Aggregation behaviour in Littorina littorea has limited thermal benefits under conditions of thermal stress

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Collection and acclimation of Littorina 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 Chanel (Chapperon and Seuront, 2009; Seuront and Spilmont, 2015; Spilmont et al., 2018). 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 5mm in diameter) held in 120-l (90×50×30 cm) tanks of running natural seawater, aerated at temperatures representative of in situ conditions at the time of collection. These perforated ‘acclimation towers’ (Seuront and Spilmont, 2015) 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. During the experiments, each individual was only used once.

Experimental conditions

The thigmotactic response of L. littorea was studied at scales pertinent to individual snails in six types of experimental containers. Specifically, we considered circular containers with three different diameters (i.e. 5, 8 and 11 cm, hereafter referred as D1, D2 and D3) and three cubic containers with three different sizes (i.e. 5, 8 and 11cm in side length, hereafter referred as C1, C2 and C3). The circular experimental containers were glass beakers (Fisherbrand) which were separated by light grey LEGO® Bricks walls to homogenize the visual field of each individuals as L. littorea used scototaxis to orient themselves (Moisez & Seuront, 2020). Cubic experimental containers were uniformly made of light grey LEGO® Bricks (Fig. 1). These experimental containers were built on a LEGO® plate (25.5 × 25.5 cm) glued on the bottom of a cubic glass aquarium and immersed in 10 cm of seawater. More fundamentally, these different experimental containers were specifically used to allow L. littorea to face two distinct forms of topographic discontinuities of three different sizes, that is (i) an uninterrupted two-dimensional discontinuity, i.e. a surface-to-wall transition in circular containers (Fig. 2A) and (ii) three-dimensional discontinuities characterized by both horizontal broken surface-to-wall transitions and vertical wall-to-wall-transitions in cubic containers (Fig. 2B). One snail was used in each replicate structure; for the cubic containers, N = 32 for C1, C2 and C3 and for the cylindric containers, N = 12 for D1 and D2 and N = 32 for D3.
The thigmotactic response of L. littorea was also studied under different salinity conditions in the two types of experimental containers previously described (5 cm diameter for the cylindric ones and 5 cm of size for the cubic ones). Specifically, four conditions of salinity and a control (hereafter referred as C) were tested in each experimental container. The control was natural seawater collected on Wimereux (France) with a salinity equal to 33.4 PSU. Natural sawater was diluted at 20% (26.7 PSU), 30% (23.4 PSU), 40% (20 PSU) and 50% (16.7 PSU). Control and dilution at 20% and 30% are realistic salinity find in the intertidal environment, whereas dilutions at 40 and 50% are extreme decreasing salinity conditions to have a pronounced answer of L. littorea. One snail was used in each replicate structure, which were further triplicated (N = 24 for the cylindric container and N = 27 for the cubic one) for each salinity condition.
The motion behaviour of L. littorea individuals was recorded every 5 s during 60 min 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 lx) measured with a digital lightmeter (Extech Instruments, 403,125; Moisez and Seuront, 2020). The resulting 720 images where subsequently assembled using Time Lapse Tool (©Al Devs) before behavioural analyses took place. We considered the end of the experiment when the individuals exit the cylinder or the cube. Between each trial, the behavioural set-up was rinsed with 70% ethanol and seawater to remove mucus cues (Erlandsson and Kostylev, 1995).

Behavioural analysis

For each snail we measured the time spent actively moving (i.e. activity time, Tact) and being inactive (Tinact). The activity time included three distinct behavioural activities: (i) the time spent displacing on the bottom of the apparatus (Tb), (ii) the time spent in discontinuity-following (Tf) and (iii) the time spent displacing on the wall of the apparatus (Tw). The intensity of discontinuity-following was assessed by the number N of revolutions that snails continuously made on the discontinuity, through the following classification: N = 0, N < 1 and N ≥ 1. The percentage of individuals that followed the discontinuity in the two experimental containers was recorded. The percentage of individuals that climbed the wall in the two experimental containers was recorded, and specifically we also recorded the percentage of individuals that climbed on the corner of the wall in the cubic experimental container.

