Come rain or shine: the combined effects of physical stresses on physiological and protein-level responses of an intertidal limpet in the monsoonal tropics


  • Gray A. Williams,

    Corresponding author
    1. The Swire Institute of Marine Science and Division of Ecology & Biodiversity, The School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, China
    Search for more papers by this author
  • Maurizio De Pirro,

    1. Academy Sea Environment, Lungomare dei navigatori 44, 58019 Monte Argentario, Italy
    Search for more papers by this author
  • Stephen Cartwright,

    1. The Swire Institute of Marine Science and Division of Ecology & Biodiversity, The School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, China
    Search for more papers by this author
  • Kiki Khangura,

    1. The Swire Institute of Marine Science and Division of Ecology & Biodiversity, The School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, China
    Search for more papers by this author
  • Wai-Chuen Ng,

    1. The Swire Institute of Marine Science and Division of Ecology & Biodiversity, The School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, China
    Search for more papers by this author
  • Priscilla T.Y. Leung,

    1. The Swire Institute of Marine Science and Division of Ecology & Biodiversity, The School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, China
    Search for more papers by this author
  • David Morritt

    1. School of Biological Sciences, Royal Holloway, University of London, Egham, TW20 0EX, UK
    Search for more papers by this author

Correspondence author.


1. Traditional approaches to understanding species responses to environmental conditions have focused on the isolated effects of single stressors, despite the fact that in nature organisms experience a variety of conditions.

2. In tropical monsoonal areas, intertidal animals can face hot desiccating conditions during emersion preceded, or followed by, intense rainfall. The combined effects of these stresses on physiological responses and protein profiles were investigated in a limpet, Cellana grata.

3. With short exposure (60 min) to single stressors, heat stressed limpets had elevated heart rates and more concentrated haemolymph and mantle water osmolalities than under normal temperatures or awash. Animals under rain had reduced haemolymph and mantle water osmolalities, but similar heart rates to unstressed animals.

4. After 120 min, unstressed animals did not differ in their physiological responses. Heat stressed limpets, however, had faster heart rates and more concentrated haemolymph and mantle water osmolalities, whilst those under rain had the lowest osmolalities, but similar heart rates to unstressed animals.

5. Limpets under rain followed by heat stress had faster heart rates, but lower haemolymph and mantle water osmolalities compared to animals under normal temperatures or heat stress. Limpets that were heat stressed, followed by rain, had similar heart rates to animals awash, under rain or normal temperatures but lower haemolymph osmolalities than other treatments, with the exception of limpets under rain.

6. There was a positive relationship between haemolymph and mantle water osmolalities, except for animals under rain, where mantle water osmolality was lower than the haemolymph, suggesting some isolation of body fluids from the external medium.

7. Haemolymph protein/peptide mass spectra of heat stressed animals (either before or after rain) were similar, while all other treatments differed, suggesting differential expression and regulation of proteins.

8. Heat stress invokes a more active physiological and protein level response than rain, but their combination had an interactive effect on limpets’ metabolism.

9. Identifying the effects of multiple stresses at a variety of biological levels highlights the interactive effects which impact species, and provides a more complete understanding of how species may respond to environmental changes in their natural habitats.


Variation in physical environmental conditions plays an important role in regulating the distribution and abundance of natural populations (Parmesan et al. 2005; Parmesan 2006) and this is especially true over steep environmental gradients, such as the rocky intertidal zone (Harley & Paine 2009). Such stressors can cause heavy mortality and limit the distribution of species when extreme events occur (e.g. Denny et al. 2009; Harley & Paine 2009). Given proposed models for climate change, how species can adapt and respond to such events is becoming an area of increasing interest in terrestrial and marine ecosystems (Walther et al. 2002; Harley et al. 2006; Helmuth et al. 2006; Parmesan 2006). Physical stresses, particularly high temperatures, are commonly invoked to explain the upper distribution of rocky intertidal species (see Little, Williams & Trowbridge 2009). In temperate areas evidence for this is derived from observations and measurements during or after extreme hot weather conditions when ‘high-shore kills’ are recorded (Wethey 1984; Hawkins & Hartnoll 1985; Kohn 1993; Harley 2008). On tropical shores, extreme environmental conditions are commonly associated with thermal stress and can cause mass mortalities of sessile species including algae (Williams 1993), barnacles (Chan et al. 2006) and mussels (Morton 1995). Mobile species also suffer such events and hundreds of individuals can be killed during calm, hot, low-tide days (Williams 1994; Ngan 2006; Firth & Williams 2009). Such observations provide snapshots of species’ responses to these stresses, but rarely allow investigators to employ a more integrated approach, investigating physiological and cellular responses to understand the importance of such stressors on individuals and the communities they inhabit (see Helmuth 2009). As such, this lack of integration limits our potential to predict species responses to climate change (see Pörtner et al. 2006; Denny & Helmuth 2009).

