A pH value between 6.5 and 9.0 is satisfactory for most freshwater animals in long-term of farming practice (Alabaster and Lloyd, 1980). Moreover, a high pH value of 9.5 is quite common in ponds for crab farming (Kong et al., 2012). In this study, there was no significant difference in growth performance parameters between any pH groups after 8 weeks, indicating the Chinese mitten crab can survive in pH 6.5–9.5. But there was also a study showing that 40% healthy crabs (about 50 g) died after exposure to pH 9.5 for 2 weeks (Pan et al., 2017). That is because smaller crabs have greater tolerance to environmental stress than bigger ones (Tood and Dehnel, 1960). Weight gain was reduced by water pH stress, and the acidic pH had more negative effect, which is consistent with the result of pH effect on protein utilization. The negative effect of pH stress may be mediated by the disruption of metabolic regulation, extra energy expenditure, and reduction of nutrient absorption (Leal et al., 2011), which may explain why weight gains in the low pH and high pH groups are low in the current study. The other possible reason may be that acidification may alter the dynamics of muscle water content, which would lead to the changes of weight gain.

Environmental stress can trigger the over production of reactive oxygen species (ROS) and result in a severe damage to cells (Du et al., 2013). MDA is one of the most known products of lipid peroxidation and is a well-established marker of oxidative stress, which can be induced by a wide range of environmental stress (Wang et al., 2004). Therefore, the increase of MDA is usually associated with various environmental stress and the pathological state of animals (Zhang Y. et al., 2015). Aquatic crustaceans lack an adaptive immune response and mostly rely on innate immune responses, among which the antioxidant defense system is important to reduce ROS damage (Muta and Iwanaga, 1996; Zhang W. et al., 2015). SOD is a key antioxidant enzyme directly participating in the removal of ROS (Kobayashi et al., 2019). ACP and AKP are highly conserved enzymes and play an important role in the non-specific immunity of crustaceans (Yang et al., 2007; Gisbert et al., 2018). Thus, higher ACP and AKP activities have a positive effect on the defense against external microbial invasion (Wu et al., 2019). Total hemocyte numbers of crustaceans can vary greatly after exposure to environmental stress and infections by bacteria, fungi, and viruses (Truscott and White, 1990; Vargas et al., 1997; Iwanaga and Lee, 2005; Vazquez et al., 2009). Any increase in THC could lead to an increase of immune defense ability (Min et al., 2015). The results of the present study reveal that all three immune parameters of the crabs were significantly affected after exposure to alkaline acid stress. Also, an increase of OH^– concentration seems to have more stimulative effects on immune function of the crabs than those at a high H⁺ concentration, which may be the reason why the MDA content in the high pH group was lower than that in the low pH group.

When suffering in environmental stress, crustaceans require additional energy to maintain homeostasis (Wang et al., 2016). Carbohydrates are often included in artificial diets to serve as an energy source, and they can also meet the high energy demand of aquatic animals under stress (Tseng and Hwang, 2008; Wang et al., 2017). Mitochondrial oxidative phosphorylation provides over 90% of the energy produced by aerobic organisms; therefore, the regulation of oxidative phosphorylation is a major issue for coping with the environment changes to meet more energy need (Bermejo-Nogales et al., 2015). However, animals would use glycolysis to produce energy in the absence of adequate oxygen. In the presence of adequate oxygen levels, the intracellular pH might be a factor that determines which way to obtain energy. For instance, oxidative phosphorylation could be driven in an acidic condition and aerobic glycolysis is driven in an alkaline condition (Khalifat et al., 2008; Calderon-Montano et al., 2011). Similar to these findings, the significant changes in oxidative phosphorylation, TCA cycle, and pyruvate metabolism pathways at low pH may be driven by the acidic medium, and the glycolysis/gluconeogenesis pathways can be driven by high pH. However, the primary response in aquatic animals is usually accompanied with high levels of plasma glucose and anaerobic glycolysis (Arends et al., 1999; Acerete et al., 2004; Fazio et al., 2015). This may explain why both oxidative phosphorylation and glycolysis/glycogenesis pathways were upregulated in the low pH group. Due to the enhanced glucose activities, the digestion of carbohydrates was increased in terms of the upregulated starch and sucrose metabolism pathways in both acidic and alkaline groups.

