PharmD Fabienne Venet

PhD; Sylvie Tissot

MD Anne-Lise Debard

PharmD Caroline Faudot

Carine Crampé

PharmD Alexandre Pachot

PhD Alfred Ayala

PhD; Guillaume Monneret
email: guillaume.monneret@chu-lyon.fr

Decreased monocyte human leukocyte antigen-DR expression after severe burn injury: Correlation with severity and secondary septic shock

B

urns remain a major cause of injury and death worldwide [START_REF] Church | Burn wound infections[END_REF][START_REF] Hettiaratchy | ABC of burns. Introduction[END_REF]. Increasing patient age, increasing burn size, and inhalation injury are factors that increase the risk of death after major burn injury. Secondary infection is also a leading factor of morbidity and mortality. After the initial resuscitation, 70% to 75% of mortality is related to infection [START_REF] Church | Burn wound infections[END_REF][START_REF] Ansermino | Intensive care management and control of infection[END_REF].

It has been postulated for a long time that severe thermal injuries may provoke a state of immunosuppression [START_REF] Schwacha | Macrophages and post-burn immune dysfunction[END_REF]. Burn injury induces the activation of a proin-flammatory response that is thought to be followed by a subsequent anti-inflammatory phase, in an effort to maintain inflammatory immune homeostasis. However, this compensatory feedback may also contribute to a state of immunosuppression (4). Among other depressed functions, monocyte alterations have been suggested to play a critical role in the development of burn-induced immunodepression [START_REF] Church | Burn wound infections[END_REF]. It has been proposed that these alterations in the innate immune system exhibit some similarities with the state of "immunoparalysis" or "leukocyte reprogramming" observed after septic shock, which is thought to decrease the natural defenses that are responsible for counteracting pathogens. As a result, burn patients would be more susceptible to wound infections, severe sepsis, and subsequent multiple organ failure [START_REF] Church | Burn wound infections[END_REF]. A diagnostic laboratory marker would therefore be desirable to help in identifying immunosuppressed patients at high risk for infectious complications after severe burn.

Decreased monocyte human leukocyte antigen-DR (mHLA-DR) expression has been demonstrated to be a reliable marker for monitoring immunosuppression in critically ill patients. Strongly diminished mHLA-DR expression has been used to identify patients with temporary immunosuppression who will present with secondary nosocomial infections after various injuries: trauma [START_REF] Ditschkowski | HLA-DR expression and soluble HLA-DR levels in septic patients after trauma[END_REF][START_REF] Muehlstedt | Increased IL-10 production and HLA-DR suppression in the lungs of injured patients precede the development of nosocomial pneumonia[END_REF], acute pancreatitis [START_REF] Satoh | Human leukocyte antigen-DR expression on peripheral monocytes as a predictive marker of sepsis during acute pancreatitis[END_REF], cardiac surgery [START_REF] Allen | Early postoperative monocyte deactivation predicts systemic inflammation and prolonged stay in paediatric cardiac intensive care[END_REF], transplantation [START_REF] Haveman | Low HLA-DR expression on peripheral blood monocytes predicts bacterial sepsis after liver transplantation: Relation with prednisolone intake[END_REF], and severe sepsis [START_REF] Tulzo | Monocyte human leukocyte antigen-DR transcriptional downregulation by cortisol during septic shock[END_REF]. Decreased HLA-DR expression has also been shown to be an independent Objective: Severe thermal injury causes immune dysfunctions involving both pro-and anti-inflammatory mechanisms. It subsequently leads to a state of immune deficiency that shares some similarities with sepsis-induced immunosuppression. A hallmark of the latter is established by decreased monocyte human leukocyte antigen-DR (mHLA-DR) measurements. The main objective of the current study was to characterize the appearance and the duration of low mHLA-DR expression after severe burn as well as to determine its correlation with mortality and septic complications.

Design: Observational study.

Setting: Burn unit (intensive care unit) in a university hospital. Patients: Severe burn patients (total burn surface area >30%, n ؍ 14) and healthy individuals (n ؍ 29).

Interventions: None. KEY WORDS: severe burn; monocyte; human leukocyte antigen-DR; immunosuppression; septic shock predictor of mortality in septic shock [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF]. However, it is unclear whether these findings extend to burn patients.

Measurements and Main Results

The aim of the present study was thus to assess mHLA-DR expression in burn patients. Plasma cytokine measurements of interleukin (IL)-6, tumor necrosis factor (TNF)-␣, and IL-10 were also determined. Subsequently, we determined whether these variables could be associated with mortality and with the development of severe septic complications.

PATIENTS AND METHODS

Patients. Burn victims admitted to the burn unit at E. Herriot University Hospital were prospectively included in the study. Exclusion criteria included all patients younger than 18 yrs and with total burn surface area (TBSA) injury Ͻ30%. Mortality was defined as death occurring within burn unit stay. The institutional ethics committee reviewed and approved this observational study.

Diagnosis of Secondary Infections. The diagnosis of infection and septic shock was based on criteria of the American College of Chest Physicians/Society of Critical Care Medicine [START_REF] Bone | Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee[END_REF]. As surface swabs and cultures cannot distinguish infection from colonization (3), wound infections are not taken into account in our routine follow-up, except in case of graft failure or when associated with a least one systemic sign of infection. Septic shock was defined by an identifiable site of infection and evidence of a systemic inflammatory response manifested by at least two of the following criteria: a) a body temperature Ͼ38°C or Ͻ36°C; b) heart rate Ͼ90 beats/min; c) respiratory rate Ͼ20 breaths/ min; d) white blood cell count Ͼ12,000/mm 3 or Ͻ4000/mm 3 ; and e) blood pressure Յ90 mm Hg despite fluid resuscitation and requiring vasopressor therapy (Ͼ5 g/kg of body weight of epinephrine and/or norepinephrine and/or dobutamine).

Samples. EDTA-anticoagulated samples were drawn in the following time period after thermal injury: day 1 (first 24 hrs), days 2-3, days 4 -6, days 7-10, and days 11-15 (one sample per period).

Plasma Cytokine Measurements. Plasma levels of IL-10, TNF-␣, and IL-6 were measured using commercial enzyme-linked immunosorbent assay kits, according to the manufacturers' recommendations (Biosource, Camarillo, CA, for IL-10 and TNF-␣; Immunotech, Marseille, France, for IL-6). The detection limits of the assays (and normal values given by manufacturers) were 1 pg/mL (Ͻ10 pg/mL) for IL-10, 3 pg/mL (Ͻ15 pg/mL) for TNF-␣, and 3 pg/mL (Ͻ7 pg/mL) for IL-6.

Monocyte HLA-DR Measurement by Flow Cytometry. Flow cytometric (FC500, Beckman-Coulter, Hialeah, FL) expression of mHLA-DR was assessed on peripheral blood. Staining and cell acquisition on flow cytometer were per-formed within 2 hrs after blood sampling (storage at 4°C). Monoclonal antibodies were used according to the manufacturers' recommendations: fluorescein isothiocyanate-labeled anti-CD14 (10 L, Immunotech, Marseille, France) and PE-labeled anti-HLA-DR or PE-labeled corresponding isotype control (20 L, Becton Dickinson-Pharmingen, San Jose, CA) per 100 L of whole blood. As previously described [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF], monocytes (1500 from each sample) were characterized on the basis of their CD14 expression. Results were expressed either as percentages of HLA-DR-positive monocytes of total monocyte population (the threshold for positivity was defined with the isotype control and settled on 2% of positive cells) or as a number of PEconjugated antibodies bound per cell (AB/C) that was determined with BD Quantibrite PE beads (Becton Dickinson) as previously standardized in a multicenter study [START_REF] Docke | Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: A multicenter standardized study[END_REF].

HLA-DRA and CIITA Messenger RNA Quantification in Whole Blood Samples. Blood samples were collected directly in PAX-Gene Blood RNA tubes (PreAnalytix, Hilden, Germany), and total RNA was extracted using the PAXGene Blood RNA kit (PreAnalytix). Before RNA elution, the residual genomic DNA was digested using the RNase-Free DNase set (Qiagen, Hilden, Germany). Total RNA was reverse transcribed into complementary DNA using the ThermoScript reverse transcriptasepolymerase chain reaction system (Invitrogen) according to the manufacturer's instructions. The messenger RNA (mRNA) expression of HLA-DRA and the transcription factor class II transactivator (CIITA) was quantified by reverse transcriptase-polymerase chain reaction using the LightCycler instrument and the Fast-Start DNA Master SYBR Green I real-time PCR kit (Roche Molecular Biochemicals, Basel, Switzerland) as previously described [START_REF] Pachot | Messenger RNA expression of major histocompatibility complex class II genes in whole blood from septic shock patients[END_REF]. HLA-DRA and CIITA mRNA level were normalized to that of the housekeeping gene peptidylpropyl isomerase B encoding for cyclophilin B as previously described [START_REF] Pachot | Peptidylpropyl isomerase B (PPIB): A suitable reference gene for mRNA quantification in peripheral whole blood[END_REF]. The complementary DNA calibrators for HLA-DRA and CIITA were prepared from purified polymerase chain reaction amplicons obtained with the primers described previously [START_REF] Pachot | Peptidylpropyl isomerase B (PPIB): A suitable reference gene for mRNA quantification in peripheral whole blood[END_REF]. Standards and ready-to-use primer mixes for peptidylpropyl isomerase B were obtained from Search-LC (Heidelberg, Germany). Using the LightCycler Software version 4, the results were expressed as a concentration ratio of mRNA/peptidylpropyl isomerase B mRNA. Twenty-nine patients from hospital staff served as control values for mRNA measurements.

Statistical Analysis. Median values are reported together with interquartile ranges. Comparisons between groups were made using a nonparametric Mann-Whitney U test for continuous variables and the chi-square test for categorical data. A p value Ͻ.05 was regarded as significant without correction of the number of tests performed.

