Oxygen delivery does not limit thermal tolerance in a tropical eurythermal crustacean

Metabolism of resting, fasting animals (standard metabolic rate, SMR) increases exponentially as temperature rises, whereas maximal oxygen uptake typically plateaus or even decreases as the structural and physiological ceilings of the cardiorespiratory systems to transport oxygen are reached at high temperatures (Fry and Hart, 1948). The capacity to increase oxygen uptake (Ṁ_O2) and delivery by the cardiorespiratory systems is often quantified as the aerobic scope, which is the difference between Ṁ_O2 of the resting fasting animal (SMR) and its maximal Ṁ_O2 (Ṁ_O2,max), typically measured during or immediately after intense exercise (Clark et al., 2013). Since SMR represents the basic cost of living, the capacity to increase Ṁ_O2 reflects the amount of aerobically derived, and hence sustainable, energy production that can be allocated to functions beyond body maintenance, i.e. physical activity, digestion, growth and reproduction (Fry and Hart, 1948; Nilsson et al., 2009; Eliason et al., 2011). These temperature relationships have led to the development of the oxygen and capacity limited thermal tolerance (OCLTT) model, proposing that aquatic animal fitness in rising temperatures is closely correlated to the capacity of the cardiorespiratory system to maintain oxygen supply, which thus provides the key mechanism defining the upper temperature limit of aquatic animals (Pörtner, 2010). In crustaceans, several studies over the past decade have reported such limitations in the oxygen supply capacity at high temperatures, revealed as a decline in cardiac and ventilatory performance as well as reductions in oxygen levels in the hemolymph with increasing temperature (Frederich and Pörtner, 2000; Frederich et al., 2009; Marshall et al., 2011; Jost et al., 2012). As further support for the OCLTT model, several marine invertebrates, including crustaceans, resort to anaerobic metabolism when approaching their upper thermal limit (Taylor et al., 1973; Taylor et al., 1977; Sommer et al., 1997; Frederich and Pörtner, 2000; Peck et al., 2002; Sokolova and Pörtner, 2003).

The majority of studies supporting the OCLTT model have been on stenothermal species (Pörtner, 2010) and it remains to be investigated whether this model also applies to eurythermal species that may have evolved a more thermoresistant cardiorespiratory system. Furthermore, numerous authors have argued that tropical species in general are close to their upper thermal limit and vulnerable to even small temperature increases (Deutsch et al., 2008; Tewksbury et al., 2008; Nilsson et al., 2009). Here we measure the effect of a 6°C increase in temperature from 27 to 33°C on growth capacity over a 3 month period in the giant freshwater shrimp (Macrobrachium rosenbergii De Man 1879), a crustacean of global importance to tropical aquaculture with annual production exceeding 400,000,000 kg per year (FAO, 2005–2013). Furthermore, we directly investigate whether increased temperature causes collapse of the oxygen supply capacity by the cardiorespiratory systems, by measuring Ṁ_O2 of resting and exercising shrimp at various temperatures, as well as heart rate, ventilation rate and hemolymph lactate during acute rises in temperature.

The growth rate of M. rosenbergii was not reduced when temperature was increased from 27 to 33°C (Fig. 1). At 27°C, mean body mass increased from 7.4±0.5 to 17.8±1.1 g, while body mass at 33°C rose from 7.6±0.7 to 18.6±1.4 g over the same period. As a result, the specific growth rates (SGR) of 0.0102 and 0.0106 g g⁻¹ day⁻¹ at 27 and 33°C, respectively, were unaffected by the 6°C rise in temperature. Mortality during this period was 43.6 and 52.7% at 27 and 33°C, respectively, both of which are in the normal range for this species in aquaculture ponds primarily due to cannibalism during molting (FAO, 2005–2013).

SMR and Ṁ_O2,max both increased exponentially when temperature was increased from 30 to 38°C (Fig. 2). However, Q₁₀ was higher

for SMR than for Ṁ_O2,max (2.3 and 1.3, respectively), such that aerobic scope decreased slightly with rising temperatures. Nevertheless, at 38°C, 2–3°C below the upper critical temperature (T_crit), the animals still retained 76% of the aerobic scope at 30°C.

