Mathilde Martin
email: mathilde.martin@universite-paris-saclay.fr

Tess Gridley

Simon Harvey Elwen

Isabelle Charrier

Feel the beat: cape fur seal males encode their arousal state in their bark rate

Keywords: Cape fur seal, vocal communication, territorial defence, arousal state, rhythm perception

Introduction 1

The use of acoustic signals for communication is widespread in animals, particularly in 2 birds and mammals. Acoustic communication is involved in most social interactions including 3 sexual interactions, parent-offspring relationships, territory defence, group coordination, and 4 foraging [START_REF] Halliday | Animal behaviour[END_REF]. Acoustic signals can convey multiple types of information 5 depending on their function and acoustic characteristics. Acoustic signals produced only in 6 specific contexts become linked to a specific signal function and thus can provide information 7 about their general environment. For example, the presence of predators can be advertised 8 through alarm calls [START_REF] Seyfarth | Vervet monkey alarm calls: Semantic communication in a free-ranging primate[END_REF]Blumstein and Armitage 1997a;Macedonia and Evans 9 2010;[START_REF] Moran | Vigilance Behaviour and Alarm Calls in a Captive Group of Meerkats, Suricata suricatta[END_REF] or detection of food through species specific foraging calls (Chapman and 10 Lefebvre 1990;[START_REF] Brown | Food-sharing signals among socially foraging cliff swallows[END_REF][START_REF] Hauser | Food-elicited calls in chimpanzees: Effects of food quantity and divisibility[END_REF][START_REF] Mahurin | Chick-a-dee call variation in Carolina chickadees and recruiting flockmates to food[END_REF]. Some 11 vocalisations can give information about the emitter's social intentions linked to agonistic or 12 affiliative interactions such as aggression or courtship calls [START_REF] Owen | Courtship Calls and Behavior in Two Species of Chorus Frogs, Genus Pseudacris (anura: Hylidae)[END_REF]Charlton et al. 13 2007;[START_REF] Ballentine | Reliable aggressive signalling in swamp sparrows[END_REF][START_REF] Faragó | The bone is mine": affective and referential aspects of dog growls[END_REF]. On a secondary level, vocalisations are further 14 shaped by anatomo-morphological differences among individuals such as the size and shape of 15 the body and/or the vocal tract which affect the voice of an animal, more formally referred to 16 as 'source-filter theory' [START_REF] Fant | Acoustic Theory of Speech Production[END_REF]Taylor and Reby 2010). These variations can encode 17 information about the emitter's species, sex, social rank, age, body size or individual identity 18 and has been reported in many species (e.g. [START_REF] Charrier | Individual identity coding depends on call type in the South Polar skua Catharacta maccormicki[END_REF][START_REF] Rendall | Acoustic correlates of caller identity and affect intensity in the vowel-like grunt vocalizations of baboons[END_REF]Reby et al. 19 2005;[START_REF] Gwilliam | Vocal identity and species recognition in male Australian sea lions, Neophoca cinerea[END_REF][START_REF] Briefer | Indicators of age, body size and sex in goat kid calls revealed using the source-filter theory[END_REF][START_REF] Matrosova | The potential to encode sex, age, and individual identity in the alarm calls of three species of Marmotinae[END_REF]Déaux et al. 20 2016). These acoustic 'voice' features constitute the 'static' component of the information 21 encoded in signals [START_REF] Taylor | Vocal Production by Terrestrial Mammals: Source, Filter, and Function[END_REF]). This plays a major role in social interactions such as 22 parent-offspring individual recognition, sexual competition, territoriality and mate selection 23 [START_REF] Bradbury | Principles of Animal Communication[END_REF]. 24

Acoustic signals can also convey information on the motivational and emotional state 25 of the emitter, called the 'dynamic' component (Ohala 1984;[START_REF] Taylor | Vocal Production by Terrestrial Mammals: Source, Filter, and Function[END_REF]. The expression 26 of emotions in signals enhances the quantity of information transmitted as it provides additional 27 cues about the internal state of the calling individual or the urgency of the situation (Briefer 28 2020). Numerous studies have shown that vocalisations produced by mammals are capable of 29 expressing emotions (for review see [START_REF] Briefer | Vocal expression of emotions in mammals: Mechanisms of production and evidence[END_REF]. The expression of emotional state in 30 communicative signals benefits both the transmitter and the receiver by reducing uncertainty 31 and eliciting appropriate behavioural responses (e.g. approach or avoidance), a crucial aspect 32 for an organism's fitness [START_REF] Dall | Information and its use by animals in evolutionary ecology[END_REF]. Conveying accurate emotional information through 33 acoustic signals can be especially advantageous in specific situations involving potentially high 34 energy expenditure such as predator avoidance or the assessment of rivals [START_REF] Charrier | What Makes an Australian Sea Lion (Neophoca cinerea) Male's Bark Threatening?[END_REF]35 Casey et al. 2015) and the general mediation of social interactions among individuals (Bradley 36 and Mennill 2009;[START_REF] Bradbury | Principles of Animal Communication[END_REF][START_REF] Brudzynski | Social Origin of Vocal Communication in Rodents[END_REF][START_REF] Papale | The Social Role of Vocal Complexity in Striped Dolphins[END_REF]. 37

Generally, the acoustic parameters mostly altered by arousal state are linked to the fundamental 38 frequency or energy distribution in the spectrum, and the temporal patterning of calls (number 39 of vocalizations/elements produced per unit of time and/or interval between those elements, 40 Briefer 2012). These dynamic variations in vocalizations are mostly involuntary, resulting from 41 changes in the somatic and autonomic nervous system which in turn lead to modifications in 42 vocal production organs as well as in respiration and salivation [START_REF] Scherer | Vocal communication of emotion: A review of research paradigms[END_REF]. Changes in the 43 call production rate within sequences have been reported to convey emotional or motivational 44 cues in signals of different species: non-song birds [START_REF] Gemard | Variation, use and informative content of burrowing petrels' male calls in Kerguelen[END_REF]), songbirds (DuBois et al. 45 2009;[START_REF] Geberzahn | How a songbird with a continuous singing style modulates its song when territorially challenged[END_REF] and multiple mammal species e.g. dogs (Yin and McCowan 46 2004), baboons [START_REF] Rendall | Acoustic correlates of caller identity and affect intensity in the vowel-like grunt vocalizations of baboons[END_REF], bats [START_REF] Bastian | Affect cues in vocalizations of the bat, Megaderma lyra , during agonistic interactions[END_REF], marmots (Blumstein and 47 Armitage 1997b), mongoose [START_REF] Manser | The acoustic structure of suricates' alarm calls varies with predator type and the level of response urgency[END_REF]. 48

Due to the diversity of their social organizations and mating strategies, Otariid species 49 are a good model system in which to study vocal signals across a range of behavioural, mating 50 and ecological contexts. By being the most colonial pinniped species and with its complex 51 social organisation, the Cape fur seal (Arctocephalus pusillus pusillus) lie at one end of this 52 spectrum. The investigation of emotional information encoded in males' vocalizations is 53 particularly interesting in this species for two reasons. First, this is a highly polygynous species 54 with territorial males competing for territories and constituting harems of up to 30 females with 55 their pups [START_REF] Wickens | Comparative population dynamics of fur seals[END_REF]. Harems are held by bulls whose age gives them sexual 56 and social maturity: males become sexually mature at around 5 years (i.e. subadults), but they 57 only reach the social maturity when they are 8 to 13 years old (i.e. adult or territorial males) 58 [START_REF] Jefferson | Marine mammals of the world: a comprehensive guide to their identification[END_REF]. Adult males are much larger than subadult males, and show 59 characteristic physical features: enlarged neck and shoulders, mane with longer guard hair 60 around the neck and shoulders [START_REF] Jefferson | Marine mammals of the world: a comprehensive guide to their identification[END_REF]. In general, territorial males fast 61 throughout the breeding period and display aggressive behaviours toward other territorial bulls 62 or subadult males [START_REF] Riedman | The pinnipeds: seals, sea lions, and walruses[END_REF]. In this context, the ability of animals to correctly identify 63 individual identity cues and emotional state are of great importance [START_REF] Insley | A review of social recognition in pinnipeds[END_REF] for 64 males to be able to differentiate neighbouring territorial bulls from unfamiliar individuals, as 65 well their arousal state in order to avoid unnecessary conflicts [START_REF] Roux | Behavioral Cues to Individual Recognition in the Subantarctic Fur Seal , Arctocephalus tropicalis[END_REF]66 Tripovich et al. 2008b, c). 67 68 Secondly, Cape fur seal subadult males are highly abundant on breeding colonies and 69 occur in close proximity to territorial males. It contrasts with most fur seal species for which 70 non-territorial males (subadults) do not haul-out on breeding colonies but form isolated groups 71 of immature individuals [START_REF] Bartholomew | Mother-young relations and the maturation of pup behaviour in the Alaska fur seal[END_REF][START_REF] Trillmich | First observations on colony structure, behavior, and vocal repertoire of the South American Fur Seal (Arctocephalus australis) in Peru[END_REF]. Cape fur seal 72 subadult males are chased and kept out of harems by territorial males through aggressive 73 behaviour and vocalizations. Subadults are quite mobile and roam over the entire colony: they 74 alternate resting periods in the colony with periods at sea to thermoregulate or to feed (unlike 75 territorial males that fast during the breeding season) [START_REF] Nowak | An ethological perspective on common cross-language utilization of F0 of voice[END_REF]. For subadult 76 males looking for mating opportunities at the edge of harems, the arousal state and behavioural 77 intentions of territorial bulls are important cues to decipher in order to adjust both their level of 78 vigilance and their behavioural response (e.g. escape in case of attack) [START_REF] Jefferson | Marine mammals of the world: a comprehensive guide to their identification[END_REF]. 79 80

