Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata

Catarina Cunha Moreira MSc in Biodiversity, Genetics and Evolution Biology Department 2015 Tutor Fernando Pádua Silva e Lima, Researcher Assistant, CIBIO Co-tutor Raquel Susana Brazão Xavier, Post-Doc Researcher, CIBIO Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata Todas as correções determinadas pelo júri, e só essas, foram efetuadas. O Presidente do Júri, Porto, ______/______/_________ FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 4 Acknowledgments Após mais uma etapa terminada tenho a agradecer todos os que estiveram de alguma maneira envolvidos nestes trabalhosos, divertidos e inesquecíveis dois anos. Em primeiro lugar, tenho a agradecer aos meus orientadores, Fernando e Raquel, pela paciência, orientação, magia e todas as oportunidades. À minha orientadora em França, Coraline, agradeço tanto a hospitalidade assim como toda a disponibilidade e a oportunidade de fazermos um projeto conjunto. Ao meu orientador da Califórnia, Jonathon, pelos acampamentos malucos, pela introdução dos deliciosos s’mores e por todo o apoio laboratorial e bioinformático. Seguidamente, tenho que agradecer a todos os meus colegas e amigos que conheci neste percurso, desde os portugueses aos franceses e americanos. Porque o carinho e as brincadeiras em momentos menos bons tornam tudo mais fácil. Destacando, a Filipa e o Rui pela paciência e por todos os ensinamentos. À minha família, em especial aos meus pais, irmão e avós pela presença e apoio incondicional porque sem isso tudo o resto seria impossível. Ao meu namorado porque mais ninguém seria maluco o suficiente para dormir no carro comigo. E porque momentos bons ou menos bons só fazem sentido quando partilhados com a pessoa certa. Por fim, todo o meu estudo foi suportado por fundos FEDER (FCOMP-01-0124- FEDER-010564, FCOMP-01-0124-FEDER-020817, FCOMP-01-0124-FEDER- 029939), pela FCT – Fundação para a Ciência e Tecnologia (PTDC/MAR/099391/2008, PTDC/MAR/117568/2010, EXPL/MAR-BIO/1034/2012), pelo "Laboratoire d'Excellence" LabexMER (ANR-10-LABX-19), por uma bolsa do governo Francês do programa "Investissements d'Avenir", por uma bolsa do Conselho Regional da Bretanha (SAD programme), e por fim por duas bolsas de mobilidade, Labexmer e ERASMUS +. FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 5 Summary Global warming is being intensively studied and temperature is considered one of the most important abiotic stress factors. The rocky intertidal, one of the most thermally complex environments on Earth, is inhabited by marine organisms which are periodically exposed to stressful terrestrial conditions during emersion. Therefore the intertidal is ideal to study the thermal response of organisms. Although this ecosystem harbours numerous microhabitats, the thermal variability between sun-exposed and shaded microhabitats has already been described. Our study uses robolimpets and thermal images to confirm that sun-exposed microhabitats are consistently hotter than shaded microhabitats. The mechanism that translates thermal stress into differential physiological and behavioral performance and fitness is poorly understood. Even though several physiologic studies have been carried out on intertidal organisms, most are not based on actual thermal data collected on the field. Our study aimed at studying the cardiac response and behavior of individuals from shaded and sunexposed microhabitats, using a realistic heat stress treatment (peaking at 40 °C). The difference on cardiac response between microhabitats was non-significant, although there was a higher proportion of individuals from shaded microhabitats having a cardiac failure than individuals from sun-exposed microhabitats. The behavior response showed a decrease on the proportion of individuals moving with the increase of temperature but an increase of the mushrooming behavior after being exposed to the maximum temperature. This highlights the importance of realistic studies to avoid under- or over-estimating the organism’s responses to thermal stress. Key words: Patella vulgata, thermal stress, cardiac activity, behavior, realistic studies. FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 6 Resumo O estudo do aquecimento global tem sido bastante aprofundado e a temperatura é considerada um dos stresses abióticos mais importantes. O intertidal rochoso, um dos ecossistemas mais complexos do planeta a nível térmico, é habitado por organismos que estão expostos periodicamente a condições terrestres stressantes durante a emersão. Daí o intertidal ser ideal para o estudo da resposta térmica dos organismos. Apesar deste ecossistema ter bastantes microhabitats, a variabilidade térmica