Etienne Frumence

Sandrine Genetet

Pierre Ripoche

Achille Iolascon

Immacolata Andolfo

Caroline Le Van Kim

Yves Colin

Isabelle Mouro-Chanteloup

Claude Lopez
email: claude.lopez@inserm.fr

Rapid Cl -/HCO 3 -exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells

Keywords: Anion exchanger 1, hereditary stomatocytosis, stopped-flow spectrofluorometry, HEK293, red blood cells

Anion Exchanger 1 or Band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis associated mutations (R730C, E758K and G796R), allowed the validation of our method.

Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared to WT AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared to WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1, and conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.

INTRODUCTION

Carbon dioxide (CO 2 ) is a by-product of oxidative metabolism. In human blood, CO 2 is carried through the venous system in three different ways. 5-10% is dissolved in the plasma, 5-10% is bound to hemoglobin and the remaining is converted to bicarbonate (HCO 3 -) ions by the red blood cells (RBCs). In all tissues except lung, CO 2 diffuses into RBCs where it is rapidly hydrated by carbonic anhydrase II (CAII) to form HCO 3 -and hydrogen ion (H + ) [START_REF] Reithmeier | A membrane metabolon linking carbonic anhydrase with chloride/bicarbonate anion exchangers[END_REF]. HCO 3 -is very weakly lipid impermeant and is transported into the plasma in exchange for extracellular chloride (Cl -) by the Anion Exchanger 1 (AE1, also known as Band 3), increasing the blood capacity to carry CO 2 . This process called the "Chloride shift" lowers the intracellular pH (pHi), which causes a conformational change in the hemoglobin and facilitates the release of oxygen (Bohr Effect) [START_REF] Jensen | Red blood cell pH, the Bohr effect, and other oxygenation-linked phenomena in blood O2 and CO2 transport[END_REF]. In the lung this process is reversed.

Plasma HCO 3 -is transported into RBCs in exchange for intracellular Cl -via AE1. Then HCO 3 -is dehydrated to CO 2 by CAII and diffuses out of RBCs to be expired by the lung. This process increases pHi (Fig. 1). AE1 belongs to the Solute Carrier 4A (SLC4A) family, is responsible for the Diego blood group system [START_REF] Alper | Molecular physiology of SLC4 anion exchangers[END_REF] and is the most abundant human RBC membrane protein (10 6 copies per cell) [START_REF] Steck | The band 3 protein of the human red cell membrane: a review[END_REF]. It binds CAII to form the "CO 2 metabolon" and is tightly associated with the chaperone-like protein glycophorin A (GPA) [START_REF] Van Den Akker | Band 3 multiprotein complexes in the red cell membrane; of mice and men[END_REF].

AE1 has been the topic of extensive experimental transport studies, particularly in the context of transport kinetics [START_REF] Knauf | In: Membrane transport and renal physiology[END_REF]. It mediates the exchange of a wide range of monovalent (nitrate, oxalate, iodide, thiocyanate) or divalent (sulfate, phosphate) anions, but chloride and bicarbonate are the physiological substrates. This Cl -/HCO 3 -exchange across the plasma membrane occurs at the fast rate of 5x10 4 ions per second at 37°C [START_REF] Jennings | Structure and function of the red blood cell anion transport protein[END_REF] and therefore cannot be measured easily. However, up to now most measurements on RBCs involved long-term (minutes to hours) influx or efflux of a radioactive anion by AE1 like chloride, bicarbonate, sulfate or phosphate [START_REF] Bruce | Monovalent cation leaks in human red cells caused by single amino-acid substitutions in the transport domain of the band 3 chloride-bicarbonate exchanger, AE1[END_REF], and were not adapted to rapid kinetics. Consequently, they provided data mixing both anion transport and metabolism, but no information corresponding to the physiological conditions of rapid electroneutral Cl -/HCO 3 -exchange. AE1 activity has also been studied in HEK293 cell lines expressing transiently the recombinant protein, because it is known to be unstable in long-term expression systems [START_REF] Cordat | Dominant and recessive distal renal tubular acidosis mutations of kidney anion exchanger 1 induce distinct trafficking defects in MDCK cells[END_REF][START_REF] Toye | Regions of human kidney anion exchanger 1 (kAE1) required for basolateral targeting of kAE1 in polarised kidney cells: mis-targeting explains dominant renal tubular acidosis (dRTA)[END_REF]. In both chloride movement and pH change measurements, experiments lasted over several minutes, far from rapid kinetics [START_REF] Morgan | Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl-/HCO3-exchanger, AE1[END_REF][START_REF] Sowah | An intramolecular transport metabolon: fusion of carbonic anhydrase II to the COOH terminus of the Cl(-)/HCO(3)(-)exchanger, AE1[END_REF].

Many different mutations of human AE1 have been described. They can either be asymptomatic or can lead to hereditary hemolytic red cell diseases (spherocytosis or stomatocytosis) [START_REF] Delaunay | The molecular basis of hereditary red cell membrane disorders[END_REF], or hereditary distal renal tubular acidosis [START_REF] Alper | Molecular physiology and genetics of Na+-independent SLC4 anion exchangers[END_REF]. Recently nine point mutations in the SLC4A1 gene (L687P, D705Y, R730C, S731P, H734R, E758K, R760Q, S762R and G796R substitutions in the protein) have been reported [START_REF] Barneaud-Rocca | Band 3 missense mutations and stomatocytosis: insight into the molecular mechanism responsible for monovalent cation leak[END_REF][START_REF] Bruce | Monovalent cation leaks in human red cells caused by single amino-acid substitutions in the transport domain of the band 3 chloride-bicarbonate exchanger, AE1[END_REF][START_REF] Guizouarn | South-east Asian ovalocytosis and the cryohydrocytosis form of hereditary stomatocytosis show virtually indistinguishable cation permeability defects[END_REF][START_REF] Guizouarn | Point mutations involved in red cell stomatocytosis convert the electroneutral anion exchanger 1 to a nonselective cation conductance[END_REF][START_REF] Iolascon | A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis[END_REF][START_REF] Stewart | Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis[END_REF][START_REF] Timmer | Inducible expression of erythrocyte band 3 protein[END_REF].

All these substitutions induce monovalent cation leaks in RBCs and are responsible for hereditary stomatocytosis (HSt) associated with hemolytic anemia [START_REF] Bruce | Hereditary stomatocytosis and cation-leaky red cells--recent developments[END_REF]. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na + and K + fluxes. Intracellular pH measurements using microelectrodes, 36 Cl -influx and 35 SO 4 2-efflux in oocytes, showed that HSt mutant AE1 proteins were no more functional, except for E758K and R760Q

mutants that kept anion exchange activity, although cation leaks were also detected in these two variants.

In the present report, we stably expressed wild type (WT) recombinant AE1 in HEK293 cells, using a tetracycline-inducible expression system to circumvent the loss of long-term expression of the protein. Three HSt mutant AE1 were also expressed, E758K (keeping anion exchange activity), R730C and G796R (inactive), the latter being also available as RBCs from two related patients. We then set up a stopped-flow spectrofluorometry assay to investigate the rapid Cl -/HCO 3 -exchange kinetics of normal and HSt mutant AE1. This assay is based on the detection of variations of pHi resulting from HCO 3 -influx by a fluorescent pH-sensitive dye, in the presence of HCO 3 -and Cl -gradients. We measured this activity in HEK293 cells expressing the recombinant proteins, and also in RBC ghosts from normal individuals and HSt patients carrying the G796R mutation at the heterozygous state. Moreover, we analyzed by flow cytometry the surface expression of WT and mutant AE1 and correlated it to Cl - /HCO 3 -exchange, allowing a reliable comparison of activities of the proteins on transfected HEK293 cells and RBCs. This study provides new insights into AE1 function in terms of kinetics, use of its natural substrate, and determination of apparent unit permeabilities for Cl - /HCO 3 -exchange.

MATERIALS AND METHODS

Reagents. pcDNA4/TO and pcDNA6/TR (T-REx system) were purchased from Invitrogen Diameter measurements of HEK293 cells. HEK293 cell diameter was measured using a CASY TTC cell counter from Roche on an average of 1,000 cells. Four measurements were performed for each cell line.

RBC ghost preparations and diameter measurements. Preparation of RBC ghosts from normal individuals and HSt patients was carried out as previously reported [START_REF] Genetet | Human RhAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells[END_REF]. Briefly 200 µl of thawed blood were washed three times in PBS and resuspended in hypotonic lysis buffer (3.5 mM K 2 SO 4 and 10 mM Hepes/KOH pH 7.2) for 40 min at 4°C, followed by resealing for 1 h at 37°C in resealing buffer (100 mM KCl, 10 mM Hepes/KOH pH 7.2, 1 mM MgCl 2 and 2 mg/ml bovine CA) containing 0.15 mM pyranine, a fluorescent pH-sensitive dye, and then washed three times in incubation buffer (100 mM KCl, 10 mM Hepes /KOH pH7.2). In some experiments resealing buffer contained various amounts of bovine CA which are detailed in Table 3. Ghost diameter measurements were performed on 300 ghosts per individual using a Zeiss Axio Observer Z1 microscope (x 1,000) equipped with an AxioCam MRm camera. For Cl -/HCO 3 -exchange analysis in RBCs, ghosts were resuspended in 3 ml of chloride buffer (100 mM KCl, 10 mM Hepes /KOH pH7.2). Then they were rapidly mixed with an equal volume of bicarbonate buffer (100 mM KHCO 3 , 10 mM Hepes/HCl pH 7.2), generating inwardly directed 50 meq HCO 3 -/CO 2 and outwardly directed 50 meq Cl -gradients. The pH-dependent fluorescence changes of pyranine were monitored at a 465 nm excitation wavelength and the emitted light was filtered with a 520 nm cut-off filter.

Stopped-flow analysis of Cl

RESULTS

Stable expression of recombinant WT and HSt mutant AE1 in HEK293 cell transfectants.

