The growth and development of crustaceans is regulated by the endocrine system, and aspects of the external environment, such as dissolved oxygen (DO), temperature, salinity, light, and pH (Castejón et al., 2015; Long et al., 2017). The level of DO is a key indicator of water quality, and partially determines the intensity of crustacean aquaculture. A suitable level of DO enables the survival of crustaceans. There are many factors that cause hypoxia in aquaculture, such as extreme weather changes, eutrophication, long-distance transportation, and human factors (Lehtonen and Burnett, 2016; Lund et al., 2017). Much research has focused on the negative effects of environmental hypoxia on crustaceans. For example, environmental hypoxia causes abnormal breathing and metabolic disorders in crustaceans, which leads to a decline in feeding and efficiency of food transformation and retarded growth (Han et al., 2017; Sun et al., 2018). Environmental hypoxia even affects the behavior, morphological characteristics, immunity, and reproduction of crustaceans (De Lima et al., 2015; Lara et al., 2017; Peruzza et al., 2018.).

The Chinese mitten crab Eriocheir sinensis is an important freshwater crustacean that is mainly cultured in the Yangtze, Yellow, and Liaohe River systems. In 2015, the total production of E. sinensis was about 823,259 t, accounting for 30% of the production of crustaceans in freshwater aquaculture (Bureau of Fisheries, Management of Chinese Ministry of Agriculture, 2016). Chinese grass shrimp Palaemonetes sinensis is the only Palaemonetes species recorded in China. This shrimp is mainly distributed in northern China and Siberia in Russia. P. sinensis not only has high ornamental and consumer value, but also has important ecological value in lakes, rivers, and reservoirs (Dong et al., 2018). In recent years, with the depletion of natural resources, artificial culture of P. sinensis has been rapidly developed. Farming models continues to innovate, and at present P. sinensis is one of the most important species cultivated in rice fields in China (Li et al., 2018).

Eriocheir sinensis and P. sinensis use an ex-gill to exchange DO in the water and CO₂ in the hemolymph to complete respiration. Under artificial breeding conditions, excessive feeding can easily cause excessive accumulation of organic matter on the bottom of culture tanks, along with excreta. The increasing density of cultures exacerbates the lack of DO, especially at night and early in the morning. At present, the solution is relatively simple, and mainly relies on an open aerator to maintain a high oxygen content in the water. However, these methods cannot solve the problem of oxygen deficiency completely. As the breeding of crustaceans intensifies, the problem of environmental hypoxia becomes increasingly prominent.

In hypoxic conditions, tissues must either increase anaerobic energy production, improve energy use, or lower energy consumption. Respiratory metabolism is the basic physiological activity of energy metabolism in living organisms, and not only reflects metabolic characteristics, physiological state, and nutritional status of the organism, but also the effects of environmental conditions on biological physiological activities (Leiva et al., 2015). Respiration is one of the important components of physiological studies of aquatic animals. Oxygen consumption rate (OCR), ammonia excretion rate (AER), and oxygen to nitrogen ratio (O:N) are important indicators of the respiratory metabolism of aquatic animals, and can be used to assess the energy use patterns of aquatic animals and their tolerance to environmental hypoxia (Zhang et al., 2014). During metabolic processes, specific enzymes accelerate the conversion of metabolites to produce energy to meet the needs of the body, and the changes in the activity of the respiratory metabolic enzymes are a direct reflection of the changes in metabolic capacity. Succinate dehydrogenase (SDH) is involved in the tricarboxylic acid cycle and oxidative phosphorylation, and its activity partially reflect levels of aerobic metabolism (Rutter et al., 2010). Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactic acid, which is a marker enzyme of anaerobic metabolism, and regarded as a stress indicator (Albert and Ellington, 1985). Therefore, SDH and LDH activities reflect the levels of aerobic and anaerobic metabolism, respectively.

