Thigmotactic behaviour plays an understated poorly known, though major, role in Littorina littorea displacements under variable topographical complexity and various salinity scenarii
Intertidal rocky shores are characterized by a highly complex and variable topography, with the presence of pits, crevices or boulders. Intertidal rocky shores are also characterized by salinity short-term variations related to both evaporation and precipitation occurring at low tide. Intertidal species, such as gastropods, are de facto constrained by the interplay between topographic complexity and salinity variations. Intertidal gastropods are able to orient themselves through the topographical complexity of their environment using thigmotaxis (i.e. movement of an organism either towards or away from the stimulus induced by a physical contact). However, less is known about how the variability in the topographical discontinuities encounter may affect gastropods displacement. Here, we investigated the thigmotactic behaviour of Littorina littorea, a common grazer typically found over the whole intertidal zone of temperate rocky shores, towards different topographic discontinuities and salinity variations. Specifically, we assessed its thigmotactic behaviour in cylindric and cubic experimental containers of three different sizes that were specifically chosen to mimic the presence of (i) an uninterrupted two-dimensional discontinuity (i.e. a surface-to-wall transition) and (ii) three-dimensional discontinuities characterized by both horizontal broken surface-to-wall transitions and vertical wall-to-wall-transitions. We further inferred the effect of decreasing salinity in the activity of L. littorea and its thigmotactic behaviour. We observed that the movement of L. littorea were not impacted by the size of the nature of the discontinuities. Moreover, the size of the discontinuities encountered by L. littorea had no impact on its thigmotactic response. However, we observed that the higher the complexity, the higher the discontinuity following. We observed that in cylindric containers, individuals consistently followed the discontinuity and subsequently switched to a negative geotactic behaviour by climbing the walls. In the cubic container, individuals also followed the horizontal discontinuities, but only climbed the wall once they reached one of the cube corner (i.e. at the point of contact between two surface-to-wall transitions and a wall-to-wall transition). These results suggest that what may be thought as a geotactic behaviour is actually a thigmotactic response to a vertical discontinuity. We also observed, in both cylindric and cubic containers, a general decrease of L. littorea activity with decreasing salinity. The occurrence of thigmotaxis was not affected by salinity, though the time individual snails spent following discontinuities decreased with decreasing salinity. Specifically, in the cubic containers, snails consistently climbed the walls mostly following wall-to-wall discontinuities. Taken together, our results suggest that thigmotaxis is one of the most important behaviour in gastropods species to navigate through topographically complex environment. They also suggest that movement behaviour of intertidal gastropods are only marginally affected by salinity changes.
Thigmotaxis is the movement of an organism either towards or away from the stimulus induced by a physical contact (Kalueff et al., 2013). This behaviour is known to contribute to the movement orientation of numerous species. For examples, some evidence for thigmotaxis behaviour were found in some terrestrial invertebrate species such as the meal worm Tenebrio molitor (Street, 1968), the earthworm Lumbricus terrestris (Doolittle, 1972) and the cockroaches Periplaneta americana (Okada and Toh, 2006). In aquatic species, thigmotaxis has been observed in the zebrafish Dario rerio (Kalueff et al., 2013) and the freshwater gastropods Lymnaea stagnalis (De Vlieger, 1968). Specifically, the intensity of the stimulus changes the thigmotactic response of L. stagnalis; positive thigmotaxis is caused by the repetition of weak tactile stimuli and negative thigmotaxis is the result of one strong stimulus (De Vlieger, 1968).
Marine species and in particular rocky intertidal species typically move in a highly complex environment (Denny and Gaines, 2007) characterized by a highly variable topography, and thus face variable stimuli. Rocky shores are characterized by the presence of pits, crevices or boulders (Raffaelli and Hawkins, 1996) and the presence of biogenic habitats typically created by species such as mussels and barnacles further contributes to increase the topographical complexity of rocky shores (Underwood and Chapman, 1989). Substrate topography affects different aspect of the ecology of intertidal gastropods such as distance travelled, population density and structure (Underwood and Chapman, 1989; Chapman and Underwood, 1994). In intertidal species, and in particular in gastropods, the ability to orientate across different habitats rely on essentially non-visual senses in environments characterized by topographically complex substrate and resource heterogeneity (Fratini et al., 2001; Keppel and Scrosati, 2004; Wyeth et al., 2006). Movements of intertidal gastropods are also triggered by different taxies such as chemotaxis (Croll, 1983; Chapperon and Seuront, 2009; Ng et al., 2013; Seuront and Spilmont, 2015), phototaxis (Newell, 1958a, 1958b; Evans, 1961; Warburton, 1973), scototaxis (Hamilton and Russell, 1982; Thain et al., 1985; Moisez and Seuront, 2020) or geotaxis (Kanda, 1916; Hayes, 1926; Newell, 1958a, 1958b; Petraitis, 1982; Moisez and Seuront, 2020). A recent study conducted on Littorina littorea suggested that the directed movements consistently observed after a dislodgement toward their original habitat resulted from a combination of geotaxis, chemotaxis and rheotaxis (Seuront et al., 2018a).
