Pierre Servais

Hélène Agogué

Claude Courties

Fabien Joux

Philippe Lebaron
email: lebaron@obs-banyuls.fr

Are the actively respiring cells (CTC+) those responsible for bacterial production in aquatic environments?

The 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining method is commonly and increasingly used to detect and to enumerate actively respiring cells (CTC+ cells) in aquatic systems. However, this method remains controversial since some authors promote this technique while others pointed out several drawbacks of the method. Using flow cytometry (FCM), we showed that CTC staining kinetics vary greatly from one sample to another.

Therefore, there is no universal staining protocol that can be applied to aquatic bacterial communities. Furthermore, using 3 H-leucine incorporation, it was shown that the CTC dye has a rapid toxic effect on bacterial cells by inhibiting protein synthesis, a key physiological function. The coupling of radioactive labelling with cell sorting by FCM suggested that CTC+ cells contribute to less than 60% of the whole bacterial activity determined at the community level. From these results, it is clearly demonstrated that the CTC method is not valid to detect active bacteria, i.e. cells responsible for bacterial production.

Introduction

In aquatic ecosystems, bacterial communities play a fundamental role in both the organic matter decomposition and the conversion of dissolved organic carbon into bacterial biomass generally transferred to higher trophic levels through the microbial food web [START_REF] Azam | The ecological role of water column microbes in the sea[END_REF][START_REF] Cole | Bacterial production in fresh and saltwater ecosystems: a cross system overview[END_REF].

Over the past 20 years, methods have been developed by microbial ecologists to quantify the biomass and activity of total bacterial communities [START_REF] Kemp | Handbook of Methods in Aquatic Microbial Ecology[END_REF]. However, as bacterial communities are made of complex assemblages of numerous species having different metabolic characteristics and because nutrients are not always distributed homogeneously at the microscale, only a fraction of bacterial cells may contribute to the overall activity at a given moment and location. Therefore, a major question to be answered now is which fraction of the bacterial community of an aquatic system is really active and contribute to the production at the time of sampling?

In order to estimate whether a single bacterial cell is active or not, different methodologies have been proposed in the literature. Microautoradiography after incubation in the presence of a labeled substrate (amino acids, glucose, thymidine, etc.) [START_REF] Tabor | Improved micro-autoradiographic method to determine individual micro-organisms active in substrate uptake in natural waters[END_REF], direct viable counts based on cell enlargement in the presence of cell division inhibitors and substrates [START_REF] Kogure | A tentative direct microscope method for counting living marine bacteria[END_REF][START_REF] Joux | Ecological implications of an improved direct viable count method for aquatic bacteria[END_REF] and activity of the electron transport system (ETS) as estimated by the reduction of the tetrazolium salt p-iodonitrotetrazolium violet (INT) [START_REF] Zimmerman | Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration[END_REF][START_REF] Dufour | The tetrazolium reduction method for assessing the viability of individual bacterial cells in aquatic environments: improvements performance and applications[END_REF] are the most common methods.

More recently, different fluorescent dyes were used for bacterial activity or viability assessment at the cellular level [START_REF] Joux | Use of fluorescent probes to asses physiological functions of bacteria at single-cell level[END_REF]. The most commonly used dyes are fluorescein diacetate and its derivatives to assess esterase activity [START_REF] Chrzanowski | Applicability of the fluorescein diacetate method of detecting active bacteria in freshwater[END_REF][START_REF] Parthuisot | Evaluation of ChemChrome V6 for bacterial viability assessment in waters[END_REF], dyes indicating the membrane potential of cells such as rhodamine 123 [START_REF] Kaprelyants | Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry[END_REF] or DiBAC4 [START_REF] Jepras | Development of robust flow cytometric assay for determining numbers of viable bacteria[END_REF] and tetrazolium salts such as 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which represents a fluorochrome analogue to INT [START_REF] Rodriguez | Use of fluorescent redox probe for direct visualization of actively respiring bacteria[END_REF].

The CTC staining method has been used increasingly in recent years to detect the single-cell activity of bacteria in aquatic systems. CTC is reduced within individual respiring cells by the ETS into a fluorescent CTC-formazan product. Fluorescent respiring cells (CTC+) are usually detected by epifluorescence microscopy sometimes coupled to an image analysis system [START_REF] Gasol | Active versus inactive bacteria: size-dependence in a coastal marine plankton community[END_REF]. More recently, CTC+ cells have been detected by flow cytometry (FCM) [START_REF] Del Giorgio | Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry[END_REF][START_REF] Sieracki | Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures[END_REF]. The fraction of CTC+ cells found in natural aquatic ecosystems is generally low and varies in the range 0-10% [START_REF] Gasol | Active versus inactive bacteria: size-dependence in a coastal marine plankton community[END_REF][START_REF] Schaule | Use of 5-cyano-2,3-ditolyltetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water[END_REF][START_REF] Lovejoy | Bacterial activity during early winter mixing (Gulf of St. Lawrence, Canada)[END_REF][START_REF] Choi | Relationship between the presenceabsence of a visible nucleoid and metabolic activity in bacterioplankton cells[END_REF][START_REF] Ulrich | Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride[END_REF][START_REF] Karner | Determination of active marine bacterioplankton: a comparison of universal 16s rRNA probes, autoradiography, and nucleoid staining[END_REF][START_REF] Pinhassi | Coupling between bacterioplankton species composition, population dynamics, and organic matter degradation[END_REF][START_REF] Bartscht | Evaluation of cell activity and of methods for the cultivation of bacteria from a lake community[END_REF][START_REF] Choi | Dead or alive? A large fraction of ETSinactive marine bacterioplankton cells, as assessed by reduction of CTC, can become ETSactive cells with incubation and substrate addition[END_REF]. In contrast, Smith [START_REF] Smith | Coherence of microbial respiration rate and cell specific bacterial activity in a coastal planktonic community[END_REF] reported some high percentages of respiring cells (47%) in the Cheasapeake bay. However, comparison between different studies is often impossible because of the lack of standardization of the method. In the literature, important differences in the CTC staining procedures are found which can influence CTC+ counts: CTC concentrations vary from 0.5 [START_REF] Coallier | The optimization and application of two direct viable count methods for bacteria in distributed drinking water[END_REF] to 10 mM [START_REF] Choi | Relationship between the presenceabsence of a visible nucleoid and metabolic activity in bacterioplankton cells[END_REF] and incubation times from 1 [START_REF] Sieracki | Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures[END_REF] to 12 h [START_REF] Gasol | Active versus inactive bacteria: size-dependence in a coastal marine plankton community[END_REF]. Today, the CTC method remains controversial since an increasing number of authors promote this technique while others recently pointed out several drawbacks of the method.

Comparative analyses have shown that the proportion of CTC+ cells in lakes tends to increase with increasing system productivity [START_REF] Del Giorgio | Increase in the proportion of metabolically active bacteria along gradients of enrichment in freshwater and marine plankton: implication for estimates of bacterial growth and production rates[END_REF]. Some authors [START_REF] Del Giorgio | Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry[END_REF][START_REF] Lovejoy | Bacterial activity during early winter mixing (Gulf of St. Lawrence, Canada)[END_REF] reported positive correlations between the abundance of CTC+ cells and bacterial production. Smith [START_REF] Smith | Coherence of microbial respiration rate and cell specific bacterial activity in a coastal planktonic community[END_REF] showed a good correlation between CTC+ cell abundances and respiration of the <3-µm planktonic community. Gasol et al. [START_REF] Gasol | Active versus inactive bacteria: size-dependence in a coastal marine plankton community[END_REF] showed that actively respiring bacteria tended to be larger than non-active bacteria leading to a strong size dependence for the probability of a bacterium to be active. In contrast, Karner and Fuhrman [START_REF] Karner | Determination of active marine bacterioplankton: a comparison of universal 16s rRNA probes, autoradiography, and nucleoid staining[END_REF] found, in a study on marine bacterial assemblages, that the abundance of metabolically active cells determined by microautoradiography (using a mixture of tritiated amino acids) was higher than the number of respiring cells detected by the CTC method. In other reports, growth rates calculated from the bacterial production and the number of CTC+ cells were very high and, therefore, generation times were very low and unrealistic [START_REF] Ulrich | Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride[END_REF][START_REF] Lebaron | Changes in bacterial community structure in seawater mesocosms differing in their nutrient status[END_REF]. These data suggest that active cell counts are underestimated by the CTC method probably due to several limitations. The detection limit of fluorescence signals may contribute to this underestimation since the most weakly active cells generally do not provide a sufficient fluorescence signal to be detectable by microscopy and visual examination. Sieracki et al. [START_REF] Sieracki | Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures[END_REF] have found that FCM counts of CTC+ cells are often higher than microscopic counts, probably due to the fact that rapidly fading cells are not detectable by microscopic examination. Choi et al. [START_REF] Choi | Dead or alive? A large fraction of ETSinactive marine bacterioplankton cells, as assessed by reduction of CTC, can become ETSactive cells with incubation and substrate addition[END_REF] showed that most of the CTC+ bacteria found in Oregon coastal waters can become CTC-after incubation in the presence of substrates, probably by increasing the fluorescence signal of the cells. Toxicity of the CTC dye may constitute a second limitation. Ulrich et al. [START_REF] Ulrich | Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride[END_REF] demonstrated by several techniques (thymidine incorporation, glucose incorporation and respiration, Microtox bioassay) that CTC at millimolar concentrations severely reduces bacterial activity. Finally, some authors showed that some bacterial strains cannot be efficiently stained with CTC [START_REF] Yamaguchi | Flow cytometric analysis of bacterial respiratory and enzymatic activity in the natural aquatic environment[END_REF] and some active bacteria cannot reduce CTC under certain conditions [START_REF] Smith | Mechanisms of INT(2-(4-iodophenyl)-3-(4nitrophenyl)-5-phenyl tetrazolium chloride and CTC (5-cyano-2,3-ditolyl-tetrazolium chloride) in Escherichia coli K-12[END_REF]. In contrast, Sherr et al. [START_REF] Sherr | Estimating abundance and singlecell characteristics of respiring bacteria via the redox dye CTC[END_REF] reported that all the bacteria isolated from this environment were able to reduce CTC.