RESULTS

Patients. Samples were obtained from 14 burn patients (five females, nine males; median age ϭ 44.5 yrs, range 18 -74 yrs). The baseline characteristics of those patients are presented in Table 1. The TBSA ranged from 32% to 85% (median 50%). Three patients presented with additional inhalation injury. Initial resuscitation was carried out on the basis of the Baxter's Parkland Hospital formula [START_REF] Maclennan | Anesthesia for major thermal injury[END_REF]. Early enteral feeding was established during initial resuscitation and progressively increased to reach a caloric intake of 35 kcal/kg/day. Silver sulfadiazine was used as topical antimicrobial. Surgical treatment, including excision and skin grafting, was usually started in the first week (median day 6, range day 3 to day 11) and then continued with a frequency of once or twice a week. Four patients died (mortality 28.6%). Before day 15, all but two patients (one survivor and one nonsurvivor) developed secondary infections: bacteremia (n ϭ 3, Staphylococcus aureus, Escherichia coli) or pneumonia (n ϭ 11, S. aureus, Pseudomonas aeruginosa, Proteus mirabilis, Haemophilus influenzae, E. coli, Klebsiella pneumoniae, Streptococcus pneumoniae). In this cohort of patients, we did not identify invasive wound infections or catheter-related infections. Septic shock was diagnosed in five patients between day 16 and day 28 after burn (incidence 36%). The bacterial species involved were P. aeruginosa (three cases), Bacteroides species (one case), and P. mirabilis (one case). Among those patients, three died. The fourth nonsurviving patient died before day 15 after burn due to multiple organ failure without any septic complications. Nonsurviving patients presented with a significantly higher Abbreviated Burn Severity Index score than survivors (Table 2). Patients presenting with delayed septic shock received significantly more red blood cell units and presented significantly more inhalation injury than nonseptic patients (Table 2). We did not observe any difference in TBSA, time of first surgery, number of surgery procedures, or number of infections before day 15 between these different groups of patients (Table 2).

Plasma Cytokine Concentrations. In the whole population, median values (and interquartile ranges) for sampling days were as follows: 2 (2-2), 4 (4 -5), 9 (7-9), and 12 [START_REF] Bone | Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee[END_REF][START_REF] Docke | Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: A multicenter standardized study[END_REF][START_REF] Pachot | Messenger RNA expression of major histocompatibility complex class II genes in whole blood from septic shock patients[END_REF] corresponding to day 2-3, day 4 -6, day 7-10, and day 11-15 periods of monitoring, respectively. There was no difference in sampling times regarding the different groups of patients. IL-6 concentrations were elevated immediately af-ter burn but then declined over the duration of the whole monitoring period (Table 3). Values tended to be higher in nonsurviving patients, although this was not statistically significant (Table 3). Alternatively, circulating TNF-␣ concentrations remained increased during the whole monitoring phase. Nonsurviving patients tended to exhibit higher TNF-␣ concentrations than survivors (Table 3). When considering maximal TNF-␣ value, it was significantly higher in nonsurviving vs. surviving patients (54 pg/mL vs. 29 pg/mL, p ϭ .034). Surprisingly, IL-10 concentrations were generally below the detection limit of the kit, except at day 1 (Table 3) and provided no prognostic information.

mHLA-DR Expression in Whole Patient Population. Results are presented either as percentages of monocytes expressing HLA-DR (normal Ͼ90%) or as number of anti-HLA-DR AB/C (normal Ͼ15,000), which is correlated with the number of HLA-DR molecules expressed on each monocyte [START_REF] Docke | Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: A multicenter standardized study[END_REF]. Burn trauma caused a pronounced decrease of TBSA, total burn surface area; ABSI, Abbreviated Burn Severity Index; RBC, red blood cell. a p Ͻ .05; b p Ͻ .01. Median and interquartile ranges are presented (Q1-Q3). Comparisons between groups were made using a nonparametric Mann-Whitney U test for continuous variable and the chi-square test for categorical data.

Table 3. Time course of cytokine measurements in 14 severe burn patients Days After Burn mHLA-DR expression. Although at days 0 -1 the percentages of monocytes expressing HLA-DR were not different from healthy individual values (median 86%, Q1-Q3 70.9 -93.2), as little as 2-3 days after burn, patients exhibited a major decrease of this percentage, and this was sustained through days 4 -6 or 7-10 (Fig. 1A). Similar results were observed when HLA-DR expression was presented as AB/C (Fig. 1A). When HLA-DRA and CI-ITA mRNA expressions were measured at days 4 -6 (a time period when mHLA-DR expression was already diminished in all patients), we found a significant downmodulation of mRNA in whole blood (Fig. 2). This was similar to what we had previously observed in septic shock [START_REF] Pachot | Messenger RNA expression of major histocompatibility complex class II genes in whole blood from septic shock patients[END_REF], in-dicating a transcriptional regulation of genes involved in HLA-DR expression. Daily variations in mRNA levels showed a similar profile to that of cell-surface mHLA-DR expression: a rapid decrease in the first days followed by a plateau at lower values (Fig. 2).

mHLA-DR Expression According to Mortality and Secondary Septic Shock.

After the first day, we observed that nonsurviving patients exhibited lower mHLA-DR values than survivors during the residual monitoring period (Fig. 1B), with a statistically significant difference reached at days 2-3 (36% vs. 53%, p ϭ .028) and at days 11-15 (26% vs. 48%, p ϭ .013). Similar results were observed when data were expressed as AB/C, with a statistically significant difference observed at days 2-3 (6500 vs. 12,300, p ϭ .028), days 7-10 (4000 vs. 7700, p ϭ .034), and days 11-15 (3200 vs. 7000, p ϭ .013). At the two time points just preceding the diagnosis of septic shock, patients with secondary septic shock presented with a significantly lower percentage of HLA-DR-positive monocytes: 26% vs. 58% (p ϭ .013) and 27% vs. 55% (p ϭ .007) at days 7-10 and days 11-15, respectively (Fig. 1C). We found comparable AB/C results: 3900 vs. 9100 (p ϭ .003) and 3500 vs. 7800 (p ϭ .005) at days 7-10 and days 11-15, respectively (Fig. 1C). The nadir of mHLA-DR expression occurred at days 7-10 after burn. Significantly lower nadir values were observed in nonsurviving vs. surviving pa- tients (Fig. 3, A-a andB-a) and in secondary septic shock patients vs. nonseptic patients (Fig. 3, A-b andB-b). Moreover, reduced nadir values were observed in patients exhibiting inhalation injury (Fig. 3, A-c andB-c) and increased Abbreviated Burn Severity Index score (Fig. 3, A-d andB-d). Finally, the mHLA-DR nadir occurred later in nonsurvivors or in patients who developed septic shock. It occurred at days 11-15 in nonsurvivors vs. days 4 -6 in survivors (p ϭ .17) and similarly at days 11-15 in septic patients vs. days 4 -6 in nonseptic ones (p ϭ .01).

DISCUSSION

Our findings show that severe thermal injury induced a marked decrease in mHLA-DR expression at both the protein and mRNA levels. Its persistence and intensity were associated with pejorative outcome and with the development of secondary septic shock.

Severe burn represents a frequent trauma in industrial countries and a ma-jor healthcare problem in developing countries [START_REF] Church | Burn wound infections[END_REF]. Mortality of severe burn patients has decreased substantially in the past few decades in specialized centers, due in large part to progress in the provision of optimized fluid resuscitation, nutritional support, and pulmonary care (1). However, despite these advances, severe sepsis and subsequent multiple organ failure remain a major source of burn morbidity and mortality. In fact, in patients with severe burns, 75% of all deaths are related to secondary sepsis [START_REF] Church | Burn wound infections[END_REF].

Initially, the immune response after thermal injury is dominated by the proinflammatory response but later becomes predominantly anti-inflammatory in nature. This latter condition is thought to lead to a state of immunosuppression (1). These alterations of the immune system may participate in increasing patients' susceptibility to infectious complications. Their development, however, is complex and likely involves multiple factors and processes that remain to be fully elucidated, including any deleterious effect of patients' treatments. For example, it is speculated that early excision may be protective although requiring more blood transfusions [START_REF] Ong | Meta-analysis of early excision of burns[END_REF]. In contrast, it has been also proposed that the number of blood transfusions may be associated with infectious complications [START_REF] Palmieri | Effect of blood transfusion on outcome after major burn injury: A multicenter study[END_REF]. Accordingly, we observed that patients who developed septic shock received more red blood cell units than nonseptic patients. However, to clearly delineate a relationship between septic onset and the different treatment strategies, further investigations and multivariate analysis in larger cohorts of patients are needed.

Regarding cytokines, consistent with previous observations [START_REF] Biffl | Interleukin-6 in the injured patient. Marker of injury or mediator of inflammation[END_REF][START_REF] Yeh | Changes in circulating levels of interleukin 6 in burned patients[END_REF], we found an initial elevation in IL-6 concentrations, which declined thereafter. Circulating TNF-␣ levels were also moderately increased in burn patients when compared with normal values. In contrast, IL-10 concentrations were generally found to be below the detection limit of the assay. The putative involvement of IL-10 in postburn immunosuppression remains unclear [START_REF] Yeh | Changes in circulating levels of an anti-inflammatory cyto-kine interleukin 10 in burned patients[END_REF][START_REF] Sachse | Association between reduced human leukocyte antigen (HLA)-DR expression on blood monocytes and increased plasma level of interleukin-10 in patients with severe burns[END_REF], and the present data do not support a role for this immunosuppressive cytokine. Taken together, these results suggest, as already proposed, that the occurrence of an initial proinflammatory response takes place rapidly after the initial burn injury [START_REF] Schwacha | The cellular basis of post-burn immunosuppression: Macrophages and mediators[END_REF]. However, we did not observe any correlation between cytokine concentrations and the development of severe septic complications, unfavorable outcome, other clinical confounders, or mHLA-DR measurements.