Environmental stresses on the shore often have interactive impacts. Thermal stress, for example, is known to be affected by a variety of climatic factors (e.g. tidal cycle, wave splash, Helmuth 2002; Harley & Helmuth 2003; Harley & Paine 2009). Many tropical areas are also affected by changes in the predominant winds, the monsoons. In some areas monsoons bring heavy rains, and many tropical areas experience ‘wet’ and ‘dry’ seasons dictated by changes in the monsoon patterns. When the wet season coincides with hot times of the year, then the physical stress on such shores is likely to be a combination of exposure to rainfall and also to heat stress, both of which can occur within a single emersion period (see Morritt et al. 2007). When this occurs, individuals can be washed from the shore by heavy rains (e.g. littorinids, Oghaki 1988) or may face osmotic challenges due to the flow and pooling of fresh (rain) water on the shore causing dilution of body fluids and swelling of soft tissues (Morritt et al. 2007). The same individuals may then face hot and drying conditions, when they dehydrate and their body fluids may become hyperosmotic. Associated with these osmotic changes will be a variety of physiological (e.g. variation in body temperature, heart rate, Wolcott 1973; Garrity 1984; Williams et al. 2005; Morritt et al. 2007) as well as biochemical changes (e.g. production of heat shock proteins, Feder & Hofmann 1999; Helmuth & Hofmann 2001; Dong et al. 2008). Opportunities to link such cellular changes (protein regulation) with physiological responses are developing with recent advances in protein profiling using MALDI-TOF mass spectrometry (MALDI-TOF MS, see W.-C. Ng, P. T. Y. Leung, D. Morritt, M. De Pirro, S. Cartwright G. A. Williams, unpublished data). Although the identification of specific proteins in molluscs is still limited, these methods allow fundamental cellular responses to be investigated by analysing the protein/peptide composition of the haemolymph (McAllen, Taylor & Freel 2005; Joseph & Philip 2007). Such approaches can generate an overall view of the haemolymph protein/peptide composition (Hortin 2006) and provide a more integrated picture of an animals’ physiological state under different environmental conditions (Pörtner et al. 2006; Denny & Helmuth 2009; Somero 2010).

Hong Kong shores are strongly affected by seasonal monsoon changes, resulting in a dry and cool season (November–March) and a hot and wet season (June–September, Kaehler & Williams 1996). During the hot and wet season average air temperatures are 28 °C (maximum 36 °C, rock temperatures can exceed 50 °C, Williams 1994) and spring low tides fall during the mid-afternoon and many species suffer from heat stress (e.g. limpets, Williams & Morritt 1995; Chelazzi, Williams & Gray 1999; chitons, Harper & Williams 2001; barnacles, Chan et al. 2006). The hot season is also the time of the greatest rainfall, receiving 75% of the annual rainfall between May and September (Kaehler & Williams 1996). During this period rainfall can be very intense, with approximately 5–6 days of the year experiencing rainfall in excess of 30 mm h−1 (Wu, Leung & Yeung 2006), with a record value of >145 mm h−1 measured in June 2008. In the Hong Kong summer, intertidal species can, therefore, experience intense thermal stress in combination with heavy periods of rainfall (see Firth & Williams 2009). These stressors have interacting effects on intertidal organisms, with brief periods of intense rainfall followed by dry, hot periods. The impact of such conditions will be especially severe on species, such as limpets, which are unable to completely isolate themselves from contact with rainwater. To investigate the possible sub-lethal impacts of such stressors, this paper tests the interactive effects of two environmental stressors, namely heat and rain stress, on physiological and protein level responses of a high shore limpet in Hong Kong. This approach, using two stressors in isolation and in combination, allows a more realistic assessment of how environmental stress may be experienced on the shore. Measuring traditional physiological responses, in combination with the novel approach of screening haemolymph proteins, also permits an insight into links between cellular and physiological responses to these stressors, and moves towards a more holistic understanding of how intertidal organisms may respond to environmental changes.

Materials and methods

Animal collections

Experiments were conducted on 3 days between 7 and 9th July 2008. On each day, more than 25 limpets, Cellana grata (Gould) (Fig. 1, 25–30 mm, representing a single cohort, G. A. Williams & S. Cartwright, unpublished data) were collected whilst awash from south facing, moderately exposed shores at Cape d’Aguilar Marine Reserve (Hong Kong 22°N, 114°E). Animals were placed on acetate sheets and quickly transferred back to the Swire Institute of Marine Science (SWIMS) ∼400 m away. In the aquarium at SWIMS, animals were placed on individually numbered and weighed circular acetate discs inside Petri dish lids (diameter 55 mm, lip depth 15 mm, drilled with small holes to allow drainage) and surrounded by plastic mesh fences. These containers were then placed under a seawater spray (∼740 mOsm kg−1) to allow animals to attach to the acetate discs and replenish their mantle water for 30 min (see Williams et al. 2005). Animals were maintained on these acetate discs and in Petri dishes for the duration of the experiments. As C. grata is a non-homing species, this method allows the animals to settle onto the acetate and accumulate mantle water. The acetate discs and Petri dishes also heat up at a similar rate to the rock tiles and permit the animals to be moved easily with minimal disturbance. After this period limpets were randomly assigned to one of ten experimental treatments.