This study aims to explore whether Cape fur seal males use acoustic signals to convey their 81 emotional state and to modulate social interactions during the breeding season. Cape fur seal 82 males produce barks in across a wide range of social interactions: from affiliative (mating 83 behaviours, adult males only) to agonistic, including both territorial defence (adult males only) 84 and general aggression (both adults and subadults) (Martin et al. in revision). Barks are very 85 short calls (mean duration: 0.12 s; Martin et al. in revision) always produced in sequences. 86

These vocalisations convey information about the sex and the age-class of the caller (Martin et 87 al. in revision), as well as individual identity [START_REF] Martin | Extreme ecological constraints lead to high degree of individual stereotypy in the vocal repertoire of the Cape fur seal (Arctocephalus pusillus pusillus)[END_REF]). Individual identity is encoded 88 in both the frequency parameters of barks (the fundamental frequency and the energy 89 distribution among the spectrum) but also in temporal parameters relative to the sequences 90 [START_REF] Martin | Extreme ecological constraints lead to high degree of individual stereotypy in the vocal repertoire of the Cape fur seal (Arctocephalus pusillus pusillus)[END_REF]. Temporal features in call sequences including the duration of a sequence, 91 the interval between barks or the bark rate has been shown to vary with behavioural context and 92 arousal state of males in several otariid species including three species of sea lion species 93 [START_REF] Peterson | Airborne vocal communication in the california sea lion, Zalophus californianus[END_REF][START_REF] Schusterman | Temporal patterning in sea lion barking (Zalophus californianus)[END_REF][START_REF] Fernández-Juricic | Vocal rates and social context in male South American Sea Lions[END_REF]Kunc and 94 Wolf 2008). In fur seals, it has been suggested in Subantarctic fur seal [START_REF] Roux | Sociobiologie de l'Otarie à fourrure d'Amsterdam, Arctocephalus tropicalis[END_REF] but 95 empirically tested for only one species, the Australian fur seal (Tripovich et al. 2008a). 96

The aim of this study was twofold. We first investigated if the acoustic characteristics 97 of Cape fur seal territorial males' barks change with the behavioural context and their arousal 98 state. We hypothesized that the bark rate could be involved in this process, with high arousal 99 states inducing an increase in bark rate, but we explored this hypothesis using multiple acoustic 100 parameters of barks. Secondly, playback experiments were performed on subadult males to 101 investigate if their behavioural response varies with the acoustic features of barks produced by 102 a territorial male. Unlike territorial males that have established territories and are likely able to 103 recognize each other by their vocalizations (demonstrated in several pinniped species : Gwilliam 104 et al. 2008;Tripovich et al. 2008;[START_REF] Casey | Rival assessment among northern elephant seals: Evidence of associative learning during male-male contests[END_REF], roaming subadults may be unfamiliar to 105 the barks of surrounding territorial males when they temporarily rest in one place. Therefore, 106

we hypothesised that subadult males may use information encoded in acoustic signals to assess 107 the general arousal state of nearby territorial males and thus determine their threat level. This 108 is the first study to experimentally investigate the mechanisms of coding-decoding the arousal 109 state in vocal signals between territorial and subadult males in pinnipeds. 110 111

Material and methods

112

Study site 113

The study was conducted at Pelican Point Cape fur seal breeding colony (25°52.2'S, 114 14°26.6'E), Walvis Bay, Namibia from 12 th December 2019 to 6 th February 2020, during the 115 breeding season. Pelican Point is a sandy peninsula with homogenous and flat topography, 116 roughly 1 km wide and 10 km north to south with most seals (and all field work) clustered in 117 the northern ~3km. There is no recent published data on the number of individuals at Pelican 118 Point but a 2011 survey reported 12.000 pups (Ministry of Fisheries and Marine Resources, 119 unpubl. data) so there would be the same number of females as each female gives birth to a 120 single pup each year. The ratio in harems averages 1 male to 30 females (harems size ranging 121 from 7 to 66 females) [START_REF] Riedman | The pinnipeds: seals, sea lions, and walruses[END_REF][START_REF] Nowak | An ethological perspective on common cross-language utilization of F0 of voice[END_REF], so the number of adult 122 territorial males (> 8-13 years old) is estimated to be around 400 on the peninsula, with likely 123 3 to 4 times as many subadult males (~3-8 years old). Throughout the breeding season, harems 124 are distributed all around the point, close to the shore, mostly on the protected waters of the 125 eastern side (MM pers obs). Some groups of non-breeding juveniles (one-or two-year old 126 individuals) and subadult males (sexually but not socially mature) are observed on the vicinity 127 of the breeding colony. to advertise his presence, and those made during interactions with females within his harem 151 (i.e., herding, nuzzling), excluding mating. ( 2) 'Confrontation' corresponded to a high arousal 152 state and involved the focal male reacting to another male approaching or entering his territory. 153

Barks were directed towards the intruder and were associated with both non-physical (chasing) 154 and physical interactions (fight) between the two individuals. (3) 'Mating' referred to barks 155 produced during a copulation event, which may act to advertise dominance/success towards 156 other males, or be directed towards the focal female. 157 Acoustic analysis -Recordings were resampled at 22.05 kHz as none of the barks' 158 frequencies exceeded 10 kHz (Martin et al. in revision). The acoustic analysis of barks was 159 performed using Avisoft SAS Lab Pro (R. Specht, version 5.2.14, Avisoft Bioacoustics, Berlin, 160

Germany) and barks were visually inspected on spectrograms calculated with a 1024-point fast 161

Fourier transform (FFT), 75% overlap and a Hamming window. Five good-quality barks (i.e. 162 with low background noise and no overlap with other vocalizations) were selected per sequence 163 and a minimum of 3 different sequences (5 barks per sequence) were analysed per behavioural 164 context for each individual (so at least 15 barks per individual per context were measured). 165

Selected sequences were individually high-pass filtered at 100 Hz because most of the 166 background wind noise was between 0 and 100 Hz. A set of nine acoustic features were 167 measured for each bark: total duration of the call (Dur; ms), fundamental frequency (f0; Hz), 168 frequency value of the 1 st , 2 nd and 3 rd energy peaks (Fmax1, Fmax2, Fmax3; Hz), quartiles of 169 energy spectrum (Q25, Q50, Q75; Hz) and the proportion of energy below 500 Hz (Ebelow500; 170 %) from the averaged energy spectrum (Hamming window, frequency range for analysis: 0-171 5000 Hz). A 10 th feature, the production rate of barks (Barkrate; Hz) was measured for three to 172 five sequences within each behavioural context for each adult male. 173 The bouts of barks used as stimuli were selected from all available bark bouts to have as wide 191 a range of bark rates between individuals (i.e. between stimuli) as possible. 192

For analysis, a set of eight acoustic features were used to investigate the potential drivers 193 of the behavioural response of subadult males. Barkrate was calculated as a single average of 194 the bark rates for all three bark sequences of a playback track. Seven spectral features (f0, 195 Fmax1, Q25, Q50, Q75, Ebelow500 and Bdw12, described in section 1) were calculated on each 196 of the three bouts of the playback track. The three values obtained for each spectral feature were 197 averaged to obtain a single value, later used in the statistical analysis. 198

Providing high quality recordings is challenging in a very noisy CFS colony. Due to 199 limited high-quality recordings some playback stimuli had to be used more than once. To limit 200 pseudo-replication a total of eight stimuli tracks from eight different adult males were used and 201 the choice of a playback track was randomized for each experiment. 202

203

Playback procedure -In this study, subadult males were not individually identifiable 204 (marked) but given that they were both numerous and highly mobile (in contrast to territorial 205 males), and there were 1-13 days between trials, we are confident that the seals tested were 206 different on each day of the experiment and would consider the broadcasted barks as from 207 unfamiliar. Playbacks were performed on non-interacting subadult males that were resting, 208 either laying down or standing still. A single observer approached closely by crawling slowly 209 into the colony. When a suitable focal animal was identified the loudspeaker was manually 210 placed at 3 to 5 meters from the animal and not directly in his field of vision i.e. on his side to 211 facilitate the evaluation of his reaction. The observer moved back to 6 to 10 metres from the 212 focal animal and remained lying on the ground to minimise any disturbance. Once the observer 213 and equipment were settled, the behaviour of the target individual was monitored for two 214 minutes to ensure the subadult male was not actively engaged in an interaction with any other 215 animals, and was not obviously responding to the presence of the observer and so his 216 behavioural response could only be due to our playback stimulus. If no interaction was observed 217 during the 2-min observation period, we would broadcast the playback track. Stimuli were 218 broadcast using a waterproof and wireless high-powered speaker (JBL Charge 3, 2 x 10W, 219 were positive for an approach towards the speaker and negative for a retreat (e.g. 0: no 236 movement, 1: walked all the way to the speaker, -0.5: half way retreat). If the tested individual 237 had been distracted/disturbed by another individual during the playback period, the experiment 238 was excluded from the analysis. Since few subadult males reacted by producing barks, variables 239 related to calling were not included in the analysis. We performed a principal component 240 analysis (PCA) on the four behavioural variables: latency to look, look duration, latency to 241

Results

257

Acoustic features of territorial adult males' barks in various behavioural contexts 258

