The seasonal lifestyle of crucian carp is characterized by winter dormancy in a hypometabolic state, which allows it to survive in a complete absence of oxygen with the energy available from large glycogen deposits [13, 44]. Low winter temperature greatly helps this lifestyle in depressing metabolic processes. However, a passive response to temperature (Q10 effect) alone is not sufficient to achieve a deep hypometabolic state in winter and therefore inherent (active) physiological adjustments with changes in gene expression are probably also required [35, 37]. The results of this study indicate that both the seasonal down-regulation of aerobic metabolic rate and the transition from aerobic to anaerobic metabolism appear to be required to achieve a deep hypometabolic state in the overwintering crucian carp.
RMR includes energy consumption in basal cellular processes (SMR) and voluntary movements [28]. Notably, RMR was significantly lower in WinA than SumA crucian carp indicating that energy consumption had been adjusted to a lower level before the oxygen-free winter by adopting the Precht type-5 inverse temperature compensation [26]. The inverse thermal compensation of RMR reported in this study is similar to that previously reported for crucian carp (Table 2). The scaling exponent for the mass-dependence of the aerobic metabolic rate (-0.19) is similar to that in goldfish (-0.15) [2, 41] and the mean of 69 teleost species (-0.21) [5]. The temperature dependence of the RMR in crucian carp (Q10 = 2.04–2.17) is also consistent with the previous observations (Q10 = 1.83) for several teleost species [5]. In the light of RMR, it is likely that naturally occurring environmental cues (e.g., temperature, day length) acclimatize crucian carp for anaerobic dormancy by inducing inverse temperature compensation of aerobic metabolic rate. Generally, inverse temperature compensation develops slowly before unfavorable conditions set in and therefore is considered to be an anticipatory physiological pattern which prepares animals for future stresses [10, 26]. At the tissue level in crucian carp, the lowering temperature is shown to induce changes in lipid composition of the brain, cardiac gene expression, and activate the build-up of glycogen stores in crucian carp [20, 37, 39].
Table 2
Comparison of aerobic (standard and routine) metabolic rate in crucian carp (Carassius carassius) and goldfish (Carassius auratus) acclimated to different temperatures. Metabolic rate is expressed as oxygen consumption in mg O2 Kg− 1 h− 1.
Species | AT | 2°C | 10°C | 18/20°C | 24/25°C | Reference |
C. carassius | 2°C | 3.06* | 8.59* | | | Present study |
C. carassius | 18°C | | 15.54* | 53.53* | | Present study |
C. auratus | 20°C | | | 48.32* | | [17] |
C. auratus | NM | | 15.9* | | | [20] |
C. auratus | NM | | 24.7* | | | [36] |
C. carassius | 2°C | 23.86† | 42.82† | 94.03† | 125.9† | Present study |
C. carassius | 20°C | | | 154.71† | 175.4 | Present study |
C. carassius | 10, 20, 25 | | 38.9† | 122.7† | 209.5† | [37] |
C. auratus | NM | | | 106† | | [38] |
C. auratus | NM | | 90† | | | [39] |
C. auratus | NM | | | | 91† | [40] |
C. auratus | NM | | 43† | | | [41] |
C. auratus | NM | | 105† | 138† | 195† | [42] |
C. auratus | NM | | 56† | | | [43] |
AT, acclimation temperature; NM, not mentioned; *, SMR; †, RMR. |
RMR consists of energy consumption in the basic cellular processes and the normal locomotor activity of fish. Therefore, the lower RMR of WinA fish may be partly due to their lower locomotor activity [21, 27, 32]. The determination of SMR was necessary to exclude the component of voluntary movements in aerobic metabolic rate. The SMR is the minimum metabolic rate needed to sustain life, and below which physiological homeostasis is impaired [4]. Reductions in SMR indicate the ability of the animal to conserve energy in basic molecular processes of its cells. SMR has not been previously measured for crucian carp (https://www.fishbase.de/home.htm), so we carefully compared it between seasonally acclimatized crucian carp. The fish was maintained in the respiratory chamber for 3 days until fully recovered from handling stress, stopped moving and reached a true resting level of metabolism as observed in goldfish [41]. Results of resting MO2 measurements confirmed marked depression (inverse thermal compensation) of SMR in WinA crucian carp. The inverse thermal compensation indicates that the aerobic metabolic machinery is downregulated in anticipation of reduced energy availability in anoxic winter. To maintain energy balance, SMR can be expected to be associated with a reduction in both aerobic ATP production and ATP consumption. All tissues of crucian carp accumulate substantial amounts (2–6% of the body mass) of glycogen for the winter, which displaces other subcellular components, as seem to happen to the mitochondrial content of the heart [42, 45]. This is associated with inverse thermal compensation in temperature-dependence of heart rate in seasonally acclimatized crucian carp [22]. Reduced mitochondrial respiration rate is typical for dormant fish and frog tissues [6, 36].