This study used E. sinensis and P. sinensis to study the changes in respiratory metabolism and respiratory metabolic enzyme activity (SDH and LDH) after environmental hypoxia stress. The results provide a theoretical basis for the development of freshwater aquaculture of E. sinensis and P. sinensis.

Oxygen consumption rates and AER are important indicators of the metabolic activity of aquatic organisms. Studies have shown that circadian rhythm, size, amount of food, temperature, and salinity can all influence the OCR and AER of organisms (Crear and Forteath, 2000). In this study, the OCR of E. sinensis and P. sinensis increased as environmental hypoxia time increased. This may explain why oxygen demand rapidly increased to compensate for the lack of oxygen supply in the body after extended hypoxia. After 12 h reoxygenation, the OCR of E. sinensis and P. sinensis was still significantly higher than that in the control group, indicating that it was difficult to recover to normal levels in such a short period of time. DO in ponds varies markedly between day and night, which may impose significant effects on E. sinensis and P. sinensis, particularly in rearing ponds without aerators during the heat of summer. E. sinensis and P. sinensis always suffered environmental hypoxia at night due to their respiration and the decomposition of accumulated organic matter from unconsumed food and feces (Cheng et al., 2003). Oxygen content gradually increases after sunrise; however, from our results, it is evident that E. sinensis and P. sinensis will consume more oxygen during the reoxygenation stage, and that OCR will rapidly increase. If oxygen supply is insufficient, it will be exhausted quickly, presenting a risk of continuing environmental hypoxia.

The nitrogen excretion products of crustaceans mainly include ammonia, uric acid, urea, and amino acids. It is generally thought that exogenous nitrogen excretion and deamination produce ammonia nitrogen, and that endogenous nitrogen protein and nucleotides decompose to produce urea. Amino acids are the main sources of ammonia production in vivo. Crustaceans can regulate the concentration of intracellular free amino acids to cope with environmental stress (Greenaway, 1991). In this study, the AER of P. sinensis after environmental hypoxia was significantly lower than that in the control group, while the AER of E. sinensis decreased gradually as environmental hypoxia time increased, and was also significantly lower than that in the control group at 8 h. After 12 h reoxygenation, the AER of R2 h and R12 h did not return to normal levels, and was still significantly lower than that in the control group. These results showed that the metabolic patterns of E. sinensis and P. sinensis changed in response to environmental hypoxia stress, while short-term reoxygenation did not enable recovery to the initial nitrogen excretion levels.

Oxygen to nitrogen ratio has been widely used to determine the nature of oxidized metabolic substrates in aquatic animals under stress conditions (Dall and Smith, 1986). Theoretically, catabolism of pure protein produces an O:N from 3 to 16, and equal proportions of proteins and lipid results in an O:N from 50 to 60. Values >60 correspond to lipids and carbohydrates (Mayzaud and Conover, 1988). Intriguingly, in crustaceans, the preferred metabolic substrate appears to differ with salinity. In general, freshwater crustaceans rely mainly on carbohydrate metabolism, while marine species tend toward protein metabolism (Clifford and Brick, 1983). In this study, the metabolic substrates of E. sinensis and P. sinensis in the control group were proteins and were more similar to seawater crustaceans. This may be related to their individual biological characteristics. E. sinensis is a migratory crab that must reproduce in seawater, while P. sinensis does not need to migrate to reproduce, but can survive for a long time in 28 ppt seawater (data unpublished). Normally, crustaceans will change their metabolite substrates to adapt to environmental change. For example, Litopenaeus stylirostris maintained at 6 mg⋅L^-1 use carbohydrates as their main energy substrate, but when exposed to an oxygen concentration of 4 mg⋅L^-1, their main energy substrate was a protein–lipid mixture, and at 2 mg⋅L^-1, protein was the main energy substrate during stress response (Re and Díaz, 2011). In this study, the O:N value increased from 11.46 to 28.83 at 4 h environmental hypoxia and to 30.59 at 8 h environmental hypoxia in E. sinensis. In P. sinensis, it increased from 7.24 to 24.75 at 4 h environmental hypoxia and to 18.31 at 8 h environmental hypoxia, which demonstrated a shift in nutrient utilization from proteins to protein–lipid mixture. These results are similar to those reported by Chandurvelan et al. (2017) for Paratya curvirostris. Cadmium induced an increased in O:N, which was explained by an increased reliance on carbohydrates and/or lipids as metabolic substrates, which were stimulated by the increased metabolic costs of environmental hypoxia. We also found that glucose level significantly increased after environmental hypoxia (data unpublished). Unexpectedly, the metabolic substrate was still a protein–lipid mixture after 12 h reoxygenation. It may be that long-term environmental hypoxia leads to excessive energy consumption, and that metabolic level cannot be restored to the initial level after such a short period of reoxygenation. Therefore, when breeding crustaceans, it may benefit healthy growth of crustaceans to adjust the proportion of nutrients in the feed, according to the oxygen conditions at different feeding times.

