Ali Houry

Michel Gohar

Julien Deschamps

Ekaterina Tischenko

Stéphane Aymerich

Alexandra Gruss

Romain Briandet
email: romain.briandet@jouy.inra.fr.

Bacterial swimmers that infiltrate and take over the biofilm matrix

Bacteria grow in either planktonic form or as biofilms, which are attached to either inert or biological surfaces. Both growth forms are highly relevant states in nature and of paramount scientific focus. However, interchanges between bacteria in these two states have been little explored. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we show that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances in nature. We hypothesized and proved that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. These findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations. M icrobial biofilms constitute the major lifestyle alternative to planktonic growth and are commonly formed on inert or living surfaces. In these biological structures, cells are held together in a 3D organization by self-produced extracellular polymeric substances (EPSs). EPS composition varies greatly depending on the biofilm ecosystem but typically contains a mixture of principally polysaccharides, as well as proteins, nucleic acids, and lipids [START_REF] Flemming | The biofilm matrix[END_REF]. This complex mixture of hydrated biomolecules is responsible for matrix strength and biofilm viscoelastic properties by means of weak physicochemical interactions (e.g., van der Waals, Lewis acid-base, and electrostatic interactions; polymers; entanglement involving flagella or pili) [START_REF] Flemming | The biofilm matrix[END_REF]. The development of this spatial organization results in molecular gradients within the matrix of nutrients, oxygen, and signaling molecules, which generate local physiological and genetic heterogeneities [START_REF] Stewart | Physiological heterogeneity in biofilms[END_REF]. The matrix can also act as a "protective shield" against the diffusion and action of antimicrobials in the bulk of the biofilm (3), thus posing a serious problem for treatment of biofilm-related infections and for microbial elimination in industrial settings [START_REF] Mah | Mechanisms of biofilm resistance to antimicrobial agents[END_REF]. According to National Institutes of Health estimates, biofilms account for over 80% of human microbial infections (http://grants.nih.gov/grants/ guide/pa-files/PA-03-047.html).

Biofilms are dynamic structures that may be subject to population shifts in response to microbial composition and environmental conditions. Numerous factors of microbial competitiveness (e.g., nutritional fitness, resistance, growth rate) may have an impact on both sessile and planktonic populations [START_REF] Litchman | Invisible invaders: Non-pathogenic invasive microbes in aquatic and terrestrial ecosystems[END_REF][START_REF] Wintermute | Dynamics in the mixed microbial concourse[END_REF]. Other factors appear to be specific to biofilms [START_REF] Stewart | Physiological heterogeneity in biofilms[END_REF][START_REF] West | Social evolution theory for microorganisms[END_REF]. Our studies led us to examine the role of motility, studied mainly in planktonic populations to date, on bacterial integrity in biofilms. Bacterial flagella were previously reported to have a positive role in nascent biofilm maturation and spreading (e.g., Bacillus subtilis, Pseudomonas sp.) [START_REF] Lemon | Flagellar motility is critical for Listeria monocytogenes biofilm formation[END_REF][START_REF] Tolker-Nielsen | Development and dynamics of Pseudomonas sp. biofilms[END_REF]. However, motile cells in mature biofilms have thus far been described as arising from a late-stage differentiation event (e.g., within hollow voids of Pseudomonas aeruginosa mushroom-like structures) and being involved in dispersion of mature biofilms [START_REF] Mcdougald | Should we stay or should we go: Mechanisms and ecological consequences for biofilm dispersal[END_REF].

Here, we report the discovery of highly motile populations within the entire biofilm matrix of several bacilli and other flagellated bacteria in early stage and mature biofilms. These movements generate short-lived pores that irrigate the biofilm and facilitate entry of macromolecules, including antimicrobials. Importantly, we illustrate how planktonic motile bacteria with high kinetic energy, such as motile bacilli, can act as invaders, leading to dissolution of heterologous biofilms and repopulation of the matrix.

Results

Motile Bacteria Generate Pores in the Biofilm Matrix. We examined biofilm formation by the motile bacterium Bacillus thuringiensis 407. The majority of cells in the biofilm matrix oscillate in a volume limited to their own cell size (a few micrometers), as expected for constrained sessile bacteria [START_REF] Rogers | Microrheology of bacterial biofilms in vitro: Staphylococcus aureus and Pseudomonas aeruginosa[END_REF]. However, timelapse confocal laser microscopy uncovered the existence of motile subpopulations in the mature biofilm, estimated to represent between 0.1% and 1% of the total population (Fig. 1 and Movie S1). Observed movements are flagella-propelled because they were not detected with flagella-deficient (Δfla) or immobile (ΔmotA) mutants (Movie S2). Bacteria moved up to 16 μm/s in all directions, following different paths through the biofilm mass. Swimming cells passing through the bulk of the biofilm created transient tunnels that lasted for about 2-5 s, highlighting the reversible elastic properties of the matrix [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Klapper | Viscoelastic fluid description of bacterial biofilm material properties[END_REF]. In addition to linear movement, rapid circular movements of bacterial chains generated local dispersion and large transient pores (∼10 μm diameter), as visible in the still frame from Movie S1 (Fig. 1). Typical chain rotation frequencies were about 0.2 Hz. The tunneling phenomenon was observed in flow cell biofilms as above and also in a microplate batch system (below), suggesting that flow of the liquid medium is not compulsory for this phenomenon.

In a second set of experiments, exponential phase GFP-labeled B. thuringiensis cells were deposited onto unlabeled B. thuringiensis biofilms. After less than 15 min, fluorescent swimming cells were visualized at the base of the biofilm (Movie S3). The rapidity of this event gives strong evidence that the swimmers detected in biofilms are supplied by the liquid planktonic phase and infiltrate the biofilm matrix. This also excludes the possibility that a mutational event is required for swimming in biofilms. We therefore consider it likely that the swimmers observed in biofilms issue from the planktonic population.

Tests for the capacity of other flagellated bacteria to generate pores within their own biofilm structures indicate that biofilm swimming may be widespread: One of four tested isolates (Table S1) of the Gram-negative motile bacterium Yersinia enterocolitica displayed a phenotype similar to that of B. thuringiensis 407 (CIP 106676; as shown in Movie S4, kindly provided by M. Naïtali, AgroParisTech). However, strains of motile species Pseudomonas aeruginosa (ATCC 1592) and Bacillus subtilis (168) did not detectably irrigate their own biofilms in our test conditions. Swimming in such biostructures might require sufficient kinetic energy to overcome the cohesive force generated by the EPS, and would thus vary according to the "swimmer" and the target biofilm [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Rogers | Microrheology of bacterial biofilms in vitro: Staphylococcus aureus and Pseudomonas aeruginosa[END_REF][START_REF] Klapper | Viscoelastic fluid description of bacterial biofilm material properties[END_REF]. It is also possible that biofilm bacteria modulate stealth swimmer activity via signaling molecules that inhibit motility within the matrix.

Impact of Biofilm Age on Tunnel Formation. The properties of tunnels generated in B. thuringiensis 407 biofilms were followed as a function of biofilm age. The time courses of biofilm macrostructure and swimmer velocities were recorded over a 3-d period (Fig. S1). The average velocity of swimmer cells, estimated by tracking ∼50 single cell trajectories in four microscopic fields, ranged from 7.3 μm/s at 24 h of biofilm growth to 4.2 μm/s at 72 h of biofilm growth. In this dataset, the prevalent proportion of swimmers traveled faster at 24 h (7 μm/s) than at 72 h (about 2 μm/s); at 72 h, short chains adopted a snake-like motion to burrow through the dense network of sessile cells. Thus, although bacterial motility within biofilms progressively slowed with the age of biofilms, a swimming subpopulation remained active within biofilms for at least 72 h.

Changes in viscoelastic properties of the B. thuringiensis matrix could account, at least in part, for reduced swimming trajectories in the older biofilms. We observed that sessile cell oscillations, which are inversely correlated with the matrix strength [START_REF] Rogers | Microrheology of bacterial biofilms in vitro: Staphylococcus aureus and Pseudomonas aeruginosa[END_REF], were 25% lower in 72-h biofilms compared with 24-h samples (P < 0.05). In the case of B. thuringiensis 407, this could be correlated with changes in the matrix exopolysaccharide density, which increased from 17 μg/mm 3 at 24 h to 49 μg/mm 3 at 72 h (P < 0.05).

To determine whether the state of the matrix is responsible for the slower speed of swimmers in older biofilms, we evaluated the behavior of freshly introduced planktonic GFP-labeled B. thuringiensis 407 cells in established 24-h-old or 72-h-old nonfluorescent biofilms. Quantification of fluorescence 2 h after depositing the motile fluorescent cells revealed that eightfold more cells integrated in the 24-h biofilm than in the 72-h biofilm (800 vs. 100 μm 3 of cells expressing GFP, respectively; P < 0.05). These results indicate that biofilm age and matrix viscoelastic properties are important determinants for swimming activity in biofilms.

Penetration of Macromolecules in Biofilms Is Facilitated by Swimmer

Bacteria. We hypothesized that pores created by motile bacilli generate microcurrents that facilitate solute access to the deeper biofilm layers. A high-molecular-weight fluorescent macromolecular tracer (FITC-dextran, 250 kDa) was used to mimic the solute. Time-lapse confocal imaging was used to follow its entry in B. thuringiensis 407 biofilms comprising motile (Bt WT) or nonmotile (Bt ΔmotA) bacteria. Note that Bt and Bt ΔmotA biofilms show similar geometrical metrics (no significant differences in biovolume, thickness, and roughness and permeability to swimmers, as determined by confocal imaging and image analysis) [START_REF] Houry | Involvement of motility and flagella in Bacillus cereus biofilm formation[END_REF]. In conditions of shear stress, the motile Bt subpopulation in WT biofilms promotes spreading on the biofilm surface, which is not observed in Bt ΔmotA biofilms [START_REF] Houry | Involvement of motility and flagella in Bacillus cereus biofilm formation[END_REF]; however, this phenomenon does not have an impact on biofilm spatial organization. Migration of FITC-dextran into the basal layer of Bt WT and Bt ΔmotA was monitored as fluorescence deep within the biofilm structure, at ∼5 μm from the inert surface (Fig. 2A). The initial slope value for fluorescence curves (reflecting FITC-dextran migration) from Bt ΔmotA biofilms reflects a 1-log lower rate of FITC-dextran penetration compared with the rate using the Bt WT swimmer strain (Fig. 2B).

These results show that mechanical movements of motile bacteria contribute to a dynamic environment within biofilm by promoting molecular exchanges between its different layers and with external medium. We suggest that swimmers would thereby facilitate solute exchange and dissipation of harsh local environments generated within biofilms.

Motile Bacilli Penetrate Biofilms Comprising a Heterologous Species.