The diminished mHLA-DR expression is proposed to be a reflection of the immunosuppressive state in critically ill patients. Indeed, it is known that monocytes from patients with low mHLA-DR are unable to produce TNF and IL-1 upon an additional bacterial challenge [START_REF] Astiz | Monocyte response to bacterial toxins, expression of cell surface receptors, and release of antiinflammatory cytokines during sepsis[END_REF] or to induce T-lymphocyte proliferation in response to recall antigen [START_REF] Manjuck | Decreased response to recall antigens is associated with depressed costimulatory receptor expression in septic critically ill patients[END_REF]. Ex vivo experiments have shown that as mHLA-DR recovers, this is accompanied by the restoration of TNF release capacity (with granulocyte macrophage colonystimulating factor) ( 26) and increased T proliferation and proinflammatory cytokine production (with granulocyte colony-stimulating factor) [START_REF] Schneider | Perioperative recombinant human granulocyte colony-stimulating factor (Filgrastim) treatment prevents immunoinflammatory dysfunction associated with major surgery[END_REF]. In clinical studies, diminished mHLA-DR expression has been correlated with septic complications after various injuries: trauma, surgery, or pancreatitis [START_REF] Ditschkowski | HLA-DR expression and soluble HLA-DR levels in septic patients after trauma[END_REF][START_REF] Muehlstedt | Increased IL-10 production and HLA-DR suppression in the lungs of injured patients precede the development of nosocomial pneumonia[END_REF][START_REF] Satoh | Human leukocyte antigen-DR expression on peripheral monocytes as a predictive marker of sepsis during acute pancreatitis[END_REF][START_REF] Hershman | Monocyte HLA-DR antigen expression characterizes clinical outcome in the trauma patient[END_REF]. In children undergoing cardiac surgery, low mHLA-DR has also been identified as an independent predictor of septic complications after adjustment for clinical variables [START_REF] Allen | Early postoperative monocyte deactivation predicts systemic inflammation and prolonged stay in paediatric cardiac intensive care[END_REF]. In septic shock patients, re-duced mHLA-DR expression has been recently demonstrated to be an independent predictor of mortality in multivariate analysis, including various clinical confounders [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF]. Only fragmented data are available regarding mHLA-DR expression after severe burn, and these were not collected using a standardized protocol [START_REF] Sachse | Association between reduced human leukocyte antigen (HLA)-DR expression on blood monocytes and increased plasma level of interleukin-10 in patients with severe burns[END_REF][START_REF] Gibbons | Reduction in HLA-DR, HLA-DQ and HLA-DP expression by Leu-M3ϩ cells from the peripheral blood of patients with thermal injury[END_REF][START_REF] Zwadlo-Klarwasser | Influence of severe burn injury on the expression of RM 3/1 and HLA-DR antigens in human blood monocytes[END_REF]. However, these data tended to indicate that low mHLA-DR expression was associated with secondary septic events. In the present study, we examined mHLA-DR expression in severe burn patients according to these recently standardized protocols [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF][START_REF] Docke | Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: A multicenter standardized study[END_REF], and we confirmed our observations at the mRNA level. While proinflammatory cytokine response rapidly developed after burn, mHLA-DR expression initially was not different from normal values. Then, we observed as early as 2-3 days after ther- mal injury a severe decrease of mHLA-DR expression. This loss of mHLA-DR expression is suggestive of a counterregulatory response. It might be thought of as a consequence of homeostatic maintenance aimed at controlling the inflammatory balance. However, this reduction in monocyte HLA-DR expression was not accompanied by increased circulating IL-10 concentrations, even though this cytokine has been found to be associated with low mHLA-DR in severe sepsis/septic shock [START_REF] Lekkou | Cytokine production and monocyte HLA-DR expression as predictors of outcome for patients with community-acquired severe infections[END_REF][START_REF] Monneret | The anti-inflammatory response dominates after septic shock: Association of low monocyte HLA-DR expression and high interleukin-10 concentration[END_REF]. This suggests that while a similar process (an mHLA-DR loss) occurs in both situations, a different mechanism may sustain the observed suppression after thermal injury. As an aside, we have also noted that the kinetics of mHLA-DR expression seem to be different in burn patients as opposed to septic shock patients. In septic shock [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF], the nadir mHLA-DR value occurred in the first 2 days, whereas in the study of thermal injury, the nadir mHLA-DR value was observed at days 7-10. Here again, while this implies that processes underlying these changes in burn differ from septic shock, specific reasons for these differences remain to be investigated.

As observed after septic shock [START_REF] Tulzo | Monocyte human leukocyte antigen-DR transcriptional downregulation by cortisol during septic shock[END_REF][START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF], the decreased monocyte HLA-DR expression provided prognostic information. It was more pronounced and persistent in nonsurviving patients and in patients developing a secondary septic shock. From days 4 to 6, mHLA-DR values increased in nonseptic patients whereas they remained very low in patients with severe septic complications. Most important, at days 7-10 and 11-15, there were significant differences between patients without septic shock and those who went on to present with septic shock after day 16. Last, lower mHLA-DR values were observed in patients with increased Abbreviated Burn Severity Index score and with inhalation injury, which constitute wellknown risk factors after burn [START_REF] Fitzwater | The risk factors and time course of sepsis and organ dysfunction after burn trauma[END_REF]. The severity of the initial burn insult may thus be associated with the intensity of the subsequent immunosuppressive state. All these putative correlations need to be further investigated in multicenter studies including large numbers of patients. For this purpose, a standardized assay for the measurement of mHLA-DR expression is now validated, and preliminary cutoff levels have been proposed: a value Ͻ5000 AB/C identifies patients with immunosuppression and enhanced risk of developing infectious complications [START_REF] Docke | Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: A multicenter standardized study[END_REF]. In previous studies, a threshold of 30% of HLA-DR positive monocytes was regularly found to differentiate survivors from nonsurvivors after severe sepsis/ septic shock [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF][START_REF] Lekkou | Cytokine production and monocyte HLA-DR expression as predictors of outcome for patients with community-acquired severe infections[END_REF][START_REF] Docke | Monocyte deactivation in septic patients: Restoration by IFN-gamma treatment[END_REF]. Accordingly, in the present study, using either 30% or 5000 AB/C for cutoff values, nonsurviving patients and patients developing secondary septic shock could be rapidly identified. In the other patients, mHLA-DR expression never declined under these thresholds.

CONCLUSIONS

We observed that after severe thermal injury, HLA-DR expression is severely decreased on circulating monocytes and that the duration and the intensity of this decrease are associated with severe septic complications and pejorative outcome. In this situation, cytokine measurements did not provide any prognostic information. After confirmation of these preliminary observational results in larger cohorts of patients (including functional testing, such as TNF intracellular staining or in vitro TNF release after stimulation), mHLA-DR expression measurement could be used as a monitoring tool to detect as early as possible immunocompromised patients who might benefit from interventional therapies that could reduce the risk of developing severe secondary septic events.

:

  Patients were immunologically monitored during 15 days. We quantified mHLA-DR expression with a standardized flow cytometry protocol. Every patient presented with decreased mHLA-DR expression at days 2-3 after burn. Then, from days 4 -6, this expression increased in patients who would survive whereas it remained low in nonsurvivors. As early as days 7-10 after burn, patients who were going to develop secondary septic shock exhibited significantly lower mHLA-DR expression in comparison with patients recovering without severe septic complications. Using quantitative reverse transcriptasepolymerase chain reaction, at days 4 -6, we found that the RNA level of the nonpolymorphic HLA-DRA chain and the transcription factor class II transactivator were also significantly decreased compared with healthy controls; however, plasma cytokines (interleukin-6, tumor necrosis factor-␣, and interleukin-10) did not provide any significant prognostic information. Conclusions: Severe burn injury induced a marked reduction in mHLA-DR expression at both protein and messenger RNA levels. Its persistent decrease was associated with mortality and the development of septic complications. (Crit Care Med 2007; 35:1910-1917)

Figure 1 .

 1 Figure 1. Time course of monocyte human leukocyte antigen-DR (HLA-DR) expression in severe burn patients. Median and interquartile ranges (Q1-Q3) are presented. Results are expressed as percentages of monocyte expressing HLA-DR (left panels) and as numbers of anti-HLA-DR antibodies bound per cell (AB/C) (right panels). The nonparametric Mann-Whitney U test was used for comparison between groups. *p Ͻ .05; **p Ͻ .01. A, monocyte HLA-DR expression in the whole burn population (n ϭ 14 severe burn patients). B, monocyte HLA-DR expression regarding the outcome (n ϭ 10 surviving patients, open columns, and n ϭ 4 nonsurvivors, filled columns). C, monocyte HLA-DR expression regarding the development of septic shock between day (D) 15 and day 28 after burn injury (n ϭ 8 nonseptic patients, open columns, and n ϭ 5 septic patients, filled columns). Dashed lines represent healthy individual values.

Figure 2 .

 2 Figure 2. Messenger RNA (mRNA) level of monocyte human leukocyte antigen-DR (HLA-DRA) (A) and class II transactivator (CIITA) (B) in peripheral blood of severe burn patients measured by quantitative reverse transcription-polymerase chain reaction. The mRNA level was normalized to that of the housekeeping gene peptidylpropyl isomerase B. The upper box plot represents mRNA levels in patients (n ϭ 9) at days 4 -6 after burn injury and healthy controls (n ϭ 29). The Mann-Whitney nonparametric U test was used for comparison between groups. **p Ͻ .01. The lower kinetics represent time-related individual variation of mRNA level in three burn patients, representative of the whole cohort.

Figure 3 .

 3 Figure 3. Box plot representations of the nadir of monocyte human leukocyte antigen-DR (HLA-DR) expression in severe burn patients. Results are expressed as percentages of monocyte expressing HLA-DR (A) and as numbers of anti-HLA-DR antibodies bound per cell (AB/C) (B). The Mann-Whitney nonparametric U test was used for comparison between groups. *p Ͻ .05; **p Ͻ .01. a, nadir of monocyte HLA-DR expression in surviving (open box plot, n ϭ 10) and nonsurviving (filled box plot, n ϭ 4) burn patients. b, nadir of monocyte HLA-DR expression in burn patients developing a septic shock (filled box plot, n ϭ 5) and nonseptic patients (open box plot, n ϭ 8). c, nadir of monocyte HLA-DR expression regarding the occurrence of an initial inhalation injury in severe burn patients. Filled box plot, patients with inhalation injury (n ϭ 3); open box plot, patients without inhalation injury (n ϭ 11). d, nadir of monocyte HLA-DR expression regarding Abbreviated Burn Severity Index score (ABSI). Open box plot, patients with an ABSI score Ͻ10 (n ϭ 7); filled box plot, patients with ABSI score Ն10 (n ϭ 7).

Table 1 .

 1 Demographic and clinical characteristics of the 14 severe burn patients included in the study

							Length
					ABSI		of Stay,		Septic
	Patient	Age, yrs	Gender	TBSA, %	Score	Inhalation	Days	Survival	Shock
	1	22	M	32	7	No	29	S	No
	2	18	M	60	9	No	32	S	No
	3	56	F	80	15	Yes	38	NS	Yes
	4	56	F	48	10	Yes	22	NS	Yes
	5	34	M	50	9	No	68	S	No
	6	47	M	51	13	No	91	S	No
	7	45	F	80	10	No	27	NS	Yes
	8	40	F	50	9	No	45	S	Yes
	9	74	M	41	10	No	24	S	No
	10	51	M	45	9	No	25	S	No
	11	27	M	45	8	No	21	S	No
	12	44	F	50	9	Yes	66	S	Yes
	13	51	M	85	12	No	11	NS	-
	14	40	M	85	12	No	17	S	No
	TBSA, total burn surface area; ABSI, Abbreviated Burn Severity Index; S, survivor; NS, nonsurvivor.

Table 2 .