Anoxic metabolic depression is a well characterized response of facultative vertebrate anaerobes like the painted turtle, goldfish, crucian carp and eel [15, 16, 41]. Ethanol is the principal end product of anaerobic metabolism in goldfish and crucian carp, and therefore ethanol production can be used for calculation of anoxic metabolic rate [17, 30]. Ethanol production of WinA crucian carp at 2°C (0.126 mmol Kg− 1 h− 1) and SumA fish at 18°C (0.76 ± 0.02 mmol Kg− 1 h− 1) are in line with results of earlier studies on this species (Table 3) and suggest anoxic metabolic depression of 78% and 92.5% from the level of SMR. Anderson [1] measured 70% and 80% anoxic metabolic depression in goldfish at 20°C and 4°C, respectively. Direct calorimetry in goldfish has confirmed those indirect measurements of Anderson (71% depression at 20°C) [41]. Prolonged anoxia in crucian carp is associated with a sustained depression of heart rate (about 60% bradycardia) [37], which is fully consistent with the close correlation between metabolic depression and decrease of heart rate in goldfish [40]. Interestingly, the anoxic metabolism of carp had a strong size dependence, and its scale exponents were much larger than that of aerobic metabolism. To our knowledge, the size dependence of anoxic metabolic rate has not previously been measured for any ectothermic vertebrate, whereas it is suggested to be independent of body size in invertebrates and prokaryotes. The strong scaling of anaerobic metabolism fits well with the size of glycogen storage, which is about 2% and 6% of body weight in 800 grams and 1 gram of crucian carp, respectively [45].
Table 3
Anoxic ethanol production of crucian carp (Carassius carassius) at different temperatures.
Acclimation temperature | Experimental temperature | Ethanol production (mmol Kg− 1 h− 1) | Reference |
2°C | 2°C | 0.126 | present study |
2°C | 10°C | 0.216 | present study |
18°C | 10°C | 0.402 | present study |
18°C | 18°C | 0.76 | present study |
2°C | 2°C | 0.17 | [44] |
2°C | 12°C | 0.69 | [44] |
2°C | 18°C | 1.37 | [44] |
8°C | 8°C | 0.30 | [45] |
15°C | 15°C | 1.40 | [32] |
All data were transformed to the fish size of 100 g using the scaling exponent of -0.412 |
The metabolic acclimatization to winter dormancy in crucian carp involves 3 steps down in energy consumption (Fig. 5). The greatest effect on low winter metabolism is exerted by the passive Q10 effect on the rate of molecular processes and reduced locomotion, which reduces RMR from the level of SumA fish by about 70% to the level of WinA fish at 2°C when using a Q10 value of 2.1. SMR of WinA fish at 2°C is about 45% lower than RMR at the same temperature further depressing the aerobic metabolic rate by active gene-based regulation of cellular metabolism. Transition from aerobic respiration to anaerobic fermentation further depresses the metabolic rate by 78.1% from the level of SMR at 2°C. Winter-time depression of both aerobic and anaerobic metabolic rate is probably an important part of the anaerobic wintering in crucian carp by conserving metabolic reserves and thereby extending the anoxic survival time. This show that crucian carp, one of the vertebrate champions of anoxia tolerance, exploits wide variety of mechanisms to enable prolonged winter dormancy.