Antioxidant responses to salinity stress in an invasive species, the red-eared slider (Trachemys scripta elegans) and involvement of a TOR-Nrf2 signaling pathway

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Highlights

  • The mRNA expression levels and activity of SOD, CAT and GSH-PX were enhanced in Trachemys scripta elegan at the early stages of salinity exposure.

  • TOR-Nrf2 pathway was activated to modulate antioxidant genes transcription in Trachemys scripta elegan in response to salinity stress.

  • MDA content significantly increased in liver, especially in the high salinity group.

Abstract

The red-eared slider (Trachemys scripta elegans), a freshwater turtle, is an invasive species in many parts of the world where it survives in both freshwater and coastal saline habitats. High salinity can induce reactive oxygen species (ROS) production and lead to oxidative damage. In this study, we investigate the antioxidant defense mechanisms of T. s. elegans in response to salinity stress. The results showed that the mRNA expression levels of superoxide dismutase (SODs), catalase (CAT) and glutathione peroxidase (GSH-PXs) were significantly increased in both 5 psu and 15 psu groups at the early stages of salinity exposure (generally 6–48 h), but typically returned to control levels after the longest 30 d exposure. In addition, hepatic and cardiac mRNA levels of the NF-E2-related factor 2 (Nrf2), showed a similar upregulation as an early response to stress, but decreased at 30 d in the 5 psu and 15 psu groups. The mRNA levels of the negative regulator of Nrf2, kelch-like ECH associating protein 1 (Keap1), exhibited the opposite pattern. Moreover, mRNA expression levels of target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) in liver and heart showed roughly similar patterns to those for Nrf2. Furthermore, the content of malondialdehyde (MDA) was significantly increased in liver, especially in the 15 psu group by ~2.5-fold. Taken together, these results indicate that T. s. elegans may activate the TOR-Nrf2 pathway to modulate antioxidant genes transcription in order to promote enhanced antioxidant defense in response to salinity stress.

Graphical abstract

The invasive species Trachemys scripta elegans, a freshwater turtle, activated antioxidant defenses to protect the body against oxidative damage in response to elevated environmental salinity by quickly up-regulating the activities and mRNA expression of antioxidant enzymes. In addition, the participation of the upstream TOR-Nrf2 axis mediated the up-regulation of antioxidant enzyme genes. The adaptation to salinity stress indicates that T. s. elegans can invade not only fresh water, but also saline habitats.

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Introduction

The red-eared slider (Trachemys scripta elegans) is native to the eastern United States and northeastern Mexico. However, as a commercially important species, T. s. elegans has been introduced into diverse aquatic habitats worldwide (>70 countries and regions in Africa, Asia, Europe, Oceanica and Australia) due to its popularity in the pet trade and in aquaculture (Kraus, 2009; Ma and Shi, 2017). Its strong ability to adapt to diverse environmental conditions and its broad ecological tolerance (Burke et al., 1995; Churchill and Storey, 1992; Krivoruchko and Storey, 2013; Willmore and Storey, 2007) has allowed T. s. elegans to become a highly invasive species (Cadi and Joly, 2004; Semenov, 2010). Although nominally a freshwater turtle (Mittermeier et al., 1943), our recent research on T. s. elegans has shown that this species can live and lay eggs in the estuary of the Nandu River (salinity: 0.1–26‰) in Hainan province, China (Hong et al., 2014; Yang and Shi, 2014), and survive for over three months in brackish water (salinity: 15‰) (Zhang et al., 2014). This indicates that T. s. elegans can invade not only fresh water, but also saline habitats, thereby creating an even greater invasive potential for this species to spread through new territories. Although some research on the adaptation of T. s. elegans to saline environments has been done in our previous study (Hong et al., 2014), the mechanisms of salinity adaptation of T. s. elegans are still unclear.