The biofilms generated by bacteria may be markedly different with respect to their viscoelastic properties, relating to differences in their EPS composition. Above a critical kinetic energy specific to each matrix, a motile swimmer can generate a reversible transient elastic response and/or an irreversible deformation [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Klapper | Viscoelastic fluid description of bacterial biofilm material properties[END_REF]. We therefore tested the capacity of motile B. thuringiensis 407 to tunnel into biofilms comprising different Gram-positive or Gramnegative pathogens (Table S1): Staphylococcus aureus RN4220 (Movie S5), Enterococcus faecalis (ATCC 700802; as shown in Movie S6), Y. enterocolitica (CIP 81.41), P. aeruginosa (ATCC 1592), or Listeria monocytogenes (ATCC BAA-679). These selected strains were confirmed to lack the "swimmer phenotype" within their same species biofilms. All these strains were permissive to the heterologous bacillus swimmers.

We tested other swimmers: four Bacillus licheniformis isolates, two B. subtilis strains, and two Bacillus cereus strains (Table S1) were used in experiments with S. aureus RN4220 biofilms (shown with B. licheniformis LMG 7560 in Movie S7). Four of the eight tested motile bacilli were obtained as isolates from surfaces in medical or industrial equipment (Table S1). Tunneling was observed with all tested motile bacilli, suggesting that stealth swimming in heterologous biofilms is a likely occurrence in natural environments.

We observed that B. thuringiensis 407 also infiltrated B. subtilis 168 biofilms. Swimming activity was also tested in a biofilm comprising a B. subtilis 168 ΔabrB mutant, in which matrix components and biofilm formation are up-regulated [START_REF] Hamon | The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis[END_REF][START_REF] Bridier | The spatial architecture of Bacillus subtilis biofilms deciphered using a surface-associated model and in situ imaging[END_REF]. The large clusters formed by this mutant were refractive to B. thuringiensis swimmers. This limit to swimming activity suggests a threshold matrix density or the existence of specific matrix components that can obstruct penetration of swimmers. More experiments are required to make definitive assignments on the matrix factors that impede swimmers.

All together, these results show that motile bacilli can perturb heterologous biofilm populations, and highlight the importance of matrix properties for swimming capacity in a given swimmerbiofilm couple.

Swimmer Infiltration Sensitizes S. aureus Biofilms to Chemical Antimicrobials. These results showed that macromolecules gain entry into biofilms by means of tunnels formed by motile bacteria. We considered that this phenomenon would facilitate penetration, and hence effectiveness, of exogenously added disinfectants. This was tested using different bacillus species and benzalkonium chloride, a biocide frequently used in hospital and industrial settings, where biofilms are a common occurrence. S. aureus RN4220 biofilms were preexposed or not preexposed to B. thuringiensis 407 and B. licheniformis LMG 7560 swimmers, either separately or in combination, followed by a 5-min exposure to the disinfectant. Compared with the treatment in the absence of bacilli, which resulted in an eightfold decrease in S. aureus counts, swimmer-pretreated biofilms showed a decrease in counts of ∼100-fold, with a synergistic effect (>300-fold decrease) in the presence of both motile strains (Table 1). The transient pores generated by motile bacteria can thus potentiate killing of biofilm bacteria by toxic substances in the environment.

S. aureus Biofilms Are Disrupted and Supplanted by B. thuringiensis Swimmers Expressing a Biocide. Numerous nonpathogenic bacteria secrete bactericidal molecules that are likely produced to reduce microbial competition (e.g., hydrogen peroxide, bacteriocins, antibiotics, autolysins) [START_REF] Abriouel | Diversity and applications of Bacillus bacteriocins[END_REF][START_REF] Selva | Killing niche competitors by remote-control bacteriophage induction[END_REF]. We reasoned that microbial motility could create a channel for in situ delivery of self-produced active bactericides to disrupt and repopulate the biofilms. As proof of principle, the integrity of biofilms formed by S. aureus (strain RN4220 expressing GFP) was challenged using either of two motile bacillus species (B. thuringiensis 407 and B. subtilis 168) expressing the S. aureus-specific cell wall endopeptidase, lysostaphin [START_REF] Gaier | Cloning and expression of the lysostaphin gene in Bacillus subtilis and Lactobacillus casei[END_REF][START_REF] Recsei | Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans[END_REF]. Biofilms that were untreated or treated with nonmotile B. thuringiensis expressing lysostaphin (BtΔfla, Fig. 3) remained essentially intact, and their biovolumes were not statistically different (P > 0.05). Remarkably, motile B. thuringiensis producing lysostaphin eradicated S. aureus within 24 h (Fig. 3). Supernatants alone or mobile non-lysostaphinproducing cells had no visible effect in the tested conditions. Equivalent results were obtained when 48-h S. aureus biofilms were exposed to the B. thuringiensis lysostaphin-producing strain. Experiments using corresponding B. subtilis strains and the motility-minus Δhag mutant expressing or not expressing lysostaphin gave results equivalent to those obtained with B. thuringiensis (Fig. S2). Moreover, similar results with lysostaphin-producing B. thuringiensis were obtained when tested against biofilms comprising other S. aureus isolates of different origins (ATCC strains 6538, 27217, and 29247). These results clearly demonstrate that motility gives toxin-carrying bacteria access deep into the biofilm layers to eradicate a preexisting population.

Counterstaining with the red nucleic acid dye SYTO 61 of the residual treated biofilms revealed a dense 3D spatial arrangement of cells visible on the surface. In our experimental conditions, B. thuringiensis 407 appear red (SYTO 61 colors all cells) and S. aureus RN4220 is GFP-tagged and appears as green or yellow (depending on relative intensities of GFP and SYTO 61). B. thuringiensis eradicates S. aureus and concomitantly builds up a new biofilm structure on the cleared surface. Eradication and replacement of the S. aureus biofilm were similarly observed when motile B. subtilis 168 was used as the bactericide carrier strain (Fig. S2).

Discussion

Microbial cells in a biofilm are generally considered as sessile and entrapped within their EPS [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Stewart | Physiological heterogeneity in biofilms[END_REF]. Discovery of high-speed swimming planktonic cells tunneling deep within the biofilm structure changes this widely accepted view of biofilm structure. Our results support a straightforward mechanism for biofilm tunneling, in which nonmutated swimmers from the general planktonic population penetrate the biofilm matrix. It is notable that several swimmer bacteria tested in these studies were initially isolated from medically or industrially related surfaces, supporting the pertinence of the reported phenomenon in nature. The physical and regulatory mechanisms that modulate stealth swimming within the biofilm matrix (e.g., to limit swimming in same-species biofilms) remain to be identified and are the subject of future studies.

The disturbances generated by tunnel formation have dual consequences: "Like" bacteria may be advantaged in that tunnels increase macromolecular transfer and may thereby improve nutrient availability and reduce harmful gradients (e.g., of pH, of secreted toxic end products). Conversely, swimming heterologous bacteria, particularly those expressing antimicrobial products, may gain a foothold in established biofilms to destroy and replace a preexisting population. In this way, bacterial swimmers may introduce rapid changes in the biofilm population.

Role of a Minor Bacterial Subpopulation in Biofilm Dynamics. The massive effects of the minor swimmer population (estimated as 0.1-1%) on biofilm structure and composition are unexpected in view of previous work. First, global approaches on biofilms would not detect expression changes or the influence of such minor subpopulations. Second, in pioneering fluorescent imaging studies of B. subtilis biofilms, expression of flagella was detected and shown to persist as a minor subpopulation in biofilm bacteria [START_REF] Vlamakis | Control of cell fate by the formation of an architecturally complex bacterial community[END_REF]. In the present study, B. subtilis swimmers in its same strain biofilm did not generate discernible perturbations, possibly explaining why this phenomenon went unnoticed.

The swimming phenomenon differs from the previously reported event of biofilm dispersal, in which motile cells differentiate within biofilm mushroom-like structures present in large clusters of mature biofilm communities. These cells spread, promoting active seeding and dispersal [START_REF] Mcdougald | Should we stay or should we go: Mechanisms and ecological consequences for biofilm dispersal[END_REF][START_REF] Marchal | Subinhibitory arsenite concentrations lead to population dispersal in Thiomonas sp[END_REF]. In contrast, stealth swimmers were observed in mature biofilms and also in early stages of biofilm formation (Fig. S1). Importantly, our results indicate that stealth swimmers originate from planktonic cells rather than from the biofilm itself. All together, these differences indicate that dispersal and stealth swimming are distinct events. We suggest that a persisting swimming subpopulation is a determinant of biofilm integrity, propagation, and turnover.

Dual Role of Motile Subpopulations in a "Biological Insurance" Hypothesis. It was previously hypothesized that diversity is a form of biological insurance that can safeguard the community in face of adverse conditions [START_REF] Boles | Self-generated diversity produces "insurance effects" in biofilm communities[END_REF]. The coexistence of sessile and motile subpopulations is an example of self-generated diversity. According to current thinking, a gradually decreasing substrate availability with time leads to slow or no growth in the deeper layers of a biofilm [START_REF] Stewart | Physiological heterogeneity in biofilms[END_REF]. In accordance with the biological insurance model, we suggest that planktonic swimmers can increase nutrient availability and gas and solute exchanges within the bulk biofilm fluid, which would favor regrowth within the deep biofilm layers by surrounding or entering cells. Alternatively, in specific niches, the existence of a motile subpopulation may be part of the equilibrium of the established community (e.g., by favoring cooperation between the microorganisms of the different layers, via solute exchanges as suggested by our work). This advantage may be reversed, however, in adverse medium, because tunneling by swimmers can also increase contact between biofilm bacteria and biocides, which would lead to accelerated cell death.

Numerous flagellated microorganisms naturally produce antimicrobial peptides, lipopeptide antibiotics, or bacteriocins, suggesting that the demonstrated biofilm assault phenomenon may occur in nature. In the case of heterologous motile species expressing antimicrobials, not only do they kill the existing biofilm bacteria but they may take over and replace them. Our findings suggest that, depending on the context, stealth swimmers can either contribute to community well-being or accelerate its destruction.