Except for conventional carbohydrates, the presence of non-glycogenic carbohydrates also contributes to the concentration of total carbohydrates in the hepatopancreas of most crustaceans (Chang and O’Connor, 1983). The amino sugar glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) are prevalent in the biosphere. For instance, amino sugars are the major component of the exoskeleton of crustaceans (Zeng et al., 2016). The changes in the amino sugar pathways in the current study indicate that acidic and alkaline stress might affect the molt activity of Chinese mitten crab. Similarly, some other studies also found that environmental stress would change molting frequency in ammonia-exposed Penaeus monodon, saponin-exposed Penaeus japonicus, copper sulfate- exposed P. monodon, nitrite-exposed P. monodon, and acid-exposed Macrobrachium rosenbergii (Chen and Chen, 1992, 2003; Chen and Lin, 1992). In the current study, the finding is further verified by the changes of pentose and glucuronate interconversions, galactose metabolism pathways, and steroid biosynthesis pathways in low pH and high pH groups.

Glycosaminoglycans are heteropolysaccharides composed by a repeat disaccharide unit in which one of the two monosaccharides always contains an amino sugar (N-acetylgalactosamine or GlcNAc) (Antonio et al., 2013). A previous study reported that the crayfish Orconectes virilis might depend on the pentose pathway during intermolt and glycolysis during premolt in carbohydrate metabolism (Mcwhinnie, 1962). The glycolytic, pentose phosphate, and glucuronate pathways are operated as catabolic pathways for glucose utilization in intermolt crayfish, Pacifastacus leniusculus (Puyear et al., 1965). Galactose also appears frequently during the premolt of period to satisfy the carbohydrate need of molting (Vernberg and Vernberg, 1972).

As a member of gonad stimulatory hormones, steroid plays a pivotal role in molting and reproduction in crustaceans (Subramoniam, 2017). Steroid hormones also have antioxidant and free radical scavenging activities and the physiological metabolisms involved in steroid in the hepatopancreas of crustacean would promote SOD activity (Cai et al., 2002; Wu et al., 2016). The SOD activity of the crab in low pH group was enhanced in the current study. Enzymes such as SOD, glutathione peroxidases (GPx), and some non-enzymatic defense such as glutathione (GSH), tocopherols, taurine, and urate constitute the first line of defense to inactive ROS and scavenge free radicals (Sharma et al., 2004; Wang et al., 2019). The thiol group in the cysteine moiety of GSH is a reducing agent and can be reversibly oxidized and reduced (Meister and Anderson, 1983). However, GSH is an acidic molecule characterized by a γ-linked amino acid, and the cysteine residue can reduce its stability in an alkaline environment (Anik et al., 2016; Giustarini et al., 2016). That is why the GSH metabolism pathways were significantly changed in the low pH group but not in high pH group. Taurine, hypotaurine, and their metabolic precursors (cysteamine, cystic acid, and cysteinesulfinic acid) may protect the organisms against a variety of oxidants (Konukoglu et al., 1999). As antioxidants could protect the crustaceans from oxidative stress, the taurine and hypotaurine metabolism pathways were significantly changed in both low and high pH groups.

Some crustaceans could also convert the linolenic acid to the highly polyunsaturated fatty acids such as eicosapentaenoic acid in the similar way to what has been reported in fish (Kabeya et al., 2018). Long chain polyunsaturated fatty acids (LC-PUFA, involved in eicosapentaenoic acid) have an important role in physiological processes such as immune and stress responses (Tocher, 2010; Norambuena et al., 2015). The changes of the linoleic acid metabolism in the high pH group indicate that alkaline stress may stimulate de novo synthesis of EPA, and improve the ability of stress resistance in crab.

In general, the crab growth was suppressed by both acidic and alkaline stress, especially at pH 6.5. The increase of OH^– concentration seems to have a more stimulative effect on antioxidant ability and immunity and stronger ability of stress resistance than the crabs under acidic stress. Environmental stress would increase the metabolic activity of animals to compensate the change and maintain homeostasis (Koehn and Bayne, 1989). In the current study, data of transcriptome reveal that the main metabolic changes were the pathways related to glucose metabolism as carbohydrates can rapidly provide energy to meet the high energy demand of aquatic animals in a stress condition. Oxidative phosphorylation might be the main source of energy under acidic stress, while the aerobic glycolysis supplies most energy during alkaline stress. The pathway analysis indicates that pH stress may affect the molting process, but this claim needs a longer term field study to confirm.