain structure for extraordinary strength-ductility synergy[END_REF].

The extensive previous work on the Al0.3CoCrFeNi alloy introduced the competition between homogeneous nucleation of metastable L12 precipitates versus defect-mediated heterogeneous nucleation of equilibrium B2 precipitates [START_REF] Gwalani | Modifying transformation pathways in high entropy alloys or complex concentrated alloys via thermo-mechanical processing[END_REF]. However, this alloy suffers from two predominant weaknesses; firstly, the solute content of 7at% Al is insufficient for achieving a substantially high phase fraction and solvus temperature of second phase intermetallic precipitates, and secondly, this alloy is prone to precipitation of the embrittling  phase at temperatures as high as 700°C. The principal objectives of this paper can be listed as follows:

i. Investigate the competition between the L12 and B2 phases in an appropriate alloy which has the propensity to precipitate a higher phase fraction of these intermetallics, based on solution thermodynamic predictions.

ii. Investigate the influence of the nature of the defect (grain boundaries versus prior deformation bands/twins) on heterogeneous B2 precipitation.

iii. Compare and contrast overall mechanical behavior and tensile properties of homogeneous FCC + L12 microstructures versus heterogeneous FCC + B2 microstructures.

Materials and Methods

Alloy with a nominal chemical composition Al0.5Co1.5CrFeNi1.5 (at%: 9.1Al-27.27Co-18.18Cr-18.18Fe-27.27Ni) was prepared using a conventional vacuum arc melter. The alloy was flipped at least three times to ensure homogenization of the melt. Microstructural characterization was done using Scanning Electron Microscopy (FEI Nova-NanoSEM 230 TM / Hitachi SU8010 FE-SEM), fixed with energy dispersive spectroscopy (EDS) and electron backscattered diffraction (EBSD) detectors. TSL OIM TM software was used to analyze the EBSD data and no clean-up procedures were employed for the maps used in this report. Transmission Electron Microscopy (FEI Tecnai G2 TF20™ operating at 200 kV/ JEOL JEM-3000F HRTEM operating at 300 kV) was used to obtain crystallographic information from the phases present in the material. For analysis of compositional partitioning, Atom Probe Tomography (APT) was performed using Cameca LEAP 5000XS 3D Atom Probe Microscope operating at a temperature of 30K with pulse energy of 50nJ, pulse rate of 100 kHz, and a detection rate of 0.5-1 in laser mode. The lift outs for TEM and APT were prepared using a FEI Nova 200 dual beam focused ion beam (FIB). Three heat treated conditions of the alloy were studied. The cast alloy was homogenized at a temperature of 1150°C for 6 hours in a vacuum furnace before annealing or rolling treatments. The homogenized condition is referred to as single phase FCC condition, and SA750 condition corresponds to the samples that have undergone subsequent annealing treatment at 750 o C for 50 hrs after homogenizing. In case of the third condition, CR750, the homogenized samples were cold rolled to 60 % reduction and annealed at 750 o C for 50 hrs. Annealing treatments at 750 o C were performed in encapsulated quartz tubes backfilled with argon. The tensile samples were machined using Kent wire saw submerged EDM with following dimensions: gauge length of 5 mm, width of 1 mm and thickness of 0.7-1 mm. The tensile samples were tested at room temperature under quasi-static conditions at a strain rate of 10 -3 s -1 using a computer controlled mini tensile testing machine. The tensile tests were performed on at least three samples for each condition. Thermodynamic predictions were performed using ThermoCalc and the TCHEA4 database.

Results

The isopleth obtained by varying Al content in CoCrFeNi, given in Fig. 1 (a) shows that increasing the Al content raises the solvus temperature and volume fraction of B2 phase. However, increasing Al beyond ~10 at% results in a change in primary solidification phases from FCC to FCC + B2.

The absence of such single phase (FCC) field deters the alloy in terms of thermomechanical processing and thus in achieving equilibrium phase compositions. Fig. 1 (b), an isopleth obtained by varying Al in Co1.5CrFeNi1.5 indicates that the single-phase FCC field can be retained at higher Al content by raising the relative contents of Co and Ni. The composition Al0.5Co1.5CrFeNi1.5 was chosen to utilize the solid solution strengthening from higher Al content and improved solvus temperature and phase fractions of intermetallic B2 phase. The temperature vs. phase fraction plots in Fig. 1 (c) and (d) compares and contrasts the extensively studied Al0.3CoCrFeNi system with Al0.5Co1.5CrFeNi1.5. It can be noted that B2 solvus temperature has improved by 70°C, and the volume fraction of B2 has increased by 20 to 80% within the temperature range of 700°C -1000°C. Chang et al. [START_REF] Chang | The evolution of microstructures and high temperature properties of AlxCo1.5CrFeNi1.5Tiy high entropy alloys[END_REF] have previously reported an FCC + (BCC based) phase field for the Al0.5Co1.5CrFeNi1.5 alloy based on x-ray diffraction results, without any detailed investigation of the phase stability, transformations, or mechanical properties.

Comparing the high temperature phase stability for a high Al containing CoCrFeNi base alloy system, Al0.5CoCrFeNi and the current alloy, Al0.5Co1.5CrFeNi1.5, SEM backscatter micrographs from 1150°C annealed samples of both alloys are shown in Fig. 2. While the Al0.5CoCrFeNi alloy exhibits a dual phase microstructure, FCC grains with substantial phase fractions of the B2 phase (Fig. 2 (a)), the Al0.5Co1.5CrFeNi1.5 alloy exhibits a single-phase microstructure (subsequently referred to as single phase FCC condition) as shown in Fig. 2 The volume fraction and average size of L12 precipitates was found to be and 12.1% and ~10.2 µm respectively. The composition of the matrix and the precipitates was determined by averaging the atoms within a 20 nm radius sphere embedded within each phase. The composition of the L12 precipitates was determined to be 21.5Al-13.9Co-6.6Fe-4.4Cr-53.6Ni (at%) whereas the composition of the FCC matrix was found to be 8Al-30.1Co-19.4Cr-18.2Fe-24.3Ni (at%). Based on the experimentally measured precipitate composition and assuming an Ni3Al (3Ni:1Al) stoichiometry, the site occupancies within this L12 or ' phase can be conjectured to be (Ni,Fe,Co)3(Al,Cr) [START_REF] Allaverdova | Atomic distribution of alloying additions between sublattices in the intermetallic compounds Ni3Al and NiAl I: Phenomenological treatment[END_REF].

Previous work by Gwalani et.al. [START_REF] Gwalani | Modifying transformation pathways in high entropy alloys or complex concentrated alloys via thermo-mechanical processing[END_REF] on the Al0.3CoCrFeNi HEA/CCA established that in the absence of a large number density of heterogenous nucleation sites within the FCC matrix, B2 precipitates cannot easily nucleate and consequently reach a near equilibrium phase fraction.

Instead, the competing mechanism of homogeneous precipitation of the metastable L12 phase might occur within the FCC matrix [START_REF] Gwalani | Modifying transformation pathways in high entropy alloys or complex concentrated alloys via thermo-mechanical processing[END_REF]. However, cold-rolling followed by directly annealing the sample at the target second phase precipitation temperature, resulted in fine recrystallized FCC grains with the recrystallization process concurrently taking place with B2 precipitation at the new grain boundaries, consequently leading to a substantially higher B2 phase fraction within the FCC matrix. The same strategy was adopted in case of the present Al0.5Co1.5CrFeNi1.5 alloy. Therefore, the homogenized alloy was cold-rolled to 60 % reduction in thickness and then directly annealed at 750°C for 50hrs, with this condition being subsequently referred to as the CR750 condition.

The partially recrystallized microstructure of this CR750 condition can be observed in the low magnification SEM micrographs shown in Figs. 5(a-b). The recrystallized regions have been outlined for convenience in these images. A montage of SEM micrographs, covering a 11 mm 2 area, was used to determine the average area fraction of recrystallized regions and was found to be ~17%. A high magnification backscatter SEM image from the recrystallized region shown in Fig.

5(c) clearly reveals fine FCC grains with grain boundary precipitates. A high magnification

backscatter SEM image from the non-recrystallized region (Fig. 5(d)) shows clearly reveals a high number density of fine scale precipitates within the coarser non-recrystallized FCC grains. These fine scale precipitates are geometrically aligned along bands (Fig. 5(d)). Both the recrystallized and non-recrystallized regions have been investigated in further detail employing SEM-EBSD, TEM and APT based techniques, for determining the identity of the second phase precipitates, their orientation relationship with the FCC matrix, and other details of the overall structure and composition of phases present.