Applications of Motility to Pathogenic Biofilm Elimination. Biofilms in industry and medicine remain a principal source of contamination and infection [START_REF] Epstein | Bacterial biofilm shows persistent resistance to liquid wetting and gas penetration[END_REF][START_REF] Mah | Mechanisms of biofilm resistance to antimicrobial agents[END_REF]. Although physical disruption has been the mainstay for industrial elimination of biofilms, biological strategies using specific molecules with antimicrobial or biofilm disruptive properties provide promising alternatives for biofilm control [START_REF] Ramage | Are we any closer to beating the biofilm: Novel methods of biofilm control[END_REF][START_REF] Kolodkin-Gal | D-amino acids trigger biofilm disassembly[END_REF][START_REF] Romero | Will biofilm disassembly agents make it to market?[END_REF]. Our studies on S. aureus biofilms show that irrigation by bacillus swimmers provides a conduit for biocides and potentiates biofilm disruption. An immediate application of swimmer-induced channels is to facilitate access to currently used surface disinfectants, such as oxidizing agents or quaternary ammonium compounds. Compared with planktonic cells, bacteria embedded in biofilms are typically 10-to 1,000-fold more resistant to these products [START_REF] Mah | Mechanisms of biofilm resistance to antimicrobial agents[END_REF][START_REF] Bridier | Resistance of bacterial biofilms to disinfectants: A review[END_REF]. Improved biocide efficacy against staphylococci in the presence of bacilli opens the door to novel treatment strategies that make use of lower amounts of environmentally polluting disinfectants. We showed that swimmers can also be put to work for in situ delivery of self-produced active antimicrobial molecules to disrupt heterologous biofilms. This strategy could be applicable to the elimination of pathogens in skin, nasal, or digestive infections (e.g., by using a motile bacteriocin-producing probiotic among B. licheniformis isolates). Natural or recombinant bacilli, or combinations of bacilli that synthesize antimicrobials, may constitute potent biofilm-disrupting mixtures that uproot and replace resident biofilm bacteria.

Materials and Methods

Biological Materials. Strains and plasmids used in this study are presented in Table S1.

Detection of Tunneling Subpopulations in the B. thuringiensis Biofilm Matrix. B. thuringiensis 407 pHT315-GFP biofilms were cultivated in BST FC81 flow cells at 30 °C (Biosurface Technologies Corporation). To initiate biofilm growth, flow chambers were inoculated with 2 mL of exponential phase cultures diluted to OD 600 = 0.01. Bacteria were allowed to attach to the substratum for 1 h without flow. LB (Difco) was then pumped continuously through the flow cell channel at 27 mL/h using a Watson-Marlow 205S peristaltic pump (Watson-Marlow Ltd.). Individual cell movements inside 48-h biofilms were examined by time-lapse confocal microscopy (Leica SP2 AOBS confocal microscope at the MIMA2 microscopy platform). Cells expressing GFP were excited at 488 nm with an argon laser, and fluorescence emission was collected on a photomultiplier detector in the range of 500-600 nm using an oil-immersion objective with a magnification of 63× (n.a. = 1.4). Individual single-cell trajectories were drawn manually from image series.

Each time series was registered with a 1.6-s interval between sequential images over a maximum period of 3 min. Images in these series were collected in four different regions deep within the biofilm matrix, about 5 μm above the coverslip. Individual cell movements in 24-, 48-, and 72-h biofilms were quantified using MATLAB 7.1 software (MathWorks). Moving cells can travel in any direction through the biofilm structure. Because recordings were made in the horizontal plane of the biofilm, only cells with sufficiently long trajectories (movement recorded through at least 2 sequential images separated by 1.6 s) in a given horizontal plane could be analyzed. The first and last positions of a given moving cell were taken into account to determine its mean velocity.

Typical cell mean velocities were determined by averaging bacterial movement over four regions of the biofilms, for ∼50 cells for each biofilm. To estimate the circular rotation frequency of cell chains, we selected cells with a dominant rotation in the observation plane. Rotation frequencies of cell chains were averaged, as were mean velocity determinations.

Evolution of Matrix Strength According to Biofilm Age. The degree of thermal oscillations of sessile B. thuringiensis 407 cells was used as a matrix compactness indicator. To compare cell oscillation frequency within biofilms of different ages, we assume that there is no preferred oscillation direction/ plane that would differ according to biofilm age. The analytical approach used to estimate cell oscillation is based on the statistical fluctuations of the cells within the analyzed regions. Selected regions were devoid of detectable motile cells within the range of time recorded. We attribute short-time variations in fluorescence intensity (FI) to the fluctuations of the number of cells in the recorded area. Fluorescence periodicity in the area, interpreted as thermal oscillation of sessile cells, was determined from the derivative of fluorescence values with time. We averaged results from experiments corresponding to each biofilm age and normalized the frequency to 1 for 24-h biofilms.

Quantification of Planktonic Fluorescent Bacilli That Infiltrate the Biofilm Matrix. Evaluation of the time necessary for pioneer planktonic cells to reach the bottom of biofilms. Biofilms of B. thuringiensis 407 were grown in 96-well microscopic-grade microtiter plates [START_REF] Bridier | The biofilm architecture of sixty opportunistic pathogens deciphered using a high throughput CLSM method[END_REF]. Bacterial suspensions were prepared as described above for flow biofilms. For each condition, a calibrated suspension (250 μL) was added in three wells and incubated at 37 °C. Nonadherent cells were removed after 1 h by rinsing wells and renewing culture medium with 250 μL of sterile trypticase soy broth (TSB, BioMérieux). After incubation at 37 °C for 24 h, the growth medium was replaced by calibrated suspensions (∼10 8 cfu/mL) of B. thuringiensis 407-GFP. Individual movements of GFPexpressing cells deep within the biofilm (5 μm above the bottom of the biofilm) were recorded by time-lapse confocal microscopy 15 min after their deposition. Visualization of matrix strength evolution according to biofilm age. Twenty-fourhour and 72-h B. thuringiensis 407 biofilms grown in flow cells were perfused with 3 mL of an exponential phase culture of B. thuringiensis 407-GFP at a concentration of 10 8 cfu/mL The flow was stopped for 1 h. After this period, the flow was resumed at 15 mL/h, and after a 2-h incubation, fluorescent cells detected inside the biofilm were quantified by confocal microscopy and their biovolumes were estimated with the PHLIP MATLAB routine [START_REF] Bridier | The biofilm architecture of sixty opportunistic pathogens deciphered using a high throughput CLSM method[END_REF].

Analysis of the Exopolysaccharide Matrix Density. B. thuringiensis 407 biofilms grown for 24 h and 72 h in flow cells were perfused for 10 min with PBS to remove loosely attached material. After the flow was resumed, the biofilm was mechanically detached from the glass slides and suspended in 1 mL of PBS. The recovered suspension was dissociated in a Potter glass homogenizer operated with 10 pestle strokes at 4 °C. The suspensions were then centrifuged at 2,700 × g for 10 min at 4 °C, and the exopolysaccharide matrix was recovered from the supernatant and frozen until use. Sugar concentration (μg/mL) was determined using the phenol-sulfuric acid assay [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF]. Sugar density within the biofilms was calculated by dividing the measured sugar concentrations by the total biovolume (μm 3 ) of cells initially present on the flow cell glass surface.

Kinetics of FITC-Dextran Penetration in B. thuringiensis Biofilms. Three biofilms of B. thuringiensis 407 (Bt WT) and ΔmotA (Bt ΔmotA) were grown in 96well microscopic grade microtiter plates as described previously. For each of the six biofilms, 2D time-lapse confocal acquisition was started just before addition of 50 μL of fluorescent tracer in each well (FITC-dextran, 250 kDa per 1 mg/mL; Sigma). Fluorescence acquisition was performed every second for 10 min through an objective with a magnification of 10× (excitation of the fluorophore at 488 nm and fluorescence acquisition in the range of 500-600 nm). The increase of FI over time was extracted from image sequences and regressed with the exponential function FI = A.(1e -kt ), where A is the maximum fluorescence intensity (FI), k a rate constant and A.k the initial slope of the curve, which reflects the initial kinetics of tracer penetration in the biofilm matrix.

Growth of Bacterial Biofilms in Microplates. S. aureus, E. faecalis, L. monocytogenes, Y. enterocolitica, and P. aeruginosa 24-h biofilms were prepared in microplates at 37 °C and in TSB as described above for B. thuringiensis 407 (strains are listed in Table S1). Disruption of S. aureus biofilms by biocides was tested using the fluorescent strain RN4220 pALC2084 expressing GFP [START_REF] Malone | Fluorescent reporters for Staphylococcus aureus[END_REF]. The resulting biofilms had a thickness of around 30 μm. For some studies, 48-h rather than 24-h S. aureus biofilms were prepared for B. thuringiensis treatments.

S. aureus Biofilm Disruption by Chemical Biocides. Two bacillus swimmer strains were used alone or in combination to irrigate S. aureus RN4220 GFP biofilms: B. thuringiensis 407 and strain LMG 7560 of B. licheniformis, a species used as a probiotic. As a negative control, biofilms were treated with the nonmotile B. thuringiensis 407 isogenic Δfla mutant. Two hundred fifty microliters of overnight cultures at ∼10 8 cfu/mL of B. thuringiensis 407, B. thuringiensis Δfla, B. licheniformis LMG 7560, or a B. thuringiensis 407 B. licheniformis LMG 7560 mixture in equal concentrations was added to 24-h S. aureus RN4220 biofilms after supernatant removal. Microplates were incubated for 4 h at 37 °C to allow swimmer infiltration, and supernatants were removed just before disinfectant testing.

Biofilms sensitized or not sensitized by swimmers were treated with benzalkonium chloride C14 (BAC; Fluka), a biocide frequently used in food and medical environments. Two hundred microliters of BAC at 750 ppm (disinfected biofilms; note that BAC concentration approximates the industrially used concentration) or 200 μL of NaCl at 150 mM (untreated biofilm) was added in the wells. After 5 min of contact at 20 °C, 200 μL of a quenching solution (3 g/L L-α-phosphatidylcholine, 30 g/L Tween 80, 5 g/L sodium thiosulfate, 1 g/L L-histidine, 30 g/L saponin) was added in wells to neutralize biocide activity. Biofilms were then mechanically detached from slides and dispersed in 5 mL of the quenching solution. S. aureus survivors were enumerated on TSA agar after serial dilution in NaCl at 150 mM and incubation for 24 h at 37 °C. The log 10 reduction in pathogen colony-forming units was calculated by comparing the ratio of survivors of the disinfected biofilms with the untreated biofilm population.

Genetic Construction of B. thuringiensis 407 Strains Producing Lysostaphin. The gene encoding lysostaphin was PCR-amplified from plasmid pWG200 (18) by tailing with 5′ XhoI and 3′ XbaI restriction sites. The aphA3 promoter (P apha-3 ) was PCR-amplified from plasmid pDG783 (30) by tailing with 5′ EcoRI and 3′ XhoI sites. Resulting fragments were digested by the corresponding restriction enzymes and ligated into EcoRI-XbaI-restricted plasmid pHT1618 [START_REF] Lereclus | Expansion of insecticidal host range of Bacillus thuringiensis by in vivo genetic recombination[END_REF]. The resulting plasmid, pHT50, carrying the P aphA-3 -driven lysostaphin gene was established using tetracycline selection in B. thuringiensis 407 and the isogenic Δfla mutant [START_REF] Houry | Involvement of motility and flagella in Bacillus cereus biofilm formation[END_REF][START_REF] Lereclus | Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis[END_REF].