Figure 1.

 Three different cohorts of the mid-high shore limpet Cellana grata on Hong Kong shores.

Laboratory experiments to simulate heat and rain stress

To investigate the individual and interactive effects of rain and heat stress on the physiology of Cellana grata, the responses of animals stressed by increased durations of rainfall or heat stress were compared with animals maintained under ambient, controlled conditions during emersion. Specifically comparisons were made between emersed animals maintained under average temperature heat conditions (H−); animals maintained under high temperature conditions (H+); animals maintained under rain in a rain simulator (R+) and animals awash in natural seawater (as a baseline group). To investigate possible interactive effects of being in rain and then heat stress, or heat stress and then rain, limpets were also switched between treatments and their responses compared to animals which were maintained under single stress conditions. Specifically ten treatments were used:

  • 1 Limpets awash in seawater for 30 min, initial, pre-emersion conditions (AW).
  • 2 Limpets under rainfall for 60 min (R+).
  • 3 Limpets emersed for 60 min under average temperatures, 30 °C (H−).
  • 4 Limpets emersed for 60 min under hot temperatures, 40 °C (H+).
  • 5 Limpets awash in seawater for 120 min (AW, AW).
  • 6 Limpets under rainfall for 120 min (R+R+).
  • 7 Limpets emersed for 120 min under average temperatures, 30 °C (H−H−).
  • 8 Limpets emersed for 120 min under hot temperatures, 40 °C (H+H+).
  • 9 Limpets emersed for 60 min under hot temperatures and then transferred to rain for 60 min (H+R+).
  • 10 Limpets under rainfall for 60 min and then transferred to being emersed under hot temperatures (R+H+).

There were seven replicates for each treatment, randomly assigned within the three different days (∑n = 10 treatments × 7 = 70). Comparison of treatments 1–4 allowed a test for differences in response to single stress conditions and unstressed animals (awash as a control group, treatment 1), whereas a comparison of treatments 5–10 allowed the effects of single stress vs. combined stress to be ascertained and compared with unstressed animals (treatment 5).

To simulate heat stress, limpets were emersed in a large Perspex tidal tank (130 × 80 × 41 cm, l × w × ht) in the aquarium, fitted with overhead heat lamps (6 × 200 W, Philips Halogen Plus Line Pro). A dark barrier separated the heat lamps so that one side of the tank received radiation from two lamps and the other from four lamps. When the lamps were turned on, experimental rock tiles (9 × 9 × 1·5 cm, l × w × d, granite tiles) heated up at a similar rate to natural rock on the shore following emersion (S.R. Cartwright unpublished data). Under four heat lamps the rock temperature reached a stable temperature at ∼40 °C, whilst under the two heat lamps the temperature stabilized at ∼30 °C.

Rainfall (<10 mOsm kg−1, 24–25 °C) was simulated in an Armfield rain simulator (see Hill, Nagarkar & Jayawardena 2002) controlled to mimic monsoonal rain conditions of ∼50 mm h−1 (see Morritt et al. 2007). Experimental rock tiles were positioned at a ∼14° angle (to allow drainage) in both the rainfall simulator and heat chamber in locations where rainfall and heat conditions were constant.

On each day after the awash phase, the mesh fences were removed and the animals carefully blotted dry and weighed (±0·00001 g) on their acetate discs. Limpets were then returned to dry Petri dishes and infrared sensors glued onto their shells directly above the heart to allow heart rate to be monitored (see Williams et al. 2005 and Morritt et al. 2007). The physiological responses of individuals for the awash phase treatment (treatment 1; AW) were then measured whilst another set of animals were maintained under awash conditions for a further 120 min (treatment 5; AW, AW). The remaining individuals were transferred in their Petri dishes onto rock tiles either in the rain simulator (R+ treatments) or the heat chamber (H− or H+ treatments).

Physiological responses

The physiological responses measured were heart rate and the osmolalities of the mantle water and haemolymph. Firstly, heart rates were measured using a portable Fluke Oscilloscope (Hz, beats s−1, mean of three readings recorded over 1 min) after which individuals were removed from the experimental areas. Limpets were then quickly dried, wet weighed on their acetate discs and then mantle water and haemolymph samples taken. Mantle water was collected by saturating a 10 mm diameter filter paper disc (Wescor Inc., Logan, UT, USA) inserted between the foot and mantle and osmolality immediately measured using a vapour pressure osmometer (Wescor 5520, Wescor Inc.). Haemolymph samples were taken by direct puncture of the pallial vein or heart using a finely drawn glass capillary after removing the remaining mantle water (see Williams & Morritt 1995 for details). Haemolymph samples were kept in closed Eppendorf tubes over ice until measurement as described above. Osmometer calibration was checked regularly against NaCl standards (Wescor Optimole).