We recorded the barks from 47 territorial males for which, 14 were recorded in at least 259 2 different behavioural contexts (standard activity, confrontation or mating). As only four adult 260 males were recorded during copulation, statistical comparisons were not possible for this 261 context. The pairwise comparison of the 10 measured acoustic features revealed that, in spite 262 of our relatively small sample size (n=10), both the variables Barkrate and f0 were significantly 263 lower during standard activity than during confrontation (Wilcoxon Signed Rank tests, n = 10, 264 V = 55, p = 0.0059 -same in both cases) (Fig. 1, Fig. 2). The bark rate increased by 17 % (0.55 265 Hz) on average between standard activity and confrontation, while the f0 value increased by 20 266 % (22 Hz) on average. Other variables such as bark duration and spectral features did not show 267 any significant differences between these two behavioural contexts. Although not statistically 268 tested barks produced by a territorial male during mating were always produced at a faster rate 269 compared to the same individual barking in standard activity (Fig. 1a). However, no general 270 trend for the fundamental frequency value as well as for other features was found (on Fig. 1b.) 271 272

Behavioural responses of subadult males to playbacks of territorial adults 273

A total of 22 playback experiments on different subadult males were included in our 274 analysis. Of these, 20 looked towards the speaker soon after they heard the playback (mean 275 latency to look: 3 ± 2.6 sec), and for a relatively long period of time (mean duration to look: 8.2 276 ± 5.2 sec). A third of tested individuals (7/22) stood up and moved in response to the playback 277 with four moving towards and three males away from the speaker. Only three subadult males 278 called back to our playback track, and one did not react at all. 279

The behavioural responses were combined into a PCA and out of the four principal 280 components generated, only the first PC (PC1) had an eigenvalue greater than 1. PC1 explained 281 48.33 % of the total variance (Table 1) and was strongly correlated with the two response 282 variables involving looking towards the loudspeaker: latency to look (negative correlation) and 283 look duration (positive correlation) (Table 1). Positive values of PC1 scores corresponded to 284 short latency to look (i.e. quick response) and a long period of looking, describing a vigilant 285 behaviour. The two response variables involving movement of the subadult males i.e. latency 286 to move and distance moved, were correlated with PC2 (Table 1) and were therefore excluded 287 from the following analysis. 288

The subadult males' responses (indicated by PC1 scores values) were investigated as a 289 function of the acoustic parameters of the barks of the territorial adult male played back during 290 the experiment using a multiple linear regression. Although only Barkrate and f0 showed 291 differences among the behavioural contexts, all acoustic variables were inspected because the 292 duration or spectral features of a call can provide clues to the physical condition of the emitter, 293 especially the body size (source-filter theory, Fant 1960; Taylor and Reby 2010) and can 294 therefore influence the behavioural response of subadult males. Of all the acoustic parameters 295 included in the model, Barkrate was the only variable with significant regression coefficients 296 (Table 2): a positive relationship was found between PC1 scores and the playback bark rate. 297

The multiple regression was therefore simplified to a simple regression between PC1 scores 298 and Barkrate and presented in Fig. 3. As PC1 was highly correlated with the latency to look 299 and the look duration, it means that as the Barkrate of the playback stimulus increased, the 300 tested subadult males looked at the loudspeaker more quickly (i.e., lower latencies) and for a 301 longer period of time. No significant relationships were found for other acoustic parameters (f0 302 and spectral features of barks, Table 2). 303 304 Discussion 305 Graded vocalisations allow animals to communicate different levels of information, emotional 306 state or urgency by modifying only a few parameters of their vocal production. For instance, 307 the rate, loudness and harshness of alarm calls vary with the type of predators or their spatial 308 proximity in several species [START_REF] Leger | Single-note Vocalizations of California Ground Squirrels: Graded Signals and Situation-specificity of Predator and Socially Evoked Calls[END_REF][START_REF] Manser | The acoustic structure of suricates' alarm calls varies with predator type and the level of response urgency[END_REF][START_REF] Leavesley | Communicating about danger: Urgency alarm calling in a bird[END_REF]. 309

The bark is the primary call type used by male Cape fur seals (Martin et al. in revision). It serves 310 in all social interactions corresponding to different arousal states: 'patrolling' their harem and 311 advertising their presence, randomly interacting with females, insistently interacting with a 312 particular female for the purpose of mating, copulating or chasing other males. In some other 313 fur seal species, males use multiple types of vocalizations (e.g. threat calls, growls or 314 submissive calls, Pierson 1987; Phillips and Stirling 2001;[START_REF] Page | Interspecific differences in male vocalizations of three sympatric fur seals (Arctocephalus spp.)[END_REF]Tripovich et al. 315 2008a) enabling them to modulate their vocal production with context and allow receivers to 316 more clearly interpret their behavioural intentions (Phillips and Stirling 2001) (Phillips and 317 Stirling 2001). For instance, in South American fur seal, the use of a full threat call is a signal 318 of high arousal state and a willingness to fight, while submissive calls rather show an attempt 319 to retreat (Phillips and Stirling 2001). For this reason, within the constraints of the restricted 320 vocal repertoire Cape fur seal males, coding arousal state in barks rates would be an advantage. 321

In colonial species, call redundancy (i.e. repetition of an identical signal) is a widely 322 used strategy allowing individuals to increase the probability of being heard in such noisy 323 environments (Mathematical theory of information, [START_REF] Shannon | Formant Modification through Vocal Production Learning in Gray Seals[END_REF]. For example, 324 colonial seabirds such as penguins use redundancy in their calls [START_REF] Lengagne | Finding one's mate in a king penguin colony: Efficiency of acoustic communication[END_REF]Aubin 325 and Jouventin 2002;[START_REF] Gemard | Variation, use and informative content of burrowing petrels' male calls in Kerguelen[END_REF], and Otariids commonly produce sequences of barks 326 [START_REF] Peterson | The Guadalupe Fur Seal: Habitat, Behavior, Population Size, and Field Identification[END_REF][START_REF] Stirling | Implications of a comparison of the airborn vocalizations and some aspectsof the behaviour of the two australian fur seals, Arctocephalus spp., on the evolution and present taxonomy of the genus[END_REF][START_REF] Roux | Sociobiologie de l'Otarie à fourrure d'Amsterdam, Arctocephalus tropicalis[END_REF][START_REF] Fernández-Juricic | Vocal communication and individual variation in breeding South American Sea Lions[END_REF]327 Phillips and Stirling 2001;[START_REF] Page | Interspecific differences in male vocalizations of three sympatric fur seals (Arctocephalus spp.)[END_REF]Tripovich et al. 2008a). Previous studies have 328

shown the high information potential of barks: with barks reported to convey information 329 relative to species recognition [START_REF] Gwilliam | Vocal identity and species recognition in male Australian sea lions, Neophoca cinerea[END_REF], individual identity (Fernández-Juricic et 330 al. 1999;[START_REF] Tripovich | Species-specific characteristics and individual variation of the bark call produced by male australian fur seals, arctocephalus pusillus doriferus[END_REF][START_REF] Gwilliam | Vocal identity and species recognition in male Australian sea lions, Neophoca cinerea[END_REF], neighbour-stranger recognition (Roux 331 and Jouventin 1987;Tripovich et al. 2008c;[START_REF] Attard | Vocal discrimination in mate guarding male australian sea lions: Familiarity breeds contempt[END_REF]) and even geographical variation 332 [START_REF] Attard | Vocal discrimination in mate guarding male australian sea lions: Familiarity breeds contempt[END_REF][START_REF] Ahonen | Adult male Australian sea lion barking calls reveal clear geographical variations[END_REF]Martin et al. 2021, in revision). To our knowledge, 333 investigations of the emotional and motivational state encoded in pinnipeds' vocalizations have 334 so far mainly focused on mother and pup calls (e.g. [START_REF] Perry | Further studies of the role of harbour seal (Phoca vitulina) pup vocalizations in preventing separation of mother-pup pairs[END_REF][START_REF] Charrier | The subantarctic fur seal pup switches its begging behaviour during maternal absence[END_REF]335 Collins et al. 2011) and little is known about the territorial or agonistic calls of males and the 336 emotional information encoded in bark rates [START_REF] Fernández-Juricic | Vocal rates and social context in male South American Sea Lions[END_REF]Charrier et al. 337 2011). 338

In the first part of this study, we reported significant changes in some acoustic features 339 and the production rate of barks depending on the context of production and the level of 340 motivation of Cape fur seal males. In line with our predictions and former assumptions among 341 Otariids, our dataset revealed an increase of the bark rate produced by males during high-342 arousal events (i.e. confrontation) compared to standard activities (i.e. spontaneous barks). Such 343 an increase of the bark rate is consistent with studies on birds and mammals which have shown 344 that the call rate is a powerful way of coding emotions and motivation states [START_REF] Rendall | Acoustic correlates of caller identity and affect intensity in the vowel-like grunt vocalizations of baboons[END_REF]345 Yin and McCowan 2004;[START_REF] Geberzahn | How a songbird with a continuous singing style modulates its song when territorially challenged[END_REF][START_REF] Gemard | Variation, use and informative content of burrowing petrels' male calls in Kerguelen[END_REF]. Repeated vocalisations 346 enhance signal detectability [START_REF] Schleidt | Tonic communication: Continual effects of discrete signs in animal communication systems[END_REF]. Moreover, an increase of the signal-to-silence ratio 347 makes the call sequence sound louder thus exhibiting a higher degree of hostility (Yin and 348 McCowan 2004). Faster barks may thus appear easier to detect in the noisy environment of the 349 colony, but could also evoke the presence of a threatening opponent. 350

