Jean-Yves Toullec
email: jean-yves.toullec@sb-roscoff.fr

Kévin Cascella

Stéphanie Ruault

Alexandre Geffroy

David Lorieux

Nicolas Montagné

Céline Ollivaux

Chi-Ying Lee

Antarctic krill (Euphausia superba) in a warming ocean: thermotolerance and deciphering Hsp70 responses

Keywords: Krill, Euphausia superba, heat shock, CTmax, Hsp70 expression

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Introduction

Global Warming constitutes a Grand Challenge for Biology as defined by Schwenk et al. [START_REF] Schwenk | Grand challenges in organismal biology[END_REF]) and [START_REF] Denny | Confronting the physiological bottleneck: A challenge from ecomechanics[END_REF]. It represents a major, unsolved problem that requires a significant improvement in capability or a fundamental change in perspective to solve. In this respect, the pivotal role of physiology has been highlighted in confronting the complex problems posed by global climatic change, and the need to strive toward a common focus, the whole organism: "It is physiology -our understanding of how individual organisms function and interact with their environmentthat presents the largest challenge. Without a better mechanistic understanding of how plants and animals work, we can never be assured of an accurate warning of what lies ahead for life on earth" [START_REF] Denny | Confronting the physiological bottleneck: A challenge from ecomechanics[END_REF].

To fit with this strategy, the choice of the biological model is extremely important. The Southern Ocean is an important breeding and/or foraging location for a wide range of charismatic megafauna such as whales, seals, penguins and other sea birds including albatrosses. Whilst these species fascinate the public, they represent the apex of a complex food chain, the keystone species of which is the Antarctic krill Euphausia superba [START_REF] Trivelpiece | Variability in krill biomass links harvesting and climate warming to penguin population changes in Antarctica[END_REF]). These shrimp-like crustaceans are not only a major prey item for these animals, but are also significant consumers, grazing on the phytoplankton bloom in the austral summer and on algae under the sea ice in winter for the most southern ones. In a more global context, the Antarctic krill has been suggested to be the most abundant eukaryotic species in the World's Oceans existing in large schools or swarms with densities that may be between 10,000 -30,000 individual animals per square meter [START_REF] Macaulay | Acoustic Characterization of Swarms of Antarctic Krill (Euphausia superba) from Elephant Island and Bransfield Strait[END_REF]. In addition, it is an increasingly profitable fisheries resource. It is therefore essential to have maximum information on the thermal resilience of these animals in order to best manage this stock [START_REF] Atkinson | Long-term decline in krill stock and increase in salps within the Southern Ocean[END_REF][START_REF] Everson | Krill biology, ecology and fisheries[END_REF][START_REF] Flores | Impact of climate change on Antarctic krill[END_REF][START_REF] Schiermeier | Ecologists fear Antarctic krill crisis[END_REF]). Given the central role of this key species in the Southern Ocean ecosystem, detailed physiological and molecular studies are essential to understand this species and its responses to a changing environment.

This work continues a previous study focused on the capacity of Antarctic krill to respond to a rise in environmental temperature [START_REF] Cascella | Diversification, evolution and sub-functionalization of 70kDa heatshock proteins in two sister species of antarctic krill: differences in thermal habitats, responses and implications under climate change[END_REF]. The Cascella study included the CT max values of two krill species from the Antarctic zone Euphausia superba and Euphausia crystallorophias, as well as the first measurements of the expression of different Hsp70 isoforms in response to thermal shocks of 3 and 6 hours to 3°C and 6 °C. These first results showed, especially in E. superba, a very weak response in terms of Hsp70 expression during the shock at these different temperatures. Furthermore, a second study on the arcto-boreal krill Thysanoessa inermis, which lives in warmer waters and should be less stenothermal, has shown a similar thermal resilience (CT max ) to the Antarctic species, but with different expression kinetics, especially in terms of isoform reactivity [START_REF] Huenerlage | Responses of the arcto-boreal krill species Thysanoessa inermis to variations in water temperature: coupling Hsp70 isoform expressions with metabolism[END_REF]).

In the current study, further experiments were performed to interrogate in more detail the question of whether E. superba is capable of an HSP response. Measurements were performed on the expression of the different Hsp70 isoforms in the recovery period (postshock), but also after short shocks of 1 and 2 hours and more intense thermal shocks at 9°C, 12°C and 15°C. In addition, a study of the expression in response to a long shock at 3 °C for 6 weeks was conducted in an aquarium under controlled conditions. Finally, organismal heat tolerance was assayed using a slower rate of warming than prior studies to provide an ecologically relevant measure of tolerance.

Material and Methods

Animal collection

E. superba were obtained from different sources, depending on the experiment (see Figure 1). E. superba for acute trials at 3°C and 6°C were fished off the French base Dumont d'Urville (DDU) in Terre Adélie just beyond the continental plateau (65.30°S, 139.57°E). Fishing was carried out using an Isaacs-Kidd midwater trawl (IKMT) from the ship « L'Astrolabe » at a maximum speed of 2 knots for 10-15 minutes. The sampling depth was determined by sonar observations of swarms, taking samples from those with a depth of between 20 and 50 meters.

After fishing, the animals were moved to a refrigerated flow-through tank on board, which was continuously supplied with seawater at -0.5 -+0.5°C. Animals were acclimated for 24 hours to aquarium conditions prior to experimentation to minimize holding stress. A circulation pump was used to set up a vertical current maintaining the animals in the water column.

A second series of acute thermal trials and longer thermal shocks were performed in the temperature-controlled aquariums of the Australian Antarctic Division in Kingston (Tasmania) [START_REF] Kawaguchi | An experimental aquarium for observing the schooling behaviour of Antarctic krill (Euphausia superba). Deep-Sea Research Part Ii-Topical Studies in[END_REF]. Animals used for these experiments were fished at 64.11°S, 111.12°E and kept in aquaria for 8 months before being challenged with heat.

Samples of

Experiments

Thermal shocks at 3°C and 6°C (onboard Astrolabe)

After around 24 hours in the main aquarium, the most mobile animals were selected and put into two aquaria containing seawater at the same temperature as the main stock aquarium. These aquaria were placed within a larger tank containing fresh water. A copper coil linked to a thermostat and pump was added to the system. The coil was used to pump hot water around the system to warm up the tank water for experiments. A circulating pump ensured that the warming was equally distributed around the tanks and air pumps were used to maintain the oxygen levels in the aquaria and also facilitate water mixing.

The protocol of the experiments is depicted in Figure 1A. For each thermal shock, animals from the same trawl were treated at the same time for 3 or 6 hours before being returned to 0°C seawater for recovery. The animals were put directly into the warmed water. At the same time, a set of control animals from the main aquarium were snap frozen in liquid nitrogen.

Animals having undergone heat shock at 3°C or 6 °C were sampled after 3 or/and 6 hours of shock and 2, 4, 6 hours of recovery (post shock (PS)) and deep-frozen. Each sampling comprised 10 animals.

Acute shocks at 3°C and 6°C (Australian Antarctic Division)

E. superba were directly transferred from the tank at 0°C to the tanks at 3°C and 6°C. Ten animals were collected after 1 and 2h and immediately snap frozen in liquid nitrogen and then kept at -80°C until required.

Acute shocks at 9°C, 12°C and 15°C for 3 hours (Australian Antarctic Division)

E. superba were directly transferred from the tank at 0°C to the tanks at 9°C, 12°C or 15°C.

Ten animals were used at each temperature. Heat shocked animals were sampled after 3h and directly frozen in liquid nitrogen before storage at -80°C.

Longer thermal trials at 3°C for 6 weeks (Australian Antarctic Division) 120 E. superba were warmed from 0°C to 3 °C at 1°C/h. Ten animals were sampled at 3, 6, 12, 24 hours and then, at the same time, every day for 2 days and finally every week until the sixth week. Animals were snap frozen and samples kept at -80°C. Ten more animals from the original tank were directly frozen and used as control (Fig 1B).

CT max experiments

CT max experiments were carried aboard the Astrolabe using increments of 1°C/10 min according to the protocol set out in Cascella et al. [START_REF] Cascella | Diversification, evolution and sub-functionalization of 70kDa heatshock proteins in two sister species of antarctic krill: differences in thermal habitats, responses and implications under climate change[END_REF]. Additional CT max experiments were carried out at rates of 1°C per day and 1°C every three days. These experiments were carried out in the AAD aquaria in Kingston (Tasmania). Animals no longer responding to a tactile stimulus during this thermal trial were removed from the aquarium.

The CT max was considered as the temperature at which 50% of experimental animals were still mobile. It was determined though the non-linear curve fitting option in JMP10 (SAS).