After 60 min, the randomly assigned individuals were measured for treatments 2–4 (H−, H+, R+) following the procedures described above, except that after removing the haemolymph animals were placed in an oven at 60 °C and dried to constant weight. The remaining experimental animals (treatments 6–10) were either left in their original treatments (H−H−, H+H+ or R+R+), or either removed from the heat chamber and transferred to the rainfall simulator (H+R+), or removed from the rain simulator and transferred to the heat chamber (R+H+). To simulate disturbance from the transfer procedure, those animals which remained in their original treatments were also picked up and moved, but then replaced back in their original treatment. Individuals were maintained for a further 60 min (making a total of 120 min experimental time) and then their physiological responses measured as described above, including the animals maintained awash for 120 min (treatment 5, AW, AW).

One way analyses of variance (anova) were used to investigate variation between treatments after 60 min (treatments 1–4) and treatments after 120 min (treatments 5–10). Despite attempts at transformation, in some cases, data failed tests for homogeneity of variances (see Table 1). Analyses were still however conducted as, given the relatively large error degrees of freedom, anova is considered to be robust to such heterogeneity (Underwood 1997). Where significant differences were found, Student-Newman-Keuls tests were used to separate means.

Table 1.   One way analysis of variance to investigate differences between physiological variables of Cellana grata after 60 min under four experimental treatments (fixed factors: awash, emersed, heated and under rain, n = 7, d.f. = 3, 24) and after 120 min under six experimental treatments (fixed factors: emersed, heated, under rain, heated and then under rain, rain and then heated and awash, n = 7, d.f. = 5, 36, except for mantle water where n = 5–7). For experiments after 60 min, variances were homogeneous for Heart Rate and Water Loss (Cochran’s test) but not for other variables; whereas for experiments after 120 min variances were homogeneous except for mantle water osmolality (Levene’s test) and Water Loss (Cochran’s test, see text for details)
TreatmentAfter 60 minAfter 120 min
Heart rate107·34<0·001134·5<0·001
Mantle water osmolality35·20<0·00124·3<0·001
Haemolymph osmolality43·85<0·001139·8<0·001
Water loss7·43<0·0017·21<0·001

Haemolymph mass spectroscopy profiling

To identify differential protein Mass Spectroscopy profiles of the haemolymph of the limpets, the same series of experiments were re-run with 30 newly collected limpets, five of which were maintained using identical experimental protocols (treatments 5–10) as for the measurements on physiological responses. After the treatments, haemolymph samples were taken from each individual as described above (n = 5). In some treatments not enough haemolymph could be collected, resulting in variable sample sizes. Haemolymph samples were snap-frozen in liquid nitrogen and stored at −80 °C for later Mass Spectroscopy (MS) profiling.

Total protein concentrations of the haemolymph samples were determined using the 2-D Quant Kit (GE Health Care, Waukesha, WI, USA) and bovine serum albumin as a reference standard. Equal amounts of protein (2 μg) from each individual of different treatment groups were used for MS profiling. Haemolymph proteins were diluted to the same concentration (1 μg μL−1) and acidity was adjusted to pH 3 using 0·1% (v/v) formic acid. Protein peptides were then concentrated and purified using pipette ZipTip® C18 (Millipore, Billerica, MA, USA). Peptides were eluted with 5 μL of 50% (v/v) acetonitrile and 0·1% (v/v) trifluoroacetic acid (TFA). The eluted sample was subsequently mixed with a matrix solution (1 μL) containing 0·01 g mL−1 CHCA (a-cyano-4-hydroxycinamic acid) and subjected to MS profiling using an ABI 4800 MALDI-TOF-TOF MS Analyzer (Applied Biosystems, Foster City, CA, USA) with a Cal Mix 4700 standard (Applied Biosystems) for external calibration. To perform MS profiling, peptide mass spectra were acquired in positive reflector mode with 750 laser shots per spectrum with a mass range of 1000–10 000 Da, under a constant laser intensity of 3000 units. To maximize the mass spectra reproducibility, three independent MS profiles were performed for each individual’s haemolymph sample using the same protocol as described above.

Mass spectra were visualized, processed, and peak definition was performed using DataExplorer® version 4.9 (Applied Biosystems). The exported peak masses for each spectrum were further combined (maximal peak shift within 200 ppm) and aligned using bioinformatic programs written in PERL. The area of each aligned peak was defined as a single variable. The peak variable data were analyzed using multivariate statistical analyses to classify the treatment groups and to identify the peaks that were responsible for treatment-specific discrimination. Only peaks which were consistently expressed in all of the three haemolymph MS profiles from individuals were screened for further statistical analysis. A data matrix composed of intensities for the screened peaks from m/z 2000 to 10 000 was generated across the experimental individuals. To visualize multivariate patterns, non-metric multidimensional scaling (nMDS) was performed on the consistently detected protein/peptides in terms of peak masses obtained from MALDI-TOF MS analysis. Tests for significant differences in MS profiles between treatments were performed by one-way Analyses of Similarity (ANOSIM).