S. aureus Biofilm Disruption and Displacement by Motile Bacilli Expressing

Lysostaphin. Growth medium of 24-h S. aureus RN4220 biofilms was replaced by calibrated suspensions (∼5 × 10 6 cfu/mL) of B. thuringiensis strains 407, 407 pHT50, or 407 Δfla pHT50 by means of sterile growth medium. The microplate was then incubated for 1 h at 37 °C, after which the biofilm supernatant was replaced by fresh sterile LB. After a 24-h incubation at 37 °C, the residual S. aureus green fluorescent biofilms were analyzed using confocal microscopy. Quantification of the residual S. aureus biofilms was performed by image analysis with the PHLIP MATLAB routine, and 3D projections were built from z-image series with the Imaris 3D function (Bitplane).

Implantation of motile bacilli in place of the destroyed staphylococcal biofilms was detected by counterstaining biofilms cells using the red nucleic acid dye SYTO 61 at 5 μM (Invitrogen). All cells are stained red; S. aureus expressing GFP is colored green or yellow, whereas bacilli are red.

  bacterial ecology | biofilm disruption | motile subpopulations | antimicrobials | time-lapse confocal imaging

Fig. 1 .

 1 Fig. 1. B. thuringiensis 407 swimmers tunnel into homologous biofilms. Motile bacilli generate pores in the 48-h biofilm matrix. The arrow in the still image indicates a large transient pore formed by the rotation of a motile chain. (Scale bar, 20 μm.) *Taken from Movie S1.

Fig. 2 .

 2 Fig. 2. Penetration of macromolecules in biofilms is facilitated by swimmer bacteria. (A) Penetration of FITC-Dextran in the basal layer of B. thuringiensis 407 biofilms comprising the motile WT strain (Bt WT in red) or a nonmotile mutant strain (Bt ΔmotA in green). Images were captured immediately after FITC-Dextran introduction in the bulk medium. The presented curves are the fits of experimental fluorescence data with the regression model: FI = A.(1e -kt ), where FI is the fluorescence intensity, A is the maximum fluorescence reached, k is a rate constant, and A.k is the initial slope of the curves. AU, arbitrary unit. (B) Initial slope (A.k) is reduced by one order of magnitude in the absence of tunneling (P < 0.05 between Bt WT and Bt ΔmotA).

Table 1 . 5 S

 15 Swimmer infiltration sensitizes S. aureus biofilms to biocide killing . aureus RN4220 24-h biofilms were submitted to a 4-h preincubation with bacillus swimmers B. thuringiensis 407 (Bt) or B. licheniformis LMG 7560 (Bl), or a mixture Bl + Bt, or with the nonmotile BtΔfla mutant. Biofilms were then disinfected with BAC. Standard errors were calculated from at least 16 values for each condition obtained in five independent experiments. The log reduction corresponds to the nontreated-to-treated ratio of S. aureus biofilms. *BAC was used at 750 ppm for 5 min. Conditions and S. aureus colonyforming unit determinations (log reduction) are provided in Materials and Methods.

Fig. 3 .

 3 Fig. 3. Biocide-carrying motile B. thuringiensis eradicates and supplants S. aureus biofilms. Confocal observations of S. aureus RN4220 GFP biofilms after treatment with motile B. thuringiensis 407 (+Bt), motile Bt expressing lysostaphin (+Bt pLysost), and nonmotile cells expressing lysostaphin (+Bt Δfla pLysost). In these experiments, S. aureus biofilms were 24 h old before being exposed to Bt. (Scale bar = 30 μm.) In the lower left-hand corner of each confocal image is the quantification of the residual biovolume (μm 3 ) of S. aureus GFP biofilms after contact with B. thuringiensis; each value is the average of ∼18 measurements performed in at least nine independent wells.

| www.pnas.org/cgi/doi/10.1073/pnas.1200791109 Houry et al.

| www.pnas.org/cgi/doi/10.1073/pnas.1200791109 Houry et al.

ACKNOWLEDGMENTS. We thank R. Vogel (Lehrstuhl für Technische Mikrobiologie) for kindly sending plasmids carrying the lysostaphin genes, A. Horswill (University of Iowa) for providing GFP-carrying plasmids, and S. Séror (Orsay University Paris XI) for the B. subtilis 168 Δfla strain. We thank E. Johansen (Chr. Hansen A/S) for valuable discussion. M. Naïtali (AgroParis-Tech) is thanked for providing Movie S4 on Y. enterocolitica biofilms. A patent application has been filed (FR 10 60945; Procedure for microbial biofilm elimination). A.H. was funded, in part, by AgroParisTech. This work was supported by Institut National de la Recherche Agronomique funding. The "Essonne department" contributed to the acquisition of the confocal microscope.


Agnès E Ricroch

Jean B Bergé

Marcel Kuntz

Evaluation of genetically engineered crops using transcriptomic

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.


T Le Naou

† N Le Floc'

I Louveau

† H Gilbert

F Gondret

Y Billon

R Janvier

V Piedvache

A Priet

P Roger

F Le Gouevec

A Chauvin

M Alix

J Liger

J F Rouault

P Ecolan

P Ganier

S Tacher

J N Thibault

C Tréfeu

Metabolic changes and tissue responses to selection on residual feed intake in growing pigs 1, 2

Keywords: energy metabolism, liver, muscle, pig, protein turnover, residual feed intake

ABSTRACT: Previous selection experiments using residual feed intake (RFI) to select pigs with a high feed effi ciency have reported that a low RFI was associated with a reduced body fat content and a greater muscle glycogen content. In the current study, growing Large White female piglets from 2 lines divergently selected for RFI were used to determine the changes in energy and protein metabolisms in key tissues and their cross talks in response to selection. Pigs of low RFI (RFI -; n = 26) or high RFI (RFI + ; n = 36) selection lines were offered free access to feed during postweaning and growing periods. Pigs of each line were then slaughtered at 19 kg (n = 8 per line) or 115 kg BW (n = 14 to 18 per line). A third group of pigs of the RFI + line was offered feed at the same level per metabolic BW (BW0.60) as n = 14). Regardless of the growth period considered, G:F was less in RFI + pigs than in RFI -pigs. At 19 kg BW, RFI + and RFI -pigs had a similar body composition and tissue lipid content. The fractional rate of protein synthesis and proteasome activity were decreased (P < 0.090) in the livers of RFI + pigs compared with RFI -pigs whereas activities of energy catabolic enzymes did not differ in the liver and LM samples. Plasma insulin was conversely greater (P = 0.049) in RFI + pigs at this stage. At 115 kg BW, enzyme activities of protein catabolism in the liver and in the LM did not differ (P > 0.10) between RFI + pigs and RFI -pigs. Both lactate dehydrogenase activity participating in glucose metabolism and hydroxylacyl-CoA dehydrogenase activity involved in fatty acid oxidation were greater (P < 0.05) in the liver and LM of RFI + pigs compared with RFI -pigs. In the liver, contrary to the LM, those differences in enzyme activities were directly associated with selection on RFI regardless of ADFI. Increased backfat depth and content and greater lipid content and adipocyte hypertrophy (P < 0.05) in subcutaneous adipose tissue were reported in RFI + pigs compared with RFI -pigs at 115 kg BW without marked changes in key lipogenic enzyme activities; these changes were directly associated with ADFI. In conclusion, the present study shows an increase of catabolic pathway activities in the liver and muscle of RFI + pigs at market weight that is likely to generate more ATP compared with RFI -pigs.

INTRODUCTION

Improving feed effi ciency to reduce feed cost and environmental impacts is one of the main goals of pig production. Feed effi ciency can be estimated by individual residual feed intake (RFI). Briefl y, RFI is the difference between the observed feed consumption of an animal and that predicted on the basis of requirements for maintenance and production of a reference population. Recent selection experiments on RFI in pigs have shown that variations in feeding behavior and daily feed intake (DFI; [START_REF] Young | Effect of selection for residual feed intake on feeding behavior and daily feed intake patterns in Yorkshire swine[END_REF] and changes in heat production due to physical activity and basal metabolic rate (Barea et al., 2010) are related to RFI. Moreover, reduced RFI is genetically correlated with a decreased body fatness [START_REF] Gilbert | Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits[END_REF] and phenotypically associated with the downregulation of several genes involved in lipid metabolism in adipose tissue [START_REF] Lkhagvadorj | Gene expression profi ling of the short-term adaptive response to acute caloric restriction in liver and adipose tissues of pigs differing in feed effi ciency[END_REF]. Greater muscle glycogen content at commercial weight and greater drip loss and lower ultimate pH in meat were reported in low RFI pigs compared with high RFI pigs [START_REF] Lefaucheur | Muscle characteristics and meat quality traits are affected by divergent selection on residual feed intake in pigs[END_REF]. Together, these studies suggest that RFI variability in pigs may be related to lipid and glucose metabolisms in key tissues. Differences in protein degradation rate or protein synthesis could also give rise to changes in the efficiency of conversion of feed to BW gain in various species [START_REF] Herd | Physiological basis for residual feed intake[END_REF]. Finally, differences in some plasma metabolites and hormones have been positively related to genetic and phenotypic measures of RFI in ruminants [START_REF] Richardson | Biological basis for variation in residual feed intake in beef cattle. 2. Synthesis of results following divergent selection[END_REF].

This study aimed to identify early and long-term modifi cations in energy or protein turnover in key tissues and their cross talks in response to a divergent selection experiment on RFI in growing pigs. Because high RFI pigs were anticipated to have a greater feed intake than low RFI pigs, subsets of pigs were also pair-fed to decipher the direct effects of RFI genetic divergence on metabolic orientation.

MATERIALS AND METHODS

The care and use of pigs were performed in compliance with the guidelines of the French Ministry of Agriculture and Fisheries for animal research.

Animals

Purebred French Large White female pigs in the course of a divergent selection experiment on RFI were produced at 2 research farms [INRA Genetique Porcine -GEPA (GEPA), Rouillé and Le Magneraud, France]. The selection program, started at INRA in 2000, was previously described [START_REF] Gilbert | Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits[END_REF]. Briefl y, DFI and ADG from 35 to 95 kg were recorded on male candidates to selection together with backfat thickness at 95 kg (UBT) averaged from 6 measures taken by ultrasonic scanning (ALOKA SSD-500 echograph; Aloka, Cergy Pontoise, France) at right and left shoulder, right and left midback, and right and left loin. A RFI selection index was computed as a linear combination of those traits to account for growth requirement as follows: RFI = DFI -(1.24 × ADG) -(31.9 × UBT), in which RFI, DFI, and ADG are measured in grams and UBT is measured in millimeters.

For genetic analysis, RFI of boars was computed us-ing their own records for DFI, ADG, and UBT and taking into account the fi xed effects of contemporary group and pen size. The R 2 of the model used to compute the predicted feed intake of P1 animals was 0.66 [START_REF] Gilbert | Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits[END_REF] and high RFI (RFI + ) lines were chosen so that their BW (8.7 ± 0.9 and 9.0 ± 1.1 kg, respectively) were representative of the average weaning BW in their contemporary groups. Piglets from a fi rst subset (n = 8 per treatment) were individually housed in metabolic cages. They were offered free access to feed until 19 kg BW to investigate early response to selection on tissue traits. They were weighed weekly, and individual feed consumption (offered food minus refusals) was recorded daily.