The survival curve used was: Survival=c/(1+(T/CT max ) b ) where c is the plateau value before the sharp decrease, CT max the temperature at which 50% of mobile animals is reached, and b a sigmoidicity coefficient. The program explores the different values of these three parameters, and calculates a Chi-square value. While exploring the different parameter values, the program aims to minimize this Chi-square value and converges towards a value for each parameter (provided with a standard error). The numbers of animals used for these CT max experiments were 43, 30 and 30 for 1°C per 10 minutes, 1°C per day and 1°C per 3 days respectively. qPCR analysis mRNA levels of the Hsp70 isoforms were determined by quantitative RT-PCR amplification, on 6 to 10 individuals for each experimental condition according to the same protocol in the previous publication [START_REF] Cascella | Diversification, evolution and sub-functionalization of 70kDa heatshock proteins in two sister species of antarctic krill: differences in thermal habitats, responses and implications under climate change[END_REF]. A dissociation curve was generated and PCR efficiency was estimated for each primer pair. All primer pairs tested generated a single peak in the dissociation curve and a PCR efficiency of 80 -100%. Data were analysed with the LightCycler 480 software. The 18S gene was chosen as a reference gene using the BestKeeper algorithm [START_REF] Pfaffl | Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper -Excel-based tool using pair-wise correlations[END_REF]) after testing EF1α, 18S, RPL8, and GAPDH as potential normalizing housekeeping sequences. Hsp70 expression was subsequently normalized to this reference. Differences of the mean normalized expression (MNE) of the five Hsp70 isoform genes (A, B, C, D and E) over the time course of the experiment were analyzed using a non-parametric Kruskal-Wallis test and a Dunn's multiple comparison test to determine significant differences between control and heat shocked samples. Relative gene expression (fold Hsp70 expression) was calculated from the MNE of test specimens (heat shock at 3°C or 6°C for 3 or 6 hours and subsequent recovery at 0°C) divided by the MNE of control specimens (kept at 0°C control temperature).

Statistical analyses were carried out using GraphPad Prism 6 (GraphPad Software, Inc., USA).

The significance level was set at p < 0.05.

Western blot

Samples of krill abdomens without their cuticle were ground in liquid nitrogen, and the powder was homogenized in 1 ml of extraction buffer (20 mM Tris/HCl, pH 7.5: 40 µl protease inhibitor cocktail (Roche, France)). The homogenates were centrifuged at 10,000 g for 15 min at 4 °C. The extracted proteins were quantified in the supernatant with the Bio-Rad Protein Assay (Bio-Rad, Marnes-la-Coquette, France) using bovine serum albumin (Sigma) as standard. Proteins of the total supernatant were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE; 7.5% acrylamide: 0.3% bisacrylamide (w/v); 20•μg•protein•well -1 ). After running the electrophoresis (15•min at 15•mA•gel -1 , 1•h at 20•mA•gel -1 ), the bands were stained with Coomassie Blue. Protein molecular mass standards from 10•kDa to 250•kDa (All Blue, Bio-Rad) were used to evaluate the apparent Mr of the separated bands.

For western blotting, the proteins were transferred from the SDS-PAGE gel (20•μg•protein•well -1 ) to a nitrocellulose membrane by semi-dry blotting at 25 mA for 2•h (Trans-Blot semi-dry cell; Bio-Rad). Membranes were blocked in milk/BSA/Tris-buffered saline pH 7.5 (TBST: Tris Buffered Saline with Tween 20) (2.5% w/v each) for 1h, rinsed in TBST and incubated overnight with a polyclonal antibody (1/1000 Mouse anti-chicken HSC70/HSP70 monoclonal antibody; Enzo Life Sciences, Lausen, Switzerland) at room temperature [START_REF] Ravaux | Heat-shock response and temperature resistance in the deep-sea vent shrimp Rimicaris exoculata[END_REF]. Subsequently, a 10•min washing step with TBST with 2.5% milk pH 7.5 was repeated four times, and the membranes were incubated with a polyclonal secondary antibody coupled to horseradish peroxydase (HRP) (1/3000 donkey anti-mouse IgG-HRP: sc-2314, Santa Cruz Biotechnology Inc.) for 1•h at room temperature. After another 4×10•min washing step with TBST, the antibody complex was detected using the Luminol reagent substrate (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific, Redford, USA) incubated at room temperature for 1•min and read with a Fusion FX (Vilber Lourmat, Marne la Vallée, France).

Results and discussion

Upper critical thermal limits vary according temperature increment rate

As a preliminary analysis to understand the thermal resilience of E. superba in the Southern Ocean, short term acute exposures were first undertaken, using a temperature increase of 0.1°C/min [START_REF] Cascella | Diversification, evolution and sub-functionalization of 70kDa heatshock proteins in two sister species of antarctic krill: differences in thermal habitats, responses and implications under climate change[END_REF]. The interpretation of the CTmax curve of E. superba gave an unexpectedly high value of 15.8 ±0.1°C for a stenothermal Antarctic ectotherm [START_REF] Peck | Animal temperature limits and ecological relevance: effects of size, activity and rates of change[END_REF]). As the thermal shocks applied in these first sets of experiments were relatively short and represented very rapid temperature increases, thermal shocks with longer increment times were conducted to confirm or refute the apparent thermal resistance highlighted by these previous experiments. Indeed, Peck et al. showed that CTmax varied with the rate of experimental warming, more precisely CTmax declined at slower rates of warming [START_REF] Peck | Animal temperature limits and ecological relevance: effects of size, activity and rates of change[END_REF][START_REF] Peck | Acclimation and thermal tolerance in Antarctic marine ectotherms[END_REF]. Thus, the time elapsed between two temperature increments of 1°C was significantly increased since the curves presented were obtained for intervals of 1 and 3 days (Figure 2). Observed CTmax values were 14.7 ± 0.1°C and 13.9 ± 0.03°C for increments of 1°C per 1 and 3 days respectively. These values appear particularly high compared to what has been regularly observed in other polar invertebrates [START_REF] Peck | Acclimation and thermal tolerance in Antarctic marine ectotherms[END_REF]). More especially, the small difference between the CTmax as a function of the time between each temperature increase was surprising. Overall, the decrease in CTmax was approximately 1°C between the different rate of change experiments. This small decrease highlights a particularly high temperature resilience. This result makes sense when comparing these values with those found with another polar eucarid, the shrimp Chorismus antarticus, a species phylogenetically closer than most of the Antarctic invertebrates studied so far, with CT max values of 11.6°C and 6.7 °C for increments of 1°C every 10 minutes or every day respectively (personal and unpublished data). In this example, the CTmax for a 1°C/10 min was already much lower than that of E. superba but drops significantly with the extension of the warming interval. These differences are certainly partially attributable to the differences in living environment, which is pelagic beyond the continental shelf for E. superba versus benthic on the continental shelf for C. antarcticus [START_REF] Peck | Animal temperature limits and ecological relevance: effects of size, activity and rates of change[END_REF].

Kinetics of Hsp70 expression

Post-shock kinetics

In a previous publication [START_REF] Cascella | Diversification, evolution and sub-functionalization of 70kDa heatshock proteins in two sister species of antarctic krill: differences in thermal habitats, responses and implications under climate change[END_REF], the expression kinetics of five E. superba Hsp70 isoforms (A, B, C, D, E) were analyzed in response to moderate thermal shocks at 3°C or 6°C for 3 or 6 hours. Results showed that, this response in Antarctic krill was very weak when compared with the fold changes observed in temperate species [START_REF] Feder | Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology[END_REF][START_REF] Ravaux | Adaptation to thermally variable environments: capacity for acclimation of thermal limit and heat shock response in the shrimp Palaemonetes varians[END_REF]. In this study, the initial heat shock experiments of [START_REF] Cascella | Diversification, evolution and sub-functionalization of 70kDa heatshock proteins in two sister species of antarctic krill: differences in thermal habitats, responses and implications under climate change[END_REF] were expanded to include a recovery period at 0°C (Figure 3). According their structural characteristics and phylogenetic position, the A, B and E isoforms were most similar to the traditional constitutive form gene cluster. The C isoform is most similar to inducible isoforms in other species. These four isoforms are cytoplasmic while the D isoform is most similar to the group of mitochondrial Hsp70s [START_REF] Baringou | Diversity of cytosolic HSP70 Heat Shock Protein from decapods and their phylogenetic placement within Arthropoda[END_REF][START_REF] Cascella | Diversification, evolution and sub-functionalization of 70kDa heatshock proteins in two sister species of antarctic krill: differences in thermal habitats, responses and implications under climate change[END_REF][START_REF] Huenerlage | Responses of the arcto-boreal krill species Thysanoessa inermis to variations in water temperature: coupling Hsp70 isoform expressions with metabolism[END_REF].

The rapid transition from 0°C to 3°C did not invoke an up-regulation of Hsp70 in E. superba (Figure 3a). Actually, there was a general trend for a reduction in Hsp70 gene expression after 6 hours compared with the controls (Figure 3b). However, if the up-regulation of expression recorded during the actual heat shock itself was weak, the analyses of post-shock showed much higher levels of expression, which increased with the length of the initial shock. The most responsive isoform was the E form, both in terms of speed of response and intensity.

Nevertheless, even though this form was the only one showing significant up-regulation during the recovery phase after 3 hours of shock (Figure 3a), the duration of the shock greatly influenced the amplitude of the responses of the other isoforms. The expression kinetics were clearly distinct according to the Hsp70 isoforms. However, similarly to the results in [START_REF] Cascella | Diversification, evolution and sub-functionalization of 70kDa heatshock proteins in two sister species of antarctic krill: differences in thermal habitats, responses and implications under climate change[END_REF], there was no correlation between the expression levels and the category of isoform group to which they belonged, i.e. the group of constitutive forms (theoretically no up-regulation to heat shock) or the group of inducible forms (theoretically strong upregulation to heat shock).

