Mylène Dutour
email: mylene.dutour@hotmail.com

Jean-Paul Léna

Adeline Dumet

Vanessa Gardette

Nathalie Mondy

Thierry Lengagne

The role of associative learning process on the response of fledgling great tits (Parus major) to mobbing calls

Keywords: Alarm call, Associative learning, Birds, Communication, Mobbing

When they detect a predator, many species emit antipredator vocalizations. In some cases, they emit mobbing calls, which are associated with the caller approaching and harassing the predator while attracting others to join it. Surprisingly, although mobbing has been widely reported in adults of numerous species, there has been no test of the role of learning in mobbing call recognition, especially during ontogeny. Here, we exposed wild great tit (Parus major) nestlings to playbacks of an unthreatening, novel sound either associated with conspecific mobbing calls (experimental treatment) or with another unthreatening novel sound (control treatment). We then tested them as nestlings and fledglings to see how responses to the novel sound compared to conspecific mobbing calls. Results revealed that fledglings in the experimental treatment behaved similarly to conspecific mobbing calls and the novel sound associated with conspecific mobbing calls. Because mobbing efficiency is often linked to interspecific communication, associative learning should be used by heterospecifics as a mobbing calls recognition mechanism. Regardless of treatment during the nestling phase, fledglings always were sensitive to the playback of conspecific mobbing calls.

However, fledglings from the control group were more likely to approach the loudspeaker than those from the experimental group when mobbing calls were played, suggesting that overexposure during the nestling phase altered mobbing learning. Overall, these results suggest that learning could play a role in the recognition of calls, like heterospecific mobbing calls, when paired with conspecific mobbing, and that mobbing is perceived as a threatening stimulus from a very young age.

Introduction

In many species of small birds and mammals, two contrasted anti-predator strategies, each associated to a specific alarm call, can be observed according to predator dangerousness: prey may flee or mob the predator [START_REF] Marler | Specific distinctiveness in the communication signals of birds[END_REF]. Mobbing behaviour is usually exhibited when predators do not represent an immediate threat and is characterized by harassment or even attacks on the predator as well as vocalizations (i.e., mobbing calls) that quickly gather neighboring conspecifics and other prey species in the mob [START_REF] Hartley | An experimental analysis of interspecific recognition[END_REF][START_REF] Curio | The adaptive significance of avian mobbing. I. Teleonomic hypotheses and predictions[END_REF]. Both the evaluation of the threat associated with the predator and the recognition of mobbing calls, including heterospecific calls, are required to make mobbing behaviour an efficient antipredator strategy.

Associative learning is a behavioural modification following reinforcement, based on associations between two stimuli, responses and events (Shettleworth 1998;[START_REF] Griffin | Cognition and personality: an analysis of an emerging field[END_REF]. This process is essential to the development of vocalizations and several behaviours such as those involved in predator avoidance [START_REF] Ferrari | Learning about non-predators and safe places: the forgotten elements of risk assessment[END_REF]. Animals can learn to flee after associating an unfamiliar sound (i.e., novel sound) with (i) the appearance of a model predator (birds: Magrath et al. 2015a;mammals: Shriner 1999;[START_REF] Wheeler | Experimental evidence for heterospecific alarm signal recognition via associative learning in wild capuchin monkeys[END_REF] or (ii) the hearing of alarm calls, without having to see the callers or a predator [START_REF] Potvin | Birds learn socially to recognize heterospecific alarm calls by acoustic association[END_REF]. In mobbing behaviour, there is ample evidence of a process of associative social learning between conspecific mobbing calls and predators (Curio et al. 1978a;[START_REF] Maloney | Historical and experimental learned predator recognition in free-living New Zealand robins[END_REF][START_REF] Maloney | Historical and experimental learned predator recognition in free-living New Zealand robins[END_REF][START_REF] Griffin | Social learning about predators: does timing matter?[END_REF][START_REF] Mcivor | Testing social learning of anti-predator responses in juvenile jackdaws: the importance of accounting for levels of agitation[END_REF] but in all cases the training phase was based on visual cues; individuals were trained about predators through pairing model predator presentations with a training stimulus (i.e., live conspecific demonstrator engaging in mobbing, mobbing calls paired with mounts of conspecifics, or mobbing calls alone). However, visual associative learning needs the inexperienced learner to be in the vicinity of the mobbing scene, so that the opportunities for learning while reducing the risk of threat may be limited, especially for species living in dense vegetation. This challenge is overcome in the case of acoustic-acoustic association, which may be efficient even when the tutor and the threat are difficult to observe, possibly resulting in the rapid spread of mobbing call recognition in natural communities.

Mobbing calls are usually characterized by loud and repetitive vocalizations, both features that should facilitate associative learning, as there is ample opportunity to hear mobbing calls while observing the threat, with less likelihood of immediate danger (Magrath et al. 2015b). In birds, such association between a predator and the expression of a mobbing behaviour could be achieved after young fledge leave their nest. In this case, young fledglings can learn from their parents and several observations are in agreement with this statement (Curio et al. 1978b;[START_REF] Griesser | Kinship modulates the attention of naïve individuals to the mobbing behaviour of role models[END_REF]. If nestlings however are able to associate several acoustic signals from their nest cavities (i.e., they are able to associate the mobbing calls emitted by parents with other vocalizations prevailing on a mobbing scene) they should also be able to respond to mobbing calls appropriately as soon as they leave the nest. To our knowledge, this capacity has never been investigated before. Indeed, until now, associative learning has been examined using the association of several sensory channels, especially visual and acoustic (e.g. [START_REF] Maloney | Historical and experimental learned predator recognition in free-living New Zealand robins[END_REF][START_REF] Mcivor | Testing social learning of anti-predator responses in juvenile jackdaws: the importance of accounting for levels of agitation[END_REF]). However, as outlined above, young could also associate unknown vocalizations with conspecific mobbing calls if both are simultaneously emitted in a mobbing scene. Such a mechanism could be particularly relevant in learning to recognize heterospecific mobbing calls.

The great tit (Parus major) is a well-suited species to study the ontogeny of mobbing behaviour in the wild. It is well known for its learning abilities [START_REF] Exnerová | Avoidance of aposematic prey in European tits (Paridae): learned or innate?[END_REF][START_REF] Cole | Individual variation in spontaneous problem-solving performance among wild great tits[END_REF][START_REF] Morand-Ferron | Taking the operant paradigm into the field: associative learning in wild great tits[END_REF] and its tendency to exhibit mobbing behaviour and emit mobbing calls when confronted with a predator [START_REF] Lind | Information, predation risk and foraging decisions during mobbing in great tits Parus major[END_REF][START_REF] Dutour | Mobbing behaviour varies according to predator dangerousness and occurrence[END_REF]Carlson et al. 2017a;[START_REF] Kalb | Subtle variations in mobbing calls are predator-specific in great tits (Parus major)[END_REF]. Furthermore, this altricial species provides a good system for studying whether naïve nestlings are able to associate mobbing calls with unfamiliar stimuli in the absence of any visual cues from adults or predators in the nest cavity.

The goal of this study was to determine whether associative learning might serve as a mechanism for the development of mobbing calls recognition for free-living animals.

Specifically, we investigated whether experiencing a novel stimulus associated with conspecific mobbing calls during the nestling period led great tit nestlings to associate this novel sound with mobbing during the fledgling period. We designed playback experiments in two groups: (i) an experimental group in which we trained individuals by broadcasting a novel sound (i.e., associative learning stimulus) with mobbing calls (i.e., functional stimulus) and (ii) a control group in which the associative learning stimulus was broadcast with another novel sound (hereafter control stimulus).

In line with previous work showing that nestlings cease begging when they hear playbacks of their parents' alarm calls [START_REF] Davies | Learning fine-tunes a specific response of nestlings to the parental alarm calls of their own species[END_REF]Hollén and Radford 2009;[START_REF] Barati | Nestlings reduce their predation risk by attending to predatorinformation encoded within conspecific alarm calls[END_REF], we predict that at the end of the learning period, only nestlings belonging to the experimental group should cease begging when they hear the associative learning stimulus. We also measured responses at the fledgling stage after the playback of the associative learning stimulus. We predict that the playback of the associative learning stimulus should elicit a stronger mobbing response from the experimental group compared to the control group (i.e., increases in scanning, calling and moving) because the experimental group should associate the unfamiliar sound with mobbing and respond to the unfamiliar sound as threatening, while the control group should not. Because it was important to ensure that fledglings were sensitive to mobbing, we also tested whether they responded to the playback of conspecific mobbing calls.