In addition, variations in the bark rate were accompanied by an increase in the 351 fundamental frequency (f0) during high arousal state contexts. Variations in motivational or 352 emotional state were claimed to lead to changes in the fundamental frequency of mammalian 353 vocalisations such as in sheep [START_REF] Sèbe | Bio-acoustic analyses to assess emotion in animals: Acoustic patterns are linked to behavioural, cardiac and hormonal responses of ewes to the separation from their lambs[END_REF], baboons [START_REF] Rendall | Acoustic correlates of caller identity and affect intensity in the vowel-like grunt vocalizations of baboons[END_REF], common marmosets 354 [START_REF] Yamaguchi | Time course of vocal modulation during isolation in common marmosets (Callithrix jacchus)[END_REF], bats [START_REF] Bastian | Affect cues in vocalizations of the bat, Megaderma lyra , during agonistic interactions[END_REF], or Weddell seals [START_REF] Collins | The influence of behavioural context on Weddell seal (Leptonychotes weddellii) airborne mother-pup vocalisation[END_REF]) 355 among many others (for full review see [START_REF] Briefer | Vocal expression of emotions in mammals: Mechanisms of production and evidence[END_REF]. The fundamental frequency 356 characterized the first step of the two-stage process of vocal production known as the 'source-357 filter theory' [START_REF] Fant | Acoustic Theory of Speech Production[END_REF]Taylor and Reby 2010). It refers to the rate of opening and closing of 358 the glottis under the pressure of an air-flow from the lungs. Primarily driven by the length and 359 mass of the vocal folds, f0 is assumed to be a relatively constant parameter, but can vary due to 360 modifications in muscular tension in the vocal folds, sub-glottal pressure or changes in airflow 361 from the lungs (Taylor and Reby 2010). In our study case, shifts in f0 between various contexts 362 of production are likely to be linked to modifications in the male's behaviour. In fact, during 363 fighting and mating, males are involved in a more active muscular effort than during standard 364 activity, and thus muscle tension might change their fundamental frequency. Therefore, these 365 f0 changes are likely produced in a less intentional way than Bark rate variations as f0 variations 366 are linked to the vocal tract anatomy. Indeed, if captive pinnipeds have been shown to be 367 capable of some vocal motor control after training and reinforcement (Reichmuth and Casey 368 2014;[START_REF] Shannon | Formant Modification through Vocal Production Learning in Gray Seals[END_REF], under natural circumstances as studied here, such variations 369 of the fundamental frequency are more likely resulting from changes in the somatic and 370 autonomic nervous system induced by emotions, and thus non-intentionally produced and can 371 be considered an honest signal of emotional state. 372

In the second part of the study, we tested if subadult male seals can perceive these 373 variations in bark series from different adult males and thus adapt their behaviour in accordance. 374

In general, the broadcasting of a territorial male's barks led to a rapid increase in the vigilance 375 of the target individuals which looked towards the sound source for an average of 3 seconds 376 after the start of the experiment. This is intuitive as bulls tend to chase subadult males away 377 from their harem, and therefore represent a real threat to subadult males that are smaller and 378 less competitive than bulls in the case of a fight. As subadult males roam in the entire breeding 379 colony, they are likely not familiar with individual territorial bulls, and thus assessing the threat 380 level from their vocalisations is the only way to adjust their behaviour, and ensure survival. 381

Here, we found that subadult males preferred to keep a low profile (i.e., remained lying down) 382 rather than an active retreat. Similar results were found in male northern elephant seals when 383 immature males were exposed to calls from mature males [START_REF] Casey | The genesis of giants: Behavioural ontogeny of male northern elephant seals[END_REF]. In colonial 384 environments where the densities of individuals are high, this strategy seems advantageous for 385 both individuals: territorial males save energy by not fighting subadults as long as they do not 386 compromise the integrity of their harem (i.e. by attempting to mate with females) and subadults 387 avoid unnecessary escape impulses. The weak reaction of subadult males to these playback 388 experiments also highlighted the importance for social species of using multi-modal signals in 389 their interactions. In this case of dominance among males, the behavioural response of subadult 390 males in response to vocalisations produced by an adult (resulting in increased vigilance) could 391 be enhanced by a visual cue (i.e. the sight of a territorial bull approaching). Acoustic signals 392 may therefore serve as an initial warning signal that would be corroborated (or not) by the 393 occurrence of a visual cue. The simultaneous presence of multiple cues is known to modulate 394 the receiver's response [START_REF] Partan | Communication goes multimodal[END_REF] and thereby may benefit communication 395 processes [START_REF] Ay | Robustness and complexity co-constructed in multimodal signalling networks[END_REF]Ratcliffe et al. 2016;[START_REF] Wierucka | Multimodal mother-offspring recognition: the relative importance of sensory cues in a colonial mammal[END_REF]. 396 Secondly, our experiments revealed that Cape fur seal subadult males' response to the 397 broadcast of territorial males' barks could vary with the bark rate of the stimulus. This 398 reinforces previous findings stating that pinnipeds have the sensory and cognitive abilities to 399 decipher rhythm in acoustic signals and are able to process them in order to modulate their 400 social behaviour [START_REF] Cook | A California sea lion (Zalophus californianus) can keep the beat: Motor entrainment to rhythmic auditory stimuli in a non vocal mimic[END_REF][START_REF] Mathevon | Northern Elephant Seals Memorize the Rhythm and Timbre of Their Rivals' Voices[END_REF]) Similar results were found in 401 Australian sea lions in which bark rate was used by males to estimate the potential threat from 402 a rival [START_REF] Charrier | What Makes an Australian Sea Lion (Neophoca cinerea) Male's Bark Threatening?[END_REF]. No other acoustic parameters such as the fundamental frequency 403 value (f0) or spectral features seemed to influence the behavioural response of subadults. Since 404 these are individualized parameters and containing information about the anatomy of the calling 405 territorial male (e.g. body size; Taylor and Reby 2010), this indicates that subadult males did 406 not specifically react to the phenotype-linked acoustic cues of territorial males but to their bark 407 rate indicating their arousal state. It contrasts with previous studies on mammals responding 408 more to vocalizations with changes in 'source-related' acoustic features [START_REF] Charlton | Giant pandas perceive and attend to formant frequency variation in male bleats[END_REF]409 Taylor et al. 2010). 410

We previously showed that the bark rate of a territorial male varied with behavioural context 411 and arousal state. In these playback experiments, all broadcast barks were recorded in the same 412 context (i.e. standard activities) to avoid confounding effects. Subadult males responded with a 413 significantly higher level of vigilance to broadcast sequences with faster bark rates. This 414 revealed that even for barks produced during low arousal standard activities, bark rate is 415 regarded as a threat level indicator. Because the fundamental frequency value is often filtered 416 by the vocal tract of the emitter and therefore difficult to perceive, the bark rate seems the 417 unique way to assess the arousal state of a territorial male for subadults. Further experiments 418

where subadult males will be tested with barks recorded in different contexts of production 419 should reveal even stronger results. 420

This study is the first investigation in pinnipeds coupling the coding of the arousal-state in 421 territorial males' vocalisations and the decoding processes by subadult males. This knowledge 422 allows us to better understand the communication network of social species and the 423 involvement of acoustic signals in their interactions. Further investigations on different 424 behavioural contexts such as male-female interactions or mating would be interesting to carry 425 out to understand whether females could use males' vocalisations to identify their harem holder 426 and facilitate orientation within the colony when they return from foraging at sea. 427

Tables

128 129

 128 This study is composed of two main componentsdescribing the acoustic features of barks and 130 sequences of barks from territorial adult males within the colony under three different levels of 131 arousal. Secondly, we used playback experiments to investigate the behavioural responses of 132 subadult males to the series of adult male barks. 133 134 Recording and describing the territorial adult males' barks in different behavioural 135 contexts 136 Recording procedure -Recordings were made of focal adult bulls identified through 137 paint marking applied for associated research questions (oil-based paint marks applied to 138 individuals using a roller and pole, Martin et al. in revision a), natural scarring, or spatial 139 distribution within the colony (GPS location). To avoid resampling the same unmarked 140 territorial males, recordings were performed at different locations within the colony. Adult 141 males' barks were recorded using a Sennheiser ME67 directional shotgun microphone 142 (frequency range: 40 -20000 Hz, sensitivity: 50mV/Pa ± 2.5 dB) using a 44.1 kHz sampling 143 rate and connected to a two-channel NAGRA LB digital audio recorder. The distance between 144 the calling animal and the microphone ranged from 3 to 6 meters (visual estimation). While 145 recording, the experimenter described the social context and the behaviour of the barking 146 individual in a lapel microphone connected to the second channel. For this study, we only used 147 behaviours and activities of territorial males during barking. Behaviours were categorized into 148 three main contexts corresponding to different arousal states: (1) 'Standard activity' referred to 149 activities with a low arousal state and included non-directed (or spontaneous) barks produced 150

