Influence of elevated CO2 concentrations on thermal tolerance of the edible crab Cancer pagurus

ARTICLE IN PRESS

Journal of Thermal Biology 32 (2007) 144–151 www.elsevier.com/locate/jtherbio

Influence of elevated CO2 concentrations on thermal tolerance of the edible crab Cancer pagurus Rebekka Metzger, Franz J. Sartoris, Martina Langenbuch, Hans O. Po¨rtner Alfred-Wegener-Institute for Polar and Marine Research, Marine Animal Ecophysiology, Postfach 12 01 61, 27515 Bremerhaven, Germany

Abstract Current trends of global climate change affect marine ectothermal animals not only through the increase in ambient temperature. Synergistic effects of carbon dioxide and temperature changes as well as more frequent hypoxia events must also be considered. As a first attempt, the combined effects of warming and elevated CO2 concentrations were investigated in the edible crab (Cancer pagurus). Arterial oxygen tension (PaO2) in the haemolymph was recorded on-line during a progressive warming scenario from 10 to 22 1C and cooling back to 10 1C. Hypercapnia (1% CO2) caused a significant reduction of oxygen partial pressure in the haemolymph as well as a large, 5 1C downward shift of upper thermal limits of aerobic scope. The present findings are the first to show that hypercapnia causes enhanced sensitivity to heat and thus, a narrowing of the thermal tolerance window of a marine ectotherm. Such interactions of ambient temperature and anthropogenic increases in ambient CO2 concentrations will need to be considered during future investigations of the effects of climate change on ecosystems. r 2007 Elsevier Ltd. All rights reserved.

1. Introduction The current trend of warming (Intergovernmental Panel on Climate Change (IPCC), 2001) and eutrophication largely caused by anthropogenic CO2 emissions exert direct effects on marine ecosystems, causing shifts in geographical distribution and increasing the risk of local extinction of species or even ecosystems like coral reefs (Parmesan and Yohe, 2003; Perry et al., 2005, Hoegh-Guldberg, 2005). These effects are expected to progressively involve direct effects of CO2 accumulating in ocean waters as well as effects of ocean acidification (Feely et al., 2004, Po¨rtner et al., 2004, Orr et al., 2005). In the past, however, mechanistic studies of physiological effects of environmental factors aimed at qualifying and quantifying the effects of individual factors rather than of combinations. Once effects of individual factors are sufficiently understood, more integrated efforts are required to comprehensively understand effects of global climate change. Corresponding author. Tel.: +49 471 4831 1307; fax: +49 471 4831 1149. E-mail address: [email protected] (H.O. Po¨rtner).

0306-4565/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2007.01.010

Studies focusing on the effects of temperature on marine invertebrates and fish led to the development of the concept of oxygen and capacity-limited thermal tolerance in ectothermal water breathers (Po¨rtner et al., 2000, 2004b; Frederich and Po¨rtner, 2000; Po¨rtner, 2001, 2002a, b). The main idea of this concept is the existence of so-called pejus-temperatures or thresholds. These borders are characterized by the transition to hypoxaemia, i.e. internal (systemic) hypoxia caused by a limited capacity of oxygen supply mechanisms (ventilation, circulation) at extreme temperatures (cf. Fig. 1). In the warmth, this mismatch arises due to progressive inability of oxygen supply to tissues to meet the temperature-dependent rise in oxygen demand. Temperature-induced hypoxaemia delineates the first line of thermal limitation and occurs in fully oxygenated waters (cf. Zakhartsev et al., 2003). These findings led to the hypothesis that thermal tolerance will be influenced by ambient factors which affect the capacity for oxygen supply to the organism like ambient hypoxia as well as increased ambient CO2 concentrations (hypercapnia). Hypercapnia as well as hypoxia may lead to the depression of metabolic rate involving several physiological processes like acid–base regulation, transmembrane ion exchange, or

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weeks before experimentation. The animals were fed twice a week with pieces of mussels (Mytilus edulis) and common cockle (Cerastoderma edule). The animals were not fed for 2 days prior to surgery and experimentation. 2.2. Surgical procedure and on-line data recording