Obviously, the CTC method requires more investigation to further understand the ecological meaning of CTC+ counts and the relationships which exist between such counts and bacterial production commonly determined in aquatic ecosystems. In this study, several points of the CTC staining protocol (CTC concentration, incubation time) were investigated using flow cytometric detection. The toxicity of CTC on bacterial activity was tested by 3 Hleucine incorporation, a classical method used to estimate bacterial production [START_REF] Kirchman | Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems[END_REF]. Finally, the relationships between CTC+ bacteria and bacterial production were investigated by coupling tritiated leucine incorporation with cell sorting of CTC+ cells by FCM. Experiments were performed on samples collected in various aquatic environments including marine, brackish and fresh waters.

Materials and methods

Water samples

Samples were collected between March and June 2000 along the Mediterranean coast (France) at sites with different physico-chemical characteristics. Seawater samples were taken in the coastal area near Banyuls-sur-Mer: in the bay at a station called SOLA (42°29'N, 3°08'E) and in the harbour (42°28'N, 3°08'E). Brackish water samples were taken in a coastal lagoon (salinity 30x): the Leucate lagoon (42°49'N, 2°9'E). Samples of freshwater were collected in the Tech river at 5 km above the river mouth (42°35'N, 2°58'E). Five liters were taken just below the surface in the different studied environments except for the SOLA station where the water was collected at 24 m depth. Samples were processed in the laboratory within 2 h after collection.

Incubation of bacteria with CTC

CTC (Polysciences Europe) reduction was used to identify active respiring cells as proposed by Rodriguez et al. [START_REF] Rodriguez | Use of fluorescent redox probe for direct visualization of actively respiring bacteria[END_REF]. Three CTC final concentrations (1, 2.5 and 5 mM) were tested on different samples. When the CTC concentration is not explicitly mentioned, a final concentration of 2.5 mM was used. A stock solution of CTC (50 mM concentration) was freshly prepared on the day of experiment and stored at 4°C. Different incubation periods ranging from 10 min to 6 h were tested. The incubation period in the presence of CTC is reported for each experiment. Incubations were performed in the dark at in situ temperature, ended by adding formaldehyde (2% final concentration) and samples were then stored in liquid nitrogen until examination.

Enumeration of CTC+ cells by FCM

Enumeration of CTC+ cells was performed with a Facs-Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with an air-cooled argon laser (488 nm, 15 mW). CTC-stained cells were excited at 488 nm, discriminated and enumerated on the basis of their orange and red fluorescence emissions collected using a 585/42 nm band pass filter and a 650 nm long pass filter, respectively. The accurate volume analyzed and subsequent cell concentration estimation were calculated by weighing the sample before and after a 5-min run of the cytometer. Fluorescent beads (1.002 Wm; Polysciences Europe) were systematically added to each analyzed sample to normalize cell fluorescence emission and light scatter values. A blank made of sterile water (seawater or freshwater depending on the salinity of the sample) filtered through 0.2-µm pore-size membrane and incubated with CTC (under the same conditions as the sample) was enumerated by FCM before each estimate of CTC+ cells in order to control the absence of non-specific signals (i.e. CTC precipitate) in the tested experimental conditions. A blank was also analyzed in the absence of CTC dye to check the absence of auto-fluorescent orange-red cells or particles in the CTC+ window. The proportion of CTC+ bacteria was calculated for each sample with regards to total cell numbers estimated by FCM (see below).

Total bacterial enumeration by FCM

For FCM analyses, 1 ml of fixed sample with formaldehyde (2% final concentration) was incubated with 0.5 µl of SYBR-II (nucleic acid stain; Molecular Probes) for 15 min at room temperature in the dark [START_REF] Lebaron | Comparison of blue nucleic acid dyes for enumeration of total bacteria in aquatic systems[END_REF]. Counts were performed with the FacsCalibur flow cytometer.

Stained bacteria, excited at 488 nm, were discriminated and enumerated according to their right angle-light scatter (SSC, related to cell size) and green fluorescence measured at 530/30 nm.

Impact of CTC on bacterial activity

The impact of CTC on bacterial activity was investigated using the 3H-leucine incorporation method commonly used to estimate bacterial production in aquatic ecosystems [START_REF] Kirchman | Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems[END_REF][START_REF] Servais | Bacterial production measured by 3H-thymidine and 3H-leucine in various aquatic ecosystems[END_REF].

Incorporation of 3 H-leucine (151 Ci mmol -1 , Amersham) was measured at 80 nM (5 nM of tritiated leucine and 75 nM non-radioactive leucine) to saturate leucine incorporation in the different aquatic systems tested (data not shown). Two types of experiments were performed to test the impact of CTC on leucine incorporation: in the first set of experiments, leucine incorporation was compared in the presence and absence of CTC during a 2-h incubation while, in the second set of experiments, CTC was added during the course of incubation of bacteria with tritiated leucine. In the first type of experiments, two subsamples (20 ml each) were incubated in the presence of leucine for 2 h in the dark at in situ temperature. One subsample was supplemented with CTC (final concentration 2.5 or 5 mM) immediately after leucine addition. After 0.5, 1, 1.5 and 2 h of incubation, 5 ml were removed from each subsample and filtered through 0.2-µm pore-size cellulose acetate membrane. After filtration, 10 ml of ice cold 5% trichloroacetic acid (TCA) was added on the filter and filtered after 10 min. Then, filters were rinsed four times with 5 ml of cold TCA. Radioactivity associated with the filters was estimated by liquid scintillation. In the second type of experiments, two subsamples (10 ml each) were incubated in the presence of leucine for 2 h in the dark at in situ temperature. In one subsample, CTC (final concentration 2.5 mM) was added after 1.5 h in order to test if the CTC addition has a direct effect on leucine incorporation. After incubation, subsamples were treated as reported for the first type of experiments.

