| A series of laboratory experiments were conducted to determine the effects of temperature on metabolic physiology of swimming crab Portunus trituberculatus in two media (air and water). The primary results were listed as below.1. Effects of different thermal levels on metabolic physiology of Portunus trituberculatus in aquatic environmentThis experiment was conducted in the laboratory to explore the adaptive features of Portunus trituberculatus to different thermal environments in terms of metabolic physiology by comparing the difference of metabolic rate and levels of substance and energy metabolism at 16,23 and 30 β. The oxygen consumption rate was shown to significantly increase with temperature increase (P< 0.05). In the hemolymph, the hemocyanin level was negatively related with temperature (P< 0.05). Levels of metabolic substances all reached the minimum at 23 β, with a significant change being observed only in glucose level (P< 0.05). The increase in temperature resulted in an elevation in urate and lactate level, and the former changed significantly (P< 0.05). A significant difference was observed in respiratory enzyme activities at different thermal levels:PK activity decreased with temperature increase (P> 0.05), which was opposite to that of LDH activity (P< 0.05), while CCO activity was maximum at 23 β (P< 0.05). No significant difference was observed in cellular energy status among thermal treatments except AMP/ATP. This preliminary study demonstrates that P. trituberculatus adapts to different thermal environments mainly depending on carbohydrates in the regulation of substance metabolism and on anaerobic metabolism and mitochondrial oxidative phosphorylation in the regulation of energy metabolism to maintain cellular energy status.2. Effects of abrupt thermal change on metabolic physiology of Portunus trituberculatus in aquatic environmentIn the laboratory, juveniles of swimming crabs Portunus trituberculatus were subject to two aquatic environments where temperature changed abruptly (i.e. 23β'16β and 23β'30 β, represented by T16 and T30 respectively). The metabolic rate and levels of substance and energy metabolism of the two treatments were determined to explore the effect of abrupt thermal change on metabolic physiology of P. trituberculatus. Compared to T16 group, T30 group was significantly higher in oxygen consumption rate (P< 0.05). A significant effect of thermal change was observed in levels of glucose, hemocyanin, urate and lactate in the hemolymph (P< 0.05). Activities of ATP-producing pathways were reduced in both treatments following thermal change, while a significant change was only observed in glycolysis pathway activity (P< 0.05). In terms of cellular energy status, T16 group was higher than T30 group (P< 0.05). The preliminary study suggests that P. trituberculatus mainly takes advantage of carbohydrates to regulate substance metabolism to adapt to thermal change. By contrast, crabs at higher temperature had a poor adaptability to environment due to a lower internal oxygen level and oxygen capacity and disrupted cellular energy status.3. Responses of ammonia loading in swimming crabs Portunus trituberculatus to aerial exposure:effects of air temperature and emersion timeAmmonia overloading is a common response for aquatic organisms to aerial exposure. This study aimed to elucidate the relationship of this phenomenon and animal death and to provide more information on the role of emersion time and temperature in affecting ammonia loading and concomitant physiological changes. In this study, swimming crabs Portunus trituberculatus were subject to aerial exposure at 16,23 and 30 β respectively followed by re-immersion (23 β) to recover for 12 h, with those immersed in acclimated seawater (23 β) as controls. At each time interval, levels of hemolymph ammonia and activities of enzymes involved in ammonia detoxification were analyzed. Groups treated with aerial exposure of 16,23 and 30 β were represented by AT16, AT23 and AT30 respectively. According to our results, crab mortality was positively related with emersion time and air temperature, while ammonia loading was alleviated in AT16 and AT30 groups compared to that in AT23 group. Following exposure to air, prolonged emersion time aggravated ammonia overloading (P< 0.05) and as a consequence, indicators related with ammonia detoxification were elevated (P< 0.05). During recovery, AT23 group showed a much reduced ammonia excretion rate in comparison with the control group (P< 0.05), which was contrary to AT16 and AT30 groups, whereas prolonged emersion time (AT23-3.0 group) elevated total free amino acids (TFAA) level and reduced urea level (P< 0.05). Statistic results showed a significant effect of emersion time on glutaminase dehydrogenase (GDH), glutamine synthase (GS) and arginase (ARG) activity. Compared with AT23 group, ammonia overloading was lower in AT 16 and AT30 group, while activities of GS and ARG were higher (P< 0.05). The preliminary results demonstrated that the emersion-induced ammonia overloading is possibly not one of the main factors leading to P. trituberculatus death during aerial exposure and subsequent recovery. When crab survival medium changed, thermal variation, in comparison with constant temperature, could reduce ammonia overloading by elevating activities of ammonia detoxification pathways and ammonia excretion