Results from the orientation image microscopy (OIM) from CR750 consisting of an inverse pole figure (IPF) and the corresponding phase map from a recrystallized region are shown in Figs. 6(ab). The inset in Fig. 6(a) shows the image quality (IQ) map of the region. These maps reveal fine recrystallized FCC grains with intragranular and intergranular precipitates which were indexed as a BCC based phase. While the FCC grain size in recrystallized regions is ~3.5 µm, the size of B2 precipitates is ~0.76 µm. Further characterization was performed using TEM. A [011] FCC zone axis electron diffraction pattern from a recrystallized FCC grain in this region, shown in Fig. 6(c), clearly indicates that there is no L12 precipitation inside these FCC grains. The electron diffraction pattern from [011] BCC zone axis, recorded from one of the second phase precipitates (Fig. 6(d The IPF and phase maps shown in Figs. 7(a-b) were recorded from the non-recrystallized region of the CR750 sample. These EBSD images show two coarse non-recrystallized FCC grains with a grain boundary and a homogeneous distribution of well-aligned fine scale needle-like precipitates within these grains. The fine-scale second phase precipitates, both at the grain boundary as well as within the grains, are indexed as BCC-based precipitates (refer to Fig. 7(b)). These precipitates have a multi-modal distribution with size ranging from 80 nm to 1.2 µm, as determined by ImageJ software. The IQ and kernel average misorientation (KAM) maps are shown in the insets of Fig. 7(a) and (b) respectively. The KAM map, which has strain-free recrystallized grains at the prior grain boundary, shows that some areas in the non-recrystallized region are relatively strain-free. This is an indication that recovery has occurred in some non-recrystallized regions. Similar to the observations from the recrystallized regions, there was no L12 precipitation within these FCC grains, and the fine-scale aligned precipitates have a B2 crystal structure, as revealed by the respective electron diffraction patterns, recorded from an FCC grain (Fig. 7(c)) and the microdiffraction pattern recorded from a fine-scale precipitate (Fig. 7(d Detailed analysis of the SEM-EBSD results from this recrystallized region in the SA750 sample, indicated the presence of multiple orientation relationships between the B2 precipitates and the adjacent FCC grains. Two distinct types of B2 precipitates are observed, those nucleating at grain boundaries or triple junctions (referred to as inter-granular) and those located within a single FCC grain (referred to as intra-granular). Fig. 8 shows pole figure analyses of two cases of intra-granular B2 precipitates within FCC grains. Based on pole figure analysis, as shown in Fig. 8, the orientation relationship (OR) between both the intra-granular B2 precipitates and the surrounding FCC matrix grain was determined to be: {111}FCC // {011}B2 and <011>FCC // <111>B2 This is referred to as the Kurdjumov-Sachs (K-S) OR [START_REF] Koumatos | A theoretical investigation of orientation relationships and transformation strains in steels[END_REF].

Another case of intra-granular B2 precipitates is shown in Fig. S1 where two conjoint B2 precipitates are located within an FCC grain. The B2 precipitate on the bottom clearly exhibits the Nishiyama-Wasserman (N-W) OR [START_REF] Koumatos | A theoretical investigation of orientation relationships and transformation strains in steels[END_REF] with the surrounding FCC matrix: {111}FCC // {011}B2 and <011>FCC // <001>B2 The B2 precipitate on the top of the same conjoint pair has only a {111}FCC // {011}B2 but no <011>FCC // <111>B2.

A set of conjoint B2 precipitates, located at a triple junction, in the recrystallized region, are shown in Fig. 9. While the B2 precipitate on top exhibits a K-S OR with the FCC grain on top, the B2 precipitate on the bottom exhibits a K-S OR with the bottom FCC grain. Finally, the SEM-EBSD analysis of a B2 precipitate located within a FCC grain of the non-recrystallized region is shown in Fig. 10. This B2 precipitate also exhibited a K-S OR with the surrounding FCC grain.

To compare the composition of B2 precipitates from the recrystallized and non-recrystallized regions in the CR750 sample, site-specific APT experiments were performed on both these regions and these results have been summarized in Fig. 11. Fig. 11(a) is an Al ion map showing the 3D atom probe reconstruction of the section of a coarser B2 precipitate and its interface with the adjacent FCC matrix from a recrystallized region. An 7.8 at.% Al iso-concentration surface together with Al ions, are shown in the same figure to clearly delineate the Al-rich B2 regions from the FCC matrix region. The proxigram in Fig. 11(b), plotted across the Al iso-surface, reveals the partitioning of elements between the matrix and B2 phases. The respective compositions of the B2 precipitate and FCC matrix, averaged over a sphere of 25 nm radius, are 22.7Al-13.3Co-11Cr-10Fe-43Ni (at%) and 4.6Al-30.5Co-21.5Cr-19.8Fe-23.6Ni (at%) respectively. Similarly, Fig. 11(c) shows the APT reconstruction from the non-crystallized region capturing a section of a fine scale B2 precipitate. A 9.8 at.% Al iso-concentration surface together with the Al ions have been plotted in the same reconstruction (Fig. 11(c)). The corresponding proxigram from this is shown in Fig. 11(d). The compositions of the B2 precipitate and the FCC matrix, determined by a similar sphere analysis as described earlier, are 23.1Al-14Co-10.2Cr-9.1Fe-43.6Ni (at%) and 5.3Al-29.5Co-21.6Cr-19.1Fe-24.5Ni (at%) respectively. Based on the experimentally determined B2 precipitate composition, and assuming an NiAl (1Ni:1Al) stoichiometry, the site occupancies within the B2 structure can be conjectured to be (Ni,Co)(Al,Co,Cr,Fe) [START_REF] Allaverdova | Atomic distribution of alloying additions between sublattices in the intermetallic compounds Ni3Al and NiAl I: Phenomenological treatment[END_REF][START_REF] Hao | Concentration of point defects and site occupancy behavior in ternary NiAl alloys[END_REF].

The tensile engineering stress-strain curves for the single-phase FCC, SA750, and CR750 conditions are shown in Fig. 12(a). The single-phase FCC condition exhibited a yield stress (YS) of 180 MPa, ultimate tensile stress (UTS) of 453 MPa with ~62% ductility. The SA750 condition which has an FCC+L12 microstructure, exhibited an yield stress of 360 MPa, UTS of 720 MPa, and 48% tensile ductility. The partially recrystallized FCC+B2 microstructure in case of the CR750 sample, exhibited the best balance of properties with the highest yield stress of 790 MPa, UTS of 1020 MPa, and 20% tensile ductility. The corresponding work-hardening curves for all three conditions are shown in Fig. 12(b). It can be observed that the hardening rate of CR750 starts at a much higher value compared to the other two conditions. However, the work hardening rate after stage I is increasing in the case of single phase FCC and CRSA750 presumably due to dynamic Hall-Petch effect [START_REF] Kusakin | Modeling the effect of deformation on strength of a Fe-23Mn-0.3C-1.5Al TWIP steel[END_REF] and decreasing in the case of CR750. Additionally, the steady state hardening or stage II which is an indication of deformation twinning appears to be prominent in single phase FCC and CRSA750 conditions. The deformation mechanisms in the three conditions associated with tensile loading is a subject of future study.

Discussion

As compared to the extensively investigated Al0.3CoCrFeNi, the Al0.5Co1.5CrFeNi1.5 HEA/CCA offers a substantially improved solvus temperature and phase fractions of strengthening intermetallic phases. The present study on the Al0.5Co1.5CrFeNi1.5 alloy, demonstrates the ability to engineer widely different microstructures; a single-phase FCC solid solution, a typical precipitation-strengthened FCC+L12 microstructure (SA750), and a partially recrystallized FCC+B2 microstructure (CR750), within the same HEA/CCA with widely varying mechanical properties. While the single-phase FCC condition exhibited a relatively low YS of 180 MPa, it had excellent strain hardenability leading to a UTS of 453 MPa and ~62% tensile ductility.

Regarding second phase precipitation in this alloy, although the ThermoCalc based solution thermodynamic modeling suggests that the equilibrium phases are FCC and B2 at 750 o C, the SA750 condition exhibited a homogenous distribution of L12 precipitates within an FCC matrix, and a small fraction of grain boundary B2 precipitates. This unexpected precipitation of a metastable L12 phase at 750°C, can be rationalized via more detailed thermodynamic analysis.

Starting from a homogeneous single-phase FCC solid solution, using ThermoCalc, the onset driving forces have been computed for all the competing intermetallic phases, B2, L12 and , for this specific alloy composition. These driving forces, as function of temperature, have been plotted in Fig. 13. While B2 is the equilibrium phase at 750°C, there is a positive driving force for the precipitation of the L12 phase at the same temperature. Despite the highest driving force for B2 precipitation at 750°C and an expected equilibrium phase fraction ~35%, there is only a very small fraction of grain boundary B2 precipitates observed in the SA750 condition. This is due to the high interfacial energy barrier associated with B2 nucleation within FCC matrix, as discussed previously [START_REF] Gwalani | Modifying transformation pathways in high entropy alloys or complex concentrated alloys via thermo-mechanical processing[END_REF]. In comparison, there is a finite onset driving force for L12 precipitation at 750°C (referring to Fig. 13), while there is likely to be a rather low nucleation barrier associated with L12 nucleation within the FCC matrix. The underlying reasoning for this difference in nucleation barriers is the substantially lower FCC/L12 interfacial energy as compared with the FCC/B2 interfacial energy. While these interfacial energies have not been determined and are very difficult to compute in case of these five component alloys, it is possible to qualitatively compare them based on the degree of structural similarity across the interface. Thus, since L12 ordering is based on the parent FCC crystal lattice, the FCC/L12 interfacial energy is typically very low for a coherent interface, ~10 -40 mJ/m 2 for Ni-base and Co-base superalloys [START_REF] Reed | The Superalloys fundamentals and applications[END_REF]. In contrast, since the B2 phase is based on a parent BCC crystal structure, the FCC/B2 interfacial energy is expected to be much higher. While there are no reports in the literature of FCC/B2 interfacial energies in any alloy system, the closest approximation can be considered to be FCC/BCC interfaces in case of austenite/ferrite microstructures which typically have interfacial energies in the 180 -660 mJ/m 2 range [START_REF] Gwalani | Modifying transformation pathways in high entropy alloys or complex concentrated alloys via thermo-mechanical processing[END_REF]. Considering the precipitation tendencies for the two competing phases at 750°C, while the driving force for the precipitation of the B2 phase is more than 2 times higher than for L12, and approximately estimating the FCC/B2 interfacial energy to be ~10 times higher than that of the FCC/L12 interface, results in a nucleation barrier ~200 times lower for the L12 phase as compared to the B2 phase. Therefore, while L12 precipitates can homogeneously nucleate within FCC, it is nearly impossible for B2 precipitates to homogeneously nucleate within an FCC matrix.