Leucine incorporation followed by cell sorting using FCM

In order to estimate the contribution of CTC+ cells to total bacterial activity, we used a procedure similar to that developed by Servais et al. [START_REF] Servais | Coupling bacterial activity measurements with cell sorting by flow cytometry[END_REF] and applied by Bernard et al. [START_REF] Bernard | Relationships between bacterial cell size, productivity and genetic diversity in aquatic environments using cell sorting and flow cytometry[END_REF] to estimate the contribution of different cell-size classes to bacterial activity in various aquatic systems. This procedure consisted of labeling bacteria with 3 H-leucine and then sorting different bacterial subpopulations by FCM in order to estimate their contribution to the activity of the whole bacterial community. In this study, CTC+ cells were sorted after 3 Hleucine labeling in order to determine the specific leucine incorporation rate of CTC+ cells. In parallel, total bacteria were sorted after similar 3 H-leucine labeling to estimate the average specific leucine incorporation rate of the total bacterial community. From a practical point of view, leucine incorporation was performed at 80 nM final concentration. Only radioactive leucine (151 Ci mmol -1 , Amersham) was added to maximize the detection limit of labeled bacteria. Two subsamples of 3 ml were incubated at in situ temperature during 2 h in the dark and in the presence of 3 H-leucine. In subsample 1, CTC (2.5 mM final concentration) was added 1.5 h after 3 H-leucine addition. The incubation of both subsamples was stopped by addition of formaldehyde (2% final concentration) so that the incubation period of subsample 1 in the presence of CTC was 30 min. Labeled CTC+ bacterial cells from subsample 1 were then sorted with the FacsCalibur flow cytometer. For all sorting experiments, the salinity of the sterile sheath fluid was adjusted to that of the sample to avoid cell lysis or protein release due to osmotic shock. The sorting window was defined on each cytogram where FL2 (585/42 nm band pass filter) was plotted against FL3 (650 nm long pass filter) (Fig. 1a). We selected on the flow cytometer the 'single sort mode' where a sort occurs only if a single target cell is identified. The result gives high purity with less emphasis on recovery and the best accurate counts of sorted cells. Sorted cells were thus enumerated and collected at the outlet of the flow cytometer directly onto a 0.2-µm pore-size membrane (acetate cellulose filter). Sorting was complete when the number of CTC+ sorted cells ranged between 300 000 and 500 000 cells to obtain a measurable radioactivity on the filter. Ten milliliters of cold 5% TCA were added to the filter in order to precipitate macromolecules and to rinse the membrane. After 10 min, TCA was eliminated by filtration and the radioactivity associated with the sorted bacteria was estimated by liquid scintillation. Immediately after the end of the incubation, bacteria of subsample 2 were stained with SYBR-II following the procedure used to determine total counts. Labeled and stained bacterial cells were then sorted using a window defined around the cells corresponding to the total bacterial community on a cytogram where green fluorescence was plotted against SSC (Fig. 1b). In a previous paper, this sorting procedure of the total bacterial population was shown to give a similar result to a direct measurement of the bacterial activity without cell sorting [START_REF] Servais | Coupling bacterial activity measurements with cell sorting by flow cytometry[END_REF]. The specific activity (SA) of respiring cells (mol leucine incorporated cell -1 h -1 ) was determined from the radioactivity incorporated in the sorted CTC+ cells. Similarly, the average SA of the total bacterial cells was calculated on the basis of the sort of total bacteria after SYBR-II staining. The total bacterial activity (mol incorporated l -1 h -1 ) and the activity of CTC+ cells were calculated by multiplying the total cell number by the average SA of the total bacterial community and the CTC+ abundance by the SA of the respiring cells, respectively. Duplicate labeling experiments were not made because of both the high cost and long time required for these experiments. However, on several samples, a coefficient of variation of 9% was estimated for the SA of the total bacterial population as determined by radioactive labeling followed by cell sorting after SYBR-II staining.

Results

Effect of CTC concentration and incubation period on the detection of respiring bacteria

The impact of different CTC concentrations on both the abundance and the fluorescence of respiring cells determined by FCM was first tested. Concentrations of 1, 2.5 and 5 mM of CTC were used (Fig. 2). Data are reported as the ratio between the number or the fluorescence of CTC+ cells measured at 2.5 and 1 mM versus the number and fluorescence values determined at 5 mM and at different incubation times. A concentration of 5 mM was used as a reference in Fig. 2 as it represents the most often used concentration reported in the literature. Our data obtained from several marine and freshwater environments suggest that CTC+ abundances were not significantly different (comparison of average values; t-test, K= 0.01) at concentrations of 2.5 M and 5 mM of CTC for all incubation times but average ratios were closer to 1 for incubation times higher than 20 min. The fluorescence of CTC+ cells was not significantly different at 2.5 and 5 mM CTC concentrations. In contrast, when 1 mM was used, the number of CTC+ cells was significantly lower at all incubation times while the average fluorescence of CTC+ cells was not significantly different to that measured at the other CTC concentrations. From these data, a concentration of 2.5 mM was applied for all subsequent CTC+ cell enumeration. In order to test the influence of the staining period on the number and fluorescence of CTC+ bacteria, kinetics of staining was performed on several samples including marine, brackish and freshwater environments (Fig. 3). The kinetics showed a first phase of increase of CTC+ counts up to a maximum followed by a second phase of decrease or stability of CTC counts. For all samples, the peak of CTC+ counts was reached after an incubation ranging from 0.5 to 2 h. The decrease after the peak was more or less pronounced from one sample to another. For instance, CTC+ cells enumerated after 4 h represented 63% of the maximum in the case of the Tech river sample (Fig. 3e) and 90% in the case of the Leucate lagoon sample (Fig. 3f). This indicates that CTC+ cells detected after a short incubation period (30 min to 2 h) can become CTC-by increasing the incubation. The fluctuations of the average fluorescence of CTC+ cells versus incubation were also very variable from one sample to another. Even within a similar environment (the marine SOLA station), the patterns in CTC+ abundances and average fluorescence observed at different sampling dates were not similar (Fig. 3a-c).

CTC toxicity

The toxicity of CTC on bacterial activity was investigated using 3H-leucine incorporation on water samples from various origins. The leucine incorporation method measures the protein synthesis rate, which is an essential and universal physiological activity in a bacterial cell, and is traditionally used to estimate bacterial production [START_REF] Kirchman | Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems[END_REF]. Fig. 4 shows the incorporation of radioactivity in the macromolecular fraction of bacteria when incubated in the presence of tritiated leucine with and without CTC. As expected in the absence of CTC, radioactivity plotted against time followed a linear pattern with a slope depending on the activity of bacterial communities. In the presence of CTC, leucine was slightly incorporated into protein during the first 30 min of incubation and then, protein synthesis was completely inhibited in all the tested samples. Data on the impact of CTC addition during the incubation with 3 H-leucine are reported in Table 1. In these experiments, one subsample was incubated for 2 h with 3 H-leucine and another was incubated for 2 h with 3 H-leucine but CTC was added during the last 30 min of incubation. The ratios of radioactivity incorporated into both subsamples were similar (close to 0.75) (Table 1) indicating that the incorporation of leucine and, thus, protein synthesis was immediately inhibited after CTC addition.

Contribution of CTC+ cells to bacterial production

A major consideration concerning the CTC technique is to gain a better understanding of the differences in metabolism between CTC+ and CTC-bacteria and their relative contributions to the whole community activity as commonly estimated by bacterial production. To investigate these contributions, the following procedure was used [START_REF] Servais | Coupling bacterial activity measurements with cell sorting by flow cytometry[END_REF] : bacteria were labeled with 3 H-leucine and different bacterial subpopulations were then sorted by FCM in order to estimate their contribution to bacterial activity. After labeling bacterial cells with tritiated leucine, total bacterial cells stained with SYBR-II were sorted from one subsample and CTC+ bacteria were sorted from another subsample stained with CTC for 30 min. This allowed us to compare the SA (leucine incorporation rate per cell) of the total community with the SA of CTC+ bacteria in a marine, a brackish and a freshwater sample (Table 2). In these samples, CTC+ cells represented from 5 to 47% of total bacterial counts.

For calculating the SA of CTC+ cells, an incubation time of 1.5 h (incubation period in the presence of 3 H-leucine and in the absence of CTC) was considered and not 2 h as for the total community because our data reported in Table 1 showed that leucine incorporation immediately stopped after CTC addition. As expected, the average SA of CTC+ bacteria was always higher than the average SA of the total community. Data reported in Table 2 allowed us to calculate the average SA of CTC-cells which were, respectively, 219x10 -21 , 47x10 -21 and 41x10 -21 mol leucine incorporated cell -1 h -1 for the Banyuls harbour, the Leucate lagoon and the Tech river. In the same samples, the ratios between SA of CTC+ and SA of CTCbacteria were 1.37, 7.47 and 1.51, respectively. From the SA data and the abundance of total and CTC+ bacteria, we found that the contribution of CTC+ cells to total bacterial activity ranged from 27 to 57%. These results showed that, on the tested samples, even if the SA of CTC+ cells was always higher than the SA of CTC-cells, the difference was sometimes not important (sample from Banyuls harbour, for example). In addition, the contribution of CTC+ cells to total bacterial production was not predominant.