It is also clear that, if the duration of the shock influences the importance of the expression, the intensity of the shock impacts this response as well. Indeed, a shock of 3h at 6°C produced a larger response for isoforms A and B than a shock of identical duration at 3°C, with a maximum observed at 6h post-shock. This response was measured paradoxically only for these so-called constitutive forms (Figure 3c). However, the up-regulation patterns are very different for 6 hours at 6°C, where the response is far more muted with a trend in overall decreases in the responses recorded so far (Figure 3d). This lack of up-regulation may indicate that the animal needs more time for recovery or has reached its limits and no longer has the ability to compensate for variation in the environment.

Be that as it may, even if the up-regulation in Hsp70 in response to thermal shock did not reach the values observed in other ectotherms, especially from temperate regions, the recovery period values were much higher than those measured during the shock itself. E. superba is therefore unquestionably capable of responding to a thermal shock via upregulation of its Hsp70s. The very low level or the absence of response observed during the heat shock is curious. Several reasons could explain the values observed: i) the timing of first samplings were too late and did not allow for a very rapid and early response, within the first three hours ii) the applied temperatures were too low to generate a response iii) Antarctic krill has a large constitutive level of Hsp70 proteins, which it can use as an available pool to cope with the molecular perturbations induced by the increase in temperature, thus explaining the late up-regulation of mRNAs. Each of these hypotheses was tested in the following experiments.

Short heat shock kinetics

In order to follow the kinetics of expression over shorter times, a new series of heat shock experiments (+3°C and +6°C) were conducted according to an identical protocol but with sampling points at 1 and 2 hours (Figure 4).

No significant up-regulation was observed except for E isoform after 2 hours of heat shock.

The values obtained were similar to the trends observed with 3 hours of heat shock. However, it is interesting to note significant down-regulation of expression after 1h for the A isoform and after 1 and 2 hours for the B isoform. The results are similar for both temperatures. This decrease in expression could be attributed to a massive recruitment of mRNAs, originally present before the heat shock, for protein synthesis. Much of the response would then partly consist of replacing the mRNAs involved in translation, when the shock remains moderate in duration and intensity. On the other hand, the potentially inducible form C does not show any significant expression variation. Again, as demonstrated throughout the heat shocks, the socalled constitutive forms are the most involved in the response of E. superba to temperature.

In summary, the hypothesis of an upregulation in Hsp70s prior to the original 3-hour sampling period was not validated.

Heat shock kinetics

In order to test the hypothesis that the weak response observed after thermal shocks at 3°C and 6°C was, or was not, related to the fact that the intensity of these shocks was insufficient, the animals were immersed for 3 hours in water at 9°C, 12°C and 15°C, then expression of Hsp70s were measured (Figure 5).

As a first observation, none of the animals immersed in water at 15°C survived the experiment and only half of the individuals resisted for 3 hours in water at 12°C, thus the 12°C results represent a resisting population, which may explain why expression levels are slightly higher at 12°C for some isoforms. This observation highlights the importance of the implementation of the thermal shock. These animals were directly immersed in water at the shock temperature, with no gradual warming. The gradual raising of temperature is an essential component in the ability of animals to manage the increase in environmental temperature, as occurs with potential acclimation. Indeed, the CT max curves (Figure 2) demonstrate for example that no loss was recorded at the temperature of 12°C with increments of 1°C every 10 minutes or even 1°C every day.

As second observation, no significant up-regulation was recorded at these temperatures. However, as observed during the acute heat shocks, there was a down-regulation of isoforms forms A and B at 9°C only. This decrease in the amount of mRNAs encoding the constitutive Hsp70s appears to be a first step in the response process. It was present at 3°C and 6°C but seems to extend to 9°C before a trend of up-regulation as observed during the recovery period (Figure 3). Finally, when the length of the heat shock is extended, there might be a neosynthesis of mRNA coding for these chaperones following the disappearance of feedback inhibition generated by native Hsp70 proteins. Thus, the absence of up-regulation during heat shocks and its appearance during post-shock recovery periods would not be linked to an absence of response or a potential recovery of animals but rather to a delayed response related to a native abundance of functional proteins, which is supported by large constitutive quantities of mRNAs of A and B isoforms in particular. These reflections lead us to postulate that E. superba has naturally high quantities of HSP70 proteins, but to date, HSP70 abundance in this species has only been evaluated at the mRNA level.

Long-term heat shock

The objective of this experiment was to estimate the ability of E. superba to acclimate to 3°C.

The aim was to follow the expression of the different isoforms during this process, which would correspond to a scenario in agreement with a future global warming or, simply to a sudden change of geographical zone or of water layer. The scenario of global warming is obviously difficult to achieve since it implies a gradual increase over several years. The second scenario is more likely, both because of the longevity of the members of the species and their migrations through geographical areas with waters of different temperatures.

The first notable result was the total absence of mortality in this experiment. Clearly, on this sole criterion of survival, the transition to 3°C seemed to have no impact on the animals and they appear to have acclimated.

Secondly, in view of the previous results, the implementation of a long heat shock was of great interest. Indeed, it should allow us to determine unequivocally if the responses observed during the post-shock recovery experiments could be due to a return to the original conditions and was therefore due to a recovery or rather to a belated response as suggested above. The isoform D was not evaluated because of the absence of a measurable signal in the majority of sampled times.

In general, the expression levels did not remain constant for the isoforms (Figure 6). Their kinetics of expression showed peaks at different times highlighting the presence of Hsp70 regulation beyond 6 hours and therefore their activity of repairing cellular damage caused by chronic temperature challenge. The most important expression variations were recorded from 12-hours onwards. It is therefore clear that post-shock conditions are not solely responsible for increasing the HSP70 response. The time factor is also fundamental.

The different isoforms showed very different kinetics in amplitude and chronology. The E isoform remained the most reactive and confirmed the data obtained during short heat shocks.

The C isoform, which is structurally affiliated to inducible Hsp70s, showed a significant increase in expression after 12 hours, which is therefore rather late compared to 3°C animals measured after acute shocks and post-shock recovery. Similarly, the so-called constitutive isoforms, A and B, underwent a significant decrease on day 1 before recording an upregulation on day 3. The comparison of the kinetics of expression of these isoforms also reveals variations in response between the animals in recovery and those maintained under extended heat shock conditions. The latter showed significant down-regulation, especially around day 1 probably related to mRNA recruitment in these chronic stress conditions, followed by up-regulation. Overall, the patterns of up-regulation were delayed, compared with acute heat shocks, probably because the isoforms were originally present in large quantities, either as protein or mRNA.

It is clear that the kinetics of expression vary on the hourly scale at least during the first day.

This observation is also valid on a day-to-day basis with respect to the differences recorded during the first three days of heat shock, although it is perfectly reasonable to think that a daily sampling smoothed the data and obscured probable short-term variations. This reflection is even truer at the scale of the week when the probability that there was a synchronism between the variations of expression and the sampling time remains low. Nevertheless, the six-week heat shock expression data appeared to show a general decrease until a slight downregulation was observed in the last week. This raises the question as to whether this overall decrease in expression was due to a metabolic decline linked to the duration of the heat shock or to a process of acclimatization that is gradually taking place, which was not possible during the course of this experiment. A posteriori, it would be interesting in the future to finalize this study by a daily, or even better, an hourly monitoring of isoform expressions.

What about constitutive HSP70 quantities?

PCR monitoring of RNA expression provides information on the impact of a heat shock, but this approach, although powerful, considers only a part of the metabolic cascade involved. It is obvious that monitoring the production of proteins in response to a heat shock would also be useful as proteins are the end production of transcription/translation. Unfortunately, technical barriers related to the difficulty of quantification but also of identification and discrimination of the different isoforms by antibodies as all the Hsp70s described here have high sequence identities. Nevertheless, a western blot approach should allow a first quantitative assessment of Hsp70 constitutive protein levels. In order to determine whether there is a large native pool Hsp70 proteins in E. superba, which could explain the delayed Hsp70 responses described above Western blots were performed abdomen extracts from different species of eucarids from the Antarctic or Arctic regions. This was performed on the krill species E. superba (fished from different areas), E. crystallorophias, Thysanoessa inermis and the shrimp Chorismus antarcticus (Figure 7).

It is clear that the strongest labeling is present in the protein sample extracted from E. superba fished near Dumont d'Urville Station (lane 3). The other extracts are weakly stained or almost absent, as shown by the results for the Antarctic shrimp (Chorismus antarcticus). These bands were globally at or slightly above the human HSPA control band estimated at 72 kDa. Due to their positions and according the amino-acid sequences deduced from transcriptomic data [START_REF] Cascella | Diversification, evolution and sub-functionalization of 70kDa heatshock proteins in two sister species of antarctic krill: differences in thermal habitats, responses and implications under climate change[END_REF][START_REF] Clark | Antarctic krill 454 pyrosequencing reveals chaperone and stress transcriptome[END_REF], the bands shown in Figure 7 can be attributed to A isoform for the Antarctic species (EusHspA = 72.46 kDa, EucHspA = 72.56 kDa, as the B isoforms molecular weight are under 72kDa, 71.64 and 71.84 kDa respectively) and isoform B for Thysanoessa inermis (72.03 kDa), in which no A isoform was characterized [START_REF] Huenerlage | Responses of the arcto-boreal krill species Thysanoessa inermis to variations in water temperature: coupling Hsp70 isoform expressions with metabolism[END_REF]. The other isoforms do not appear to have been translated in sufficient quantity to allow visible labelling by the antibody. This observation, without constituting definitive proof, supports the hypothesis that E. superba maintains high constitutive levels of Hsp70 protein. The labeling is all the weaker as the species of krill considered have a significant early response in terms of expression of the mRNAs encoding these isoforms. In fact, T. inermis showed much faster and more Hsp70 intense responses to thermal shocks than Antarctic species, even though the corrected CT max values were very similar [START_REF] Huenerlage | Responses of the arcto-boreal krill species Thysanoessa inermis to variations in water temperature: coupling Hsp70 isoform expressions with metabolism[END_REF]). In the same way, E. crystallorophias appeared to be more reactive to temperature than Antarctic krill. The kinetics of response to thermal shock have not yet been carried out on C. antarcticus, however the CT max values were found to be much lower than those measured on E. superba and was particularly sensitive to the time factor, demonstrating a greater sensitivity to temperature variation (unpublished personal data).