Succinate dehydrogenase and LDH are very important enzymes in respiratory metabolism. SDH is a connection oxidative phosphorylation and an electron transfer center and is the only enzyme in the TCA cycle mosaic in the mitochondrial membrane. As a symbol of mitochondrial enzyme activity, size directly affects the rate of the TCA cycle. In general, SDH activity is used as an important indicator to measure the aerobic capacity of the body (Simon and Robin, 1971). These results showed that significantly lower SDH activity occurred in E. sinensis and P. sinensis after environmental hypoxia, indicating a lower level of aerobic metabolism, similar to a previous study of Macrobrachium nipponense (Sun et al., 2018). LDH is a key regulatory enzyme in anaerobic glycolysis. It exists in the cytoplasm and can control and catalyze the transformation from pyruvate to lactic acid. Therefore, LDH activity is often used as an important indicator of anaerobic metabolic capacity (Viru, 1994). In this study, the content of SDH gradually decreased and LDH content gradually increased as environmental hypoxia time increased. The aerobic capacity of E. sinensis and P. sinensis decreased and anaerobic respiration ability began to rise after environmental hypoxia. These findings suggest that environmental hypoxia results in a shift from aerobic to anaerobic metabolism, and that sufficient ATP can be generated only by upregulating anaerobic metabolism in hypoxic crustaceans. Other studies have also shown that the hypoxic environment at high altitude can lead to decreased SDH activity and increased LDH activity in Qinghai lizards (He et al., 2013). Valarmathi and Azariah (2003) exposed the estuarine crab Sesarma quadratum to copper and found that the activity of LDH increased and SDH decreased in the muscle, gill, and hepatopancreas. After 12 h reoxygenation, the content of SDH gradually increased and the content of LDH gradually decreased, which restored them to the level of the control group. These results showed that the activity of SDH and LDH rapidly recovered to normal levels after reoxygenation.

This study showed that environmental hypoxia had a dramatic influence on the respiratory metabolism and activities of related enzymes of E. sinensis and P. sinensis. The OCR of E. sinensis and P. sinensis increased as environmental hypoxia time increased. However, the AER of P. sinensis after hypoxia was significantly lower than that in the control group. The energy substrate shifted from protein to a protein–lipid mixture. As environmental hypoxia time increased, the content of SDH gradually decreased while LDH content gradually increased. Therefore, when breeding crustaceans, environmental hypoxia should be avoided as much as possible. If hypoxia occurs, OCR will significantly increase after reoxygenation, and secondary environmental hypoxia due to rapid consumption of oxygen should also be avoided.

JB and HJ designed the research and wrote the manuscript. HY and JB performed the majority of the experiments, data processing, analysis, and interpretation. XL assisted in sample collection and detection of indicators. HJ and XL revised the manuscript.

XL was employed by Panjin Guanghe Crab Industry Co., Ltd., Panjin, Liaoning, China.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.