  Statistical analysis -Pairwise comparisons of the ten measured acoustic variables 174 between the different behavioural contexts were carried out using a Wilcoxon Signed Rank test 175 for paired samples in R (R Development Core Team 2020). 176 177 Playbacks: Behavioural responses of subadult males to the playback of adult barks 178 We tested the subadult male's response to barks produced at different rates through 179 playback experiments. Playback experiments were performed over the course of 17 days with 180 one to three experiments per day. Trials consisted of observing the behavioural response of 181 resting subadult males to the broadcast of barks recorded from a territorial adult male (i.e. the 182 stimulus). 183 184 Creation of stimuli -We built eight playback tracks with bark sequences from eight 185 different territorial males. Each playback track consisted of three bouts of barking (average 186 duration 4.1 sec ± SD 0.7 sec), recorded from the same territorial male and spliced together with 187 a separation of 3 seconds between bouts. Note that no manipulation was made to the natural 188 rate of each bark bout. We only used recordings made from individuals barking during standard 189 activities to avoid any confounding effect of arousal state which may reflect in voice features. 190

  frequency response: 65 HZ-20 kHz) connected to a Bluetooth sound player (Sony NW-A35). 220 The amplitude level of the playback tracks was adjusted to the natural amplitude of male barks 221 at the range of 3 to 5 m (84 ± 2 dB SPL at 1 m, measured with a 'Testo 815' sound level meter, 222 A-weighting, fast-response). 223 224 Behavioural analysis -Since there are many seals in the colony and interactions among 225 individuals frequently occur, tested individuals were constantly exposed to external stimuli 226 from neighbouring animals. To minimise these complications, the behavioural response of the 227 target individual was only observed for 20 seconds from the start of the playback. If the tested 228 individual was distracted/disturbed by another individual during the playback period, the 229 experiment was excluded from the analysis. Response variables recorded were: latency (all 230 latency measures in seconds) to look towards the speaker, look duration towards the speaker, 231 latency to move (posture change or movement), distance of movement (away or towards the 232 speaker, estimated by eye), latency to bark and the number of barks if produced. An absence of 233 response was assigned a default value of 20s for latencies. The distance to move was relative 234 and estimated as a ratio of the initial distance between the speaker and the focal animal. Values 235

  move and distance moved, to obtain a score of the behavioural response[START_REF] Mcgregor | Quantifying Responses to Playback: One, Many, or Composite Multivariate Measures?[END_REF]. The 242 PC scores of the principal components (PC) with eigenvalues greater than 1 were used to 243 quantify the level of response of subadult males to playbacks. 244245We investigated the relationships between the behavioural response of subadult males 246 (i.e. PC scores) and the eight acoustic features measured from the played back bark sequences 247 of adult males. As an initial test, the acoustic variables from adult males (Barkrate and the set 248 of 7 spectral features) were tested for multicollinearity at the level of 0.8. The variables Q25 249 and Q50 were excluded because of their high correlation with other variables. All other 250 variables included in the analyse were normalized to account for differences in units of 251 measurement and ranges of values. Using a multiple linear regression in R (lm function, base 252 package, R Development Core Team 2020), we investigated the relationship between PC scores 253 and the final set of acoustic parameters (6 variables: Barkrate, f0, Fmax1, Q75, Ebelow500 and 254 Bdw12). The normality and homoscedasticity of the residuals were visually verified. 255 256

Figure captions Fig. 1 Fig. 2 Fig. 3 2 =

 captions1232 Figure captions

  Figure 2

Table 1

 1 Summary of the principal component analysis (PCA) performed with four behavioural responses variables. Bold values show behavioural variables highly correlated to the first two principal components

	PC1	PC2	PC3	PC4

Table 2

 2 Multiple linear regression analysis of barks' acoustic parameters broadcast during the playback on subadult males' behavioural response (PC scores)

			PC1 scores
		β	SE	p
	Barkrate	1.024	0.303	0.0041*
	f0	0.011	0.482	0.982
	Fmax1	-0.396	0.479	0.421
	Q75	-0.166	0.441	0.712
	Ebelow500	-0.396	0.391	0.327
	Bdw12	0.834	0.489	0.109

Acknowledgments

We acknowledge the Namibian Ministry of Fisheries and Marine Resources and the Namibian Chamber of Environment for their support of this research. We thank Dorothy Fourie for her assistance in the field. Special thanks to Naude Dreyer, Kevin van Schalkwyk and Craig Gibson for their knowledge on the species, logistical support in the field, and their enthusiasm for the project. Special thanks to two anonymous referees for their helpful comments on the first version of our manuscript.

The research was supported by CNRS through the MITI interdisciplinary programs and the Sea Search -Namibian Dolphin Project. MM is funded by a PhD scholarship from the French Ministry of Higher Education and Research. TG was awarded by a sub-committee B post-doctoral fellowship from the University of Stellenbosch.

Conflicts of interest/by the Namibian Ministry of Fisheries and Marine Resources (MFMR).

for this paper are deposited in the Zenodo repository (http://doi.org/10.5281/zenodo.4630682).


Barbara Oliveira Mendes

Malak Alameh

Beatrice Ollivier

Jerome Montnach

Nicolas Bidere

Frederique Souaze

Nicolas Escriou

Flavien Charpentier

Isabelle Baro

Michel De Waard
email: michel.dewaard@univ-nantes.fr

Beatrice Ollivier

Jerome Montnach

Béatrice Ollivier

Jérôme Montnach

Nicolas Bidère

Frédérique Souazé

Isabelle Baró

Gildas Loussouarn
email: gildas.loussouarn@univ-nantes.fr

SARS-CoV-2 E and 3a proteins are inducers of pannexin currents

Introduction

SARS-CoV-2 is the third virus of the genus Beta-coronavirus of the Coronaviridae family to be responsible for a Severe Acute Respiratory Syndrome in this century, after SARS-CoV-1 in 2002-2003 [START_REF] Drosten | Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome[END_REF] and MERS-CoV in 2012 [START_REF] Zaki | Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia[END_REF]. As a result, it is of great importance to best characterize coronaviruses and the associated pathophysiology, with the hope that new treatments will emerge to complement vaccine approaches for people who cannot access the vaccines or are not responsive to them. In addition to paxlovid which is already available but associated with bothersome side-effects (3), many potential anti-COVID-19 treatments are in development but it is too soon to tell how efficient they will be, namely with regard to the continuous emergence of new variants, and if the cost will be reasonable [START_REF] Ledford | Hundreds of COVID trials could provide a deluge of new drugs[END_REF].

Viroporins, i.e. ion channels encoded by a virus genome, are potential targets by antiviral agents, as demonstrated by the case of amantadine which inhibits the acid-activated M2 channel of Influenza A virus [START_REF] Ison | Antivirals and resistance: influenza virus[END_REF]. Several studies led to the suggestion that two proteins of SARS-CoV are viroporins. SARS-CoV-2 Envelop (E) protein, is a one-transmembrane-domain membrane protein (75 amino-acids) almost identical to SARS-CoV-1 Envelop protein (95% identity). The SARS-CoV-2 ORF3a (3a) protein is a larger three-transmembrane-domain membrane protein (275 aminoacids) relatively similar to the SARS-CoV-1 3a protein (73% identity).

Regarding the ion channel function of these proteins, there are clearly several contradicting studies: some of them raising intriguing issues and others not confirming these reports. Concerning in-vitro membrane incorporation of purified E or 3a protein in lipid bilayers, the presence of ion channel activity was reportedly associated with these viral proteins [START_REF] Wilson | SARS coronavirus E protein forms cation-selective ion channels[END_REF][START_REF] Wilson | Hexamethylene amiloride blocks E protein ion channels and inhibits coronavirus replication[END_REF][START_REF] Torres | Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein[END_REF][START_REF] Verdiá-Báguena | Coronavirus E protein forms ion channels with functionally and structurally-involved membrane lipids[END_REF][START_REF] Nieto-Torres | Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome[END_REF][START_REF] Castaño-Rodriguez | Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis[END_REF]. However, a review article soundly outlined the lack of robust data and raised ethical concerns casting doubts on the validity of the scientific messages [START_REF] Mcclenaghan | Coronavirus Proteins as Ion Channels: Current and Potential Research[END_REF]. Concerning viral protein expression in cells, the expression of SARS-CoV-1 E protein also led to conflicting results [START_REF] Pervushin | Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel. Baric RS, éditeur[END_REF][START_REF] Nieto-Torres | Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein[END_REF]. Pervushin et al managed to identify plasma membrane currents generated by heterologous expression of SARS-CoV-1 E protein in HEK-293 cells [START_REF] Pervushin | Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel. Baric RS, éditeur[END_REF] but not Nieto-Torres et al [START_REF] Nieto-Torres | Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein[END_REF]. More recently, expression of wild-type (WT) SARS-CoV-2 E protein did not lead to interpretable ionic currents in HEK-293S or Xenopus laevis oocytes [START_REF] Cabrera-Garcia | The envelope protein of SARS-CoV-2 increases intra-Golgi pH and forms a cation channel that is regulated by pH[END_REF] despite their high homology among SARS-CoV viruses. In an attempt to favor plasma membrane targeting and reveal a putative current, a Cterminal predicted ER retention signal of SARS-CoV-1 E protein was replaced by a Golgi export signal from Kir2.1 channel. Expression of this chimera could then be associated with the generation of a non-rectifying and cation-selective current. This current was thus quite different from the outwardly rectifying current observed by Pervushin and collaborators [START_REF] Cabrera-Garcia | The envelope protein of SARS-CoV-2 increases intra-Golgi pH and forms a cation channel that is regulated by pH[END_REF]. Furthermore, another study using a membrane targeting sequence, fused to the N-terminus of the SARS-CoV-1 E protein, provided a non-rectifying current that was 100-fold larger than the one observed in the two previous studies [START_REF] Breitinger | Viroporins: Structure, function, and their role in the life cycle of SARS-CoV-2[END_REF]. This suggests that such modification of either Nor C-terminus are too drastic to faithfully report the actual activity of the native proteins. SARS-CoV-1 3a protein was also investigated and expression of the WT protein in HEK-293 cells [START_REF] Chan | The ion channel activity of the SARS-coronavirus 3a protein is linked to its pro-apoptotic function[END_REF] or Xenopus laevis oocytes [START_REF] Lu | Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release[END_REF][START_REF] Schwarz | Emodin inhibits current through SARSassociated coronavirus 3a protein[END_REF][START_REF] Schwarz | Kaempferol Derivatives as Antiviral Drugs against the 3a Channel Protein of Coronavirus[END_REF] was associated with a poorly selective outwardly rectifying current in both models, resembling to the one observed upon expression of the E protein.