The remaining piglets (n = 46), which were littermates of those retained for the fi rst subset, were housed in collective pens (1 pen per line) at their arrival in the postweaning facilities. They were offered free access to feed during this period. At 67 ± 1 d of age (corresponding to 25 ± 2 kg BW in the RFI -group and 29 ± 5 kg BW in the RFI + group, respectively), piglets were moved to growing facilities, where they were individually housed until commercial slaughter weight (115 ± 3 kg BW). Pigs had free access to transition diets the fi rst week and were then allocated in 3 experimental treatments from the second week onwards: a fi rst treatment composed of the RFI -pigs having free access to feed (n = 18), a second treatment composed of RFI + pigs having free access to feed (n = 14), and a third treatment composed of RFI + pigs offered feed at the same level per metabolic BW (BW 0.60 ) as RFI -pigs (RFI +R ; n = 14). Sisters were allocated into RFI + and RFI +R treatments so that their BW at entering in growing facilities did not differ. Pigs were weighed weekly and in the morning on the day of slaughter. Feed consumption was recorded daily. Daily feed allocated to RFI +R pigs was calculated weekly, based on the average daily feed consumption recorded during the week before in RFI -group, the average BW of RFI -and RFI +R pigs at the day of calculation, and the predicted ADG of RFI -pigs for the week after. Throughout the experiment, animals had free access to water and were fed standard diets formulated for postweaning and growing periods (Table 1). The ADG, DFI, total feed intake, and G:F were then computed for the postweaning and growing periods.

Animal Slaughtering and Sample Collection

Pigs for studying short-or long-term responses to RFI selection were slaughtered at 19 ± 1 and 115 ± 3 kg BW, respectively. Pigs were killed 2 h after their last morning meal, by jugular exsanguination after electronarcosis. At bleeding, a blood sample (10 mL) was collected on EDTA (1.95 mg/mL) and centrifuged for 15 min at 2,500 × g at 4°C. Plasma was stored at -20°C until analysis.

Dorsal subcutaneous adipose tissue (SCAT), comprising all fat layers, was sampled immediately after death by an incision along the dorsal right side of the body at the last rib. The carcass was then prepared by removing digestive, urogenital, and respiratory tracts. Liver and kidneys were weighed, and the liver was sampled. Perirenal fat was removed and weighed. A sample of the LM was collected within 20 min of death at the last rib on the right carcass side, just below the adipose tissue sampling location. All tissue samples were cut into small pieces, snap frozen in N 2 , and stored at -75°C until biochemical analyses. Another SCAT sample was restrained onto fl at sticks and frozen in N 2 for histological measurements. For pigs killed at 115 kg, backfat and muscle thicknesses (means of measurements between the third and fourth lumbar vertebra and the third and fourth last rib) were obtained by ultrasonic measure-ments on the hot carcass using a Fat Lean Meter (CGM, Sydel, Lorient, France). These parameters were also used to estimate the lean meat content [START_REF] Daumas | Taux de muscle des pièces et appréciation de la composition corporelle des carcasses[END_REF]. The carcass was then chilled at 4°C. The next day, the left-half carcass side was weighed and dissected into its main cuts (backfat, belly, loin, ham, and shoulder). The LM was dissected from the loin and individually weighed. Weights of all cuts were then expressed as percentage of the left chilled-carcass weight.

Tissue Lipid Content

Concentration of total lipids was determined in SCAT, LM, and the liver samples using a chloroform and methanol (2:1) extraction procedure [START_REF] Folch | A simple method for the isolation and purifi cation of total lipides from animal tissues[END_REF]. Total amounts of lipids in those tissues were calculated by considering the weight of tissues before sampling.

Adipose Tissue Cellularity

Histological methods were used to determine adipocyte mean diameter. Frozen SCAT was sectioned at 12-μm thickness on a cryostat (2800 Frigocut; Reichert-Jung, Francheville, France) at -30°C, mounted on slides, and stained with Oil Red O solution to reveal lipids within cells [START_REF] Gardan | Cellular and biochemical features of skeletal muscle and subcutaneous adipose tissue in pigs differing in IGF-II genotype[END_REF]. Cross sections were counterstained with a crystal violet solution (0.05%) to reveal adipocyte membranes. Images of adipocytes were obtained at a 10-fold magnifi cation with a digital camera system (CV-M90; Jai, Glostrup, Denmark). Five slides were prepared per SCAT sample, and images with the best membrane integrity were retained for cellular analyses (at least 200 adipocytes per sample). Cross-sectional area of each adipocyte in the image was measured using a digitizing table and image analysis software (Visilog 6.0 Imaging software; Noesis, Courtaboeuf, France). The mean diameter (μm) was then calculated considering adipocytes as spherical cells. The number of adipocytes per gram of tissue was estimated by dividing lipid content per gram of tissue by the estimated mean volume of adipocytes in the sample.

Enzyme Activities

Activities of several enzymes involved in lipid synthesis, energy catabolism, or protein degradation were monitored in tissues. To calculate specifi c enzyme activities, protein content in the assayed supernatants was measured using Bradford reagent with BSA as standard [START_REF] Bradford | Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding[END_REF].

Activities of enzymes participating in de novo lipogenesis were assessed in SCAT and the liver. Tissue samples (500 mg for SCAT and 200 mg for the liver) were homogenized in 0.25 mol/L ice-cold sucrose solution containing EDTA (1 mmol/L) and dithiothreitol (DTT; 1 mmol/L). Mixtures were ultracentrifuged at 100,000 × g for 1 h at 4°C. The resulting supernatants containing cytosolic proteins were collected and frozen at -75°C until use. Specifi c activities of malic enzyme, glucose-6-phosphate dehydrogenase (G6PDH), and fatty acid synthase (FAS) were assayed spectrophotometrically at 340-nm absorbance, as described previously (Bazin and Ferre, 2001). Activities of lactate dehydrogenase (LDH) involved in glucose metabolism, β-hydroxylacyl-CoA dehydrogenase (HAD) involved in fatty acid β-oxidation, and citrate synthase (CS) participating to terminal oxidation of nutrients within the Krebs cycle were also assayed in the LM and the liver. Samples (approximately 300 mg) were homogenized in a 0.1 mol/L phosphate buffer (pH = 7.5) containing EDTA (2 mmol/L). Mixtures were sonicated (60 s at 50 Hz) and centrifuged (1,500 × g for 13 min at 4°C). Supernatants were stored in ice. Specifi c activities were assayed spectrophotometrically at 340-nm absorbance for LDH [START_REF] Vassault | Lactate dehydrogenase: UV-method with pyruvate and NADH[END_REF]HAD (Bass et al., 1969) and at 405-nm absorbance for CS [START_REF] Srere | 4.1.3.7. Citrate oxaloacetate-lyase (CoA-acetylating)[END_REF].

Activities of the proteasome, the key enzyme of the ubiquitin-dependent proteolysis, and of calpains (μ-calpain and m-calpain) involved in Ca 2+ -dependent proteolysis were assayed in the LM and the liver. Samples (50 mg) were homogenized in a 20 mmol/L Tris-HCl buffer (pH = 7.2) containing DTT (1 mmol/L) and Triton X100 (1%; Sigma, Steinheim, Germany). Mixtures were then centrifuged for 15 min at 12,000 × g at 4°C. Activities were assayed by fl uorometry at 355 nm for excitation and 460 nm for emission [START_REF] Jamin | Accelerated growth rate induced by neonatal high-protein milk formula is not supported by increased tissue protein synthesis in low-birth-weight piglets[END_REF], using clasto-lactacystin-β-lactone as proteasome inhibitor (VWR, Fontenay sous Bois, France) and MDL-28170 as calpain inhibitor (Tebu-Bio, Le Perray en Yvelines, France).

Plasma Metabolite and Hormone Concentrations

Commercial kits were used to determine plasma concentrations of glucose (Glucose RTU; bioMérieux, Craponne, France), lactate (Lactate PAP; bioMérieux), NEFA (NEFA-HR(2); Wako Chemicals GmbH, Neuss, Germany), urea (Urea UV 250; bioMérieux), proteins (Protéines-kit; bioMérieux), triglycerides (Triglycérides Enzymatique PAP 150; bioMérieux), β-hydroxybutyrate (Thermo Fisher, Illkirch, France), and albumin (Albuminkit; bioMérieux). These parameters were measured with a Konelab 20i device (Thermo Fisher Scientifi c, Courtaboeuf, France). Intra-assay CV were less than 5%.

Plasma concentrations of insulin were measured using a RIA kit (INSULIN-CT; CisBio Bioassays, Codolet, France). Plasma leptin concentrations were quantifi ed us-ing the multispecies RIA kit (Millipore, St. Charles, MO) previously validated for use in porcine plasma [START_REF] Qian | Leptin mRNA expression and serum leptin concentrations as infl uenced by age, weight, and estradiol in pigs[END_REF]. Intra-assay CV for these hormones were <6%. Plasma concentrations of total triiodothyronine (TT3) and total thyroxin (TT4) were determined using commercial RIA kits (MP Biomedicals, Illkirch Graffenstaden, France). Plasma IGF-I concentrations were determined using a double antibody RIA [START_REF] Louveau | Effect of a growth hormone infusion on plasma insulin-like growth factor-I in Meishan and Large White pigs[END_REF] after an acid-ethanol extraction [START_REF] Daughaday | Inhibition of access of bound somatomedin to membrane-receptor and immunobindings sites -A comparison of radioreceptor and radioimmunoassay of somatomedin in native and acid-ethanol-extracted serum[END_REF]. The intra-assay CV for plasma sample with a mean IGF-I concentration of 302 ng/mL was 7.3%.

Protein Synthesis

The day before slaughter at 19 kg BW, piglets were fi tted with a catheter in the jugular vein as described by [START_REF] Matte | A rapid and non-surgical procedure for jugular catheterization of pigs[END_REF]. On the day of slaughter, blood samples (3 mL) were collected on heparin (50 UI/mL) from the catheter just before, 7 min after, and 14 min after an injection of a fl ooding dose of valine (1.50 mmol/kg BW of a solution composed of 20% L- 15 [N]-Val and 80% L-Val) to measure protein synthesis [START_REF] Hamard | A moderate threonine defi ciency differently affects protein metabolism in tissues of earlyweaned piglets[END_REF]. Piglets were killed 15 min after valine injection. Tissue protein synthesis rate was then determined in sampled liver and LM. Plasma-free valine enrichment (S A ) was determined by GC-MS [gas chromatography (GC) 7890A coupled with a mass spectrometer (MS) 5975C; Agilent Technology, Santa Clara, CA] after derivatization with N-tert-butyldimethylsilyl N-methyl-trifl uoroacetamide. Protein-bound valine enrichment (S B ) was measured by GC-C-IRMS [gas chromatography 8131 coupled to an isotopic Isochrom mass spectrometry (IRMS); Fisons Instrument (GV Instruments, Manchester, UK)] after derivatization with ethylchloroformate. Fractional protein synthesis rate (K S ; %/d), corresponding to the percentage of protein newly synthesized per day, was calculated as follows:

K S = (S B × 100)/(S A × t),
in which S B represents plasma enrichment, S A represents the tissue protein enrichment, and t represents the time elapsed between the injection of valine and animal bleeding.