 2 Demographic and clinical characteristics of the 14 severe burn patients regarding issue and development of septic shock between day 15 and day 28 after burn

			Gender,					Time of First	No. of	No. of
			Male/	TBSA,	ABSI	RBC,	Inhalation,	Surgery,	Surgical	Infections	Length of
		Age, yrs	Female	%	Score	Units	Yes/No	Days	Procedures	Before Day 15	Stay, Days
	Survivors (n ϭ 10) 40 (29-46)	8/2	50 (45-51) 9 (9-9.75) 16 (10-37)	1/9	6 (4-8)	3.5 (3-4)	1 (1-1)	31 (24-61)
	Nonsurvivors	53.5 (50-56)	1/3	80 (72-81) 11 (10-13) a 30 (20-44)	2/2	5.5 (3.5-7)	4 (2.5-4) 1 (0.5-1.5) 24.5 (19-30)
	(n ϭ 4)
	Septic shock after
	day 15
	No (n ϭ 8)	37 (26-48)	8/0	47.5 (44-53) 10 (9-10) 12.5 (10-19)	0/8	7.5 (4.5-9)	3 (2.5-4) 1 (1-1)	27 (23-41)
	Yes (n ϭ 5)	45 (44-56)	0/5 b	50 (50-80) 9 (9-11)	57 (36-68) b	3/2 a	4 (3.75-5.5) 4 (4-4)	1 (1-2)	38 (27-45)

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ACKNOWLEDGMENTS

Dr. Monneret thanks the Immunology Laboratory staff at Hôpital Neurologique (Lyon, France) for their help in supporting his clinical research group on stressinduced immunosuppression.


Valérie-Inés De

La Ville

Nathalie Nicol

Shopping from a child's perspective: an anxiety-generating experience?

Purpose:

The aim of this communication is to give some insights about how the children, aged between 4 and 12, live theirs shopping experiences. The research enables us to reveal the 'shopscapes' of young consumers.

Design / Methodology / Approach: Built on a comprehensive approach, our research is based on data collected from 15 pairs of siblings during interviews where the children collectively produced a draw of a shop they chose. The drawing was a support for the verbal exchange and doing it in pairs encourages each child to clarify his or her intentions about the drawing.

Findings:

In this paper, by developing four arguments, we demonstrate that shopping is an anxiety-generating experience when children are concerned.

Research limitations / implications: Our data were collected among children who were asked about the buying of a present for a friend's birthday. Children have consequently given information about mainly toy's stores.

Practical implications:

There are great opportunities for retailers to modify this feeling by welcoming children in the stores and enhancing them to behave as consumers.

Social implications:

There could be opportunities for retailers to invest the consumption education area.

Original / Value: Our research is child-centered and thanks to the concept of shopscapes, we reveal new and surprising insights about children' shopping experiences.

Because of their increasing spending power, children are targeted by retailers who consider them as a new way to enlarge their existing markets. Children are in the stores from their youngest age, with their parents, with friends, … or alone sometimes. The first research conducted about children as consumers have focused on the products bought by children, their preferred stores, their influence on the family's buying and the skills they have to master to become competent consumers [START_REF] Mcneal | The child consumer, a new market[END_REF][START_REF] Mcneal | Children as Consumers, Insights and Implications[END_REF][START_REF] Mcneal | Kids as Consumers, A Handbook of marketing to children[END_REF][START_REF] Wells | Direct observation of purchasing behavior[END_REF][START_REF] Fady | L'observation directe des comportements des clients en magasin libreservice[END_REF][START_REF] Reece | Children and shopping: some public policy questions[END_REF]Brée, 1993 ;[START_REF] Acuff | What kids buy and why, the psychology of marketing to kids[END_REF][START_REF] Mcneal | A study of children's consumer socialization process in Hong-Kong over a five-year period: Income, spending and saving[END_REF][START_REF] Roedder- | Children Consumer Socialization of -A Retrospective look at twenty-five years of research[END_REF][START_REF] Valkenburg | The development of a child into a consumer[END_REF][START_REF] Ozgen Ö | An analysis of child consumers in Turkey[END_REF][START_REF] Engelhart | Store visits and information sources among urban Chinese children[END_REF]…). The aim of our research is about understanding how children, from their point of view, live their shopping experience.

In the first part, we present the academic literature about children in the retails areas. Some insights are given and but doesn't help us to understand what is the experience lived by children when they are shopping. That's why we have to build a new way of approaching the shopping experience lived by children. We present this concept and our methodology in a second part. We gained very rich and strong data about children in the stores. We discuss in this paper the sole results we had about anxiety felt by children while shopping.

Children in the stores:

Children accompany their relatives in the stores and start to behave on the market from their youngest age. In their research, [START_REF] Dely-Gray Z | Perception of young children of the ideal shopping experience[END_REF] show how young children, aged between 3 and 6, are very active and involved in their parents' shopping process and that they appreciate this shared activity. Children consequently gain information during their visits about the store and its features, the products and the brands, the packaging, the store's layout. Throughout observation and participation in the buying process, they develop their consumer skills [START_REF] Mcneal | Kids as Consumers, A Handbook of marketing to children[END_REF]Roedder-John, 2001) and become more and more competent as consumers. The stores can then be seen as places and spaces that contribute to the socialization of the children into consumers.

Kids have positive feelings about retailers and shops they perceive as "warehouses of satisfactions" (McNeal, 1992, p. 108) where a lot of products are available for buying and where children can discover new brands and new products. The profusion of goods can arouse children's envy and drive them to adopt negative behaviors called pester power such as crying and nagging [START_REF] Valkenburg | The development of a child into a consumer[END_REF][START_REF] Nash | Pester Power: it's all in "the game[END_REF]. [START_REF] Valkenburg | The development of a child into a consumer[END_REF] show that 70% of the parents of 5-years-ols children have experienced those negative behaviors while shopping with their children. [START_REF] Ayadi | Exploring children's response to store atmosphere[END_REF] explain that the hedonic dimensions of a store can enhance conflicts between children and parents because children will be tempted to play and explore the store and won't be as much cooperative as in a more basic store.

To help the parents to manage these situations, retailers can offer edutainment activities (Feenstra et al., 2015, p. 48), which can be defined as places where children can play and learn throughout activities (workshops where they can create, customize products and cook, birthday parties and events organized by brands) while their parents are shopping. [START_REF] Andrieu | Hypermodernité et distribution : le cas du West Edmonton Mall[END_REF] explain how the West Edmonton Hall propose to the customers many playgrounds and activities for their children, in order to make customers feel free in the mall, free to walk around and to buy whatever they want. It is a kind of "social contract" between the mall, the retailers and the customers (Andrieu et al, 2004, p. 41), because children can be seen as well as barriers as incentives during shopping (Heliès-Hassid, 2007).

As stores can be seen as spaces conceived and designed by adults and for adults [START_REF] De | Responsabilité sociale de l'entreprise et responsabilité du chercheur en gestion : essai de mise en abyme à propos de la consommation enfantine[END_REF], children try to re-appropriate the commercial spaces by giving them new meanings (Ironico, 2012). Through games but also through the products and the commercial stimuli, children assign to the stores new features. Because they are creative and thanks to their physical capabilities, they access to spaces which are not conventionally reachable such as windows or cash desk. The space offered by the stores are negotiated by the children as space they can discover and where they can play. To Ironico (2012), this kind of behaviors is also a way for the children to get socialized in the stores because it contributes to enhance the children's familiarity of the stores.

Children have a spending power through their pocket money; they can influence the choice of a product or of a brand and they represent for the retailers, their future customers [START_REF] Mcneal | Kids as Consumers, A Handbook of marketing to children[END_REF]. If stores and retailers are familiar to the children, even to the youngest of them, there is a need for both academic researchers and retailers, to understand how the childre n live their shopping experiences.

Methodology of the research:

To gain access to and to grasp children's shopping experiences, it is crucial for researchers to put them at the center of the research, by considering them as valid informants who create the research with the researcher [START_REF] Christensen | Working with ethical symmetry in social research with children[END_REF]. In our research, we wanted to have a deep child-centric approach to hear children's voices [START_REF] Banister | Exploring innovative methodologies for childcentric consumer research[END_REF][START_REF] Ezan | Positionnement interprétatif et ethnographique: pou r un nouveau regard porté sur l'enfant consommateur[END_REF]. This approach can challenge the researcher and consequently, the researcher has to anticipate about the relationship to establish with the children. This question can't be avoided by the researcher who intends to gain access to the childish cultures, to hear the children's voices and to reduce (ideally to cancel) the power balance between the researcher and the children. Consequently, we enhance to develop a positive and friendly relationship (Fine and Sandström, 1988) with the children and to exchange with them in a collaborative and trustful manner. As suggests [START_REF] Raffety | Minimizing social distance: participatory research with children[END_REF], we consider ourselves as a "cultural novice" and try to reduce the cultural distance the children and us.

Our research develops the concept of 'shopscapes' as a conceptual tool to understand children's experience of shopping. Based on the 'scapes' (Appaduraï, 1996), the 'foodscapes' [START_REF] Brembeck | Foodscapes and children's bodies[END_REF][START_REF] Wenzer | Eating out and practices among swedish youth, Gothenburg area foodscapes[END_REF] and the French Theory [START_REF] Deleuze | Capitalisme et Schizophrénie[END_REF][START_REF] Deleuze | Critique et Clinique[END_REF], the 'shopscapes' are the imaginary geographies each person or group of people builds based on his or her daily experiences and practices with reference to retail environments and activities [START_REF] Nicol | The shopscapes: a tool and a methodology to better grasp kid's experiences of retailing[END_REF]. By definition, 'shopscapes' are built unconsciously by each person, are dynamic and culturally grounded.

Therefore, the researcher has to design innovative methodologies to collect the data to enable the non-verbal techniques to transcend the verbal and adult-centric techniques [START_REF] Corsaro | The sociology of childhood[END_REF][START_REF] Robinson | Introduction: developing creative methods with children and young people[END_REF]. [START_REF] Punch | Research with children: the same or different from research with adults?[END_REF] also recommends to use techniques that are specific to children, such as drawing [START_REF] Ezan | Le dessin comme langage de l'enfant : Contributions de la psychologie à l'enrichissement des méthodolo gies de recherche appliquées aux enfants consommateurs[END_REF]. Child-centered-techniques encourage the children to share their own images and representations they built about their lives and experiences [START_REF] Corsaro | The sociology of childhood[END_REF].

