Echinolittorina malaccana (Philippi 1847) occurs abundantly on rocky-shores throughout the Indo-Pacific (Reid, 2007). The local shores of Brunei Darussalam sustain two morphologically-distinct ecotypes, occupying different vertical zones. A brown ecotype inhabits the upper intertidal zone (roughly 1–2.5 m Chart Datum) and experiences tidal wetting, whereas a pale blue ecotype inhabits the supratidal zone (2.5- above 5 m Chart Datum) and is only wetted during high seas and monsoon swells. After periods of wetting and feeding, snails stop moving as the tide recedes, glue their shells to the rock surface, and withdraw into the shell (Marshall et al., 2011; Monaco et al., 2017). Because avoidance of desiccation while moving over hot dry rocks supersedes behavioral selection of thermally suitable resting (aestivating) sites, individuals often settle under direct sunlight (Marshall and Chua, 2012; Marshall et al., 2013; Monaco et al., 2017). Isolated resting snails undergo temperature-insensitive metabolic rate depression to overcome the energetic problem of high temperature exposure (Marshall and McQuaid, 2011; Marshall et al., 2011).

This study considered the intertidal brown ecotype. We determined the thermal regimes experienced at their upper distribution on a cool and a warm shoreline at Pantai Tungku, Brunei Darussalam (4.974°N, 114.867°E), between 21 June and 22 July 2018 (30 days during the warmest time of the year; Supplementary Figure S1). Pantai Tungku comprises a man-built promontory with artificial seawalls having diametrically-opposed orientations and carrying very different heat-loads. Study sites were established on the seawalls around 2 km apart, along the contour of the coast; the cool shoreline (CS) comprised a north-west-facing seawall exposed to prevailing monsoon winds and swells (#1, Supplementary Figure S1), whereas the warm shoreline comprised a north-east-facing sheltered seawall (#2, Supplementary Figure S1). To assess the potential range of temperature conditions experienced in snail microhabitats on either shoreline, temperature-loggers (see details in Monaco et al., 2017) were deployed on rocks under direct solar exposure (a 45° angled surface), or in total shade under the rocks. Loggers were set to record temperatures every 30 min.

Snails (7–9 mm) were collected from both shorelines within the proximity of the temperature loggers, while awash and feeding. In the laboratory they were rinsed in freshly-collected seawater to rehydrate the snails before starting the acclimation treatments. Prior to field-fresh thermal tolerance determinations, snails were exposed to blown air (30°C for 20 min; Memmert UFE 500, Schwabach, Germany) to inactivate them, dry shells and induce withdrawal into the shell (Marshall et al., 2018). Field-fresh tolerance experiments were carried out within 12 h of collection of the snails.

Four (4) primary laboratory temperature acclimation treatments were conducted on both warm-shore (WS, warm-acclimatized) and cool-shore (CS, cool-acclimatized) snails. The first set of experiments involved using a programmable Memmert Peltier-cooled (IPP400) incubator to cool-acclimate (CA) one group of randomly selected snails in air at 22–23°C, which approximates the coolest thermal regimes naturally experienced by tropical populations of E. malacccana (Marshall et al., 2010; Monaco et al., 2017; Figure 2 and Supplementary Figure S2). A second group was warm-acclimated (WA) to a daily 25–45°C thermal cycle, holding the temperature at 45°C for 4 h (midday) and at 25°C for 12 h (night-time), to mimic hot daily field conditions (Figure 2 and Supplementary Figure S2). Notably, whereas sun-exposure associates with high daily temperature fluctuations, cool-shaded conditions are relatively stable. These acclimations proceeded for 10 d, with snails, unfed and resting, immersed each day in flowing seawater for 5 min, to simulate tidal wetting, maintain full hydration and prevent deep aestivation (Marshall and McQuaid, 2011). Another set of experiments, which tested whether complete acclimation occurred during the above thermal treatments, involved 10 d more severe cooling [near-constant 20°C; extra-cool acclimation (ECA)] and warming (25–50°C cycle, 2 h at 50°C; extra-warm acclimation, EWA; Supplementary Figure S2). We further tested whether full acclimation occurred within a 10 d period, by assessing the effect of WA for 20 d.