Only heterogeneous nucleation of B2 precipitates is possible and in case of the SA750 condition, grain boundaries are the only available heterogeneous nucleation sites. However, the small fraction of grain boundary surface area per unit volume available in the coarse-grained SA750 condition limits the B2 nucleation density and results in a small phase fraction of this phase, much lower than the expected equilibrium phase fraction of ~35%. Instead, the strong chemical ordering tendency introduced via the introduction of Al in the Co1.5CrFeNi1.5 base alloy results in the homogenous precipitation of a metastable L12 phase. The yield stress in SA750 is thus nearly doubled (enhanced by over 180 MPa) when compared to the single FCC phase condition, due to the homogeneous L12 intermetallic precipitation in the former case.

Recently, there has been a lot of interest on heterogeneous microstructures in HEAs, with multiple studies reporting very good combinations of strength and ductility resulting from such microstructures [START_REF] Shukla | Hierarchical features infused heterogeneous grain structure for extraordinary strength-ductility synergy[END_REF][START_REF] Yang | Dynamically reinforced heterogeneous grain structure prolongs ductility in a medium-entropy alloy with gigapascal yield strength[END_REF]. Heterogeneous microstructures in HEAs can have different connotations, such as a mixture of different grain size distributions in a single-phase microstructure, or a mixture of different phases in the microstructure, or a combination of both of these. CR750 exhibits such a heterogeneous microstructure with a mixture of ~17% recrystallized regions and ~83% nonrecrystallized regions. From the SEM micrographs (Fig. 3(a-b)), as expected, the recrystallization primarily occurs at prior grain FCC boundaries and at sites of high dislocation activity. While the recrystallized regions consisted of fine grains of FCC with B2 precipitates, on the same size scale as the grains, the non-recrystallized region contained a homogenous distribution of refined B2 precipitates, which were crystallographically aligned along deformation bands and twins. Thus, the heterogeneous microstructure in case of the CR750 condition of the Al0.5Co1.5CrFeNi1.5 alloy is a complex mixture of two different grain size distributions of the parent matrix FCC phase, together with multiple scales of distribution of intermetallic B2 precipitates. Within the recrystallized regions, concurrent recrystallization and second phase B2 precipitation take place due to the competition between, recovery followed by strain-free grain nucleation dictated by the prior strain arising from cold-rolling, and the large thermodynamic driving force for B2 precipitation at the same temperature. This phenomenon restricts the size of recrystallized FCC grains due to the grain boundary pinning effect arising from the B2 precipitates. On the other hand, in the non-recrystallized regions, the alignment of B2 precipitates is presumably along specific crystallographic directions, dictated by the deformation bands and twins produced by prior cold rolling. Due to the availability of large number of heterogenous nucleation sites in CR750, the volume fraction of B2 is substantially higher than that observed in case of the SA750 condition.

Interestingly, nearly identical compositions of B2 precipitates are observed in both recrystallized and non-recrystallized regions, as determined using APT. Any minor differences are within the local composition variations. The grain boundary diffusion in case of the recrystallized regions, and the pipe diffusion at deformation bands and twins helped attain the equilibrium composition of B2 precipitates in both recrystallized regions as well as non-recrystallized regions. This heterogeneous microstructure exhibited the highest yield stress of ~790 MPa, nearly double that of the SA750 (FCC + L12) condition, primarily arising from the high phase fraction of B2 precipitates coupled with the mixture of fine and coarse FCC grains. The stress-strain curves shown in Fig. 12(a), and the corresponding work hardening curves shown in Fig. 12(b), also indicate that the work hardening is significantly higher in case of the CR750 condition, as compared to the other two conditions, single phase FCC and SA750. This could be related to the effect of the complex multi-phase heterogeneous microstructure consisting of two distinct regions; recrystallized refined FCC grains with B2 precipitates at grain boundaries and triple junctions, and the partially recovered coarser prior FCC grains with intra-granular B2 precipitates. The refined recrystallized FCC grains presumably exhibits a high strength due to the combined effect of Hall-Petch hardening together with the B2 precipitation at grain boundaries and triple junctions. The prior FCC grains in the non-recrystallized region have at least partially recovered and softened due to the 750°C annealing treatment, however, the high density of deformation bands and twins, decorated with B2 precipitates, act as barriers to dislocation movement, consequently increasing the strength of these coarse FCC grains. While it is difficult to assess which of these two regions is stronger, the overall yield strength of this composite heterogeneous microstructure is substantially higher. Subsequent to yielding, the deformation produced in the relatively softer regions of this microstructure is restricted by the surrounding plastically hard material, thereby producing back stresses at the interface. This phenomenon often termed back stress hardening strengthens the softer regions, raising the work-hardening rate of the material [START_REF] Wu | Heterogeneous materials: a new class of materials with unprecedented[END_REF]. Further detailed experimental investigations coupled with computational modeling are required for a better assessment of the relative strengths of the two different regions in this heterogeneous microstructure, which will be the subject of future research. Conclusions 1. Al0.5Co1.5CrFeNi1.5 is a candidate alloy to study FCC+B2 microstructures in HEA/CCAs because of the high B2 solvus temperature (~1200 o C) and phase fractions (~35% at 750 o C), as predicted by solution thermodynamic modeling.

2. A lack of heterogeneous nucleation sites for the equilibrium B2 phase, results in this alloy precipitating metastable L12 precipitates within the FCC matrix grains during annealing at 750 o C. The relatively lower thermodynamic nucleation barrier for L12 precipitation as compared to B2 precipitation drives this phase transformation.

3. Annealing the alloy directly after cold-rolling produces a large number of heterogenous B2 nucleation sites, and results in a complex multi-phase heterogeneous microstructure consisting of two distinct regions; recrystallized refined FCC grains with B2 precipitates at grain boundaries and triple junctions, and partially recovered coarser prior FCC grains with intragranular B2 precipitates decorating the prior deformation bands and twins resulting from coldrolling. This condition exhibited a ductility of ~20% and a yield strength of ~780 MPa, which is approximately a 400% increase from the single phase FCC condition of the same alloy. mechanical properties, Mater. Res. Lett. (2017). https://doi.org/10.1080/21663831.2017.1343208.

Figure Captions

  (b). Due to the long-term homogenizing treatment, the FCC grains in this condition have reached a size of ~ 253.3 µm. The discrepancy in the predicted single phase FCC field at 1200°C (refer to Fig. 1(d)) and the experimentally verified temperature of 1150 o C is possibly due to the inaccuracies in solution thermodynamics models for these complex concentrated alloys. The precipitation anneal temperature of 750°C was selected for a high B2 volume fraction based on the ThermoCalc predicted isopleth and phase fraction diagrams shown in Figs. 1 (b) and (d) respectively. The micrographs in Fig. 3 (a-b) shows the backscatter images of SA750 condition. A coarse FCC grain structure with grain boundary precipitates is visible in the low magnification image shown in Fig. 3(a). The backscatter high magnification image, shown in Fig. 3(b), reveals fine scale precipitation within the FCC matrix grains and a bright region around the grain boundary precipitates, presumably due to depletion of Al in matrix surrounding the precipitates. An SEM micrograph from an etched condition of the SA750 sample is shown in Fig. 3(c) revealing both grain boundary precipitates, as well as finer scale homogeneously distributed intragranular precipitates (inset in Fig. 3(c)). The crystal structures of these two types of precipitates, coarse grain boundary and fine scale intra-granular, were revealed by TEM characterization. A brightfield TEM image from the SA750 sample is shown in Fig. 3(d), revealing a collection of coarse grain boundary precipitates and refined intragranular precipitates. The high symmetry selected area diffraction pattern (SADP) in Figure 3(e) is recorded from one such grain boundary precipitate and is unambiguously indexed as [001] BCC zone axis. The superlattice reflection at {001} positions clearly establish the ordering of BCC lattice in the precipitates (B2 phase). An SADP from [001] FCC zone axis, recorded from one of the matrix grains is shown in Fig. 3(f). The {001} superlattice reflections in this diffraction pattern confirm that the intragranular precipitates are ordered FCC phases also known as L12 or ' (gamma prime) precipitates. APT was carried out to determine the composition of L12 precipitates in the SA750 sample. The superimposed ion map consisting of all ions, shown in Fig. 4 (a), which is a ~2 nm thick slice of the atom probe 3D reconstruction, clearly shows the enrichment of Ni and Al within the L12 precipitates. Isoconcentration surfaces at 13.13 at.% Al along with Al ions, have been plotted in the 3D reconstruction shown in Fig. 4(b). This reconstruction illustrates the 3D distribution of these precipitates within the FCC matrix. The proxigram constructed across the same iso-surface reveals the preferential partitioning of Ni and Al to L12 precipitates and Co, Cr and Fe to the FCC matrix.

  )) clearly reveals superlattice reflections at {001} locations, confirming that these BCC based precipitates are ordered B2 precipitates. A bright-field image and a {001} superlattice B2 darkfield TEM image, recorded from a precipitate, are shown in Figs. 6(e) and (f) respectively.

  )). Corresponding brightfield and {001} B2 superlattice dark-field TEM images, recorded from the needle-like precipitate are shown in Figs. 7(e) and (f) respectively.