That adequate capacity for oxygen supply is maintained at high temperatures is also supported by the lack of transition to anaerobic metabolism even when temperatures approached T_crit (Fig. 3). This was not due to an inability of M. rosenbergii to perform anaerobic metabolism, as exercise at 30°C was accompanied by an immediate and almost fourfold (t-test, P<0.001) rise in hemolymph lactate concentration (Fig. 3).

Each heartbeat consisted of a double-spiked top and each gill ventilation cycle consisted of a short spike followed by a longer spike, corresponding to the elevation and depression of the scaphognathite (Fig. 4A). At low temperatures, shrimps exhibited prolonged heartbeats associated with spontaneous cardiac arrest, and intermittent breathing patterns where ventilatory periods of around 10 s were interspersed amongst apneas of ~20 s. These patterns were replaced by short heartbeats and continuous ventilation when temperatures exceeded ~34°C. From 30 to 40°C, both heart rate (Fig. 4B) and gill ventilation rate (Fig. 4C) increased exponentially (Q₁₀ values of 2.3 and 4.8 between 30 and 40°C, respectively). Only at temperatures immediately below T_crit (~41°C) did the shrimp exhibit a sudden and very pronounced drop in these rates of convective oxygen transport. The collapse of cardiovascular and respiratory functions coincided with the loss of buoyancy and the shrimp became clearly moribund.

Our observations that aerobic scope, as well as heart rate and ventilation were maintained at temperatures immediately below T_crit, and that anaerobic metabolism is not utilized in resting animals, document that the upper thermal tolerance of resting M. rosenbergii is not determined by inadequate oxygen delivery. These results are in striking contrast to data on the European spider crab (Maja squinado) during acute temperature elevation (1°C h⁻¹), showing an initial increase in heart rate and ventilation rate from 0 to 17°C, followed by a plateau phase from 17 to 30°C and finally a decrease from 30 to 40°C until death at 40°C (Frederich and Pörtner, 2000). In M. squinado, the plateau phase was accompanied by a decrease in arterial hemolymph P_O2 consistent with the decline in heart and ventilation rates from 30 to 40°C, and the subsequent death. Although resting heart and ventilation rates in M. rosenbergii increased exponentially with temperature (Fig. 3B,C), a drop in stroke volume of either system could reduce cardiac or gill output and with these the overall oxygen transport capacity of the cardiorespiratory system. However, both SMR and Ṁ_O2,max also increased exponentially from 30 to 38°C (Fig. 2), indicating that the overall capacity of the cardiorespiratory system was fully sufficient to meet the rise in oxygen demand by the tissue. Aerobic scope was measured in South Vietnam during the dry season where the normal water temperature in the tanks was around 30°C. This temperature was therefore chosen as the starting temperature. Furthermore, growth rates at 27 and 33°C did not differ, indicating that performance was not compromised at 30°C. In a previous study of the effects of salinity on metabolism in M. rosenbergii, SMR in freshwater at 27°C was estimated to be 79.8±3.1 μmol kg⁻¹ min⁻¹ (Ern et al., 2013). Extrapolating the exponential equation fitted to SMR values between 30 and 38°C in the present study back to 27°C and rescaling for the differences in animal size (White et al., 2006), we estimate a 27°C SMR of around 125 μmol kg⁻¹ min⁻¹. Animals in the present study seemed quiescent for the entire 9 h of measuring. The SMR value in the present study may nevertheless be slightly higher than found by Ern et al. (Ern et al., 2013), which may be a result of our protocol, kept short to avoid the very rapid increase in background Ṁ_O2 due to the bacterial growth that inevitably becomes very evident above 33°C. The Q₁₀ for SMR in the present study is 2.3 and is within the range 2.0–2.4 reported on M. rosenbergii in the literature (Chen and Kou, 1996; Manush et al., 2004). We conclude therefore that while the SMR values reported here may be slightly overestimated, this overestimation would have resulted in a similar underestimation of aerobic scope and that the conclusion that this species retains a large percentage of its aerobic scope up to a few degrees below T_crit is valid. The lack of reduced oxygen transport capacity at high temperatures may in part be due to the cardiovascular anatomy of crustaceans. The single-chambered crustacean heart is situated within the pericardial sinus and supplied with oxygen-rich hemolymph entering the heart through ostia from the gills (McGaw, 2005). This is in contrast to the cardiovascular layout in fish where most of the cardiac oxygen supply derives from the oxygen-depleted venous return (Pörtner and Farrell, 2008). Given the different position of the heart relative to the respiring tissue, the crustacean heart may be less vulnerable to the lowering of venous P_O2 arising from the elevated metabolic demand argued to be responsible for cardiac failure in fish at high temperatures (Heath and Hughes, 1973; Wang and Overgaard, 2007; Farrell, 2009). Hence as long as the diffusive conductance of gills can meet oxygen demands, the heart will be perfused with oxygen-rich hemolymph and low venous oxygen levels would therefore not limit the ability to maintain pressure and cardiac output. However, while this argument can explain why M. rosenbergii appears to maintain cardiac work up to temperatures immediately below T_crit, it does not provide a satisfactory explanation of why other invertebrates resort to anaerobic metabolism when approaching their thermal thresholds (Frederich and Pörtner, 2000; Peck et al., 2002; Sokolova and Pörtner, 2003).