We assayed the effect of acute heating on mortality using the median lethal temperature (LT₅₀; Marshall and McQuaid, 2011; Marshall et al., 2011, 2015, 2018). Acute overheating, rather than chronic temperature conditions that involve energetics, is the most likely cause of thermal stress related mortality in high-shore animals (Williams and Morritt, 1995; Marshall and McQuaid, 2011; Marshall et al., 2011). The critical thermal maximum (CT_max), the temperature at which neuromuscular co-ordination fails, which is commonly used in ectotherm experiments, is unsuitable for determining gastropod lethality. This is because inactivity in high-shore littorinid snails relates to desiccation-risk-avoidance, and snails with shells glued to rock surfaces withstand temperatures well above those limiting foot physiological performance (Marshall and McQuaid, 2011; Marshall et al., 2013, 2015; Monaco et al., 2017). Much controversy surrounds the effects of ramping on acute heat tolerance determination (Terblanche et al., 2011). We selected a ramp rate of 0.25°C.min^-1 or slightly slower, appropriate to the heating experienced in the field (Figure 2; Marshall et al., 2011). Prior to determining heat tolerance of acclimated snails, individuals were rehydrated and their shells dried as in the case of the field-fresh snails.

To determine heat tolerance, acclimated snails were placed in dry 50 ml glass tubes in a programmable bath (Grant TXF200, Cambridge, United Kingdom) and equilibrated at 30°C for 10 min, before being heated at 0.25°C.min^-1. To maintain water bath temperature stability during the LT₅₀ experiment, the heating rate was slowed to 0.12°C.min^-1 between 50 and 60°C. Naturally, the apparent lethal temperature will be affected by time at different temperatures. Temperatures inside the test tubes were recorded every minute using calibrated K-type thermocouples connected to a TC-08 interface and Picolog software (Pico Technology, Cambridge, United Kingdom). Lethality (LT₅₀) was determined for groups of 10 snails that were randomly removed from the water bath at 1°C intervals between 55 and 60°C, and allowed to recover at 28°C in wetted Petri dishes. Snails that emerged from their shells, extended their foot, and remained attached to the surface after 12 h were scored as alive. Alive but unattached snails, which are ecologically non-functional and vulnerable, were scored dead. All experiments were repeated either three or six times based on logistic constraints, with numbers of individuals limited for conservation purposes. One thousand nine hundred individual snails were used in the experiments. The study was approved by the Faculty of Science Ethics Committee, Universiti Brunei Darussalam. The effect of acclimation on LT₅₀ was statistically compared between paired treatments using Generalized Linear Models (GLZM) for a binomial distribution, with a logit-link function (Statistica v12, StatSoft, New York, United States). Actual values of LT₅₀ were computed from three parameter logistic regressions, which were plotted using Sigmaplot v14 (Systat Software, Inc., New York, United States).

The daily temperature conditions and thermal frequencies for four habitats, the coolest in the shade and the hottest in the sun, are shown for each shoreline in Figure 2. There was little difference between the habitats in average temperature for this period (28.5–30.1°C; Table 1). Likewise, absolute and mean daily minimum temperatures were largely invariable among the habitats (21.7–23.7 and 24.2–26.0°C, respectively). However, the maxima differed greatly (34.3–53.3 and 30.6–45.8°C, respectively), being markedly elevated in the sun-exposed habitats. Notably, in the three cooler habitats, daily temperatures did not rise above 45°C, the temperature around which a heat shock response (HSR) is induced (Marshall et al., 2011; Han et al., 2019), whereas peak temperatures in the warmest habitat surpassed 45°C on 18 (of the 30) days (Figure 2). Daily temperature variation (ΔT) was greatest in the warmest habitat (sun-exposed, WS; 21.6°C) and lowest in the coolest habitat (shade, CS; 4.6°C; Table 1); the warmest habitat exhibited the highest maximum and the lowest minimum temperatures (Figure 2 and Table 1). The stark difference in temperatures between the shores is highlighted by similar measures (means, maxs, mins, and ΔT) for the sun-exposed, CS and the shaded, WS habitats (Figure 2 and Table 1). Although several factors influence long-term temperature variations in these tropical habitats, including seasonal and El Nino effects, our recordings for a narrow timeframe are representative of relative daily thermal regime differences between the habitats and shores.