The potential Hsp70A band could be preferentially involved in the response to cold shock in the natural environment [START_REF] Peck | Acclimation and thermal tolerance in Antarctic marine ectotherms[END_REF]. Interestingly, there is less native Hsp70A in E. crystallorophias, a species adapted to the colder environment of the Antarctic continental shelves. On the other hand, this protein is absent in the surveyed T. inermis, which lives in the warmer waters of the Arctico-boreal region. But most interesting is the labelling observed on the protein extract from E. superba specimens fished on the edge of the Antarctic Peninsula (Figure 7, lane 7). The water temperature in this region is regularly positive and it would have been reasonable to expect, on the basis of the stated hypothesis, that this Hsp70A band would have been less stained if the high quantity of Hsp70A in the sample in lanes 3 and 4 is due to cold acclimation. However, the difference is much more drastic, since the A band disappeared in favor of the potential band B (Figure 7, lane 7), similar to T. inermis (Figure 7, lane 6).

General discussion and conclusion

The high quantity of these Hsp70 isoforms in Euphausia superba constitutes a significant advantage in response to either a heat or cold shock. The hypothesis that ectothermic animals living in cold environments favor the accumulation of Hsps to correct the damage directly linked to the problems of protein folding at low temperatures could be an explanation for this physiological behavior [START_REF] Peck | A Cold Limit to Adaptation in the Sea[END_REF][START_REF] Place | Comparison of Hsc70 orthologs from polar and temperate notothenioid fishes: differences in prevention of aggregation and refolding of denatured proteins[END_REF]. Thus, the delay in gene expression would find its origin in the presence of large constitutive amounts of Hsp proteins in Antarctic animals, in quantities that would be sufficient to manage the immediate damage caused by a moderate or an acute temperature shock. When the shock is prolonged and quantities and/or degradation of available Hsp70s become critical to correct cell damage caused by a thermal shock, the available mRNAs could be recruited via translation and at that moment new mRNAs would need to be transcribed. As a consequence, the higher the amounts of Hsp present as either protein or mRNA within the cell under normal conditions, the longer delay expected in the heat shock response [START_REF] Bedulina | Sub-littoral and supra-littoral amphipods respond differently to acute thermal stress[END_REF]). These assumptions correspond well with what was observed with acute heat shocks. Short shocks of 1, 2, 3 or 6 hours did not cause significant up-regulation of Hsps whatever the intensity of the heat treatment. These results showed a general decrease in the mRNAs encoding the constitutive forms, probably involved in the synthesis of new Hsp70 proteins. However, there was significant up-regulation during post-shock recovery. This up-regulation of HSP70 transcripts, which could, in the first instance, be attributed to recovery, were as likely to be due to the natural abundance levels of Hsp70s that have delayed the response, as demonstrated by the western blot results.

Nevertheless, the presence of large amounts of Hsp70, particularly in comparison with the benthic shrimp (Chorismus antarcticus), questions its justification as a universal adaptation of Southern Ocean endemic species. Reasoning that the large amount of Hsp70 protein in control animals would be linked to a potential protection against the effects of cold, Antarctic krill fished near the French base at Dumont d'Urville would suffer a permanent cold stress if an optimum temperature at 0.5 ± 1°C is considered [START_REF] Atkinson | Natural growth rates in Antarctic krill (Euphausia superba): II. Predictive models based on food, temperature, body length, sex, and maturity stage[END_REF][START_REF] Brown | Temperature effects on the growth and maturation of Antarctic krill (Euphausia superba). Deep-Sea Research Part Ii-Topical Studies in[END_REF][START_REF] Cuzin-Roudy | Southern Ocean Euphausiids[END_REF][START_REF] Mackey | Antarctic macrozooplankton of the southwest Atlantic sector and Bellingshausen Sea: Baseline historical distributions (Discovery Investigations, 1928-1935) related to temperature and food, with projections for subsequent ocean warming[END_REF]. Similarly, the labelling shown in E. superba from the Antarctic Peninsula could be attributed to a heat shock. Each of the animals studied would be outside its comfort zone and would therefore be subject to thermal stress. This result is a new evidence highlighting the role of HSPs in the adaptation of organisms to temperature other than through thermotolerance [START_REF] Banerjee | Seasonal variation in expression pattern of genes under HSP70 : Seasonal variation in expression pattern of genes under HSP70 family in heat-and cold-adapted goats (Capra hircus)[END_REF][START_REF] Ulmasov | Heat-Shock Proteins and Thermoresistance in Lizards[END_REF]). The speciesspecific differences in HSP70 isoforms may be more likely related to variations in thermal tolerance [START_REF] Yamashita | Characterization of multiple members of the HSP70 family in platyfish culture cells: Molecular evolution of stress protein HSP70 in vertebrates[END_REF]), and isoform expression may vary with regard to thermotolerance [START_REF] Hightower | Heat shock responses of closely related species of tropical and desert fish[END_REF]. In this context, the comparison of the two sister species, E. crystallorophias and E. superba, illustrates this duality in adaptive choices [START_REF] Cascella | Diversification, evolution and sub-functionalization of 70kDa heatshock proteins in two sister species of antarctic krill: differences in thermal habitats, responses and implications under climate change[END_REF][START_REF] Papot | Divergent ecological histories of two sister Antarctic krill species led to contrasted patterns of genetic diversity in their heat-shock protein (hsp70) arsenal[END_REF]. Indeed, there are differences in terms of CT max , response and antibody staining intensity of Hsp70s and therefore of the amount of proteins, but also in terms of selection pressure experienced by the two sister species E. superba and E. crystallorophias [START_REF] Papot | Divergent ecological histories of two sister Antarctic krill species led to contrasted patterns of genetic diversity in their heat-shock protein (hsp70) arsenal[END_REF]). These differences may be related to their geographic distribution, with ice krill preferring cold continental shelf waters with low temperature variation [START_REF] Mackey | Antarctic macrozooplankton of the southwest Atlantic sector and Bellingshausen Sea: Baseline historical distributions (Discovery Investigations, 1928-1935) related to temperature and food, with projections for subsequent ocean warming[END_REF], whereas E. superba lives in deep oceanic waters [START_REF] Atkinson | Oceanic circumpolar habitats of Antarctic krill[END_REF] and is more exposed to variable temperatures. E. crystallorophias would favor molecular thermotolerance to cold and thus a weak degradation of proteins in a cold environment and a limited involvement of Hsp70, whereas E. superba, due to a wider and more variable distribution, would be less cold adapted and would have developed a greater HSP response.

A similar case has been reported in amphipod species with different vertical distributions and thermotolerance capacities [START_REF] Bedulina | Expression patterns and organization of the hsp70 genes correlate with thermotolerance in two congener endemic amphipod species (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal[END_REF][START_REF] Bedulina | Sub-littoral and supra-littoral amphipods respond differently to acute thermal stress[END_REF]. The hypothesis put forward was that one of the two isoforms would only be synthesized under normal physiological conditions, whereas the second could be synthesized under normal conditions but also induced by heat stress. It seems similar in the case of krill, but the difference is more pronounced since although the two isoforms Hsp70A and B can be expressed together, they are mostly represented in the transcriptomes, they do not seem to be translated simultaneously.

It was surprising, in view of the similar values of the FPKMs recorded for the two isoforms in the transcriptome of E. superba [START_REF] Cascella | Diversification, evolution and sub-functionalization of 70kDa heatshock proteins in two sister species of antarctic krill: differences in thermal habitats, responses and implications under climate change[END_REF], that the western blot revealed only one band in each of the samples of E. superba from east or west Antarctica. This should not be a recognition problem with the western blot since clearly both Hsp70 A and B isoforms can be recognized by the heterologous antibody. This difference could be attributed to the fact that the original E. superba transcriptome was made from a mixture of control and thermally stressed individuals. Nevertheless, the values obtained for qPCR for the controls are comparable for the two isoforms A and B. It could also be envisaged that the differences could be related to the genetic diversity of two populations. However, Antarctic krill is particularly genetically homogeneous despite its large population size and wide range, limiting differences potentially related to local conditions [START_REF] Deagle | Antarctic krill population genomics: apparent panmixia, but genome complexity and large population size muddy the water[END_REF]. Based on these observations, differential translation as a function of environmental temperature would therefore remain a reasonable explanation for the results in this study. The experiment carried out by western blot was conducted on control animals only, i.e. without the application of a heat shock. It will obviously be interesting to continue this study on animals having undergone heat stress in order to validate the hypothesis and to estimate the kinetics of appearance of the different isoforms. The E. superba sample (Figure 7, lane 4) was kept in an aquarium for 8 months at a temperature of 0.5°C before freezing, which corresponds to its theoretical thermal optimum [START_REF] Kawaguchi | An experimental aquarium for observing the schooling behaviour of Antarctic krill (Euphausia superba). Deep-Sea Research Part Ii-Topical Studies in[END_REF]. Although the Hsp70A staining seems less strong than for krill fished near Dumont d'Urville on the east coast, which is to be confirmed on a larger sample of individuals, it is nevertheless present and well-marked.

