Behavioural adaptation to heat stress: shell lifting of the hermit crab Diogenes deflectomanus.

Behavioural responses to heat and desiccation stress in ectotherms are crucial for their survival in habitats where environmental temperatures are close to or even exceed their upper thermal limits. During low tide periods when pools in intertidal sediments heat up, a novel shell lifting behaviour (when hermit crabs crawl out of pools and lift up their shells) was observed in the hermit crab, Diogenes deflectomanus, on tropical sandy shores. On-shore measurements revealed that the hermit crabs left pools and lifted their shells predominantly when pool water exceeded 35.4 °C. Standing on emersed substrates above the pool water, the hermit crabs maintained their body temperatures at 26 - 29 °C, ∼ 10 °C lower than temperatures at which their physiological performances (as measured using heart rate) reached the maximum. This mismatch between preferred body temperatures and temperatures at maximal physiological performance was also observed under a laboratory controlled thermal gradient, where hermit crabs spent more time at 22 - 26 °C as compared to > 30 °C. These behaviours suggest a thermoregulatory function of the shell lifting behaviour, where the hermit crabs can avoid further increase in body temperatures when pools heat up during low tide periods. Such a behavioural decision allows the hermit crabs to be less prone to the strong temporal fluctuation in temperatures experienced during emersion periods on thermally dynamic tropical sandy shores.

Status Quo and Future Perspectives of Molecular and Genomic Studies on the Genus Biomphalaria-The Intermediate Snail Host of Schistosoma mansoni.

Schistosomiasis, or also generally known as bilharzia or snail fever, is a parasitic disease that is caused by trematode flatworms of the genus Schistosoma. It is considered by the World Health Organisation as the second most prevalent parasitic disease after malaria and affects more than 230 million people in over 70 countries. People are infected via a variety of activities ranging from agricultural, domestic, occupational to recreational activities, where the freshwater snails Biomphalaria release Schistosoma cercariae larvae that penetrate the skin of humans when exposed in water. Understanding the biology of the intermediate host snail Biomphalaria is thus important to reveal the potential spread of schistosomiasis. In this article, we present an overview of the latest molecular studies focused on the snail Biomphalaria, including its ecology, evolution, and immune response; and propose using genomics as a foundation to further understand and control this disease vector and thus the transmission of schistosomiasis.

Behaviour broadens thermal safety margins on artificial coastal defences in the tropics.

Tropical species are predicted to be among the most vulnerable to climate change as they often live close to their upper limits to thermal tolerance and in many cases, behavioural thermoregulation is required to persist in the thermal extremes of tropical latitudes. In concert with warming temperatures, near-shore species are faced with the additional threat of shoreline hardening, leading to a reduction in microhabitats that can provide thermal refuges. This situation is exemplified in Singapore, which lies almost on the equator and so experiences year-round hot temperatures, and much of its coastline is now seawall. To investigate the thermal ecology of a common intertidal gastropod, Nerita undata, on these artificial structures, we measured thermal conditions on two seawalls, the temperatures of habitats occupied by the snail, and compared these with the snail's thermal tolerance by measuring heart rate and behavioural thermoregulation (as preferred temperature, Tpref). At one of the two seawalls (Tanjong Rimau), temperatures experienced by N. undata exceeded all measures of thermal tolerance in the sun, while at the other (Palawan Beach), they did not. Temperatures in habitats occupied by the snails on the seawalls were similar to their measured Tpref in the laboratory and were lower than all measures of thermal tolerance. Behavioural thermoregulation by the snails, therefore, significantly increased the thermal safety margins of N. undata on the relatively homogenous seawalls in Singapore, and at one of the two seawalls were necessary to allow snails to survive. Accordingly, to facilitate motile species to maintain broad thermal safety margins through behavioural regulation, the provision of additional refuges from thermal stress is recommended on artificial coastal defences such as seawalls.

Why are 'suboptimal' temperatures preferred in a tropical intertidal ectotherm?