Physiological responses

The heart rates of animals under hot temperatures (H+, 40 °C) after 60 mins were almost twice as fast as animals kept awash (AW), under rain (R+) or under average temperatures (H−, 30 °C, Table 1, Fig. 2). Mantle water and haemolymph osmolality showed a similar pattern, with animals which were maintained under hot temperatures having more concentrated mantle water and haemolymph osmolalities than all other treatments. The osmolalities of these fluids in animals under rain were lower than in animals which were maintained awash or under average heat stress, which were similar (Table 1, Figs 3 and 4). In general, limpets in most treatments lost a similar amount of weight (∼5–10% of their body water) during the course of the experiment, with the exception of the animals maintained under rain (R+) for 60 mins which gained weight (Table 1, Fig. 5).

Figure 2.

 Mean heart rate (Hz, beats s−1, + SD, n = 7) of limpets maintained for 60 min under rain (R+), awash with seawater spray (Aw), under average temperatures (H−) or heat stress (H+) and limpets maintained for 120 min under rain (R+R+), average temperatures (H−H−), heat stress (H+H+) or transferred after 60 min under rain to under heat stress (R+H+) or 60 min heat stressed to under rain (H+R+). Treatments with different letters are significantly different (P < 0·05, Student-Newman-Keuls Test).

Figure 3.

 Mean mantle water osmolality (mOsm kg−1, + SD, n = 7) of limpets maintained for 60 min under rain (R+), awash with seawater spray (Aw), under average temperatures (H−) or heat stress (H+) and limpets maintained for 120 min under rain (R+R+), average temperatures (H−H−), heat stress (H+H+) or transferred after 60 min under rain to under heat stress (R+H+) or 60 min heat stressed to under rain (H+R+). Treatments with different letters are significantly different (P < 0·05, Student-Newman-Keuls Test).

Figure 4.

 Mean haemolymph osmolality (mOsm kg−1, + SD, n = 7) of limpets maintained for 60 min under rain (R+), awash with seawater spray (Aw), under average temperatures (H−) or heat stress (H+) and limpets maintained for 120 min under rain (R+R+), average temperatures (H−H−), heat stress (H+H+) or transferred after 60 min under rain to under heat stress (R+H+) or 60 min heat stressed to under rain (H+R+). Treatments with different letters are significantly different (P < 0·05, Student-Newman-Keuls Test).

Figure 5.

 Mean percentage water loss (%, + SD, n = 7) of limpets maintained for 60 min under rain (R+), awash with seawater spray (Aw), under average temperatures (H−) or heat stress (H+) and limpets maintained for 120 min under rain (R+R+), average temperatures (H−H−), heat stress (H+H+) or transferred after 60 min under rain to under heat stress (R+H+) or 60 min heat stressed to under rain (H+R+). Treatments with different letters are significantly different (P < 0·05, Student-Newman-Keuls Test).

After 120 min, heart rates were similar between animals continuously maintained under rain, awash, average temperatures and heat and then rain treatments (Table 1, Fig. 2). Heart rates were, however, significantly elevated in animals under hot conditions, and even more so in animals initially maintained under rain and then transferred to hot conditions (R+H+, Table 1, Fig. 2). Animals which had either been maintained under rain (R+R+) or heated and then placed under rain (H+R+) had similar, low mantle water and haemolymph osmolality (Table 1, Figs 3 and 4). The mantle water of animals maintained under rain for 60 min and then transferred to hot conditions (R+H+) was similar to animals maintained under average heat conditions (H−H−) or awash, whereas it was higher in limpets maintained for 120 min under hot conditions (H+H+, Table 1, Fig. 3). Haemolymph osmolality changes showed a similar trend, being lowest in limpets under rain and then increasingly significantly from animals transferred from hot conditions to rain (H+R+), rain to hot conditions (R+H+), to animals under average heat conditions (H−H−) and awash which were similar, and finally to animals under hot temperatures (H+H+) which had the most concentrated haemolymph (Table 1, Fig. 4).

There was a general positive relationship (Pearson’s correlation, r= 0·75, P < 0·01, Fig. 6) between mantle water osmolality and haemolymph, with highest concentrations occurring in animals which were heat stressed, and lower concentrations in animals which had been under rain. This relationship was particularly strong where osmolalities recorded were >700 mOsm kg−1. Below this value, however, haemolymph values were higher than would be predicted if there was a strict positive relationship between mantle water and haemolymph osmolality (points above the isosmotic line, Fig. 6) for animals maintained under rain (R+R+) for 120 min, or under heat for 60 min and then transferred under rain (H+R+).

Figure 6.

 The relationship between haemolymph and mantle water osmolality in limpets under different experimental treatments (see legend). The solid line represents the iso-osmotic line.