Material and methods

Study sites and species

We chose great tits, a common territorial passerine which breeds in secondary holes and wooden artificial nest boxes throughout Europe and parts of Asia and North Africa [START_REF] Perrins | Population fluctuations and clutch-size in the Great Tit, Parus major L[END_REF]). The study was conducted in the Pierre Vérots Foundation, a private natural reserve located in the south-east France (4°91'E, 45°95'N). The study area covers approximately 150 ha and contains 120 nest boxes separated at minimum by 50 m and mainly occupied by great tits. This distance corresponds to the minimum distance separating tit territories during the breeding season in our study area and is far enough to ensure that the playbacks are not heard by neighbours (at 50 meters, the amplitude levels fluctuate from 33 to 39 dB, obtained from the loudspeaker, Solo 01dB Metravib, Z weighting, re: 20 μ m Pa; these levels are lower than the natural background noise levels in forest areas, fluctuate from 35 to 45 dB in quiet situations, 52 to 70 dB in noisy situations). The study took place during the breeding season (April-July 2017). We visited nest boxes at least every two days from the beginning of the breeding season onwards to determine the laying and hatching dates. When nestlings were 8 days old, they were ringed with a unique combination of color bands for individual identification. Nestlings spent 19.3 ± 1.4 days in the nest. Nestlings from the same nest all hatched on the same day and subsequently also fledged on the same day.

Experimental procedures: presentation of stimuli

The aim of our experiment was to assess whether chicks were able to associate a novel stimulus with conspecific mobbing calls. In 120 nest boxes, 31 were unoccupied, 14 occupied by the blue tit (Cyanistes caeruleus) and one was occupied by the willow tit (Poecile montanus). Each of the remaining 74 nest boxes occupied by great tits was randomly assigned to one of the two treatments (balanced for laying date to ensure equal representation of both treatment groups over the breeding season). In the first treatment (38 nests), the associative learning stimulus was associated with conspecific mobbing calls (hereafter experimental group) while in the second treatment (36 nests) the associative learning stimulus was associated with unfamiliar stimulus (control sequence, hereafter control group). Behavioural tests were conducted both during the development of nestlings and the fledging day.

Playbacks were always broadcasted once adults were silent and away from the nest (at least 15 m) or from the focal fledgling (at least 5 m). If parents responded to playback during tests, we stopped the playback and restarted at least 30min after, but such cases were rare (~5% of total tests).

Choice of experimental stimuli and playback materials

Three kinds of stimuli were used during playback experiments. First, we used mobbing calls produced by four great tits in response to the Eurasian pygmy owl (Glaucidium passerinum) and to conspecific mobbing calls. Calls were recorded with a Fostex FR2LE digital recorder connected to a Sennheiser ME67-K6P microphone. We also used mobbing calls produced by ten great tits obtained from the Xeno Canto online database (http://www.xeno-canto.org) (see online supplement, Appendix 1). For the associative learning and the unfamiliar stimulus, we used two musical instrument sounds (clarinet and trumpet sequences) obtained from an online sound bank (http://www.universal-soundbank.com) to ensure the stimuli used had no biological value and were unfamiliar. In order to ensure that control sounds were unfamiliar, we first conduced a pilot study to assess the response of adult great tits (n = 9). None of them approached and mobbed the loudspeaker. We matched the frequency characteristics and temporal structure of the associative learning and the unfamiliar stimulus to obtain stimuli of the same duration and the same frequency bandwidth using Avisoft-SASLab Pro. For the experimental treatment, we built playbacks containing the associative learning stimulus (i.e., clarinet) with mobbing calls. For the control treatment, the associative learning stimulus was associated with the unfamiliar stimulus (i.e., trumpet) (spectrograms of mobbing calls, associative learning stimulus and unfamiliar stimulus are available in supplementary material Appendix 2 Fig. A2). Each playback track consisted of 10 seconds of associative learning stimulus followed by 10 seconds of the mobbing calls or the unfamiliar signal, and this sequence was repeated three times in order to reach one-minute in total, time used in a recent study conducted on nestling passerines in response to calls [START_REF] Wheatcroft | Repetition rate of calls used in multiple contexts communicates presence of predators to nestlings and adult birds[END_REF]. In order to avoid habituation to playbacks, we constructed 21 one-minute playback samples for each group and each sample was used only once (i.e., as nestlings spent 19.3 ± 1.4 days in the nest, we used one stimulus by day) [START_REF] Hurlbert | Pseudoreplication and the design of ecological field experiments[END_REF][START_REF] Kroodsma | Pseudoreplication in playback experiments, revisited a decade later[END_REF]). Because we used mobbing calls produced by 14 great tits in total to construct 21 playback sequences, some soundtracks are therefore only imperfectly independent from other ones, but the risk of pseudoreplication was clearly minimized. We also built two additional 30-second playback samples containing associative learning stimulus or mobbing calls to test the response of nestlings and fledglings. We decided to keep the test duration shorter than was usual in previous studies conducted on adults' great tit (including our own Dutour et al. 2017b; see also [START_REF] Suzuki | Referential mobbing calls elicit different predator-searching behaviours in Japanese great tits[END_REF]) in order to limit the risk of interference from parents during tests. The average amplitude used for these playbacks was obtained with a sound level meter placed 1 m from the loudspeaker (83.9 ± 3.51 dB, mean ± SD, Solo 01dB Metravib, Z weighting, re: 20 μ m Pa). We broadcast sound tracks using a Shopinnov 20 W loudspeaker (frequency response

Behavioural responses of nestlings

Begging calls suppression is described as a good measure of whether nestlings interpret sounds as cues of danger [START_REF] Magrath | Calling in the face of danger: how predation risk affects acoustic communication by parent birds and their offspring[END_REF]). To determine whether playback treatment (i.e., associative learning stimulus with unfamiliar stimulus or associative learning stimulus with mobbing calls) influenced nestling calling activity, every day, between 7am and 1pm, and between hatching day and fledging day, we measured nestling production of begging calls within the nest box during 30 seconds (begging call = 1, no begging call = 0) before and after broadcasting the treatment, 1 minute being time by which passerine nestlings' behaviour return to normal [START_REF] Wheatcroft | Repetition rate of calls used in multiple contexts communicates presence of predators to nestlings and adult birds[END_REF]. To measure this behaviour, one observer was placed ∼ 50 cm from each nest to listen to the calls of the nestlings, which were clearly heard at short distance. Although there were multiple observers (n = 8) to measure begging call propensity, we did not detect an observer effect (GLMM: P > 0.05). Additionally, at the end of the nestling phase, between 19 days post hatch and fledging day, begging call propensity was measured before and after playback of the associative learning stimulus. To create as natural a situation as possible, the loudspeaker was placed on the top of the nest box for all trials. The average amplitude used for the playback was obtained with a sound level meter placed in the nest boxes (67.98 ± 0.94 dB, mean ± SD, Solo 01dB Metravib Z weighting, re: 20 μ m Pa).

Behavioural responses of fledglings

To test whether associative learning is involved in the development of mobbing calls recognition, we studied fledglings' responses to the playback of either the associative learning stimulus alone (control: n = 30 from 22 nests; experimental: n = 31 from 19 nests) or the conspecific mobbing calls (control: n = 19 from 14 nests; experimental: n = 26 from 16 nests).

Tests were performed during the day of fledging (except 4 tests conducted the second day after fledging corresponding to 3.8% of total tests). Each test was conducted by two field assistants. One was assigned to the soundtrack preparation and playback operation, while the other was kept unaware of the selected soundtrack (although they could hear it) and assigned to the observation of the focal bird. In all experiments and before the playback, the identity of the focal fledgling was determined using the unique leg color bands fitted to each fledgling using binoculars. Neither of the two field assistants knew whether the fledging was in the control or experimental group. Once a focal fledging was identified, the loudspeaker used to broadcast the acoustic stimulus was placed ∼ 20 m from the fledgling at the base of a tree at 1.5 meter high. All observations were done using binoculars. Observations were made ∼10 m from the loudspeaker and the focal fledging, a distance from which the tits' behaviour was not disturbed. Trials were conducted mostly around 12am (range 8am -5pm) to ensure good light, in calm and dry weather. Playbacks were started when no bird was visible close to the speaker and no bird was calling. To test fledglings' responses, we recorded the following behavioural variables during the 30 s of playbacks: (i) number of horizontal scans (i.e., the number of movements that birds made with their heads from left to right or right to left), (ii) calling (i.e., producing any mobbing vocalizations; call propensity: mobbing vocalization = 1; no mobbing vocalization = 0), (iii) approaching the loudspeaker (i.e., we recorded whether birds approached within 15 m of the loudspeaker during playback; see Dutour et al. 2017b for more details), and (iv) fleeing (i.e., we recorded whether birds moved away more than 10 m from their initial position). We measured these behaviours because they are good indicators of perceived danger in birds (Curio et al. 1978a) and are common during mobbing events [START_REF] Suzuki | Experimental evidence for compositional syntax in bird calls[END_REF]Carlson et al. 2017a;Carlson et al. 2017b;Dutour et al. 2017b;[START_REF] Suzuki | Wild Birds Use an Ordering Rule to Decode Novel Call Sequences[END_REF]. There was 10-minute gap between playback of the associative learning stimulus and playback of mobbing calls.