Built on a comprehensive approach, our research is based on data collected from 15 pairs of children during interviews where children draw a store they chose. Children were proposed a script about the buying of a birthday present for a friend of them. Working in pairs, with a child older than the second one, allows us the work in the zone of proximal development (ZPD) [START_REF] Vygotsky | Thinking and Speaking[END_REF][START_REF] Vygotsky | Thinking and Speaking[END_REF][START_REF] Vygotsky | Mind in society, The development of higher psychological processes[END_REF] and requires the two children exchange verbally and explicit theirs intentions during the shared drawing activity [START_REF] Bodrova | Les outils de la pensée, l'approche vygotskienne dans l'éducation à la petite enfance[END_REF]. The researcher consequently collects rich and fruitful data during (1) the exchange between the two children and (2) the exchange between the children and her.

To have pairs of children, with one older than the other, the first pairs of children we interviewed was constituted of siblings. We saw that facing siblings really reduce the power balance: the researcher is alone and facing two children who well know each other and get on well when facing someone they do not know. We consequently decide to collect our data exclusively among siblings.

In our research, drawing is an excuse to make children speak about their shopping activities and experiences. The drawing has the advantage of structuring the verbal exchange and to get the children focused on the activity [START_REF] Yuen | It was fun…I liked drawing my thoughts" Using drawings as a part of the focus group process with children[END_REF]. Drawing in a group of two is moreover a way to renew the activity [START_REF] Bodrova | Les outils de la pensée, l'approche vygotskienne dans l'éducation à la petite enfance[END_REF]. Upon the typology of drawing proposed by [START_REF] Ezan | Le dessin comme langage de l'enfant : Contributions de la psychologie à l'enrichissement des méthodolo gies de recherche appliquées aux enfants consommateurs[END_REF], the oriented-drawing was chosen to give the children more freedom in their drawing: organization of the drawing, viewpoint, type, size and name of the store…When collecting data via oriented drawings, the number of drawings is reduced and the presence of the researcher during the drawing activity is required in order to have a real verbal exchange with the informants.

All the interviews were written down and the comparison between all of all them was the key to get the meanings children attribute to their shopping experiences. As the drawing activity was just an alibi and as it is a very risky activity to interpret, the drawing itself has not been analyzed. [START_REF] Cambier | Ce que dessiner signifie[END_REF] warns the researchers using drawing to collect data with children, about the risk to consider the drawing as an autonomous object, independent from the context where it has been produced. She argues that the circumstances in which the drawing was realized and the process which conducted the children to draw, have to be taken into consideration when analyzing.

The 15 shopping experiences the children we met let us discover, happen in different kind of stores: 10 big toys stores with national or international brand-names, 2 supermarkets, 1 sport items store, 1 bazaar and 1 bookshop.

Findings:

The interviews realized with the children shows that children integrate in their 'shopscapes' four components as specific parts of the imaginary geographies they develop about stores and retail.

The thieves are in the place:

In the stores described by the children, there are thieves whose intention is to take away some products of the store without paying them: "thieves" "the bad", "the bandit".

And some children can give a lot of details about this phenomenon: they know that valuable products and expensive ones can attract thieves.

Arnaud (boy -7 years old): You know, the ones who steal items because they are valuable...they take the goods and run away!' Children know that the stores try to face this threat. Therefore, the stores have many solutions at their disposal and children can describe those solutions: the presence of security guards, the use of antennas and of close-circuit cameras. They explain how those solutions operate: the antenna can ring when a product is stolen, the security guard can check the customer's bag or look on the screen the images of the CCTV. They show a real consciousness of the technical and human solutions in the shop to prevent theft. For the children, it is a part of the store a s well as the products or the tills. They are very familiar with this equipment and have internalized it presence in the stores. Amélie (girl -10 years old): And then, there is something here but there is often a man just here… Researcher: There is a man… What is he doing? Amélie (girl -10 years old): He is watching the entrance and the exit.

Marin
Arnaud (boy -7 years old): … And also the camera. In fact, she is filming the people to check there is no bandit.

A specific behavior is expected from children in the store:

Children also demonstrate how well they have integrated that a specific behavior is expected when they entered a store. The entrance of the stories very often materialized by a fence on which there a no-entry way (See Table 3). This equipment acts as a cognitive and technical discrimination and conveys the idea of control and interdiction [START_REF] Cochoy | Progressive Grocer, ou la « petite distribution » en mouvement[END_REF]. It is an incentive to behave in the appropriate manner. If children do not behave in the appropriate and expected manner within the shop, they know that the security guards can warn them. They remember situations where being warned or situations where they know that the security guards are busy and cannot see them (for instance in a store where security guards mainly stay on the ground floor and children can play hideand-seek on the first floor).

Mathilde (girl -12 years oldremembering a shopping trip with her mummy and they didn't find in the store the product they were looking for): So we wanted to get out of the store. And it was forbidden. So we say 'no worry, we go where there are fences'. And then a man came to us and says we were not allowed to go that way. So mummy got angry and said 'I'm fed up with the obligation of going that way…'. And then, the security guard let us go… Amélie (girl -10 years old): In fact, upstairs, we have fun. We are playing a sort of hide-andseek; it's too funny! Researcher: You are playing hide-and-seek in the garments? Amélie (girl -10 years old): Yes Marine (girl -8 years old): And what is nice, it's that the guard men don't come very often upstairs! So it's easy! Amélie (girl -10 years old): There is only one and he is watching the entrance Marine (girl -8 years old): And sometimes there is one walking around. But just one. Once, one of them came and told: "hey girls, stop playing!"

All the fences at the entrance and the exit of the store are a way to channel the customers in the store but also to direct their behaviors. Going through the entrance fence of a store implies for the children the acceptance that a specific behavior is expected from them. There are lots of rules to respect when shopping in a store: to be polite, to pay the items, to respect the fence and the flows…

A labyrinth where you can easily get lost:

The children also describe the stores as a labyrinth where it seems easy to get lost. The shelves and the aisles organize a kind of maze customers have to go through in order to discover the store's offer. Children have stated that risk during the interviews but also in their drawings (see Tables 4 and5). Mathilde (girl -12 years old): I really find that those stores are a kind of labyrinth. To get to this point, we have to go around the whole store Fear of getting lost is really present during some interviews. All the stores drawn by the children are quite big. Even the bookshop is a 4-floor building. And two children have drawn the welcome desk where someone can be called if you gest lost in the store (see Table 5). Stores are considered as dangerous places for children (James et al., 1998, p. 11), where their primary evil-instincts are reactivated. Children are tempted to disobey to their parents when they are shopping together. Are they tempted to explore the store by themselves? Consequently, they can get lost…

Table 5: Calling at the welcome desk

Eloïse (girl -12 years old): Well, it's the welcome desk…. And if the store opens or closes, or if there is a problem: « Mister …Madam… is expected at the welcome desk ». I'm going to draw a speech bubble.

Children's books reinforce the risk of getting lost in a store. The theme of children lost in stores is often concerned in children's books. Children's books about children getting lost always take place in stores (see Table 6). In their research in 2016 about representations of shopping in the children's books, Badot et al. show that large stores are described and presented to the children as "places of stress and distress" (p. 982) where children can encounter dangers among which the risk of getting lost or the risk of being separated from their parents.

Table 6: Some children's books about getting lost in a store

The absence of contact with people:

In their 'shopscapes', the children we met describe that customers are also present in the stores. Among these customers, friends of them can also be shopping in the same store. Concerning the staff working in the store, the children identify easily the checkout assistants. Paying the product they have chosen is an obligation and consequently, unless of a selfcheckout-system, the children have to interact with the checkout assistants. Except this category of staff, children have hardly been in position of giving information about sales assistants. Some children even consider there is no sales assistant in the stores.

Beatrice and Michel hate department stores. Today, while they go to the bathroom, Mummy and Daddy have disappeared! Where are they? Beatrice and Michel try to join the exit but they arrive too late: the store is closed. The lights are turned off and there is nobody in the store. They are prisoners… Max et Lili have lost their gran-mother in a department store; Hopefully Lili has the idea to go back to the point when they get separated…but Max doesn't want to follow to sister… Today, Tom goes to shop with her mother. In the department store, there are a lot of people and lots of products to look at. Tom has an obsession: find the new trousers he wants. But when he turns around, her mother is no more closed to him. He runs in all the department store. Her mother has disappeared! Tom is lost and is really afraid among all those unknown people… Lulu-Grenadine is having fun in the department store. But, suddenly, she cannot see her mother! MUMMYYYYYY ! Researcher: Is there staff working in the store? Jules (boy -5 years old): No.

Children, when shopping interact with the checkout assistants and have noticed the presence of the security guards. But they have not noticed any sales assistant in the store. We conclude that this situation is the consequence of a lack of interaction between children and sales assistants. Children are invisible for the retailer's staff and maybe, for the retailers also. Are sales assistants trained to discuss, to interact and to help children when they are shopping? This absence of link and of interaction could lead to reinforce the feeling of anxiety felt by children when they are in stores. The stores are big and there is nobody to help them or to take care of them.

Discussion:

Shopping, as far as children are concerned, is an activity where people can get lost in the stores, where children can meet thieves but no sales assistant and where a certain kind of behaviour is expected by the store in order to consider the child as a "normal" shopper. Retail environments express moral injunctions to children in order them to adopt an appropriate behaviour in the stores and especially to prevent them from shoplifting. Shopping in these conditions is an anxiety-generating experience for children. Shops try to « discipline », in [START_REF] Foucault | Surveiller et punir, Naissance de la prison[END_REF] acceptation, the young shoppers and stores become micro-institutions of power [START_REF] Foucault | Surveiller et punir, Naissance de la prison[END_REF] by editing norms of behaviours and demanding people to adopt them.

It seems that children when shopping are exploring a potentially dangerous and mysterious space. They are like explorers who are discovering a new land, following Cochoy's comparison, where the market is a new land to discover and the shopper, a traveller discovering a new space (Cochoy, 2002, p. 5).

Can a child learn to become a consumer when acting in such a stressful environment? How could the retailers welcome the children and offer them the conditions to learn to become a consumer? Shops are conceived by and made for adults [START_REF] De | Responsabilité sociale de l'entreprise et responsabilité du chercheur en gestion : essai de mise en abyme à propos de la consommation enfantine[END_REF] and as [START_REF] Cook | Spatial biographies of children's consumption[END_REF] argues, it is retailer's responsibilities to adopt the child perspective ('pediocularity') and to see and examine the stores with children's eyes. It's not only a matter of edutainment activities [START_REF] Feenstra | Edutainment experiences for children in retail stores, from a child's perspective[END_REF] proposed to the children to get them busy whilst their parents are shopping; it's about allowing the children to act, to behave as a creative consumer in the retail areas and to make then possible the stores become appropriated by the children. Through events and specific displays, the stores can offer to children an opportunity of acting and then to get appropriated by the children. Through awareness campaign and maybe specific trainings, the store's staff can participate actively in better welcoming children by considering children and recognizing them as valuable consumers.

