Effect of Different Environmental Stresses on the Antioxidant Capability of Air-Breathing Loach, Paramisgurnus dabryanus (Sauvage, 1878)

Yaqiu Liu1, Zhijian Wang 2, Xinhui Li 1*, Jianrong Zhao2 and Weitao Chen 1

1Pearl River Fisheries Research Institute, Chinese Academy of Fishery Science, Guangzhou, China

2Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Southwest University, Chongqing 400715, China

ABSTRACT

The effect of oxidative stress on gill and gut enzyme activity in Paramisgurnus dabryanus was studied under different breathing restraint treatments. A total of 150 healthy mature individuals (mean initial body weight = 16.1 ± 3.7 g and mean initial total fork length = 13.1 ± 1.2 cm) were selected for the experiment. The experimental specimens were separated into three modes: control group (n = 50) without any stress treatments; inhibited group (n = 50) with gut respiratory inhibition; and an air-exposed group (n = 50) with gill respiratory inhibition. After a 7 d acclimation for the inhibited and air-exposed groups, individuals were returned to the same conditions as the control group for a further 3 d acclimation. The catalase (CAT) and superoxide dismutase (SOD) activity in the gill and gut in P. dabryanus were examined in all three groups after the 7 d stress acclimation, 1 d later, and after a 3 d recovery. It was found that CAT and SOD activity in the gill, foregut, and midgut in the air-exposed group were much lower than in the control group. After recovery for 1 d, CAT activity in the gill and middle gut increased rapidly and was higher than in the inhibited and control groups. The SOD activity in the gill and gut also significantly increased, although there was no significant difference between the air-exposed and control groups. After a 3 d recovery, CAT and SOD activity in the gill and gut of P. dabryanus did not differ significantly between groups. There was a high viability of P. dabryanus under the air-exposed condition (in anaerobic conditions), while the level of oxidative stress in the gill, anterior, and middle gut clearly decreased. When recovering under normoxia conditions, the physical level of oxidative stress quickly returned to normal levels.

Article Information

Received 29 October 2019

Revised 02 December 2019

Accepted 27 December 2019

Available online 08 January 2021

Authors’ Contribution

YL and ZW designed the study. YL

and JZ performed the experiments and analyzed the data. ZW and WC helped in experimental work and data analysis. YL and XL wrote the article.

Key words

Air-exposed, Gut, Respiratory inhibition, Oxidative stress, Paramisgurnus dabryanus

DOI: https://dx.doi.org/10.17582/journal.pjz/20191029011014

* Corresponding author: [email protected]

0030-9923/2021/0002-0431 $ 9.00/0

Copyright 2021 Zoological Society of Pakistan

INTRODUCTION

The oxygen concentration in aquatic ecosystems has a direct impact on fish. It is often affected by diurnal changes in environmental factors, water temperature changes, and drought. Many fish are very sensitive to small changes in the oxygen concentration. To supplement oxygen consumption during aquatic hypoxia, some fish have evolved several adaptive strategies in terms of structure, physiology, and behavior (Catling et al., 2001; Gonzales et al., 2006; Huang et al., 2015). For example, members of the Loricariidae and the Callichthyidae families rely on specific tissues, such as vascularized stomach or gut tissue, for aerial gas exchange under hypoxic conditions (Bickler et al., 2007; Pelster et al., 2016).

It has been reported that there are more than 400 species of fish that have an accessory organ for air-breathing (Graham, 2011). Some accessory air-breathing organs are located in the digestive tract, such as the esophagus, stomach and gut (Podkowa and Goniakowska-Witalinska, 2002). Gut air-breathing as an accessory respiratory approach are mainly subordinate to Cobitidae and Callichthyidae (Nelson, 2014). Studies of the gut air-breathing of fish such as Misgurnus anguillicaudatus (Goncalves et al., 2007), Lepidocephalichthys guntea (Moitra et al., 1989), Hoplosternum thoracatum (Huebner et al., 1978), Corydoras aeneus (Podkowa et al., 2002) have been reported. Paramisgurnus dabryanus relies on the gut for assisted respiration, and its accessory organs are located in the posterior part of the gut, which implies regional modifications of the gut for gas exchange. Like many other air-breathing fish, the respiratory structures of P. dabryanus are vascularized and have a thin diffusion distance between the external and internal mediums (Moitra et al., 1989; Goncalves et al., 2007).