Data analysis

Analyses were done using SAS 9.4 (SAS Institute Inc., Cary, NC, U.S.A.) and R v.2.15.1 softwares (The R Foundation for Statistical Computing, Vienna, Austria, http://www.rproject.org). We first examined whether begging call propensity varied between treatment groups during the nestling period. For this purpose, we analyzed call propensity recorded each day before broadcasting the treatment using a logistic linear mixed model (LLMM) for which the call propensity was treated as a repeated dependent binary variable. To this aim, nest was considered as a random effect and the correlation between successive observations recorded at the same nest was specified using an autoregressive covariance structure. The treatment group, the time elapsed since hatch (i.e., nestlings age) as well as their interactive effect, were introduced as fixed effects. The significance of each fixed effect was tested using a nonsequential F test and the Kenward-Roger method was used to approximate the denominator degrees of freedom (SAS 9.4). Non-significant terms were successively dropped to obtain the final model. We used a similar model framework to compare the propensity of nestlings to stop begging after the onset of the broadcasting treatment according to the treatment group and the time elapsed since hatching. For this purpose, all observations for which nestlings were not calling before the onset of the playback treatment were discarded from the analysis.

We used a Fisher exact test to test whether the begging calls propensity just after the playback of the associative learning stimulus only when nestlings were between 19 days post hatch and fledging day varied according to the group (experimental or control).

To investigate the behavioural responses of fledglings, we first examined whether the number of horizontal scans varied between treatment groups and playback during the fledgling period (n = 106). For this purpose, we ran generalized linear mixed models (GLMM) which included the treatment group, the playback (associative learning stimulus or mobbing calls) and their interactive effect as a fixed terms and nest as a random term. We used a negative binomial error distribution and log-link function for this analysis (glmer.nb in the package lme4; [START_REF] Bates | lme4: Linear mixed-effects models using Eigen and S4. R package version 1[END_REF]). To investigate the call propensity, we used a binomial error distribution and logit-link function (glmer in the package lme4; [START_REF] Bates | lme4: Linear mixed-effects models using Eigen and S4. R package version 1[END_REF] (calling response = 1; no calling response = 0). As above, nests were included in the model as a random effect. The treatment group, the playback and their interactive effect were introduced as fixed effects. For both scanning and call propensity, we further conducted pairwise comparisons (glht in the package multcomp). Finally, because approaching and fleeing behaviours (two behaviours linked to movements) were opposed to immobility behaviour (see online supplement Appendix 3, Fig. A3), all approaching and fleeing individuals were regrouped within a unique category (hereafter moving individuals) and compared to the individuals who stayed still. To compare the propensity of fledglings to move vs individuals who stayed still according to treatment group and playback, we used a binomial error distribution and logit-link function. We then focused our analysis on moving individuals only (i.e., fleeing and approaching individuals; n = 23). Because only a small number of fledglings moved, Fisher's exact test was used to investigate the variation in mobbing propensity (i.e., approach) according to the treatment group in response to the mobbing call playbacks (n = 17). Because only one of the control fledglings moved when exposed to the playback of the associative learning stimulus, we discarded this group and we used a Fisher's exact test to investigate the variation in mobbing propensity of individuals from the experimental group between the two playbacks (n = 16).

Data availability

The datasets generated during the current study are available from the corresponding author on request.

Results

Behavioural responses of nestlings

The calling behaviour before the broadcasting treatment did not significantly vary in function to the time elapsed since hatching up to fledging but it was significantly higher for the experimental group than the control group (n = 1420, treatment effect: F 1,341 = 4.17; P = 0.042 ; time effect: F 1,426 = 0.66; P = 0.417; interaction: F 1,425 = 0.40; P = 0.529). The propensity to stop calling after the onset of the broadcasting treatment significantly increased in function to the time elapsed since hatching but did not vary between treatment groups although call suppression tended to increase with time slightly more for the control group than for the experimental group (n = 353, treatment effect: F 1,156 = 0.11; P = 0.745 ; time effect: F 1,176. = 46.19; p < 0.0001; interaction: F 1,170 = 3.69; P = 0.057). We did not detect a treatment group effect on the nestlings responses to the playback of the associative learning stimulus (n = 11;

P = 0.608).

Behavioural responses of fledglings

Scanning was significantly affected by the interaction between the stimuli that were broadcast (associative learning stimulus or mobbing calls) and the treatment group (experimental and control) (Table 1; Fig. 1). In response to mobbing calls playbacks, the rate of horizontal scans did not differ between the two groups (P = 0.99; Fig. 1). It was higher during playback of mobbing calls than during playback of the associative learning stimulus for the control group (P = 0.004; Fig. 1) while it did not differ for the experimental group (P = 0.99; Fig. 1). Similar variations were found for call propensity as indicated by a significant interaction effect between group and playback (Table 1; Fig. 2), although these variations were not statistically different between both groups in response to the associative learning stimulus playback (P = 0.95; proportion of calling: associative learning = 22.5%; control = 6.7%).

Furthermore, there was a significant effect of playback on the probability of moving (i.e., approaching and flight behaviours), whereas group had no significant effect (Table 1; Fig. 3).

In response to mobbing calls, fledglings were more likely to move than in response to the associative learning stimulus playback. Results indicate that (i) in response to the mobbing call playbacks, individuals from the control group were more prone to approach the loudspeaker than experimental individuals (n = 17; Fisher test: P = 0.049; Fig. 3) (ii) approaching behaviour of individuals from the experimental group did not differ between the two playbacks (n = 16; Fisher test: P = 0.999; Fig. 3).

Discussion

We have investigated whether nestlings submitted to a novel stimulus associated with mobbing calls enabled them to recognize this stimulus as an indicator of threat proximity when becoming fledglings. This experiment, involving classical conditioning, showed that fledglings learned to associate a novel stimuli with mobbing calls and exhibited vigilance behaviours in response to the novel stimuli whereas they do not as nestlings.

Effect of the associative learning during the nestling phase

Several studies have shown that nestlings become silent when they hear playbacks of their parents' alarm calls [START_REF] Davies | Learning fine-tunes a specific response of nestlings to the parental alarm calls of their own species[END_REF][START_REF] Platzen | Parental alarm calls suppress nestling vocalization[END_REF][START_REF] Madden | Nestling responses to adult food and alarm calls: 1. Species specific responses in two cowbird hosts[END_REF][START_REF] Haff | Learning to listen? Nestling response to heterospecific alarm calls[END_REF][START_REF] Barati | Nestlings reduce their predation risk by attending to predatorinformation encoded within conspecific alarm calls[END_REF]. However, most of these studies have compared alarm calls to background noise or sympatric species stimuli (i.e., stimuli known).

In the present study, nestlings suppressed calling when submitted to associative learning stimulus equally to mobbing call playbacks, suggesting that novelty (i.e., a stimulus never heard before) alone may be used as a signal of danger [START_REF] Schaller | The development of visual discrimination patterns in the crouching reactions of nestling grackles[END_REF]Curio et al. 1978b). We found that, contrary to young nestlings, older nestlings reduced begging calls in response to the associative learning stimulus and mobbing calls, suggesting that older nestlings are less prone to recognition errors than younger ones [START_REF] Davies | Cuckoos versus reed warblers: adaptations and counteradaptations[END_REF][START_REF] Davies | Learning fine-tunes a specific response of nestlings to the parental alarm calls of their own species[END_REF]). This pattern of gradual acquisition is in accordance with previous studies conducted on nestling great tits [START_REF] Rydén | Differential responsiveness of great tit nestlings, Parus major, to natural auditory stimuli[END_REF]. We did not detect a group effect on nestling' responses to playbacks of the associative learning stimulus at the end of the learning period, but the low sample size may have reduced our ability to detect a significant pattern. A detailed study of nestlings' behaviour would be insightful to actually address this question (e.g., if they crouched down inside their nest cavity, suppressed their movements, increase their heart rate and electromyographic activity after parental alarm calls ;[START_REF] Rydén | Differential responsiveness of great tit nestlings, Parus major, to natural auditory stimuli[END_REF]Ryden 1980;[START_REF] Suzuki | Parental alarm calls warn nestlings about different predatory threats[END_REF]. Finally, another interesting aspect is related to the sound degradation. As we used hole nesters, a sound degradation may occur that a sound will appear different within and outside the nest box. However, quite low-frequency sounds have been used in this study, with probably low alteration, and the sounds were broadcast using a loudspeaker placed on the top of the nest box for all trials, with the only obstacle being the wall thickness of the nest box.