Discussion

A review of literature on the protocols used to estimate the fraction of actively respiring cells in natural aquatic systems by the CTC technique revealed large differences in both the concentration of CTC and the incubation time. These different protocols have been established from staining kinetics performed at various CTC concentrations [START_REF] Rodriguez | Use of fluorescent redox probe for direct visualization of actively respiring bacteria[END_REF][START_REF] Sieracki | Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures[END_REF][START_REF] Schaule | Use of 5-cyano-2,3-ditolyltetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water[END_REF][START_REF] Lovejoy | Bacterial activity during early winter mixing (Gulf of St. Lawrence, Canada)[END_REF][START_REF] Choi | Dead or alive? A large fraction of ETSinactive marine bacterioplankton cells, as assessed by reduction of CTC, can become ETSactive cells with incubation and substrate addition[END_REF][START_REF] Coallier | The optimization and application of two direct viable count methods for bacteria in distributed drinking water[END_REF].

The differences in the proposed protocols may be explained by the fact that the method was applied to different types of environments (pure culture of bacteria, lake water, drinking water, marine systems) which induced differences in the bacterial SA and thus in the CTC staining. Discrepancies between protocols may also be due to the use of different instruments for the detection of fluorescent cells since microscopy and FCM have different detection limits [START_REF] Del Giorgio | Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry[END_REF][START_REF] Sieracki | Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures[END_REF]. The enumeration by FCM minimises the effect of fading which constitutes one of the most important limitations when using microscopy. In addition, FCM offers the advantage of quantifying the fluorescence of an important number of cells in a few minutes.

Del Giorgio et al. [START_REF] Del Giorgio | Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry[END_REF] estimated that flow cytometric counts of CTC+ cells were five-to 10fold more accurate than microscopic counts.

In this paper, we showed using FCM that for both natural marine and freshwater systems, CTC+ counts and cell fluorescence intensities obtained using a CTC concentration of 2.5 mM were similar to those obtained at 5 mM, the most often used concentration reported in the literature [START_REF] Choi | Dead or alive? A large fraction of ETSinactive marine bacterioplankton cells, as assessed by reduction of CTC, can become ETSactive cells with incubation and substrate addition[END_REF][START_REF] Smith | Coherence of microbial respiration rate and cell specific bacterial activity in a coastal planktonic community[END_REF][START_REF] Sherr | Estimating abundance and singlecell characteristics of respiring bacteria via the redox dye CTC[END_REF]. In contrast, CTC+ counts were underestimated with 1 mM of CTC. Concentrations higher than 5 mM were not tested since several papers clearly showed a decrease of CTC+ counts at these concentrations [START_REF] Rodriguez | Use of fluorescent redox probe for direct visualization of actively respiring bacteria[END_REF][START_REF] Sieracki | Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures[END_REF]. For the incubation time, kinetic experiments performed on various aquatic systems (including fresh, brackish and marine waters) and at different periods for a given environment revealed different patterns from which no general rule could be defined. From our data, it appears clearly that it is impossible to define a standard staining protocol to enumerate CTC+ cells, probably due to the toxic effect of CTC on viable cells (see below). Therefore, it seems necessary to perform a kinetics study of CTC staining before each analysis and to consider the maximum of CTC+ counts as the correct number of actively respiring cells but this procedure is extremely expensive and time-consuming.

The decrease of CTC+ counts observed by increasing the incubation time suggests that CTC may be released or lost by respiring cells after a short period of incubation (30 min to 2 h). This loss of fluorescence may be the result of a passive efflux of formazan due to the toxicity of the dye leading to cell death and to the permeabilisation of membranes which may occur more or less quickly depending on species. This observation has important consequences since interpretations of results from other studies that used long and variable incubation periods are questionable [START_REF] Gasol | Active versus inactive bacteria: size-dependence in a coastal marine plankton community[END_REF][START_REF] Sieracki | Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures[END_REF][START_REF] Smith | Coherence of microbial respiration rate and cell specific bacterial activity in a coastal planktonic community[END_REF].

The inhibitory effect of CTC on bacterial activity measured by 3 H-thymidine incorporation, the uptake and respiration of 14 C-glucose, growth on plate count and Microtox bioassay was demonstrated by Ulrich et al. [START_REF] Ulrich | Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride[END_REF][START_REF] Ulrich | Is the CTC dye technique an adequate approach for estimating active bacterial cells ?[END_REF]. The two types of experiments presented here demonstrated the drastic toxic effect of CTC on protein synthesis at CTC concentrations (2.5 and 5 mM) which are commonly used to estimate the fraction of respiring bacteria. In addition, the toxic effect of CTC was very rapid as protein synthesis was immediately inhibited after CTC addition (Table 1). This toxic effect of the CTC dye on major physiological activities (substrate uptake and respiration, DNA and protein synthesis) probably leads to an underestimation of active cell counts estimated by this technique. This underestimation may explain why Karner and Fuhrman [START_REF] Karner | Determination of active marine bacterioplankton: a comparison of universal 16s rRNA probes, autoradiography, and nucleoid staining[END_REF] found higher numbers of active cells by microautoradiography than by the CTC technique. It could also explain why unrealistically high production values per cell and short generation times were found in natural environments when determined from production measurements and CTC+ counts [START_REF] Ulrich | Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride[END_REF][START_REF] Lebaron | Changes in bacterial community structure in seawater mesocosms differing in their nutrient status[END_REF][START_REF] Ulrich | Is the CTC dye technique an adequate approach for estimating active bacterial cells ?[END_REF].

Several authors have suggested that CTC+ bacteria are responsible for the bulk of total bacterial activity [START_REF] Del Giorgio | Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry[END_REF][START_REF] Lovejoy | Bacterial activity during early winter mixing (Gulf of St. Lawrence, Canada)[END_REF][START_REF] Smith | Coherence of microbial respiration rate and cell specific bacterial activity in a coastal planktonic community[END_REF]. These assumptions were based on positive correlations found between the total bacterial production [START_REF] Del Giorgio | Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry[END_REF][START_REF] Lovejoy | Bacterial activity during early winter mixing (Gulf of St. Lawrence, Canada)[END_REF][START_REF] Sherr | Estimating abundance and singlecell characteristics of respiring bacteria via the redox dye CTC[END_REF] or respiration of the 63-µm planktonic community [START_REF] Smith | Coherence of microbial respiration rate and cell specific bacterial activity in a coastal planktonic community[END_REF] and the absolute number of CTC+ bacteria but never on a quantitative estimation of the contribution of CTC+ cells to total activity. Sherr et al. [START_REF] Sherr | Estimating abundance and singlecell characteristics of respiring bacteria via the redox dye CTC[END_REF] suggested that CTC+ cells represent bacteria with a high level of metabolic activity whereas some CTCcells have sufficient RNA content and the ability to assimilate substrates to be identifiable as active cells by indices of cell-specific RNA content and microautoradiography. These authors also pointed out the fact that quantitative differences in metabolism between `highly active' CTC+ cells and `less active' CTC-cells have still to be determined. To our knowledge, this is the first report in which the absolute activity of CTC+ and CTC-cells has been quantitatively estimated and compared to total bacterial activity. The data obtained by cell sorting after radioactive labeling showed that, even if the SA of CTC+ bacteria is higher than that of CTCcells, some CTC-cells significantly contribute to the total bacterial activity. In our samples, the contribution of CTC-cells to total activity ranged between 43 and 73%. This means that we cannot consider CTC-cells as inactive cells nor as weakly active cells. Thus, some active bacteria are probably not stained by CTC because of its toxicity and/or because some cells are not able to reduce the CTC in the conditions met in natural aquatic environments as suggested by Smith and McFeters [31] while they are able to do so after an incubation in the presence of substrate as shown by Choi et al. [START_REF] Choi | Dead or alive? A large fraction of ETSinactive marine bacterioplankton cells, as assessed by reduction of CTC, can become ETSactive cells with incubation and substrate addition[END_REF]. Although only a few samples from different environments were analysed by the time-consuming cell sorting procedure, these data alone suggest that CTC+ cells are not responsible for the bulk of the bacterial production in natural aquatic systems.