HEK293 cells, although of renal origin, were previously shown to lack an endogenous expression of kAE1 ( 8) but not of CAII [START_REF] Sterling | A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers[END_REF]. As it has been shown that the generation of cell lines that constitutively overeproduce AE1 leads to cell death (30), we established permanent HEK293 cell lines with inducible expression of human AE1. HEK293 cells were primarily stably transfected with the pcDNA6/TR vector encoding the Tet repressor and then with the pcDNA4/TO vector bearing either WT AE1 cDNA or the three HSt (R730C, E758K and G796R) mutant cDNAs constructed by site-directed mutagenesis. Before analysis of HEK293 cell transfectants, AE1 production was induced with tetracycline. Flow cytometry experiments revealed AE1 at the surface of transfected cells (Fig. 2, A and B). More than 97% of the WT AE1 transfected cells expressed the protein after three weeks of zeocin selection.

An apparent surface site number of about 10 5 molecules per cell was calculated from the binding capacity of the BRIC6 monoclonal antibody, using Qifikit calibration beads. As expected, no labeling could be detected on parental HEK293 cells. Mutant AE1 proteins were significantly expressed at the cell surface but at a lower level than WT AE1 (R730C, G796R, and E758K; 66%, 40% and 15% of WT molecules, respectively). Their comparison using a one-way ANOVA analysis followed by the Dunnett's multiple comparison test from GraphPad Prism software revealed statistical significance (WT AE1/R730C, P < 0.05; WT AE1/G796R and WT AE1/E758K: P < 0.01). By immunofluorescence confocal microscopy on intact and permeabilized transfected HEK293 cells (Fig. 2C, left and right, respectively), WT, R730C, G796R, and E758K AE1 expression was localized at the plasma membrane and also in intracellular compartments, especially for E758K mutant. These results show that WT AE1, and R730C, G796R and E758K mutants can traffic to and be stably expressed at the plasma membrane of HEK293 cells, with however a significant internal retention of the protein.

Cl -/HCO 3 -exchange in WT and HSt mutant AE1 transfected HEK293 cells. The Cl -/HCO 3 exchange capacity of human AE1 proteins, stably expressed in HEK293 cells, was investigated by stopped-flow spectrofluorometry using BCECF as an intracellular pH-sensitive probe, in the presence of inwardly directed 10 meq HCO 3 -/CO 2 and outwardly directed 67.5 meq Cl -gradients at 30°C. This temperature (instead of the more physiological 37°C) was used to standardize experimental conditions in this study, because in RBC ghosts, kinetics at 37°C are too rapid (less than 1 s) to precisely measure Cl -/HCO 3 -exchange (see below). Fig. 3A indicates the time-course of the fluorescence increase after submission of WT AE1 expressing cells to the gradients at pH 7.2. These fast kinetics allowed the calculation of alkalinization rate constants k, which correspond to the constants of the mono-exponential functions that fit to the experimental curves (Table 1). The fluorescence variation for WT AE1 cells exhibited a rapid increment corresponding to intracellular alkalinization, whereas almost no increase of fluorescence was observed in parental HEK293 cells, lacking AE1 expression (k were 0.38 ± 0.04 s -1 versus 0.03 ± 0.02 s -1 , respectively). Alkalinization in WT AE1 cells reached a plateau within about 10 s. After incubation of WT AE1 transfected cells with the anion exchanger inhibitor DIDS, Cl -/HCO 3 -exchange was reduced at the parental HEK293 cell level (k = 0.03 ± 0.02 s -1 ). Transport recorded in absence of DIDS is therefore totally specific of the AE1 protein. The Cl -/HCO 3 -exchange capacity of recombinant cells expressing the HSt E758K and G796R AE1 mutants was then investigated (Fig. 3B). The alkalinization process was five to six times faster for AE1 WT (k = 0.38 ± 0.04 s -1 ) compared to E758K (k = 0.07 ± 0.03 s -1 ) and similar to parental HEK293 cells for G796R (k = 0.02 ± 0.01 s -1 ) and R730C (k = 0.01 ± 0.01 s -1 ) (Table 1). However, since flow cytometry analysis revealed heterogeneous expression levels of the AE1 mutants, it was essential to correlate these alkalinization rate constants to the number of molecules at the surface of HEK293 cell transfectants (Fig. 3C). This experiment clearly showed that the E758K mutant exhibited the same relative transport activity as WT AE1 (98% versus 100%), whereas R730C and G796R

were inactive. DIDS treatment of E758K mutant also completely inhibited its Cl -/HCO 3 exchange capacity (k = 0.01 ± 0.01 s -1 ), indicating the specificity of the AE1 transport activity. Determination of the number of recombinant molecules per cell was used to calculate the apparent unit permeabilities of AE1 for Cl -/HCO 3 -at 30°C, which were equivalent for WT AE1 and E758K mutant (9.81 x 10 -3 m 3 /s and 11.96 x 10 -3 m 3 /s, respectively; Table 2), thus confirming that E758K mutant was fully active.

Comparison of AE1 protein expression from control and HSt G796R mutant erythrocytes. In order to assess AE1 transport activity in RBCs from natural variants, we used blood from two normal individuals and from the two HSt patients carrying the G796R mutation in the AE1 protein, previously described [START_REF] Iolascon | A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis[END_REF]. Evaluation by flow cytometry, using the BRIC6 antibody, of AE1 amount in RBC membrane did not show significant differences between control and HSt patients (1.5 x 10 5 molecules per RBC), indicating neither traffic deficiency nor defective AE1 protein in G796R red cells (Fig. 4). AE1 is known to be the most abundant integral membrane protein with 10 6 copies per normal RBC; the much lower number of molecules from our result is probably attributable to steric hindrance of the BRIC6 antibody on RBCs which might prevent access to all available epitopes, as previously described [START_REF] Smythe | Monoclonal antibodies recognizing epitopes on the extracellular face and intracellular Nterminus of the human erythrocyte anion transporter (band 3) and their application to the analysis of South East Asian ovalocytes[END_REF]. As GPA is tightly associated with AE1, we used an antibody against the GPA/AE1 epitope, Wr b , to assess their co-expression in both normal and HSt patients. G796R mutant RBCs exhibited roughly the same number of molecules as control RBCs (2.8 x 10 5 versus 2.5 x 10 5 copies per cell) (Fig. 4).

Cl -/HCO 3 -exchange in WT and G796R AE1 RBC ghosts. To measure the Cl -/HCO 3 -exchange kinetics on RBCs, we used ghosts resealed in the presence of pyranine, a fluorescent pHsensitive dye, and submitted to inwardly directed 50 meq HCO 3 -/CO 2 and outwardly directed 50 meq Cl -gradients at 30°C. As mentioned above, experiments were not performed at 37°C, because anion exchange lasts much less than 1 s at this temperature, precluding an accurate determination of alkalinization rate constants. RBCs contain high levels of cytosolic carbonic anhydrase I (CAI) that is removed during the ghost preparation. They also harbor 10 6 copies of CAII directly bound to the carboxyl-terminal tail of membrane AE1 [START_REF] Reithmeier | A membrane metabolon linking carbonic anhydrase with chloride/bicarbonate anion exchangers[END_REF]. This interaction is weak and pH dependent, CAII therefore can be readily removed during membrane ghost preparation, precluding any control of the CAII left amount. Indeed, our first stopped-flow experiments generated great variations of alkalinization rate constants from one ghost preparation to the other, for a same individual (not shown). WT RBC ghosts were therefore resealed in the presence of varying quantities of bovine CA and tested for Cl -/HCO 3 exchange by stopped-flow analysis (Table 3). CA clearly appeared to be rate-limiting to AE1 transport activity measurement since the highest alkalinization rate constant was recorded with ghosts resealed in the presence of 2 mg/ml CA (k = 4.87 ± 0.56 s -1 ). pHi variations inside ghosts were only dependent on transmembrane Cl -/HCO 3 -exchange at this CA concentration, all the following experiments were therefore performed under this condition. A preliminary optimization of stopped-flow methodology on RBC ghosts showed that modifications of either HCO 3 -or Cl -gradients did not significantly alter alkalinization rate constants, whereas the amplitude of alkalinization was directly proportional with the Cl -gradient only (not shown). Consequently, although Cl -/HCO 3 -exchange is measured indirectly by pHi variations generated by HCO 3 -influx, Cl -efflux actually reflects AE1 activity. Analysis of the HSt variant activity (Fig. 5A and Table 4) indicated that the alkalinization process was two to three times faster in WT ghosts than in ghosts carrying the G796R mutation (k were 4.87 ± 0.56 s -1 versus 1.86 ± 0.17 s -1 ) and lasted only 1s and 2 to 3 s, respectively. Since WT AE1 and G796R AE1 were expressed at the same level, correction of activity for surface expression of the protein was not necessary. Recorded k constants thus directly indicate around 60% reduction of activity of G796R ghosts compared to WT ghosts (Fig. 5B). Apparent unit permeability (p' unit ) for Cl -/HCO 3 -was 1.7 fold lower for G796R AE1 compared to WT AE1

(2.04 x 10 -3 m 3 /s versus 3.49 x 10 -3 m 3 /s) (Table 2). Of note, diameter of G796R ghosts was slightly larger than that of WT ghosts (6.8 ± 0.50 m versus 5.9 ± 0.35 m, n = 600), with statistical difference (t test, P < 0.0001) and explained the smaller difference of p' unit than of constant k between WT and mutant ghosts. This result was not surprising since macrocytosis was observed on RBCs from both patients harboring the G796R mutation [START_REF] Iolascon | A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis[END_REF].

To ascertain that these results were directly correlated to AE1 exchange, WT and HSt ghosts were incubated with 20 µM DIDS. Cl -/HCO 3 -exchange was abolished in both WT and HSt ghosts (k = 0.02 ± 0.01 s -1 versus 0.03 ± 0.01 s -1 ) (Fig. 5 and Table 4). Similar results were obtained after incubation of WT ghosts with 100 µM dipyridamole (Dipy) (k = 0.02 s -1 ) or 100 µM DiBAC (k = 0.04 s -1 ), other known anion exchanger inhibitors (Table 4). Replacing

Cl -with sulfate, a poorly transported organic anion, also blocked exchange activity (not shown). After incubation with 100 µM acetazolamide (ATZ), a sulfonamide that inhibits CA enzymatic activity without direct effect on anion exchange, more than 90% of Cl -/HCO 3 exchange was inhibited (k = 0.33 s -1 ), confirming the crucial role of CA in our system.