Gut air-breathing, evolved as a physiological behavior and affects the fish’s normal physiological and behavioral activities and its metabolic patterns and oxidative stress when repressed. The oxygen concentration required for aerobic metabolism may also cause the production of reactive oxygen species (ROS) (Pelster et al., 2016). The accumulation of ROS may result in oxidative stress, which is harmful for organisms (Lushchak, 2014). ROS concentrations vary with changes in oxygen availability, and organisms have developed sophisticated defense systems to eliminate reactive oxygen molecules when exposed to hypoxic and hyperoxic conditions (Welker et al., 2013). Hypoxia is one of the most common conditions that can cause oxidative stress (Welker et al., 2013). Catalase (CAT) and superoxide dismutase (SOD) play an important role in removing ROS. In addition, previous studies have clearly shown that the defense system against ROS is not static but is highly responsive to changing environmental conditions (Lushchak and Bagnyukova, 2006; Welker et al., 2013). For example, a significant increase in ROS degrading enzyme activities and in the concentration of antioxidants is frequently observed following periods of hypoxia.

However, the response of air-breathing fish to oxidative stress under different environmental stresses has not been reported in the literature. Therefore, we studied the changes of antioxidative capacity under water and respiratory gas respiration inhibition in separate treatments to determine the effect of environmental stress on oxidative stress in air-breathing fish. Basic adaptive evolutionary studies were consulted to provide basic information.

 

MATERIALS AND METHODS

Experimental fish and holding conditions

Loach (Paramisgurnus dabryanus) specimens used in the experiment were collected from a local market in Dianjian, Chongqing, China (32°20′N; 107°21′E). The body weight was 15.2 ± 2.3 g and fork length was 13.3 ± 1.2 cm. The experimental specimens were kept in a re-circulating water tank (180 × 120 × 80 cm) in the laboratory for two weeks. In the tanks the fish had free access to air and could therefore breathe air voluntarily. During this period, the temperature of the de-chlorinated freshwater used was maintained at 21.3 ± 1.7°C the oxygen level was kept above 7.0 mg L-1, the pH ranged from 6.7 to 7.5. The photoperiod was maintained at 12 h light: 12 h dark and fish were fed with a commercial fish food (Xiwang Group, Chongqing, China).

Experimental protocol

At the end of domestication, 150 healthy fish of a similar size were selected and randomly divided into three groups (Fig. 1): (1) inhibited group (n = 50), in which the P. dabryanus were kept active below the water surface for intestinal respiration; (2) air-exposed group (n = 50), in which P. dabryanus were exposed to the air, although their bodies were kept; and (3) a control group, without any inhibition (n = 50). The details of the experimental protocol are shown in Figure 2.

 

 

Sample collection

Six fish from each group were randomly selected for the determination of antioxidant enzyme activity. Body weight and fork length were measured after anesthesia with 0.1 g / L ethyl m-aminobenzoate. The fish were placed on an ice tray and the gills, foregut, midgut, and hindgut were quickly removed into liquid nitrogen. The organs were then placed in a cryogenic refrigerator at −80°C for the determination of further enzyme activity.

Determination of enzyme activity

The samples were removed from the refrigerator and placed on ice to thaw. After thawing was completed, each sample was weighed and homogenized. The homogenate was diluted with a F6 / 10 FLUKO hand-held

 

Table I. Comparison of basic morphological parameters among different group (n=9).

	Control group	Inhibited group	Air-exposed group
Weight (g)	15.90 ± 4.29	16.02 ± 4.10	16.19 ± 3.24
Fork length (cm)	13.04 ± 0.82	12.35 ± 1.20	13.76 ± 1.05
Gut mass (g)	0.27 ± 0.08	0.21 ± 0.05	0.25 ± 0.06
Gut length (cm)	6.36 ± 0.84	5.47 ± 0.62	6.48± 0.37
Gut length/fork length	0.49 ± 0.05	0.45 ± 0.06	0.47 ± 0.05
Gut mass/body mass	0.017 ± 0.01	0.013 ± 0.003	0.016 ± 0.004
Gill mass (g)	0.25 ± 0.02	0.19 ± 0.03	0.20 ± 0.02
Gill mass/body mass	0.016 ± 0.004	0.014 ± 0.005	0.013 ± 0.003