To summarize, there is no unequivocal evidence that SARS-CoV E and 3a proteins are viroporins active at the plasma membrane of the host cell. However, it was recently reported that SARS-CoV-2 E and 3a proteins can promote cell death [START_REF] Ren | The ORF3a protein of SARS-CoV-2 induces apoptosis in cells[END_REF][START_REF] Xia | SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target[END_REF], on one hand. On the other hand, apoptosis is associated with the increase of an outwardly-rectifying current conducted by pannexins [START_REF] Bao | Pannexin membrane channels are mechanosensitive conduits for ATP[END_REF][START_REF] Senthivinayagam | Adaptive thermogenesis in brown adipose tissue involves activation of pannexin-1 channels[END_REF][START_REF] Bhat | Human Pannexin 1 channel: Insight in structure-function mechanism and its potential physiological roles[END_REF]. This led us to reinvestigate the actual function(s) of SARS-CoV-2 E and 3a proteins in mammalian cells in the frame of the cell toxicity of these proteins.

In this study, CHO cells expressing either CoV-2 E or 3a proteins tended to develop into a roundshaped form with a tendency to detach from the Petri dish, a process exacerbated compared to control conditions. This cell phenotype is consistent with cell death [START_REF] Uchida | Induction of Apoptosis by Phosphatidylserine[END_REF][START_REF] Crea | Over-expression of hTERT in CHO K1 results in decreased apoptosis and reduced serum dependency[END_REF] and we confirmed by flow cytometry experiments that expression of E or 3a proteins does indeed promote cell death. Transfected cells, still attached to the Petri dish (adhering cells), had unchanged basal currents indicating that E and 3a proteins are unlikely to act as plasma membrane channels. In contrast, recording whole-cell currents on round-shaped and detached cells, we observed large outwardly-rectifying currents only in E or 3a protein-expressing cells but not in control dying cells. This current is reminiscent of those observed in previous publications using HEK-293 cells and oocytes expressing the SARS-CoV-1 proteins [START_REF] Pervushin | Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel. Baric RS, éditeur[END_REF][START_REF] Lu | Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release[END_REF][START_REF] Schwarz | Emodin inhibits current through SARSassociated coronavirus 3a protein[END_REF][START_REF] Schwarz | Kaempferol Derivatives as Antiviral Drugs against the 3a Channel Protein of Coronavirus[END_REF]. Application of carbenoxolone, a pannexin channel inhibitor, suggests for the first time that these currents are pannexinmediated conductances, potentially activated in apoptotic cells. In conclusion, both SARS-CoV-2 E and 3a proteins are most likely triggers of endogenous conductance.

Results

We first focused on native E and 3a proteins. To maximize the chance of observing E and 3a protein-induced ionic currents, we chose to use pIRES plasmids, in which the protein of interest situated in the first cassette is more expressed than the eGFP reporter in the second cassette, thereby guaranteeing expression of a high level of the protein of interest in fluorescent cells [START_REF] Mizuguchi | IRES-Dependent Second Gene Expression Is Significantly Lower Than Cap-Dependent First Gene Expression in a Bicistronic Vector[END_REF][START_REF] Choveau | KCNQ1 Channels Voltage Dependence through a Voltage-dependent Binding of the S4-S5 Linker to the Pore Domain[END_REF]. For the purpose of this study, we also selected CHO rather than HEK-293 cells because they express minimal endogenous currents [START_REF] Yu | Endogenous voltage-gated potassium channels in human embryonic kidney (HEK293) cells[END_REF]. We compared whole-cell currents recorded during a ramp protocol, in cells transfected either with a control pIRES2-eGFP plasmid (pIRES), or the same plasmid containing the cDNA of the SARS-CoV-2 E protein (pIRES -E) or 3a protein (pIRES -3a). Unexpectedly, we did not observe any difference in the currents recorded for the SARS-CoV-2 proteins expressing cells compared to control pIRES condition (Figure 1A). However, many cells transfected with either E-or 3a-encoding plasmids were developing altered morphology, shifting from spindle-like cells to more round cells (Figure 1B), similar to what was previously observed in MDCK cells heterologously expressing SARS-CoV-1 E protein [START_REF] Teoh | The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis[END_REF]. Analysis with Fiji tool confirmed an increase in cell roundness (Figure 1C and suppl. Figure 1). In particular, in patch-clamp experiments, some cells were coming off from the dish bottom by losing adhesion. Cell counting indicated that slightly more cells were losing adhesion when E or 3a protein were expressed (3.4 ± 0.6 % in non-transfected cells, 5.2 ± 1.0% in pIRES condition, 6.6 ± 0.7% in pIRES -E, 6.0 ± 1.2% in pIRES -3a, n=3-5). As classically performed, currents shown in Figure 1A were recorded from adhering cells while non-adhering cells were disregarded in this initial investigation. Noteworthy, in each condition, both spindle-like and round adhering cells were studied (pIRES: 21 spindle-like and 17 round cells; pIRES E: 9 and 13, pIRES 3a: 18 and 11). Since both E and 3a proteins are promoting cell death [START_REF] Ren | The ORF3a protein of SARS-CoV-2 induces apoptosis in cells[END_REF][START_REF] Xia | SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target[END_REF], we hypothesized that the various cell morphological patterns (spindle-shaped, round-adhering, and round non-adhering) may correspond to the development of cell death, as described earlier in CHO and other cells [START_REF] Uchida | Induction of Apoptosis by Phosphatidylserine[END_REF][START_REF] Crea | Over-expression of hTERT in CHO K1 results in decreased apoptosis and reduced serum dependency[END_REF]. Flow cytometry analysis performed on the eGFP-positive CHO cells (Figure 2) showed that expression of E and 3a proteins increases the percentage of dying cells, more significantly late cell death, revealed by propidium iodide permeability (suppl. Figure 2). The effect of 3a protein was greater than the effect of the E protein. E protein-induced cell death could be reduced by the pan-caspase inhibitor QVD-Oph, while 3a protein-induced cell death was not (suppl. Figures 3 &4), suggesting that E protein induces apoptosis, while 3a protein activates non-conventional caspase-independent cell death.

Both E and 3a proteins possess a C-terminal PDZ binding motif (PBM). E protein PBM has been suggested to be a virulence factor [START_REF] Castaño-Rodriguez | Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis[END_REF] and binds to host cells PDZ domains, leading to abnormal cellular distribution of the targeted proteins [START_REF] Teoh | The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis[END_REF]. 3a PBM interacts with at least five human PDZcontaining proteins (TJP1, NHERF3 & 4, RGS3, PARD3B), suggesting that it also alters cellular organization [START_REF] Caillet-Saguy | Host PDZcontaining proteins targeted by SARS-CoV-2[END_REF]. We thus evaluated whether deletion of these domains impacts E and 3a proteins propensity to trigger cell death. Two C-terminal deletions used in previous studies to remove E protein PBM, ∆4 for the last 4 amino-acids (31) and ∆8 for the last 8 residues (11) abolished the pro-apoptotic effect of E protein (Figure 2). When looking individually at early and late cell death, we observed that both truncation of E and 3a protein decreased late cell death (suppl. Figure 2). Since both E and 3a proteins promote cell death, we hypothesized that the cells starting to come off the surface may express currents induced by cell death such as pannexin currents [START_REF] Senthivinayagam | Adaptive thermogenesis in brown adipose tissue involves activation of pannexin-1 channels[END_REF][START_REF] Bhat | Human Pannexin 1 channel: Insight in structure-function mechanism and its potential physiological roles[END_REF][START_REF] Poon | Unexpected link between an antibiotic, pannexin channels and apoptosis[END_REF]. We thus compared patch-clamp recordings of adhering cells vs. non-adhering cells in the 3 conditions: control pIRES, pIRES -E and pIRES -3a plasmids (Figure 3). In the control pIRES condition, focusing on non-adhering cells in the 35-mm dish and using the ramp protocol, we observed an outwardly rectifying current with a mean current density of 8.5 ± 3.1 pA/pF at +70 mV, slightly higher than in spindle-or round-shaped adhering cells (3.4 ± 0.8 pA/pF). On the other hand, in non-adhering cells expressing either the E or 3a protein, currents were much larger in the E protein condition (I +70mV = 31 ± 9 pA/pF, two-way ANOVA test on the ramp-evoked currents: p<0.0001), but also in the 3a protein condition (I +70mV = 44 ± 13 pA/pF, two-way ANOVA test on the ramp-evoked currents: p<0.0001), as compared to non-adhering cells in the control pIRES condition. Noteworthy, only a fraction of the cells was exhibiting large rectifying currents, as shown in Figure 3: 4 out of 43 in the control pIRES condition, 14 out of 46 in the E protein condition and 16 out of 41 in the 3a protein condition. These experiments suggest that cell death and/or change in morphology induced by expression of E and 3a proteins may lead to an increased membrane permeability by enhancing the expression or activity of an endogenous ion channel.