entre os microhabitats expostos ao sol e os microhabitats na sombra já foi descrita. Este estudo usa robolapas e imagens térmicas para confirmar que os microhabitats expostos ao sol são mais quentes que os da sombra. O mecanismo que traduz o stress térmico a uma performance fisiológica e comportamental é pouco conhecido. Apesar de muitos estudos fisiológicos terem sido elaborados com organismos intertidais, a maioria não é baseada em dados térmicos recolhidos no campo. Este estudo tem como objetivo estudar a resposta cardíaca e comportamental de indivíduos de microhabitats expostos ao sol e microhabitats na sombra, usando um stress térmico realista (com um máximo de 40 °C). A diferença da resposta cardíaca entre microhabitats não foi significativa, no entanto uma maior proporção de indivíduos provenientes de microhabitats na sombra tiveram um falha cardíaca comparativamente aos provenientes de microhabitats expostos ao sol. A nível comportamental, se por um lado houve uma diminuição na proporção de indivíduos em movimento com o aumento da temperatura, por outro lado houve um aumento do comportamento de “mushrooming” após estarem expostos à temperatura máxima. Este estudo destaca a importância de estudos realistas de maneira a evitar a sub- ou sobreestimação da resposta térmica dos organismos. Palavras-chaves: Patella vulgata, stress térmico, atividade cardíaca, comportamento, estudos realistas. FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 7 Table of Contents 1. Introduction ................................................................................................................... 10 2. Materials and Methods ................................................................................................. 12 2.1. Field measurements of P. vulgata body temperatures ...................................... 12 2.2. Collection of individuals ........................................................................................ 13 2.3. Experimental Set-up ............................................................................................. 13 2.4. Heart rate measurements .................................................................................... 13 2.5. Behavior analyses ................................................................................................. 15 2.6. Statistical analyses ............................................................................................... 15 3. Results ........................................................................................................................... 15 3.1. Robolimpets........................................................................................................... 15 3.2. Thermal images .................................................................................................... 18 3.3. Heart Beat measurements ................................................................................... 18 3.4. Behavior ................................................................................................................. 19 4. Discussion ..................................................................................................................... 20 5. Bibliographic References ............................................................................................. 23 FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 8 List of Figures Fig.1. Data from robolimpets in shaded (blue) and sun-exposed (red) microhabitats. (a) Daily cumulative heat of shaded (238.05 ± 7.71 ºC, mean ± SE) and sun-exposed (292.88 ± 12.69 ºC, mean ± SE); (b) Maximum temperature of shaded (15.09 ± 0.23 ºC, mean ± SE) and sun-exposed (22.65 ± 0.83 ºC, mean ± SE); (c) Heating rate of shaded (0.65 ± 0.04 ºC/h, mean ± SE) and sun-exposed (2.63 ± 0.22 ºC/h, mean ± SE); (d) Cooling rate of shaded (0.80 ± 0.07 ºC/h, mean ± SE) and sun-exposed (2.73 ± 0.28 ºC/h, mean ± SE). Fig.2. Body temperature obtaining from thermal images of individuals from shaded (blue) (27.46 ± 0.37 ºC, mean ± SE, n = 283) and sun-exposed (red) microhabitats (15.05 ± 0.27 ºC, mean ± SE, n= 317). Fig.3. Data obtained from the cardiac activity of individuals from shaded (blue) and sun-exposed (red) microhabitats during the simulated thermal stress. (a) Arrhenius break temperatures (ABT) of heart rates of individuals from shaded (27.60 ± 1.24 ºC, mean ± SE) and sun-exposed microhabitats (29.38 ± 0.94 ºC, mean ± SE) were not significantly different (ANOVA, P = 0.399, df = 11, n = 6 for each species); (b) Proportion of individuals reaching ABT from shaded (75.00 %, n=16) and sun-exposed microhabitats (53.8 %, n=26) were significantly different (Chi-square test, P = 