Substantial heat tolerance plasticity was observed in E. malaccana snails. The overall magnitude of their LT₅₀ adjustment was 2.9°C, when accounting for all laboratory-acclimated and field-acclimatized conditions (mean LT₅₀ ranged from 56.1 to 59.0; Figures 3, 4 and Table 2). Whereas snails from either shore exhibited similar magnitudes in reversible acclimation (∼2°C), the thermal bands over which acclimatory adjustments were made differed between the shores. The acclimation band for WS snails was shifted to a hotter temperature range compared to that for CS snails (Figure 4).

Cool acclimation (CA) significantly lowered the mean LT₅₀ of CS snails below that of WS snails (p < 0.001; Table 2). Because further cooling (ECA) of CS snails did not reduce the LT₅₀ further, we consider the mean hard lower limit to heat tolerance of this snail population to be 56.1°C (p = 0.177; Table 2). Similarly, the hard lower limit for WS snails is apparently 57.1°C (Figure 4 and Table 2). Warm acclimation (WA) markedly raised the mean LT₅₀ of CS snails (58.1°C), but no further increase in heat tolerance was observed for this population with further warming (EWA, extra-warm acclimation; p = 0.065; Table 2), suggesting that their hard upper heat tolerance boundary is reached under WA (Figure 4). There was also no significant difference between the two shoreline populations under WA (p = 0.127; Table 2). However, WA apparently did not result in complete thermal acclimation of the WS snails, as their heat tolerance rose following extra-warm temperature acclimation (EWA, LT₅₀ = 59.0°C; p < 0.001, Table 2).

Mean field-fresh LT₅₀ was greater in WS snails (57.7°C) compared to CS snails (57.0°C; p < 0.001; Table 2), reflecting the different recent thermal histories experienced on the different shorelines. There was no difference in LT₅₀ between the 10 d and 20 d WA treatment for warm shore snails, indicating that 10 d is sufficiently long to produce complete compensation (p = 0.295). When the cool and warm habitat data were combined (CS and WS), and compared with the data for WS snails, the combined data set showed significantly lower LT₅₀ values for ECA, CA, and EWA (p < 0.007; Table 3), but not for the WA laboratory treatments (p = 0.369; Table 3). While it was not investigated in the present study, individual variation can be as important an endpoint as the mean value in any study investigating temperature responses.

Reversible plasticity enables individual organisms to adjust performances and tolerances in response to cycling temperatures for timeframes from seasons to days. In comparative studies, laboratory acclimation is typically performed to eliminate the effect of recent field temperatures on the physiological performance of individuals. Our observed persistence of phenotypic differences after laboratory acclimation in populations from thermally-different shorelines (Figure 3 and Table 2) suggests an effect of heritable differences between the populations or non-reversible plasticity. Because the snail larvae settling on either shoreline were randomly drawn from the same planktotrophic pool, comprising individuals derived from multiple different parents, we disregard local adaptation or other inherited effects as the cause of the observed population difference (Schmidt and Rand, 1999; Williams and Reid, 2004; Kelly et al., 2011; Foo and Byrne, 2016). Non-reversible plasticity has been described as transgenerational, such as non-genetic heat-hardening transferred to the embryo from the parent, or as developmental, such as post-embryonic heat-hardening (Angilletta, 2009; Donelson et al., 2011, 2012; Reusch, 2014). Using the same argument as above for random shoreline recruitment of larvae, the shoreline (population) differences in thermal plasticity can also not be explained by a transgenerational response to heat exposure (Williams and Reid, 2004). Our findings therefore suggest that non-reversible heat tolerance is most likely founded after larval snails have settled on the shore; thus, shoreline differences are best described as developmental plasticity (Angilletta, 2009). Because the crawling snails occupy meter-size habitats, cross-shore migrations can be discounted. Notably, the apparent developmental plasticity should be reinforced by the shoreline-specific temperature conditions during the lifetime of each snail, from the crawling juvenile to the adult (Angilletta, 2009; see Slotsbo et al., 2016 on reversibility of developmental plasticity).