The outwardly rectifying currents that we observed resemble apoptosis-induced and stretchinduced pannexin currents [START_REF] Bao | Pannexin membrane channels are mechanosensitive conduits for ATP[END_REF][START_REF] Senthivinayagam | Adaptive thermogenesis in brown adipose tissue involves activation of pannexin-1 channels[END_REF][START_REF] Bhat | Human Pannexin 1 channel: Insight in structure-function mechanism and its potential physiological roles[END_REF]. Thus, we applied the pannexin inhibitor carbenoxolone (CBX) on non-adhering cells that display large outwardly rectifying currents (Figure 4). We observed that CBX, applied at 50 µmol/L, inhibits the observed current, restoring current amplitudes similar to the ones observed in the control cells. Altogether, these observations suggest that the current triggered by the expression of E and 3a protein is a pannexin current. We reported above (Figure 2) that deleting the last 4 amino-acids of E protein (∆4) drastically reduced its pro-apoptotic effect. Cells expressing the ∆4 E protein showed an average roundness similar to cells expressing the WT protein, suggesting that deletion did not prevent its effect on cell morphology (Figure 5A and suppl. Figure 5). Also, when focusing on round and non-adhering ∆4 E protein expressing cells, we could still record large outwardly rectifying currents (5 out of 20 cells), suggesting that C-terminal deletion of E protein does not abolish the induction of pannexin currents, despite prevention of apoptosis probed by flow cytometry experiments (Figure 5B).

We also reported in Figure 2 that the deletion of the last 10 amino-acids of 3a protein (∆10) also decreased cell death albeit to a lesser extent. As for E protein deletion, cells expressing the truncated 3a protein showed an average roundness similar to cells expressing the WT protein (Figure 5A and suppl. Figure 5). Focusing on the non-adhering cells, we could still record large outwardly rectifying currents (9 out of 14 cells), suggesting that deletion of the last 10 aminoacids is not sufficient to completely abolish the induction of pannexin-like currents (Figure 5C).

Discussion

The concept that SARS-CoV E or 3a proteins could be viroporins expressed at the plasma membrane is a seducing one as it could help the identification of new therapeutic drugs against COVID-19 by setting up a screening program on the channel activity. However, this concept was controversial and part of the reasons that explain the controversy about the function of E and 3a proteins is likely linked to the fact that these proteins also trigger morphological alterations and cell death. Cell death is associated to important changes in lipid composition and membrane permeability which suffices to fuel the controversy. One could imagine for instance that cell death would be a way to activate the function of E and 3a proteins at the plasma membrane, but an alternative hypothesis could be simply that cell death triggers an endogenous cell conductance unrelated to the cell function of E and 3a viral proteins. The only way to address these issues was to confirm the cell toxicity, to characterize the impact of the expression of these proteins on cell shape and behavior, to measure plasma membrane conductance, and to characterize them to get an idea of their nature and probe a pharmacological agent that would match the conductance identity. We manage to solve these issues by carefully characterizing the membrane conductances triggered by both E and 3a proteins. The fact that both proteins trigger the same conductance independently of each other was a first indication that they could not be viroporins at the plasma membrane. The second hint was that adhering cells, whether they had a round-shape or not, did not exhibit any outward conductance in spite of E or 3a protein expression. Finally, the sensitivity to carbenoxolone of the outward currents triggered by E or 3a proteins in non-adhering cells was an indication that these viral proteins trigger cellular alterations, such as morphological changes and cell death that are inducers of pannexin-like current. Globally, these observations remain consistent with previous observations that both E and 3a proteins are mainly localized in intracellular compartments in various cell types [START_REF] Nieto-Torres | Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein[END_REF][START_REF] Yuan | Subcellular localization and membrane association of SARS-CoV 3a protein[END_REF][START_REF] Nal | Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E[END_REF][START_REF] Liao | Biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein[END_REF][START_REF] Cohen | Identification of a Golgi Complex-Targeting Signal in the Cytoplasmic Tail of the Severe Acute Respiratory Syndrome Coronavirus Envelope Protein[END_REF][START_REF] Kern | Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs[END_REF]. Therefore, it is fair to mention that we cannot fully conclude on the viroporin nature of these viral proteins as their localization in subcellular organelles prevents us for clearly testing their intrinsic potential for channel activity.

We showed that expression of either of these two proteins in CHO cells induces an increase in cell death, as quantified by flow cytometry experiments. It is likely, although we did not investigate this point in details, that this cell death accompanies the change in cell morphology and Petri dish detachment. As such, our observation that pannexin-like currents are mainly observed in detached round-shaped cells indicates that major cell morphology changes, up to level of surface detachment, is required for the induction of pannexin-like currents. Whatever the exact mechanism, the upregulation of pannexin channels upon cell death was already observed earlier [START_REF] Chekeni | Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis[END_REF]. It is thus not so surprising in fact that other reports faced problems reporting and identifying the conductances triggered by E and 3a viral proteins. The conditions for observing them are indeed quite drastic and require examining cells that are in the combined dying and detachment process, something that is not naturally pursued by researchers, especially if one hopes to detect a viroporin activity. To reconcile our data with earlier publications, we noticed that whole-cell currents observed by others after SARS-CoV-1 E or 3a protein expression in HEK 293 cells [START_REF] Pervushin | Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel. Baric RS, éditeur[END_REF][START_REF] Chan | The ion channel activity of the SARS-coronavirus 3a protein is linked to its pro-apoptotic function[END_REF] were also very similar to pannexin currents: outwardly rectifying current, with a reversal potential close to 0 mV at physiological ion concentrations indicating a poor ion selectivity, and amplitude of a few 100 pA.

One possibility is that the pannexin-like currents that we observed are due to the classical caspase-induced cleavage of pannexin [START_REF] Navis | Pannexin 1 Channels as a Therapeutic Target: Structure, Inhibition, and Outlook[END_REF]. Intriguingly, deletion of the C-terminal PBM of E protein abolished its pro-apoptotic effect but cell morphology alteration and pannexin-like currents were still present. Regarding the 3a protein, deletion of its PBM domain only decreased and did not completely abolish the promoted cell death, but again cell morphology alteration and pannexin-like currents were preserved. Altogether, these results suggest that cell morphology modification and pannexin induction may be linked and these processes are not necessarily accompanied by cell death. One has to keep in mind that pannexin currents are activated by many stimuli in addition to cell death [START_REF] Navis | Pannexin 1 Channels as a Therapeutic Target: Structure, Inhibition, and Outlook[END_REF]. In particular, pannexin currents are also stretch-activated and may be enhanced in the detached cells which are undergoing major morphological alterations [START_REF] Bao | Pannexin membrane channels are mechanosensitive conduits for ATP[END_REF]. If pannexins are already activated by stretch, they would not be overactivated by their cleavage by caspase, which would explain the fact that E and 3a proteins truncation do no prevent pannexin current, but only cell death.

In conclusion, SARS-CoV-2 native E and 3a proteins, and probably SARS-CoV-1 ones, do not act as plasma membrane ion channels, but trigger the activity of plasma membrane pannexin channels, most likely through morphological alteration of the cells. However, our study does not rule out potential channel activity in intracellular membranes leading to cell death. Future studies will give more insights on the role of pannexin channels in COVID-19 physiopathology and treatment [START_REF] Swayne | Consideration of Pannexin 1 channels in COVID-19 pathology and treatment[END_REF][START_REF] Luu | Pannexin-1 channel opening is critical for COVID-19 pathogenesis[END_REF][START_REF] Nadeali | Possible role of pannexin 1 channels and purinergic receptors in the pathogenesis and mechanism of action of SARS-CoV-2 and therapeutic potential of targeting them in COVID-19[END_REF].

Materials and Methods

Cell culture

The Chinese Hamster Ovary cell line, CHO, was obtained from the American Type Culture Collection (CRL-11965) and cultured in Dulbecco's modified Eagle's medium (Gibco 41966-029, USA) supplemented with 10% fetal calf serum (Eurobio, EU), 2 mM L-Glutamine and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin, Corning, EU) at 5% CO 2 , maintained at 37°C in a humidified incubator. This cell line was confirmed to be mycoplasma-free (MycoAlert, Lonza).

Drugs

Carbenoxolone disodium salt was purchased from Sigma and 100 mmol/L stock solution was prepared in H 2 O. Drugs used for flow cytometry experiments were QVD-Oph (#OPH001, R&D Systems, 10 mmol/L stock solution in DMSO), S63-845 (Chemieteck, 10 mmol/L stock solution in DMSO), ABT737 (Selleckchem, 10 mmol/L stock solution in DMSO).

Construction of E and 3a proteins encoding plasmids

SARS-CoV-2 E and 3a nucleotide sequences, containing a Kozak sequence added right before the ATG (RefSeq NC_045512.2) were synthesized by Eurofins (Ebersberg, EU) and subcloned into the pIRES2 vector with eGFP in the second cassette (Takara Bio Europe, EU). Truncated ∆4 and ∆8 E proteins, as well as ∆10 3a protein constructs, lacking the last 12, 24 and 30 last nucleotides, respectively, were also synthesized by Eurofins. Plasmid cDNAs were systematically resequenced by Eurofins after each plasmid in-house midiprep (Qiagen, EU).

E and 3a cDNA transfection

The Fugene 6 transfection reagent (Promega, WI, USA) was used to transfect WT and mutant E and 3a plasmids for patch-clamp, morphology analysis and flow cytometry experiments according to the manufacturer's protocol. Cells were cultured in 35-mm dishes and transfected, at a 20% confluence for patch clamp experiments and 50% confluence for flow cytometry assay, with a pIRES plasmid (2 µg DNA) with the first cassette empty or containing wild-type or truncated SARS-CoV-2 E or 3a protein sequence. For morphology analysis, cells were cultured in ibidi μ-Slide 8 well dishes and transfected at a 20% confluence with the same plasmids. In pIRES2-eGFP plasmids, the second cassette (eGFP) is less expressed than the first cassette, guaranteeing expression of high level of the protein of interest in fluorescent cells [START_REF] Mizuguchi | IRES-Dependent Second Gene Expression Is Significantly Lower Than Cap-Dependent First Gene Expression in a Bicistronic Vector[END_REF][START_REF] Choveau | KCNQ1 Channels Voltage Dependence through a Voltage-dependent Binding of the S4-S5 Linker to the Pore Domain[END_REF].