DISCUSSION

AE1, associated with CAII, is responsible in erythrocytes for efficient transport of CO 2 , and its hydrated form, HCO 3 -, by exchange with Cl -. In the vast majority of AE1 mutations, there is a deficiency of this protein which causes hereditary spherocytosis, whereas if the missense mutation brings about a variant of AE1 that is inserted in the plasma membrane, the defect is associated with dominant HSt and hemolytic anemia. In the latter situation, affected individuals have an increase in RBC membrane permeability to cations and in most cases a reduction of anion movements through AE1. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na + and K + fluxes [START_REF] Barneaud-Rocca | Band 3 missense mutations and stomatocytosis: insight into the molecular mechanism responsible for monovalent cation leak[END_REF][START_REF] Bruce | Monovalent cation leaks in human red cells caused by single amino-acid substitutions in the transport domain of the band 3 chloride-bicarbonate exchanger, AE1[END_REF][START_REF] Guizouarn | South-east Asian ovalocytosis and the cryohydrocytosis form of hereditary stomatocytosis show virtually indistinguishable cation permeability defects[END_REF][START_REF] Guizouarn | Point mutations involved in red cell stomatocytosis convert the electroneutral anion exchanger 1 to a nonselective cation conductance[END_REF][START_REF] Iolascon | A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis[END_REF][START_REF] Stewart | Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis[END_REF][START_REF] Stewart | The GPA-dependent, spherostomatocytosis mutant AE1 E758K induces GPA-independent, endogenous cation transport in amphibian oocytes[END_REF]. These data were consistent with the concept that the substitutions convert the protein from an anion exchanger into an unregulated cation channel, although, so far, no study has clearly demonstrated that the cation leaks are mediated through AE1. In addition, HSt AE1 proteins exhibited no anion exchange activity, except for E758K and R760Q mutants.

We established permanent HEK293 cells with tetracycline-inducible expression of WT and

HSt mutant AE1, thereby avoiding the issue of loss of this protein in long-term expression systems. Human AE1 has already been stably expressed in HEK293 cells, using muristerone A induction on cells constitutively expressing ecdysone and retinoic acid receptors [START_REF] Timmer | Inducible expression of erythrocyte band 3 protein[END_REF].

However, AE1 expression level was only roughly estimated by immunoblotting and immunofluorescence, which precluded a precise correlation of activity (measured by 36 Cl - efflux and 35 SO 4 2-influx) with protein expression level and a quantitative comparison of these parameters between different samples. Stewart and colleagues reported that AE1 E758K mutant surface expression in amphibian oocytes was enhanced by co-expression of GPA ( 29), while membrane expression of R730C mutant was not [START_REF] Stewart | Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis[END_REF]. These results fit to our data in the HEK293 kidney cell line lacking erythroid proteins such as GPA, since E758K mutant was expressed in significant lesser amounts than R730C mutant (Newman-Keuls multiple comparison test, P < 0.05). Induced expression of AE1 in our system was nevertheless sufficient for exploring its transport activity without co-expression of exogenous GPA.

The Cl -/HCO 3 -exchange through AE1 across the plasma membrane is very rapid. We could observe by stopped-flow spectrofluorometry a rapid intracellular alkalinization corresponding specifically to this exchange in HEK293 expressing WT AE1, whereas no activity was detectable in parental cells. Our results also showed that E758K mutant was fully active, as previously reported in Xenopus oocytes [START_REF] Barneaud-Rocca | Band 3 missense mutations and stomatocytosis: insight into the molecular mechanism responsible for monovalent cation leak[END_REF][START_REF] Stewart | The GPA-dependent, spherostomatocytosis mutant AE1 E758K induces GPA-independent, endogenous cation transport in amphibian oocytes[END_REF]. Indeed, although alkalinization rate constant was five to six times lower for E758K mutant than for WT AE1 (0.07 s -1 versus 0.38 s -1 ), normalization of transport values with surface expression level indicated comparable relative activities (98% versus 100%) and apparent unit permeabilities (11.96 x 10 -3 m 3 /s versus 9.81

x 10 -3 m 3 /s) of both E758K mutant and normal proteins. The defect in Cl -/HCO 3 -transport activity of G796R and R730C mutants was not due to reduced expression or processing of the proteins to the cell surface and also corroborates their inactivity reported in Xenopus oocytes (4, 15, 28). The inducible system used is a valuable tool to control AE1 expression in HEK293 cells, and the heterogeneous amounts of the protein at cell surface from one sample to the other actually validated our method based on the comparison of activities.

We showed that stopped-flow methodology is also applicable to measure Cl -/HCO 3 -exchange in RBCs, using ghost preparations from blood of normal individuals and HSt patients carrying the G796R mutation in the AE1 protein. In erythrocytes there is 20-fold more CAII activity than anion exchange activity [START_REF] Sterling | A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers[END_REF], CAII is therefore not rate-limiting in physiological conditions. However, as we could not control the amount of CAII left after ghost preparation, it appeared that this enzyme was a limiting factor in our system. Thus we resealed the RBC ghosts in the presence of an excess of bovine CAII (2 mg/ml), in order to overcome this issue and allow an accurate measurement of Cl -/HCO 3 -exchange by AE1. HSt G796R ghosts exhibited a 60% reduction of activity, however calculation of apparent unit permeabilities for Cl -/HCO 3 -showed barely a 2-fold decrease for G796R AE1 compared to WT AE1 (2.04 x 10 - 3 m 3 /s versus 3.49 x 10 -3 m 3 /s). This discrepancy is explained by the higher diameter of mutant ghosts than that of WT ghosts, which raises the p' unit value of G796R protein. This result also strengthens our findings that an accurate comparison of activity between samples not only depends on alkalinization rate constant measurement, but also on the surface expression level of AE1 (as seen above for HEK293 cells), as well as the size of the cells or RBC ghosts harboring these proteins. An approximate 50% reduction of AE1 activity in G796R ghosts was expected since this mutation is present in the heterozygous state on HSt RBCs [START_REF] Iolascon | A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis[END_REF], and we showed here that G796R AE1 mutant protein is inactive in HEK293 transfectants, as previously reported using pHi measurements in oocytes [START_REF] Barneaud-Rocca | Band 3 missense mutations and stomatocytosis: insight into the molecular mechanism responsible for monovalent cation leak[END_REF][START_REF] Iolascon | A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis[END_REF]. Apparent permeability in WT ghosts for Cl -/HCO 3 -at 30°C (4.79 m/s) is close to permeability for Cl - measured at 38°C in RBCs (5 m/s) at the same pH 7.2 [START_REF] Brahm | Temperature-dependent changes of chloride transport kinetics in human red cells[END_REF]. Although HEK293 cells and RBC ghosts are not comparable, since RBCs contain neither a nucleus nor the different organelles found in all other mammalian cells, WT AE1 exhibited the same range of apparent unit permeability for Cl -/HCO 3 -in both cell systems (9.81 x 10 -3 m 3 /s and 3.49 x 10 -3 m 3 /s, respectively). Even though the number of AE1 molecules at plasma membrane is underestimated because of steric hindrance of BRIC6 antibody on RBCs [START_REF] Smythe | Monoclonal antibodies recognizing epitopes on the extracellular face and intracellular Nterminus of the human erythrocyte anion transporter (band 3) and their application to the analysis of South East Asian ovalocytes[END_REF] and most probably on HEK293 cells too, the calculated permeabilities indicate that AE1 activity measurements lead to reliable results in both cell systems. We found about two times more AE1 molecules on RBCs using Wr b antibody, it is thus required to always use the same monoclonal antibody to establish an accurate comparison of transport activities.

Taken together, the data reported here show that stopped-flow spectrofluorometry is a powerful technique to investigate the rapid transport kinetics of AE1 on both stably transfected HEK293 cells and RBC ghosts, using the natural substrate of the protein, Cl - /HCO 3 -. Moreover, conjugated with flow cytometry, this methodology allows for the first time a precise correlation of fast Cl -/HCO 3 -exchange to surface expression of AE1, and therefore a fine comparison of activity between different samples. It sheds new light on the anion transport features in red blood cells presenting pathological alterations associated with AE1 abnormalities. For example, Cl -/HCO 3 -exchange function can be potentially studied for any other AE1 mutant and in RBCs exhibiting pathologies such as AE1 clustering and/or modifications of tyrosine phosphorylation in sickle cell disease [START_REF] Corbett | Band 3 and glycophorin are progressively aggregated in density-fractionated sickle and normal red blood cells. Evidence from rotational and lateral mobility studies[END_REF][START_REF] Liu | Red cell membrane remodeling in sickle cell anemia. Sequestration of membrane lipids and proteins in Heinz bodies[END_REF], in G6PD deficiency [START_REF] Pantaleo | Irreversible AE1 tyrosine phosphorylation leads to membrane vesiculation in G6PD deficient red cells[END_REF] and senescent RBCs [START_REF] Antonelou | Aging and death signalling in mature red cells: from basic science to transfusion practice[END_REF]. Regulation of AE1 activity will also be assessed in stomatindeficient RBCs since a physical link between stomatin and AE1 has recently been demonstrated [START_REF] Rungaldier | Stomatin interacts with GLUT1/SLC2A1, band 3/SLC4A1, and aquaporin-1 in human erythrocyte membrane domains[END_REF]. These future investigations will hopefully lead to the development of a new diagnosis tool for several red cell pathologies by measurement of AE1 function. Data from three time courses were averaged and fitted to a mono-exponential function using the simplex procedure of Biokine software (Bio-logic), from which alkalinization rate constants (k, s -1 ) were calculated (see Table 1). C: transport activity in HEK293 cells expressing WT and mutant AE1 was represented as relative values of alkalinization rate constants correlated to the number of molecules at the surface of HEK293 cell transfectants and after subtraction of the non-transfected HEK293 cells constant. Values are means  SE. were calculated (see Table IV). The time-courses were similar for both WT ghosts and both G796R ghosts, respectively. B transport activity in WT AE1 and G796R AE1 ghosts was represented as relative values of alkalinization rate constants. Values are means  SE.