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Fig. 1. Simplified graphical model of oxygen and capacity limited thermal tolerance (top) and associated temperature-dependent aerobic performance (bottom) in aquatic metazoa. Aerobic scope decreases beyond pejus limits and is lost beyond critical limits once anaerobic metabolism sets in during excessive oxygen deficiency and hypoxaemia of body fluids (Po¨rtner, 2001). Accumulated CO2 (hypercapnia) and ambient hypoxia have been hypothesized to cause a concomitant shift (reflected by arrows) of upper and lower thermal limits and a narrowing of the thermal window as well as a reduction in performance capacity and rates (modified after Po¨rtner et al., 2005a).

accumulation of inhibitory neurotransmitters like adenosine (for review see Po¨rtner et al., 2004, 2005a). We hypothesized that the net effect would be a narrowing of the thermal window of aerobic scope as depicted in Fig. 1 (Po¨rtner et al., 2005a). The present study was designed to test the hypothesis of synergistic effects of temperature and CO2 in a marine crustacean, the edible crab Cancer pagurus, from a physiological point of view. More specifically, we tested whether elevated CO2 concentrations in fact cause a shift of upper thermal limits to cooler temperatures. Cancer pagurus lives in a thermal range between about 4 and 15 1C in the North Sea around Helgoland (mean habitat temperature: 5–8 1C). Previously, recordings of haemolymph oxygen tensions proved adequate to qualify the thermal window of another crustacean, Maja squinado (Frederich and Po¨rtner, 2000), therefore, we chose oxygen recordings in arterial haemolymph of Cancer pagurus to monitor effects of temperature as well as of temperature combined with elevated CO2 concentrations on thermal tolerance. To our knowledge the present study is the first to investigate the effects of increased CO2 concentrations on thermal tolerance of a marine ectothermal animal species. 2. Materials and methods 2.1. Animals Edible crabs (Cancer pagurus) with a carapace width of 11.7–18.3 cm and 260–1165.6 g fresh weight were caught in October and November 2004 in the area around Helgoland, Germany. The animals were kept in tanks and 12 h light cycle with aerated natural seawater at 1070.2 1C and 32–33% salinity at the Alfred Wegener Institute in Bremerhaven, Germany. They were held for at least 6–8

Animals were prepared for experimentation by attaching the carapace to a grid; however, walking legs and chelipeds remained unrestrained. Following the procedure by Frederich and Po¨rtner (2000) a small hole was drilled through the carapace directly behind the heart, avoiding injury to the hypodermis. The hole was covered by a latex dam to prevent haemolymph loss. For measurements of oxygen partial pressure (arterial oxygen partial pressure, PaO2) in the haemolymph, microoptodes (NTH-L5-NS 12  0.40PA0.9-TF-PSt1-YOP, PreSens GmbH, 93053 Regensburg, Germany) were used. The data were recorded on-line by use of temperature compensation through TX3 oxygen monitors and software (V 5.20) (PreSens Regensburg). Optodes were calibrated in air-saturated seawater (100%) and in seawater containing 2% ascorbic acid (0% air saturation). After calibration, the tip of the optode was inserted through the latex dam into the pericardial sinus and fixed with dental wax. No animal died during surgical procedures. 2.3. Measurements under normocapnia and hypercapnia After implantation of the optodes, animals were allowed to recover for at least 20–24 h prior to any temperature change. During recovery from surgery and during experimental periods animals were kept in a tank containing 100 l of seawater (32–33 %). The water was filtered, aerated and the tank covered by a lid. The oxygen tension of the water was always maintained close to air saturation with a minimum level of 17 kPa recorded at 22 1C. After recovery at control temperature (10 1C) the water was warmed continuously from 10 to 22 1C at a rate of 1 1C h1. During the subsequent 16 h, the water was cooled to 10 1C. Experiments under hypercapnia were carried out with animals exposed to water equilibrated with 1% CO2 in air. Water pH had fallen from 7.9 to 7.06. Animals were exposed to hypercapnia during 24 h at 10 1C prior to surgery, during 24 h of recovery from surgery and, finally, during experimental temperature changes. The 48 h period prior to temperature change should have allowed the animals to reach new acid–base equilibria in their body fluids (Cameron and Iwama, 1987). Haemocyanin concentrations were monitored following the procedure by Nickerson and van Holde (1971). 2.4. Statistics PO2 data were analyzed by use of GraphPad Prism 4. Data are given as means7SD. Significant differences (po0.05) in PaO2 values between normocapnia and