Fig. 1 :

 1 Fig. 1: (a) Pseudo-binary isopleth for AlxCoCrFeNi system (b) Pseudo-binary isopleth for

Fig. 2 :

 2 Fig. 2: (a) Backscatter SEM micrograph of Al0.5CoCrFeNi showing a two-phase microstructure.

Fig. 3 :

 3 Fig. 3: (a-c) SEM of CRSA750 condition, (a) Backscatter micrograph of CRSA750 condition (b)

Fig. 4 :

 4 Fig. 4: Atom Probe Tomography (APT) of CRSA750 condition. (a) A 2 nm slice of superimposed

Fig. 5 :

 5 Fig. 5: Backscatter SEM of CR750 condition (a-b) Low magnification pictures showing the

Fig. 6 :

 6 Fig. 6: (a) IPF map obtained from a fully recrystallized region in CR750 condition using EBSD

Fig. 7 :Fig. 12 :

 712 Fig. 7: (a) IPF map obtained from a non-recrystallized region in CR750 condition using EBSD

Fig. 13 :

 13 Fig. 13: Normalized onset driving force vs temperature plots for the three predicted intermetallic

Fig. S1 :

 S1 Fig. S1: Orientation relationship between intragranular conjoint B2 precipitates and FCC matrix

Acknowledgements

The work was supported by the US Air Force Office of Scientific Research under grant FA9550-17-1-0395. The authors acknowledge the Materials Research Facility (MRF) at the University of North Texas for use of microscopy facilities.

Competing Interests

None zone axis (e) Bright field micrograph showing a layer of B2 precipitates in FCC matrix. (f) Dark field micrograph obtained from {001} superlattice reflection in (d).


Xiao-Wei Zhang

Chun Chen

Rui-Wu Wang

Finn Kjellberg

The cost of parasitism: High larval developmental mortality following attacks by a parasitoid fig wasp on a fig pollinating wasp

Keywords: Ficus racemosa, parasitoid, demography

Parasitoids contribute to controlling phytophagous insect populations. Their action on a host is often measured by numbers of emerging adult parasitoids. However, parasitoid oviposition attempts may lead to host mortality and sometimes both host and parasitoid die during larval development. Therefore, establishing the effect of parasitoids on host populations requires estimating total effect of parasitoids.

The communities of wasps developing in figs (the closed inflorescences of Ficus) have become a model system to investigate micro-hymenopteran interaction networks. A galled ovule produces either a single galling wasp or a single parasitoid providing a simple way to measure costs. However, some galled flowers produce no wasps. We show that about one quarter of galled flowers fail to produce pollinator offspring in the absence of parasitoids. In the presence of parasitoids, the number of empty galls is increased. For each emerging parasitoid, 1.9 pollinator larvae were killed. Hence, host mortality induced by parasitoids is much larger than actual parasitoid production, a feature documented in some other host-parasitoid interactions and that may be pervasive in nature

Introduction

Parasitoid hymenoptera represent the most diversified lineage of insects [START_REF] Forbes | Quantifying the unquantifiable: why Hymenoptera, not Coleoptera, is the most speciose animal order[END_REF]. They can exert strong control of host populations [START_REF] Godfray | Parasitoids: Behavioral and Evolutionary Ecology[END_REF]. As such, they provide natural ecological services [START_REF] Losey | The economic value of ecological services provided by insects[END_REF] and they are largely used as biological control agents [START_REF] Wang | Parasitoid wasps as effective biological control agents[END_REF]. The effect of parasitoids on a host may be measured as numbers of emerging parasitoids. However, actual parasitism levels may be much higher as joint mortality of host and parasitoid larva is increasingly documented, especially using nextgeneration sequencing [START_REF] Sow | Deciphering host-parasitoid interactions and parasitism rates of crop pests using DNA metabarcoding[END_REF]. Other factors, such as parasitoid ovipositor probing, may also result in increased mortality [START_REF] Abram | Nonreproductive effects of insect parasitoids on their hosts[END_REF]). An extreme situation was observed for Itoplectis conquisitor (Say) ovipositing on Lymantria dispar, with 200 hosts killed by oviposition attempts for one successfully developed parasitoid offspring [START_REF] Campbell | Some ichneumonid-sarcophagid interactions in the gypsy moth Porthetria dispar (L.)(Lepidoptera: lymantriidae)[END_REF]. Such mortality has mainly been investigated for some invasive host species and for some parasitoids that are bred for biological control [START_REF] Abram | Parasitoid-induced host egg abortion: an underappreciated component of biological control services provided by egg parasitoids[END_REF]. It is difficult to obtain a global vision on the prevalence of host mortality that does not lead to parasitoid production in natural host-parasitoid communities as they are generally complex with a diversity of more or less specialized parasitoids feeding on a set of hosts [START_REF] Stone | Reconstructing community assembly in time and space reveals enemy escape in a Western Palearctic insect community[END_REF].

Fig trees and their host-specific associated communities of fig wasps constitute a tractable model system to investigate the ecology and evolution of wasp communities [START_REF] Cook | Mutualists with attitude: coevolving fig wasps and figs[END_REF]. Because fig wasps are fig specialists, it is possible to collect the complete regional diversity of fig wasps by collecting wasps on the whole set of Ficus species. Levels of host specificity can be established [START_REF] Farache | Insights into the structure of plant-insect communities: specialism and generalism in a regional set of nonpollinating fig wasp communities[END_REF]. Unique insights may be obtained. For instance, observations throughout the range of a Ficus species has evidenced the presence of series of mutually exclusive parapatric sister species, demonstrating competition between species in a saturated community [START_REF] Darwell | Cryptic diversity in a fig wasp community-morphologically differentiated species are sympatric but cryptic species are parapatric[END_REF].

Ficus are characterized by their enclosed urn-shaped inflorescence whose inside is lined by uniovulate pistillate flowers and some male flowers. Pollinating wasps enter figs and oviposit in the pistillate flowers. A pistillate flower will either give a seed or turn into a gall that will eventually produce one wasp. Usually, a Ficus species is pollinated locally by a single pollinating fig wasp species while it can host multiple nonpollinating fig wasps [START_REF] Borges | How to be a fig wasp parasite on the fig-fig wasp mutualism[END_REF]. Non-pollinating fig wasp (NPFW) communities include gallers, inquilines, kleptoparasites, parasitoids of the pollinators, parasitoids of other non-pollinating wasps, and hyperparasitoids and can be particularly diverse, with up to 30 or more species present on a single tree [START_REF] Compton | Determinants of species richness in southern African fig wasp assemblages[END_REF]. As the pollinators, non-pollinating fig wasps oviposit into ovules and, whatever the species, a single non-pollinating wasp individual emerges from an ovule. Most non-pollinating wasp species have very long ovipositor that allow them to lay their eggs from outside the fig through the figwall. The timing of oviposition of the different species is scattered throughout the development of the fig [START_REF] Wang | Structure of a fig wasp community: temporal segregation of oviposition and larval diets[END_REF].

Despite the interest for non-pollinating fig wasps biology, most studies are based on observations of timing of oviposition and correlations between the number of wasps of different species produced within a fig [START_REF] Bronstein | The nonpollinating wasp fauna of Ficus pertusa: exploitation of a mutualism?[END_REF][START_REF] Cardona | Non-pollinating fig wasps decrease pollinator and seed production in Ficus andicola (moraceae)[END_REF][START_REF] Kerdelhue | Non-pollinating Afrotropical fig wasps affect the figpollinator mutualism in Ficus within the subgenus Sycomorus[END_REF][START_REF] Krishnan | Parasites exert conflicting selection pressures to affect reproductive asynchrony of their host plant in an obligate pollination mutualism[END_REF][START_REF] West | The ecology and evolution of the New World non-pollinating fig wasp communities[END_REF]. However, the results of correlation analysis can be very misleading as demonstrated by [START_REF] Raja | Interactions between pollinator and nonpollinator fig wasps: correlations between their numbers can be misleading[END_REF]: more productive figs produce more pollinators and more parasitoids resulting in a positive correlation between pollinator and parasitoid production [START_REF] Raja | Interactions between pollinator and nonpollinator fig wasps: correlations between their numbers can be misleading[END_REF]. Lack of direct evidence for feeding regime may also obscure interpretations.

Ficus racemosa is one of the rare species for which the feeding regimes of the associated community of wasps is well described [START_REF] Wang | Structure of a fig wasp community: temporal segregation of oviposition and larval diets[END_REF][START_REF] Yadav | Host-parasitoid development and survival strategies in a non-pollinating fig wasp community[END_REF]. Using the pollinator of F. racemosa, Ceratosolen fusciceps and its single parasitoid, Sycophaga agraensis, we addressed the following questions: i) do we have evidence for larval mortality in Ceratosolen fusciceps in the absence of parasitism; ii) do we have evidence for reduced total wasp production in the presence of parasitoids iii) and is increased pollinator mortality affecting equally male and female pollinating wasps?

Material and method

The work was carried out in and around the Xishuangbanna Tropical Botanic Garden (21°41′ N, 101°25′ E), Yunnan province, China. The altitude is approximately 600 m and the climate includes a wet and dry season. In Xishuangbanna, the wet season lasts from May to October.