Electrophysiology

Two days after transfection, CHO cells were mounted on the stage of an inverted microscope and bathed with a Tyrode solution (in mmol/L: NaCl 145, KCl 4, MgCl 2 1, CaCl 2 1, HEPES 5, glucose 5, pH adjusted to 7.4 with NaOH) maintained at 22.0 ± 2.0°C. Patch pipettes (tip resistance: 2.0 to 2.5 MΩ) were pulled from soda-lime glass capillaries (Kimble-Chase, USA) with a Sutter P-30 puller (USA). A fluorescent cell was selected by epifluorecence. The pipette was filled with intracellular medium containing (in mmol/L): KCl, 100; Kgluconate, 45; MgCl 2 , 1; EGTA, 5; HEPES, 10; pH adjusted to 7.2 with KOH. Stimulation and data recording were performed with pClamp 10, an A/D converter (Digidata 1440A) and a Multiclamp 700B (all Molecular Devices) or a VE-2 patch-clamp amplifier (Alembic Instruments). Currents were acquired in the whole-cell configuration, low-pass filtered at 10 kHz and recorded at a sampling rate of 50 kHz. First, series of twenty 30-ms steps to -80 mV were applied from holding potential (HP) of alternatively -70 mV and -90 mV to subsequently off-line calculate Cm and Rs values from the recorded currents. Currents were then recorded using a 1-s ramp protocol from -80 mV to +70 mV, every 4 s (cf. Figure 1). Regarding non-adhering cells, we considered them as with large current density when the current density measured at +70 mV was superior to mean + 2 x standard deviation of the current density in adhering cells, in the same condition.

Figure 1 :Figure 1 :

 11 Figure 1: Expression of E and 3a protein is accompanied by altered cellular morphology but no modification in whole-cell currents in adhering cells. (A) Average current densities (± sem) recorded during the ramp protocol (inset) in adhering CHO cells expressing either eGFP (pIRES), SARS-CoV-2 E and eGFP proteins (pIRES -E) or SARS-CoV-2 3a and eGFP proteins (pIRES -3a). (B) Superimposed brightfield and eGFP fluorescence images of CHO cells in the same 3 conditions as shown in A. (C) Analysis of cell roundness in cell automatically detected by Fiji, cf. suppl. Figure 1. Plot of individual cells (pIRES, n = 1922; pIRES -E, n = 1198; and pIRES -3a, n = 1283), median ± interquartile range. **** p<0.0001 as compared to pIRES control, Kruskal-Wallis test.

Figure 2 :

 2 Figure 2: Expression of E and 3a protein induces cell death. (A) Flow cytometric analysis of eGFP-positive CHO cells expressing only eGFP (pIRES), or co-expressing eGFP and one of the following SARS-CoV-2 cDNA: the full-length E protein (pIRES -E), C-terminally deleted E protein (pIRES -E Δ 4 or pIRES -E Δ 8), the full length 3a protein (pIRES -3a) or the C-terminally deleted 3a protein (pIRES -3a Δ 10). After 48 h of expression, cells were stained with annexin V AlexaFluor 647 (APC)/propidium iodide (PI, Perc-P). (B) Mean ± sem of the percentage of Annexin V+ cells among eGFP-positive CHO cells expressing only eGFP (pIRES), or eGFP and fulllength or truncated E protein. *** p<0.001 as compared to pIRES control, one-way ANOVA, ## p<0.01 as compared to E protein, t-test. (C) Mean ± sem of the percentage of Annexin V+ cells among eGFP-positive CHO cells expressing only eGFP (pIRES), or eGFP and full-length or truncated 3a protein. ** p<0.01 as compared to pIRES control, one-way ANOVA.

Figure 3 .

 3 Figure 3. Expression of E and 3a protein is accompanied by outwardly rectifying currents in non-adhering CHO cells only. Left, average current densities (± sem) recorded during the ramp protocol in adhering (empty circles) or non-adhering (filled circles) CHO cells expressing either eGFP (A, pIRES), SARS-CoV-2 E and eGFP proteins (B, pIRES -E) or SARS-CoV-2 3a and eGFP proteins (C, pIRES -3a). Right, plot of the individual adhering cells (darker color) or non-adhering cells (lighter color). **** p<0.0001, as compared to adhering cells, two-way ANOVA.

Figure 4 .

 4 Figure 4. Currents due to E and 3a protein expression in non-adhering CHO cells are suppressed by the pannexin inhibitor carbenoxolone (CBX). (A) Recording of the current amplitude in a CHO cell expressing SARS-CoV-2 E and eGFP proteins that displays large outwardly rectifying currents, in absence and presence of CBX. (B) Left, average current densities recorded during the ramp protocol in non-adhering CHO cells, expressing SARS-CoV-2 E and eGFP proteins (pIRES -E), in absence (Ctrl, lighter color) and presence of CBX (darker color). Right, plot of the individual non-adhering cells in absence (lighter color) and presence of CBX (darker color). (C) Recording of the current amplitude in a CHO cell expressing SARS-CoV-2 3a and eGFP proteins that displays the outwardly rectifying currents, in absence and presence of CBX. (D) Left, average current densities recorded during the ramp protocol in non-adhering CHO cells, expressing SARS-CoV-2 3a and eGFP proteins (pIRES -E), in absence (Ctrl, lighter color) and presence of CBX (darker color). Right, plot of the individual non-adhering cells in absence (lighter color) and presence of CBX (darker color). *** p<0.001 or **** p<0.0001, as compared to Ctrl, two-way ANOVA with repeated measures.

Figure 5 .

 5 Figure 5. C-terminal deletion of E and 3a protein does not prevent cell characteristic changes. (A) Cell roundness measured in CHO cells expressing eGFP (pIRES) alone, or in combination with WT (pIRES -E) or the C-terminally deleted E protein (pIRES -E ∆4) or in combination with WT (pIRES -3a) or the C-terminally deleted 3a protein (pIRES -3a ∆10). Plot of individual cells, median ± interquartile range. **** p<0.0001 as compared to pIRES control, Kruskal-Wallis test. (B) Left, average current densities (± sem) recorded during the ramp protocol in adhering (empty circles) or non-adhering (filed circles) CHO cells expressing the C-terminally deleted E protein (pIRES -E ∆4). Right, plot of the individual adhering (darker color) or non-adhering cells (lighter color). *** p<0.001, as compared to adhering cells, two-way ANOVA. (C) Left, average current densities (± sem) recorded during the ramp protocol in adhering (empty circles) or non-adhering (filled circles) CHO cells expressing the C-terminally deleted 3a protein (pIRES -3a ∆10). Right, plot of the individual adhering (darker color) or non-adhering cells (lighter color). **** p<0.0001, as compared to adhering cells, two-way ANOVA.

Acknowledgements

We thank the Agence Nationale de la Recherche for its financial support to the Région Pays de la Loire (ANR FLASH Covid-19 -CoV2-E-TARGET) and the laboratory of excellence 'Ion Channels, Science and Therapeutics' (grant No. ANR-11-LABX-0015). We acknowledge the IBISA MicroPICell facility (Biogenouest), member of the national infrastructure France-Bioimaging supported by the French national research agency (ANR-10-INBS-04). We thank the Cytometry Facility Cytocell from Nantes for expert technical assistance. We thank Hugues Abriel and Jean-Sebastien Rougier (Institute of Biochemistry and Molecular Medicine, university of Bern) for fruitful discussions and support and critical reading of the manuscript.

Cell morphology assay

Cell roundness was estimated using the Analyze Particle function of the Fiji software, as described in suppl. Figure 1.

Flow cytometry assay

Two days after transfection, CHO cells were prepared to cell death detection following the user guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/mp13199.pdf), to measure annexin V binding and propidium iodide (PI) intake. The cells were washed with cold PBS, trypsinized, collected by centrifugation and gently resuspended in annexin-binding buffer (V13246, Invitrogen, USA) at 1 × 10 6 cells/mL. To each 300-µL cell suspension were added 0.5 µL of annexin V AlexaFluor 647 (A23204, Invitrogen) and 1 µL of propidium iodide (PI) at 100 µg/mL (P3566, Invitrogen). CHO cells were incubated at room temperature for 15 minutes in the dark, and then maintained on ice until flow cytometry analysis within one hour.

To study the cell death pathways induced by E and 3a protein expression, non-transfected or transfected CHO cells were treated with inhibitors or inducers of apoptosis (inhibitor: 5 µmol/L QVD-OPh incubated for 48 h; activators: 3 µmol/L S63-845 + 8 µmol/L ABT737 incubated for 3 h).

The cytometer BD FACSCanto (BD Biosciences, USA) was used to sample acquisition. CHO cells transfected with an empty plasmid were used to determine the population to be analyzed. Monolabeled cells were used to establish the photomultiplier voltage of each channel (PMT) and to proceed with fluorescence compensation after the acquisitions. In order to detect cell death, only eGFP-positive CHO cells (FITC) were analyzed to Annexin V AlexaFluor 647 (APC) and PI (Perc-P) labeling. Analyses were performed using FlowJo software.