0.002). Fig.4. Behavior of individuals from shaded (blue) and sun-exposed (red) microhabitats during three phases of the simulated thermal stress: Before (B), on the temperature peak (P) and after (A). (a) Proportion of individuals from shaded (B: 1.96 ± 0.86; P: 0.00 ± 0.00; A: 0.00 ± 0.00, mean ± SE) and sun-exposed microhabitats (B: 2.26 ± 0.67; P: 0.00 ± 0.00; A: 0.00 ± 0.00, mean ± SE) performing rotation; (b) Proportion of individuals from shaded (B: 22.56 ± 6.15,; P: 0.00 ± 0.00; A: 0.00 ± 0.00, mean ± SE) and sun-exposed microhabitats (B: 18.19 ± 8.68; P: 0.00 ± 0.00; A: 0.00 ± 0.00, mean ± SE) performing displacement; (c) Proportion of individuals from shaded (6.80 ± 4.40; P: 10.00 ± 5.96; A: 9.64 ± 3.49, mean ± SE) and sun-exposed microhabitats (B: 3.57 ± 3.19; P: 6.67 ± 5.96; A: 14.44 ± 5.63, mean ± SE) performing mushrooming. FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 9 List of Abbreviations ABT : Arrhenius brake temperature HSP: Heat shock protein IR: Infra-red FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 16 The thermal profile of both microhabitats was characterized by plotting the daily cumulative heat (Fig. 1.a), the maximum temperature (Fig. 1.b), heating rates (Fig. 1.c) and cooling rates (Fig. 1. d). All these analyses showed that sun-exposed microhabitats are hotter and have higher heating and cooling rates than shaded microhabitats. The difference in maximum temperatures between microhabitats was 8 ºC and the difference in daily cumulative heat between microhabitats was 55 ºC. The heating and cooling rates were substantially higher in sun-exposed microhabitats when compared with their shaded counterparts. Heating rates were four times higher and cooling rates were three times higher (Fig 1c and d). FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 17 Fig.1. Data from robolimpets in shaded (blue) and sun-exposed (red) microhabitats. (a) Daily cumulative heat of shaded (238.05 ± 7.71 ºC, mean ± SE) and sun-exposed (292.88 ± 12.69 ºC, mean ± SE); (b) Maximum temperature of shaded (15.09 ± 0.23 ºC, mean ± SE) and sun-exposed (22.65 ± 0.83 ºC, mean ± SE); (c) Heating rate of shaded (0.65 ± 0.04 ºC/h, mean ± SE) and sun-exposed (2.63 ± 0.22 ºC/h, mean ± SE); (d) Cooling rate of shaded (0.80 ± 0.07 ºC/h, mean ± SE) and sun-exposed (2.73 ± 0.28 ºC/h, mean ± SE). FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 18 3.2. Thermal images Thermal images of a total of 283 and 317 limpets from shaded and sun-exposed microhabitats, respectively, were taken in three different days in order to assess the body temperature of P. vulgata. Thermal imaging confirmed that sun-exposed microhabitats are hotter than shaded microhabitats. Temperature from individuals inhabiting sun-exposed microhabitats was, in average, 1.82 times higher than the body temperature of individuals living in shaded microhabitats (Fig.2). Fig.2. Body temperature obtaining from thermal images of individuals from shaded (blue) (27.46 ± 0.37 ºC, mean ± SE, n= 283) and sun-exposed (red) microhabitats (15.05 ± 0.27 ºC, mean ± SE, n= 317). 3.3. Heart Beat measurements The cardiac activity of a total of 26 individuals from sun exposed microhabitats and 16 individuals from shaded microhabitats was assessed. A total of six individuals from each microhabitat reached the ABT. The value of ABT in sun-exposed and shaded microhabitats was 29.4 and 27.6 ºC, respectively (Fig.3a), but they were not statistically different (p = 0.399, df = 11, ANOVA). The proportion of occurrence of ABT in shaded microhabitats was 20% higher than in sun-exposed microhabitats (p = 0.002, chi-square test) (Fig.3b). 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Body temperature (°C) Shaded microhabitats Sun-exposed microhabitats FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 19 Fig.3. Data obtained from the cardiac activity of individuals from shaded (blue) and sun-exposed (red) microhabitats during the simulated thermal stress. (a) Arrhenius break temperatures (ABT) of heart rates of individuals from shaded (27.60 ± 1.24 ºC, mean ± SE) and sun-exposed microhabitats (29.38 ± 0.94 ºC, mean ± SE) were not significantly different (ANOVA, P = 0.399, df = 11, n = 6 for each species); (b) Proportion of individuals reaching ABT from shaded (75.00 %, n=16) and sun-exposed microhabitats (53.8 %, n=26) were significantly different (Chi-square test, P = 0.002). 