To reinforce a child-centric culture, retailers could become partners in the consumption education programs as [START_REF] Drasigh | Les centres d'éducation à la consommation responsable, L'expérience des Coop en Italie[END_REF] describes. The mission of the Italian cooperative system 'Coop' is to help people to understand the "rules in the merchant world" (Drasigh, 2004, p. 199). The cooperative system has not only commercial and financial objectives; it promotes responsibility, solidarity and transparency. In the Italian 'Coop', those values are implemented in a program to help the teachers to teach consumption to children. The retailer offers its shops as real playgrounds where children can discover, learn and experiment consumption. The retailer also proposes its expertise to the teachers and has developed programs teachers can choose, to deploy all the skills children need to master to become consumers that are more competent. The retailer become then a real and engaged partner in training children to consumption.

Even if our research is much contextualized (children were in the situation of buying a birthday present for a friend of them and most of the situations they described and drawn happened in toys' stores), our data reveal that shopping is for children an anxiety-generating experience. Retailers are responsible of this feeling and they have opportunities to act in a responsible and ethic manner, to diminish or extinguish, this negative feeling. Welcoming children in the stores, recognizing them as valuable consumers, giving them the opportunity to behave and to interact with the sales assistants are quite simple actions retailers can implement in their stores.

  (boy -6 years old): And now I'm going to draw another thieve! Table 2: The thief is arriving to the store; he is on the parking but a policewoman has noticed his presence: Marin (boy -6 years old): She is watching the entrance. No she is a policewoman! Researcher: And what is the policewoman doing at the entrance of the shop? Marin (boy -6 years old): Because in fact, she is watching when the shop is about to close. Then if thieves are searching for… want to steal toys…She can see them in 5 minutes! From the end of the corridor, she has a spyglass, binoculars…then she looks and she can see the thieves immediately!

Table 2 :

 2 Antitheft Antennas in the shop (Extracts from interview and drawing N° 2) Interview: Marie (girl -9 years oldlooking at what her younger sister is drawing): What is that? Louise (girl -4 years old): But you know…it's when there is a thief, well, you can hear « beep beep ». Marie (girl -9 years old): Thieves? Louise (girl -4 years old): Yes, if there are thieves! Drawing : Louise (girl -4 years old): But you know, it's when there is a thief, and well you can hear "beep beep" Mathilde (girl -12 years old): Oh he is watching the entrance! Researcher: So he is watching the entrance… Mathilde (girl -12 years old): In case there are some thefts maybe. In case people open the…. How can I say? The boxes?

Table 4 :

 4 Stores as labyrinths Léa (girl -12 years old) Mathieu (boy -8 years old) Mathilde (girl -12 years old) Luc (boy -10 years old) Marc (boy -6 years old): And then the third, there is something with car and circuits. And then the fourth first, well there are spinning-tops. Marine (girl -8 years old): Oh yes… it's like this, it's true. Sorry, I get lost. Mathilde (girl -12 years old): We are going to draw a labyrinth I believe…

de

  Saint Mars D. et Bloch S. (2004), Max et Lili se sont perdus, Editions Alligram. Gillot L. et Durbiana L. (2014), Lulu-Grenadine se perd dans un grand magasin, Editions Nathan. Le Masne C. et Bawin M.A. (1998), Tom est perdu, Editions Mango. Cau V., Phipson J et Kiefé L. (2013), Une nuit au grand magasin, Editions Bayard Jeunesse.


Catherine Fernandez
email: catherine.fernandez@imbe.fr

Amélie Saunier

Henri Wortham

Elena Ormeño

Magali Proffit

Caroline Lecareux

Stéphane Greff

Dao Van Tan

Mai Sy Tuan

Huynh Duc Hoan

Bui Nguyen

The Kiet

Dounia Dhaou

Virginie Baldy

Anne Bousquet-Mélou

Mangrove's species are weak isoprenoid emitters

Keywords: BVOC emissions, Isoprenoids, Mangroves, Flooded ecosystems, extreme environments Highlights

Mangroves are ecosystems interfacing terrestrial and marine environments submitted to extreme abiotic factors (e.g. anoxia, flooding, salinity) producing stress on vegetation. Due to these stresses, we hypothesized that mangroves potentially emit biogenic volatile organic compound (BVOC), particularly isoprenoids as they are defense compounds. Despite mangroves cover only about 5% of the forest areas of the world, their emissions could impact air quality at the continental-ocean interface. As a result, it is important to fill the gap in the knowledge about BVOC emissions from the canopy of the major mangrove trees. The aim of this study was thus to screen isoprenoid emissions of the mangrove species. In this study, we sampled isoprenoid emissions of 15 species foliage among the 38 core species existing in the Indo-West Pacific (IWP) and the Atlantic Est Pacific (AEP) regions.

Sampling was performed using a branch-bag dynamic enclosure system and analyzed with gas chromatography coupled to mass spectrometry. Our analysis showed that mangrove tree species are very low emitters suggesting that mangrove ecosystems would not strongly influence atmospheric chemistry and air quality.

Introduction

Vegetation (croplands, shrublands, woodlands etc) is the most important source of volatile organic compound (VOC) as it accounts for 1015 TgC.yr -1 , about 90% of total biogenic emissions [START_REF] Guenther | A global model of natural volatile organic compound emissions[END_REF]. VOCs gather a wide variety of chemical compounds having a high vapor pressure (at least 0.01 KPa) at ambient temperature. Among them, isoprenoids (including isoprene but also monoterpenes and, to a lesser extent, sesquiterpenes), as well as highly volatile compounds such as methanol, acetaldehyde, or formaldehyde, are mainly emitted (e.g. Saunier et al., 2017a). These compounds play a major environmental role on air quality and indirectly modulate climate conditions. Indeed, in the presence of sunlight and nitrogen oxides (NOx), biogenic VOCs (BVOCs) contribute to the formation of tropospheric ozone [START_REF] Fehsenfeld | Emissions of volatile organic compounds from vegetation and the implications for atmospheric chemistry[END_REF]) and secondary organic aerosols (Claeys et al. 2004;Ng et al. 2006), which in turn act on cloud formation and albedo [START_REF] Makkonen | BVOCaerosol-climate interactions in the global aerosol-climate model ECHAM5. 5-HAM2[END_REF]).

Among BVOCs, isoprenoids are therefore key compounds for chemistry-model-transport and climatechange models that aim to forecast photo-chemical pollution episodes and their contribution in the future climate change [START_REF] Makkonen | BVOCaerosol-climate interactions in the global aerosol-climate model ECHAM5. 5-HAM2[END_REF]. These two types of models rely on thousands of publications that describe, since the 1980s, BVOCs emissions from plant species [START_REF] Keenan | Process based inventory of isoprenoid emissions from European forests: model comparisons, current knowledge and uncertainties[END_REF], their dependence on environmental factors such as light and temperature (Guenther et al. 1993). Since isoprenoid emissions are species-specific, it is crucial to investigate isoprenoid emissions from the main vegetal formations at the global scale. For this purpose, temperate environments (e.g. Mediterranean, continental, oceanic) have been widely studied in terms of BVOCs emissions [START_REF] Owen | Volatile organic compounds (VOCs) emitted from 40 Mediterranean plant species: VOC speciation and extrapolation to habitat scale[END_REF][START_REF] Ormeno | Water deficit stress induces different monoterpene and sesquiterpene emission changes in Mediterranean species. Relationship between terpene emissions and plant water potential[END_REF]Saunier et al. 2017b), but plant species from extreme environments such as mangroves have been clearly neglected [START_REF] Rinnan | Plant volatiles in extreme terrestrial and marine environments[END_REF][START_REF] Exton | Uncovering the volatile nature of tropical coastal marine ecosystems in a changing world[END_REF].

Mangroves are extreme ecosystems interfacing terrestrial and marine environments. These forests are key elements of tropical coastal ecosystems as they provide many ecosystem services such as limiting erosion (e.g. cyclone, wave) and saline intrusion [START_REF] Brander | Ecosystem service values for mangroves in Southeast Asia: A meta-analysis and value transfer application[END_REF][START_REF] Hilmi | Correlation Analysis Between Seawater Intrusion and Mangrove Greenbelt[END_REF], providing raw materials, food and habitat for fauna (e.g. migration, breeding, spawning), or being important carbon (C) sinks [START_REF] Jakovac | Costs and Carbon Benefits of Mangrove Conservation and Restoration: A Global Analysis[END_REF].They are found in two distinct regions: the Atlantic Est Pacific (AEP) and the Indo-West Pacific (IWP) (Spalding 2010) and account for 147,359 km2 (127,925-168,895) in 2020 [START_REF] Bunting | Global Mangrove Extent Change 1996-2020: Global Mangrove Watch Version 3.0[END_REF]. In both geographical areas, there are several common genera but no common species. To handle their particular changing environmental conditions (e.g. high salinity, flooding, anoxia), mangrove species have developed several specific defense mechanisms, including physical barriers (e.g. waxes, [START_REF] He | Compositions and isotopic differences of iso-and anteiso-alkanes in black mangroves (Avicennia germinans) across salinity gradients in a subtropical estuary[END_REF] or chemical protections (e.g. secondary metabolites, [START_REF] Naskar | Anatomical and physiological adaptations of mangroves[END_REF]. Indeed, specialized compounds can be produced and/or emitted by plants to cope flooding and salinity as demonstrated for other species [START_REF] Copolovici | Environmental impacts on plant volatile emission[END_REF][START_REF] Yang | Response of plant secondary metabolites to environmental factors[END_REF]. However, to our knowledge, only a few studies have investigated BVOC emissions from mangrove despite the large distribution of this unique ecosystem over the world. For instance, [START_REF] Barr | Red mangroves emit hydrocarbons[END_REF] focused on volatile compounds emitted by foliage of Red mangroves (Rhizophora mangle L.) in Florida, and found very low emissions of isoprene (< 0.1 µg.g DW -1 .h -1 ). Despite these reported low emissions, mangrove trees could have a non-negligible effect on atmospheric processes since mangroves represent one of the most biomass-productive biomes on our planet and cover about 75% of the coasts and estuaries of tropical regions [START_REF] Ellison | Mangrove communities[END_REF][START_REF] Fao | The world's mangrove 1980-2005[END_REF].

The aim of this study was to perform for the first time a screening of isoprenoid emissions from 15 mangrove trees located in two geographical areas: the IWP (Vietnam) and the AEP (French Guyana)

regions. We hypothesized that some mangrove species could be isoprenoid emitters in response to extreme conditions in mangrove ecosystems.