An increase of environmental salinity often causes a variety of physiological stress responses by organisms, including plasma hormone changes, abnormal energy metabolism and electrolyte imbalance (Choi et al., 2008; Fiess et al., 2007), as well as elevated reactive oxygen species (ROS) generation (Hossain et al., 2016; Liu et al., 2007; Wang et al., 2106). High ROS levels can cause oxidative damage, such as increased lipid peroxidation, protein oxidation, and DNA damage, all affecting cell viability by mechanisms including membrane damage and enzyme inactivation (Oldham and Bowen, 1998; Xing et al., 2008). To protect against ROS-generated oxidative stress, aerobic organisms have evolved antioxidant defense systems (Choi et al., 2008; Mcfarland et al., 1999) that include both enzymatic and non-enzymatic components (McFarland et al., 1999; Wedderburn et al., 2000). Antioxidant enzyme systems including glutathione peroxidase (GSH-PX), glutathione S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT) can remove excess ROS to mitigate the damage (Xing et al., 2008). Hence, the antioxidant system is important for aquatic animals to deal not only with salinity stress but also other dangers in the aquatic environment (e.g. heavy metals, pollutants, etc.) (Deng et al., 2016; Li et al., 2012).

The NF-E2-related factor 2 (Nrf2) transcription factor, as one of the upstream regulators of antioxidant enzyme expression, is a vital part of the cytoprotective apparatus of the cellular detoxification system (Maher and Yamamoto, 2010). Nrf2 itself is regulated by large number of upstream protein kinases responding to diverse signals, including mitogen activated protein kinases (MAPKs), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), PKR-like endoplasmic reticulum kinase (PERK) and glycogen synthase kinase-3 (GSK-3) (Huang et al., 2015; Shay et al., 2012). Among these are the target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) (Shay et al., 2012). From these, we hypothesized that T. s. elegans may activate the Nrf2 regulated antioxidant defense system in response to elevated environmental salinity to support a successful invasion of brackish or saline waters. The present study examines the antioxidant enzymes and the antioxidant defense mechanisms in T. s. elegans responding to salinity challenge. These data further illuminate the physiological tolerances of this invasive turtle species to a range of saline conditions, and also help to assess the capability of this invasive species for future expansion in aquatic environments.

Section snippets

Animals

Healthy T. s. elegans (mean mass 392.3 ± 20.6 g, 3 years old) were obtained from a local turtle farm in Hainan, China. Turtles were held in cement pools in freshwater and fed a commercial diet each Monday and Thursday; 24 h after feeding, unused feed was siphoned out followed by replacement of one-third of the water volume in each pool. Water quality parameters were monitored 2–3 times a week with steady values of pH 7.5–7.9, total ammonia nitrogen (<0.02 mg L−1) and temperature (26–28 °C).

Effect of salinity stress on activity and gene expression of SOD

The mRNA expression levels of SOD1, SOD2 and SOD3 in liver significantly increased after 24 h and 48 h of exposure to 5 psu and 15 psu (Fig. 1A–C). However, the levels were significantly reduced below the controls at 30 d of exposure to either salinity challenge. Cardiac SOD1, SOD2 and SOD3 mRNA levels were significantly elevated at 6 h of salinity exposure but then dropped to levels at or below 50% of the control at 24 h exposure. Then, values rose again at 48 h for the 5 psu. Notably, hepatic

Discussion

In the present study, we demonstrated that T. s. elegans exhibited higher gene expression and activities of SOD, CAT and GSH-PX in liver and heart in response to elevated environmental salinity. In addition, the mRNA levels of Nrf2, a main upstream regulator of antioxidant gene expression, showed a similar upregulation as an early response to stress, and the negative regulator of Nrf2, Keap1, exhibited the opposite pattern. Moreover, TOR and S6K1 mRNA levels showed roughly similar patterns to

Conflict of interest statement

We do not have any conflicts of interest to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31760116, 31772486) and the Natural Science Foundation of Hainan Province (318MS046), Hainan Science Research Project of Higher Education (Hnky2016-15).

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