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hypercapnia were detected by Students t-test. Paired t-tests were used when comparing control values at 10 1C and values reached after cooling. The progressive development of PaO2 values was analysed by regression analysis. Discontinuities in the relationship between PO2 and temperature were determined from intersections of fitted two-phase regressions according to the minimum sum of squares (Nickerson et al., 1989). Normocapnic data were ln-transformed for linearization and to reduce the influence of variability. 3. Results PaO2 values recorded in the haemolymph were unrelated to body weight or size of the animals. Haemocyanin concentrations were similar between normocapnic (61.27 25.7 mg ml1 haemolymph, n ¼ 6) and hypercapnic (55.67

23.2 mg ml1, n ¼ 10) groups. In each individual analysed, haemolymph PaO2 recordings displayed large fluctuations over time (Fig. 2), likely caused by oscillations in cardiac and ventilatory activity as previously seen in the spider crab, Maja squinado (Frederich and Po¨rtner, 2000). Evaluation of means either included all PaO2 values or maxima of oxygen tensions at each temperature. Significant differences between data collected under normocapnia and hypercapnia resulted from both analyses. Because temperature-dependent oxygen supply capacity is reflected more by maximum oxygen tensions rather than by the means of all oxygen tensions at each temperature, results depicted in Fig. 3 only show maximum PaO2 values. The haemolymph PaO2 of animals grouped under normo- and hypercapnia were significantly different over the whole temperature range (Fig. 3). Control values at the beginning of the experiment were 10.972.5 kPa (normocapnia; n ¼ 6)

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Fig. 2. Examples of original data recordings of PaO2 (kPa) in the haemolymph of individual animals (A–E) during warming (and cooling, D, E) protocols under normocapnia. The data depict the variability in haemolymph oxygen tensions of individual animals over time. For one animal (C) the graph displays two different time resolutions (recording rate 1 min1. The graphs also depict the process of temperature change (—).

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10 12 14 16 18 20 22 20 18 16 14 12 10 Temperature (°C) Fig. 3. Dynamic changes in temperature-dependent maximum PaO2 values in the haemolymph of Cancer pagurus during warming at 1 1C h1 and subsequent cooling at 1–1.5 1C h1 (& ¼ aminals under normocapnia, n ¼ 6; n ¼ animals under hypercapnia, n ¼ 8. Values are means+SD, normocapnia or SD, hypercapnia, for steps of 0.1 1C).

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versus 8.474.4 kPa (hypercapnia; n ¼ 8, excluding two animals that died during experimentation), similar to values found at the end of the cooling phase which were 10.775.2 kPa (normocapnia; n ¼ 6) and 5.873.7 kPa (hypercapnia; n ¼ 8), respectively. Lowest values reached at 21 1C were 2.170.3 kPa under normocapnia and 0.970.9 kPa under hypercapnia. Fig. 4a demonstrates a delayed drop in haemolymph PaO2 under normocapnia compared to the early exponential decrease seen under hypercapnia, evidenced by different regressions as a result of best fits. The decrease in PaO2 values induced by warming was reversed during cooling under both normocapnia and hypercapnia (Fig. 3). The respective increase in PaO2 values during cooling followed an exponential pattern in both cases but PaO2 values were again maintained at higher levels under normothan under hypercapnia (Fig. 4b). However, while all animals survived the warming and cooling protocol under normocapnia, two animals exposed to hypercapnia died during the cooling phase (excluded from the data set) and four more, previously hypercapnic animals died within 1 week after the analyses had been terminated. Following the approach by Frederich and Po¨rtner (2000) the evaluation of two-phase regressions revealed significant discontinuities. An example is depicted in Fig. 5. Ln transformation of PaO2 data reveals a clear discontinuity and a shift to steeper slopes upon warming. This discontinuity is interpreted to indicate the onset of falling aerobic scope at the upper pejus temperature TpII. Such an early breakpoint was not visible under hypercapnia, when PaO2 fell upon the onset of warming. Further discontinuities result, where PaO2 data begin to follow an asymptotic decline phase towards zero and are interpreted to reflect critical temperatures (cf. Frederich and Po¨rtner,