In thermally extreme environments, it is challenging for organisms to maximize performance due to risks associated with stochastic variation in temperature and, subsequently, over evolutionary time minimizing the exposure to risk can serve as one of the mechanisms that result in organisms preferring suboptimal temperatures. We tested this hypothesis in a slow-moving intertidal snail on tropical rocky shores, where temperature variability increases with time from 30 min to 20 hr when recorded at 30 min intervals (due to short-term environmental autocorrelation where temperatures closer in time are more similar as compared to temperatures over a long period of time). Failure to accommodate temporal variation in thermal stress by selecting cool habitats can result in mortality. Thermal performance curves for different traits (heart rate and locomotion) were measured and compared to the snail's thermal preferences in both the field and laboratory. Predicted performances of the snails were simulated based on thermal performance curves for different traits over multiple time-scales and simulated carryover effects. A strong mismatch was found between physiological and behavioural thermal maxima of the snails (physiological thermal maximum being higher by ~7°C), but the snails avoided these maxima and sought temperatures 7-14°C cooler. Such a risk-averse strategy can be explained by their predicted performances where the snails should make decisions about preferred temperatures based on time periods ≥5 hr to avoid underestimating the temporal variation in body temperature. In extreme and stochastic environments, where the temporal variation in environmental conditions can lead to substantial divergence between instantaneous and time-averaged thermal performances, 'cooler is better' and 'suboptimal' body temperatures are preferred as they provide sufficient buffer to reduce mortality risk from heat stress.

Temperature-buffering by oyster habitat provides temporal stability for rocky shore communities.

Intertidal rocky shores are considered among the most thermally stressful marine ecosystems, where many species live close to their upper thermal limit and depend on access to cool microclimates to persist through heat events. In such environments, the provision of cool microclimates by habitat-forming species enables persistence of associated species during high temperature events. We assessed whether, by maintaining cool microclimates through heat events, habitat formed by rock oysters (Saccostrea cucullata) provides temporal stability to associated invertebrate communities over periods of extreme temperatures. On three tropical rocky shores of Hong Kong, which experiences a monsoonal climate, we compared changes in microclimates and invertebrate communities associated with oyster and bare rock habitats between the cool and hot seasons. Oyster habitats were, across both seasons, consistently characterised by lower maximum temperatures and greater thermal stability than bare rock habitats. Invertebrate communities in the bare rock habitat were less diverse and abundant in the hot than the cool season, but communities in the cooler habitats provided by oysters did not display temporal change. These results demonstrate that microclimates formed by oysters provide temporal stability to associated communities across periods of temperature change and are key determinants of species distributions in thermally stressful environments. The conservation and restoration of oyster habitats may, therefore, build resilience in associated ecological communities subject to ongoing environmental change.

Behavioral repertoire of high-shore littorinid snails reveals novel adaptations to an extreme environment.

Species that inhabit high-shore environments on rocky shores survive prolonged periods of emersion and thermal stress. Using two Hong Kong high-shore littorinids (Echinolittorina malaccana and E. radiata) as models, we examined their behavioral repertoire to survive these variable and extreme conditions. Environmental temperatures ranged from 4°C in the cool season to 55.5°C in the hot season, with strong seasonal and daily fluctuations. In the hot season, both species allocated >35% of their activity budgets to stress-mitigating thermoregulatory behaviors (e.g. standing, towering) and relatively small proportions to foraging (<20%) and reproduction (<10%). In the assumedly benign cool season, greater proportions (>70%) of activity budgets were allocated to stress mitigation behaviors (crevice occupation, aggregation formation). Both species exhibited multifunctional behaviors that optimized time use during their tidally-constrained activity window in the hot season. Females mated while foraging when awash by the rising tide, and some males crawled on top of females prior to ceasing movement to form 'towers', which have both thermoregulatory benefits and reduce searching time for mates during subsequent activity. The function of such behaviors varies in a state-dependent manner, for example, the function of trail following changes over an activity cycle from mate searching on rising tides, to stress mitigation on falling tides (aiding aggregation formation), and to both functions through tower formation just before movement stops. Many of these behavioral responses are, therefore, multifunctional and can vary according to local conditions, allowing snails in this family to successfully colonize the extreme high-shore environment.

Integrating mechanistic models and climate change projections to predict invasion of the mussel, Mytilopsis sallei, along the southern China coast.