Materials and methods

Species

From the 73 species recognized as "true" mangroves, we investigated 15 of the dominant mangrove species among the 38 core species ( monthly average in September (300-400mm). To the sampling site, not directly connected to the to the main estuarine channel, the salinity ranges from 16 to 23 [START_REF] Vinh | Changes in Leaf Litter Quality During Its Decomposition in a Tropical Planted Mangrove Forest (Can Gio, Vietnam)[END_REF]).

The second experimental site was located in French Guyana, along the Mahury River (04° 51.4429'N 052°15.8035'W). In this site, the climate is humid equatorial climate with 2 wet seasons and 2 dry seasons. French Guiana coast is mesotidal with semi-diurnal tides [START_REF] Ray | Mangrove-Derived Organic and Inorganic Carbon Exchanges Between the Sinnamary Estuarine System (French Guiana, South America) and Atlantic Ocean[END_REF]) with a tide elevation ranged from 0.92 m and 3.06 m in this site. The average annual temperature is 26.5 °C with a thermal amplitude of 3 °C at the most between the hottest and the coldest month and the average annual rainfall ranges from 2500 to 3000 mm y -1 . In the site the salinity ranges from 16 to 26 [START_REF] Marchand | Pore-water chemistry in mangrove sediments: relationship with species composition and developmental stages (French Guiana)[END_REF]).

Samplings were performed in the dry season during 2 field campaigns in April 2019 in Vietnam and October 2019 in French Guyana. The environmental conditions in terms of temperature (°C), relative humidity (%) occurring during the field campaigns were recorded using a HOBO device (Onset, USA). Photosynthetically Active Radiation (PAR) was measured using a quantum sensor (PAR-SA 190®, LI-COR, USA) (Table 1).

Isoprenoid sampling

For each species, five mature trees (2-5m) were sampled during each field campaign and sampling site (only exception: 6 Avicenia alba were sampled), for a total of 76 trees sampled (see Table 2 for details). The sampling procedure was described in detail in Saunier et al. (2017). On each sampled tree, one branch sun-exposed and without flower and having about 6 leaves was enclosed in a polytetrafluoroethynele (PTFE) chamber of about 10 L. A continuous atmospheric air flow, filtered with VOC and ozone scrubbers, flowed through the chamber at 1 L.min -1 . After a 30 min system stabilization period, air from the enclosure was collected during 30 min at 0.2 L.min -1 on cartridges (two beds of adsorbants: tenax and cartotrap B), one for isoprene and one for other isoprenoids. Inlet and outlet air flows were controlled by mass flow controllers (Porter Instrument, USA). Cartridges

were then stored at -20°C until analysis (portable freezer in the field and classical one in the laboratory). Samplings were performed between 10 h and 15 h (local time) on fully developed leaves.

Twice a day, blanks were performed to control inlet air quality.

One test was performed in April 2019 to check the reliability of our protocol of transport and storage.

According to this test, no significant difference was detected between an analysis right after the sampling or after a period of transport/storage (Fig. S1 in supplementary files).

GC-MS analyses

Cartridge analyses were conducted by gas chromatography (model 6890N, Agilent technologies, USA) coupled to mass spectrometry (model 5973, Agilent Technologies, USA). VOC were extracted using thermodesorption system (TDS3/CIS, Gerstel, Germany) using helium as carrier gas (50 mL.min -1 ) at 250 °C during 10 min. The extracted compounds were concentrated on-line by cryotrapping at -50 °C using liquid nitrogen. The sample was then injected by rapid heating to 250°C at 10°C/min. Initial oven temperature was 40 degrees hold for 5 minutes and then increasing by 3 degrees per minute to 250 °C maintained for 15 min.

Two distinct chromatographic methods were used, one to detect and quantify isoprene (C5) and another one to analyze C10-C15 compounds. Isoprene was analysed using a capillary Al/KCl column (Agilent Technologies, USA; 30 m x 0.25 mm x 5 µm) as described in Saunier et al. (2017b) whereas other isoprenoids were separated on a HP-5MS column (Agilent Technology, USA; 30 m x 0.25 mm x 0.25 µm) as shown in [START_REF] Ormeño | Effect of intraspecific competition and substrate type on terpene emissions from some Mediterranean plant species[END_REF]. For both analyses helium was the carrier gas at a constant flow rate of 1 mL.min -1 . Isoprenoids were identified based on the co-injection of analytical standards (Sigma-Aldrich, Germany), the Kovàts' index calculation through the injection of alkanes (C 8 -C 20 ) as well as the comparison of mass spectra to the NIST library (2011). BVOC emission rate (ER) were calculated according to Saunier et al. (2017).

Statistical analyses

The statistical analyses were performed with R software (version 4.2.1). After having checked the normality of the data set, one way-ANOVA or Kruskal-Wallis tests were performed for isoprene emissions and other BVOC emissions according to the species. Dunn post-hoc tests were performed to detect the species with statical differences in their emissions.

Results

The 15 studied mangrove species are low isoprenoid emitters (< 1.200 ng.gDW -1 .h -1 , Table 2). In details, species from the IWP region are the lower isoprene emitters with emissions rates ranging between 0.3 and 1.6 ng.gDW -1 .h -1 while species from the AEP region present a larger range of isoprene emission rate with values ranging between 0.1 and 2.1 ng.g DW -1 .h -1 . Isoprene emissions represent 0.3, 0.5 and 2.1% of total emissions for A. germinans, L. racemosa and R. racemosa, respectively.

Besides, those species are also low emitters of other BVOC (200 -1.2 ng. g -1 DW.h -1 , Table 2 and3) and the main BVOC detected are monoterpenes but also benzenoids as well as methyl salicylate.

Sesquiterpene emissions are very low, closed to the detection limit of the analyzers.

Other BVOC such as benzaldehyde, benzeneacetaldehyde, acetophenone or methyl salicylate contribute (Table 3) significantly to the total BVOC emission specifically for A. germinans and L.

racemosa with 76 and 82% of the total emissions, respectively.

Rhizophora racemosa is the highest emitter of the mangrove species with an average emission rate of about 1200 ng.g -1 DW.h -1 . Contrary to the two other species from AEP region, R. racemosa emissions are dominated by monoterpenes which represent about 93% of its total emissions. The three dominant emitted monoterpenes of R. racemosa are α-pinene, sabinene and β-pinene.

Discussion

The low isoprenoids emissions observed for the 15 mangrove species sampled in the two geographical regions (IWP and AEP) are in agreement with previous findings [START_REF] Barr | Red mangroves emit hydrocarbons[END_REF]). In addition, [START_REF] Guenther | Natural volatile organic compound emission rate estimates for U.S. woodland landscapes[END_REF] reported values below 100 ngC.g -1. h -1 for emissions from the genera Avicennia, Laguncularia and Rhizophora in the United States of America, referring to the works of Zimmernan (1979) and [START_REF] Evans | Estimates of isoprene and monoterpene emission rates in plants[END_REF]. Moreover, the dominant monoterpenes α-pinene and β-pinene detected here are also present in the study of [START_REF] Barr | Red mangroves emit hydrocarbons[END_REF]. Sabinene was detected in the essential oil of Melaleuca cajuputi, a species not directly related to the mangrove but often found in the back of mangroves (Sharif et al. 2009). The presence of monoterpenes but also benzenoids as well as methyl salicylate in emissions from mangrove species is not surprising as they are observed in many other biogenic emissions [START_REF] Rodriguez-Saona | Field responses of predaceous arthropods to methyl salicylate: a meta-analysis and case study in cranberries[END_REF][START_REF] Gentner | Emissions of terpenoids, benzenoids, and other biogenic gas-phase organic compounds from agricultural crops and their potential implications for air quality[END_REF].

The two geographic areas of mangroves have no species in common but share Avicennia L. and Rhizophora L., the most characteristic genera of mangrove systems worldwide [START_REF] Robert | A safe hydraulic architecture as wood anatomical explanation for the difference in distribution of the mangroves Avicennia and Rhizophora[END_REF]. In our study, several species belonging to these two genera were sampled and show qualitatively different but always small emissions. These results agree with those of Lorto ( 2002) who showed that within the genus Quercus, that have a wide geographical distribution, non-emitting species and emitting species of isoprenoids (isoprene and/or terpenes) can be found.

Although they share a common ecological niche and must adapt to similar environmental conditions, mangroves are nonetheless a genetically diverse group of flowering plants [START_REF] Duke | Global distribution and genetic discontinuities of mangrovesemerging patterns in the evolution of Rhizophora[END_REF]. This may explain the observed differences in their emission spectra, which are also driven by internal factors such as genetics [START_REF] Peñuelas | The Complexity of Factors Driving Volatile Organic Compound Emissions by Plants[END_REF].

This emission rate from mangroves is low compared to the emission rates of most other tropical plant species. For instance, [START_REF] Geron | Biogenic volatile organic compound emissions from a lowland tropical wet forest in Costa Rica[END_REF] performed a screening of BVOC from twenty species in a lowland wet forest located in Costa Rica. Half of these common species emitted isoprene at a rate ranging from 10 to 126 µg.gDW -1 .h -1 or about 100 times higher than the isoprene emissions of the 15 mangrove species under study. The same trend is observed for the other BVOCs that are generally emitted by tropical species at higher rates than mangrove species [START_REF] Singh | Emissions of monoterpene from tropical Indian plant species and assessment of VOC emission from the forest of Haryana state[END_REF][START_REF] Bracho-Nunez | Leaf level emissions of volatile organic compounds (VOC) from some Amazonian and Mediterranean plants[END_REF].

We can confirm that mangroves invest in isoprenoid emissions as chemical defences against the extreme environmental conditions that typically occur in their environment (e.g. flooding, salinity) but to a small extent. Indeed, mangroves have other strategies that ensure very good adaptation to these extreme conditions. They possess strategies such as osmolyte production which absorb salt as previously observed in Avicennia genus and other halophytes [START_REF] Khan | Metabolic implications of salt induced osmolyte accumulation in Avicennia marina[END_REF]) [START_REF] Slama | Diversity, distribution and roles of osmoprotective compounds accumulated in halophytes under abiotic stress[END_REF].

However, samplings were performed only on a short time. As a result, it cannot be excluded that we missed plant response to salinity and or flooding that could have caused emission puffs. For example,

Eucalyptus globulus, a non-halophyte plant, exhibits puffs of isoprene only when salt stress is relieved [START_REF] Loreto | Emission of isoprene from salt-stressed Eucalyptus globulus leaves[END_REF]. [START_REF] Velikova | The impact of winter flooding with saline water on foliar carbon uptake and the volatile fraction of leaves and fruits of lemon (Citrus× limon) trees[END_REF] also observed important emissions of volatile isoprenoids from Citrus leaves, mainly limonene, during flooding with saline water.