In summary, this differentiation of Hsp70 isoform protein expression between animals from West and East Antarctica raises more questions than it answers. However, it represents a new opportunity in the study of Hsp70s, which until now were not considered as strong markers of thermal stress in Antarctic krill species. Understanding the differential mechanisms of these thermoregulatory actors in E. superba is a prerequisite in the search for indicators of the biological impact of climate change on this global source of fisheries protein.

Legends

Figure 1: A) Experimental protocol used during the thermal shock kinetics: 3 and 6 hours at 3°C or 6°C (on Astrolabe board). B) Experimental protocol used for long term thermal shock at 3°C (AAD aquarium) Figure 2: Curves representing the loss of mobility of a population of krill subjected to gradual temperature increase (0.1 °C / min, blue curve, n=43; 1°C / day, red curve, n=30; 1°C / 3days, green curve, n=30). CT max is the temperature at which 50% of mobile animals are reached Figure 3: Mean normalized expression ratios of five hsp70 isoforms in the muscle tissue of adult E. superba (n=6) during 3 and 6 hours continuous heat shock at 3°C or 6°C followed by 6 hours of recovery at control temperature (0°C). Thermal shocks are presented on a white background, post shocks on a grey background. Values relate to the control group (specimens continuously kept at 0°C). Asterisk (*): significant MNE difference to 0°C control temperature evaluated with a Kruskal-Wallis test. Figure was created using GraphPad Prism 6.0 Figure 4: Mean normalized expression levels of five hsp70 genes obtained by qPCR in E. superba during a heat shock of 3°C and 6°C whereas 0-hour group is control at 0°C. Heat shocks were carried out during 1 and 2 hours, and hsp70 expressions measured at those times on n=6 individuals per group. Hsp70 expressions were normalized by the 18S gene expression. To compare values, a Kruskal-Wallis test followed by a Dunn's test were used to compare control groups (0h) and shocked groups (1h and 2h). Significant differences between mean normalized expressions are indicated by asterisks (*). Figure was created using GraphPad Prism 6.0 Figure 5: Mean normalized expression levels of five hsp70 genes obtained by qPCR in E. superba during a heat shock of 9°C and 12°C whereas 0h group is control at 0°C. Heat shocks were carried out during 3h, and hsp70 expressions measured at those time on n = 6 individuals per group. Hsp70 expressions were normalized by the 18S gene expression. To compare values, a Kruskal-Wallis test followed by a Dunn's test were used to compare control groups (0h) and shocked groups (9°C and 12°C). Significant differences between mean normalized expressions are indicated by asterisks (*). Figure was created using GraphPad Prism 6.0

  E. superba from western Antarctica (62.29°S, 139.59E) were kindly provided by Drs. Juan Höfer (IDEAL, Chile) and Marcelo Gonzalez-Aravena (INACH, Chile) for the Western blots. This project (IPEV-1039) was approved by IPEV (Institut Paul Emile Victor, the French Polar Institute) review committee and was declared to, and approved by, the ''Terres Australes et Antarctiques Françaises'' in 2009 according the Annex I of the Madrid Protocol and the French Decret No 2005-403. No endangered or protected species were used.

Figure 6 :Figure 7 :

 67 Figure 6: Mean normalized expression ratios of four hsp70 isoforms in the muscle tissue of adult E. superba (n = 6) during continuous heat shock at 3°C for 6 weeks. Values relate to the control group (specimens continuously kept at 0°C). Asterisk (*): significant MNE difference to 0°C control temperature evaluated with a Kruskal-Wallis test. Figure was created using GraphPad Prism 6.0 Figure 7: Western blot bands obtained using an anti-chicken monoclonal antibody for the detection of Hsp70 proteins in abdomen muscle of E. superba. Species and origin are listed under the figure. At least 3 samples from each origin were tested.

KC received a PhD grant from the Emergence-UPMC 2011 research program and the "Région Bretagne". JYT benefited from funding provided by Institut Paul Emile Victor (IPEV) (KREVET program) and also from the "Région Bretagne" (SAD-1 -DRAKAR program). JYT would like to thank all the IPEV staff at the base Dumont d'Urville and Astrolabe's crew for their help during different Antarctic campaigns in Terre Adélie. JYT would also like to thank Dr. So Kawaguchi and Rob King for their invaluable advice and support in setting up the aquarium experiments at the AAD in Kingston, Tasmania. Many thanks to Drs. Juan Höfer (IDEAL, Chile) and Marcelo Gonzalez-Aravena (INACH, Chile


Yolande Piris
email: yolande.piris@univ-ubs.fr

Anne-Cécile Gay
email: annececile.gay@gmail.com

Customer satisfaction and natural language processing (NLP)

Customer satisfaction and natural language processing 1. Introduction

As early as 1999, Pine and Gilmore viewed the evolution of the economy as a pyramid showing the reduction of goods and services and the centralization of experiences. More recently, [START_REF] Klaus | Towards a better measure of customer experience[END_REF] have shown that the customer experience is a better predictor of loyalty or recommendations to third parties than satisfaction measures. Customer delight is now considered alongside satisfaction to best understand consumers' judgment of an entire consumption experience, which can engender loyalty if positive [START_REF] Ahrholdt | Enhancing loyalty: When improving consumer satisfaction and delight matters[END_REF]. Understanding the customer journey and fostering a positive and memorable experience constitute major challenges for companies, but important ones especially given the positive link between loyalty programs and financial performance [START_REF] Lee | The financial impact of loyalty programs in the hotel industry: A social exchange theory perspective[END_REF]. Essential to meeting these challenges are data and data marketing, as the Adobe and eConsultancy (2019) digital trends report shows. The report indicates that 24% of respondents prioritize customer data ahead of customer experience optimization (19%).

At the same time, the development of the Internet and the emergence of e-commerce have considerably expanded the customer journey. The number of contact points between customers and companies has increased, as have the opportunities for customers to air their views. This situation encourages both the development of omni-channel strategies [START_REF] Verhoef | From multi-channel retailing to omni-channel retailing: Introduction to the special issue on multi-channel retailing[END_REF] and the proliferation of customer data, which brings up questions about measuring and processing customer satisfaction. Satisfaction is closely linked to customer experience, but it is also influenced by the omni-channel context in which customers operate. Both issues of customer experience, understood as the cognitive and affective treatment of the direct and indirect links between companies and their customers [START_REF] Klaus | Towards a better measure of customer experience[END_REF][START_REF] Verhoef | Customer experience creation: Determinants, dynamics and management strategies[END_REF], and customer satisfaction are commonly dealt with from a quantitative perspective. Customer satisfaction is measured as the gap between preexisting expectations and actual experience [START_REF] Anderson | Consumer dissatisfaction: The effects of disconfirmed expectancy on perceived product performance[END_REF]; in contrast, experience is not measured directly as a performance gap but in a more holistic way [START_REF] Verhoef | Customer experience creation: Determinants, dynamics and management strategies[END_REF].

Hence, this study brings into question the limitations of quantitative approaches to measure and process customers' experience and satisfaction. As the customer journey has become increasingly complex, and experiences become increasingly globalized, it is necessary to use qualitative data and natural language processing (NLP) methods to handle them. In this study, we used a bank's customer database of over 12,300 respondents, which enabled us to investigate over 6,800 qualitative answers and Net Promoter Score (NPS) indicators. This controlled database (as opposed to diverse data collected on the Internet) shows the need to use qualitative data for mass processing through NLP in order to better reveal the factors influencing customer satisfaction and to provide both automatic and individualized processing.

Theoretical framework

Customer experience and satisfaction

Very early, the literature recognized the need to foster a strong and positive customer experience [START_REF] Pine | The experience economy: Work is theater and every business a stage[END_REF][START_REF] Hogan | Linking customer assets to financial performance[END_REF], to which is now added the need to take into account the multiplication of contact points [START_REF] Lemon | Understanding customer experience throughout the customer journey[END_REF]. As defined by [START_REF] Schmitt | Experiential marketing[END_REF] as a multidimensional variable, experience involves five dimensions: sensorial, affective, cognitive, physical, and identity-related. [START_REF] Meyer | Understanding customer experience[END_REF] indicate that experience has become so globalized that it needs to incorporate all direct and indirect contacts (e.g. advertising, services, uses) with customers.

In their review article on the customer experience, [START_REF] Lemon | Understanding customer experience throughout the customer journey[END_REF] propose a literature review and a synthetic model showing the complexity of the customer experience. The authors detail the habitual stages of the purchasing process and identify for each of them four possible "touch points": brand-owned touch points, controlled and initiated by the brands; partner-owned touch points, partly controlled by the brands with their partners (e.g. distributors); customer-owned touch points, controlled by neither brands nor their partners and resulting from consumers' decisions; and social/external touch points, also controlled by neither brands nor partners, but created by role players in the social environment of consumers.