FIGURE CAPTIONS

  Glostrup, Denmark) according to the manufacturer's instructions. The results were expressed as specific antibody-binding-capacity units that proved to be directly proportional to the number of molecules bound per cell.Immunofluorescence confocal microscopy. As previously reported[START_REF] Zidi-Yahiaoui | Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells[END_REF], HEK293 transfectants were cultured on poly-L-lysine coverslips (BD Biosciences) for 2 days before immunostaining. Cells were fixed in 4% (w/v) paraformaldehyde in PBS for 20 min and washed in PBS. Free aldehyde groups were blocked in 50 mM NH 4 Cl/PBS for 10 min. Cells were then washed in PBS and permeabilized in 1% (w/v) SDS/PBS for 15 min or left untreated. After washes in PBS, non-permeabilized cells were incubated with mouse monoclonal (anti-AE1) BRIC6 (1/200), and permeabilized cells with rabbit polyclonal anti-AE1 (1/5,000) diluted in background immunostaining reducing buffer (Dako) for 1 h. Cells were washed three times in PBS/BSA (0.5%) and incubated for 1 h with Alexa Fluor 488 goat anti-mouse or donkey anti-rabbit IgG diluted in PBS/BSA (1/200). Samples were examined by wide-field microscopy using a Nikon Eclipse TE300 inverted confocal microscope equipped with a 60× oil-immersion objective, numerical aperture 1.4.

3 -/

 3 -/HCO 3 -exchange. For Cl -/HCO 3 -exchange analysis on HEK293 cells, 6 x 10 6 cells were washed twice in PBS and incubated for 30 min at 30°C in 1 ml of PBS containing 20 µM of the fluorescent pH sensitive probe BCECF-AM. Cells were washed twice in PBS, then equilibrated and resuspended in chloride buffer (130 mM NaCl, 5 mM KCl, 10 mM Hepes/KOH pH 7.2) at a concentration of 2 x 10 6 cells/ml. Cl -/HCO 3 -exchange was measured at 30°C using a stopped-flow instrument (SFM400, Bio-Logic, Grenoble, France). Cells were rapidly mixed with an equal volume of bicarbonate buffer (110 mM Nagluconate, 5 mM K-gluconate, 20 mM NaHCO 3 and 10 mM Hepes/KOH pH 7.2), generating inwardly directed 10 meq HCO CO 2 and outwardly directed 67.5 meq Cl -gradients. The pH-dependent fluorescence changes of BCECF were monitored at a 485 nm excitation wavelength and the emitted light was filtered with a 520 nm cut-off filter. Data from three to four time-courses were averaged and fitted to a mono-exponential function using the simplex procedure of the Biokine software package (Bio-Logic).

Fig. 1 .Fig. 2 .

 12 Fig. 1. The CO 2 transport in RBCs. In all tissues, carbon dioxide (CO 2 ) diffuses into RBCs where it is rapidly hydrated by carbonic anhydrase (CAII) to form bicarbonate (HCO 3 -) and hydrogen ion (H + ). HCO 3-is transported into the plasma in exchange for extracellular chloride (Cl -) by the Anion Exchanger 1 (AE1), increasing the blood capacity to carry CO 2 . In lungs, plasma HCO 3 -is transported into RBCs in exchange for intracellular Cl -via AE1. Then HCO 3 -is dehydrated to CO 2 by CAII and diffuses out of RBCs to be expired by the lung.

Fig. 3 . 3 -/

 33 Fig. 3. Time course of fluorescence changes in HEK293 cells expressing WT and HSt mutant AE1. Cells loaded with the fluorescent pH-sensitive probe BCECF-AM were rapidly mixed with an equal volume of buffer containing NaHCO 3 , generating inwardly directed 10 meq HCO 3 -/CO 2 and outwardly directed 67.5 meq Cl -gradients. pHi-dependent fluorescence changes were monitored at a 485 nm excitation wavelength and the emitted light was filtered with a 520 nm cut-off filter, using a stopped flow spectrofluorometer. Typical time courses of fluorescence changes in HEK293 expressing WT (A) or mutant AE1 (B) are represented. In A,

Fig. 4 .

 4 Fig. 4. Cell surface expression of AE1 in WT and HSt RBCs. Surface expression of AE1 by quantitative flow cytometry on two WT and two G796R RBCs, using monoclonal BRIC6 anti-AE1 and Wr b anti-GPA/AE1 association, and mouse IgG-coated calibration beads Qifikit as standards. All experiments were repeated at least three times to obtain mean values ( SE).

Fig. 5 . 3 -/

 53 Fig. 5. Time course of fluorescence changes in WT and HSt RBCs. RBC ghosts were prepared from two normal individuals and the two HSt G796R patients. Ghosts resealed in the presence of pyranine, a fluorescent pH-sensitive probe, and bovine CA at 2 mg/ml, were rapidly mixed with an equal volume of buffer containing KHCO 3 , generating inwardly directed 50 meq HCO 3 -/CO 2 and outwardly directed 50 meq Cl -gradients. pHi-dependent fluorescence changes were monitored at a 465 nm excitation wavelength and the emitted light was filtered with a 520 nm cut-off filter, using a stopped flow spectrofluorometer. A typical time courses of fluorescence changes in WT AE1 and G796R AE1 RBC ghosts incubated or not with the anion exchanger inhibitor DIDS (20 µM) for 15 min are represented. Data from three time courses were averaged and fitted to a mono-exponential function using the simplex procedure of Biokine software (Bio-logic), from which alkalinization rate constants (k, s -1 )

  salt] and dipyridamole were purchased from Sigma-Aldrich and DiBAC [bis(1,3-dibutylbarbituric acid) pentamethine oxonol] from Invitrogen. The mouse monoclonal antibodies anti-AE1 BRIC6 and Wr b against the GPA/AE1 epitope were from the

	transfected with the relevant pcDNA4/TO-AE1 expression vectors and selected in culture
	medium supplemented with 5 µM blasticidin and 25 µM zeocin (Invitrogen). Expression of
	WT or mutated AE1 was induced with tetracycline and pools of cells were sorted using a
	International Blood Group Reference Laboratory (IBGRL, Bristol, UK). The rabbit Fluorescence-activated cell sorter (FACS) Influx 500 (BD BioSciences, Bedford, MA) at the
	polyclonal antibody anti-AE1 was kindly provided by Dr. C. Wagner (University of Zürich, Institut Jacques Monod (Paris, France). Cells exhibiting the best AE1 expression were kept.
	Switzerland). Secondary antibodies used in flow cytometry analysis were goat anti-mouse Resistant cells were analyzed by flow cytometry for AE1 expression.
	FITC or PE-conjugated F(ab')2 fragments from Beckman Coulter (Villepinte, France). Blood samples. RBCs from two patients (mother and son) carry the G796R mutation in the Secondary antibodies used in confocal microscopy were Alexa Fluor 488 donkey anti-rabbit protein AE1 and were previously described (15). RBCs were collected in Naples (Italy) and or goat anti-mouse IgG from Invitrogen. sent to and kept frozen in the Centre National de Référence pour les Groupes Sanguins
	Construction of the AE1 expression vectors and site-directed mutagenesis. Full-length cDNA (CNRGS, Paris, France). Control RBCs were provided by the CNRGS. All samples were
	encoding erythroid AE1 was amplified by PCR from the plasmid pUC18-eAE1 with addition obtained after informed consent for the studies, according to the Declaration of Helsinki. The
	of a Kozak sequence at its 5'end, and subcloned in the pcDNA4/TO vector. Point mutations Institutional Review Board of the Federico II University from Naples approved the study.
	leading to R730C, E758K, and G796R substitutions were introduced in AE1 cDNA with the Flow cytometry analysis. Expression of AE1 on HEK293 cells or RBCs and GPA/AE1 QuikChange II XL site directed mutagenesis kit from Stratagene (La Jolla, CA) according to epitope on RBCs was detected using a FACSCanto II flow cytometer (BD BioSciences, the supplier's instructions. Wild-type (WT) and mutant AE1 constructs were sequenced by Bedford, MA), after staining with the primary mouse antibodies BRIC6 (1/40) and Wr b (1/8) the GATC company (Konstanz, Germany). (IGBRL, Bristol, UK), respectively, and secondary FITC or PE-conjugated goat anti-mouse
	Cell culture and transfection. Human embryonic kidney (HEK) 293 cells (American Type antibody (1/100) (Beckman Coulter, Villepinte, France). The cell-surface antigen expression
	Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's was quantified using as standards mouse IgG-coated calibration beads from Qifikit (Dako,
	medium/F12/Glutamax I (Invitrogen) supplemented with 10% (v/v) fetal calf serum
	tetracycline-free (Dutscher, Brumath, France), nonessential amino acids 1x (Invitrogen),
	antibiotic-antimycotic 1x (Invitrogen), 25 mM sodium bicarbonate (Invitrogen), and 25 mM
	Hepes (Invitrogen). T-REx (Invitrogen) is a Tet-regulated mammalian expression system
	based on the binding of tetracycline (Tet) to a Tet repressor and derepression of the promoter
	controlling the expression of the gene of interest. HEK293 cells were first transfected with the
	regulatory plasmid pcDNA6/TR expressing the Tet repressor using FuGene 6 transfection
	reagent (Roche, Meylan, France), selected in culture medium supplemented with 5 µM
	blasticidin (Invitrogen) and cloned by limiting dilution. Individual clones were transiently
	transfected in duplicate with the pcDNA4/TO-AE1 vector, and 24 h later one half was
	(Leek, The Netherlands). Primers used in PCR and mutagenesis experiments were from incubated for 16 h with 1 g/ml tetracycline (Invitrogen) (induced cells), the other half was
	MWG Biotech (Ebersberg, Germany). BCECF-AM [2',7'bis-(2-carboxyethyl)-5(6)-left untreated (uninduced cells). HEK293-TR clones were analyzed by flow cytometry (see
	carboxyfluorescein acetoxymethyl ester] and pyranine [8-hydroxypyrene-1,3,6-trisulfonic below) and one of them was selected, according to the best repression of AE1 expression in
	non induced cells and the best AE1 expression level in induced cells. HEK293-TR cells were

acid] fluorescent probes, and bovine carbonic anhydrase (CA) were obtained from Sigma-Aldrich (Seelze, Germany). AE1 inhibitors DIDS [4,4 2-Diisothiocyanatostilbene-2,2 2-disulfonic acid disodium

Table 1 .