The crustaceans in which high temperatures cause collapse of the cardiorespiratory systems and increases in anaerobic metabolism are primarily temperate or arctic species with relatively low T_crit values (reviewed in Pörtner, 2010). In these species, the cardiorespiratory system is adapted to relatively low temperatures, and at least the intertidal crabs of the genus Petrolisthes from cooler habitats have increased temperature sensitivity of heart function compared with warmer habitats (Stillman and Somero, 1996). Thus a parsimonious explanation may be that the OCLTT model (Pörtner, 2010) adequately applies to species where T_crit is well below the debilitating effects of temperatures around 40°C where proteins denature and where cell membrane lipid function deteriorates (Somero, 2012). In these species animals are unable to sustain oxygen transport capacity (i.e. aerobic performance) below and above the ‘pejus’ temperatures (T_p), leading to critical temperatures, beyond which oxygen delivery is insufficient to sustain aerobic energy production causing mobilization of anaerobic metabolism. As a result animal fitness is determined by limitations in oxygen transport capacity of the cardiorespiratory system and the two therefore correlate over the entire temperature range of the animal (Fig. 5A). However, in tropical species such as M. rosenbergii and the cane toad (Rhinella marina) (Overgaard et al., 2012), the cardiorespiratory systems are adapted to function at high temperatures and these animals, therefore, do not experience a gradual decrease in aerobic performance at high temperatures.

We therefore propose a modification to the OCLTT model for the relationship between oxygen supply capacity, animal fitness and thermal tolerance in tropical species (Fig. 5B). Here aerobic performance is maintained until denaturation temperatures (T_d) and the primary determinants of T_crit are therefore not inadequate oxygen delivery from organ malfunction, but rather a more generalized heat-induced loss of protein structure or membrane integrity leading to a very sharp decline in metabolic and physiological functions. Fitness-related parameters decline in a number of crustaceans when approaching T_crit (Brown et al., 1992; King, 1994; O'Brien, 1994). Growth rates did not differ at 27 and 33°C in M. rosenbergii (Fig. 1), but animals die within a few days after being exposed to 38°C after a gradual rise in temperature (0.33°C day⁻¹) (R. Ern, unpublished observation). Thus given that acute aerobic performance was maintained up to T_crit, future studies should address whether this performance is amendable to acclimation to higher temperatures. It is also imperative to establish how aerobic scope correlates with the decline in growth in M. rosenbergii between 33 and 38°C, as well as other physiological and biochemical functions immediately below T_crit.