Statistical Analysis

Data were processed using SAS Software (SAS Inst. Inc., Cary, NC) and analyzed within each slaughtering weight (19 or 115 kg BW). Data were processed by ANOVA using the GLM procedure considering the fi xed effects of the experimental treatments, replicates, and the interaction between treatment and replicate (treatment × replicate). For performance traits during the grow-ing period, BW at 67 d of age was also included as an additional covariate in complementary analyses. The fi nal BW was added as covariate for carcass traits, tissue lipid composition, and cellular and biochemical data determined at slaughter. Litter within experimental group was fi rst considered as a random effect but was removed from the models as it was generally not signifi cant. When a statistical difference was found for the effect of experimental treatments, least square means were compared. Differences were considered signifi cant for P ≤ 0.05 whereas 0.05 < P ≤ 0.10 was discussed as a trend.

RESULTS

Growth Performance, Carcass Composition, and Tissue Composition

From 28 d of age to 19 ± 1 kg BW at slaughter (53.2 ± 0.8 d of age on average), ADG did not differ (P > 0.10) between RFI -and RFI + treatments (370 g/d on average). Daily feed consumption was less (P = 0.03) in RFI -piglets (551 ± 59 g/d) than in RFI + piglets (625 ± 59 g/d). Moreover, G:F tended (P = 0.09) to be greater in RFI -than RFI + pigs (0.74 vs. 0.70 for RFI -and RFI + treatments, respectively). At postweaning period, carcass weight (13.6 ± 0.3 kg), the liver and kidneys weights, and the perirenal fat proportion (relative to BW) did not differ (P > 0.10) between the 2 treatments (data not shown).

From 67 ± 1 d of age to 115 ± 2 kg BW at slaughter (growing period), ADG was lesser in RFI -pigs compared with RFI + pigs (-37 g/d; P < 0.001; Table 2); however, ADG did not differ (P > 0.10) between those 2 treatments when adjusted for respective BW at 67 d of age. Both DFI and total feed consumption during this period were lesser in RFI -pigs than RFI + pigs (-8 and -13%, respectively). Consequently, G:F was improved (P < 0.001) in RFI -pigs compared with RFI + pigs. As expected according to experimental design, feed metabolic consumption was similar (P > 0.10) in RFI -and RFI +R pigs (182 g•d -1 •kg -1 BW on average). Feed restriction in RFI +R pigs lowered ADG (P < 0.001) when compared with their RFI + littermates but did not modify G:F (P > 0.10; 0.35 kg gain/kg feed in both treatments). At slaughter, carcass weight did not differ (P > 0.05) among the 3 treatments (Table 2). The RFI -pigs had the lowest backfat depth and content and a lower proportion of belly when compared with RFI + pigs (P < 0.05). Conversely, they displayed a greater lean meat content and increased proportions of loin and shoulder compared with RFI + pigs (P < 0.05). However, LM weight did not differ (P > 0.10) between RFI -and RFI + pigs. Carcass composition of RFI +R pigs was similar (P > 0.10) to that of RFI -pigs, except for shoulder proportion that was greater (P < 0.01) in RFI -pigs than in RFI +R pigs.

Lipid-Related Traits

At 19 ± 1 kg BW, lipid contents of SCAT (60.2 g ± 5.5 g per 100 g of fresh tissue weight on average), LM (1.5 g ± 0.2 g/100 g on average), and the liver (3.9 g ± 0.2 g/100 g on average) did not differ (P > 0.05) between the 2 RFI treatments (data not shown). A 3-fold greater (P = 0.05) activity of malic enzyme was found in the liver of RFI -pigs compared with RFI + pigs whereas the specifi c activities of FAS and G6PDH did not differ (P > 0.05) between the 2 treatments in that organ (Table 3). In SCAT, there were no differences (P > 0.05) in the activity levels of the 3 lipogenic enzymes under investigation.

At 115 kg ± 2 kg BW, decreased concentration (-5%; P < 0.05) and total amount (-19%; P < 0.01) of lipids were reported in SCAT from RFI -pigs compared with RFI + pigs (Table 4) whereas the same traits were rather similar in RFI -and RFI +R pigs. Mean diameter of adipocytes was smaller (-7%) in RFI -pigs than RFI + pigs, and it was the least in RFI +R pigs (P = 0.04). The number of adipocytes was conversely greater in SCAT of RFI +R pigs when compared with RFI + pigs and RFI - pigs (P = 0.08). The specifi c activities of key lipogenic enzymes in SCAT did not differ (P > 0.10) among the 3 treatments. In the liver and LM, the lipid concentration did not differ (P > 0.05) among the 3 treatments; however, the total amount of lipids in the liver was slightly lower (P = 0.07) in RFI -pigs than RFI + pigs (Table 4). Hepatic lipogenic enzyme activities did not differ (P > 0.10) among the 3 treatments.

Energy Catabolism and Protein Turnover

At 19 kg ± 1 kg BW, activity levels for LDH, HAD, and CS in LM as well as in the liver did not differ (P > 0.10) between RFI -and RFI + piglets (Table 3). Proteasome activity and the K S in the liver tended to be greater (P < 0.10) in RFI -piglets than in RFI + piglets. The activities of key proteolytic enzymes and the rate of protein synthesis in the LM did not differ (P > 0.05) between the 2 treatments.

At 115 ± 2 kg BW, the specifi c activity of LDH was lower in the LM (-13%; P < 0.05) and the liver (-7%; P < 0.05) of RFI -pigs than RFI + pigs (Table 5). Similarly, HAD activity was reduced in LM (P < 0.001) and the liver (P < 0.05) of RFI -pigs compared with RFI + pigs. The specifi c activity of CS in LM was also less (P < 0.01) in RFI -pigs than in RFI + pigs but did not differ (P > 0.05) between those treatments in the liver. Feed restriction in RFI +R pigs did not change (P > 0.05) the activity of LDH, HAD, and CS in the liver, but it decreased the specifi c activities of those enzymes in LM to a level similar to RFI - pigs (for LDH) or intermediate between RFI -and RFI + 2 Activities of fatty acid synthase (FAS), malic enzyme, or glucose-6-phosphate dehydrogenase (G6PDH) are expressed as nmol•min -1 •mg -1 protein.

3 SCAT = subcutaneous adipose tissue. 4 Activities of lactate dehydrogenase (LDH), β-hydroxylacyl-CoA dehydrogenase (HAD), or citrate synthase (CS) are expressed as μmol•min -1 •mg -1 protein.

5 Activities are expressed as milliRFU •min -1 •mg -1 protein. RFU = relative fl uorescent unit. 6 Fractional rate (K s ) is expressed as percent per day. pigs (for HAD and CS). Proteasome and calpain activities did not differ (P > 0.10) among the 3 treatments either in LM or in the liver.

Nutrient and Hormone Plasma Concentrations

At 19 ± 1 kg BW, insulin concentration was lower (P = 0.05) in RFI -piglets than in RFI + piglets (36.0 and 51.8 μUI/mL, respectively). However, there were no differences (P > 0.10) in plasma concentration of nutrients and hormones between RFI + and RFI -groups (data not shown).

At 115 ± 2 kg BW, there was notably a reduced (P = 0.04) plasma triglyceride concentration in RFI -pigs than in RFI + pigs (Table 6), with RFI +R pigs having triglyceride concentration intermediate between the 2 other treatments. Conversely, TT4 plasma concentration tended to be greater (P = 0.10) in RFI -pigs than in RFI + pigs and intermediate in RFI +R pigs. Plasma concentrations of other nutrients and hormones did not differ (P > 0.10) among the 3 treatments.

DISCUSSION

Growth Performance Divergences Associated to Selection on Residual Feed Intake

The present study shows that a divergent selec-tion on RFI had commensurate effects on G:F in the 2 growth periods considered; however, the difference was amplifi ed at the latest stage. This is in accordance with previous results of Barea et al. (2010) obtained under the fourth generation of the same selection experiment. In our study, these selection-associated differences in G:F resulted mainly from the decreased feed consumption of RFI -pigs compared with RFI + pigs, rather than to ADG, which was similar in the 2 treatments during the postweaning period and during the growing period when adjusted for difference in BW at 67 d of age. Similarly, pigs of a line selected for RFI -had consumed less feed over a period of 6 wk with a starting BW of 20 kg compared with a randomly selected control line (Boddicker et al., 2011b), but G:F at early stage was not affected by selection on RFI -in this aforementioned study. Moreover, in the present study, there was no RFI-associated difference in plasma IGF-I concentration assessed at the end of each test period whereas [START_REF] Hoque | Genetic associations of residual feed intake with serum insulin-like growth factor-I and leptin concentrations, meat quality, and carcass cross sectional fat area ratios in Duroc pigs[END_REF] using Duroc pigs found a weak genetic correlation between RFI and IGF-I concentration in the growing period from 30 to 105 kg BW. Our results are in accordance with [START_REF] Bunter | Insulin-like growth factor-I measured in juvenile pigs is genetically correlated with economically important performance traits[END_REF] using Large White × Landrace pigs, who suggested that only juvenile IGF-I concentration (around weaning to d 35) is strongly correlated Table 5. Activities of key energy and protein catabolic enzymes in tissues of pigs divergently selected on low RFI (RFI -) or high residual feed intake (RFI + ) and having either free access to feed or offered feed at the same level per metabolic BW (BW0.60) as RFI-pigs (RFI +R ) access to feed from 67 d of age to slaughter a,b Within a row, means without a common superscript differ (P < 0.10).

1 Values are least square means. RSD = residual SD (corresponding to the root-mean-square error of the full ANOVA model).