Molecular processes underlying thermal plasticity are cued by different components of the thermal regime (mean, maxima, and minima) for different durations of thermal cycling (daily or seasonal). Whereas seasonal acclimatization, regulated by isozyme expression (Hochachka and Somero, 2002), is typically cued by mean temperature change (Angilletta, 2009), acute daily heating of rocky-shores elicits thermal plasticity through a HSR, triggered by peak (maximum) temperatures (Hofmann and Somero, 1996; Feder and Hofmann, 1999; Tomanek and Somero, 1999; Somero et al., 2017). Although HSRs are complex, involving upregulation of multiple heat shock proteins (Hsps), much information can be gleaned from individual gene thermal expression profiles (relative levels and thermal ranges of expression) and thermal thresholds (Tomanek and Somero, 1999; Wang et al., 2017; Han et al., 2019). Previous studies on E. malaccana showed that hsp70 expression profiles differ among geographically-separated populations (Wang et al., 2017; Han et al., 2019), such that reduced expression correlates with populations from cooler locations and vice versa (Wang et al., 2017; Han et al., 2019). The same mechanism in a spatially scaled-down form could underlie the heat tolerance differences observed between our cool and warm shore populations. Whereas these findings imply plasticity of hsp expression profiles, studies for diverse populations of E. malacanna suggest that the HSR thermal threshold for this species may otherwise be fixed at around 45°C (Marshall et al., 2011; Han et al., 2019; see also Hoffmann et al., 2003). Interestingly, whereas our cool and warm shore snails adjusted heat tolerance to the same level when acclimated to a daily maximum of 45°C, only the WS snails that commonly experience temperatures above 45°C responded positively to the extra-warm acclimation treatment (EWA; for which the daily thermal peak was 50°C; Figure 3 and Table 2).

Whereas daily maximum temperatures varied greatly across the snail habitats, mean field temperatures (which typically drive seasonal acclimatization) were largely similar across habitats (sunned and shaded) and shorelines (cooler and warmer; Table 1). Clearly, mean temperatures contribute insignificantly to heat tolerance plasticity selection in tropical Echinolittorina snails. Likewise, minimum field temperatures were largely invariable across the habitats and shores, which excludes these temperatures as potential determinants of the lower boundary for heat tolerance plasticity. The limit to this boundary, assessed during CA for both shores, possibly relates to the loss of heat-hardening, a mechanism which should be beneficial by eliminating costs associated with the upregulating and functioning of Hsps (Angilletta, 2009).

Plastic responses involving Hsps incur significant energetic and fitness costs, a topic extensively critiqued in the framework of the beneficial acclimation hypothesis (Leroi et al., 1994; Huey et al., 1999; Wilson and Franklin, 2002; Woods and Harrison, 2002; Deere and Chown, 2006). In their high-shore habitat, Echinolittorina snails face severe energy intake restrictions due to limited food availability and limited time in which to feed. Consequently, they have evolved multifaceted behavioral and physiological mechanisms to conserve energy resources (Marshall et al., 2011, 2013; Marshall and Chua, 2012). Among the most impressive energy-conserving mechanism is deep temperature-independent metabolic rate depression (10% of resting metabolism; Marshall et al., 2011; Verberk et al., 2016). In view of their energetic constraints, advantage should be gained by minimizing the use of costly physiological processes, including HSRs.