 1 Cl -/HCO 3 -exchange in WT and HSt mutant SE.  n values in parentheses †10 M DIDS for 30 min.

	AE1transfected HEK293 cells
	_________________________________________
	Cell lines	Alkalinization rate constant
		k (s -1 )
	_________________________________________
	HEK293	0.03 ± 0.02 (5) 
	WT AE1	0.38 ± 0.04 (5)
	WT AE1 + DIDS †	0.03 ± 0.02 (3)
	E758K	0.07 ± 0.03 (3)
	E758K + DIDS †	0.01 ± 0.01 (3)
	G796R	0.02 ± 0.01 (3)
	R730C	0.01 ± 0.01 (3)
	_________________________________________
	Values are means ±

Table 2 .

 2 Apparent permeability of AE1 for Cl -/HCO 3 cells, around 1,000 cells measured per cell line. Diameter of ghosts, 300 ghosts measured per individual. V, volume; SA, surface area; N, number of AE1 copies at surface; k, alkalinization rate constant; P', apparent permeability for Cl -/HCO 3 -= k x V/SA, where k is the alkalinization rate constant, V is volume and SA surface area of cells or ghosts; p' unit , apparent unit permeability for Cl -/HCO 3 -at 30°C = P' x SA/N, where N is number of AE1 copies calculated using Qifikit in flow cytometry.  Values are means ± SE

			-
	___________________________________________________________________________
		HEK293	RBC ghosts
		WT AE1	E758K	WT AE1	G796R
		_______________________	________________________
	Diameter, m	17.0 ± 0.40 	16.9 ± 0.15	5.9 ± 0.35	6.8 ± 0.50
	V, m 3	2572.45	2527.32	107.54	164.64
	SA, m 2	907.92	897.27	109.36	145.27
	N	100,000	15,000	150,000	150,000
	k, s -1	0.38	0.07	4.87	1.86
	P', m/s	1.08	0.20	4.79	2.11
	p' unit , m 3 /s	9.81 E -03	11.96 E -03	3.49 E -03	2.04 E -03
	___________________________________________________________________________
	Diameter of HEK293

Table 3 .

 3 Cl -/HCO 3 -exchange in WT RBC ghosts in the presence of various amounts of CA _______________________________________ CA  (mg/ml) Alkalinization rate constant k (s -1 ) _______________________________________ ghosts resealed in the presence of bovine CA. †n values in parentheses.

	0	0.73 ± 0.50 (18) †
	0.1	2.58 ± 0.24 (3)
	0.5	4.32 ± 0.03 (3)
	1	4.58 ± 0.77 (3)
	2	4.87 ± 0.56 (6)
	3	4.68 ± 0.18 (4)
	5	4.42 ± 0.15 (3)
	_______________________________________
	Values are means ± SE.



Table 4 .

 4 Cl

	-/HCO 3	-exchange in WT and G796R AE1 RBC ghosts
	____________________________________________________
	RBC ghosts		Alkalinization rate constant
			k (s -1 )
	____________________________________________________
	WT AE1		4.87 ± 0.56  (6) †
	WT AE1 + DIDS ‡	0.02 ± 0.01 (3)
	G796R		1.86 ± 0.17 (3)
	G796R + DIDS ‡		0.03 ± 0.01 (3)
	WT AE1 + Dipy ‡		0.02 (2)
	WT AE1 + DiBAC ‡	0.04 (2)
	WT AE1 + ATZ ‡		0.33 (2)
	____________________________________________________
	 Values are means ± SE
	†n values in parentheses
	‡ghosts were incubated for 15 min with the appropriate inhibitor

ACKOWLEDGMENTS

The authors would like to thank Nicole Boggetto (Flow cytometry platform, Institut Jacques-Monod, Paris, France) for cell sorting experiments, Julien Picot and Sylvain Bigot (Institut National de la Transfusion Sanguine, Paris, France) for flow cytometry analysis, and Eliane Véra (Centre National de Référence des Groupes Sanguins, Paris, France) for providing thawed blood samples. Funding: This work was financed by Institut National de la Transfusion Sanguine, Institut National de la Santé et de la Recherche Médicale and Université Paris Diderot. E.F. was supported by fellowships from Conseil Régional de la Réunion and Institut National de la Santé et de la Recherche Médicale. I.A. was supported by grant GGP 09044 from the Italian Telethon Foundation. Current address of E.F.: Université de la Réunion, F-97490 Sainte Clotilde, France DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s).


Isabelle Tritsch

François-Michel Le Tourneau

Population densities and deforestation in the Brazilian Amazon: New insights on the current human settlement patterns 

Keywords:  Population density,  Urbanization,  Spatial analysis,  Brazilian Amazon

Highlights

•Low overall relations between population density and deforestation.

•One third of Amazon deforestation is associated with only 1.5% of the population.

•In contrast, 13% of the rural population live in preserved forests.

•'Discrete urbanization' of rural areas: multiplication of small towns.

Introduction

The impact of population settlement on natural resources has been a central issue in population and environmental studies. In the tropical forests, a number of case studies have shown a positive relation between population density and deforestation (Ehrhardt-Martinez, 1998, Laurance et al., 2014and López-Carr and Burgdorfer, 2013). However, since the 2000s, this paradigm has been changing, and human population density is no longer pointed out as the principal determinant of tropical deforestation in land-use change studies. Instead, it is viewed as a factor interacting with complex social, economic, and political processes at local and global levels (DeFries et al., 2010, Geist and Lambin, 2002, Pacheco et al., 2011and Rudel et al., 2009).

For the Brazilian Amazon, which represents 40% of the world's remaining tropical rainforests (FAO, 2011) and supplies important ecosystem (Foley et al., 2005) and climate (Malhi et al., 2008 andNobre, 2014, p. 42) services, such insights may be of great relevance, since the demographic growth during the last decades has been impressive: the overall population of the Legal Amazon rose from about 2.5 million in 1960 to over 24,3 million today, a 10-fold increase (IBGE, 2010). In the same period, almost 71 million hectares were deforested, equivalent to 18% of the original forest cover, with important annual rates of deforestation, even if since 2009 the phenomenon has been lowered to about 5000 km 2 per year (Le Tourneau, 2016, Nepstad et al., 2009and Instituto Nacional de Pesquisas Espaciais (INPE), 2015). If a lot of studies have shown the influence of different factors on deforestation, like the presence of roads (Kirby et al., 2006;Laurance et al., 2002 andPerz et al., 2008) and land tenure issues (Araujo et al., 2009, Ostrom and Nagendra, 2006, Paneque-Gálvez et al., 2013and de Espindola et al., 2012), few recent studies have addressed the relationships between population densities and deforestation in the Brazilian Amazon. This paper investigates the mixed relationship between human population density and deforestation in the Amazon for the 2000 to 2010 period. Our research hypothesis is that there is a low overall relation in the Amazon for population and deforestation because local conditions vary widely. For example, strong deforestation rates may be observed in areas of low population densities because of expansion of large-scale agriculture and cattle ranching. This issue of the heterogeneity of population settlement patterns and their impacts on natural resources in the Amazon is critical for policy makers concerned with land-use planning.

First, we examine the partition of urban and rural population and its variations across the Amazon, identifying human settlement patterns (from dispersed rural population to dense city centers). Then, we investigate the transformations of such settlement patterns between 2000 and 2010 and analyze possible relationships between such transformations and the evolution of deforestation during the same period.

The basis of our analysis are the database on deforestation released by the Brazilian space research center (INPE) (INPE, 2015) and the population census data at the census sector level published by the federal geographical and statistical agency IBGE (IBGE, 2010). Using such fresh data at the sub-municipality scale, we aim at bringing an accurate vision of the human occupation of the Amazon both in the land use frontier and in its remotest parts.

State of the art

Deforestation processes and human settlements in the Brazilian Amazon

The large-scale colonization process of the Brazilian Amazon since the 1960s resulted in different human settlement patterns with different combinations of population density and deforestation.

In-migration and Amazonian land use frontier consolidation

In the 1960s and 1970s, the military government began to promote rural colonization of the Amazon in order to populate and to integrate it to the Brazilian economy. Several public policies were implemented to accelerate immigration and economic development in the region and have contributed to the appearance of large-scale deforestation (Fearnside, 2005). Construction of major transport infrastructures began and ambitious colonization programs (both private and state sponsored) tried to attract smallholders in the region under the motto "Lands without men for men without lands" (Le Tourneau & Bursztyn, 2011). Hundreds of thousands of families arrived in the region and began to convert the forest into agricultural lands (Morton et al., 2006). In these areas of small-scale family farming, the underlying human settlement pattern tends to be a combination of medium to high population density and medium to high deforestation, except where land speculation takes place, giving way to the installation of latifundios.

In the 1970s and 1980s, generous fiscal incentives were also implemented in order to attract large firms into the Amazon, leading in a vast majority of cases to the establishment of largescale cattle ranching and to the conversion of forest areas to pasture (Carvalho et al., 2002). Last, several big infrastructure projects (Tucurui dam, Transamazônica highway, etc.) were also responsible for the migration of workers to the Amazon and the further installation of these in public lands and local towns (Browder & Godfrey, 1997).

In the 1980s, forest conversion to pasture continued to expand because of land speculation: creating pasture in forest areas was a cheap way to create "productive land", to claim land titles and to sell the new properties with high profits in a context of rapid rise of land prices (Nepstad et al., 2006 andNepstad et al., 2006). This system of extensive, low-input cattle ranching motivated by land speculation resulted in creating large areas of degraded land (Bowman et al., 2012 andCarvalho et al., 2002) with few human settlements. Indeed, large scale ranching has contributed to the evolution of the human settlement pattern with the emergence of large areas with relatively low population density and high deforestation.