Species invasion is an important cause of global biodiversity decline and is often mediated by shifts in environmental conditions such as climate change. To investigate this relationship, a mechanistic Dynamic Energy Budget model (DEB) approach was used to predict how climate change may affect spread of the invasive mussel Mytilopsis sallei, by predicting variation in the total reproductive output of the mussel under different scenarios. To achieve this, the DEB model was forced with present-day satellite data of sea surface temperature (SST) and chlorophyll-a concentration (Chl-a), and SST under two warming RCP scenarios and decreasing current Chl-a levels, to predict future responses. Under both warming scenarios, the DEB model predicted the reproductive output of M. sallei would enhance range extension of the mussel, especially in regions south of the Yangtze River when future declines in Chl-a were reduced by less than 10%, whereas egg production was inhibited when Chl-a decreased by 20-30%. The decrease in SST in the Yangtze River may, however, be a natural barrier to the northward expansion of M. sallei, with colder temperatures resulting in a strong decrease in egg production. Although the invasion path of M. sallei may be inhibited northwards by the Yangtze River, larger geographic regions south of the Yangtze River run the risk of invasion, with subsequent negative impacts on aquaculture through competition for food with farmed bivalves and damaging aquaculture facilities. Using a DEB model approach to characterise the life history traits of M. sallei, therefore, revealed the importance of food availability and temperature on the reproductive output of this mussel and allowed evaluation of the invasion risk for specific regions. DEB is, therefore, a powerful predictive tool for risk management of already established invasive populations and to identify regions with a high potential invasion risk.

Timing Metabolic Depression: Predicting Thermal Stress in Extreme Intertidal Environments.

AbstractAnticipatory changes in organismal responses, triggered by reliable environmental cues for future conditions, are key to species' persistence in temporally variable environments. Such responses were tested by measuring the physiological performance of a tropical high-shore oyster in tandem with the temporal predictability of environmental temperature. Heart rate of the oyster increased with environmental temperatures until body temperature reached ∼37°C, when a substantial depression occurred (∼60%) before recovery between ∼42° and 47°C, after which cardiac function collapsed. The sequential increase, depression, and recovery in cardiac performance aligned with temporal patterns in rock surface temperatures, where the risk of reaching temperatures close to the oysters' lethal limit accelerates if the rock heats up beyond ∼37°C, coinciding closely with the body temperature at which the oysters initiate metabolic depression. The increase in body temperature over a critical threshold serves as an early-warning cue to initiate anticipatory shifts in physiology and energy conservation before severe thermal stress occurs on the shore. Cross-correlating the onset of physiological mechanisms and temporal structures in environmental temperatures, therefore, reveals the potential role of reliable real-time environmental cues for future conditions in driving the evolution of anticipatory responses.

High thermal stress responses of Echinolittorina snails at their range edge predict population vulnerability to future warming.

Populations at the edge of their species' distribution ranges are typically living at the physiological extreme of the environmental conditions they can tolerate. As a species' response to global change is likely to be largely determined by its physiological performance, subsequent changes in environmental conditions can profoundly influence populations at range edges, resulting in range extensions or retractions. To understand the differential physiological performance among populations at their distribution range edge and center, we measured levels of mRNA for heat shock protein 70 (hsp70) as an indicator of temperature sensitivity in two high-shore littorinid snails, Echinolittorina malaccana and E. radiata, between 1°N to 36°N along the NW Pacific coast. These Echinolittorina snails are extremely heat-tolerant and frequently experience environmental temperatures in excess of 55 °C when emersed. It was assumed that animals exhibiting high temperature sensitivity will synthesize higher levels of mRNA, which will thus lead to higher energetic costs for thermal defense. Populations showed significant geographic variation in temperature sensitivity along their range. Snails at the northern range edge of E. malaccana and southern range edge of E. radiata exhibited higher levels of hsp70 expression than individuals collected from populations at the center of their respective ranges. The high levels of hsp70 mRNA in populations at the edge of a species' distribution range may serve as an adaptive response to locally stressful thermal environments, suggesting populations at the edge of their distribution range are potentially more sensitive to future global warming.

Multifunctional behaviour in a sandy shore crab enhances performance in extreme intertidal environments.