Future work could investigate which sound characteristics of the parent calls could be perceived by offspring in their nest according to their nestling conditions (hole nesters vs open nesters).

Mobbing calls recognition and associative learning in fledglings

Fledglings exhibited a higher rate of scanning when hearing the associative learning stimulus but only if it was associated with mobbing calls during the nestling phase. A similar trend was also observed for calling activity and the propensity to move. Together, these results indicate that individuals associated the artificial sound with conspecific mobbing calls without having to see the mobbing scene. Scanning rate is a good proxy of vigilance effort [START_REF] Lendrem | Predation risk and vigilance in the blue tit (Parus caeruleus)[END_REF][START_REF] Huang | Heterospecific information about predation risk influences exploratory behavior[END_REF][START_REF] Creel | Effects of predation risk on group size, vigilance, and foraging behavior in an African ungulate community[END_REF] suggesting that chicks actually perceived the associative learning stimulus as an indicator of threat proximity only if it was associated with mobbing calls during the learning phase. Whether fledglings associated the artificial stimulus as the source of threat itself or as heterospecific mobbing calls (indicating the presence of a threat) and whether the ontogeny of mobbing behaviour only relies on learning processes, remain open questions. Positive correlation between the rate of response development and the magnitude of exposure to heterospecific alarm calls suggest a learning process. For instance, infant vervet monkeys (Cercopithecus aethiops pygerythrus) develop responses to superb starling (Lamprotornis superbus) mobbing calls more quickly on territories where these birds are common, suggesting that these young have more opportunities to learn about the calls than young on territories where starlings are less common [START_REF] Hauser | How infant vervet monkeys learn to recognize starling alarm calls: the role of experience[END_REF]. Concerning adults, they can associate novel sounds with a chorus of conspecific and heterospecific aerial alarm calls [START_REF] Potvin | Birds learn socially to recognize heterospecific alarm calls by acoustic association[END_REF]. One may thus expect that a learning process where individuals associate the threat and/or heterospecific mobbing calls with the conspecific mobbing calls can operate in the wild. Such mechanisms could make the set of recognized heterospecific mobbing calls adjusted to the local composition of prey communities, which is also congruent with field studies reporting local variations of the rate of responses towards heterospecific mobbing calls [START_REF] Wheatcroft | Learning and signal copying facilitate communication among bird species[END_REF].

In addition to such associative learning processes, our study also reveals that fledglings responded to conspecific mobbing calls despite the treatment they experienced. Indeed, in both groups, hearing conspecific mobbing calls resulted in increased vigilance, a higher proneness to call as well as to move. Since all these tests were performed on fledging day, we can safely assume that no fledgling could have had the opportunity to observe a mobbing scene before the tests. Our results therefore indicate that fledglings do not need to observe mobbing events to respond to mobbing calls, suggesting that conspecific mobbing calls are inherently perceived as indicating a threat. However, two hypotheses, not mutually exclusive, could explain the responses to mobbing calls: the response ( 1) is at least partly innate, this result has been found in adult great tits in response to allopatric mobbing calls [START_REF] Randler | A possible phylogenetically conserved urgency response of great tits (Parus major) towards allopatric mobbing calls[END_REF], Dutour et al. 2017b), or (2) is due to the impregnation of acoustic signals heard during the nestling phase. Mobbing calls are composed of frequency modulated elements, which are involved in vigilance, and D notes involved in foraging flocks or to recruit partners during the breeding season, to which receivers respond by approaching the caller [START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF]). In the present study, tests were performed during fledgling when great tits form family groups and often use D notes to maintain group cohesion (Dutour, pers. observation). The responses of fledglings could be related to the context (i.e., presence of family members in the vicinity), or could be due to sensitivity to the D notes. Furthermore, contrary to fledglings belonging to the control group, those belonging to the experimental group were more prone to flee than approach the loudspeaker. It could be from the playbacks which were always presented to fledglings in the same order (i.e., associative learning sound, then mobbing calls), creating biases that differed between treatments. Indeed, this could be a carry-over from the experimental group hearing the unfamiliar sound, which is threatening to them, before mobbing calls, while the control group heard a non-threatening sound and then the mobbing calls. The situation seemed more dangerous for the experimental group if the two cues had an additive effect. However, this result suggests that reinforcement with mobbing calls during the nestling phase altered mobbing learning. Such impairment could be either due to call overexposure, as previously shown in vocalization learning [START_REF] Tchernichovski | Vocal imitation in zebra finches is inversely related to model abundance[END_REF][START_REF] Brainard | Translating birdsong: songbirds as a model for basic and applied medical research[END_REF], or because of the absence of a threat associated with the mobbing call during exposure. An alternative explanation is that maybe fleeing is the adaptive response to mobbing calls for young juveniles. This should be addressed in further studies. It would be feasible in relatively resident birds like great tits where dispersal distances are small. Flee would not be an impairment when expressed by a young, inexperienced, and physically clumsy individual compared with more experienced, agile and older juveniles (~ 120 days post fledging) [START_REF] Kullberg | An experimental study of predator recognition in great tit fledglings[END_REF] or adults. In that case, being exposed to additional mobbing via playbacks may have sped up the development of this response.

Conclusion

This study shows that nestling great tits do not discriminate between two different acoustic signals but demonstrates that they associate mobbing calls with a novel sound when exiting the nest cavity as fledglings. Nevertheless, fledglings did not respond with typical mobbing behaviour (i.e., approach and harass), which likely require experience of parental visual response (i.e., cultural transmission) [START_REF] Kullberg | An experimental study of predator recognition in great tit fledglings[END_REF]. The present findings raise questions about how nestlings extract information about the nature of predators and how finely they can discriminate between different acoustic stimuli.

Table 1

Generalized linear mixed models (GLMM) results for playback (associative learning stimulus or mobbing calls) and group (experimental or control) as predictors of variation in fledglings response behaviours (scanning, calling, approaching + fleeing). The "*" character indicates the interaction between the explanatory terms. Significant P values are indicated in bold.

Behaviours

Fixed

FIGURE CAPTIONS Fig. 1 Fig. 2 Fig. 3

 CAPTIONS123 FIGURE CAPTIONS

Acknowledgments

We warmly thank the "Fondation Pierre Vérots" (PVF) for giving us access to the field site. We are indebted to JP Rabatel (PVF) for his assistance. We express our gratitude to several students who assisted with the field work. We are grateful to Bernard Kaufmann for English corrections. We thank the editor, Christoph Randler, and anonymous reviewers for constructive comments on an earlier version of this manuscript.

Funding This work was supported by French Ministry of Research and Higher Education funding (to M.D. PhD grants 2015-2018).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval


Emilie Moisez

Laurent Seuront
email: laurent.seuront@cnrs.fr

Deciphering the known unknowns in the behavioural ecology of the intertidal gastropod Littorina littorea

Keywords:

A considerable amount of attention has been devoted to understand the role of chemoreception in the behavioural ecology of gastropods. There is still, however, a limited amount of information related to their sensory ecology and their subsequent ability to navigate through topographically complex landscapes, such as intertidal rocky shores, despite the documented role of substrate spatial structure in the movement rates and directionality of gastropods.

Responses to gravity and visual cues have been documented in a range of intertidal gastropods, but often led to contradictory conclusions. We examined the response of the gastropod Littorina littorea, a keystone grazer on most temperate intertidal ecosystems of the northern hemisphere, to substrate slope, topographic discontinuities and light using purpose-designed experimental set-ups under laboratory-controlled conditions. In contrast to previous results, we showed that L. littorea did not exhibit a strict geotactic behaviour. Instead, they were predominantly thigmotactic following an encounter with a topographic discontinuity, and we suggest that the observed seemingly negative geotactic responses are only a by-product of thigmotaxis, i.e. a thigmotactically-induced negative geotactic response. L. littorea also showed a strong preference for dark areas and shapes. Taken together, our results suggest that thigmotaxis and scototaxis are evolutionary advantageous to navigate through topographically complex and thermally stressful environments, and that the behavioural repertoire of gastropod may be much wider than previously thought.