After 120 min, animals which experienced rain (either rain for 120 min or rain then heat and vice versa) lost little water, with the continuous rain treatment and the heat followed by rain (H+R+) treatments losing only ∼2% of their body water (Table 1, Fig. 5). Animals under rain and then subject to heat stress (R+H+) lost more water (∼5%). There was, however, no significant difference between this treatment and those which were awash or experienced 120 min of average heat conditions (H−H−) which lost almost twice as much water (∼10%). The animals maintained under hot conditions (H+H+), lost most water (∼17%), but because of the large between individual variation in the H−H− treatment it was not possible to separate these treatments statistically (Fig. 5).

Haemolymph mass spectroscopy profiling

A total of 155 peaks were recorded among the mass spectra of the samples, of which 66 peaks were detected consistently in the three MS profiling replicates and hence subjected to multivariate analysis. The mass spectra peaks showed an overall significant difference among the six treatment groups (Table 2). In general, limpets maintained awash (AW), under rain (R+R+) or average temperature (H−H−) were separated from each other (pairwise R = 0·477–0·576, Table 2, Fig. 7), while those which experienced hot temperatures (either H+H+, H+R+ or R+H+) were not well separated (pairwise R = 0·069–0·250). The major differences were between the three heat treated groups and the other treatments (pairwise R = 0·667–1), suggesting a more substantial change in the haemolymph MS profiles after high temperature exposure (Table 2, Fig. 7).

Table 2.   Matrix showing R-statistic values (One-way ANOSIM) for pairwise comparisons between MS profiles in Cellana grata under experimental treatments (H−H− = emersed, H+H+ = heated, R+R+ = under rain, H+R+ = heated and then under rain, R+H+ = rain and then heated and AW = awash, below the diagonal) and within-group similarity percentages (SIMPER, on the diagonal). Global R = 0·618, P < 0·001
Figure 7.

 Multivariate analysis. nMDS plot to illustrate the separation protein expression profiles detected by mass spectrometry of the haemolymph of Cellana grata under experimental treatments (H−H− = emersed, H+H+ = heated, R+R+ = under rain, H+R+ = heated and then under rain, R+H+ = rain and then heated and AW = awash, n = 2–5). The protein expression profiles represent the relative occurrence of the 66 consistently detected protein/peptides in terms of peak masses obtained from MALDI-TOF MS analysis.


This is the first attempt to link cellular and physiological level responses of tropical intertidal organisms to multiple environmental stressors. As such, the interactive effects of being stressed by heat, rainfall or combinations of the two has shown that limpets respond differentially at the cellular and physiological levels to these scenarios, and that this will have implications for the strategies species’ employ to survive in the physically challenging environment of tropical intertidal shores.

Physiological response to either heat or rain stress

Differences in physiological responses between treatments after 60 min were not great, as the short duration of the treatments did not severely stress the limpets (see Williams et al. 2005). After 60 min, however, limpets showed predictable responses to their respective experimental conditions. Heat stressed limpets showed elevated heart rates and mantle water and haemolymph concentrations compared with other treatments. A positive relationship between limpet heart rates and increasing temperatures is well documented (Marshall & McQuaid 1992; Chelazzi, Williams & Gray 1999) and desiccation is clearly associated with heat stress, resulting in evaporation of water and increasing osmotic concentrations of both mantle and body fluids (Williams & Morritt 1995; Williams et al. 2005).

Limpets under simulated rain showed similar heart rates to those awash or emersed under average temperatures, although their mantle water and haemolymph concentrations were lower. Limpets under rain also gained water slightly, whereas there was a similar small loss of body water in all other treatments. Under prolonged rain limpets can exhibit reduced heart rates, including periods of bradycardia or acardia, and also gain weight due to osmotic uptake of water, although weight gain is variable between individuals, resulting in lower osmotic concentrations of mantle water and haemolymph (Morritt et al. 2007). As expected for animals with limited osmoregulatory ability, and which are unable to completely isolate themselves from their environmental conditions, there was a positive relationship between mantle water and haemolymph osmolality in all treatments (Segal & Dehnel 1962).

Physiological response to heat and rain stress

Responses after 120 min showed more pronounced differences between treatments. Heat stressed limpets showed predictably fast heart rates and the highest recorded mantle water and haemolymph concentrations, in keeping with responses exhibited by heat stressed limpets of similar size in previous studies (Williams & Morritt 1995; Chelazzi, Williams & Gray 1999; Williams et al. 2005). These limpets were not, however, severely stressed as there were no indications of brady- or acardia and no animals died during the experiment. Limpets in this treatment also lost the most water, but the amount was similar to animals maintained under average temperatures or under rain and then heat stressed (R+H+). During the course of the experiments extraction of mantle water became increasingly difficult in heat stressed animals (H+H+ and R+H+) as compared to other treatments, resulting in variable sample sizes. With increasing mantle water loss, limpets lose the ability to exchange gases as their gills collapse, and as a result they are unable to excrete waste products (especially CO2) and their haemolymph becomes increasingly concentrated (especially hypermagnesic, Chaisemartin 1970 and acidic, Marshall & McQuaid 1992; Williams & McMahon 1998).