In the giant freshwater shrimp (Macrobrachium rosenbergii) the current worst-case scenario predicting a 3°C increase in tropical water temperature by the end of this century (Nilsson et al., 2009) does not cause a negative effect on its growth performance. Furthermore, oxygen transport capacity of the cardiorespiratory system does not seem to be universally coupled to animal fitness in aquatic organisms as previously proposed (Pörtner, 2010). Too heavy a reliance on the relatively easily measured aerobic performance characters such as heart rate, gill ventilation rate, aerobic scope and anaerobic metabolites will therefore yield inaccurate predictions on the impact of climate change in these species.

Physiological measurements were performed on giant freshwater shrimp (Macrobrachium rosenbergii) obtained from a local hatchery outside Can Tho University (Vietnam). The shrimp were held in 1 m³ freshwater tanks (29±1°C) at Can Tho University and fed to satiety on dry feed and freshly thawed shrimp every second day. Water was changed regularly to ensure that NH₄⁺-N, NO₂⁻ and NO₃⁻ never exceeded 0.25, 0.3 and 20 mg l⁻, respectively. All measurements were performed in the intermolt period in animals fasted for 4 days.

Maximum oxygen uptake (Ṁ_O2,max) at 30, 34 and 38°C was measured in 30 animals, 10 at each temperature. The shrimp was placed in a water tank at 30±0.1°C and the temperature either maintained at 30±0.1°C or elevated to 34±0.1 or 38±0.1°C (2°C h⁻¹). At the target temperature, a vigorous escape response was induced by nudging the carapace with a soft brush. After 3–4 min, the shrimp was deemed exhausted, indicated by the inability to induce escape responses, and immediately moved to a respirometer and Ṁ_O2 measured over 20 min. The decline in P_O2 was divided into 3 min bins and the bin with the highest δP_O2 used to estimate Ṁ_O2,max. Randomly distributed blind tests to measure bacterial respiration after the shrimp had been removed from the respirometers showed that background Ṁ_O2 due to bacterial respiration never exceeded 5% of SMR.

Hemolymph lactate concentrations were measured in resting and exhausted shrimp at 30°C and in resting shrimp at 34 and 38°C, using a handheld lactate analyzer (Accutrend Plus, Cobas-Roche, Mannheim, Germany). At all three temperatures, shrimp were initially placed overnight in a water tank (radius: 60 cm) at 30±0.1°C. The following morning temperature was either maintained at 30±0.1°C or elevated to 34±0.1 or 38±0.1°C (2°C h⁻¹). At the target temperature a hemolymph sample was taken by inserting a needle into the pericardial space in gently handled shrimp where escape responses were avoided. At 30°C the shrimp was exhausted (as described above) after the first sampling and another hemolymph sample was taken for lactate measurements after exercise.

Heart and ventilation rates in M. rosenbergii were measured using reflective infrared sensors (AMP03, Newshift Ltd, Leiria, Portugal) fitted into plastic tubes and glued onto the carapace above either the heart or the scaphognathites (Fig. 4A). The animal was submerged in an aerated water tank at 30±0.1°C and left overnight to obtain resting values. The following morning water temperature was elevated (2°C h⁻¹), while heart or ventilation rate were recorded using a Biopac (Varna, Bulgaria) MP100 data acquisition system at 200 Hz.

One-way analysis of variance (P<0.05) was used to test the effect of acclimation temperature on growth rate, the effect of temperature on hemolymph lactate concentrations and the effect of temperature on SMR and Ṁ_O2,max, respectively. Student's t-test (P<0.05) was used to test the effect of exercise on hemolymph lactate concentrations in resting shrimp at 30°C. All statistical analyses were performed using JMP statistical Discovery from SAS (www.jmp.com).

The authors would like to thank Heidi M. Jensen, Per G. Henriksen and Rasmus Buchanan for animal husbandry, as well as John S. Jensen, Niels S. Bøgh, Niels U. Kristensen and Morten L. Hedegaard for technical assistance.