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Fig. 4. (a) Regression analyses (best fits) of changes in PaO2 under normocapnia (’; n ¼ 6) and hypercapnia (m; n ¼ 8) during progressive warming from 10 to 22 1C. Normocapnia: f ðxÞ ¼ ð18:8  0:61Þþ ð0:762  0:362Þnx; r2 ¼ 0.3682, po0,05. Hypercapnia: f ðxÞ ¼ ð111:0  27:31Þne½ð0:25260:0249Þnx þ ð0:23  0:26Þ; r2 ¼ 0.4986, po0.05. (b) Regression analyses (best fits) of changes in PaO2 (kPa) under normocapnia (’; n ¼ 6) and hypercapnia (m; n ¼ 8) during progressive cooling from 22 to 10 1C. Normocapnia: f ðxÞ ¼ ð84:3  30:96Þne½ð84:3 30:96Þnx þ ð3:06  0:50Þ; r2 ¼ 0.3773, po0,05. Hypercapnia: f ðxÞ ¼ ð53:4  16:29Þne½ð0:23120:0299Þnx þ ð0:43  0:17Þ; r2 ¼ 0.7549, po0.05.

2000), characterized by the minimization of aerobic scope and transition to anaerobic metabolism. 4. Discussion Minimized time periods of dynamic experimental temperature change (Fig. 3) ensured that the present data represent the acute thermal window of the species when acclimated to 10 1C. Effects of acclimation which would likely cause shifts in thermal limits were thereby minimized. The highest PaO2 values recorded under normocapnia at 10 1C were similar to those found in other Cancer species (Johansen et al., 1970; McMahon and Wilkens, 1977;

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f(x)=(3,083 ± 0,1019) + (-0,06930 ± 0,007895)*x r2=0,7197; p ∠ 0,05

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Temperature (°C) Fig. 5. Example of an analysis for discontinuities (in this case: upper pejus values) in the development of haemolymph PaO2 during warming from 10 to 22 1C under normocapnia. PaO2 values were ln-transformed prior to analysis. Depicted are the means of PaO2 and the mean breakpoint evaluated from analyses for individual specimens.

McMahon et al., 1978; Houlihan et al., 1980; Bradford and Taylor, 1982). Frederich and Po¨rtner (2000) also recorded highly variable individual PaO2 levels in Maja squinado, where values ranged from 4.4 to 17.9 kPa at 12 1C. A similarly high variability was found in the present study and is most likely caused by intermittent respiration cycles and associated pauses in cardiac and ventilatory scaphognathite activity which cause intermittent reductions of haemolymph PaO2 levels (Ansell, 1973, McMahon and Wilkens, 1977, Houlihan et al., 1980, McMahon, 1985, Frederich and Po¨rtner, 2000, cf. Fig. 2). Previous studies had shown that CO2 concentrations of 1% as used here cause initial acid–base disturbances, which are compensated to various degrees during long-term hypercapnia, depending on the species and phyla examined. The level of compensation is also different for intracellular and extracellular values (pHi and pHe). Disturbances of pHi in various tissues and species are usually fully compensated within 24–48 h as seen especially in muscle tissue of selected species of sipunculids, crustaceans, fish and amphibians (Heisler, 1980; Heisler et al., 1982; Toews and Heisler, 1982; Gaillard and Malan, 1983; Larsen et al., 1997, Po¨rtner et al., 1998, McKenzie et al., 2003). In contrast, the drop in pHe is commonly compensated to a lesser extent in marine invertebrates than in fish (see Po¨rtner et al., 2004 for review). As a result, the functionality of the haemocyanin and, therefore, oxygen supply might be affected. A permanent extracellular acidosis as seen under hypercapnic conditions would cause a right-shift of the O2-dissociation curve in crustacean haemolymph due to the Bohr effect (Truchot, 1992; Burnett, 1997) leading to a reduced PaO2 as shown by Booth et al. (1982). However, considering available data for the reduction of pHe under 1% CO2 (Cameron, 1978; Gaillard and Malan, 1983) and the Bohr factor in the haemolymph of Cancer pagurus (Danford et al., 2002), a maximum right-shift of affinity (P50) by approximately 0.07 kPa would result. In conclusion, the influence of