3.4. Behavior After removing all individuals that were not present, a total of 26 individuals from shaded microhabitats and 19 from sun-exposed microhabitats were considered for the behavior analyses. While before the temperature peak the behavior was mostly displacement and a higher proportion of individuals from shaded microhabitats were active, on the peak and after the peak, all the individuals exclusively performed mushrooming. Individuals from sunexposed started to be more active after the peak than individuals from the shaded microhabitat (Fig.4 a-c). FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 20 Fig.4. Behavior of individuals from shaded (blue) and sun-exposed (red) microhabitats during three phases of the simulated thermal stress: Before (B), on the temperature peak (P) and after (A). (a) Proportion of individuals from shaded (B: 1.96 ± 0.86; P: 0.00 ± 0.00; A: 0.00 ± 0.00, mean ± SE) and sun-exposed microhabitats (B: 2.26 ± 0.67; P: 0.00 ± 0.00; A: 0.00 ± 0.00, mean ± SE) performing rotation; (b) Proportion of individuals from shaded (B: 22.56 ± 6.15,; P: 0.00 ± 0.00; A: 0.00 ± 0.00, mean ± SE) and sun-exposed microhabitats (B: 18.19 ± 8.68; P: 0.00 ± 0.00; A: 0.00 ± 0.00, mean ± SE) performing displacement; (c) Proportion of individuals from shaded (6.80 ± 4.40; P: 10.00 ± 5.96; A: 9.64 ± 3.49, mean ± SE) and sun-exposed microhabitats (B: 3.57 ± 3.19; P: 6.67 ± 5.96; A: 14.44 ± 5.63, mean ± SE) performing mushrooming. 4. Discussion This study aimed at determining the ability of individuals from different microhabitats to cope with thermal stress. Using robolimpets and thermal imaging, both microhabitats were characterized showing that sun-exposed microhabitats are consistently hotter than shaded microhabitats. Although there were differences in the physiological (cardiac activity) response to acute stress by Patella vulgata originating from different habitats, there were no differences in the behavior. FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 21 Microhabitat variability in thermal stress Data from present work confirm that Patella vulgata experience different thermal regimes according to their position in the shore. Observed temperatures in shaded and sun exposed microhabitats were different, especially regarding heating and cooling rates. Previous studies already showed differences in temperature from high to low shore as well as at the microhabitat level, between crevices and flat rocks. Furthermore, thermal stress experienced by organisms was shown to be higher in sunexposed than in shaded microhabitats (Stirling 1982, Tomanek and Somero 1999, Stillman and Somero 1996, Davenport and Davenport 2005, Stenseng et al. 2005, Nguyen et al. 2011, Prusina et al. 2014, Chapperon et al. submitted). The present data unequivocally demonstrate that the maximum temperature (a proxy for chronic stress), the daily cumulative heat (a proxy for the degree of acute stress) and the heating and cooling rates of sun exposed and shaded microhabitats, are consistently higher in sun exposed habitats. The observed differences are likely to be responsible for the higher expression of heat shock proteins by P. vulgata from sun-exposed microhabitats described for Atlantic coast of Europe (see Lima et al., 2015). Physiological response to thermal stress Experimental temperature profiles based on realistic heating and cooling rates allowed the determination of the proportion of individuals from different microhabitats that are able to cope with thermal stress. However, a significantly higher proportion of individuals from shaded microhabitats reached Arrhenius breaking point temperature (ABT), the value of ABT were not significantly different from individuals from sun-exposed microhabitats. Still, these results seem to indicate that sun-exposed individuals are more acclimated and therefore are likely to better cope with acute heat stress than individuals from shaded microhabitats. Since intertidal organisms are daily exposed to stressful conditions and consequently are close to their thermal limit, an increase of temperature can lead to high mortalities rates as reported before (see Harley, 2008). Furthermore, these results indicate a potential for acclimation what can also suggest that individuals from sunexposed microhabitats, which are already exposed to higher temperatures than individuals from shaded microhabitats, have the capacity to cope with an acute stress, as also have been studied ( Chapperon et al. submitted). Behavioral response to thermal stress FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 22 Short-term acclimation and variability in heat stress are likely to be translated in different behavioral patterns. We hypothesize that a behavior such as mushrooming (shell raising), which has an important role in evaporative cooling (Denny and Harley, 2006) and in reducing thermal stress (Williams et al., 2005), should be more frequent in individuals less acclimated to high temperatures. This behavior is considered a