As the CTC dye is not suitable for the detection and enumeration of active bacteria (i.e. cells contributing to bacterial production), other fluorescent dyes should be used for this purpose. For instance, when nucleic acids dyes such as TOTO, SYTO 13, SYBR green dyes are applied to natural bacterial communities, they allow us to discriminate two cellular subpopulations characterized by a high DNA content (HDNA) and a low DNA content, respectively [START_REF] Li | DNA distributions in planktonic bacteria stained with TOTO or TO-PRO[END_REF][START_REF] Gasol | Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria[END_REF]. Interestingly, it was recently hypothesized that cells with a high DNA content are responsible for the total bacterial activity [START_REF] Li | DNA distributions in planktonic bacteria stained with TOTO or TO-PRO[END_REF][START_REF] Gasol | Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria[END_REF]. The approach based on radioactive labeling followed by cell sorting applied in this paper has been recently used to validate this hypothesis [START_REF] Lebaron | Does the high nucleic-aid content of individual bacterial cells allow to discriminate active cells in aquatic systems?[END_REF].

Conclusions

The results reported in this study clearly indicate that the CTC procedure is not valid for estimation of the fraction of active cells in natural aquatic environments. Firstly, CTC+ bacteria are not responsible for a predominant part of the total bacterial activity because CTCbacteria significantly contribute to this total activity. Secondly, CTC has a strong toxic effect on key metabolic functions. Finally, because CTC staining varies greatly from one sample to another, it requires expensive and time-consuming staining kinetics to optimize the staining protocol.

Figure 1 .

 1 Figure 1. (a) Cytogram of red (FL3) versus orange (FL2) fluorescence used for cytometric detection of CTC+ cell. The window used for cell sorting is also reported. (b) Cytogram of green fluorescence (FL1) versus right angle-light scatter (SSC) used for flow cytometric detection of total cell number after stainng with SYBR-II. The window used for cell sorting is also reported. Sample collected in the Banyuls harbor (29/06/2000).

Figure 2 .

 2 Figure 2. Effect of CTC concentration on the detection of respiring bacteria. (a). Ratio at different times of incubation between the number of CTC+ cells enumerated at 2.5 mM (black symbols) and 1 mM (grey symbols) versus CTC+ enumerated at 5 mM. (b) Ratio at different times of incubation between the fluorescence of CTC+ cells measured at 2.5 mM (black symbols) and 1 MM (grey symbols) versus fluorescence of CTC+ cells measured at 5 mM.

Figure 3 .

 3 Figure 3. Effect of time of incubation on the number (•) and the average fluorescence (○) of CTC+ bacteria corresponding to (a, b and c) samples from the marine SOLA station (collected, on 8 of March, 11 of April and 9 of May, respectively) ; (d) sample from Banyuls harbor (29 of June) ; (e) sample from Leucate lagoon (30 of May) ; (f) sample from Tech river (15 of May).

Figure 4 .

 4 Figure 4. Leucine incorporation as a function of incubation time without CTC (•) and with a CTC concentration of 2.5 mM (○) and 5 mM (∆). Samples from (a) seawater (marine SOLA station), (b) brackish water (Leucate lagoon) and (c) freshwater (Tech river).

Table 2 .

 2 Contribution of the CTC+ cells to total bacteria activity determined by 3 H-leucine labeling followed by cell sorting in 3 water samples

	Sample (date)
	Banyuls harbour	Leucate lagoon	Tech river
	(29/6/00)	(30/5/00)	(15/5/00)

Acknowledgements

The FACSCalibur cytometer was funded by the Région Languedoc-Roussillon (France), the Centre National de la Recherche Scientifique (CNRS-INSU-SDV, France) and the EU Eloise Project, PL95049. At the time of this study, P.S. was working at the Observatoire Océanologique of Banyuls-sur-Mer and was temporally employed by the CNRS (INSU) and the University Pierre et Marie Curie.

Freshwater samples 3 0.75±0.04

Subsample 1 was incubated for 2 h with 3 H-leucine. Subsample 2 was incubated for 2 h with 3 Hleucine and CTC was added 1.5h after the beginning of the incubation. Data are presented as ratios of radioactivity incorporated in subsample 2 and 1 for different (n) marine and freshwater samples.


Nathalie Ortar
email: nathalie.ortar@entpe.fr

Félicie Drouilleau

High-tension choices: an analysis of everyday energy-related practices among Lyon-based families

This article examines how personal habits related to energy consumption both in a domestic setting and during day-to-day transport use are initially acquired and how they subsequently develop; it also focuses on the nature of the changes that have occurred since the latest jump in energy prices. The analysis presented here draws on the findings of the socio-anthropological component of

Introduction

This article examines how personal habits related to energy consumption both in a domestic setting and during day-to-day transport use are initially acquired and how they subsequently develop; it also focuses on the nature of the changes that have occurred since the latest jump in energy prices. The analysis presented here draws on the findings of the socio-anthropological component of the TransEnergy research project, financed by the Agence Nationale de la Recherche as part of the Villes Durables (Sustainable Cities) programme, which examined the impact of energy price rises on households living and working in the peri-urban fringe zone of France's second city, Lyon. This research focused on two areas: firstly, the extent to which socio-economic factors influences energy consumption and, secondly, how awareness of energy consumption changes according to an individual's stage of life. It thus feeds into a more comprehensive socio-anthropological account of behaviours relating to energy consumption and the processes of change at work in this area. For while, as Isabelle Garabuau-Moussaoui has remarked, "energy is a resource that has, since the end of the 19 th century, been perceived as both abundant and a basic necessity" [START_REF] Garabuau-Moussaoui | Chapitre 3 -L'énergie est-elle un enjeu de pouvoir dans la famille ?[END_REF], a notion that the use of nuclear power has helped perpetuate in France, this perception of energy as an abundant and cheap resource has nonetheless begun to change in recent years. The rise in energy prices, set against a background of economic crisis and higher living costs, has done much to bring about this shift.

The parallel analysis of "energy consumption associated with being 'indoors' and 'outdoors', in other words associated with housing and practices of mobility" carried out by Xavier Desjardins and Lucile Mettelal [START_REF] Desjardins | L'habiter périurbain face à l'enjeu énergétique[END_REF] has demonstrated the importance of this relationship to the ways in which periurban residents relate to their lived spaces: the central position of the house for these residents allows certain carry-forward mechanisms applied in the context of energy spending to be understood. We have decided to draw on their approach in our study, which widens the scope of this investigation and adopts a systematic methodological framework allowing energy use in the domestic and transport sectors to be examined in tandem. This project thus integrates an analysis of spending on transport and housing with a wider examination of the overall cost of energy and of consumption more generally, its aim being to establish how attitudes to energy-related spending change over an individual's lifetime and whether or not phenomena of carrying forward and compensation occur between different areas of spending. The survey was given to employees of companies identified as producing high levels of CO2 by the statistical study (see the article by Patricia Lejoux in this dossier) and consisted of a questionnaire. The companies in question are all situated within the city's commuter belt in zones with poor or nonexistent public transport links. Interviewees were selected on the basis of their socio-professional category -office and blue-collar workers were given priority -and of the distance between their home and place of work (equal to or greater than 25 km (15 miles) or involving a journey of at least 25 minutes each way). 27 individuals from the Lyon area were interviewed. The survey was carried out either in the home, at the place of work (office, meeting room, cafeteria), or in a neutral space. 21 interviewees used a car as their sole means of transport to get to work. The others used public transport or cycled (in one case) for a journey of around one hour each way.

Those surveyed live both in peri-urban zones and within the conurbation. Peri-urbanisation has been associated in many studies with the 'middle classes' [START_REF] Berger | Les périurbains de Paris. De la ville dense à la métropole éclatée ?[END_REF]. However, recent studies based on qualitative research in this area [START_REF] Cartier | Enquête sur la banlieue pavillonnaire[END_REF], along with statistical analyses of rural and peri-urban populations, have built up a more nuanced picture, emphasising the strong presence of the socio-professional categories represented by blue-collar and office workers [START_REF] Mishi | Pour une sociologie politique des mondes ruraux[END_REF] who together form what A. Bihr and R. Pfefferkorn have termed a "stable, salaried working class" [START_REF] Bihr | Le renouveau d'une société de classes[END_REF]. We chose to focus our survey on these population segments because they are likely to be particularly vulnerable in the short term owing to the upward trend in the prices of energy and of everyday consumer products b . Indeed, Jean-Pierre Nicolas et al. [START_REF] Bozonnet | Socialisation et engagement écologiste en Europe. L'école, la famille et l'environnementalisme en héritage[END_REF] have demonstrated the increasing vulnerability of people on low and middle incomes living in the peri-urban zones around Lyon. Their comments on this vulnerability chiefly concern the budgetary efforts required to offset increased spending on transport, as living on the outer edges of the conurbation or in Lyon's peri-urban zone usually involves a "high rate of car use and particularly high mileage being driven daily" [START_REF] Nicolas | Mobilité quotidienne et vulnérabilité des ménages[END_REF]. In addition to this, "the mobility practices of households identified as being vulnerable are particularly dependent on cars. Before the recent rise in petrol prices they were already spending close to 10% of their income on their day-to-day transport needs" [START_REF] Nicolas | Mobilité quotidienne et vulnérabilité des ménages[END_REF]. However, while families may often move away from the city centre, jobs have also become less geographically concentrated as companies relocate towards the periphery (see Lejoux), as "in the contemporary city, jobs and housing are increasingly dispersed, all the more so in the case of low-skilled jobs and workers" [START_REF] Nicolas | Mobilité quotidienne et vulnérabilité des ménages[END_REF]. These relocation zones are in most cases very poorly served by public transport, so reaching them involves, de facto, the use of a car. The present research was carried out within these zones where access to work is difficult. In order to test the hypothesis that stress is being placed on households due to the location of their place of work as well as where they live, employees living in the city centre were also surveyed. b In France, energy precarity is considered to begin when the proportion of the household budget spent on energy reaches 10%. According to the ADEME (Agency for Energy Management and the Environment) (2008), "The poorest households spend 15% of their income on energy-related expenditure as coompared to 6% for the wealthiest" (see ADEME & Vous, Stratégie et Études n° 3, 03/04/2008); families are classed as being in energy poverty when their expenditure rises above 15%.