Soft sediment shores in the tropics are highly dynamic environments, where behavioural patterns of organisms are constrained by tidal conditions, and environmental temperatures during an organisms' activity periods can exceed their thermal tolerance levels. In such extreme habitats, behavioural responses to environmental changes are key to survival, driving differential performance. We investigated sponging behaviour (water uptake from sediments) of the deposit-feeding crab, Scopimera intermedia, on tropical sandy shores to determine its thermoregulatory function. The thermal physiology of the crabs and their habitat conditions were quantified by measuring thermal performance curves and recording environmental temperatures during the crabs' activity periods. Environmental temperatures were combined with experimental data to investigate the role of sponging on the thermal performances of the crabs by simulating field body temperatures. Sponging rate was strongly and positively correlated with feeding rate, as sponging replenishes water for flotation feeding. Sponging, however, also reduced body temperatures on average by 1.3 °C. Simulated populations of crabs which were unable to sponge had more variable body temperatures, which exceeded the critical thermal maximum of the crabs (~ 39 °C) nearly 2000 times more often than crabs able to sponge. Sponging is, therefore, a multifunctional behavioural trait important for both feeding and thermoregulation. The evolution of such multifunctional traits is likely to be a widespread, but overlooked phenomenon in intertidal species, as maintaining a functional body temperature is energetically costly in habitats where environmental conditions fluctuate strongly such as on tropical shores.

Combined effects of thermal conditions and food availability on thermal tolerance of the marine bivalve, Perna viridis.

Organisms can mitigate the effects of long term variation in environmental conditions through acclimation, which involves changes in various physiological responses. To elucidate the possible effects of temperature and food concentrations on acclimation capacity, physiological responses of the mussel, Perna viridis, were measured after individuals were held for six weeks under varying temperatures and food availability. Warm-acclimated mussels experiencing higher food levels had significantly greater upper thermal limits than those maintained on lower food levels. In contrast, the upper thermal limits of cold-acclimated mussels were not affected by food levels. For warm-acclimated mussels, differences in upper thermal limits were likely due to rapid depletion of energy storage as predicted by Dynamic Energy Budget model simulations for P. viridis exposed to lower food levels. Clearance rates of cold-acclimated mussels were significantly lower than warm-acclimated mussels, regardless of food availability. The impacts of lower food acquisition on energy storage, however, could be compensated by lower metabolic rates of the cold-acclimated mussels. The availability and the ability to acquire food are not, therefore, the main drivers differentiating between the upper thermal tolerances of cold- and warm-acclimated mussels, but these differences are driven by the past thermal history the mussels experienced. The temperature tolerance range of P. viridis showed a positive shift to tolerate higher temperatures after acclimation. Such flexibility in thermal tolerance implies P. viridis has high capacity to acclimate to novel environments, which will enhance its future success given its commercial importance as an aquaculture species.

Correction: Comparative genomics and the nature of placozoan species.

[This corrects the article DOI: 10.1371/journal.pbio.2005359.].

Differential sensitivity of larvae to ocean acidification in two interacting mollusc species.

Anthropogenically-induced ocean acidification (OA) scenarios of decreased pH and altered carbonate chemistry are threatening the fitness of coastal species and hence near-shore ecosystems' biodiversity. Differential tolerances to OA between species at different trophic levels, for example, may alter species interactions and impact community stability. Here we evaluate the effect of OA on the larval stages of the rock oyster, Saccostrea cucullata, a dominant Indo-Pacific ecosystem engineer, and its key predator, the whelk, Reishia clavigera. pH as low as 7.4 had no significant effect on mortality, abnormality or growth of oyster larvae, whereas whelk larvae exposed to pH 7.4 experienced increased mortality (up to ∼30%), abnormalities (up to 60%) and ∼3 times higher metabolic rates compared to controls. Although these impacts' long-term consequences are yet to be investigated, greater vulnerability of whelk larvae to OA could impact predation rates on intertidal rocky shores, and have implications for subsequent community dynamics.

Comparative genomics and the nature of placozoan species.

Placozoans are a phylum of nonbilaterian marine animals currently represented by a single described species, Trichoplax adhaerens, Schulze 1883. Placozoans arguably show the simplest animal morphology, which is identical among isolates collected worldwide, despite an apparently sizeable genetic diversity within the phylum. Here, we use a comparative genomics approach for a deeper appreciation of the structure and causes of the deeply diverging lineages in the Placozoa. We generated a high-quality draft genome of the genetic lineage H13 isolated from Hong Kong and compared it to the distantly related T. adhaerens. We uncovered substantial structural differences between the two genomes that point to a deep genomic separation and provide support that adaptation by gene duplication is likely a crucial mechanism in placozoan speciation. We further provide genetic evidence for reproductively isolated species and suggest a genus-level difference of H13 to T. adhaerens, justifying the designation of H13 as a new species, Hoilungia hongkongensis nov. gen., nov. spec., now the second described placozoan species and the first in a new genus. Our multilevel comparative genomics approach is, therefore, likely to prove valuable for species distinctions in other cryptic microscopic animal groups that lack diagnostic morphological characters, such as some nematodes, copepods, rotifers, or mites.