Distinguishing these different contact points is all the more important as consumers operate in an omni-channel context. Numerous studies have focused upon the development of consumer behaviors in the present environment, in which virtual spaces (e.g. Internet sites, e-commerce) are added to the traditional purchasing process [START_REF] Avery | Adding bricks to clicks: Predicting the pattern of cross-channel elasticities over time[END_REF][START_REF] Verhoef | Consumer connectivity in a complex, technology-enabled, and mobile-oriented world with smart products[END_REF] and the customer journey is influenced by new strategies for information search and purchasing [START_REF] Grewal | Retailing in a connected world[END_REF][START_REF] Verhoef | Multi-channel customer management: Understanding the research-shopper phenomenon[END_REF]. In this context, [START_REF] Vanheems | Navigating between real and virtual spaces: An exploration of the hybrid shopping experience[END_REF] show an absence of fragmentation; consumers switch between real and virtual spaces without any apparent difficulty and view their purchasing experience as a whole, despite its blending very diverse aspects. Consumers may search for information on a range of websites or on social media [START_REF] Bronner | Social media and consumer choice[END_REF] and may be interrupted by popup ads, flash sales, e-mail alerts, and so on, while keeping contact with the brand or company and retrieving information. Exchanges with other consumers may sometimes impact on the journey [START_REF] Leeflang | Identifying the direct mailprone consumer[END_REF][START_REF] Libai | Customer-to-customer interactions: Broadening the scope of word of mouth research[END_REF]. Even individuals' future time perspective may affect the link between satisfaction and loyalty [START_REF] Kuppelwieser | Exploring the influence of customers' time horizon perspectives on the satisfaction-loyalty link[END_REF]. All these elements modify the process and complexity of the customer journey [START_REF] Grant | Modelling real-time online information needs: A new research approach for complex consumer behaviour[END_REF] as well as consumers' overall experience [START_REF] Schmitt | From experiential psychology to consumer experience[END_REF].

The increasing attention paid to the customer experience is due to its increasing complexity and its major role in consumer satisfaction [START_REF] Fornell | A national customer satisfaction barometer: The Swedish experience[END_REF]. The customer experience is viewed as preceding satisfaction [START_REF] Shankar | Customer satisfaction and loyalty in online and offline environments[END_REF], which is a determining factor explaining loyalty, number of purchases, average amount spent, and company growth [START_REF] Anderson | The antecedents and consequences of customer satisfaction for firms[END_REF][START_REF] Anderson | Strengthening the satisfaction profit chain[END_REF] as well as recommendations [START_REF] Hosany | Patterns of tourists' emotional responses, satisfaction and intention to recommend[END_REF]. Satisfaction is defined on two levels: horizontal and vertical [START_REF] Oliver | Satisfaction: On a behavioral perspective on the consumer[END_REF]; whereas the former refers to satisfaction arising from a given action (e.g. a transaction), the latter refers to the accumulation of experiences associated with a brand or company. Thus, transaction-specific satisfaction needs to be distinguished from relational satisfaction involving a synthetic evaluation.

Relational satisfaction, just like the customer experience, is influenced by customer journeys that are increasingly dense and complex. Brands cannot control all the contact points or, hence, the overall customer experience [START_REF] Lemon | Understanding customer experience throughout the customer journey[END_REF], but they still have to ensure their customers' satisfaction, all the more so as consumer satisfaction is a determining variable companies may act upon [START_REF] Ho | Incorporating satisfaction into customer value analysis: Optimal investment in lifetime value[END_REF]. It is thus useful to investigate existing approaches to knowledge of and processing of customer satisfaction.

Analysis and processing of customer satisfaction

The complexity of the customer journey is reflected in the numerous theoretical approaches to it, which have been interweaving and following each other for several years [START_REF] Lemon | Understanding customer experience throughout the customer journey[END_REF].

Consumer data processing is known to be a complex and dynamic task [START_REF] Davenport | Managing customer support knowledge[END_REF].

Central to this type of work is the literature on satisfaction, viewed as essential to apprehend the consumer experience, and loyalty [START_REF] Kumar | Revisiting the satisfaction-loyalty relationship: Empirical generalizations and directions for future research[END_REF][START_REF] Wu | Destination loyalty modeling of the global tourism[END_REF][START_REF] Yoo | The effects of e-mass customization on consumer perceived value, satisfaction, and loyalty toward luxury brands[END_REF].

Work on satisfaction measurement is extensive (e.g. [START_REF] Hubbert | Service expectations: The consumer versus the provider[END_REF][START_REF] Oliver | Satisfaction: On a behavioral perspective on the consumer[END_REF]. However, in light of the context detailed in the preceding section, it is difficult to expect that consumers may provide anything else than a very general evaluation, more or less influenced, depending upon the situation and the moment, by the horizontal and/or vertical elements. [START_REF] Llosa | L'analyse de la contribution des éléments de service à la satisfaction : Un modèle tétraclasse[END_REF] distinguishes between different product attributes to increase understanding of satisfaction, identifying four families of attributes that correspond to various combinations of contributions to satisfaction and consumer perception:

• The "basic" elements contribute to customer dissatisfaction and are not determining factors of satisfaction;

• The "plus" elements influence only customers' overall satisfaction and do not influence their dissatisfaction;

• The "key" elements strongly influence the degree of satisfaction, regardless of the customers' evaluation of satisfaction, and they are always determining factors;

• The "secondary" elements do not play a role in overall customer satisfaction and are never determining factors.

Customers' journeys and experiences are dotted with numerous contact points of various natures, and a traditional approach based upon attributes appears to be rather complex: how can one isolate and take into account the elements arising from consumers' actions (customer-owned touch points) or their environment (social/external touch points) [START_REF] Lemon | Understanding customer experience throughout the customer journey[END_REF]? Satisfaction reflect judgments that capture an entire consumption experience [START_REF] Ahrholdt | Enhancing loyalty: When improving consumer satisfaction and delight matters[END_REF]. This also explains interest in delight, a key emotional response [START_REF] Finn | Customer delight: Distinct construct or zone of nonlinear response to customer satisfaction[END_REF] that also matters for the understanding of loyalty [START_REF] Ahrholdt | Enhancing loyalty: When improving consumer satisfaction and delight matters[END_REF].

Furthermore, these approaches entail measures that aim either at identifying problematic attributes [START_REF] Anderson | Strengthening the satisfaction profit chain[END_REF] or at understanding satisfaction level from a general perspective through indirect measures such as the NPS [START_REF] Reichheld | The one number you need to growth[END_REF]. The former are limited by the non-symmetrical and non-linear nature of the relationship between attributes and consumer satisfaction [START_REF] Anderson | Strengthening the satisfaction profit chain[END_REF]. It is difficult to generalize quantitative approaches since, depending upon the situation (e.g. type of company, sector), some attributes may contribute to maintaining satisfaction without necessarily fostering it and others vice versa [START_REF] Anderson | Strengthening the satisfaction profit chain[END_REF]. The NPS could be the main indicator to show the link between satisfaction (as measured by the propensity to recommend the brand) and company growth. Although companies use this indicator to assess overall satisfaction, its capacity to predict growth has been called into question [START_REF] Keiningham | A longitudinal examination of net promoter and firm revenue growth[END_REF]. In short, traditional approaches do not yield generalizable and transferable quantitative tools or operational solutions for satisfaction assessment. [START_REF] Lemon | Understanding customer experience throughout the customer journey[END_REF] have also pointed out the limitations of traditional approaches to customer experience, and after reviewing the main trends relating to knowledge of customer experience, particularly concerning satisfaction, have suggested exploring the potential of big data. The goal of such an approach would be to collect numerous sources of information to take into account the complexity, dynamism, and specific characteristics of the customer experience. Indeed, we contend that masses of qualitative data, used alone or combined with other measures, could account for all the determining factors from the perspective of consumers, and that experiences have become so globalized that a qualitative approach giving free run to consumers' views could help reveal both horizontal and vertical aspects of satisfaction, which we believe are linked rather than independent. In addition, this approach could allow automatic customized processing, in contrast to the current approaches using general or even non-existent processing, especially since we know the importance of recovery efforts after complaining evaluations by consumers [START_REF] Maxham | A longitudinal study of complaining customers' evaluations of multiple service failures and recovery efforts[END_REF].

Design of the study and methodology

The first goal of this study is the automatic exploration of customer voices based on identification of the main themes mentioned by customers. In contrast to traditional approaches focusing upon either a horizontal or a vertical approach [START_REF] Oliver | Satisfaction: On a behavioral perspective on the consumer[END_REF], we contend that the customer experience is made of an optimal blending of vertical and horizontal elements. Hence, we used an NLP method, Latent Dirichlet Allocation, which is a probability model that can categorize documents using themes whose number has been determined beforehand. NLP combines linguistic, computing, and artificial intelligence resources with a view to conducting specific processing of customers' qualitative discourse.

The second goal of this study is to link this analysis to NPS results so as to generate automatic processing of satisfaction. To do this, we tested different hypotheses in order to 1) understand whether the number of themes evoked by the respondents is different according to their NPS score and 2) explore whether the nature of topics evoked is different according to the respondent's NPS score.