Learning energy-management skills

Energy management involves a complex array of instrumental activities, self-representations and collective performances that cannot be reduced to questions of individual rationality and choice. In her analysis of daily energy consumption, Elizabeth Shove [START_REF] Shove | Comfort, Cleanliness and Convenience : The Social Organization of Normality[END_REF] refers in particular to two concepts, comfort and convenience, which she sees as the drivers of consumption. Comfort is a normatised concept that has evolved over time according both to arbitrary regulations and the practical character of its use by the individual, a mode of practice that is in turn dependent on context.

Individual consumption is thus determined by choices made at an international and subsequently a governmental level, then by collective choices over which the individual has little control, such as the type of heating system used in an apartment building, before coming down to an individual level, which itself depends not only on the availability of a choice of technical solutionswhether in terms of insulation, vehicles or electrical goodsbut also on collective and family-based norms [START_REF] Subrémon | Le climat du chez-soi. Une fabrication saisonnière[END_REF].

Understanding how habits are acquired and built into the full range of daily routines is essential if we are to analyse the origins of the learning process relative to energy management, as well as ongoing processes and the role of public policy in influencing the decisions taken by households. The rather ambivalent status of energy management in western societies is also important in this respect.

Two periods in individuals' lives exert a key influence over how they learn to manage energy. The first occurs in the context of socialisation within the family, whereas the second involves learning through experience after leaving the parental home. As Isabelle Garabuau-Moussaoui et al. [START_REF] Garabuau-Moussaoui | Chapitre 3 -L'énergie est-elle un enjeu de pouvoir dans la famille ?[END_REF] have shown, this socialisation within the family occurs on various levels according to the situation in question and the roles being performed. The degree of care taken in relation to consumption thus drops when children are asked to wash, for instance, or when parents resort to using electronic games to keep them quiet. The handing down of ecologically sound actions and attitudes is only partially due to environmentalist discourse, as economic concerns relating to reduced consumption play an important role, as does a more general dislike of waste. A careful attitude towards energy is a constant which cuts across social and political space, even if the desire for economy is as much a part of a wider set of practices (associated principally with the lower classes) targeting all types of waste as it is part of an ecological effort more typical of social milieux whose members have been through higher education [START_REF] Bozonnet | Socialisation et engagement écologiste en Europe. L'école, la famille et l'environnementalisme en héritage[END_REF]. This is shown by a comparison of the attitudes of the parents of Emmanuelle (24, technician, single), who were conscious of environmental issues: "My father would always say: in the winter, when you're in the house, you wear a pullover.[…] My parents are very conscious of environmental issues. Sometimes they can come across as a bit, you could say, like eco-warriors! So that rubbed off on me, too", with those of the mother of Hubert (46, manager, married, 2 children), who was concerned with saving money: "I was brought up like that. Out of a need to save money. But it was … it wasn't about saving on energy, it was about saving on my mother's purse".

So, in spite of their sometimes ambivalent attitudes and motivations, parents play a fundamental role in inculcating the everyday actions and habits that relate to energy consumption. Passing down a legacy of energy-efficient behaviour is seen as a duty to the next generation, as Gérard (45, divorced, technician, 2

children) reflects: "Our parents taught us to be economical, to keep an eye on energy use. So we do keep an eye on it; and we carry on doing so and we pass it on. Yes, of course, it's a way of life. It's part of our way of life."

Routines may be classed as everyday rituals facilitating action, which itself consists of intentionally chosen activities, the objectives of which, along with their "way of doing things", the individual in question is able to explain. The intentionality behind this internalization relates in particular to a need to reduce uncertainty during the decision-making process [START_REF] Ilmonen | Sociology, consumption and routine[END_REF], and to structure everyday existence [START_REF] Ehn | The Secret World of Doing Nothing[END_REF]. Routines function as bodily memories. Their disadvantage, however, is that they thus limit action [START_REF] Ilmonen | Sociology, consumption and routine[END_REF] as changing them requires effort. In our interviews, it was the routines surrounding the use of water and the reflex actions acquired with respect to electricity, such as turning out the lights when leaving a room, that were spontaneously mentioned. These concerns end up becoming partially disconnected from questions of cost, and having more to do with a rejection of all forms of waste.

Moving into adulthood

The experience of studying and/or living away from home for the first time constitutes an intense period of learning which significantly influences future energy consumption behaviours. Unlike in childhood, when learning consisted in following an example and involved a degree of duress, moving out of the parental home is a period of experimentation. Time spent studying at university constitutes a particularly important stage in the development of an awareness of environmental issues, whether or not the studies in question relate specifically to this subject, and this is especially true for individuals belonging to those generations who were less exposed to notions of sustainable development through the primary-and secondary-school curriculum of the time [START_REF] Garabuau-Moussaoui | Chapitre 3 -L'énergie est-elle un enjeu de pouvoir dans la famille ?[END_REF][START_REF] Garabuau-Moussaoui | Energy-related logics of action throughout the ages in France: historical milestones, stages of life and intergenerational transmissions[END_REF]. Even those interviewees who refer to the handing-down of a concern for energy from their parents are quick to point out that the development of their personal awareness of this issue was linked to their studies. Nicolas (27, manager, living with a partner) "My course had an environmental element, so through my studies I became aware of various aspects of my energy consumption. And my parents had done a bit to raise my awareness as well. My course reinforced my consciousness of this issue and my desire to do something positive." J.-P. Bozonnet [START_REF] Bozonnet | Socialisation et engagement écologiste en Europe. L'école, la famille et l'environnementalisme en héritage[END_REF] has shown that the development of an ecological consciousness in a social context through contact with peers and teachers is closely linked to the subsequent integration and transmission of this consciousness to the following generation. University studies are thus a key period for the socially-determined development of an awareness of energy consumption.

Living away from home for the first time also brings with it a realisation of how much energyrelated costs contribute to the price of accommodation when these costs are borne individually. Even when young people do keep up practices inherited from their parents in terms of cooking and/or energy consumption [START_REF] Garabuau-Moussaoui | Chapitre 3 -L'énergie est-elle un enjeu de pouvoir dans la famille ?[END_REF][START_REF] Garabuau-Moussaoui | Energy-related logics of action throughout the ages in France: historical milestones, stages of life and intergenerational transmissions[END_REF], having to deal with their first fuel bills creates an awareness of energy consumption and heating methods that may not have been present before. Independent living forces one to reconsider practices which had previously been given little or no thought, such as whether or not to heat bedrooms or lower the thermostat. In a home setting, these are usually parental decisions, and it is rare for children to be involved in taking them [START_REF] Garabuau-Moussaoui | Chapitre 3 -L'énergie est-elle un enjeu de pouvoir dans la famille ?[END_REF][START_REF] Garabuau-Moussaoui | Energy-related logics of action throughout the ages in France: historical milestones, stages of life and intergenerational transmissions[END_REF].