Deforestation driven by distal demand for agricultural products

In the 1990s, soybean began to expand into the Amazon thanks to the development of new adapted technologies (Fearnside, 2001) and to the rise of soybean prices on the international market. With the growing international demand for soybean, large-scale mechanized agriculture expanded into the southern and eastern extent of the Brazilian Amazon and modified the rural dynamics of the region. The soybean industry expansion contributed to a new valorization of land, leading ranchers to sell their properties and seek new areas further north where land prices were lower, participating to the land use frontier advance (Nepstad et al., 2006 andNepstad et al., 2006) and to a trend toward depopulation of the rural areas (Lapola et al., 2014). Thus large-scale crop production has added a new pressure on the forest due to direct deforestation for cropland implantation and to intensified use of lands previously cleared for cattle ranching (Arvor et al., 2012 andMorton et al., 2006). Thereby, the human settlement pattern consisting in low population density and high deforestation has been expanding through the Amazon frontier.

Moreover, as for the big infrastructure projects of the 1970s and 1980s, the magnitude of investments for the commodities production and exportation led to a rapid growth of the cities located along one of the Amazon's export corridors (Richards & VanWey, 2015). This has contributed to the attraction of rural population and to the multiplication of quite densely populated urban areas.

Toward the conciliation of forest conservation and human settlement: the progressive recognition of the land rights of Amazonian "traditional" peoples

In parallel, since the 1990s, the "traditional" peoples of the Amazon, including Indigenous peoples, but also Quilombolas communities (communities formed by descendant of fugitive slaves) and other communities whose subsistence historically depends of non-timber forest products (ex. Rubber-tappers) have gained collective land rights on relative large portion of their ancestral territories. Today, about 21.7% of the Brazilian Amazon is classified as Indigenous land and 14.5% is under diverse protected areas aiming at conciliating the presence of rural communities and the conservation of the Amazon forest ( Veríssimo, Rolla, Vedoveto, Futada, & de, 2011, p. 87). In these areas of forest extractivism and slash and burn agriculture, the underlying human settlement pattern is a combination of low to medium population density and low deforestation.

Adding to this, 8.9% of the Amazon is under integral protected areas where human presence is forbidden (therefore theoretically registering zero population and zero deforestation).

As this quick historical description shows, the Amazon consists in a vast mosaic of diverse land tenures and human settlement pattern, both terms having influenced each other (Nasuti, Tritsch, & Eloy, 2015).

New trends of Amazonian deforestation

Since 2009, the efforts of the Brazilian government to curb deforestation induced a spectacular diminution on the annual deforestation rates and the progressive decoupling of agricultural expansion and deforestation (Gollnow and Lakes, 2014, Le Tourneau, 2016, Macedo et al., 2012and Nepstad et al., 2009), suggesting that it is possible to manage the advance of the Amazon frontier (Nepstad et al., 2014). A number of recent studies have addressed the question of the current diminution of deforestation. These studies underlined the role of ambitious and effective command-and-control environmental public policies (such as the Action Plan for Prevention and Control of Deforestation in the Amazon, PPCDAm) (Nepstad et al., 2014), increased market regulations such as the Soy and Beef Moratorium (Gibbs et al., 2015, Rudorff et al., 2011and Soares-Filho et al., 2014), economic incentives such as payments for environmental services (Börner, Marinho, & Wunder, 2015), but also agricultural intensification process (Arvor et al., 2012 andMorton et al., 2006). Concerning the responsibility of different social actors on deforestation, Godar, Gardner, Tizado, and Pacheco (2014) showed that during the 2004-2011 period, the contribution of the largest landholders to annual deforestation decreased over time whereas that of smallholders went up by a similar amount, particularly because command and control policies have focused on larger properties in deforestation hotspots. However, Lapola et al. (2014) stress that these news trends in the Amazon land use induce a commoditization of the land and tend to reinforce the long history of inequality in land ownership. This contributes to the rise of rural-urban migrations and thus to the transformation of the human settlement dynamics (Richards & VanWey, 2015).

Human settlement pattern theory of the land use frontier

The deforestation processes and human settlements pattern in the Brazilian Amazon are consistent with the theories of frontier cycle and land use transition (DeFries et al., 2004 andFoley et al., 2005). The frontier can be conceptualized as a moving fringe separating forested areas from areas where population settlements and human activities are consolidated (Arvor et al., 2012). At the beginning of the cycle, increased population density from in-migration tends to induce forest conversion and farmland expansion in a process of extensification. As the frontier evolves, population density increases both from in-migration and fertility and can lead to some agricultural intensification in order to take advantage of the labor force and because of waning forest resources. Then, land consolidation usually induces a rise of the price of land which encourages small farmers to sell their properties to bigger ones, leading to lower population densities because of the expansion of large landholdings (latifundio). This frontier dynamic is known as the "hollow" frontier ( Rudel et al., 2002 andWalker, 2004). Consequently, the less capitalized smallholders either move forward to other frontier areas or migrate to urban areas where they hope to find better living standards, thus contributing to the urban population growth ( Browder andGodfrey, 1997 andPerz et al., 2005). In both case, despite the decline of rural population, deforestation tends to persist since the lands sold by the smallholders are converted by big farmers for commodity crops or pastures ( Carr, 2004 andFearnside, 2008). Thus, these theories suggest that the demographic aspects of the tropical deforestation are dynamic and time-dependent in relation with the stage of the land use frontier. These different population-deforestation patterns can be viewed as situated at different points along a curve that describes the process of frontier settlement.

Materials and methods

Demographic data

Brazil publishes high-precision data on its population and socio-economic conditions through the decennial population census (IBGE, 2010). The census data are public and freely available on the IBGE site, in the form of aggregated indices at the municipality scale and, since the 2000 census, in the form of an extensive list of variables at the census sectors scale, for which geo-referenced shapefiles are also available. The census sector level presents a spatial scale 48 times more spatially refined than the municipality level which is generally used in studies looking at demographic and socio-economic factors for the Brazilian Amazon. In fact, IBGE raw data are produced for 37,295 census sectors covering the entire Amazon while there are only 775 Amazonian municipalities. It must be noted however that the IBGE census sectors have changed between the 2000 and 2010 censuses, which makes comparisons between the two periods problematic, unless the data are projected in a uniform grid.

Deforestation data

Brazil has extensive data on the Amazon forest area cleared annually through its PRODES program of the INPE, based on the remote sensing analysis of images of multiple satellites (LANDSAT, CBERS, UK2-DMC, RESOURCESAT) (INPE, 2013). The PRODES is the world's most complete and enduring tropical forest monitoring program, offering exhaustive data used by several studies analyzing the Amazon deforestation dynamics (Börner et al., 2015 andNolte et al., 2013). The minimum deforested area mapped by the PRODES is 6.25 ha, and deforestation is defined as the complete removal of forest cover thus not including forest degradation. Reforestation and forest regrowth processes are not considered either: a polygon deforested at year N will always continue classified as 'deforested'. Finally, the PRODES only considers areas originally covered by forest vegetation (ie. areas forested in the pre-1970s). So there is no deforestation data available for all the savannas (Cerrado) included in the Brazilian Legal Amazon (near 1 million square kilometers south but also in Roraima and Amapá, in the northern part of the region). For this reason, our analysis does not consider the areas covered by non-forest vegetation (Cerrado).

GIS data processing

In order to link the deforestation data and the demographic data from the census sectors of 2000 and 2010, using a GIS, all the data were projected in quadrats of 10 × 10 km, covering the whole Brazilian Legal Amazon (5,000,000 square kilometers). Although the PRODES deforestation data would allow for a finer resolution, 10 × 10 km was judged suitable because of the large size of most rural census sectors. For each quadrat, deforestation and demographic variables were calculated for the year 2000 and 2010. Four steps were performed to accurately project the PRODES and the census sectors data into the 10 × 10 km grid: 1) calculation of the surface of original forest for each quadrat by subtracting nonforest areas and waterways, 2) calculation of the quantity of deforestation for each quadrat, 3) calculation of the estimated density of demographic variables according to the surface of the IBGE census sectors, 4) calculation of the absolute values of demographic variables for each quadrat (Fig. 1). Step 1 Calculation of the surface of original forest for each quadrat by subtracting non-forest areas and waterways.

The surface originally covered by forest vegetation was calculated for each quadrat. It refers for the INPE to the areas covered by forest in the early 1970, and can be obtained on GIS by subtracting the areas that are not covered by water bodies and by savannas and other nonforest vegetation according to the INPE shapes. This surface in original forest is our reference surface for the study and will be used to calculate the quadrats' total deforestation in 2000 and 2010. In order to do not distort our interpretations of the dynamics of deforestation, we excluded from our analysis all quadrats situated along the margins of the Legal Amazon and those that had less than 70% of their surface covered by original forest.

We therefore consider in our analysis an Amazonian area of 4 million square kilometers, inhabited by 18.4 million people in 2010, representing 40,408 quadrats in our database.

Step 2 Calculation of the quantity of deforestation for each quadrat.

In the step 2, all the deforestation polygons were intersected with the 10 × 10 km grid. At that point, the surface of all the fragments of deforestation polygons included in each quadrat was summed and the total deforestation of each quadrat was calculated in function of the surface in original forest obtained in step 1.

Step 3 Calculation of the estimated density of demographic variables according to the surface of the IBGE census sectors.

Demographic and socio-economic data released by the IBGE use individuals or households as units. We converted them in densities by dividing them by each sector's size in km 2 . By doing so, we presumed that population density within a sector is equal throughout the sector, which is a necessary assumption in any population density study. This assumption is obviously much more valid for small urban sectors than for big rural ones. However, as the data used are the most detailed data available, we think that apart from some misrepresentations in extreme cases, it is statistically sound.

Step 4 Calculation of the absolute values of demographic variables for each quadrat.

Once the demographic and socio-economic variables expressed as estimated density, census sectors were intersected by the grid of 10 × 10 km quadrats. For each fragment of census sectors, the absolute values of these variables were calculated according to the area of the fragment. Then the absolute values of all the fragments included in the same quadrat were summed, allowing the calculation of the demographic and socio-economic data in 2000 and 2010 for each quadrat. -Territorial indicators on land tenure (type of protected areas) and distance to the major roads, to major navigable rivers and to state capitals, calculated from GIS data of the Brazilian minister of environment (http://mapas.mma.gov.br/i3geo/datadownload.htm).

Data processing was carried using a GIS developed on the free software QGIS 2.6.1 and GRASS GIS 6.4.3.