To do so, we formulated, for different couples (three populations corresponding to "promotors", "neutral", and "detractors") of NPS scores and for the different topics revealed by NLP, the following hypotheses: 1) For each population: H0: average number of topic (population 1) ≤ average number of topic (population 2) H1: average number of topic (population 1)>average number of topic (population 2) 2) For each population and each topic: H0: proportion of topic (topic 1) (population 1) ≤proportion of topic (topic 1) (population 2) H1: proportion of topic (topic 1) (population 1)>proportion of topic (topic 1) (population 2)

Data collection

The data used for this analysis stem from a controlled satisfaction survey sent by a bank to its customers on a random basis (10% of total customers), to ensure internal validity [START_REF] Campbell | Experimental and Quasi Ex-perimental Designs for Research[END_REF]. Customers may not be asked more than once a year to answer a questionnaire, and they were all questioned in the same short period of time without any interaction between them. This controlled data collection, as distinct from diverse data collected on the Internet, ensures homogeneity in the customers' response topics and prevents collection from contaminating information. Customers provided two simple items of information that were used in the study:

• A recommendation score ranging from 0 to 10, used to calculate the NPS;

• Free comments explaining the score (optional field).

Over 12,000 customer feedback answers were analyzed, among which over 6,800 (i.e. 55%) included qualitative comments concerning the respondents' intention to recommend the brand or not.

Data pre-processing

Data pre-processing constitutes an essential and systematic step in any text modeling. Its purpose is to facilitate algorithmic processes by decreasing noisy data and optimize computation time by reducing vocabulary size upstream.

Identification of multi-word items

The first data pre-processing step involves defining a list of multi-word items that form meaningful and inseparable units. In addition to compound words (e.g., credit crunch, give up) and fixed expressions (e.g., arriving after the battle, in the medium term), the list contains the specific and technical vocabulary pertaining to the banking domain (e.g., bank card, Livret A [French savings account], plan d'épargne logement [French housing savings plan]).

Segmentation (or tokenization)

Segmentation (also called tokenization) involves cutting up text into units composed of single words and multi-word items in order to facilitate the next pre-processing step. Punctuation and capitals are deleted.

Filtering out (removing) the stop words

Stop words are common words bringing little contextual information, such as articles, pronouns, and prepositions. Their frequency of occurrence is high, and removing them can reduce considerably the volume of data before modeling, with little loss of information. There are dictionaries of stop words that can be completed by the specificities of each corpus of documents. For the study, we used the list of stop words in the natural language toolkit (NLTK) in Python.

Stemming

Finally, we used stemming to reduce the complexity of the corpus vocabulary. This step involves removing the prefix and/or suffix of each word to reduce it to its stem, to factor in words belonging to the same stem or lemma (e.g. "retrieval", "retrieved", and "retrieves" reduce to the stem "retrieve"). We used the Snowball stemming algorithm developed by Martin Porter and implemented in NLTK in Python (Table 1).

Data representation

Bag-of-words representation

Machine learning algorithms use digital data rather than text; to use these algorithms, it is thus necessary to transform a textual corpus into numbers. A technique widely used in NLP employs the bag-of-words (BoW) method: the textual corpus is represented by an "items/documents" matrix containing the customer comments in rows and the words used in columns.

Each cell ci,j of the BoW matrix shows the number of occurrences of item j in comment i (Figure 1). After the implementation of the comment pre-processing stage (segmenting, removing stop words, stemming), the corpus of comments may be represented in the BoW matrix (Figure 1).

Term Frequency -Inverse Document Frequency weighting

To increase document differentiation, a Term Frequency-Inverse Document Frequency (TF-IDF) statistical measure is applied to the items/documents matrix. The purpose of this analysis is to underweight items that appear in most documents and thus bring little information upon each occurrence while over-weighting rare words that appear in few documents and are informative.

Hence, in a corpus composed of D documents, the TF-IDF weighting of the word wi in the document dj is calculated as follows:

TF-IDF(wi , dj) = TF(wi , dj) ₓ IDF(wi), with TF(wi , dj)=frequency of use of the word wi in document dj and IDF(wi)= ( )with ni the number of documents containing word wi.

Identifying the corpus topics: Latent Dirichlet Allocation modeling

Probabilistic methods for thematic identification

Document topic modeling is a major issue in text mining. Different NLP techniques have been developed to automatically identify the main topics present in large document corpora and the words that characterize these topics. Probabilistic methods are those that use probability theory to identify topics.

Probabilistic methods of topic identification are based upon the following assumptions:

• Each document is a probabilistic mixture of topics among a set of k topics. This mixture uses a multinomial law. Thus, given a document d, the topic z is present in this document with the probability P(z|d).

• Each topic is a probabilistic mixture of words. This mixture also follows a multinomial law.

Given a topic z, the word w is present in this topic with the probability P(w|z).

Latent Dirichlet Allocation modeling

Latent Dirichlet Allocation (LDA) is a common probabilistic model for topic identification. LDA modeling is a generative probabilistic modeling approach (i.e. the model can infer topics on any new document) proposed by [START_REF] Blei | Latent Dirichlet Allocation[END_REF]. It is a non-supervised method that can model the contents of a document using a set of topics previously unobserved. These topics are defined by word similarities.

The method works as follows.

It is supposed that the document corpus is constituted of k topics. The number k is fixed ahead of the algorithmic process using expert advice and/or a posteriori validation of the consistency of the links between topics and words.

We assume the following:

• Each document is a mixture of topics among a set of k topics. This mixture follows a Dirichlet parameter law α;

• Each topic is a mixture of words. This mixture follows a Dirichlet parameter law β.

The purpose of LDA is to learn the topics present in each document and the words associated with these topics. It is an iterative algorithm:

• Initialization: for each verbatim statement, a set of topics is identified following a Dirichlet parameter distribution α. For each topic, a set of words is identified following a Dirichlet parameter distribution β. The resulting model at this stage is rather improbable because randomly generated.

• Learning: Modeling involves finding the topic and word distributions that have generated answers with the highest probability.

Each word of each document is viewed, and its related topic is updated. This new topic is the one that would have the highest probability of generating the word in this document. Hence, the assumption is that all topics are correct except for the word in question.

Thus, for each word w of each document d, the calculation is: These steps are repeated until stabilization of the estimates.

P(topic z | document d) = probability
These different steps give rise to the mixture of topics present in each document by counting each representation of a topic associated with the words in the document and to the words associated with each topic by counting the words associated with it in the corpus.

Hence, LDA models document words through a set of topics and topics through a set of words.

Results

The first outcome of the analysis is that writing a comment, over and above giving a recommendation score, indicates a lower level of customer satisfaction: whereas the ratio of promoters remains relatively constant, the ratio of detractors is significantly higher when a verbatim statement is entered (Table 2).

Analysis of the corpus topics

Eight major topics emerge from the qualitative data analysis (Table 3). To interpret these topics, we use the notion of relevance as presented by [START_REF] Sievert | LDAvis: A method for visualizing and interpreting topics[END_REF].

With V the number of words composing the vocabulary of the corpus.

With w a word in the corpus (w є {1,…,V}).

We observe Φzw the probability of occurrence of the word w in topic z.

We observe pw the probability of occurrence of the word w in the corpus.

The relevance of the word w in topic z given the parameter f λ (0 ≤ λ ≤ 1) is written:

r(w, z | λ) = log(Φzw) + (1-λ) log(Φzw / pw),
with λ the weighting attributed to the probability of occurrence of the word w in topic z in relation to its frequency of occurrence in the whole corpus.

Thus, if λ=1, the relevance of the word indicates its presence in topic z, regardless of its presence in the rest of the corpus. High relevance shows high presence of the word in topic z, without analyzing its presence in the rest of the corpus.

If λ=0, the relevance of the word indicates the difference in frequency of occurrence of the word between topic k and the rest of the corpus; high relevance shows a much higher presence of the word in topic z than in the rest of the corpus.

Topics and NPS: Five families of elements accounting for satisfaction

Although the ability of the NPS to predict company growth has been called into question [START_REF] Keiningham | A longitudinal examination of net promoter and firm revenue growth[END_REF], this score remains a good overall evaluation of customer satisfaction level. The links between the different topics and the NPS are used to determine whether some topics are significantly associated with a greater number of detractors or promoters; hence, we observe that the analysis of the NPS shows large disparities depending upon the topics mentioned by the customers (Table 4).

A detailed analysis of the significant differences shows that all topics do not appear to play the same role in customer satisfaction, which is congruent with the work of [START_REF] Llosa | L'analyse de la contribution des éléments de service à la satisfaction : Un modèle tétraclasse[END_REF] on categories of attributes and their impact upon satisfaction. Four categories of topics may be slotted into the four families of attributes identified by Llosa:

1. "Basic" topics, significantly associated with more detractors or fewer promoters, which contribute to customer dissatisfaction only and are not determining factors of satisfaction:

• The relational and human dimension

• The bank's brick-and-mortar branches 2. "Key" topics, significantly associated with more promoters but also fewer detractors, which foster satisfaction:

• The bank's Internet site and mobile applications • Mortgages 3. One "plus" topic, significantly associated with more promoters, which plays a role in overall customer satisfaction only, and does not influence dissatisfaction:

• Means of communication 4. "Secondary" topics, not significantly associated with more or fewer detractors or promoters, whose role does not appear to be a determining one:

• Day-to-day operations of accounts and/or contracts

• Pricing policy

Topic 5, which corresponds to the customers' attitude elements, has not been categorized, inasmuch as it corresponds mainly with customers expressing their satisfaction or lack thereof.