rates during the recovery period.4. Responses of metabolic physiology in swimming crabs Portunus trituberculatus to aerial exposure:effects of air temperature and emersion timeThe internal hypoxia usually occurs in aquatic animals during transport out of water. In this process, temperature is an important abiotic factor. To explore the effect of air temperature and emersion time on metabolic physiology of Portunus trituberculatus, levels of hemocyanin, urate, glucose and lactate in the hemolymph and levels of glycogen, metabolic enzyme activity and adenylate in the muscle were determine at different air temperature (16,23 and 30 β) and emersion time (0.5 and 3 h) treatments. Results showed that media thermal inconsistence stimulated oxygen consumption rate of crabs to a significant higher level during re-immersion (P< 0.05). All hemolymph parameters increased with emersion time increase and most of them were significantly influenced by air temperature (P< 0.05). During the entire experiment, glycogen level decreased especially for AT30 group and metabolic enzyme acticity also decreased. A significant effect of air temperature was recorded in PK, CCO and LDH activity (P< 0.05), while emersion time significantly influenced PK and LDH activity (P< 0.05). With respect to cellular energy status, it correlated with air temperature:During aerial exposure, there was a significant reduction in AT23 and AT30 groups (P< 0.05), while following re-immersion, it appeared in ATI 6 group (P< 0.05). This preliminary study demonstrates an inhibitory effect of aerial exposure on energy-producing capability of P. trituberculatus. In response to air temperature increase, cellular energy status may decline and emersion-induced internal hypoxia is aggravated in crabs, which are two possible reason of the crab death during aerial exposure and subsequent recovery.5. Metabolic response of swimming crab Portunus trituberculatus to thermal variation:implication for crab transport methodTemperature variation and air exposure are two predominant abiotic stressors during the transport and storage of Portunus trituberculatus. To understand the effects of these factors on P. trituberculatus, we examined the hemolymph (hemocyanin, glucose and lactate level) and muscle indicators (glycogen content, metabolic enzymes activity and energy status) through laboratory experiments. Energy status was evaluated by measuring changes in total adenine nucleotide (TAN) concentration, ATP, ADP, AMP, ADP/ATP, AMPATP, adenylate energy charge value (AEC) and the apparent equilibrium constant for adenylate kinse (KβADEN)-It was found that thermal variation significantly influenced (P< 0.05) the energy metabolism of crabs by increasing lactate level, ADP and AMP content, cytochrome c oxidase (CCO) and lactate dehydrogenase (LDH) activity and the ratio of ADP/ATP and AMP/ATP and by decreasing glucose and ATP content, AEC and KβADEN value at high temperature. At lower temperature, all parameters except the hemocyanin and lactate level were similar between immersed and aerial exposed crabs (P> 0.05), indicating that the maintenance of energy homeostasis was independent of environment media. However, at higher temperature, air exposure resulted in a more perturbation of energy metabolism in the crabs compared to immersion, as a greater decrease in hemocyanin, ATP, AEC and KβADEN level (P< 0.05), and a greater increase in glucose, ADP, AMP, ADP/ATP and AMP/ATP level (P< 0.05) were observed in aerial exposed crabs. In summary, high temperature especially during air exposure could lead to a severe stress in P. trituberculatus, and the perturbation of energy status as a result of mismatch between energy demand and supply is a possible inducer for crab death.6. Effects of low temperature on metabolic physiology in swimming crabs Portunus trituberculatus during aerial exposureTransport out of water is an alternative method in transporting Portunus trituberculatus, while what range of air temperature is optimum during the process is unclear. To address this issue and to explore effects of low air temperature on crab survival and metabolic physiology of P. irituberculatus, crabs were subject to different thermal conditions (4,8,12 and 16 β) in air. Besides, crabs were treated with dipping in 16 β seawater for 3 h prior to aerial exposure, to validate what role this step plays during exposure of crabs to air. Results showed that there was a longest survival time (35 h) for crabs in AE12 and SA16 groups. Following exposure to air, a significant elevation was observed in glucose and lactate level in all treatments (P< 0.05), while there was no significant difference among them (P> 0.05). With respect to ATP level, AE8 was lower than other treatments. All groups, in particular AE4 and/ or AE8 groups, upregulated the expression of Na+/K+-ATPase and HSP90 (P< 0.05). No significant effect of temperature was observed in the expression of CCO (P> 0.05). According to this preliminary study, temperature at approximately 12 β is suitbale for crab transport out of water, which may be attributed to the relatively higher capability to exchange ions, to repair cells and to utilize stored ATP. Dipping in cold seawater is a suggested pre-tranport process as it can obviously improve the survival of P. trituberculatus during transport out of water. |
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