2 Activities of lactate dehydrogenase (LDH), β-hydroxylacyl-CoA dehydrogenase (HAD), or citrate synthase (CS) are expressed as μmol•min -1 •mg -1 proteins3 Activities are expressed as milliRFU •min -1 •mg -1 proteins Table 6. Nutrient and hormone plasma concentrations of pigs divergently selected on low residual feed intake (RFI -) or high residual feed intake (RFI + ) and having either free access to feed or offered feed at the same level per metabolic BW (BW0.60) as RFI-pigs (RFI +R ) access to feed from 67 d of age to slaughter with RFI. Besides IGF-I, IGF-II and insulin-like growth factor-binding proteins (IGFBP) should be considered in further studies for their important roles in tissue development and postnatal growth. In the present study, restricted feeding (about 90% of ad libitum) during the growing period in pigs selected for RFI + caused a slight decrease (-11%) in ADG, but it did not affect G:F during this period and plasma IGF-I concentration at market weight. A restriction of feed intake by less than 20% is likely not enough to affect these variables, as previously described [START_REF] Combes | Moderate food restriction affects skeletal muscle and liver growth hormone receptors differently in pigs[END_REF][START_REF] Lee | Effects of restricted feeding, low-energy diet, and implantation of trenbolone acetate plus estradiol on growth, carcass traits, and circulating concentrations of insulin-like growth factor (IGF)-I and IGF-binding protein-3 in fi nishing barrows[END_REF] whereas a more severe restriction (65% of DFI) may decrease G:F [START_REF] Heyer | Compensatory growth response in pigs: Effects on growth performance, composition of weight gain at carcass and muscle levels, and meat quality[END_REF]. Importantly, modifi cations in lean meat content, body fat depth, and adipose tissue lipid content were manifested exclusively at commercial slaughter weight. This confi rms that the magnitude of the association between body composition and variation in RFI is infl uenced by age and stage of maturity of the tested animals as previously suggested in beef cattle [START_REF] Herd | Physiological basis for residual feed intake[END_REF]. The novel fi nding of the present study is that adaptations of tissue metabolisms in response to RFI occurred in pigs; however, the differences were also mainly observed at market weight.

Changes in Protein Metabolism Occurred at Short Term

At 19 kg BW (postweaning period), both protein synthesis rate and the specifi c activity of the proteasome involved in protein catabolism were decreased in the liver of RFI + pigs compared with RFI -pigs. This suggests that hepatic protein turnover might be reduced in the less effi cient piglets. This might be partly related to the greatest plasma insulin concentration measured in RFI + piglets during the postprandial phase. Indeed, insulin participates together with AA to the inhibition of hepatic proteolysis [START_REF] Chotechuang | Down-regulation of the ubiquitinproteasome proteolysis system by amino acids and insulin involves the adenosine monophosphate-activated protein kinase and mammalian target of rapamycin pathways in rat hepatocytes[END_REF]. However, insulin has generally no effect on hepatic protein synthesis, as shown in neonate piglets [START_REF] Davis | Differential effects of insulin on peripheral and visceral tissue protein synthesis in neonatal pigs[END_REF] or in growing mice (Bark et al., 1998). In addition, enzyme activities related to protein catabolism in the liver did not differ between RFI lines at commercial weight. Moreover, there was no difference in protein synthesis or proteolytic enzyme activities in the LM, irrespective of the test period. This is somewhat surprising, considering that differences in the rates of protein degradation and protein synthesis within the muscle are assumed to have a signifi cant role in variability of energy expenditure (Herd and Arthur, 2009, for review). With the fi nding of a greater calpastatin activity in muscle of RFI -pigs compared with controls at 50 kg BW [START_REF] Smith | Effects of selection for decreased residual feed intake on composition and quality of fresh pork[END_REF], it is then possible that the activity of this inhibitor of proteolysis was affected in LM of RFI + pigs compared with RFI -pigs, which may contribute to the decreased loin proportion recorded in the carcass of the formers compared with the latter pigs at market weight. However, this deserves specifi c studies.

Long-Term Alterations of Energy Catabolism in Tissues

Whereas enzyme activity levels related to energy metabolism in liver and LM did not differ between RFI - and RFI + piglets during postweaning, our study shows elevated enzyme activities for both tissues of RFI + pigs compared with RFI -pigs at market weight. Our study provides new evidence that these differences were mainly associated to a direct effect of RFI genetic divergence in the liver whereas they were more associated with selection-induced difference in daily feed consumption in the LM. One modifi ed pathway in response to RFI selection likely concerned glucose metabolism, with the pivotal role of LDH in the cross talk between skeletal muscle and the liver. Indeed, LDH activity was found greater in RFI + pigs than in RFI -pigs in LM where this enzyme is involved in the terminal anaerobic conversion of glucose into lactate as well as in the liver where this enzyme participates to the neosynthesis of glucose from lactate. These concomitant increases suggest an acceleration of the Cori cycle [START_REF] Katz | Recycling of glucose and determination of the Cori cycle and gluconeogenesis[END_REF] in RFI + pigs compared with RFI -pigs. This might be a way for RFI + pigs to generate ATP faster within muscle cells without adverse effects on glycemia, as illustrated by the lack of change in plasma glucose concentration between RFI + and RFI -groups. Because gluconeogenesis from lactate needs 6 nucleotide triphosphates whereas anaerobic glycolysis produces only 2 ATP from 1 molecule of glucose, this accelerated cycle involving glucose and lactate between skeletal muscle and the liver could then contribute to the greater maintenance requirements calculated for pigs selected for RFI + compared with RFI -pigs (Barea et al., 2010).

The greater activity of HAD, a key enzyme in the mitochondrial and peroxisomal fatty acid β-oxidation, in liver and LM of RFI + pigs compared with RFI -pigs indicates that fatty acid oxidation might be another energy metabolic pathway altered by RFI selection. There was also a greater activity of CS in the LM of RFI + pigs, where this fi rst enzyme in the Krebs cycle is implicated in the conversion of acetyl CoA for ATP generation. In agreement, variation in the energetic effi ciency of mitochondria previously was suggested to be associated with differences in feed efficiency (for review, see [START_REF] Herd | Physiological basis for residual feed intake[END_REF]. Thus, in the liver where CS activity was similar in both RFI lines, the excess of acetyl CoA generated from the incomplete fatty acid oxidation can be converted to ketone bodies, which are exported in blood to be used as an energy source in skeletal muscles [START_REF] Williamson | Activities of enzymes involved in acetoacetate utilization in adult mammalian tissues[END_REF]. Whereas a positive correlation between RFI and plasma β-hydroxybutyrate concentrations were reported in heifers [START_REF] Kelly | Effect of divergence in residual feed intake on feeding behavior, blood metabolic variables, and body composition traits in growing beef heifers[END_REF], plasma β-hydroxybutyrate concentration did not differ between the RFI -pigs and RFI + pigs under study. In nonruminants, ketogenesis primarily takes place in fasted animals [START_REF] Fernandez-Figares | The role of insulin, glucagon, dexamethasone, and leptin in the regulation of ketogenesis and glycogen storage in primary cultures of porcine hepatocytes prepared from 60 kg pigs[END_REF]. This likely explains why plasma β-hydroxybutyrate concentrations were particularly low in the present study where blood was sampled a few hours after the last morning meal. Therefore, kinetics investigations would be helpful to determine the responses of ketone body production to RFI selection. Altogether, the decreased activities of energy-producing enzymes in the liver and LM of RFI -pigs compared with RFI + pigs support the assumption that pigs selected on RFI -require less energy to maintain a constant BW (Boddicker et al., 2011a).

Considering that thyroid hormones promote heat production and stimulate both anabolic and catabolic processes [START_REF] Freake | Thermogenesis and thyroidfunction[END_REF], greater plasma concentrations in thyroid hormones could have been expected in RFI + pigs compared with RFI -pigs, as described in cockerels [START_REF] Gabarrou | Glucose-insulin relationships and thyroid status of cockerels selected for high or low residual food consumption[END_REF]. However, in the present study, TT4 plasma concentration tended to be lower in those pigs compared with RFI -pigs at market weight. Similarly, [START_REF] Lefaucheur | Muscle characteristics and meat quality traits are affected by divergent selection on residual feed intake in pigs[END_REF] found lower plasma concentrations of TT4 but also depressed concentrations of TT3 and free triiodothyronine (T3) in RFI + pigs compared with RFI - pigs. Moreover, feed restriction in RFI +R pigs did not affect plasma thyroid hormone concentrations whereas caloric restriction was shown to decrease both plasma TT3 and TT4 concentrations independently of RFI status [START_REF] Lkhagvadorj | Gene expression profi ling of the short-term adaptive response to acute caloric restriction in liver and adipose tissues of pigs differing in feed effi ciency[END_REF]. This discrepancy may result from the difference in restriction level between the 2 experiments.

Adipose Tissue Traits Associated to Differences in Carcass Fat Content

Although numerous studies reported positive phenotypic and genetic correlations between RFI and carcass fat content in pigs in the range of 50 to 114 kg BW [START_REF] Gilbert | Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits[END_REF]Boddicker et al., 2011a;[START_REF] Smith | Effects of selection for decreased residual feed intake on composition and quality of fresh pork[END_REF], the cellular and metabolic bases of these associations remain largely to be clarifi ed. Our study fi rst extends these fi ndings by showing that differences in adiposity traits between RFI + and RFI -lines at market weight disappeared when RFI + pigs were fed the same level of feed as RFI -pigs during the growing period. Similarly, [START_REF] Young | Effect of selection for residual feed intake on feeding behavior and daily feed intake patterns in Yorkshire swine[END_REF] showed that backfat content was positively correlated with daily feed intake when comparing a selection line for RFI -with a control line. In the present study, differences in backfat depth and content between RFI + and RFI -treatments were parallel to enlarged adipocytes in SCAT of RFI + pigs. However, there were no marked changes in specifi c activity levels of key lipogenic enzymes in SCAT and in the liver of the pigs under study. This was quite unexpected because [START_REF] Lkhagvadorj | Gene expression profi ling of the short-term adaptive response to acute caloric restriction in liver and adipose tissues of pigs differing in feed effi ciency[END_REF] revealed a downregulation of numerous genes participating in lipid metabolism in backfat of RFI -pigs compared with RFI + pigs. As adipocyte volume is infl u-enced by the balance between lipolysis and lipogenesis, it is possible that lipolytic rate was modifi ed in response to both RFI and feed restriction. Another explanation that may account for differences in adiposity between RFI lines could be a greater uptake of circulating lipids by adipose tissues in RFI + pigs, as we reported a greater plasma concentration in circulating triglycerides in those pigs compared with RFI - pigs at market weight. Altogether, further research using high-throughput gene expressions and proteomic profi ling on adipose tissue, liver, and muscle should be conducted to reveal other pathways associated with RFI.

Finally, although circulating leptin concentration was positively correlated with RFI in pigs [START_REF] Hoque | Genetic associations of residual feed intake with serum insulin-like growth factor-I and leptin concentrations, meat quality, and carcass cross sectional fat area ratios in Duroc pigs[END_REF][START_REF] Lefaucheur | Muscle characteristics and meat quality traits are affected by divergent selection on residual feed intake in pigs[END_REF], there were no differences in leptinemia between RFI groups in the current study. We have no defi nitive explanation for this discrepancy because the breed and slaughtering stage were rather similar in our study and that of [START_REF] Lefaucheur | Muscle characteristics and meat quality traits are affected by divergent selection on residual feed intake in pigs[END_REF] who focused on a former (fourth) generation of selection. Similarly, [START_REF] Lkhagvadorj | Gene expression profi ling of the short-term adaptive response to acute caloric restriction in liver and adipose tissues of pigs differing in feed effi ciency[END_REF] did not observe any modifi cations in leptinemia in relation to RFI in growing pigs, but they have suggested that abundance in leptin transcripts may be a more sensitive marker to evaluate feed effi ciency-associated traits than serum leptin concentration. This remains to be investigated.