hypercapnia on oxygen transport is considered small in the present study. Few studies have been carried out in crustaceans of how hypercapnia affects metabolic rate (Batterton and Cameron, 1978; O0 Mahoney and Full, 1984). O’Mahoney and Full showed that Callinectes sapidus experiences a small but significant drop in oxygen consumption under acute and severe hypercapnia, associated with reduced ventilation volume and O2 extraction efficiency. It can be assumed that this effect was not caused by a drop of pHi due to complete compensation of the intracellular acidosis within 24–48 h (see above). In contrast, an incomplete compensation of extracellular acidosis might cause a reduction in metabolic rate as seen in invertebrates and also in fish tissues or cells (Po¨rtner et al., 2000, Langenbuch and Po¨rtner, 2002, 2003). An extracellular acidosis likely also causes dissolution of carapace components and elicits an extracellular accumulation of Mg2+ which in turn is well known to elicit muscle relaxation and anaesthesia in crustaceans, especially in the cold (Morritt and Spicer, 1993, Frederich et al., 2001). Elevated haemolymph Mg levels may thus contribute to an anaesthetizing effect of CO2. Further contributing mechanisms might include changes in the concentrations of neurotransmitters like adenosine (Reipschla¨ger et al., 1997). The respective situation is unclear in crustaceans where an increase of ventilation rate, heart rate and in the velocity of haemolymph circulation was observed in response to adenosine infusions (Stegen and Grieshaber, 2001). However, O0 Mahoney and Full (1984) found no increase in the motor activity of different crustacean species under hypercapnia. This was also true in the present study with Cancer pagurus. As a corollary, the reduction of PaO2 values under hypercapnia as found in the present study is in line with other findings of narcotic effects of elevated CO2 concentrations in marine invertebrates (Po¨rtner et al., 1998, Michaelidis et al., 2005, cf. Po¨rtner et al., 2004, 2005a). Frederich and Po¨rtner (2000) showed in Maja squinado that PaO2 in the haemolymph fluctuates depending on ventilatory and circulatory activity. Due to increased metabolic rate, elevated temperatures cause increased rates of ventilation and cardiac activity. Beyond the upper pejus temperature (TpII, Fig. 1), a decrease in PaO2 occurs because ventilation and circulation systems reach their capacity limits and can no longer increase oxygen supply (cf. Frederich and Po¨rtner, 2000). Regression analysis indicates a TpII of Cancer pagurus at about 15–16 1C. This is the maximum temperature value that adult Cancer pagurus experience in their habitat in the North Sea (Cuculescu et al., 1998). Therefore, the upper limit of thermal tolerance as quantified by the upper pejus temperature TpII matches the maximum habitat temperature of Cancer pagurus. This leads to the prediction that oxygen limitations resulting from a mismatch between oxygen supply and demand will be the first thermal limits experienced by the species during future warming

