Antioxidant and non-specific immune defenses in partially freeze-tolerant Xizang plateau frogs, Nanorana parkeri
Introduction
The Xizang plateau frog, Nanorana parkeri, has the highest altitudinal distribution of all frogs in the world (Lu et al., 2016). Many studies have been conducted to explore its adaptions for survival on the Tibetan Plateau (Sun et al., 2015; Wang et al., 2018; Zhang et al., 2016), that is known as the “Roof of the World”, the highest plateau on Earth (mean elevation ∼5000 m above sea level). These frogs experience up to six months of winter under conditions of severe cold weather and low/no food availability (Niu et al., 2020). To prolong survival time and conserve body fuel reserves at low temperature, overwintering N. parkeri use adaptations to actively suppress metabolism at multiple levels: e.g. whole animal, mitochondria, and expression of key enzymes (Niu et al., 2020). These frogs also endure relatively brief exposures to subzero temperatures in the locations where they overwinter. They have been shown to survive freezing for 12 h at −2 °C with 39.9% ± 5.4% of body water frozen (Niu et al., 2021). Furthermore, levels of oxidative stress and oxidative damage increased significantly in winter-collected frogs relative to summer-collected individuals, whereas antioxidant capacity decreased (Niu et al., 2018). However, little else is known about the physiological regulatory mechanisms of N. parkeri during freezing, especially about the antioxidant defense and non-specific immune systems. Thanks to their ability to survive in freezing and hypoxic environments, overwintering frogs are regarded as an ideal subjects for studies of the adaptability of ectothermic vertebrates. Elucidating these mechanisms will not only reveal the adaptations supporting life in this extreme environment but could potentially aid further development of cryopreservation technologies for cells, tissues and organs.
Temperature is one of the most crucial abiotic factors; daily thermal fluctuations or seasonal changes as well as extreme low or high temperature can significantly affect the growth, development, reproduction, geographic distribution, and survival of ectotherms (Angilletta et al., 2002; Huey and Berrigan, 2001). Low temperature generally depresses many physiological and biochemical processes, such as metabolic pathways, enzymes activity, respiratory rate and signal transduction, and can impose stress on ectotherms (DeVries, 2019; Gracey et al., 2004). To survive low temperature stress, ectothermic vertebrates can adopt physiological strategies, such as freeze tolerance and/or freeze avoidance. Freeze tolerance is one of the most amazing phenomena in the animal kingdom, with some species living at high latitudes or altitudes able to withstand whole body freezing for weeks or months at a time during winter (Storey and Storey, 2019). To survive, ice formation is restricted to only extracellular and/or extra-organ spaces since freezing inside cells is lethal. However, most physiological activities are slowed down or even stopped due to extracellular freezing, such as breathing, heartbeat, blood flow, and nerve transmission. As a result, freeze-tolerant animals are subjected to collateral ischemic and anoxic stresses once the body is frozen. Oxidative stress is usually related to oxygen availability and hence frozen frogs can suffer ischemia-reperfusion injury during thawing. Previous studies have confirmed that freeze-tolerant animals have well-developed antioxidant defenses that can minimize oxidative stress and oxidative damage during recovery after freezing episodes (Hermes-Lima et al., 2001; Hermes-Lima and Storey, 1993; Joanisse and Storey, 1996a; Storey, 2006). Such elevation of antioxidant defenses is a sign of their ability to tolerate environmental stressors. The phenomenon of maintaining high levels of antioxidant defenses at all times or reinforcing them in advance has been termed “preparation for oxidative stress (POS)” (Giraud-Billoud et al., 2019; Moreira et al., 2016).
Reactive oxygen species (ROS) are produced in the normal aerobic metabolism of living organisms and include superoxide radicals, hydroxyl radicals, and hydrogen peroxide (Birben et al., 2012). Environmental stress also induces ROS production, disturbing steady-state ROS concentrations, leading to oxidative stress, under conditions such as hypoxia, anoxia, freezing, dehydration, estivation, and hibernation (Afifi and Alkaladi, 2014; Bocchetti et al., 2008; Fangyu et al., 2011; Lushchak, 2011; Hermes-Lima et al., 2015; Giraud-Billoud et al., 2019; Niu et al., 2018; Storey et al., 2021). Appropriate levels of ROS play a vital role in cell growth, apoptosis, and signaling (Redza-Dutordoir and Averill-Bates, 2016; Sauer et al., 2001). However, high levels of ROS can directly attack and damage macromolecules including lipids, protein, and DNA. Common markers of increased oxidative damage include elevated levels of lipid peroxides, carbonyl proteins and thiobarbituric acid reactive substances (Hermes-Lima et al., 2015). Overproduction of ROS can be counteracted by the action of enzymatic and non-enzymatic antioxidants. Antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione-S-transferase (GST) (Joanisse and Storey, 1996a). Common non-enzymatic antioxidants include vitamin C and E, uric acid, and reduced glutathione (Joanisse and Storey, 1996a). Both types of antioxidants are important scavengers of ROS in cells and play a major role in preserving cell function (Birben et al., 2012). A third type of antioxidant system has also been found in amphibian skin, novel antioxidant peptides (Yang et al., 2009). High levels of ROS can also cause apoptosis (programmed cell death), which plays a significant role in the host immune system and cellular development (Redza-Dutordoir and Averill-Bates, 2016). The immune system, mediating phagocytic removal of dying or infected cells, is an indispensable component of various cellular processes and can be affected by temperature and other abiotic factors in ectotherms (Wright and Cooper, 1981). Moreover, effects of temperature on ectotherm immunity are particularly pronounced and far-reaching in winter since cold temperatures and overwintering are important triggers and regulators of immune activity (Ferguson et al., 2018). Acid phosphatase (ACP) and alkaline phosphatase (AKP) are well known representatives of non-specific immunity, participating in degrading foreign proteins, carbohydrates, and lipids, as well as acting in metabolic processes such as detoxification (Liu et al., 2004; Rahman and Siddiqui, 2004). ACP is often used as a marker to detect lysosomes within cell fractions and even to assess environmental pollution (Rajalakshmi and Mohandas, 2005). AKP as an intrinsic plasma membrane enzyme has been found in the membranes of almost all animal cells (Mazorra et al., 2002). Previous studies have demonstrated that non-specific immune defenses play an important protective role in freeze tolerance of amphibians (Storey and Storey, 2017).