Non-reversible plasticity should be energetically beneficial by ensuring that the range for reversible heat tolerance plasticity matches the habitat temperatures of a particular shore. Such matching should prevent snails on warmer shores from experiencing excessive HSR induction and hsp overexpression (Feder and Hofmann, 1999; Sørensen et al., 2003). Non-reversible heat tolerance plasticity should enable reversible acclimatization to occur in similar ways and at similar costs on thermally-different shores under the same regional change in ambient air temperature. Furthermore, this plasticity should yield species-level benefits by enabling colonization of a broader range of shores (warmer and cooler shores) along a coastline. Importantly, these findings accounting for non-reversibility supersede an earlier suggestion that E. malaccana is unlikely to benefit from reversible plasticity (Marshall et al., 2018).

Our understanding of thermal acclimation of rocky-intertidal animals in the context of contemporary theory (Angilletta, 2009) is encompassed by the influential work of Somero and Stillman (see Stillman, 2002; Somero, 2005, 2010). This work suggesting a reduced acclimation capacity in higher-shore species compared to their lower-shore congeners, arises from generalization of data for porcelain crabs (Stillman, 2002; Somero, 2005, 2010). These crabs, however, behaviourally thermoregulate by sheltering in the shade under rocks during air emersion, limiting their exposure to the full spectrum of the shoreline’s thermal heterogeneity. The substantial heat tolerance plasticity observed in high-shore Echinolittorina snails contradicts this theory, and we suggest that the difference between the crab and snail responses relates to the snails using a broader spectrum of the shoreline thermal heterogeneity. They experience relatively great habitat temperature variation through the behavior of settling when air-exposed in thermally-divergent microhabitats, including those under direct solar heating (Marshall et al., 2010, 2013). This habitat temperature variation persists despite the phenomenal variety of morphological and behavioral thermoregulatory attributes of littorinid snails (Miller and Denny, 2011; Marshall and Chua, 2012; Marshall et al., 2013; Ng et al., 2017), primarily because the most effective thermoregulatory behavior, shade-seeking, can be undermined by desiccation risk avoidance behavior (Monaco et al., 2017).

An arising question is whether capacity for non-reversible plasticity (the shoreline effect) is restricted to high-shore species or whether it is independent of vertical distribution on the shore. Because lower-shore habitats are strongly stabilized by the seawater temperature, and because the seawater temperature is largely invariable across nearby shorelines, the shoreline effect should be lessened in the lower-shore. In addition to suggesting that seawater temperature stability is likely to restrict thermal acclimation selection in lower-shore tropical species, we propose as a testable hypothesis that non-reversible plasticity should also be more constrained in lower-shore compared to higher-shore species.

Acclimation capacity (the degree of change in a trait following cool or warm laboratory acclimation) is becoming a key measure of the vulnerability of ectothermic animals to future warming (Gunderson and Stillman, 2015; Rohr et al., 2018). An earlier study revealed that laboratory experiments alone (without field-referencing) may underestimate this vulnerability in animals living in near-completely acclimatized states, which are unable to improve heat hardening with further warming (Marshall et al., 2018). The present study adds to this caveat by showing that inaccuracies in determining heat-tolerance acclimation capacity can potentially arise from not accounting for shoreline-specific temperature differences. Whereas the capacity for reversible acclimation was similar for each shoreline (∼2°C), this became greater when the data for the shores were combined (∼2.9°C; Table 3 and Figure 4). This highlights the importance, when assessing acclimation capacities, of prior knowledge of the habitat thermal heterogeneity of experimental animals.

Emerging from this study is a second methodological issue relating to diel cycling of the laboratory acclimation temperature. This is not only important considering that such cycling occurs naturally, but also in terms of initiating an acclimatory HSR. If the primary heat-hardening response requires that exposure temperature surpasses an HSR induction threshold, then acclimation temperatures that are stable or fluctuate below this threshold will conceivably not yield an acclimatory response, leading to erroneous conclusions. This further highlights the need to determine the HSR threshold temperature prior to developing acclimation protocols in marine intertidal animal studies.