Moreover, it would be worth investigating the emission of highly volatile organic compounds such as acetaldehyde and methanol since mangroves face anoxic conditions that could lead to alcoholic fermentation processes and, as a result, ethanol could be produced and transformed to acetaldehyde and to a lesser extent to acetic acid [START_REF] Oikawa | Catabolism of volatile organic compounds influences plant survival[END_REF]. More generally, plants can also emit methanol through growth process [START_REF] Hüve | Simultaneous growth and emission measurements demonstrate an interactive control of methanol release by leaf expansion and stomata[END_REF]) and acetone through acetoacetate decarboxylation [START_REF] Fall | Abundant oxygenates in the atmosphere: a biochemical perspective[END_REF]. Acetaldehyde and acetic acid impact air quality by contributing to the atmospheric oxidative capacity and forming toxic secondary pollutants such as formaldehyde [START_REF] Oikawa | Catabolism of volatile organic compounds influences plant survival[END_REF]. In addition, other halophytes have been shown to be a source of methyl halides and dimethylsulphoniopropionate (DMSP) [START_REF] Otte | Dimethylsulphoniopropionate (DMSP) and related compounds in higher plants[END_REF][START_REF] Rhew | Gross production exceeds gross consumption of methyl halides in northern California salt marshes[END_REF]. One experimental study conducted under greenhouse conditions also demonstrated that mangrove trees emit methyl halides with possible atmospheric impact [START_REF] Manley | Methyl halide emissions from greenhouse-grown mangroves[END_REF]). However, this study emphasizes the need for field experiments to check for the occurrence of these emissions from mangroves. Further investigations are thus required to explore if mangroves are a source of halide VOC.

Conclusion

Our results show that mangrove trees are very low isoprenoid emitters. Only R. racemosa can be regarded as a non-negligeable source though monoterpene emissions still low (~ 1000 ng.gDW.h -1 ).

Such results suggest that mangrove does not strongly affect local or regional atmospheric chemistry through BVOC emissions despite the large distribution of this ecosystem over the globe. However, despite the low-rate emission, the data from this study can be used in atmospheric chemistry models to study the impact of biogenic VOC emissions on air quality in areas where mangroves are present.

It is still worthy to note that we only made punctual samplings and that continuous monitoring of isoprenoid emissions should be carried out to check for potential seasonal variations. Moreover, as far as our results showed important differences between plant species, it could be interesting to broaden the sampling design including other sites (African west coast and Caribbean Islands) as they include mangrove species that have not been investigated.

It could be also interesting to focus on other compounds than isoprenoids such as highly volatile or halogenated compounds which might be tackled in future studies focused on BVOC emissions from mangroves.

  Ho Chi Minh City in the South of Vietnam (10°30'N 106°51'E). The Can Gio mangrove is formed by the deltaic confluence of the Saigon, Dong Nai and Vam Co Rivers and it is the first mangrove biosphere reserve in Vietnam (UNESCO/MAB Project 2000). This estuary is subject to semi-diurnal tides, and the tidal elevation ranges from 2 to 4 m. The climate is monsoonal, with a dry season lasting from December to April and a wet season from May to November. In this

	Engl. (Lythraceae). Due to the large surface area they occupy, these species could be the most
	important contributors to the biosphere atmosphere relationship.
	The 15 species were sampled in two distribution areas: 11 in the IWP (Vietnam) and 3 in the AEP
	(French Guyana) regions. In Vietnam, the experimental site was located in the botanical garden of Can
	Gio District (CGBG),
	Spalding 2010): Avicennia alba Blume, Avicennia germinans
	(L.)L., Avicennia marina (Forsk) Vierh (Acanthaceae), Aegiceras corniculatum (L.) Blanco, Aegiceras
	floridum Roem. & Schult. (Myrsinaceae), Bruguiera gymnorrhiza Lam., Ceriops zippeliana Blume,
	Rhizophora apiculate Salvoza, Rhizophora racemose (Hochr.) Salvoza, Rhizophora stylosa Griff.
	(Rhizophoraceae), Laguncularia racemose (L.) C.F. Gaertn., Lumnitzera littorea (Jack) Voigt,
	Lumnitzera racemosa Wild. (Combretaceae), Sonneratia alba Sm. and Sonneratia caseolaris (L.)

site, the annual mean temperature is 27.4°C with the highest (37.6•C) occurring in October and the lowest (21.7°C) in February. The annual average rainfall ranges from 13000-14000mm with highest

Table 1 :

 1 Means (± SE) of temperatures (T, °C), relative humidity (RH, %) and Photosynthetic Active Radiation (PAR, µmol.m -2 .s -1 ) between 10 h and 15 h (sampling time for BVOC, local time) during each field campaign. PAR in March 2019 could not be calculated due to technical problems with the HOBO sensor.

	Site	Sampling	T	RH	PAR
	Vietnam	April 2019	33.0 ± 0.2	66.0 ± 0.5	301 ± 23
	French Guyana	October 2019	35.0 ± 0.3	60.0 ± 0.6	603 ± 55

Table 2 :

 2 Emission rates (ER, ng.gDW -1 .h -1 ) of 15 mangrove species in terms of isoprene and other 425 BVOC (see Table 3 for details) detected with GC-MS, n.d.: no compound detected. Values are means 426 ± SE. Statistical test are presented for each BVOC type and site. Lower case letters denote post-hoc 427 test differences at 95% confidence level (Due to a problem during the analysis, we lost a series of 428 samples from French Guyana and therefore only have 4 samples with results)

	429
	Sites	Species	ER isoprene*	n	ER other BVOC	n
			C 5		(C 10 -C 15 )
	Vietnam	Aegiceras corniculatum	1.5 ± 1.0 a	5	n.d.
		Aegiceras floridum	1.0 ± 0.4 a	5	n.d.
		Avicennia alba	0.3 ± 0.1 a	6	n.d.
		Avicennia marina	0.2 ± 0.1 a	5	n.d.
		Bruguiera gymnorrhiza	n.d.	5	n.d.
		Ceriops zippeliana	0.8 ± 0.5 a	5	n.d.
		Lumnitzera racemosa	1.6 ± 0.8 b	5	n.d.
		Lumnitzera littorea	0.1 ± 0.1 a	5	n.d.
		Rhizophora apiculata	0.3 ± 0.2 a	5	n.d.
		Rhizophora stylosa	0.7 ± 0.4 a	5	n.d.
		Sonneratia alba	0.3 ± 0.2 a	5	n.d.
		Sonneratia caseolaris	1.3 ± 0.4 a	5	n.d.
			Kruskall-Wallis test p=0.02
	French	Avicennia germinans	0.5 ± 0.3 a	5	212 ± 38
	Guyana	Laguncularia racemosa	0.3 ± 0.3 a	5	222 ± 97
		Rhizophora racemosa	2.1 ± 0.8 b	4	1176 ± 626
			Kruskall-Wallis test p=0.04	Anova test p=0.09
	430
	431

Table 3 :

 3 Emissions rates (ng.gDW -1 .h -1 ) of Avicennia germinans, Laguncularia racemosa and Rhizophora racemosa. Asterisks indicate the co-injection of analytical standards to perform compound identifications and quantifications. Means ± SE.

	Compounds	A. germinans	L. racemosa	R. racemosa
	Monoterpenes
	α-pinene*	18.5 ± 3.1	3.9 ± 2.1	393.6 ± 220.0
	camphene	0.1 ± 0.1	n.d.	32.8 ± 25.4
	sabinene	0.6 ± 0.6	n.d.	393.3 ± 268.0
	β-pinene*	0.4 ±0.4	0.1 ±0.1	102.5 ± 68.2
	β-myrcene	19.9 ±6.2	7.3 ± 4.1	13.0 ± 2.7
	α-terpinene	n.d.	n.d.	4.9 ± 2.9
	p-cymene*	3.5 ± 2.0	3.4 ± 1.1	8.4 ± 3.6
	Limonene*	2.8 ± 1.7	4.7 ± 1.9	72.8 ± 43.9
	β-ocimene (Z)	n.d.	2.1 ± 1.6	n.d.
	β-ocimene (E)	n.d.	10.9 ± 4.6	0.4 ± 0.4
	γ-terpinene	n.d.	n.d.	12.3 ± 6.8
	sabinene hydrate	n.d.	n.d.	0.4 ± 0.3
	E-DMNT	0.5 ± 0.5	2.0 ± 1.8	n.d.
	linalool	n.d.	3.4 ± 3.4	3.4 ± 2.4
	allo-ocimene	n.d.	0.3 ± 0.3	0.2 ± 0.2
	camphor	n.d.	n.d.	2.1 ± 1.3
	pinocarvone	n.d.	n.d.	5.3 ± 3.6
	borneol	0.4 ± 0.4	0.6 ± 0.6	40.1 ± 20.9
	terpinen-4-ol	n.d.	n.d.	1.1 ± 0.5
	myrtenal	n.d.	n.d.	4.9 ± 3.1
	verbenone	n.d.	n.d.	0.9 ± 0.5
	Sesquiterpenes
	β-caryophyllene	3.9 ± 3.9	n.d.	n.d.
	α-bergamotene (Z)	0.1 ± 0.1	n.d.	0.7 ± 0.7
	Other BVOC
	benzaldehyde	73.6 ± 11.3	89.1 ± 49.3	42.2 ± 8.3
	benzeneacetaldehyde	2.1 ± 0.4	1.2 ± 1.1	0.8 ± 0.2
	acetophenone	83.6 ± 14.2	90.2 ± 36.0	38.1 ± 5.7
	methyl salicylate	2.6 ± 2.6	2.7 ± 2.5	0.4 ± 0.4
	n.d. = not detected, E-DMNT = (E)-4,8-Dimethyl-1,3,7-nonatriene

Acknowledgements

We thank Sylvie Dupouyet (IMBE), Mr. Le Van Sinh, from Can Gio Mangrove Protection Forest Management Board of Ho Chi Minh City, Vietnam for help with sampling emissions. This study was funded by the ECCOREV research Federation, by the CNRS through the French National program EC2CO (Ecosphère Continentale et Côtière), PEPS (Projets Exploratoires Premier Soutien) and the IRP-CNRS France-Vietnam (Tropical Ecology Laboratory) 2018-2021. This work was also supported by the Pépinière Interdisciplinaire de Guyane and the Foundation "Ecologie d'Avenir" from "Académie des Sciences".