Introduction

The understanding of animal movements is a key determinant of population viability and species dynamics [START_REF] Tilman | Competition and biodiversity in spatially structured habitats[END_REF][START_REF] Chapman | Poor design of behavioural experiments gets poor results: examples from intertidal habitats[END_REF][START_REF] Morales | Scaling up animal movements in heterogeneous landscapes : the importance of behavior[END_REF]. Typically, animal search for food, mates and hosts, while avoiding predators in spatially and temporally complex, structured environment [START_REF] Levin | Patchiness in marine and terrestrial systems: from individuals to populations[END_REF][START_REF] Tilman | Competition and biodiversity in spatially structured habitats[END_REF][START_REF] Morales | Scaling up animal movements in heterogeneous landscapes : the importance of behavior[END_REF][START_REF] Haynes | Interpatch movement and edge effects : the role of behavioral responses to the landscape matrix[END_REF]. This issue is particularly relevant in tidepools and rocky shores that are exemplary complex environments [START_REF] Denny | Encyclopedia of tidepools and rocky shores[END_REF]. Habitat selection and the ability of invertebrates, in particular gastropods, to make decisions about moving across different habitats rely on essentially non-visual senses in environments characterized by topographically complex substrate and resource heterogeneity [START_REF] Fratini | Feeding clusters and olfaction in the mangrove snail Terebralia palustris (Linnaeus) (Potamididae : Gastropoda)[END_REF][START_REF] Keppel | Chemically mediated avoidance of Hemigrapsus nudus (Crustacea) by Littorina scutulata (Gastropoda): effects of species coexistence and variable cues[END_REF][START_REF] Wyeth | Orientation and navigation relative to water flow, prey, conspecifics, and predators by the nudibranch mollusc Tritonia diomedea[END_REF]. Noticeably, the presence of biogenic habitats typically created by species such as mussels and barnacles further contributes to the complex topography of rocky shores [START_REF] Underwood | Experimental analyses of the influences of topography of the substratum on movements and density of an intertidal snail, Littorina unifasciata[END_REF]. Understanding the determinism of gastropod navigation is hence an absolute prerequisite to explain the dispersion, distribution and local biodiversity of other sessile and mobile species [START_REF] Chapman | Poor design of behavioural experiments gets poor results: examples from intertidal habitats[END_REF].

A considerable amount of attention has been devoted to understand the role of chemoreception in driving the trail-following behaviour involved in the homing and aggregation behaviour of intertidal gastropods, and the localization of sexual partners, preys and predators, and preferential habitats [START_REF] Croll | Gastropod chemoreception[END_REF]Chapperon andSeuront, 2009, 2011a, b;[START_REF] Ng | Snails and their trails: the multiple functions of trail-following in gastropods[END_REF]. There is still, in turn, a limited amount of information related to their sensory ecology and their subsequent ability to navigate through topographically complex landscapes, despite the documented role of substrate spatial structure in the movement rates and directionality of gastropods [START_REF] Evans | Responses to disturbance of the periwinkle Littorina punctata (Gmelin) on a shore in Ghana[END_REF][START_REF] Emson | An experimental investigation into the effect of crevice availability on abundance and size-structure in a population of Littorina rudis (Maton): Gastropoda: Prosobranchia[END_REF][START_REF] Underwood | Movements of intertidal gastropods[END_REF][START_REF] Petraitis | Occurrence of random and directional movements in the periwinkle, Littorina littorea (L. )[END_REF][START_REF] Underwood | Multifactorial analyses of directions of movement of animals[END_REF][START_REF] Jones | State-dependent habitat selection by an intertidal snail: the costs of selecting a physically stressful microhabitat[END_REF][START_REF] Fraser | Trying to fit in: are patterns of orientation of a keystone grazer set by behavioural responses to ecosystem engineers or wave action?[END_REF][START_REF] Fraser | Orientation in a keystone grazer: interactions between habitat and individual identity drive patterns of resting behaviour[END_REF]. Responses to gravity and visual cues have nevertheless been documented in a range of intertidal gastropods. For instance, negative geotaxis (i.e. orientation away from a gravitational force) has been observed in Littorina littorea [START_REF] Kanda | Studies on the geotropism of the marine snail, Littorina littorea[END_REF]. This response has further been shown to increase with the slope of the substrate [START_REF] Kanda | Studies on the geotropism of the marine snail, Littorina littorea[END_REF], to be more pronounced in highshore than in low-shore snails [START_REF] Hayes | The negative geotropism of the periwinkle: a study in littoral ecology[END_REF], and to be habitat-related (Newell, 1958a,b). Snails originating from horizontal and vertical surfaces respectively showed positive and negative geotaxis (Newell, 1958b), and this response was more pronounced in featureless environments (Newell, 1958b) -though contradictory results exist [START_REF] Petraitis | Occurrence of random and directional movements in the periwinkle, Littorina littorea (L. )[END_REF]. Intertidal gastropods exhibit oriented responses to the light of the sun in L. littorea (Newell, 1958a,b), L. punctata [START_REF] Evans | Responses to disturbance of the periwinkle Littorina punctata (Gmelin) on a shore in Ghana[END_REF] and Nerita plicata [START_REF] Warburton | Solar orientation in the snail Nerita plicata (Prosobranchia: Neritacea) on a beach near Watamu, Kenya[END_REF] and unidentified celestial objects in Aplysia brasiliana [START_REF] Hamilton | Celestial orientation by surface-swimming Aplysia brasiliana Rang (Mollusca: Gastropoda)[END_REF], and to a range of visual cues such as black walls [START_REF] Thain | Return of the prosobranch Gibbula umbilicus (Da Costa) to the littoral region after displacement to the shallow sublittoral[END_REF] shore-line topographic features and nocturnal silhouettes of vegetation (Evans, 1961, Chelazzi and[START_REF] Chelazzi | Researches on the coast of Somalia. The shore and the dune of Sar Uanle. 9. Coastward orientation in Nerita textilis Dillwyn (Gastropoda Prosobranchia)[END_REF], and to large areas of vegetation and individuals plant stems [START_REF] Hamilton | Daily movements and visual relocation of plant stems by Littorina irrorata (Mollusca: Gastropoda)[END_REF][START_REF] Hamilton | The role of vision in adaptive oriented movements of Littorina irrorata (Mollusca: Gastropoda) when displaced from their natural habitat[END_REF]. These contradictory observations suggest, however, the presence of both positive phototaxis (i.e. the orientation and movement toward a source of light) and scototaxis (i.e. a natural preference for dark areas) in intertidal snails. Despite the acknowledged role of topographic features, such as boulders and pebbles, crevices, cavities and pits in the ecology of intertidal ecosystems (Little et al., 1998) and early suggestions on the existence of a kinaesthetic memory in limpets, which determine their location based on previous movements and local topography [START_REF] Pieron | Sens de l'orientation et mémoire topographique de la Patella[END_REF], the role of topographic complexity and the related alternation of shaded and unshaded areas on the motion behaviour of intertidal gastropod is still unknown. In particular, no evidence exists for thigmotaxis (i.e. movement towards or away from the stimulus induced by a physical contact) in intertidal gastropods.

In the present study, we chose the intertidal herbivorous gastropod, Littorina littorea, as a model species due to its abundance on most Western and Northern European coasts where it can reach densities up to hundreds of individuals per metre square [START_REF] Petraitis | Occurrence of random and directional movements in the periwinkle, Littorina littorea (L. )[END_REF][START_REF] Bertness | The Ecology of Atlantic Shorelines[END_REF][START_REF] Seuront | Heavy-tailed distributions in the intermittent motion behaviour of the intertidal gastropod Littorina littorea[END_REF], and its role in controlling algal growth [START_REF] Stafford | Spatial patchiness of epilithic biofilm caused by refugeinhabiting high shore gastropods[END_REF], sediment dynamics [START_REF] Kamimura | Effects of opportunistic feeding by the intertidal gastropods Batillaria zonalis and B. flectosiphonata on material flux on a tidal flat[END_REF] and the recruitment of both algae and invertebrates [START_REF] Buschbaum | Direct and indirect effects of Littorina littorea (L.) on barnacles growing on mussel beds in the Wadden Sea[END_REF][START_REF] Lotze | Complex interactions of climatic and ecological controls on macroalgal recruitment[END_REF]. The net distance travelled by L. littorea between two low tides in its rocky shore habitats ranged from 1 cm to 10-15 metres [START_REF] Seuront | Heavy-tailed distributions in the intermittent motion behaviour of the intertidal gastropod Littorina littorea[END_REF]. Over this range of scales, L. littorea is typically exposed to a broad range of topographic features, suggesting that it may have adapted its senses to navigate in such a complex environments. In this regard, we aimed to elucidate the behavioural response of L. littorea to substrate slope, topographic discontinuities and light using purpose-designed experimental set-ups under laboratory-controlled conditions. Specifically, we assessed the presence of negative geotaxis in L. littorea, and we further tested the alternative hypothesis that geotaxis may either be positive or negative following the encounter with a topographic discontinuity. We also assessed the behavioural response of L. littorea to light using two distinct though complementary approaches based on the ability to actively (i) choose between a dark and a light area and (ii) orient towards a dark shape. If L. littorea demonstrate such behavioural adaptations to structurally complex environments, the behavioural repertoire and resilience of this keystone species may respectively be much wider and stronger than previously thought.