NPS and average number of topics: Overall satisfaction

On average, customers mentioned 2.2 topics in their comments. The NPS analysis of the number of topics indicates that low satisfaction is significantly associated with a higher average number of topics.

Conversely, satisfaction is significantly associated with a lower average number of topics (Tables 5 and6). These results come from unilateral tests, allowing us to test the non-inferiority of the average number of themes as a measure of low customer satisfaction (Table 6). These results are consistent with the notion that evaluation is vertical [START_REF] Oliver | Satisfaction: On a behavioral perspective on the consumer[END_REF], that is, general, rather than horizontal, that is, focused on one event only, and all the more so as customer satisfaction is low.

Hence, customers' free comments are general: more satisfied customers express fewer comments, or even none, as mentioned earlier, whereas less satisfied customers express more comments. Positive and negative comments are thus clearly distinct in terms of both content and form.

NPS and topics mentioned: The critical role of the relational or human dimension

Finally, the different unilateral tests were conducted; the analysis of the topics mostly mentioned in relation to the NPS, and hence to the satisfaction level, also reveals significant differences (Table 7).

We observe that topic 5, which focuses on the elements of attitude towards the bank, is significantly present for the promoters and the detractors, but not for neutral customers. This result indicates that customers express their opinion more readily when they are either satisfied or dissatisfied.

The promoters mention topic 1, relating to the Internet site and mobile applications, significantly more than the other customers, which confirms the "key" role of this topic in the construction of satisfaction. At the same time, promoters mention theme 3, relating to the relational and human dimension, significantly less. This result confirms the significant influence of the topic on dissatisfaction. The relational and human aspect appears to be expected by customers, but as something taken for granted. Hence, it tends to be detractors who mention the theme, and their frequent mention of it indicates considerable unmet expectations in this domain.

Finally, neutral customers mention topic 6 significantly more often; it corresponds to the brick-andmortar branches, and was classified as a "plus", as it is positively correlated with satisfaction. This result might indicate comments relating to recommendations rather than negative opinions.

Discussion

The main contribution of this study lies in its methodology, which demonstrates the value of using NLP analysis to identify the nature of the attributes that influence consumer satisfaction and to overcome the limits of traditional approaches to reveal key elements of satisfaction and the nature of their impact [START_REF] Anderson | Strengthening the satisfaction profit chain[END_REF]. The messages of customer voices and the topics identified can be classified following [START_REF] Llosa | L'analyse de la contribution des éléments de service à la satisfaction : Un modèle tétraclasse[END_REF] tetraclass model, which enhances the effectiveness of the method for the identification and analysis of the attributes contributing to consumer satisfaction level.

The study also provides a theoretical contribution in that it complements studies that contrast specific (horizontal) satisfaction and relational (vertical) satisfaction [START_REF] Oliver | Satisfaction: On a behavioral perspective on the consumer[END_REF] by drawing attention to the significant differences in the number and types of topics mentioned depending upon whether customers are promoters, detractors, or neutral. This study promotes considering satisfaction from that perspective of promotion/detraction/neutrality, which shows that satisfaction is not vertical or horizontal but may involve a more or less significant combination of topics. In the case of the data examined in this study, the lower the satisfaction level, the higher the number of topics mentioned. The study also calls into question the traditional approaches used to measure satisfaction, which do not distinguish between the discursive characteristics of satisfied and dissatisfied customers.

The main advantages of NLP lie in the spontaneous and exhaustive character of the data collected and processed, whereas traditional methods presuppose having identified beforehand the determining attributes based on a representative sample. These methods cannot be necessarily associated with any operational processing of satisfaction and/or dissatisfaction mentioned by the respondents. This is particularly the case with the NPS, which is nevertheless used extensively by companies. When customers indicate they would not recommend their bank, they are often met with silence, which can be disastrous and can worsen even further their dissatisfaction level. In contrast, combining NPS and qualitative data processed at both individual and mass levels makes it possible to devise a customized response that can properly address customers' satisfaction issues. In addition, the processing rapidity and automation of this method prove crucial to deal with some situations of dissatisfaction.

Hence, this study calls for the development of artificial intelligence tools for the automation of qualitative data processing and the establishment of models of response to customers. The method used in this study has identified critical topics, which require extensive processing and a high-valueadded human response, and differentiated them from secondary topics that may be dealt with more automatically. Artificial intelligence may thus help provide a response to all customers while making the best use of the valuable-human-time of operational teams, whom it will allow to focus upon the more sensitive cases.

Furthermore, working on data arising from a controlled collection can generate customer knowledge and expertise that may lead to more effective processing of any other qualitative mass-generated materials, such as comments found on social networks or the Internet more generally. At a time of increasingly globalized and omni-channel consumers, these methods prove to be highly promising for customer knowledge. They could also reduce the risk of rupture in the customer journey, enhance the customer experience, and increase the company's profitability. Illustration of the different pre-processing steps for a text corpus.

Step Example

-Acquiring customer comments

Figure 2

 2 Figure2shows an illustration of a BoW transformation of a customer corpus:

  that the document d is concerned by the topic z; P(word w | topic z) = probability that the topic z in the corpus is assigned to the word w.The new topic z is then chosen with the following probability: P(topic z | document d) * P(word w | topic z) = probability that the topic z will generate the word w in the document d.

Fig. 1 .

 1 Fig. 1. Representation of a textual corpus using the bag-of-words method.

  Fast and useful answers given to customers' requests, advisors who know their products very well, particularly the different types of bank cards 2 -Segmenting (tokenization) fast -and -useful -answers -given -to -customers -requests -advisors -who -know -their -products -very -wellparticularly -the -different -types -of -bankcards 3 -Removing stop words fast -useful -answers -given -customers -requests -advisors -know -products -well -particularly -different -types -bank cards 4 -Stemming fast -use -answer -give -customer -request -advisor -know -product -well -particular -different -type -bank card

Table 1

 1

Table 2

 2 Ratio of promoter and detractor customers depending upon the verbatim statements entered.

	Free verbatim statements	Ratio of promoter customers	Ratio of detractor customers
	Verbatim statement not entered	25.1%	35.8%
	Verbatim statement entered	27.6%	45.0%
	Equality of proportions test (p-value)	0.109	<.00001

Table 3

 3 Topics emerging from LDA modeling.

	Topics	Frequent words (λ=1)	Frequent words in this topic only (λ=0)
	1	Internet site and mobile applica-tions	account, site, Internet, manage-ment, products, facilitate, mo-bile, propose	product, mobile, simplicity, trans-parence, informative, experience, service, tool, web
	2	Day-to-day operations of the ac-counts and contracts	account, brand name, request, month, customer, card, fees, service, change, transfer	euros, closing, payment, salary, authorization, transfer, debit, oppo-sition, incident
			customer, listening, advisor,
	3	The relational and human dimen-sion	reception, responsiveness, reac-tivity, availability, rapidity, solu-	listening, availability, rapidity, efficiency, access, friendly, efficient
			tion, staff
			consulting response, request	response, email, contact, answer,
	4	Means of communication	email, telephone, contact, an-	send impossibility, email, interview,
			swer, quick	urgent
	5	Attitude towards the institution	brand name, bank, branch, con-sulting customer, recommend, problem	recommend, satisfied, dissatisfied, work, director, today, replace
	6	The brick-and-mortar branch	branch, reception, opening hours, person, opening, midday, counter	opening hours, midday, counter, morning, hour, machine, Saturday
			loan, real estate, rate, credit,	loan, real estate, manager, renegoti-
	7	Mortgages	request, project, file, manager,	ation, reduce, redemption, exces-
			agreement	sive
	8	Pricing policy	tariff, high, expensive, reduction, cost, diminish, exorbitant, price	fees, tariff, too much, banking, high, expensive, less, management, reduction, more, card

Table 4

 4 Link between topics and customer satisfaction.

		Detractor customers	Promoter customers	NPS
	Topic	Rate	Significance	Rate	Significance
	1 Internet site and mobile	22%	0.001	58%	<0.00001	36
	applications
	2 Day-to-day operations of the	49%	0.219	19%	0.069	-29
	accounts and contracts
	3 The relational and human	49%	0.014	25%	0.153	-24
	dimension
	4 Means of communication	42%	0.472	41%	0.001	-1
	5 Attitude towards the institution	46%	0.490	26%	0.503	-20
	6 The brick-and-mortar branch	43%	0.509	19%	0.041	-23
	7 Mortgages	30%	0.008	46%	<0.00001	16
	8 Pricing policy	44%	0.741	22%	0.180	-22

Table 5

 5 Average number of topics and NPS.

	Customer typology	Score	Average number of topics per comment
			0	2.392
		Detractors 0 to 2	1	2.218	2.352
			2	2.360
	Detractors		3	2.375		2.314
		Detractors 3 to 6	4 5	2.322 2.254	2.287	2.197
			6	2.240
	Neutral	Neutral	7 8	2.190 2.094	2.135	2.135
	Promoters	Promoters	9 10	2.070 2.064	2.067	2.067

Acknowledgements

The authors would like to thank the Bred for making the data available. They would also like to thank the different members of the DCC Chair (Data and customer knowledge). They also thank Nicole Geslin for the translation.

P-value