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Statistical analysis

As the distribution of measured parameters was non-normally distributed (Shapiro-Wilk test, p > 0.05), non-parametric statistics were used throughout this work. The inactivity time (Tinact), activity time (Tact) and the durations which composed the activity time (Tb, Tf, Tw) were compared using a Kruskal-Wallis (hereafter K-W test) test between each size condition for each experimental container and also between each salinity conditions for each experimental containers; when necessary a subsequent post-hoc test was performed using a Dunn test (Zar, 2010). These different times were also compared between circular and cubic experimental container of the same size or of same salinity conditions using a Mann-Whitney (hereafter M-W test) pairwise test.

Thigmotactic and salinity experiment

In C, 26.7 and 23.4 PSU conditions, 100% of L. littorea individuals followed the discontinuity in the cylindric container. In 20 and 16.7 PSU conditions, 75 and 70.8% of individuals followed the discontinuity, respectively. In the cubic container, 100% of the individuals followed the discontinuity in C and 26.7 PSU conditions. In 23.4, 20 and 16.7 PSU conditions, 92.6, 77.8 and 66.7% of L. littorea individuals respectively followed at the discontinuity. There was no significant difference in the number of individuals that followed the discontinuity between the cylindric and the cubic containers (Chi², p > 0.05). The individuals that did not follow the discontinuity, did not avoid it, but instead either directly climbed the wall without discontinuity-following or did not move or only on the bottom of the container. In the cubic container, geotaxis without discontinuity-following (i.e. thigmotaxis) occurred only for 7.4 and 3.7% of L. littorea individuals respectively in 23.4 and 16.7 PSU conditions. In the cylindric container, geotaxis without thigmotaxis only occurred in the 20 and 16.7 PSU conditions for respectively 4.16 and 16.7% of the individuals.

Table of contents :

Summary
Remerciements
List of Tables
List of Figures
Chapter I. General Introduction
Chapter II. Thigmotactic behaviour plays an understated poorly known, though major, role in Littorina littorea displacements under variable topographical complexity and various salinity scenarii
Abstract
1. Introduction
2. Methods
2.1 Collection and acclimation of Littorina littorea
2.2 Experimental conditions
2.3 Behavioural analysis
2.4 Statistical analysis
3. Results
3.1 Behavioural activity of Littorina littorea
3.1.1 Thigmotactic experiment
3.1.2 Thigmotactic and salinity experiment
3.2 Discontinuity-following behaviour
3.2.1 Thigmotactic experiment
3.2.2 Thigmotactic and salinity experiment
3.3 Frequency of occurrence of climbing behaviour
3.3.1 Thigmotactic experiment
3.3.2 Thigmotactic and salinity experiment
4. Discussion
4.1 Increase in topographical complexity increase Littorina littorea thigmotactic behaviour
4.2 Decreasing salinity decrease activity and intensity of thigmotactic behaviour in Littorina littorea
Conclusion
Chapter III. Microhabitats choice in intertidal gastropods is species-, temperature- and habitat-specific
Abstract
1. Introduction
2. Methods
2.1 Study site
2.2 Thermal imaging
2.2.1 Thermal properties of the four habitats
2.2.2 Body temperatures (BT) and microhabitat substrate temperatures (STμhab)
2.3 Aggregation status
2.4 Statistical analysis
3. Results
3.1 Environmental conditions
3.2 Habitat distribution
3.3 Abundance and occurrence of Patella vulgata and Littorina littorea
3.4 Substrate temperature (ST)
3.5 Body temperatures (BT) and microhabitat temperatures (STμhab) .
3.6 Frequency of thermal microhabitat choice
4. Discussion
4.1 Aggregation
4.2 The selection of a macrohabitat as refuge
4.3 Microhabitat selection: a way to escape from unfavourable temperature?
Conclusion
Chapter IV. Aggregation behaviour in Littorina littorea has limited thermal benefits under conditions of thermal stress
Abstract
1. Introduction
2. Methods
2.1 Field survey
2.2 Laboratory experiments
2.3 Thermal analysis
2.4 Statistical analysis
3. Results
3.1 Field survey
3.2 Laboratory experiments
3.2.1 Substrate temperatures
3.2.2 Body temperatures
4. Discussion
4.1 Aggregation behaviour does not lead to a thermal benefit in Littorina littorea
4.2 Aggregation behaviour of Littorina littorea: to an obsolete behaviour under warming climate?
Conclusion
Chapter V. General Discussion and Perspectives
Orientation behaviour in intertidal gastropods: the case of thigmotaxis
Thermal heterogeneity of intertidal shores
Microhabitat selection: as a way to compensate temperature variations
Aggregation behaviour: as a non-adaptive response to thermal stress
Conclusion
References.

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