Methods

Collection and Acclimation of L. littorea

Littorina littorea individuals were collected from the Fort de Croy (Wimereux, France; 50°45'48"N, 1°35'59''E) an intertidal reef typical of the rocky habitats found along the French coasts of the eastern English Channel [START_REF] Chapperon | Cue synergy in Littorina littorea navigation following wave dislodgement[END_REF][START_REF] Seuront | The smell of sex: water-borne and air-borne sex pheromones in the intertidal gastropod Littorina littorea[END_REF][START_REF] Spilmont | First records of Ptilohyale littoralis ( Amphipoda : Hyalidae ) and Boccardia proboscidea ( Polychaeta : Spionidae ) from the coast of the English Channel : habitat use and coexistence with other species[END_REF]. Before any experiment took place, L. littorea individuals (10 to 15 mm in length) were acclimatized for 24 h in the laboratory in acrylic glass (i.e. polymethyl methacrylate, PMMA) cylinders (50 cm tall and 20 cm in inner diameter, riddle with holes 5 mm in diameter) held in 120-l ( 90´ 50 ´ 30 cm) tanks of running natural seawater, aerated at temperatures representative of in situ conditions. These perforated 'acclimation towers' [START_REF] Seuront | The smell of sex: water-borne and air-borne sex pheromones in the intertidal gastropod Littorina littorea[END_REF] allow both seawater to be continuously renewed and captive snails to move freely in and out of the water without being able to escape. No food was available during the acclimation period. Each individual was only use once. Between each trial the behavioural set-up was rinsed with 70% ethanol and seawater to remove mucus cues [START_REF] Erlandsson | Trail following, speed and fractal dimension of movement in a marine prosobranch, Littorina littorea, during a mating and a non-mating season[END_REF]. In each of the behavioural assays described hereafter, the motion behaviour of L. littorea individuals was recorded every 5 seconds during 120 minutes using a Raspberry Pi NOiR camera overlooking the experimental set-up and operated through a Raspberry computer under homogenous dim light conditions (i.e. 168 lux) measured with a digital lightmeter (Extech Instruments,403125). The resulting 1480 images where subsequently assembled using Time Lapse Tool (©Al Devs) before behavioural analyses took place.

Geotaxis Assay

Geotactic experiments were conducted in glass aquaria (length: 34.5 cm, width: 25 cm, depth: 18 cm) where Littorina littorea were immersed under 10 cm of seawater. To assess the potential geotactic response of L. littorea, behavioural assays were conducted in aquaria tilted with three different angles, i.e. 6, 13 and 25°. These angles were specifically chosen to cover the range of slopes reported to induce a geotactic response in this species [START_REF] Kanda | Studies on the geotropism of the marine snail, Littorina littorea[END_REF].

Control experiments were conducted in aquaria lying horizontally. One individual was placed in the middle of 5 separate aquaria, with their head initially oriented randomly, and each experimental condition replicated 10 times (N = 50). Treatments and control experiments were randomized.

The time taken by L. littorea to resume movement (tmove) was estimated for each snail. The time of activity, tactive, was defined as the time between the first displacement at location (x0, y0) and the moment snails reached a maximum net distance dmax = W/2, where W is the width of the aquarium. Movement speed and direction were respectively calculated as v = dmax/tactive.

Direction was the angle of the vector between a snail starting point (x0, y0) and the point where snails reached a maximum net distance dmax = W/2 (xW/2, yW/2). The behaviour of snails that encountered the wall was also recorded, and the percentage of individuals that followed and/or climbed the wall of the aquarium was also recorded.

Thigmotaxis Assay

The response of Littorina littorea to topographic discontinuities was studied at a scale pertinent to individual snails through the development of a LEGO-based behavioural apparatus.

More fundamentally, the experimental set-up was designed to assess if the previously reported observations of snail moving up upon encountering the wall of an aquarium [START_REF] Kanda | Studies on the geotropism of the marine snail, Littorina littorea[END_REF][START_REF] Petraitis | Occurrence of random and directional movements in the periwinkle, Littorina littorea (L. )[END_REF] were actually an evidence of negative geotaxis, and not an idiosyncratic artefact due to the impossibility for the snails to move downward. Our apparatus was specifically designed to associate the encounter of a discontinuity (i.e. a surface-to-wall transition or a surface-to-drop-off transition) with the possibility of moving upward or downward. Note that the height of the walls is identical to the height of the drop-offs, i.e. 3 cm. The apparatus was uniformly made of light grey LEGO ® Bricks (Fig. 1A), and included four replicate cubic structures (length: 11 cm, height: 6 cm), constituted of vertical walls where snail can move up or down, that were built on a LEGO plaque (25.5 ´ 25.5 cm) glued on the bottom of a cubic glass aquarium and immersed in 10 cm of seawater. This design was specifically chosen to offer the same amount of discontinuities leading to upward and downward surfaces to L. littorea individuals.

One snail was used in each replicate structure and 10 replicates were done (N = 40). L. littorea behaviour was dichotomized in four behavioural displays following the encounter with a discontinuity: (i) discontinuity avoidance (i.e. the behaviour by which a snail actively avoids a discontinuity upon contact, through a drastic change in direction, without following it; Fig. 3A), (ii) discontinuity following (i.e. the behaviour by which a snail follows a discontinuity at a distance less than one bodylength; Fig. 3B), (iii) moving upward, and (iv) moving downward.

The time allocated to each behaviour was noted for each individual and the frequency of occurrence of the four behaviours was subsequently calculated. The total time spent in our LEGO apparatus (ttotal) and the time of activity (tactive) were recorded for each snail as well as the time of initial scanning behaviour (tscan; i.e. non-oriented movements of a snail spinning around its vertical axis before heading into a specific direction), the time to resume movement and the activity index. A behavioural complexity index I was also calculated for each snail as I = Ni ´ factive, where Ni is the total number of observed behaviours and factive the percentage of activity.

Scototaxis Assay

Two distinct but complementary approaches were used to infer the presence of scototaxis in Littorina littorea. First, we used a binary choice assay conducted in a PVC container (length: 51 cm, width: 11 cm, depth: 20 cm) half black and half white with two removable vertical panels used to isolate a central area from the rest of the apparatus. Five Littorina littorea were placed in circle at the centre of the container filled with 10 cm of seawater. To enhance the visibility of individuals when they crawled on the black section, they were marked with white nail polish. After marking the snails, the nail polish was allowed to dry for 30 minutes and the snails were kept in flowing seawater aquaria until the next day (Seuront et al., 2018a). The experiment was repeated four times (N = 20). Control experiments were run in totally black and totally white version of our experimental container to ensure no bias intrinsic to the experimental set-up was present. The parameters considered for the behavioural analysis were the position of each snail after two hours of experimentation, the proportion of individuals that left the aquaria and the total time (ttotal) spend in each section. This time was decomposed into activity (tactive) and inactivity (tinactive) times in each section of the aquaria.

Second, we used a target-based assay that was conducted using a modification of the aforementioned LEGO thigmotaxis assay. Specifically, four replicate cubes (length: 11 cm, height: 6 cm) were built on a LEGO plaque (25.5 ´ 25.5 cm), and each cube was either made of three white walls and a target black wall (W3B1 assay) with a white bottom or three black walls and a target white wall (B3W1 assay) with a black bottom (Fig. 1B,C). We used black and white LEGO bricks to maximise the contrast between the target wall and the other walls.

The position of the black and white walls was assigned randomly and the supporting LEGO plaque was glued at the bottom of a cubic glass aquarium and immersed in 10 cm of seawater.