Experience matters: context-dependent decisions explain spatial foraging patterns in the deposit-feeding crab Scopimera intermedia.

Behavioural decisions are often context-dependent, where information from immediate experience is incorporated into an individual's decision-making, particularly in complex environments. To test whether such mechanism is adopted by foragers in heterogeneous environments, we investigated the foraging behaviour of the deposit-feeding sand-bubbler crab, Scopimera intermedia An individual-based model was constructed, based on an optimal-patch selection criterion, which implicitly assumed that individuals adjust foraging decisions based on immediate past experience. The model's predictions were tested on the shore by manipulating the location of food patches, where the crab showed a strong context-dependent foraging pattern. When resources were randomly distributed, the crab responded by spending 56% of time in enriched patches compared with only 28% in the same area when patches were composed of natural sediments. Shore manipulations varying resource distribution supported the underlying principles of the model mechanism, and highlighted the benefits of such a strategy in heterogeneous environments such as intertidal sediments where food resources vary at different spatial and temporal scales. The proposed model therefore provides a mechanistic process, based on optimal foraging, to predict foraging decisions and movement patterns of animals feeding in heterogeneous landscapes.

A novel method for estimating the strength of positive mating preference by similarity in the wild.

Mating preference can be a driver of sexual selection and assortative mating and is, therefore, a key element in evolutionary dynamics. Positive mating preference by similarity is the tendency for the choosy individual to select a mate which possesses a similar variant of a trait. Such preference can be modelled using Gaussian-like mathematical functions that describe the strength of preference, but such functions cannot be applied to empirical data collected from the field. As a result, traditionally, mating preference is indirectly estimated by the degree of assortative mating (using Pearson's correlation coefficient, r) in wild captured mating pairs. Unfortunately, r and similar coefficients are often biased due to the fact that different variants of a given trait are nonrandomly distributed in the wild, and pooling of mating pairs from such heterogeneous samples may lead to "false-positive" results, termed "the scale-of-choice effect" (SCE). Here we provide two new estimators of mating preference (Crough and Cscaled) derived from Gaussian-like functions which can be applied to empirical data. Computer simulations demonstrated that r coefficient showed robust estimations properties of mating preference but it was severely affected by SCE, Crough showed reasonable estimation properties and it was little affected by SCE, while Cscaled showed the best properties at infinite sample sizes and it was not affected by SCE but failed at biological sample sizes. We recommend using Crough combined with the r coefficient to infer mating preference in future empirical studies.

Untangling the roles of microclimate, behaviour and physiological polymorphism in governing vulnerability of intertidal snails to heat stress.

Biogeographic distributions are driven by cumulative effects of smaller scale processes. Thus, vulnerability of animals to thermal stress is the result of physiological sensitivities to body temperature (Tb), microclimatic conditions, and behavioural thermoregulation. To understand interactions among these variables, we analysed the thermal tolerances of three species of intertidal snails from different latitudes along the Chinese coast, and estimated potential Tb in different microhabitats at each site. We then empirically determined the temperatures at which heart rate decreased sharply with rising temperature (Arrhenius breakpoint temperature, ABT) and at which it fell to zero (flat line temperature, FLT) to calculate thermal safety margins (TSM). Regular exceedance of FLT in sun-exposed microhabitats, a lethal effect, was predicted for only one mid-latitude site. However, ABTs of some individuals were exceeded at sun-exposed microhabitats in most sites, suggesting physiological impairment for snails with poor behavioural thermoregulation and revealing inter-individual variations (physiological polymorphism) of thermal limits. An autocorrelation analysis of Tb showed that predictability of extreme temperatures was lowest at the hottest sites, indicating that the effectiveness of behavioural thermoregulation is potentially lowest at these sites. These results illustrate the critical roles of mechanistic studies at small spatial scales when predicting effects of climate change.

The importance of thermal history: costs and benefits of heat exposure in a tropical, rocky shore oyster.