The role of antioxidant defenses in freezing survival has received attention in various species including insects (Joanisse and Storey, 1996b), frogs (Joanisse and Storey, 1996a), turtles (Krivoruchko and Storey, 2010) and a lizard (Voituron et al., 2006). However, as yet, there are no data to comprehend the biochemical adaptations associated with freeze tolerance in high-altitude frogs, N. parkeri. Although our previous study demonstrated that overwintering N. parkeri can tolerate a partial freezing of their body, little is known about the protective role of antioxidant defenses or about non-specific immune systems in freezing survival.
Therefore, this study undertook an analysis of the activities of antioxidant defense enzymes (SOD, CAT, GPX, GST, and GR) and non-specific immune enzymes (AKP and ACP) in five tissues (heart, brain, liver, kidney, and muscle) of frogs, N. parkeri, during freezing induced by exposure to −2 °C for 12 h. To assess the disturbance of the balance of prooxidant/antioxidant systems, we also measured the levels of malondialdehyde (MDA) and carbonyl groups (CG) in the above-mentioned tissues, that are indicators of oxidative damage. Ascorbic acid or vitamin C (Vc), a small molecule antioxidant, and total antioxidant capacity (T-AOC) were also analyzed. Our present study expands our understanding of freeze tolerance and antioxidant defense strategies in the high altitude frog, Nanorana parkeri.
Section snippets
Animals
Adult male frogs Nanorana parkeri (n = 36) were collected from ponds in Damxung County (30.28° N, 91.05° E, 4280 m above sea level), Xizang Autonomous Region, China, in mid-December 2018. The mean snout-vent length of frogs was 3.93 ± 0.03 cm (n = 36), and the mean body mass of frogs was 4.18 ± 0.09 g (n = 36). At Lanzhou University, animal pretreatment and subsequent freezing experiments was performed as described previously (Niu et al., 2021). Briefly, frogs were placed into a wet plastic box
Oxidative damage
Freezing exposure induced a significant increase in the content of MDA in all tissues investigated except heart. Compared to controls, the MDA content increased significantly by ∼412% (P < 0.001) in brain, 79% (P < 0.001) in liver, 118% (P = 0.002) in kidney and 54% (P = 0.001) in skeletal muscle of frozen frogs (Fig. 1a). There were also significant differences in MDA contents among different tissues. MDA levels were highest in the brain and lowest in the kidney of both control and
Discussion
Oxidative stress can be induced by environmental factors that change tissue oxygenation with ROS levels showing significant increases during stress or recovery from stress (Granger and Kvietys, 2015). This can be important for freeze-tolerant ectotherms, that in the frozen state, may not breathe for days, weeks or months. However, after thawing, heartbeat and breathing are soon restored and tissues are quickly reoxygenated. Moreover, biological functions, including immune defenses, can be
Conclusions
In conclusion, freezing exposure disturbed the redox state and was accompanied by oxidative stress and oxidative damage in most tissues of the high-altitude frog, N. parkeri. A remarkable upregulation of SOD, CAT and GPX activities contributes to rising T-AOC in liver and brain during freezing. These changes indicate that antioxidant defenses play a significant part in the adaptive machinery for the survival of partially freeze-tolerant Xizang plateau frogs under subzero temperatures. Moreover,
Author statement
Yonggang Niu: Conceptualization, Investigation, Formal analysis, Visualization, Data curation, Writing – original draft, review & editing, Funding acquisition. Xuejing Zhang: Conceptualization, Investigation, Formal analysis, Writing – original draft. Haiying Zhang: Methodology, Investigation, Project administration. Tisen Xu: Investigation, Resources. Shengkang Men: Investigation, Resources. Kenneth B. Storey: Conceptualization, Writing – review & editing. Qiang Chen: Conceptualization,
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (no. 32001110 and no. 31971416), the Project of Scientific Research Foundation of Dezhou University (2019xjrc315) and the Training Program for Cultivating High-level Talents by China Scholarship Council (2021lxjjw01). The authors are grateful to Qimeng Jiao, Minghui Tan and Hairong Liu for their help in this study.
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