short-term and a high-risk strategy since when raising their shell organisms lose water and therefore may become unable to maintain adhesion to the rock surface (Williams et al., 2005). Individuals that need to perform this behavior are more likely to not survive in extreme events. Our results show that at the beginning of the experiment a higher proportion of individuals were moving around when the rock of the aquarium was still wet. At this point the temperature was not restricting their behavior. With the increase of temperature and consequently the increase of heart rate all individuals changed their behavior. A low proportion of moving individuals on the temperature peak and after the peak is possibly due to some of them reaching the ABT. When the temperature reached its maximum, all individuals changed their behavior to mushrooming. Since a 40ºC day is not common at the location where individuals were collected, it is not surprising that a high proportion of individuals would perform this behavior. There was a higher proportion of individuals from sun-exposed microhabitats performing mushrooming, although its significance cannot be assessed. Further prospects With ongoing climate change there is a major concern to understand the thermal stress mechanisms. The importance of performing realistic studies depends on having confidence to really understand these mechanisms. Therefore, studies that are not realistic can be misleading and may provide erroneous results. Even though our study used realistic heating and cooling rates, we acknowledge that the way we computed them (i.e., pooling temperature data from most of the distribution range of the species) may not be the most appropriated for the population that was used in the experimental trials. This study would undoubtedly benefit from temporal, and, especially, from spatial replication. It would be interesting to compare the responses of populations from the middle of the distribution range with those from the range edges, especially since recent studies have found that this species displays regional variations in thermal stress response (measured via Hsp expression, see Lima et al., 2015). Other FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 23 molecular tools, such as transcriptomic analysis could also help us to understand the mechanisms driving the thermal response of this species. 5. Bibliographic References Bennie J, Huntley B, Wiltshire A, Hill M O, Baxter R (2008). Slope, aspect and climate: spatially explicit and implicit models of topographic microclimate in chalk grassland. Ecological Modelling 216(1): 47-59. Burnett NP, Seabra R, de Pirro M, Wethey DS,Woodin SA, Helmuth B, Zippay ML, Sarà G, Monaco C, Lima FP (2013). An improved noninvasive method for measuring heartbeat of intertidal animals. Limnology oceanography 11: 91–100. Burrows MT, Schoeman DS, Buckley LB, Moore P, Poloczanska ES, Brander KM et al. (2011). The pace of shifting climate in marine and terrestrial ecosystems. Science 334(6056): 652-655. Chapperon C, Volkenborn N, Clavier J, Séité S, Seabra R, Lima FP (submitted). Thermal sensitivity and tolerance limits of an intertidal ectotherm under heat stress: not all vulnerable to climate warming. Chapperon C, Seuront L (2011a). Space–time variability in environmental thermal properties and snail thermoregulatory behaviour. Functional Ecology 25(5): 1040- 1050. Chapperon C, Seuront L (2011b). Behavioral thermoregulation in a tropical gastropod: links to climate change scenarios. Global Change Biology 17(4): 1740-1749. Coumou D, Rahmstorf S (2012). A decade of weather extremes. Nature Climate Change 2(7): 491-496. Davenport J, Davenport JL (2005). Effects of shore height, wave exposure and geographical distance on thermal niche width of intertidal fauna. Marine Ecology Progress Series 292: 41-50. Denny MW, Harley CD (2006). Hot limpets: predicting body temperature in a conductance-mediated thermal system. Journal of Experimental Biology 209(13): 2409-2419. Dong Y, Dong S, Meng X (2008). Effects of thermal and osmotic stress on growth, osmoregulation and Hsp70 in sea cucumber (Apostichopus japonicus Selenka). Aquaculture 276(1): 179-186. FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 24 Gardner JL, Peters A, Kearney MR, Joseph L, Heinsohn R (2011). Declining body size: a third universal response to warming?. Trends in ecology and evolution 26(6): 285- 291. Gedan KB, Bernhardt J, Bertness MD, Leslie HM (2011). Substrate size mediates thermal stress in the rocky intertidal. Ecology 92(3): 576-582. Harley, CD (2008). Tidal dynamics, topographic orientation, and temperature-mediated mass mortalities on rocky shores. Marine Ecology Progress Series 371: 