Conclusion

Despite immediate modifi cation in G:F in response to RFI selection, there were no changes in carcass composition and tissue energy metabolism during the postweaning period, with the exception of decreased protein synthesis and proteolysis in the liver of piglets selected for RFI + compared with piglets selected for RFI -. At market weight, the main modifi cations concerned specifi c activity levels of energy catabolic enzymes in the liver, where they were directly associated with genetic divergence on RFI as well as in the LM where they were more related to differences in daily feed intake. Greater catabolic activities in glucose and fatty acid metabolisms in those tissues for pigs selected for RFI + may notably generate greater ATP to sustain daily BW gain and maintenance requirements in those pigs.

LITERATURE CITED

Barea, R., S. Dubois, H. Gilbert, P. Sellier, J. van Milgen, and J. Noblet. 2010

  . In the present study, pigs from the sixth and seventh generations of this divergent selection experiment were considered. Average genetic values of RFI

	were 67.16 (SE = 2.79) g/d for the high RFI line and
	-70.34 (SE = 3.31) g/d for the low RFI line, respective-
	ly, which were not different from the average values of
	these generations.
	At weaning (28 d of age), 36 and 26 female piglets
	from the high and low RFI lines, respectively, were trans-
	ferred to the experimental facilities of INRA Physiologie,
	Environnement et Genetique pour l'Animal et les Sys-
	tèmes d'Elevage (PEGASE; Saint-Gilles, France) in 2
	successive replicates. Piglets from the low RFI (RFI -)

Table 1 .

 1 Ingredients and chemical composition of diets (as-fed basis) fed to growing pigs

	Item	Starter 1	Starter 2	Growing
	Ingredient, %
	Wheat		23.2	26.2
	Corn		25.0	16.0
	Barley	45.3	24.1	25.5
	Wheat bran			5.0
	Soybean meal	17.5	22.6	19.0
	Soy protein concentrate	2.5
	Vegetable (corn) oil	2.3	0.5	2.0
	Molasses			3.0
	Whey	20.0
	Lactose	8.0
	Calcium carbonate	1.41	1.13	1.29
	Monocalcium phosphate	0.80	0.97
	Dicalcium phosphate			0.50
	Salt		0.40	0.45
	Vitamin-mineral premix	0.50	0.50	0.50
	Chemical composition and nutritional values, %
	CP	19.0	18.0	16.5
	Crude fats	6.7	2.8	4.2
	Digestible Lysine, %	12.8	11.4	8.4
	NE, kJ/g	10.63	9.67	9.67

Table 2 .

 2 Growth performance and carcass traits in pigs divergently selected on low residual feed intake (RFI -) or on high residual feed intake (RFI + ) having either free access to feed or offered feed at the same level per metabolic BW (BW0.60) as RFI-pigs (RFI +R ) access to feed from 67 d of age to slaughter 1

	Item	RFI -	RFI + RFI +R	RSD P-value
	N	1 8	1 4	1 4
	Growth traits
	67-d-old BW, kg	25.2 a	29.2 b	29.3 b	3.8	<0.01
	Final BW, kg	114.9	114.8	114.7	3.2	0.99
	Final age, d	170 b	162 a	173 b	8	<0.01
	ADG, g/d	877 b	914 c	813 a	52	<0.001
	Feed intake
	Total feed intake, kg	228 a	248 b	245 b	16	<0.01
	Daily feed consumption,	182 a	209 b	183 a	14	<0.001
	g•d -1 •kg -1 BW 0.60
	G:F	0.39 b	0.35 a	0.35 a	0.02 <0.001
	Organ weight, g
	Liver	2,049 a 2,251 b 2,009 a	179	<0.01
	Kidneys	394 a	445 b	393 a	51	0.03
	Perirenal fat	1,113 a 1,248 b 1,043 a	211	0.08
	Carcass traits
	Chilled carcass	88.1	88.4	87.8	1.7	0.63
	weight, kg
	Mean backfat	16.8 a	19.0 b	16.7 a	2.5	0.03
	thickness, mm
	Lean meat content, %	59.3 b	57.6 a	58.9 b	1.7	0.02
	Carcass composition, % 2
	Backfat	6.7 a	7.7 b	7.0 a	1.0	0.04
	Belly	13.7 a	15.0 b	13.6 a	1.2	<0.01
	Shoulder	24.8 b	24.1 a	24.2 a	0.5	<0.01
	Ham	23.9	23.9	24.0	0.8	0.92
	Loin	27.8 b	26.4 a	28.3 b	1.5	<0.01
	LM	3.5	3.0	3.1	0.7	0.17

a,b Within a row, means without a common superscript differ (P < 0.10).

Table 3 .

 3 Specifi c activities of key enzymes involved in lipid synthesis, energy catabolism or protein degradation, and rate of protein synthesis in tissues of piglets divergently selected on low residual feed intake (RFI -) or high residual feed intake (RFI + ) and killed at 19 kg BW 1

	Item		RFI -	RFI +	RSD	P-value
	N		8	8
	Lipogenic enzyme activities 2
	SCAT 3	FAS	13.7	13.7	3.9	0.97
	SCAT	Malic enzyme	156	239	108	0.15
	SCAT	G6PDH	167	193	46	0.29
	Liver	FAS	19.9	18.7	3.5	0.54
	Liver	Malic enzyme	34.3	11.1	20.1	0.05
	Liver	G6PDH	41.8	35.8	11.3	0.31
	Enzyme activities of energy catabolism 4
	Liver	LDH	1.26	1.07	0.53	0.48
	Liver	HAD	0.022	0.025	0.012	0.61
	Liver	CS	0.23	0.23	0.10	0.92
	LM	LDH	14.9	14.2	3.6	0.73
	LM	HAD	0.049	0.052	0.015	0.66
	LM	CS	0.104	0.098	0.028	0.71
	Enzyme activities of protein catabolism 5
	Liver	Proteasome	109	91	18	0.08
	Liver	Calpains	211	206	31	0.76
	LM	Proteasome	14.1	15.2	5.0	0.66
	LM	Calpains	4.0	5.3	4.5	0.60
	Protein synthesis 6
	Liver	K S	102	89	11	0.06
	LM	K S	10.0	9.2	1.5	0.35
	1 Values are least square means. RSD = residual SD (corresponding to the
	root-mean-square error of the full ANOVA model).

Table 4 .

 4 Lipid-related characteristics of pigs divergently selected on low residual feed intake (RFI -) or high residual feed intake (RFI + ) and having either free access to feed or offered feed at the same level per metabolic BW (BW0.60) as RFI-pigs (RFI +R ) access to feed from 67 d of age to slaughter 1

	Item		RFI -RFI + RFI +R RSD P-value
	N		18	14	14
	Lipid content, g/100 g fresh weight
	SCAT 2		79.0 a 83.4 b 80.2 ab 4.1	0.03
	LM		1.8	2.1	1.8	0.4	0.13
	Liver		4.2	4.3	4.5	0.5	0.15
	Total tissue lipid amount, g
	SCAT		2263 a 2791 b 2325 a 413 <0.01
	LM		202	232	216	46	0.26
	Liver		87	97	91	11	0.07
	Adipocyte cellularity
	SCAT	Mean diameter, μm	88.1 ab 94.8 b 80.3 a 1.37 0.04
	SCAT	Adipocyte number,	2.6 a	2.3 a	3.5 b	1.2	0.08
		billion per gram of tissue
	Lipogenic enzyme activity 3
	SCAT	FAS	17.8 15.1 20.1	8.3 0.29
	SCAT	Malic enzyme	309	386	376	123	0.17
	SCAT	G6PDH	323	342	283	96	0.26
	Liver	FAS	8.7	8.4	8.5	1.7	0.89
	Liver	Malic enzyme	4.7	5.6	5.8	3.0	0.57
	Liver	G6PDH	48.4 45.4 48.3 10.8	0.70

a,b Within a row, means without a common superscript differ (P < 0.10).

  . Energy utilization in pigs selected for high and low residual feed intake. J. Anim. Sci. 88:2062-2072. Bark, T. H., M. A. McNurlan, C. H. Lang, and P. J. Garlick. 1998. Increased protein synthesis after acute IGF-I or insulin infusion is localized to muscle in mice. Am. J. Physiol.-Endoc. M. 275:E118-123. Bass, A., D. Brdiczka, P. Eyer, S. Hofer, and D. Pette. 1969. Metabolic differentiation of distinct muscle types at the level of enzymatic organization. Eur. J. Biochem. 10:198-206. Bazin, R., and P. Ferre. 2001. Assays of lipogenic enzymes. Methods Mol. Biol. 155:121-127.

Corresponding author: fl orence.gondret@rennes.inra.fr Received February 21, 2012. Accepted July 22, 2012. at INRA Institut National de la Recherche Agronomique on September 11, 2013 www.journalofanimalscience.org Downloaded from

at INRA Institut National de la Recherche Agronomique on September 11, 2013 www.journalofanimalscience.org Downloaded from

Values are least squares means. RSD = residual SD (corresponding to the root-mean-square error of the full ANOVA model).

Proportions expressed as percentage of the chilled carcass weight. at INRA Institut National de la Recherche Agronomique on September 11, 2013 www.journalofanimalscience.org Downloaded from

Values are least square means. RSD = residual SD (corresponding to the root-mean-square error of the full ANOVA model).

SCAT = subcutaneous adipose tissue.

Activities of fatty acid synthase (FAS), malic enzyme, or glucose-6-phosphate dehydrogenase (G6PDH) are expressed as nmol•min -1 •mg -1 protein. at INRA Institut National de la Recherche Agronomique on September 11, 2013 www.journalofanimalscience.org Downloaded from

Values are least square means. RSD = residual SD (corresponding to the root-mean-square error of the full ANOVA model).

β-OH = β-hydroxybutyrate.

TT3 = total triiodothyronine.

TT4 = total thyroxin. at INRA Institut National de la Recherche Agronomique on September 11, 2013 www.journalofanimalscience.org Downloaded from

This article has been cited by 3 HighWire-hosted articles: at INRA Institut National de la Recherche Agronomique on September 11, 2013 www.journalofanimalscience.org Downloaded from

We are also grateful to L. Lefaucheur for critical reading of the manuscript. 2 The study was funded by the French National Research Agency (Agence Nationale de la Recherche ANR, ANR-08-GENM038 PIG_FEED). T. Le Naou was supported by a scholarship from INRA and the research fund of Region Bretagne (France).