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scenarios. The abundance of the species will decrease unless it is able to shift its thermal tolerance limits to warmer temperatures by acclimation. However, the capacity to acclimate is limited. Results obtained in a recent study of marine fishes, the eelpout Zoarces viviparus in the Wadden Sea, in fact demonstrated that temperature extremes during hot summers exceed pejus temperatures and the acclimation capacity of the species and cause a decrease in population density (Po¨rtner and Knust, 2007). Under normocapnia maximum haemolymph PaO2 values of Cancer pagurus were kept high within a wide thermal window. This was also shown for Maja squinado (Frederich and Po¨rtner, 2000). Under hypercapnia maximum and overall PaO2 levels remained below normocapnic values and, furthermore, decreased earlier, i.e. at cooler temperatures during progressive warming. This indicates a downward shift of the upper pejus limit to cooler temperatures, most likely due to mechanisms that cause a depression of functional capacity (see above). Heat limits are thus reached at cooler temperatures under hypercapnia than under normocapnia (Fig. 6). At more extreme temperatures, the transition to an asymptotic decline phase in PaO2 indicates the critical temperature (Frederich and Po¨rtner, 2000). This asymptotic decline phase was reached sooner upon warming under hypercapnia than under normocapnia and reflects a downward shift of the upper critical temperature. According to the observed patterns of PaO2 during cooling after heat exposure, animals seemed to fully recover under both normo- and hypercapnia. No significant differences were found between PaO2 values at the beginning and the end of the experimental period. This might indicate full reversibility of heat stress on short time Tp

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Temperature (°C) Fig. 6. Modelled depiction of the change in heat tolerance under hypercapnia superimposed on original data depicted in Fig. 3. Discontinuities in the PaO2 curve under normocapnia were quantified as in Fig. 5 and named pejus temperature TpII (according to Frederich and Po¨rtner 2000). This threshold had likely shifted by about 5 1C to cooler temperatures under 1% hypercapnia. Similarly, the general lowering of haemolymph PO2 under hypercapnia caused a downward shift of critical temperatures by about 4.5 1C.

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scales. However, mortality reached 60% under hypercapnia during and after the cooling phase. An increased mortality under moderate hypercapnia was also seen in Octopus vulgaris (Kumagai et al., 2003) and in Sipunculus nudus (Langenbuch and Po¨rtner, 2004). No increase in mortality was evident in Callinectes sapidus during 48–72 h under 2%, 4% and 6% CO2 (Cameron and Iwama, 1987). However, Yamada and Ikeda (1999) found enhanced mortality (50%) in the copepod Paraeuchaeta elongata after 1 week at moderately reduced water pH values (0.2 units). In conclusion, hypercapnia alone can lead to increased mortality at least in some crustacean species. Elevated mortality may involve a reduction in protein synthesis rates as seen in S. nudus under elevated CO2 concentrations and at low extracellular pH (Langenbuch and Po¨rtner, 2002, Langenbuch et al., 2006) causing reduced survival on long time scales. In the present study, the incubation period under 1% CO2 lasted about 96 h. The fact that all animals survived the warming and subsequent cooling protocol under normocapnia but not under hypercapnia, corroborates the conclusion that sensitivity to warming was enhanced by hypercapnia and the associated reduction in tissue performance. Mortality was likely exacerbated by the downward shift of thermal limits and the extended period of more severe hypoxaemia experienced by hypercapnic animals in the warmth. As a corollary, the present study is the first to investigate the combined influences of elevated CO2 concentrations and temperature change on a marine ectothermal species. It could be demonstrated that 1% hypercapnia elicits a large shift of heat tolerance limits in Cancer pagurus to cooler temperatures (Fig. 6). The present data fully support our earlier hypothesis that elevated CO2 concentrations, in fact, cause a narrowing of thermal tolerance windows (cf. Po¨rtner et al., 2005a, Fig. 1). For a better mechanistic understanding, further investigations should address how the various mechanisms involved in CO2 effects respond to warming and also how the narrowing of thermal windows develops with rising concentrations of ambient CO2. However, the large degree of the observed shift in pejus and critical limits under 1% CO2 suggests that a shrinking of thermal windows begins to develop at CO2 concentrations much below 1%, possibly including those expected from anthropogenic CO2 accumulation in the atmosphere (Caldeira and Wickett, 2003). In a simplified scenario, a linear relationship may exist between CO2 concentrations and the downward shift of upper, and upward shift of lower thermal limits (cf. Fig. 1). Assuming a similar, parallel shift of both upper and lower limits the downward shift of the upper limit by 5 1C would suggest an extreme narrowing of the thermal window of Cancer pagurus by about 10 1C (starting from a width of between 10 and 13 1C, similar to Maja squinado, Frederich and Po¨rtner, 2000) at 1% CO2 (10,000 ppm). The narrowing would still occur by 0.5 1C at 500 ppm or by 1 1C at 1000 ppm. With unabated anthropogenic CO2 release into the atmosphere CO2 concentrations of 500 ppm would be reached in about