Sensations of warmth and cold, along with dryness and dampness, are typically associated with different types of housing and heating systems. These notions derive from childhood memories and early experiences of living away from home which bring greater awareness both of different heat sources and of the varying quality of buildings. The degree of attention paid to energy consumption rests on a foundation of experiences and knowledge accumulated over the years which is called upon when the time comes to move to new accommodation, as Sébastien (42, supervisor, married, 2 children) relates: "We always had apartments with electric heaters when we were students and it always made the winters unbearable. We tried just leaving the heating off completely but, whew! … Sometimes it was just, you know, horrible. And then there are… we had books going mouldy because we left the heating off, we never opened the windows… so,you know, whew!… we had mezzanines to try to sleep as high up as possible and keep as warm as possible. But then when you had to climb down, it was just the pits. […] So that's why we're… […] we're trying to find a compromise."

Evolution of energy-awareness over accommodation trajectories

We will classify the memory of these sensations, along with the habits picked up in different types of accommodation, as habitational experiences which will determine subsequent housing choices by focusing the individual's attention on one particular type of energy. This notion of a 'trajectory' does not apply to all individuals, as some may not have been sufficiently marked by negative experiences, but it nevertheless helps account for the development of increased alertness in this respect.

In Sébastien's case, like that of most of the people interviewed, his initial habitational experience was acquired in low-quality privately-rented accommodation, and was characterised by uncomfortable extremes of temperature due to the building's poor insulation and heating system. The second stage involves gaining access to higher-quality rented accommodation, whether in the private or government/council housing sector. When government/council accommodation is chosen, accessibility and the availability of shared heating are key criteria.

Alexandra (30, telephone operator, living with a partner) describes how she took careful note of the type of heating when she moved into her apartment: she was absolutely set against having an electric system, and saw shared gas-fired heating as far preferable. Her main concern, however, was not so much the billsshe cannot recall off-hand what her heating costs areas a certain notion of comfort and a desire for reliability which stems from her past experiences. In her previous rented accommodation she had found herself without any heating and was keen not to repeat the experience: "We'd had a real nightmare with the hot-water tank in our last apartment, and with the heating as well… we even ended up taking the rental agency to court because they didn't want to do anything about it. We had no heating. […] So I… I did look to see if the boiler was fairly new. […] I was pleased to see that it was a shared gas-fired system with big cast-iron radiators, as you can see, they're really nice."

With shared heating, energy bills tend to become less visible, disappearing among the other items in the monthly charges. Boudjemaa (37, telephone operator, married, 2 children) rents a council flat with shared gas-fired heating. When he was looking for accommodation, his priority had been to find a council property, as this would allow him to make substantial rent savings compared to the private sector. He even goes so far as to declare that his heating is free, before correcting himself: "Well, you can say it's free. Well, we do pay… When I say free, I mean it's covered by the monthly charges". This lack of attention is reflected in an inability to distinguish the heating bill from amongst the other monthly charges. This 'invisibility' of heating costs is in stark contrast to the attention paid to the electricity bill, which is paid separately: "With the electricity, the lights all have low-energy bulbs… […] We try to run the washing machine at off-peak times, in the evening. It depends, sometimes… I turn off the immersion heater. Because it's electric. In the evenings I turn off the modem, anything to do with the computer, etc. These are just little things, you know. To bring the bill down a bit".

For insulation improvements, tenants have to refer to their landlord. What counts the most in the choice of privately rented accommodation is the type of heating system installed. Concern for the quality of construction is only shown by the wealthiest individuals, along with those who own a car and are thus able to live outside the town centre. This distinction based on access (or not) to a car, whether out of choice or out of inability to afford one, is a determining factor in these accommodation trajectories.

What matters for young people is not so much proximity to their place of work as general accessibility. The markedly unstable character of many career paths makes being located at the centre of a network of transport links more important in their choice of location than enjoying easy access to the job they happen to be in when they are looking for accommodation. For couples living in council/government housing, public transport links were also a major consideration, as those interviewed only had access to one car per household. Therefore, even if only one member of the couple commutes by car, access to public transport remains a decisive factor in choosing where to live. Overall, there was a low level of car ownership among employees living in the town centre, and even when they did have access to a vehicle they tended to use public transport to get to work While the next step along this trajectory is generally the purchase of an apartment or house, a return to renting may occur in cases of divorce or separation. The need to start living apart rapidly, not to mention cheaply, becomes a priority. Shared custody of children imposes new constraints on location which take priority over all other considerations. Habitational experiences are thus not linear and do not necessarily determine future behaviour. For most people, however, they are a decisive factor.

Buying a property, in particular a house, thus constitutes the third stage in these accommodation trajectories. Several factors are involved concurrently in the choice of location, including the budget available, car ownership in the household, networks of family and friends, the jobs held by the couple and the proportion of income spent on transport at the time of purchase. Houses are bought on the basis of more or less accurately estimated costs, maintenance and transport costs alike. The choice of heating system and the amount of attention paid to insulation depend to a large extent on previous experiences. Except for those cases when one of the members of the couple has acquired relevant technical knowledge through specialised training, decisions are based on negative past experiences and the information available to the general public. The thermal efficiency of the property and the performance of the heating system depend in these cases on the quality of information available or, for self-builds, on current regulations.

Since people move home less frequently once they have bought a property, budgeting for transport costs, or under-estimating these, can put a long-term strain on finances. A shift in behaviour can be seen to correlate with the date of purchase, namely that the families living furthest from their places of work bought their properties in the 2000s or earlier. This acceptance of travelling large distances was not found in the survey on households' current criteria for buying a property. Rather, those households unable to access housing in their chosen location were unwillingly to buy owing to the recent and apparently permanent rise in the cost of petrol.

The experience related by Adriana (33, administrator, married, 1 child) is typical in that she has had to make various concessions with respect to the distance travelled to work over the course of her accommodation trajectory. She has been working for the same company, currently located 45 km (29 miles) from her home, for the last 15 years. "[When I started work there] I lived 4 km [2.5 miles] away. Then I moved to a new place 10 km [6 miles] away. Then the company moved offices. I was 20 minutes away by car. And then after that I had to buy a place and I had an opportunity to get somewhere cheaper and at the time that added an extra quarter of an hour, you know, a good quarter of an hour." Although Adriana begins by talking in terms of kilometres, it is telling that as the distances increase she begins to measure them in time spent driving, as if this were more acceptable than the number of kilometres covered. Since the traffic on her journey to Pusignan is becoming heavier and heavier, it now takes her 50 minutes to get there.

Company relocations, an element over which couples have no control, retrospectively re-shuffle the cards as far as the latter's choice of where to live is concerned. Catherine (40, married, 2 children) has been working as a transit agent in a logistics firm since 1997. When she started, she was living with her partner in a two-bedroom apartment in Feyzin, southern Lyon. In 2001, shortly after the birth of their first child, the couple had a house built in the city's eastern suburbs, in Saint-Just-Chaleyssin, around 30 km (20 miles) from her place of work at that time (Genas, south-east of Lyon). Shortly afterwards, Catherine's firm relocated to a site a further five kilometres (3 miles) away, in Pusignan. At the time, her partner was working in Saint-Quentin-Fallavier, less than 15 km (9 miles) from their house; he later changed company and began working in Corbas, 20 km (12 miles) from Saint-Just-Chaleyssin. Successive adjustments of this kind are typical of these constantly evolving trajectories. Economic instability is thus combined with professional instability, exacerbating increases in transport costs.

Mobility

Like the routines governing energy use in the home, habits of mobility are learnt in the context of the family [START_REF] Vincent-Geslin | Altermobilités, mode d'emploi. Déterminants et usages de mobilités alternatives au tout voiture[END_REF]. For most of our interviewees, even the youngest, getting their driving licence and then a car are part of a process from which it is difficult to escape. Cars are consumer items which, as they wear out, are replaced by older generations, who then pass on any surplus vehicles to younger relatives. This gift of a first car is often what causes driving habits to develop on a family-based model [START_REF] Rocci | De l'automobilité à la multimodalité ? Analyse sociologique des freins et leviers au changement de comportements vers une réduction de l'usage de la voiture[END_REF]. Our study shows that the location of the place of work has a powerful impact on the mode of transport chosen by employees, even among those who had consciously decided that they did not want to drive. The amount of time spent on buses ends up overcoming the willpower of most employees. Julien (31, manager, living with a partner) would have preferred not to have a car. When he first came to Lyon, he found a job on the city's outskirts, on a site not served by public transport. At first, he tried to travel to work using environmentally-friendly methods: "I had to take the metro [underground], then the bus and after that, because the place where I was working didn't have a stop, I had to resort to using a kick scooter, so I bought myself a scooter; in the depths of winter, 20 minutes on a scooter! Morning and evening. In the evening… that meant I was spending two and a half, three hours getting to work each day. That's quite a long time." Very quickly, he decided to buy a small car, with good fuel economy, a Renault 'Twingo Orange'. Julien justifies his purchase by his desire to respect his ecological principles as far as possible.