Data analysis

Classification of quadrats according to their human settlement patterns

The 40,408 quadrats were classified into ten human settlement patterns (A to J) according to the thresholds of deforestation extent and population density presented in Fig. 2. The rationale for defining these classes of human settlement patterns was first of all based on the level of population density. Hence, all quadrats with less than 1 inhabitant per square kilometer in the period analyzed were considered as "desert", those with 1-10 were considered as "populated", those with 10-50 as "densely populated" and those with more than 50 inhabitant per square kilometer (i.e. sheltering more than 5000 persons) were considered as very densely populated "urban". Deforestation extent in 2010 was then used to situate quadrats in relation to the land use frontier, with those with low deforestation extent (less than 5%) considered "forested", those with deforestation extent 5-40% considered "deforested" and those with more than 40% deforestation extent considered "heavily deforested". The thresholds applied were defined attempting to capture the variability in the population and deforestation variables.

Statistical analysis

Statistical analyses were performed on the R statistical free software, version 3.1.0 (R Core Team, 2015). We used non-parametric tests as the variables are not normally distributed. We evaluated the statistical relationships among the classes of human settlement patterns and the demographic and territorial variables using the Kruskal-Wallis test and the Tuckey's honestly significant different test.

Results: characterization of the human settlement patterns in the Brazilian Amazon

Our results show very distinct patterns of human settlement with low-low and high-high classes of population density and deforestation, and also low-high and high-low classes as illustrated in Fig. 3 and Table 1. There is a low overall relation in the Amazon for population and deforestation with four main contrasted patterns that we detail bellow. Note: All variables present significant variation across the human settlement pattern classes (Kruskal-Wallis: p < 0.0001). * Not Applicable because the roads network considered does not take into account the urban and municipality roads.

Densely populated urban areas are increasing (pattern U)

A large proportion of the Amazonian population is concentrated in towns or cities: 71.5% of the population (13.1 million people) live in urban areas in 2010 (quadrats with a population density greater than 50 inhabitants/km2, pattern U, Table 1). They are spread over 526 quadrats in 2010, corresponding to less than 1.2% of the overall original forest area of the Amazon (46,082 km2).

This urban population has increased by 30.3% in 10 years, meaning an increase of more than 3 million inhabitants between 2000 and 2010. In 2000, there was only 442 densely populated urban quadrats against 526 in 2010 (19% increase) and the proportion of urban population was 'only' 66.4%. This trend of increasing urbanization is sustained by a strong rural exodus: if the urban population show a 30.3% increase in 10 years, the whole rural population only grew from 2.4% in the same period (passing from 5,100,714 to 5,223,061 inhabitants). Cities attract people because they offer better access to jobs and public services such as school and healthcare (Nasuti et al., 2015 andTritsch et al., 2015). Moreover, rapid midsize cities growth is closely tied with traded commodities production because these cities capture capital and value before it is extracted from the region (Richards & VanWey, 2015).

Preserved forests have different human settlement patterns: from "empty" forests to populated forests (patterns A to C)

In areas where forest cover is well preserved (quadrats with less than 5% deforestation in 2010), contrasted situations are observed. On the one hand, the "empty" forests cover a large surface: 61.6% (2,405,551 km2) of the Amazon forest have less than 1 inhabitants/km2 (24,623 quadrats) (Table 1) and 42.1% (1,642,725 km 2 ) have a population density inferior to 0.1/km 2 . Not surprisingly, such areas are often located far from roads and main waterways (Table 1, Fig. 3). Their average population density is only 0.13 people/km2. On the other hand, the number of people living in those well preserved forest is not as low as one can imagine. A total of 688,380 people (13% of the rural population) live in areas where deforestation is less than 5% (patterns A, B and C), showing that an important fraction of the Amazonian rural population still has a way of life consistent with the maintenance of forest cover. Moreover, the numbers of inhabitants of the "populated forests" (pattern B) and "densely populated forests" (pattern C) have augmented between 2000 and 2010, with respectively a 23% and 46% increase.

Low population densities and deforested areas: the increase of "deforested human deserts" (pattern D and G)

In contrast, some heavily deforested areas have very low population densities: 2554 quadrats have more than 40% of their area deforested in 2010 but have population densities lower than 1 inhabitants/km2 (pattern G). They represent a deforested area of 162,171 km 2 , accounting for 13% of the 2010 whole deforestation, for only 0.6% of the Amazonian population (115,316 inhabitants) (Table 1). These "heavily deforested human deserts" areas are situated next to the main roads, in average 18 km (median = 10 km) and mainly in the south and southeast of the Amazon (Table 1, Fig. 3).

The tendency of heavily deforested areas to have low population densities seems to be accelerating in the Amazon: in 2000, the pattern G had only 1415 quadrats against 2554 in 2010, which represents an increase of 80.5% in ten years (Table 1). Moreover, the pattern D (deforestation extent between 5 and 40% and population densities smaller than 1 inhabitants/km2) already counts 3871 quadrats in 2010 (Table 1). If the deforestation in such areas persists, these quadrats will certainly turn into "heavily deforested human deserts" (pattern G) and so will contribute to the expansion of the pattern consisting of low population densities living in heavily deforested areas.

Moreover, settlement patterns D and G have registered a decrease in their total population between 2000 and 2010, while during the same period their total deforested area has been rising. The population of pattern G decreased by 24% between 2000 and 2010 though, over the same period, deforestation has risen by 38.5%. For the pattern D, population decreased from 21% in ten years while deforestation has risen by 117% in the same period.

High number of inhabitants in heavily deforested rural areas: patterns E, F, H and I

On the other hand, some deforested rural areas are heavily populated (at least according to Amazonian standards). In 2010, the pattern I (population densities from 10 to 50 inhabitants/km2 and deforestation higher than 40%) represented only 998 quadrats but sheltered 2.0 million inhabitants. The pattern H, which has population densities from 1 to 10 inhabitants/km2 and deforestation higher than 40%, represented 4211 quadrats for 1.4 million inhabitants. Concerning patterns E and F, they together represent 2100 quadrats for 850,611 inhabitants (Table 1).

Discussion: main dynamics of human settlement in the Brazilian Amazon

Trends towards rural population concentration in growing villages or small towns and 'discrete urbanization'

In rural areas of the Amazon, our analysis shows a trend toward rural population concentration in more densely populated rural areas and, correlatively, a trend toward depopulation of the other ones. Rural quadrats with 10-50 inhabitants/km2 are experiencing a 15% increase in their population (going from 2.1 to 2.4 million inhabitants between 2000 and 2010) whereas quadrats with less than 10 inhabitants/km2 face a 6.6% decrease in their population. This concentration of rural population in some big growing villages reflects the trend toward rural Amazon 'villagization', defined by Eloy, Brondizio, and Do Pateo (2015) as a process of demographic growth and spatial expansion of the main rural villages. This 'discrete urbanization' of the Amazon occurred especially in downstream areas near state capitals and roads and around the secondary education poles (Costa andBrondízio, 2011 andParry et al., 2010). On the one hand, in remote areas, the displacement of people toward more densely populated areas may reflect the lack of infrastructures and the importance of transportation costs (especially on rivers) (Parry et al., 2010 andTenkanen et al., 2015). Nowadays, most families feel the need to go to town at least one time a month in order to receive social benefits from cash transfer programs (ex. Bolsa familia and rural retirement), or even more frequently in case there are youngster studying at the college ( Eloy et al., 2015 andNasuti et al., 2015). On the other hand, in rural areas situated close to the main road network, the improved access to road facilities has simplified the rural mobility (Théry, 2014).

Such shifts greatly change the profile of the Amazon region, currently mainly viewed by public policies as a rural region with only two big metropoles, Belém and Manaus. The multiplication of small towns and second-order cities modifies the needs and should provoke a major overhaul in policies in order to respond to them. For instance, water treatment and waste collection are failing in most Amazonian cities, turning them into emitters of different types of pollution which, in turn, will threat the environment of vast regions surrounding them.

Finally, the shortening of fallow lengths around these growing villages, due to higher population densities and scarcity of available land, challenges the land use sustainability in these areas since agricultural systems are still mainly based on extensive slash and burn agriculture. The question of subsistence hunting in these rural areas of higher population densities is also complicated (Tritsch, Gond, Oszwald, Davy, & Grenand, 2012). In consequence, more and more households stop their subsistence agricultural activity and local food (ex. Cassava) tends to be substituted by imported food (congealed chicken and food made from wheat), threatening local food independence (Peralta & Kainer, 2008). On the other hand, cash transfer programs and local jobs available in these growing villages and small towns enable households to buy imported food, which help stabilize forest frontiers because households become less reliant on subsistence agricultural production (van Vliet, Adams, Vieira, & Mertz, 2013). For all these reasons, agro-ecological programs promoting alternative ways of sustainable production, such as agroforestry systems, must be implemented in order to support farmers in the adaptation of their production systems to these news social and environmental conditions.

"High deforestation -low population": expansion of large-scale agriculture and depopulation of deforested rural areas

We showed that high deforestation may be observed in areas of low population densities: almost one third of the Amazonian deforestation in 2010 is associated with less than 1.5% of the Amazonian population, representing what we have called "deforested human deserts" (pattern G and D). The population density of these areas declined between 2000 and 2010 while the deforestation increased, indicating that the amount of deforestation on a perinhabitant basis increased. Thus, these areas present a trend toward rural depopulation and a demographically "hollow" frontier. This suggests that smallholders are selling their land to cattle ranchers and large scale farmers. As emphasized by Fearnside (2008) and López-Carr and Burgdorfer (2013), in these deforested areas of low population density, large-scale farmers and cattle ranchers are most likely responsible for deforestation.

The increase in the extent of these areas (pattern G and D) between 2000 and 2010 shows that deforestation does not necessarily go along with settlement of fixed population. The activities implemented in the arc of deforestation have failed in fixing human settlements, at least in recent times, despite the common discourse on the Amazon linking deforestation to local demographic expansion (Kirby et al., 2006;Laurance et al., 2002). In fact, the Amazonian land use frontier has always been multifaceted, mixing small-scale colonization and large-scale latifundios. However, today, the relative weight of each term may have shifted. The process of agrarian colonization, as idealized in the years 1970 and pursued until the late 1990s, which aimed to settle rural families, has largely stepped back today, except for a few regions. At the same time, a complex process of large-scale mechanized agriculture and cattle ranching implementation has occupied a growing proportion of the Amazon. Such large estates employ and harbor small numbers of people in favor of an even smaller number of landowners, a characteristic situation of the concentration of large tracts of land in some few hands ( Pacheco et al., 2011).