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20 years and of 1000 ppm by the end of this century. These values require experimental verification; however, this simplified back-of-the-envelope calculation emphasizes the importance of future studies which consider the synergistic effects of temperature, hypoxia and CO2 where negative influences of either parameter are amplified by the other (Po¨rtner et al., 2005a). At the ecosystem level the present data and analysis suggest that future scenarios of combined global warming and ocean acidification induced by anthropogenic CO2 imply a narrowing of geographical distribution ranges of aquatic species due to the narrowing of thermal windows under the effect of CO2. Effects of accumulated CO2 on thermal tolerance will become effective first at southern (and northern, cf. Fig. 1) distribution limits of a species. Such range retractions have been discussed to increase extinction risks in the face of gobal warming (e.g. Thomas et al., 2004, 2006). While temperate zone ectotherms acclimate to ambient temperature and its oscillations on seasonal time scales (cf. Po¨rtner et al., 2005b) it is presently unclear to what extent elevated CO2 concentrations affect the capacity of marine ectotherms to undergo thermal acclimation. These considerations emphasize that further research is urgently needed to complement the present preliminary data set. References Ansell, A.D., 1973. Changes in oxygen consumption, heart rate and ventilation accompanying starvation in the decapod crustacean, Cancer pagurus. Neth. J. Sea Res. 7, 455–475. Batterton, C.V., Cameron, J.N., 1978. Characteristics of resting ventilation and response to hypoxia, hypercapnia and emersion in the blue crab, Callinectes sapidus (Rathbun). J. Exp. Zool. 203, 403–418. Booth, C.E., McMahon, B.R., Pinder, A.W., 1982. Oxygen uptake and the potentiating effects of increased hemolymph lactate on oxygen transport during exercise in the blue crab, Callinectes sapidus. J. Comp. Physiol. 148, 111–121. Bradford, S.M., Taylor, A.C., 1982. The respiration of Cancer pagurus under normoxic and hypoxic conditions. J. Exp. Biol. 97, 273–288. Burnett, L.E., 1997. The challenges of living in hypoxic and hypercapnic aquatic environments. Am. Zool. 37, 633–640. Caldeira, K., Wickett, M.E., 2003. Anthropogenic carbon and ocean pH. Nature 425, 365. Cameron, J.N., 1978. Effects of hypercapnia on blood acid–base status, NaCl fluxes, and trans-gill potential in freshwater blue crabs, Callinectes sapidus. J. Comp. Physiol. B 123, 137–141. Cameron, J.N., Iwama, G.K., 1987. Compensation of progressive hypercapnia in channel catfish and blue crabs. J. Exp. Biol. 133, 183–197. Cuculescu, M., Hyde, D., Bowler, K., 1998. Thermal tolerance of two species of marine crab, Cancer pagurus and Carcinus maenas. J. Thermal. Biol. 23, 107–110. Danford, A.R., Hagerman, L., Uglow, R.F., 2002. Effect of emersion and elevated haemolymph ammonia on haemocyanin–oxygen affinity of Cancer pagurus. Mar. Biol. 141, 1019–1027. Feely, R.A., Sabine, C.L., Lee, K., Berelson, W., Kleypas, J., Fabry, V.J., Millero, F.J., 2004. Impact of anthropogenic CO2 on the CaCO3 system in the oceans. Science 305, 362–366. Frederich, M., Po¨rtner, H.O., 2000. Oxygen limitation of thermal tolerance defined by cardiac and ventilatory performance in the spider crab Maja squinado. Am. J. Physiol. 279, R1531–R1538.

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