The other major trigger for the purchase of a car is the birth of children. Once bought, the car is used by the member of the couple with the longest commute. As Boudjemaa (37, telephone operator, married, 2 children) relates: "The twins were born premature. One time it happened that they were both ill and we didn't have a car and so we said to ourselves: "and what if they got ill in the evening…?" The amazing thing was, as soon as we got the car, a week later, they got ill! And that's when I realised that you can't do without it, you really can't do without it. No way. When you're living on your own, well… you can get by, you can muddle through." Having a car is reassuring, and it gives the owner the ability to help others. Owning a car justifies using it for other purposes, such as travelling between home and work, a journey which Boudjemaa had previously tolerated despite the considerable difficulties it presented: "I had to take the tram, the metro, line A and line D, then the bus… you just had to have a problem on one line… and your whole journey would be messed up… It meant that if I was starting work at 8.00, the bus would be there at 7.30 sharp. It would leave at 7.30. That meant that if I turned up at 7.31, I'd have to wait until 8.00. So there's no… all it took was some technical problem on the metro. That happened a lot, metro, technical problem, five minutes late, that's it! I'm not five minutes late, but half an hour late…" A car, then, may be used for purposes other than that for which it was originally purchased. The demonstrable amount of time saved on the journey to work and the ease of day-to-day driving in peri-urban zones are both elements which encourage the daily use of a vehicle.

Two major groups are identifiable among public transport users according to their transport practice and their socio-professional status, broadly reflecting the split between those who have access to a car and those who do not.

The mode of practice exhibited by those whose journey is direct and do not live far from a bus route may be characterised as 'unproblematic'. They have completed at least two years of study after the baccalauréat (A-Level), are for the most part single and are able to live where they choose. They take the bus even when they own a car, as much for ecological reasons as for reasons of economy and convenience. This practice is the opposite of what we will term the 'ordeal by bus' experienced by Julien and Boudjemaa. The latter are employees on short-term contracts living in those urban districts that are least well served by public transport. This group works non-standard hours early in the morning and late in the evening, as well as at weekends, outside normal service times. Most of them also work shifts, which reduces their ability to adapt their routine. While the former group take an average of three quarters of an hour to get to their place of work, the average for these employees is more like an hour and a half. This 'ordeal by bus' thus equates to a doubling of journey times in comparison to how long they would take by car, coupled with a likely lack of punctuality owing to the many changes required which has further repercussions on their work.

Car-sharing is one possible solution, in particular for those who work shifts. The call centre based in an industrial estate north of Lyon is, out of the companies surveyed, the one which has best implemented a system of car-sharing in order to make up for the shortcomings of the public transport service. The company works day and evening shifts. The employees on fixed-term contracts are young, are either still studying or are recent graduates, and are single without children. The car-share system is also open to permanent staff who live a long way from the site, and is presented to potential recruits at their job interviews as a solution to the problem of getting to and from the site outside normal hours of service. However, the system is based on employees 'sorting things out' between themselves, as the company does nothing to organise and facilitate this. Those car owners who live in the city centre are for the most part former public transport users who are well aware of the difficulties involved and are thus happy to offer their services. Nevertheless, car-sharing to and from the city centre is a non-reciprocal service which leads to an imbalanced exchange. The situation makes passengers feel awkward on account of its unilateral character. While the solidarity among employees that the employers rely upon does exist, the situation is nonetheless a rather delicate one for staff who do not own cars, as the system is based on 'muddling through' and goodwill, and there are no clearly defined rules regarding remuneration. Journeys to and from work are also subject to various work-and family-related needs and constraints, making car-sharing on a daily basis difficult when the individuals involved do not work the same hours or do not even know which hours they will be working. Car-sharing means having to adapt to the needs of others and, if one of the parties has children, being able to rely on the availability of a partner. It also relies on companies being ready to play their part by ensuring that employees' working hours coincide wherever possible.

Adapting to rising costs

The survey was conducted in 2012, following several years of consecutive rises in energy prices across the board. Our aim was to establish how households were dealing with this, what changes were occurring in their routines, and what sort of trade-offs were being made between savings achievable within the home and savings on transport costs. The first adjustment made by all these households within the family home was to turn the thermostat down in all or some of the rooms. All homeowners, regardless of their social status and income, as well as when their house was built, had lowered the average temperature in their homes. It is rare that a desire to save money is the only reason given to justify this reduction. Heating in bedrooms may be turned off for the sake of comfort and healthy living, while the thermostat is turned down in the daytime so as not to waste energy. In all these cases, this change in behaviour had occurred over the five years preceding the interview, which is to say since the major rise in fuel prices in 2008. Turning down the thermostat has an immediate effect on the heating bill and does not require making any investment. Its downside is a reduction in comfort and an increased dependence on the vagaries of the weather.

After turning down the thermostat, the second step is to look for alternative, cheaper heating solutions. For those living in houses, this consists in installing a wood-burning stove, the purchase of which had either been made or was being planned for when the interview was conducted. Improving the building's insulation was rarer because of the cost involved. While installing double glazing was considered desirable by most of the homeowners who had not already invested in it, insulating the loft and walls remained less of a priority and only interested those who had the financial means required. It tends to be carried out either when the work is covered by a grant, or at key stages in the individual's life-cycle, such as retirement.

For most homeowners, adapting to the rise in costs does not reflect a pre-planned strategy. Homeowners react on an ad hoc basis as needs and problems arise and force decisions to be taken, or as a reaction to rising costs, with no really concerted attempt to optimise efficiency. Their decisions have a rather improvisatory character, and as a result they come to the conclusion that, despite all the efforts and investments they have made, the rise in costs canot be reversed. Hubert (46, manager, 2 children): "We changed the windows so we "shed" so to speak half a tank [of fuel oil] a year. But what we saved relatively quickly, well it was… It got eaten up as the litre went up, you know. The price per litre". This whole set of behaviours does little to change established habits and does not demand any major changes in the same way that, for example, changes in habits related to transport might do.

While the costs of commuting tend to be calculated very precisely, indicating greater awareness and concern in the face of rising prices, for the moment this has had relatively little impact on individual habits and lifestyle choices. One indication of change and of increasing tension with respect to energy costs, however, is that employees are actively involved in car-sharing for leisure activities, especially where transporting children is concerned, an area where this practice is preferred whenever possible. Strategies have also been implemented in order to bring down the costs of journeys. When looking for a new car, most people now choose a vehicle with lower fuel consumption. Those who do not do so generally lack the financial means to invest in an energy-efficient vehicle. Toll motorways are avoided as far as possible and average speeds reduced. Finally, whereas the commute from home to work has so far not been seriously affected by the rise in energy costs, households say that are paying more attention to expenditure in other areas, such as leisure activities and food.

Conclusion

Adaptation to the rise in energy costs is largely being carried out by trial and error on an ad hoc, dayto-day basis. It relies on the application of old-fashioned family values and routines acquired during childhood, along with knowledge gleaned from the media, colleagues and friends. This often patchy knowledge base is called upon as opportunities and/or tensions arise surrounding the family budget, although for the time being this does not involve housing choices being called into question, whether in a peri-urban or urban context. In the report they produced for the CREDOC, B. Maresca et al. [START_REF] Maresca | La consommation d'énergie dans l'habitat entre recherche de confort et impératif écologique[END_REF] have noted, on the basis of surveys conducted before and after the first oil crisis in 1973, that whereas before the crisis interviewees said that they would be prepared to invest in order to lower their energy consumption, in reality this translated into 63% of interviewees turning down the heating in the homes, a short-term response to an abrupt price change that was reversed as soon as the oil price went down again. The situation today, however, is different in that a range of indicators are leading even the least well-informed households to conclude that energy prices are not going to drop significantly.

The as yet limited development of shuttle bus services for commuting workers is evidence of the difficulties involved in changing these practices, which is not to say that this area of expenditure is not under pressure, as shown by the savings sought in other sectors of family budgets, nor that employees would not be prepared to modify their behaviour, and in particular to car-share, if real opportunities for this were opened up, such as through the reorganisation of working hours within companies or the improvement of on-site amenities.

the TransEnergy research project, financed by the Agence Nationale de la Recherche as part of the Villes Durables (Sustainable Cities