Limpets under rain had the slowest heart rates (Morritt et al. 2007), most similar to those originally under heat stress and then rain (H+R+), but also not significantly different from those maintained under average temperatures or awash with seawater, and similar to treatments after 60 min. A reduction in heart rate with emersion duration has been interpreted as a sign of metabolic depression (McMahon 1988) and has also been recorded in mussels and chitons during rapid changes in water salinity (Stickle & Sabourin 1979). Limpets under rain did not become immersed by the rainwater which was allowed to drain over them, and so were not severely osmotically stressed. As a result, they did not exhibit brady- or acardia which has been recorded in limpets under low salinity immersion (Marshall & McQuaid 1993; Chelazzi, De Pirro & Williams 2001), prolonged rain (Morritt et al. 2007) as well as those experiencing extreme thermal stress (Chelazzi, Williams & Gray 1999; Williams et al. 2005). Metabolic rate (as measured by O2 consumption) in limpets is known to be positively linked with heart rate (Marshall & McQuaid 1992) and so reduced heart rates, bradycardia and acardia under environmental stress would suggest either some form of metabolic depression (Chelazzi, Williams & Gray 1999; Chelazzi, De Pirro & Williams 2001) or the initiation of a coma state.

In some cases limpets can exhibit elevated heart rates, above rates expected due to increasing environmental temperatures. Increasing heart rates suggest an increase in metabolism in response to physiological needs, as suggested for limpets lifting their shells to reduce thermal stress (‘mushrooming’, Williams et al. 2005). Limpets maintained under rain and then heat stressed exhibited the highest heart rates of all treatments, higher than would be predicted as a result of elevated temperatures when heat stressed (as in the H+H+ treatment). It would appear, therefore, that being transferred from rain, and then drying and being heated, invokes an extra energy requirement and hence an increase in metabolism, which may be related to recovering from hypo-osmotic conditions, or an active response to thermal stress. Limpets which experience the reverse transfer, from a hot, dry environment to being under rain (H+R+), showed the opposite response and had a lower heart rate when compared to animals initially maintained under hot conditions (H+), with heart rates being similar to non-stressed animals awash or emersed under average temperatures.

Limpets transferred from the rain (where mantle fluid and haemolymph osmotic concentrations were low) to heat stress conditions (R+H+) showed a rapid recovery of their mantle water and haemolymph osmolality to values similar to limpets maintained under average temperatures. A similar rapid change in osmolality, but in the reverse direction, was seen in limpets transferred from heat to rain (H+R+). This suggests rapid osmotic adjustment by animals when transferred to different conditions as recorded by Morritt et al. (2007), when hypo-osmotically stressed animals were sprayed by seawater. Such a rapid adjustment of osmotic concentrations is likely to be highly adaptive to surviving prolonged emersion, as wave splash or inundation by the rising tide will allow animals to recover rapidly.

There is some speculation as to whether limpets can exhibit a degree of regulation of their haemolymph relative to their mantle water, either maintaining their haemolymph concentration below the concentration of the mantle water under hyper-osmotic stress (high temperatures, Williams et al. 2005) or conversely higher than the mantle water under hypo-osmotic stress (rain, Morritt et al. 2007). Limpets maintained under rain, or transferred from the heat treatment to rain (H+R+) were able to maintain their haemolymph hyper-osmotic to their mantle water concentrations. Whether this involves active regulation or simply reflects a time lag establishing equilibrium between the two fluid compartments is unclear. In contrast to the elevated heart rates of animals transferred from rain to heat (R+H+), this osmotic response was not associated with an increase in heart rates as animals transferred from heat to rain (H+R+) had similar heart rates to animals maintained in rain, awash or under average temperatures.

Protein level responses to heat and rain stress

With increasing temperatures, limpets have elevated heart rates and often induce heat shock proteins as a response to protein degradation (Dong et al. 2008). There are also other protein level changes in their metabolism, which invoke energetic costs to up- or down-regulate certain proteins involved in thermal resistance or osmotic balance (Sokolova & Pörtner 2001; Kultz et al. 2007). The response to rainfall is different, where under heavy rainfall limpets have depressed heart rates (Morritt et al. 2007; and this study). Under rainfall, therefore, limpets appear to limit their metabolic activities. Desiccation is not an issue for these individuals but there must be physiological effects associated with osmotic stress, such as a reduction in respiration and possible anaerobiosis (as for littorinids which can isolate themselves from their environment, Sokolova, Bock & Pörtner 2000). Consequently, under this ‘low risk’ scenario, there does not seem to be an increase in energy expenditure in an attempt to synthesize new metabolites to balance osmotic potentials.