A single crop on a single tree was used for the experimentation in order to reduce sources of variation. Indeed, our aim was to demonstrate the presence of an effect. We did not try to quantify the consequences of this effect on wasp population dynamics, as this will require much larger experimentation. Each fig was longitudinally bisected twice along the ostiole-peduncle axis, so that the wasps still inside the fig cavity, the empty galls from which wasps had emerged, and galls containing dead wasps could be counted. We also counted the number of "bladders''. These are empty, unperforated galls (bladders) similar in size and form to normal galls but lacking insects [START_REF] Galil | Studies on mutualistic symbiosis between syconia and sycophilous wasps in monoecious figs[END_REF]. They are generally interpreted as having been galled by ovipositing wasps but have become hollow due to early mortality of the wasp egg or embryo, [START_REF] Galil | Studies on mutualistic symbiosis between syconia and sycophilous wasps in monoecious figs[END_REF][START_REF] Jansen-González | Mutualism from the inside: coordinated development of plant and insect in an active pollinating fig wasp[END_REF][START_REF] Verkerke | Structure and function of the fig[END_REF]. Total gall numbers were calculated by summing numbers of exited galls, bladders, and those containing dead, nonemerged wasps.

Data analysis

We used the Shapiro-Wilk test to test the normality of the data of two treatments. The number of galls, wasps, and the sex ratio of pollinator offspring for the two treatments were normally distributed. Therefore, we used Student independent sample t-tests comparing gall numbers, pollinating wasp numbers and sex ratio of pollinating wasp between the two treatments. To analyze the relationship between the total number of wasps and the number of galls, two linear models in which the total number of wasps was set as explanatory variable and the gall number as the variable to be explained were used separately for the two treatments. Three other linear regression models were used to explore the quantitative effects of the presence of Sycophaga agraensis in this study. One linear model in which total gall number was set as explanatory variable and the number of Sycophaga agraensis as the variable to be explained was used to explore whether the number of offspring Sycophaga agraensis increases with gall number. To determine the effect of Sycophaga agraensis on the probability that a gall would give a wasp, we used a linear model in which the proportion of galls that produced a Sycophaga agraensis in each fig was set as explanatory variable and the proportion of the galls in which a wasp developed to become adult as the variable to be explained variable. To describe the relationship between offspring number of Sycophaga agraensis and pollinator, a linear model in which number of Sycophaga agraensis was set as independent variable and pollinator offspring number as dependent variable was used. The statistics were carried out with R 3.5.3.

Results

The number of galls did not differ between the two treatments (with and without parasitoid wasp) (893.82 ± 45.97 versus 939.82 ± 45.91 (Mean ± SE), t = 0.696, df = 37, P = 0.491), while fewer wasps developed in parasitized figs than in figs into which only pollinators oviposited (720.94 ± 36.55 versus 893.82 ± 31.88 (Mean ± SE), t = 3.239, df = 37, P < 0.01). The proportion of galls that produced a wasp was reduced in figs exposed to Sycophaga agraensis (0.63 ± 0.02 versus 0.77 ± 0.02 versus (Mean ± SE), t = 0.379, df = 37, P < 0.001). The sex ratio (number of males divided by total number of pollinating wasps) of pollinating wasps did not differ between the two treatments (0.291 ± 0.012 versus 0.286 ± 0.008 (Mean ± SE), t = 0.311, df = 37, P = 0.758).

The number of pollinator offspring that developed in figs exposed to Sycophaga agraensis was reduced by one third (472.18 ± 29.99 versus 720.94 ± 36.55versus(Mean± SE), t = 5.309, df = 37, P < 0.001; Fig. 3). Both female pollinator offspring (338.73 ± 22.84 versus 513.00 ± 28.75 versus (Mean ± SE), t = 4.811, df = 37, P < 0.001; Fig. 3) and male pollinator offspring numbers declined in figs exposed to S. agraensis (133.45 ± 8.16 versus 207.94 ± 12.65 (Mean ± SE), t = 5.147, df = 37, P < 0.001; Fig. 3). The number of empty galls (bladders) was much higher in figs exposed to parasitoids (329.82 ± 23.60 versus 218.88 ± 19.18 (Mean ± SE), t = -3.495, df = 37, P < 0.01).

The total number of wasp offspring produced per fig correlated strongly with the total number of galls in both treatments, but the regressions were significantly different between the two treatments (Sycophaga agraensis absent: y = 0.7297x + 35, N = 17, R 2 = 0.84 p < 0.001; S. agraensis present: y = 0.6087x + 20, N = 22, R 2 = 0.7703, p < 0.001; test of difference between the two regressions, F2,35 = 16.6, P < 10 -4 ; Fig. 1). For the two correlations, the intercepts were not significantly different from zero. The number of Sycophaga agraensis produced correlates positively with the total number of galls: more productive figs produced more parasites (y = 0.1224x -17.595, N = 22, R 2 = 0.3555, p < 0.01, intercept was not significantly different from zero, Fig. 2). The proportion of galls producing an insect correlated negatively with the ratio number of Sycophaga agraensis divided by total number of galls (y = -0.897x + 0.725, N = 22, R 2 = 0.23 p < 0.05; Fig. 3). The intercept (0.725) is the proportion of galls giving a wasp in the absence of parasitism. The slope gives the decrease in the proportion of galls giving a wasp as a function of the abundance of Sycophaga agraensis. For each additional Sycophaga, the total number of emerging wasps decreases by 0.897, and hence the number of emerging pollinating wasps decreases by 1.897.

In summary, figs without the parasitoid produced on average, 721 pollinators, 219 bladders, for a total of 940 galls while figs with parasitoids produced on average, 472 pollinators, 92 parasitoids, 330 bladders, for a total of 894 galls.

Discussion

In the absence of parasitoids, 27% of the galled flowers became bladders. These bladders could result from larval mortality or from gall initiation without oviposition. In Ficus montana, counts of the proportion of flowers into which pollinators had oviposited have shown i) that bladders corresponded to flowers into which a pollinator had oviposited and ii) that in the absence of parasitoids over 1/3 of the flowers that had received a pollinator egg turned into bladders (Ghana et al., We therefore accept as a provisional working hypothesis that there is a 27% larval mortality in Ceratosolen fusciceps. Similarly, in Ficus hirta figs into which one or two foundresses had been introduced, over 25% of the galled flowers were bladders, suggesting high larval mortality [START_REF] Yu | Spatial variation in pollinator gall failure within figs of the gynodioecious Ficus hirta[END_REF]. Hence, despite 70 Ma coevolution between Ficus and their pollinating agaonid wasps [START_REF] Cruaud | An extreme case of plantinsect codiversification: figs and figpollinating wasps[END_REF], and despite aligned interests of plant and insect [START_REF] Anstett | Figs and fig pollinators: evolutionary conflicts in a coevolved mutualism[END_REF], it appears that larval mortality remains remarkably high.

In the presence of parasitoids, the proportion of bladders (empty galls) was increased. This suggests either an increase in wasp developmental mortality or the initiation of new galls by the parasitoids. Initiation of new galls is unlikely because female flowers wilt rapidly after fig visitation by pollinators. Therefore, when Sycophaga agraensis oviposits into figs, 1-2 weeks after pollinator entry [START_REF] Yadav | Host-parasitoid development and survival strategies in a non-pollinating fig wasp community[END_REF], female flowers can probably no longer be galled. Further, the total number of galls did not increase in the presence of parasitoids confirming that no additional galls were initiated. The regressions show that for each additional Sycophaga produced, 0.9 additional bladder was present in the fig. Hence, the development of one parasitoid results in 1.9 fewer pollinator offspring. In the literature it is often implicitly assumed that the impact of pollinator-sized NPFWs is equal to the number of occupied ovaries, a galler larva occupying a single ovary and each parasitoid/kleptoparasite larva developing at the expense of one host wasp larva [START_REF] Conchou | The non-pollinating fig wasps associated with Ficus guianensis: community structure and impact of the large species on the fig/pollinator mutualism[END_REF]. Indeed, the weight of a female individual of Sycophaga agraensis is identical to that of an individual Ceratosolen fusciceps, confirming that this parasitoid feeds on a single host [START_REF] Ghara | Comparative life-history traits in a fig wasp community: implications for community structure[END_REF]. Nevertheless, the data presented here suggests high larval mortality, even in pollinators, and one parasitoid replacing two pollinator larvae. Hence, there is no simple proxy to determine the cost to the mutualism of the different non pollinating fig wasps involved.

While we show that the reduction in total wasp production due to parasitism should not be neglected, our result do not provide an estimate of induced mortality values in natural populations and of the factors influencing this mortality. This will require investigating variation among crops within trees and among trees. Nevertheless, a similar result was obtained in Ficus sur: figs protected against parasitoid oviposition produced 1.5 time more wasps than those that were exposed to parasitoids [START_REF] Compton | Complex interactions between mutalisms: ants tending homopterans protect fig seeds and pollinators[END_REF]. Hence the phenomenon described here is probably general in fig wasps .  The results also show that we cannot estimate a cost of parasitism simply correlating host and parasite production. Indeed, in our data, despite each parasitoid costing two pollinators, the correlation between number of parasitoids produced and number of pollinators produced was not significant. This is because figs that initially contained more pollinator larvae were more parasitized.

The sex ratio strategies of pollinating fig wasps have been extensively studied [START_REF] Herre | Sex-ratio adjustment in fig wasps[END_REF][START_REF] Raja | The mechanism of sex ratio adjustment in a pollinating fig wasp[END_REF]. Sex ratio adjustment is achieved by females beginning by laying many males, and progressively reducing the proportion of males throughout oviposition [START_REF] Raja | The mechanism of sex ratio adjustment in a pollinating fig wasp[END_REF]. Females are selected to lay a higher proportion of males at the beginning of an oviposition sequence if the pollinating wasps have incomplete control over the number of male and female offspring [START_REF] Kjellberg | Clutch size: a major sex ratio determinant in fig pollinating wasps?[END_REF]. The large pollinator larval mortality documented here could be an important factor limiting this control. Asymmetric effect of parasitoids on male and female pollinator offspring mortality could also affect sex ratio. In Ficus hirta, male pollinator offspring have been shown to be less exposed to parasitoids than female offspring, probably because their galls are located further from the fig wall, a factor that may limit the risk of a fig containing insufficient numbers of males to ensure female fertilization [START_REF] Yu | Moving your sons to safety: galls containing male fig wasps expand into the centre of figs, away from enemies[END_REF]. This was not the case in our study and may relate to figs of Ficus racemosa generally containing many more wasp offspring than F. hirta figs. Larger broods automatically result in reduced sex ratio variance [START_REF] Kjellberg | Clutch size: a major sex ratio determinant in fig pollinating wasps?[END_REF], and hence reduced necessity to protect male offspring.

