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.