The per capita environmental cost of such latifundio areas is obviously very high, but is offset in public discourse by the economic return of commodities exports. However, there is a growing pressure toward diversification of production, and toward intensification and value aggregation of the agricultural production (turning, for instance, from soybean to hog and chicken meat production). But the fitness of the current demographic profile for such purpose is not guaranteed.

Public policies could thus take advantage of the precise mapping which may be realized by studies such as ours in order to promote focused land-reform policies and generally increase demographic densities in the already deforested areas, thus alleviating part of the pressure felt in other areas. In this sense, the restoration of degraded areas should be prioritized. Indeed, Martini, Moreira, Formaggio, and Dalla-Nora (2015) recently mapped an amount of 200,000 km 2 of already converted areas in the Brazilian Amazon which could support human settlement and agricultural expansion without promoting the advance of deforestation.

Efforts such as the recent agriculture program ABC (Plano para uma Agricultura de Baixo Carbono) which aims to ensure a transition toward a more environmental friendly agricultural model, for example by promoting the restoration of degraded pastures, should be pursued.

"Low deforestation -high population": role of land tenure and public policies to encourage forest conservation and human settlement

According to our data, 688,380 people (around 13% of the Amazon rural population) lives in forest without causing massive deforestation. The inhabitants of these "preserved" forests (deforestation <5%) show that, even today, human presence may be associated with forest conservation. Indigenous peoples are part of those 600,000 people who live in the forest without cutting it down. However, they are not the only ones, because the indigenous people living inside indigenous territories do not represent more than 250,000 people in the Brazilian Amazon, or about 42% of those inhabitants.

Much of these "preserved" forest areas are characterized by specific forms of land tenure: 69.2% are located in indigenous lands or different types of protected areas. This reinforces the idea that the recognition of specific land tenure types such as indigenous lands and protected areas participate in curbing deforestation, especially in areas of high pressure on natural resources (Nolte et al., 2013 andPfaff et al., 2015), the former probably being the most efficient (Nepstad et al., 2006 andNepstad et al., 2006). It also means that 30.8% of these preserved forests (representing 783,280 km2) are not registered under any special land tenure and so have an uncertain future concerning their future land use.

The presence of population in the Amazon necessarily implies a certain proportion of deforestation to practice small scale agriculture. But deforestation remains low in these "preserved" forests: on average only 0.18% of the overall forest cover of the quadrats were removed between 2000 and 2010 (9992 km2 deforested in 2010). However, if these areas present a low amount of forest clearing on a per-inhabitant basis and are good examples of how to combine the maintain of rural population and the conservation of forest cover, it is important to emphasize that they are vulnerable nowadays because of the falling prices of many non-timber forest products, which result in a decline of traditional economic activities.

In this context, the attractiveness of the more environmentally destructive economic activities (like cattle ranching) increases due to a lack of other options (Schwartzman & Zimmerman, 2005). Therefore, there is currently an important issue about local economic opportunities for the younger generations. Indigenous territories constitute another important example about the tensions between conservation and development (Le Tourneau, 2015).

Hence, while this "low deforestation -high population" pattern is undeniably environmentally and theoretically important, its social and economic challenges remain significant as they complicate the picture of the Amazonian sustainability. This highlights the need for appropriate public policies and economic incentives, such as Payments for ecosystem services (PES), to support these rural populations ( Börner et al., 2015 andPacheco et al., 2011). Different PES initiatives have already been implemented in the Brazilian Amazon, for example the Bolsa Floresta program, which encompasses a set of integrated interventions aiming at conserving forests and improving the welfare of residents in selected human-occupied protected areas of sustainable development or the REDD + pilot program PAS (Projeto Assentamentos Sustentaveis na Amazonia) in the Trans-Amazonian Highway. However, it still lacks evaluations of the impacts of these PES initiatives which are necessary to improve the sustainability of these programs and promote their wider implementation. So more studies like Simonet, Subervie, Ezzine-de-Blas, Cromberg, and Duchelle (2015) on the cost-effectiveness and additional and leakage effects of these initiatives should be encouraged.

Conclusion

As the Brazilian Amazon remains a moving frontier, the interactions between demography and deforestation are complex and changing. Since the launch of large scale development projects in the 1970s they have shaped different human settlement patterns which cannot anymore be treated by homogeneous public policies for the whole 'Legal Amazon'.

Two patterns are especially interesting. In one hand, in 2010, the "deforested human deserts" pattern accounted for almost one third of the total deforestation, highlighting that the Amazon deforestation cannot be viewed as a result of a demographic pressure. On the other hand, other regions have relatively important population densities and little deforestation, like in the "populated forest" pattern. These latter areas represent a series of experiments combining human presence and conservation of biodiversity and forest cover, which should be preserved by the implementation of supportive public policies.

However, the recent context of political and economic crisis could weaken the institutional capacity of the Brazilian government to ensure continuity with the ambitious and effective environmental and socio-economic policies that have significantly reduce the Amazon deforestation (Rodrigues-Filho, Verburg, Bursztyn, Lindoso, Debortoli, & Vilhena, 2015) while enabling several millions of Brazilian to leave poverty behind (ex. Bolsa Familia, Fome zero, Luz para todos, and rural retirement programs) ( Barrientos, 2013 andFenwick, 2013). In the Amazonian "populated forests", part of the current rural economy does rely on these social benefits ( Nasuti et al., 2015 andTritsch et al., 2015). So, changes in the economic priorities of the Brazilian government can foster new transformations in the Amazonian human and land use settlement patterns.

Our data also confirm the rapid growth of urban areas as shown by Richards and VanWey (2015). An important process of 'discrete urbanization' of the Brazilian Amazon must also be underlined: the rural population tends to concentrate in big growing villages or small towns with, correlatively, a trend towards depopulation of remote areas. A number of medium to big rural nuclei now constitute the backbone of the rural world in the Amazon, even in riverine areas. Thus, the problems associated with urban areas, in particular urban planning and waste treatment should gain more attention in the definition of public policies for the Amazon, as urban populations now encompass the majority of the Amazonian population. In the same way, the problems related with depopulation of rural landscapes such as increased fire activity (Uriarte et al., 2012) should also seriously be taken into account, especially as the projection of the United Nations predicts a major decline in rural population for Brazil until 2050 (United Nations, 2015).

The crossing of deforestation and demographic spatial data was useful in order to pinpoint the existence of different patterns and to quantify their extent. Our analysis of the relationship of population and deforestation in the Brazilian Amazon reveals a set of distinct combinations across the basin. Those combinations in turn require distinct policies to be effective in conserving forest where it still stands, taking into account varying population densities. The macro-zoning policy developed by the Brazilian Federal Government for the Amazon is a step toward recognizing the specificity of such regions, but the different regions are not detailed enough and the way it will be put concretely into effect is not clear at this stage.

Fig. 1 .

 1 Fig. 1. GIS processing of the data: projection of PRODES and IBGE census sectors data into a 10 × 10 regular grid.

  These data were associated to the deforestation data in a database composed of 40,408 quadrats with the following variables: -Demographic indicators in 2000 and 2010: the estimated number of inhabitants and the population density, -Total deforestation (see definition in Section 3.2) in 2000 and 2010: the total area deforested registered by PRODES for year 2000 (i.e. summing up the deforestation between the 1970s and 2000) and for year 2010 (i.e. summing up the deforestation between 1970s and 2010).

Fig. 2 .

 2 Fig. 2. Definition of human settlement pattern classes (A to J) according to population density in 2010 and deforestation extent in 2010.

Fig. 3 .

 3 Fig. 3. Human settlement patterns in the Brazilian Amazon in 2010.

Table 1 .

 1 Comparison of selected variables of each human settlement pattern.

	Human settlement pattern	Nb. of cells in 2000	Nb. Of cells in 2010	Inhabitants in 2000	Inhabitants in 2010	Population variation (2000-2010) (%)	Percent of the 2010 whole population (%)	Total deforestation in 2000 (km 2 )	Total deforestation in 2010 (km 2 )	Percent of the 2010 total deforestation (%)	Mean distance from major roads (km)
	U: densely
	populated urban	442	526	10,081,791 13,134,369 30.28	71.55	30,196	33,603	4.85	NA*
	areas
	A: Empty forests 26,636 24,623 419,533	302,272	-27.95	1.65	4646	8158	1.18	117.60
	B: Populated forests	2013 1467 251,148	309,502	23.23	1.69	864	1759	0.25	99.96
	C: Densely
	Populated	119	58	52,598	76,606	45.64	0.42	77	55	0.01	47.80
	forests
	D: Deforested human deserts	3492 3871 159,466	125,780	-21.12	0.69	31,726	68,782	9.93	42.25
	E: Deforested
	populated rural	1974 1907 449,605	515,376	14.63	2.81	21,201	37,144	5.36	45.35
	areas
	F: Deforested
	densely populated rural	202	193	323,664	335,235	3.58	1.83	2549	3576	0.52	33.56
	areas
	G: Heavily
	deforested	1415 2554 151,856	115,316	-24.06	0.63	117,070	162,171	23.41	17.92
	human deserts
	H: Heavily
	deforested populated rural	3192 4211 1,556,802 1,422,535 -8.62	7.75	238,847	300,301	43.36	13.52
	areas
	I: Heavily
	deforested
	densely	923	998	1,736,042 2,020,439 16.38	11.01	68,336	77,098	11.13	7.25
	populated rural
	areas
	TOTAL	40,408 40,408 15,182,505 18,357,430 -	100.00	410,149	692,647	100.00	-

Acknowledgments

We thank the University Sorbonne Nouvelle, Paris 3 and the CNRS -UMR CREDA for their funding sources. We are also grateful to the two anonymous reviewers for their constructive comments, which helped to improve the manuscript. The authors declare no competing interests.