Conclusion

The study presented here confirms the necessity to take into account parasitoid effects on host mortality that do not simply result from the development of a parasitoid up to adult stage as suggested by [START_REF] Abram | Nonreproductive effects of insect parasitoids on their hosts[END_REF]. Neglecting these effects provides for a highly skewed vision of the effect of parasitoids on host populations.

  The experiment consisted of two treatments. In one, we introduced 5 pollinating fig wasps per fig, and in the other treatment we introduced 5 pollinating wasps and then we exposed the fig to 10-20 parasitoid wasps. We located trees that presented a crop of pre-receptive figs and enclosed randomly selected figs in nylon bags in order to prevent natural wasp colonization. The figs were monitored every three days. When the figs were close to the receptive stage (the stage at which pollinators are attracted and enter the figs), they were checked daily. When the figs were receptive, we deposited 5 pollinating wasps on each fig and let them enter. We used pollinating wasps that had emerged naturally from other trees. The figs were then enclosed individually in nylon bags to prevent further pollinator entries or uncontrolled parasitism by non-pollinating fig wasps. When the figs were ready for parasitoid oviposition (8-10 days after pollinator oviposition), we replaced the nylon bag enclosing the fig by a bag containing 10-20 parasitoid wasps and let the parasitoids oviposit. Several days later, when the parasitoid wasps had ceased oviposition, we replaced the bag with a new empty protective bag. After pollinating wasp introductions, we let the treated figs develop to maturity and collected the figs just after the wasps had emerged from the figs into the bag. Wasps and fig were then preserved in alcohol.

Fig. 2 .

 2 Fig. 2. Linear regression forcing the intercept to zero between the number of Sycophaga agraensis offspring and the total number of galls when S. agraensis is present.

Fig. 1 .Fig. 3 .

 13 Fig. 1. Linear regression forcing the intercept to zero between the number of wasp offspring and the total number of galls when Sycophaga agraensis is absent or present. Open diamonds: S. agraensis absent; crosses: S. agraensis present

Fig. 4 .

 4 Fig. 4. Lack of correlation between number of pollinator offspring and number of Sycophaga agraensis offspring.

Acknowledgments

We thank Adrien Taudières for advices for the statistics. We thank XTGB for permission to work in the gardens and for access to the laboratory facilities of the research centre. We also thank Professor Yanqiong

Peng for her hosting XWZ during the experiment at XTBG.

Funding

This work was supported by the NSFC-Yunnan United fund [grant number U1302267]; the National Science Fund for Distinguished Young Scholars [grant number 31325005]; and the National Natural Science Foundation of China grants [grant number 31270433 to R.W. Wang, grant number 31760105 to C Chen].

Role of authors

XWZ devised the experiments, did the experiments, analysed the data and co-wrote the paper. CC participated in the experiments. RWW devised the experiments, analysed the data and co-wrote the paper. FK contributed to data analysis and co-wrote the paper.

Declaration of competing interest

The authors declare that they have no competing interests.


PhD Beverley Balkau
email: beverley.balkau@inserm.fr

MSc Leila Mhamdi

MD Jean-Michel Oppert

MD, FRCPI John Nolan

MD Alain Golay

MD Francesca Porcellati

MD Markku Laakso

MD Ele Ferrannini

R J Heine

J Dekker

S De Rooij

G Nijpels

Boorsma Athens

Greece : A Mitrakou

S Tournis

K Kyriakopoulou

Thomakos Belgrade

N Lalic

K Lalic

A Jotic

L Lukic

Civcic Dublin

Ireland : J Nolan

T P Yeow

M Murphy

C Delong

G Neary

M P Colgan

M Hatunic

T Konrad

H Böhles

S Fuellert

F Baer

Zuchhold Geneva

E Harsch Bobbioni

V Barthassat

V Makoundou

Tno Lehmann

Merminod Glasgow

now Dundee J R Petrie

C Perry

F Neary

C Macdougall

K Shields

Malcolm Kuopio

U Salmenniemi

A Aura

R Raisanen

U Ruotsalainen

T Sistonen

M Laitinen

England: SW Saloranta London

N Coppack

J Mcintosh

L Ross

Pettersson

Khadobaksh Lyon

F. Bonnet (now Rennes M Laville

Brac De La Perriere

C Louche-Pelissier

C Maitrepierre

J Peyrat

S Beltran

Serusclat Madrid

: R Spain

E M Gabriel

R Sánchez

A Carraro

B Novella Friera

Sweden Malmö

P Nilsson

M Persson

G Östling

O Melander

Italy: PM Burri Milan

L D Piatti

E Monti

E Setola

F Galluccio

A Minicucci

Newcastle-Upon-Tyne Colleluori

Newcastle, England M Walker

I M Ibrahim

M Jayapaul

D Carman

K Short

Y Mcgrady

Richardson Odense

: H Denmark

P Beck-Nielsen

K Staehr

V Hojlund

C Vestergaard

Olsen

Hansen Perugia

G B Bolli

C Fanelli

P Lucidi

F Calcinaro

A Saturni Pisa

A Natali

E Muscelli

S Pinnola

Kozakova Rome

G Mingrone

C Guidone

A Favuzzi P Di

Rocco Vienna

C Anderwald

M Bischof

M Promintzer

M Krebs

M Mandl

A Hofer

A Luger

W Waldhäusl

M Roden

P Gaffney

G Boran

Hormones Odense

Denmark

C Olsen

L Hansen

H Beck-Nielsen

A Kok

S Patel

A Mari

L Landucci

S Hills

L Mota

G Pacini

C Cavaggion

Physical Activity and Insulin Sensitivity. The RISC Study

Keywords:

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

RESEARCH DESIGN AND METHODS-This cross-sectional study is of 346 men and 455 women, aged 30 to 60 years, without cardiovascular disease and not treated by drugs for diabetes, hypertension, dyslipidaemia or obesity; they were recruited in 18 clinical centres from 13 European countries. Insulin sensitivity was measured by hyperinsulinaemic euglycaemic clamp. Physical activity was recorded by accelerometry for a median of six days. We studied the relationship of insulin sensitivity with total activity (number of counts/min), percent time spent sedentary, percent tine in light activity and activity intensity (whether the participant recorded some vigorous or some moderate activity).

RESULTS-In both men and women, total activity was associated with insulin sensitivity (P < 0.0001). Time spent sedentary, in light activity, and activity intensity were also associated with insulin sensitivity (P < 0.0004/0.01; 0.002/0.03; 0.02/0.004 respectively for men/women), but lost significance once adjusted for total activity. Adjustment for confounders, such as adiposity, attenuated the relations with total activity; there were no interactions with confounders. Even in the 25% most sedentary individuals, total activity was significantly associated with better insulin sensitivity (P < 0.0001) CONCLUSIONS-Accumulated daily physical activity is a major determinant of insulin sensitivity. Neither time spent sedentary, in light-activity, nor bouts of moderate or vigorous activity impacted on insulin sensitivity independently of total activity. Physical activity is now recognised as a major component of type 2 diabetes prevention: cohort studies have documented the lower risk of incident diabetes even for everyday activities such as walking [START_REF] Joen | Physical activity of moderate intensity and risk of type 2 diabetes: a systematic review[END_REF][START_REF] Hu | Walking compared with vigorous physical activity and risk of type 2 diabetes in women: a prospective study[END_REF]. In a post hoc analysis of the Finnish Diabetes Prevention Study, walking for exercise for at least two and a half hours a week in comparison to less than one hour, was associated with a 63-69% lower risk of incident diabetes (3). Physical activity is a complex behaviour, characterised by intensity, duration and frequency [START_REF]Physical Activity and Health: A Report of the Surgeon General[END_REF]. Various consensus groups recommend physically active lifestyles for adults, with an accumulation of at least 30 min of moderate-intensity aerobic physical activity five or more days a week or vigorous intensity aerobic physical activity for at least 20 min three days a week [START_REF] Haskell | Physical Activity and Public Health. Updated Recommendation for Adults From the American College of Sports Medicine and the American Heart Association[END_REF]. Another important dimension is the time spent in sedentary occupations [START_REF] Hamilton | Role of low energy expenditure and sitting in obesity, metabolic syndrome, type 2 diabetes, and cardiovascular disease[END_REF]: in the Nurses Health Study, the number of hours spent sedentary was related with incident diabetes even after adjusting for total physical activity [START_REF] Hu | Television watching and other sedentary behaviors in relation to risk of obesity and type 2 diabetes mellitus in women[END_REF].

Physical activity may decrease the risk of diabetes by increasing insulin sensitivity [START_REF] Joen | Physical activity of moderate intensity and risk of type 2 diabetes: a systematic review[END_REF]. Insulin sensitivity has been shown to increase with physical activity, as assessed by questionnaire [START_REF] Mayer-Davis | Intensity and amount of physical activity in relation to insulin sensitivity: the Insulin Resistance Atherosclerosis Study[END_REF].