Although thermal performance is widely recognised to be pivotal in determining species' distributions, assessment of this performance is often based on laboratory-acclimated individuals, neglecting their proximate thermal history. The thermal history of a species sums the evolutionary history and, importantly, the thermal events recently experienced by individuals, including short-term acclimation to environmental variations. Thermal history is perhaps of greatest importance for species inhabiting thermally challenging environments and therefore assumed to be living close to their thermal limits, such as in the tropics. To test the importance of thermal history, the responses of the tropical oyster Isognomon nucleus to short-term differences in thermal environments were investigated. Critical and lethal temperatures and oxygen consumption were improved in oysters that previously experienced elevated air temperatures, and were associated with an enhanced heat shock response, indicating that recent thermal history affects physiological performance as well as inducing short-term acclimation to acute conditions. These responses were, however, associated with trade-offs in feeding activity, with oysters that experienced elevated temperatures showing reduced energy gain. Recent thermal history, therefore, seems to rapidly invoke physiological mechanisms that enhance survival of short-term thermal challenge but also longer term climatic changes and consequently needs to be incorporated into assessments of species' thermal performances.

Genetic Basis of Differential Heat Resistance between Two Species of Congeneric Freshwater Snails: Insights from Quantitative Proteomics and Base Substitution Rate Analysis.

We compared the heat tolerance, proteomic responses to heat stress, and adaptive sequence divergence in the invasive snail Pomacea canaliculata and its noninvasive congener Pomacea diffusa. The LT50 of P. canaliculata was significantly higher than that of P. diffusa. More than 3350 proteins were identified from the hepatopancreas of the snails exposed to acute and chronic thermal stress using iTRAQ-coupled mass spectrometry. Acute exposure (3 h exposure at 37 °C with 25 °C as control) resulted in similar numbers (27 in P. canaliculata and 23 in P. diffusa) of differentially expressed proteins in the two species. Chronic exposure (3 weeks of exposure at 35 °C with 25 °C as control) caused differential expression of more proteins (58 in P. canaliculata and 118 in P. diffusa), with many of them related to restoration of damaged molecules, ubiquitinating dysfunctional molecules, and utilization of energy reserves in both species; but only in P. diffusa was there a shift from carbohydrate to lipid catabolism. Analysis of orthologous genes encoding the differentially expressed proteins revealed two genes having clear evidence of positive selection (Ka/Ks > 1) and seven candidates for more detailed analysis of positive selection (Ka/Ks between 0.5 and 1). These nine genes are related to energy metabolism, cellular oxidative homeostasis, signaling, and binding processes. Overall, the proteomic and base substitution rate analyses indicate genetic basis of differential resistance to heat stress between the two species, and such differences could affect their further range expansion in a warming climate.

Snails and their trails: the multiple functions of trail-following in gastropods.

Snails are highly unusual among multicellular animals in that they move on a layer of costly mucus, leaving behind a trail that can be followed and utilized for various purposes by themselves or by other animals. Here we review more than 40 years of experimental and theoretical research to try to understand the ecological and evolutionary rationales for trail-following in gastropods. Data from over 30 genera are currently available, representing a broad taxonomic range living in both aquatic and terrestrial environments. The emerging picture is that the production of mucus trails, which initially was an adaptation to facilitate locomotion and/or habitat extension, has evolved to facilitate a multitude of additional functions. Trail-following supports homing behaviours, and provides simple mechanisms for self-organisation in groups of snails, promoting aggregation and thus relieving desiccation and predation pressures. In gastropods that copulate, trail-following is an important component in mate-searching, either as an alternative, or in addition to the release of water- or air-borne pheromones. In some species, this includes a capacity of males not only to identify trails of conspecifics but also to discriminate between trails laid by females and males. Notably, trail discrimination seems important as a pre-zygotic barrier to mating in some snail species. As production of a mucus trail is the most costly component of snail locomotion, it is also tempting to speculate that evolution has given rise to various ways to compensate for energy losses. Some snails, for example, increase energy intake by eating particles attached to the mucus of trails that they follow, whereas others save energy through reducing the production of their own mucus by moving over previously laid mucus trails. Trail-following to locate a prey item or a mate is also a way to save energy. While the rationale for trail-following in many cases appears clear, the basic mechanisms of trail discrimination, including the mechanisms by which many snails determine the polarity of the trail, are yet to be experimentally determined. Given the multiple functions of trail-following we propose that future studies should adopt an integrated approach, taking into account the possibility of the simultaneous occurrence of many selectively advantageous roles of trail-following behaviour in gastropods. We also believe that future opportunities to link phenotypic and genotypic traits will make possible a new generation of research projects in which gastropod trail-following, its multitude of functions and evolutionary trade-offs can be further elucidated.