One snail was put in the middle of each replicate cube with the head oriented randomly and each assay replicated 8 times (N = 32). L. littorea behaviour was recorded as described for the geotactic and thigmotactic experiments. In the B3W1 assay, the 32 individuals were marked with white nail polish to be able to see them on the black background. Control experiments were run in both uniformly black and white LEGO structures. The behavioural analysis was based on the scanning behaviour (i.e. non-oriented movements of a snail spinning around its vertical axis before heading into a specific direction) and the net direction.

Statistical Analyses

As the distribution of measured parameters was non-normally distributed (Shapiro-Wilk test, p = 0.17), non-parametric statistics were used throughout this work. In the geotaxis assay, the potential differences between inclination angles and the time taken by Littorina littorea to resume movement (tmove), their time of activity (tactive), and their movement speed were assessed using a Kruskal-Wallis test (hereafter referred to as KW test) and when necessary a subsequent post-hoc test was performed using a Dunn test [START_REF] Zar | Biostatistical analysis[END_REF]. The Rayleigh test was further used to assess the circular uniformity of the snail dispersal for each inclination angle and control [START_REF] Zar | Biostatistical analysis[END_REF]. The initial orientation of L. littorea in the thigmotaxis assay (i.e. toward upward or downward surfaces) was compared to a theoretical equi-repartition of the individuals using a c 2 test [START_REF] Zar | Biostatistical analysis[END_REF]. The presence of a causal link between the time of activity and (i) the number of behaviour and (ii) the activity index (i.e. percentage of activity) was further tested using the Pearson correlation coefficient.

In the control experiments of the binary scototaxis assay, the proportion of individuals found on each half of the uniformly coloured containers (i.e. black or white) was compared to a theoretical equi-repartiton using a c 2 test.

A Wilcoxon test for paired samples was performed to compare the proportion of time spend in the black and the white section. The percentage of activity and inactivity times were also compared for the two sides and between the wall and the bottom using a Wilcoxon test for paired samples. The proportion of activity and inactivity on the walls and on the bottom were compared for each side using a Friedman test. A Mann-Whitney pairwise test for paired samples was performed when necessary.

The uniformity of the net direction followed by Littorina littorea in the control target-based scototaxis assay run in uniformly black and white LEGO set-up were compared using a Rayleigh test. The net direction of the 32 snails was recorded (i.e. toward black or white wall) and the time of displacement between the initial position and the first wall touched (i.e. black or white) was compared using a Wilcoxon-Mann-Whitney U-test (hereafter called U-test).

Results

Geotaxis Assay

The time taken by Littorina littorea to resume movement (tmove) did not significantly differ (KW test, p = 0.1295) between the different treatments (Fig. 2A). The time of activity (tactive, i.e. the time between the first displacement and the moment snails reached a maximum net distance dmax = W/2, where W is the width of the aquarium) was significantly different between treatments (KW test, p = 0.00329). More specifically, tactive was significantly shorter for individuals in aquarium tilted by 25° angles than for all the others treatments (Fig. 2B).

The distribution of the net orientation of Littorina littorea did not significantly differ from a uniform distribution (Rayleigh test, p = 0.2146) under all experimental treatments (Fig. 2D). In contrast, L. littorea speed significantly differed between treatments (KW test, p < 0.0001). Specifically, L. littorea moved significantly faster on surface tilted by 25° (Dunn test, p < 0.0001) than for the surfaces tilted to 6 and 13° and the control, where speed did not significantly differ (Fig. 2C). Finally, all the snails that encountered the wall systematically followed it, before 78 to 100% of them climbed it.

Thigmotaxis Assay

Respectively 18 and 22 individuals moved toward the vertical walls and the sections of our LEGO-based structure without wall. There was no significant difference between this distribution and a theoretical equi-repartition of the individuals (c 2 , p = 0.6578). The most frequent behaviour after the first encounter with either a surface-to-wall or a surface-to-dropoff discontinuity was discontinuity-following (67.5%), in contrast to discontinuity-avoidance which was only marginally observed (2.5%). Climbing directly downward and upward after encountering a topographical discontinuity (i.e. a surface-to-drop-off discontinuity and a surface-to-wall discontinuity, respectively) represented respectively 10% and 20%. After following a discontinuity, 100% of the individuals moved up after following a surface-to-wall discontinuity or down after following a surface-to-drop-off discontinuity.

Scototaxis Assay

Control experiments run in either uniformly black or uniformly white versions of both the binary container and target-based LEGO structure respectively showed that the proportion of individuals found in each half of the aquaria did not significantly differ from a theoretical equirepartition ( c 2 test, p = 0.177), and that the net directions followed by Littorina littorea were uniform (p = 0.295), hence that no intrinsic directionality related to the experimental set-up was present.

In the binary choice assay, any group effects or inter-individual interactions such as trailfollowing between snails in a group trial have never been observed. All individuals can then be considered as being independent. The time spent in the black section was more than twice as high as the time spent in the white section (Wilcoxon test, p = 0.00195). In contrast, there is no significant difference in the percentages of activity and inactivity in the two different sections (Wilcoxon test, p = 0.51).

In the W3B1 assay (i.e. experimental set-up with three white wall, a black wall and a white bottom), 78% of the individuals oriented towards the black wall, whereas only 22% oriented towards a white wall (Fig. 3C,D, Fig. 4). Similarly, in the B3W1 assay (i.e. experimental setup with three black wall, a white wall and a black bottom) a vast majority of the snails (90.6%) moved towards a black wall, while only 9.4% moved towards the white wall (Fig. 4).

Discussion

The ability to navigate through structurally complex environments is a key determinant of the ecological success of a species. Intertidal gastropods are no exception, especially given the highly complex nature of their environment, and the heavy constraints exerted by a range of stressors such as thermal and desiccation stresses, wave exposure and the related risk of dislodgement by wave action [START_REF] Denny | Encyclopedia of tidepools and rocky shores[END_REF]. However, despite their critical role in the structure and function of intertidal ecosystems, little is still known -with the noticeable exception of trail-following, see [START_REF] Ng | Snails and their trails: the multiple functions of trail-following in gastropods[END_REF] for a review -on the role of sensory mechanisms and the related navigational capabilities of intertidal gastropods in general, and Littorina littorea in particular. Based on the existing and often contradictory literature, this work has inferred the presence of geotaxis, thigmotaxis and scototaxis in L. littorea under laboratory-controlled conditions.

Geotaxis as a by-product of thigmotaxis

Despite the presence of significant differences between treatments in the time taken by L. littorea to resume movement (tmove), their time of activity (tactive) and their speed, no significant relationship was found between any of these variables and the slope of their substrate. These results suggest that Littorina littorea may behave similarly on substrates of different inclination.

Specifically, the lack of dependence of L. littorea movement direction to substrate slope further suggests an absence of geotactic behaviour in L. littorea investigated on substrates inclined from 6 to 25°. L. littorea was also observed to move consistently significantly faster on surface tilted by 25°. These results suggest that despite the absence of geotactic response, L. littorea exhibited a kinetic response triggered by the conditions and the angles of the substratum. In addition, the high proportion of L. littorea that climbed the wall of the experimental aquarium after his encounter (78 to 100%) may have indicated a high expression of geotaxis. However, in all instances, this behaviour consistently occurred after the snails followed the discontinuity between the bottom and the wall of the aquarium. These observations further suggest that the observed seemingly negative geotactic responses may instead be triggered by a discontinuity encounter, i.e. a thigmotactically-induced negative geotactic response.

Thigmotaxis as a way to navigate

The predominant behaviour of the thigmotactic assay was by far discontinuity-following, before 100% of Littorina littorea individuals actually moved up or down, hence switched to a geotactic behaviour. This switch from thigmotaxis to geotaxis consistently happened along both surface-to-drop-off and surface-to-wall discontinuities. However, though these results have not been included in the present work, L. littorea have also been observed to switch from following a surface-to-wall discontinuity to following a surface-to-drop-off discontinuity and vice versa, and these transitions were consistently followed by a geotactic response (Moisez, personal observations). Even though geotaxis has been observed in the absence of thigmotaxis in 30% of our observations, overall our observations suggest that previously reported positive or negative geotactic response may actually have been essentially triggered by a thigmotactic response.

Though the following may be considered as highly speculative, the fact that discontinuityfollowing was the predominant behaviour following an encounter with a vertical discontinuity and was consistently followed by a geotactic response may also be though as an obstacle response of individual L. littorea which were attempting to go around an obstacle before actually choosing to moving up or down. Additionally, the mechanical constraints of vertical crawling (up or down) due to e.g. gravity and the shape of L. littorina shell may be such that it may be easier to begin by turning and follow a discontinuity rather than crawling directly downward or upward after encountering a surface-to-drop-off discontinuity and a surface-towall discontinuity, respectively. In particular, the hypothesis of a mechanical constraint related to the shape of L. littorina shell is consistent with the observed 2-fold difference between the frequency of climbing downward after encountering a surface-to-drop-off discontinuity (i.e. 20%) and the frequency of climbing upward after encountering a surface-to-wall discontinuity (i.e. 10%). This observation may indicate a higher mechanical constraint associated to a surfaceto-wall.