The haemolymph MS profiles revealed changes in overall protein/peptide composition in the haemolymph of the animals under different treatments. The most profound difference was between the treatments which experienced hot temperatures (H+H+, H+R+ or R+H+) and other treatments. The lack of strong differentiation among the heat treatments (H+H+, H+R+ or R+H+) suggests that the response to heat is likely to dominate other stressors. The clear separation in haemolymph MS profiles among H−H−, R+R+ and AW treatments also reflected the different physiological states of animals under these treatments, which may be associated with more subtle adjustments in haemolymph composition, although this response was not linked to a change in heart rate. These observations suggest that different cascades of metabolic pathways are likely to operate with respect to limpets’ responses to environmental conditions. The physiological state of intertidal animals is known to be well correlated to different phases of the tidal cycle (see Gracey et al. 2008). In this study, changes in haemolymph MS profiles of Cellana grata reflected a variety of responses to combat different environmental stressors. As the genome of more species are sequenced, further elucidation of the specific proteins involved in these changes will be possible and help identify the most important metabolic pathways involved, and possibly reveal limiting factors to species tolerance (see Denny & Helmuth 2009; Somero 2010).

Differential responses to environmental stresses

Responses to heat and rain may, therefore, represent two strategies. Rainfall does not apparently represent a significant threat to intertidal limpets. Whilst there are records of dislodgement of smaller gastropods (e.g. littorinids, Oghaki 1988), there is very little documentation of heavy mortality of limpets due to rain (although large animals on vertical surfaces can detach under heavy rainfall, Morritt et al. 2007; and if rock pools become inundated for long time periods mortality may occur, Firth & Williams 2009). Limpets therefore adopt a ‘passive’ response to rainfall and tolerate the conditions, adopting a sit-and-wait strategy. Under rain, limpets exhibit normal or depressed heart rates and their osmotic concentrations reflect environmental conditions, there is however no risk of desiccation and mortality is low.

Hot sunny days, however, represent a high risk of mortality and at some sites >80% of individuals can be killed during summer (Ngan 2006). Heat and desiccation stress are, therefore, very real threats to intertidal limpets (Wolcott 1973; Kohn 1993; Harley 2008; Miller, Harley & Denny 2009). As a result, animals ‘actively’ respond to these stresses, invoking behavioural (microhabitat choice, Williams & Morritt 1995; mushrooming, Williams et al. 2005) and physiological mechanisms to combat these environmental stresses and to ensure survival. Wolcott (1973) has argued that concentration of body fluids was the main cause of mortality in North East Pacific limpets, highlighting the importance of water loss and subsequent hyper-osmotic stress in these animals and the need to address this problem through active management of metabolites.


The combined effects of different stresses are, therefore, likely to play an important role in individual success. In the present study the strongest response in terms of an increase in heart rate (and therefore metabolism) was seen in rain and then heat stressed limpets (R+H+). These animals are responding to the sequence of the combined effects of rain and then heat stress, and this response has a high cost in terms of energy expenditure, greater than in limpets experiencing heat stress alone. This response illustrates the rapid response of these animals to cope with the extremely dynamic environment of the rocky intertidal zone, swiftly switching from a low metabolic state when not at risk (e.g. under rain) when animals can tolerate hypo-osmotic changes without the risk of desiccation, to a high metabolic state when rain stops and animals are in the hot, drying sun. Such a change represents a shift into a highly risky scenario, when limpets have to respond rapidly to address possible metabolic debts incurred whilst under rain (as suggested by Chelazzi, De Pirro & Williams 2001), but also to defend against thermal and hyper-osmotic stresses which pose significant threats to their survival.

The effects of increasing temperatures on the survival of many intertidal animals may, therefore, not be dependent solely on periods of perceived elevated stress as their microclimate warms, but is more likely to be influenced by interactive effects of other environmental conditions, and more importantly the sequence in which they occur (Denny & Helmuth 2009; Harley & Paine 2009). Energetic costs of coping with more variable environmental conditions may have chronic long-term effects on individuals’ survival (Somero 2010), adding to the potential acute effects commonly seen during mass mortalities of intertidal organisms on hot, calm spring tide days. Identifying these chronic effects are, however, more difficult as their influences are potentially insidious. Using a combination of ecological and molecular approaches could prove the best means of detecting such effects; and such an integrated approach may play an important role in the successful prediction of species’ responses to environmental changes.


We are grateful to the Agriculture, Fisheries and Conservation Department, Hong Kong Government for permission to work in the Cape d’Aguilar Marine Reserve. Excellent technical assistance was provided by Ms Cecily Law and laboratory assistance from Ms June Leung and Ms Vera Shan. We thank colleagues at the Swire Institute of Marine Science for their active involvement in discussions on experimental design and analysis especially Drs Kenny Leung and V. ThiyagaRajan. Prof Ron Hill advised on the use of the rain machine and Mr Juilian Yeung (Department of Psychiatry, HKU) wrote the peak alignment program using PERL programming language. Priscilla Leung was partially supported by the Strategic Research Theme of Sustainable Environment (Sustainable Water) and the Faculty of Science of the University of Hong Kong. This project was partly supported by Small Project Funding from The University of Hong Kong (200707176090 and 200807176202).