Objective assessment of physical activity is now possible with unobtrusive accelerometer-based motion sensors [START_REF] Freedson | Calibration of the Computer Science and Applications, Inc. accelerometer[END_REF][START_REF] Ekelund | Physical activity and metabolic risk in individuals with a family history of type 2 diabetes[END_REF].

The aim of this study was to describe the relationship between insulin sensitivity -as measured by the "gold standard" hyperinsulinaemic euglycaemic clamp -and habitual physical activity assessed by accelerometer: total activity, its intensity, and time spent in light and sedentary activities.

RESEARCH DESIGN AND METHODS

Participants and protocol. In 2002-2004, healthy adults aged 30-60 years, without diabetes, hypertension or dyslipidaemia, were recruited into the European RISC (Relationship between Insulin Sensitivity and Cardiovascular risk) Study [START_REF] Hills | EGIR-RISC Study Group: The EGIR-RISC STUDY (The European group for the study of insulin resistance: relationship between insulin sensitivity and cardiovascular disease risk): I. Methodology and objectives[END_REF][START_REF] Ferrannini | RISC Investigators: Insulin resistance, insulin response, and obesity as indicators of metabolic risk[END_REF]. Each centre had Ethics Committee approval and participants signed an informed consent.

Participants had a clinical examination, an oral glucose tolerance test and were fitted with an accelerometer, which they returned one week later when they presented for their hyperinsulinaemic euglycaemic clamp.

We report on 346 men and 455 women from 18 clinical centres, with data available on insulin sensitivity and accelerometer-measured physical activity.

Physical activity -accelerometer. Physical activity was measured objectively by a small single-axis accelerometer, (ACTIGRAPH, AM7164-2.2 Computer Science and Applications Inc, Florida USA) [START_REF] Freedson | Calibration of the Computer Science and Applications, Inc. accelerometer[END_REF][START_REF] Ekelund | Physical activity and metabolic risk in individuals with a family history of type 2 diabetes[END_REF]. The acceleration signal was digitized with 10 samples per s, registered as counts over 1-min intervals. The accelerometer was worn for up to eight days, on a belt in the small of the back, from waking to going to bed except during water-based activities. We analysed participants with at least three days of data, including days when the device was worn more than 10 hours; we assumed it was not worn if there were 60 consecutive min with no counts. Accelerometer data were processed with custom software developed for this project, using SAS Version 9. Data was checked for spurious recording -high counts >20,000 counts/min or repeated counts [START_REF] Masse | Accelerometer data reduction: a comparison of four reduction algorithms on select outcome variables[END_REF]. Our software provided:

• total activity: average number of counts/min when accelerometer was worn;

• activity intensity group: participants were classed as having on any day when accelerometer was worn (a) some vigorous activity (>5724 counts/min for at least 10 consecutive min); (b) some moderate activity (1952-5724 counts/min for at least 10 consecutive min); (c) neither moderate nor vigorous activity on any day (9);

• percent time sedentary: with <100 counts/min when accelerometer worn [START_REF] Ekelund | Physical activity and metabolic risk in individuals with a family history of type 2 diabetes[END_REF];

• percent time in light activity: not sedentary nor in moderate or vigorous activity. Anthropometric measurements. On lightly clad participants we measured: body weight and fatfree mass by bipodal bio-electric impedance (Tanita, TBF 300), height with a stadiometer, waist circumference with a horizontally placed tailor's tape-measure mid-way between the lower costal margin and the iliac crests.

Smoking and alcohol. Never smokers, ex-smokers, current smokers, family history of diabetes, menopause and alcohol intake were from a self-administered questionnaire.

Analytical methods. Local laboratory data were used for study inclusion criteria. Blood collected was stored at -20°C and centrally analysed in Odense, Denmark: plasma glucose was measured by the Glucose Oxidase Technique (Roche Cobas Integra) and serum insulin by a specific timeresolved fluroimunoassay (AutoDELFIA TM Insulin kit, Wallac Oy, Turku, Finland).

Insulin sensitivity. A 2-hour hyperinsulinaemic euglycaemic clamp used a primed-continuous

infusion rate of 240 pmol . min -1. m -2 and a variable dextrose infusion adjusted every 5-10 min to maintain the plasma glucose level within 0.8 mmol/l (±15%) of target glucose (4.5-5.5 mmol/l).

The procedure was standardised across centres, with a written protocol and a video demonstration; data from the clamp were quality controlled centrally [START_REF] Hills | EGIR-RISC Study Group: The EGIR-RISC STUDY (The European group for the study of insulin resistance: relationship between insulin sensitivity and cardiovascular disease risk): I. Methodology and objectives[END_REF][START_REF] Ferrannini | RISC Investigators: Insulin resistance, insulin response, and obesity as indicators of metabolic risk[END_REF].

Insulin sensitivity is expressed as the ratio of the M value -averaged over the final 40 min of the clamp and normalised by fat-free mass to the mean plasma insulin (I) over the final 40 min of the clamp (M/I, in µmol . min -1. kg ffm -1. nM -1 ) [START_REF] Ferrannini | RISC Investigators: Insulin resistance, insulin response, and obesity as indicators of metabolic risk[END_REF].

Statistical analysis. SAS version 9 was used and statistical significance refers to P<0.05.

Logarithms of insulin sensitivity, fasting insulin, 2-hour plasma insulin and average number of counts/min worn were used in statistical testing; data are presented transformed to the original units. Participant characteristics are described by means (SD) or %, and compared between genders by t-and χ 2 tests. MIXED linear models were used to predict insulin sensitivity, adjusted for age classes (<40, 40-49, ≥ 50 years) and for recruitment centre as a random factor, and for gender when men and women were combined. Mean insulin sensitivity (95% confidence interval) is shown according to evenly spaced classes of total activity, percent time sedentary, and activity intensity, and tested for linear trends. Beta coefficients quantify the relations between insulin sensitivity and activity variables; additional adjustments were made for other activity variables and for potential confounding factors (BMI, waist, fasting glucose, alcohol intake, smoking, diabetes in family, menopause). The relations between insulin sensitivity and activity variables were linear, as quadratic terms were non-significant; interactions with centre, age class and confounders were nonsignificant.

As gender interactions were non-significant, men and women were combined. Total activity and percent time sedentary were divided into quartiles, and the mean insulin sensitivity in the resulting 16 categories estimated and trend tests were used to compare across quartiles.

RESULTS

On average, men were 43 years, women 45 years, with BMI of 25.9 and 24.4 kg/m² respectively (Table 1). The accelerometers were worn for a median of 6 days, a total of 89 hours in men, 87 hours in women. While total activity was similar between genders, men spent more time sedentary and in vigorous or moderate intensity activity than women (Table 1). Women spent more time than men in light intensity activity.

Insulin sensitivity was positively related with total activity, with percent time spent in light activity and with activity intensity, and negatively with time spent sedentary, in both men and women (Fig. 1, Table 2). However, after adjustment for total activity, the relations with insulin sensitivity were no longer significant for other activity variables. Adjustment for other potential confounding factors attenuated the relations between insulin sensitivity and total activity.

In the most sedentary quartile group, those with more than 68% time sedentary, there was a highly significant relation between insulin sensitivity and total activity (P trend <0.0001) (Fig. 2); for the other quartiles, there was a trend for a positive relation (P trend <0.1, 0.03, 0.2 respectively).

In contrast, for a given quartile group of total activity, there was no significant trend between insulin sensitivity and time spent sedentary (P trend 0.2, 0.4, 0.9, 0.5 respectively).

DISCUSSION

This is the first study to report the relation between physical activity and insulin sensitivity using the "gold standard" method for determining insulin sensitivity and an objective measure of activity. Our results show that in both men and women, total physical activity is the key parameter positively related with insulin sensitivity: neither the percent time spent sedentary, in light-activity or the intensity of activity were associated with insulin sensitivity after adjusting for total activity.

The percent time spent in moderate or vigorous activity was less than 2%, with 38% in light activity. Thus, it is the accumulation of physical activity, over the day, that appears to be the determinant of insulin sensitivity. The relation between total activity and insulin sensitivity remained after adjusting for overall (BMI) or abdominal adiposity (waist circumference) or other potential confounders.

In the Insulin Resistance Atherosclerosis Study, insulin sensitivity was quantified by the frequently sampled intravenous glucose tolerance test and physical activity by questionnaires; insulin sensitivity was positively associated with the frequency of vigorous physical activity and also with the energy expended in vigorous and non-vigorous activities [START_REF] Mayer-Davis | Intensity and amount of physical activity in relation to insulin sensitivity: the Insulin Resistance Atherosclerosis Study[END_REF]. Other studies have measured physical activity objectively, but used surrogate measures of insulin sensitivity. Total activity as measured by accelerometer has been shown to be more strongly related to fasting insulin than either sedentary or moderately intense activity [START_REF] Ekelund | Physical activity and metabolic risk in individuals with a family history of type 2 diabetes[END_REF]. In line with our results, after one-year follow-up in the ProActive trial, fasting insulin was significantly associated with change in total body movement, as measured by accelerometer [START_REF] Simmons | On behalf of the ProActive Research Team: Increasing overall physical activity and aerobic fitness is associated with improvements in metabolic risk: cohort analysis of the ProActive trial[END_REF]. Fasting glucose has been shown to be related to total activity (counts/min worn), but not to time spent in sedentary, light or moderate to vigorous intensity activities [START_REF] Healy | Objectively measured sedentary time, physical activity, and metabolic risk: the Australian Diabetes, Obesity and Lifestyle Study (AusDiab)[END_REF]; in contrast in the same population, 2-hr glucose was related to time spent in all three activities [START_REF] Healy | Objectively measured light-intensity physical activity is independently associated with 2-h plasma glucose[END_REF].