Despite the fundamental importance of substrate topography in the ecology of intertidal organisms [START_REF] Denny | Encyclopedia of tidepools and rocky shores[END_REF], only a limited amount of work has been devoted to the understanding of the mechanisms relating substrate topography and the navigational abilities of intertidal gastropods. For instance, the complexity of surface topography has been shown to affect different aspects of the ecology of intertidal snails such as distance travelled, population density and structure [START_REF] Underwood | Experimental analyses of the influences of topography of the substratum on movements and density of an intertidal snail, Littorina unifasciata[END_REF][START_REF] Chapman | Dispersal of the intertidal snail, Nodilittorina pyramidalis, in response to the topographic complexity of the substratum[END_REF]. In addition, the presence of species such as mussels or barnacles further creates biogenic habitats that contribute to increase the complexity of surface topography and in turn affect different aspects of mobile fauna biology and ecology. For instance, in gastropods the pattern of limpet orientation was affect by this complex biogenic habitat [START_REF] Fraser | Trying to fit in: are patterns of orientation of a keystone grazer set by behavioural responses to ecosystem engineers or wave action?[END_REF]. In the early 20 th century, [START_REF] Pieron | Sens de l'orientation et mémoire topographique de la Patella[END_REF] introduced the idea of a kinaesthetic memory, which allowed homing limpets to determine their location based on previous movement and local topography. In contrast to limpets, littorinids do not exhibit homing behaviour, though evidence exist for navigation back to a preferred shore height or resting habitat following dislodgment or transplantation [START_REF] Evans | Responses to disturbance of the periwinkle Littorina punctata (Gmelin) on a shore in Ghana[END_REF][START_REF] Bock | The role of behavior in determining the intertidal zonation of Littorina planaxis Philippi, 1847, and Littorina scutalata Gould, 1849[END_REF][START_REF] Gendron | Habitat selection and migratory behaviour of the intertidal gastropod Littorina littorea ( L .)[END_REF][START_REF] Thain | Return of the prosobranch Gibbula umbilicus (Da Costa) to the littoral region after displacement to the shallow sublittoral[END_REF][START_REF] Rolán-Alvarez | The maintenance of a cline in the marine snail Littorina saxatilis: the role of home site advantage and hybrid fitness[END_REF][START_REF] Miller | Dislodged but not dead: survivorship of a high intertidal snail following wave dislodgement[END_REF][START_REF] Chapperon | Cue synergy in Littorina littorea navigation following wave dislodgement[END_REF]Seuront et al., 2018a). As such, the consideration of thigmotaxis, and in particular both the prevalent discontinuity-following behaviour and the thigmotactically-induced negative geotaxis observed in the present work, may be a first step into furthering our understanding of how these organisms may navigate through complex landscapes and is a plausible candidate to explain how the aforementioned putative kinaesthetic memory may work.

Scototaxis assay as an adaptation to a contrasted environment

Littorina littorea orient toward black areas, hence exhibit a strong positive scototaxis (Fig. 4). This behaviour is consistent with early suggestions that eyes of Littorina could be used for monitoring light intensity and for orientation [START_REF] Stoll | On the role of the eyes and non-ocular light receptors in orientational behavior of Lymnaea stagnalis (L)[END_REF], and with littorinids visual abilities as L. littorea have simple eyes which are optimum for under water vision during the day [START_REF] Seyer | Resolution and sensitivity in the eye of the winkle Littorina littorea[END_REF]. Our results are also consistent with previous evidence that a range of gastropod species orient towards black forms, with the strength of the response increasing with their dimensions [START_REF] Thain | Return of the prosobranch Gibbula umbilicus (Da Costa) to the littoral region after displacement to the shallow sublittoral[END_REF]Hamilton andWinter, 1982, 1984). This behaviour may reflect the fact that objects with ecological importance essentially appear as vertical for benthic organisms [START_REF] Hamilton | Behavioural responses to visual stimuli by the snails Tectarius muricatus, Turbo castanea, and Helix aspersa[END_REF]. Though further work is needed to untangle the details of the mechanisms involved, the capacity to distinguish and orient toward black areas could be important for snails to allow them to find their way back after feeding excursions or dislodgement as recently demonstrated for L. littorea (Seuront et al., 2018a).

Note that the orientation towards dark areas is also likely to be relevant to gastropods thermal ecology and predation avoidance. Indeed, crevices, pits and the lower part of boulders typically appear as darker areas in comparison to the more exposed flat substrates, hence they can exploit topographical complexity of the rocky shore to find darker microhabitats which provide shelter from desiccation and thermal stresses [START_REF] Jackson | Effects of topography on the environment[END_REF]Chapperon and Seuront, 2011b;Seuront et al., 2018b) This hypothesis is also consistent with the reported decreased activity of L. littorea in dark environments.

It is finally stressed that because the presence of other individuals in the scototaxis assays may implicitly generate a time-dependent dark cue on a light background depending on the relative position of individual snails respective to each other and to the dark and black walls, it is likely that the snails considered in each scototaxis assay

Conclusion

This study suggests that the geotactic behaviour of Littorina littorea, previously reported in the literature [START_REF] Kanda | Studies on the geotropism of the marine snail, Littorina littorea[END_REF][START_REF] Hayes | The negative geotropism of the periwinkle: a study in littoral ecology[END_REF]Newell, 1958a,b;[START_REF] Petraitis | Occurrence of random and directional movements in the periwinkle, Littorina littorea (L. )[END_REF] is more likely to be a by-product of the thigmotactic response induced by a contact with topographical discontinuities. The new evidence of the existence of the prevalence of thigmotactic response in L. littorea presented in the present work as well as demonstration that this species actively chooses dark areas where they consistently decreased their level of activity provide new insights into our understanding of intertidal gastropods may navigate through complex landscapes.

Much work is, however, still needed to entangle the complex relationship that exist between the structure and functions at play in their environment, the cognitive abilities of intertidal snails and the adaptive value of using different taxes as a response to environmental cues. In particular, increasing the complexity of our LEGO-based structure designed to assess the presence of thigmotaxis, to be more coherent with the complex landscape of the rocky shore would contribute to generalise our results. In addition, because our experiments were consistently undertaken under laboratory conditions, underwater and on substrates devoid of food, further work is needed to entangle the role of e.g. food availability, temperature or humidity gradient, as well as different heat and air exposure stress may play in the observed behavioural patterns.

Fig. 1 .

 1 Fig. 1. Illustration of the LEGO-based structures designed to assess (A) the presence of geotaxis and thigmotaxis in Littorina littorea and the presence of scototaxis in L. littorea based on experiments run on four subsections made of (B) 3 white walls and 1 target black wall (W3B1) or (C) 3 black walls and 1 target white wall (B3W1). The white points from the panel C correspond to the Littorina littorea individuals marked with white nail polish.

Fig. 2 .

 2 Fig. 2. Behavioural measurement of Littorina littorea for the different treatments in the geotactic experiment, (A) time to resume movement (tmove), (B) time of activity (tactive), (C) movement speed (v) and (D) the net orientation travelled by L. littorea for the experiment tilted to 6°. The limits of the box are the 25 and 75 percent quartiles. The median is shown with a horizontal line inside the box and the minimal and maximal values are shown with

Fig. 3 .

 3 Fig. 3. Illustrations of representative tracks from our thigmotactic assay (A) representing the discontinuity avoidance behaviour, (B) the discontinuity following behaviour, and from ours scototaxis assay (C) W3B1 assay and (D) B3W1 assay.

Fig. 4 .

 4 Fig. 4. Frequency of occurrence of target-based positive and negative scototaxis in Littorina littorea. The black

Acknowledgements

The authors thanks P. Notez for his help in developing the software environment to operate both the Raspberry computer and Raspberry Pi NOiR camera, and D. Menu for building the experimental set-up used in our scototaxis assay. N. Spilmont is acknowledged for his constructive comments and criticisms on a previous version of this work. This work has been financially supported by a joint PhD fellowship from the Région Hauts-de-France and the Université of Lille to E.M. This work is a contribution to the CPER research project CLIMIBIO.

The authors thank the French Ministère de l'Enseignement Supérieur et de la Recherche, the Hauts de France Region and the European Funds for Regional Economical Development for their financial support for this project.