37-46. Harley CD, Denny MW, Mach KJ, Miller LP (2009). Thermal stress and morphological adaptations in limpets. Functional ecology 23(2): 292-301. Helmuth B, Broitman BR, Blanchette CA, Gilman S, Halpin P, Harley CDG, O’Donnell J, Hofmann GE, Menge B, Strickland D (2006). Mosaic patterns of thermal stress in the rocky intertidal zone: implications for climate change. Ecology Monographs 76: 461-479. Huey RB, Kingsolver, JG (1989). Evolution of thermal sensitivity of ectotherm performance. Trends in Ecology and Evolution 4(5): 131-135. Lathlean JA, Minchinton TE (2012). Manipulating thermal stress on rocky shores to predict patterns of recruitment of marine invertebrates under a changing climate. Marine Ecology: Progress Series 467: 121-136. Lathlean JA, Seuront L. (2014). Infrared thermography in marine ecology: methods, previous applications and future challenges. Marine Ecology: Progress Series 514: 263-277. Lima FP, Wethey DS (2009). Robolimpets: measuring intertidal body temperatures using biomimetic loggers. Limnology and Oceanography: Methods 7(5): 347-353. Lima FP, Wethey DS (2012). Three decades of high-resolution coastal sea surface temperatures reveal more than warming. Nature Communications, 3. Lima FP, Gomes F, Seabra R, Wethey DS, Seabra I, Cruz T, Santos AM, Hilbish TJ (2015). Loss of thermal refugia near equatorial range limits. Global Change Biology. Marcott SA, Shakun JD, Clark PU, Mix AC (2013). A Reconstruction of Regional and Global Temperature for the Past 11,300 Years. Science, 339: 1198–1201. Marshall DJ, Baharuddin N, McQuaid CD (2013). Behaviour moderates climate warming vulnerability in high-rocky-shore snails: interactions of habitat use, energy consumption and environmental temperature. Marine biology 160(9): 2525-2530. Meng XL, Ji TT, Dong YW, Wang QL, Dong SL (2009). Thermal resistance in sea cucumbers (Apostichopus japonicus) with differing thermal history: The role of Hsp70. Aquaculture 294(3): 314-318. FCUP Does thermal microhabitat variability modulate thermal stress responses? A study focusing on the physiology and behavior of Patella vulgata 25 Miller LP, Harley CDG, Denny MW (2009). The role of temperature and desiccation stress in limiting the local-scale distribution of the owl limpet Lottia gigantea. Functional Ecology 23: 756-767. Nguyen KDT, Morley SA, Lai CH, Clark MS, Tan KS, Bates AE, Peck LS (2011). Upper temperature limits of tropical marine ectotherms: global warming implications. PLoS One 6(12): e29340-e29340. Pincebourde S, Sanford E, Helmuth B (2008). Body temperature during low tide alters the feeding performance of a top intertidal predator. Limnology and Oceanography 53(4): 1562-1573. Pörtner HO, Bennett AF, Bozinovic F, Clarke A, Lardies MA, Lucassen M, Pelster B, Schiemer F, Stillman JH (2006). Trade-off in thermal adaptation: the need for a molecular to ecological interaction. Physiological and Biochemical Zoology 79, 295- 313. Pörtner, H. O. (2010). Oxygen-and capacity-limitation of thermal tolerance: a matrix for integrating climate-related stressor effects in marine ecosystems. The Journal of experimental biology 213(6): 881-893. Prusina I, Sarà G, De Pirro M, Dong YW, Han GD, Glamuzina B, Williams GA (2014). Variations in physiological responses to thermal stress in congeneric limpets in the Mediterranean Sea. Journal of Experimental Marine Biology and Ecology, 456, 34- 40. Sala OE, Chapin FS, Armesto JJ, Berlow E, Bloomfield J, Dirzo R, et al. (2000). Global biodiversity scenarios for the year 2100. Science 287(5459): 1770-1774. Seabra R, Wethey DS, Santos AM, Lima FP (2011). Side matters: microhabitat influence on intertidal heat stress over a large geographical scale. Journal of Experimental Marine Biology and Ecology 400, 200-208. Seabra R, Wethey DS, Santos AM, Gomes F, Lima FP (submitted). Equatorial limits of na intertidal ectotherm are more linked to water than air temperature. Sinclair ELE, Thompson MB, Seebacher F (2006). Phenotypic flexibility in the metabolic response of the limpet Cellana tramoserica to thermally different microhabitats. Journal of Experimental Marine Biology and Ecology 335, 131-141. Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB et al. (2007). IPCC, 2007: summary for policymakers. Climate change: 93-129. Somero GN (2010). The physiology of climate change: how potentials for acclimatization and genetic adaptation will determine “winners” and “losers”. The Journal of Experimental Biology 213, 012-920. Stenseng E, Braby CE, Somero GN (2005). Evolutionary and acclimation-induced variation in the thermal limits of heart function in congeneric marine snails (Genus Tegula): implications for vertical zonation. The Biological Bulletin, 208(2), 138-144.