Recently, evidence for a thigmotactic behaviour was found in the intertidal gastropod Littorina littorea (Moisez and Seuront, 2020). Specifically, individuals followed the topographical discontinuities of their environment and their thigmotactic behaviour induced a geotactic response. These observations suggest that previously reported positive or negative geotactic response (Kanda, 1916; Hayes, 1926; Newell, 1958a, 1958b; Petraitis, 1982) may actually have been essentially triggered by a thigmotactic response (Moisez and Seuront, 2020). Abiotic factors such as light (Newell, 1958a, 1958b; Charles, 1961; Evans, 1961), temperature, (Garrity, 1984) or salinity (Perez, 1969; Moser et al., 1989; Jury et al., 1994, 1995) can modify behaviour and in particular movements in intertidal gastropods. For example, to escape salinity variations, motile organisms can move in areas with optimal conditions (Perez, 1969; Moser et al., 1989; Jury et al., 1994, 1995). However, salinity stress can also reduce motility; for example, in the gastropods species, Batillaria attramentaria, a diminution in salinity lead to a diminution of the distance travelled by the individuals (Ho et al., 2019). The impact of salinity variations on taxies in intertidal gastropods is still unknown. Specifically, how diminution in salinity can affects thigmotaxis behaviour in intertidal gastropods?
In the present study, we choose the intertidal gastropod Littorina littorea as a model species due to its abundance on Western and Northern European coasts and its role in controlling algal growth (Stafford and Davies, 2005a), sediments dynamics (Kamimura and Tsuchiya, 2006) and the recruitment of both algae and invertebrates (Buschbaum, 2000; Lotze and Worm, 2002). L. littorea is found on rocky shore from the upper shore into the sublittoral and thus is submitted to large salinity variations. L. littorea is exposed to a broad range of topographic features, suggesting that it may also have adapted its senses to navigate in such a complex environment. In this context, we aimed to elucidate the behavioural response of L. littorea to different topographic discontinuities and salinity variations. Specifically, we assessed the thigmotactic response of L. littorea in cylindric and cubic experimental containers that were specifically chosen to mimic the presence of (i) an uninterrupted two-dimensional discontinuity (i.e. a surface-to-wall transition) and (ii) three-dimensional discontinuities characterized by both horizontal broken surface-to-wall transitions and vertical wall-to-wall-transitions. We also tested the thigmotactic response of L. littorea in the two experimental containers previously described of three different sizes. The behavioural activity and thigmotactic response of L. littorea were also considered under five conditions of salinity to assess the effect of this major intertidal stressor on their behavioural and navigational abilities.
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.
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.
Fig. 2. Illustration of ours two different experimental containers were specifically used to allow Littorina littorea individuals to face two distinct forms of topographic discontinuities, that is (A) an uninterrupted two-dimensional discontinuity and (B) three-dimensional discontinuities characterized by both horizontal broken surface-to-wall transitions and vertical wall-to-wall-transitions.
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 seawater 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).
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.
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.
Table of contents :
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
2.1 Collection and acclimation of Littorina littorea
2.2 Experimental conditions
2.3 Behavioural analysis
2.4 Statistical analysis
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
4.1 Increase in topographical complexity increase Littorina littorea thigmotactic behaviour
4.2 Decreasing salinity decrease activity and intensity of thigmotactic behaviour in Littorina littorea
Chapter III. Microhabitats choice in intertidal gastropods is species-, temperature- and habitat specific
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
3.1 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
4.2 The selection of a macrohabitat as refuge
4.3 Microhabitat selection: a way to escape from unfavourable temperature?
Chapter IV. Aggregation behaviour in Littorina littorea has limited thermal benefits under conditions of thermal stress
2.1 Field survey
2.2 Laboratory experiments
2.3 Thermal analysis
2.4 Statistical analysis
3.1 Field survey
3.2 Laboratory experiments
3.2.1 Substrate temperatures
3.2.2 Body temperatures
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?
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