Among the strengths of the RISC study is the quality control procedure used to evaluate each clamp as it was completed in clinical centres [START_REF] Hills | EGIR-RISC Study Group: The EGIR-RISC STUDY (The European group for the study of insulin resistance: relationship between insulin sensitivity and cardiovascular disease risk): I. Methodology and objectives[END_REF]. Only healthy individuals, from a wide spectrum of countries, were selected for the study, with no drug treatment for diabetes, hypertension, lipids or obesity and a healthy clinical and biological profile.

We have used accelerometer decision rules written specifically for this project, and these rules may affect some of the results [START_REF] Masse | Accelerometer data reduction: a comparison of four reduction algorithms on select outcome variables[END_REF]. We assumed that the accelerometer was not worn if 60 min was recorded with no activity, thus we may record longer percentages of sedentary time than other studies. For moderate or vigorous activity, we required at least 10 min of this activity, in line with current physical activity recommendations that refer to "bouts lasting 10 or more min" [START_REF] Haskell | Physical Activity and Public Health. Updated Recommendation for Adults From the American College of Sports Medicine and the American Heart Association[END_REF]. Activity intensity may be underestimated as it has been studied in three groups: more than a third of the population recorded no moderate or vigorous activity, thus intensity is difficult to study as a continuous variable. Further, it has been reported that the accelerometer underestimates higher intensity movements [START_REF] Brage | Reexamination of validity and reliability of the CSA monitor in walking and running[END_REF].

Physical activity can influence insulin sensitivity in many ways, among which are: (a) enhancing both GLUT4-dependent and hypoxia-dependent glucose transport in skeletal muscle [START_REF] Holloszy | Exercise-induced increase in muscle insulin sensitivity[END_REF]; (b) increasing skeletal muscle vascularization, mitochondrial neobiogenesis, and eventually, tissue mass [START_REF] Joseph | Control of gene expression and mitochondrial biogenesis in the muscular adaptation to endurance exercise[END_REF]; (c) repartitioning intracellular fat, thereby improving its utilisation [START_REF] Horowits | Exercise-induced alterations in muscle lipid metabolism improve insulin sensitivity[END_REF]; (d) fat mass loss. We and others have shown that aerobic exercise has a dose-and intensity-related effect to increase insulin signalling and glucose transporter content in skeletal muscle [START_REF] Sriwijitkamol | Effect of Acute Exercise on AMPK signalling in Skeletal Muscle of Subjects with Type 2 Diabetes[END_REF][START_REF] O'gorman | Exercise training increases insulin-stimulated glucose disposal and GLUT-4 (SLC2A4) protein content in patients with Type 2 diabetes[END_REF]. Exercise training increases insulin-stimulated glucose disposal and GLUT-4 (SLC2A4) protein content in obese patients with type 2 diabetes [START_REF] O'gorman | Exercise training increases insulin-stimulated glucose disposal and GLUT-4 (SLC2A4) protein content in patients with Type 2 diabetes[END_REF]. High levels of sedentary time produce the reverse effect. However, inactivity physiology may be qualitatively different to exercise physiology, with different cellular mechanisms [START_REF] Hamilton | Role of low energy expenditure and sitting in obesity, metabolic syndrome, type 2 diabetes, and cardiovascular disease[END_REF]. For example, experimental data from animals show that reducing lowintensity activity had a greater effect on skeletal muscle lipoprotein lipase regulation than adding intensive exercise [START_REF] Hamilton | Role of low energy expenditure and sitting in obesity, metabolic syndrome, type 2 diabetes, and cardiovascular disease[END_REF]. This parallels an Australian study which found that the negative effects on hyperinsulinaemia of 14 h per week of TV viewing, were similar to the beneficial effects of 2.5 hours of physical activity (walking and more vigorous activities) [START_REF] Dunstan | AusDiab Steering Committee: Associations of TV viewing and physical activity with the metabolic syndrome in Australian adults[END_REF].

Our study emphasizes that activity has beneficial effects on insulin sensitivity. In this population of men and women aged 30-60 years, the total accumulated activity was the important factor rather than the intensity of the activity. Even in those who spent most of their time sedentary, more movement during the day from work, household, commuting and also from leisure and sporting activities, accumulated to exert a beneficial effect on insulin sensitivity, and so decrease the risk of type 2 diabetes and other diseases associated with insulin resistance. These results highlight the importance of even light activity, which should be taken into account in recommendations for increasing the total amount of physical activity in the general population to prevent diabetes.

TABLE 1

 1 Anthropometric, activity and metabolic characteristics: mean (SD) and percentages. The RISC Study

	Men (n=346)	Women (n=455)	P value

TABLE 2

 2 Relations between activity parameters and insulin sensitivity*: beta regression coefficients (standard errors) adjusted for age class and recruitment centre, and for gender when men and women combined, and then additionally adjusted, one by one, for other activity parameters and potential confounders. The RISC Study.

	Men	Women	Men & Women
	n=346	n=455	n=801

time in light activity (% time not sedentary, nor in moderate or vigorous activity)

	Activity intensity (
	adjusted: age, centre	0.0083 (0.0027)	0.002	0.0043 (0.0019)	0.03	0.0061 (0.0016)	0.0002
	adjusted: age, centre, total activity*	0.0037 (0.0032)	0.3	-0.0029 (0.0024)	0.2	0.0001 (0.0019)	0.9
	adjusted: age, centre, % time sedentary	-0.0177 (0.0112)	0.1	-0.0150 (0.0095)	0.1	-0.0149 (0.0072)	0.04
	adjusted: age, centre, activity intensity	0.0088 (0.0027)	0.001	0.0045 (0.0019)	0.02	0.0064 (0.0016)	0.0001
	(3 classes)

in three classes, some vigorous, some moderate, neither moderate nor vigorous activity) some moderate vs neither moderate nor vigorous some vigorous vs neither moderate nor vigorous

  Mean insulin sensitivity (age, sex and recruitment centre adjusted) and physical activity measured by accelerometer (in men and women combined) by quartiles of average number of counts/min worn and quartiles of percent time sedentary.Table gives the distribution of participants according to total-activity and time-sedentary quartiles. The RISC Study.

	adjusted: age, centre adjusted: age, centre, total activity * adjusted: age, centre, % time sedentary adjusted: age, centre, % time light activity 120 120 120 sedentary time (B) and by groups according to some moderate and some vigorous intensity activity 0.070 (0.067) 0.22 (0.08) 0.02 0.091 (0.042) 0.23 (0.07) 0.004 0.017 (0.069) 0.088 (0.092) 0.6 0.028 (0.043) 0.11 (0.08) 0.4 0.055 (0.066) 0.19 (0.08) 0.04 0.081 (0.042) 0.22 (0.07) 0.006 0.064 (0.066) 0.23 (0.08) 0.01 0.088 (0.041) 0.24 (0.07) 0.003 140 140 140 180 women, according to: mean number of counts/min that the accelerometer was worn (A), by percent 160 160 160 Figure legends FIG. 1. Mean insulin sensitivity (95% confidence interval), adjusted for age and clinical recruitment centre, and characteristics of physical activity measured by accelerometer in men and 180 200 220 A Insulin sensitivity, M/I (µmol min-1 kgffm-1 nM-1) 200 220 13% B 220 C Insulin sensitivity, M/I (µmol min-1 kgffm-1 nM-1) Insulin sensitivity, M/I men, p trend=0.0004 women, p trend=0.005 200 (µmol min-1 kgffm-1 nM-1) Insulin sensitivity: men, p trend=0.0001 women, p trend=0.0001 180 180 men, p trend=0.007 women, p trend=0.0009 M/I (	0.083 (0.036) 0.22 (0.05) 0.022 (0.038) 0.070 (0.036) 0.20 (0.05) 0.078 (0.036) 0.23 (0.05) 0.085 (0.057)	0.004 0.0001 0 0.3
	* logarithm of variable (C). The RISC Study. 100 100 100 160
	30/06/2008 30/06/2008 30/06/2008 30/06/2008 quartiles: percent time sedentary FIG. 2. RISC RISC RISC RISC 80 60 80 80 140 60 60 men 11% 27% 31% 13% 6% women 11% 29% 27% 20% 8% 200 300 400 500 average number of counts per minute worn men 5% 8% 8% 18% 21% 16% women 9% 10% 16% 18% 16% 13% 45 50 55 60 65 70 no moderate some percent of time sedentary men 33% 46% women 38% 52% or vigorous activity moderate activity 4 3 2 53% 61% 68% 80 100 120	1	12% 6% some 75 12% 600 8% 21% 10% vigorous activity 11% 9% 1 261	19/22 20/22 21/22 22/22 minute 4 428 3 quartiles: 341 2 average counts per

µmol.min -1 kgffm -1 nM -1 )

		distribution of participants according to total activity and sedentarity
	13% 8% 3% 1%	6% 7% 9% 2%	4% 6% 8% 6%	2% 2% 5% 16%	>428 341-428 261-341 <261	quartiles: average counts per minute worn
	<53%	53-61%	61-68%	> 68%

quartiles: percent time sedentary

ACKNOWLEDGMENTS

The RISC Study received the EU grant QLG1-CT-2001-01252. Additional finances have been provided by AstraZeneca (Sweden). The EGIR group is supported by Merck Santé, France. L Mhamdi was financed in part by a grant from La Fondation de France. C Lange is thanked for her help with the accelerometer data.