 

homogenizer for 5 min in an ice bath to ensure that the sample was fully ground. The volume ratio of the sample mass to the volume of the homogenate diluent was set to 1:9. The resulting homogenate was centrifuged using a cryogenic ultracentrifuge and the supernatant was used for the determination of antioxidant enzyme activity. CAT (EC1.11.1.6) and SOD (ECl.15.1.1) activity were measured in gill and gut tissue using a CAT assay kit (NO: A007, Nanjing Jiancheng Bioengineering Institute, PR China) and SOD assay kit (NO: A001, Nanjing Jiancheng Bioengineering Institute). All assays were performed on duplicate samples using a Thermo Multiskan spectrophotometer (Thermo Fisher Scientific, MA, USA). UV-permeable Corning 96-well microplates were used for all assays. All reactions were run at the saturating substrate concentrations determined for each enzyme (Li et al., 2013).

Statistical analysis

IBM SPSS 19.0 was used for all statistical analysis of the recorded data. The effect of acclimation group (inhibited, air-exposed, control) and days on CAT and SOD activity were determined by a two-way analysis of variance (ANOVA). The effect of acclimation group (inhibited, air-exposed, control) on morphological parameters were determined by a one-way ANOVA followed by Dunnett’s test. Any difference at P < 0.05 was regarded as statistically significant. All values were expressed as mean ± SD.

 

RESULTS

Morphological changes

After domestication for 7 d, there were no significant differences in the morphological parameters of P. dabryanus between groups (Table I).

Gill antioxidant activity

After domestication for 7 d, the gill CAT activity in the air-exposed group was significantly lower than that in the control and inhibited group (P < 0.05), while there was no significant difference in CAT activity between the control and inhibited group (Fig. 3a). After recovery for 1 d, the CAT activity in P. dabryanus was significantly increased in the air-exposed group (P < 0.05), which was higher than that in the control and inhibited groups (P < 0.05) (Fig. 3a). After recovery for 3 d, the CAT activity in P. dabryanus was significantly decreased (P < 0.05), and there was no significant difference in CAT activity between the two groups (Fig. 3a). The gill SOD activity in the air-exposed group was much lower than that in the control and inhibited group (P < 0.05), while the SOD activity in P. dabryanus in the control and inhibited group were not significant different (Fig. 3b). After recovery for 1 d, the SOD activity in P. dabryanus increased significantly in the air-exposed group (P < 0.05), and after recovery for 3 d, there was no significant difference in SOD activity between the groups (Fig. 3b).

Foregut antioxidant activity

After domestication for 7 d, the foregut CAT activity in P. dabryanus in the air- exposed group was much lower than that in the control and inhibited groups (P < 0.05) (Fig. 4a). The CAT activity in the foregut of P. dabryanus was significantly increased (P < 0.05) after recovery for 1 d, and there was no significant difference in CAT activity between the groups (Fig. 4a). The SOD activity in P. dabryanus in the air-exposed group was lower than that in the control and inhibited groups (P < 0.05) (Fig. 4b). After a 1 d recuperation, the SOD activity in P. dabryanus was significantly increased (P < 0.05) in the air-exposed group and there was no significant difference in CAT activity between groups (Fig. 4b).

Midgut antioxidant activity

CAT activity in the midgut of P. dabryanus specimens in the air-exposed group was lower than that of the control group after domestication for 7 d (P < 0.05) (Fig. 5a). After recovery for 1 d, the CAT activity in the midgut of P. dabryanus sharply increased in the air-exposed group, and was much higher than that in the control and inhibited groups (P < 0.05) (Fig. 5a). The midgut SOD activity in P. dabryanus in the air-exposed group was significantly lower than that in the control and inhibited groups (P < 0.05) (Fig. 5b). After a 1 d recovery, SOD activity was significantly increased (P < 0.05) in the midgut of P. dabryanus specimens that were exposed to air (Fig. 5b). After recovery for 3 d, the SOD activity in P. dabryanus decreased significantly in the air-exposed group (P < 0.05). There was no significant difference in the CAT activity between groups (Fig. 5b).

 

Hindgut antioxidant activity

After domestication for 7 d, the CAT activity in the hindgut of P. dabryanus in the inhibited group was higher than that in the control and air-exposed groups (P < 0.05), and there was no significant difference in CAT activity between groups after recovery for 1 d (Fig. 6a). The SOD activity in P. dabryanus in the air-exposed group was lower than that in the control and inhibited groups (P < 0.05) (Fig. 6b). SOD activity in P. dabryanus in the air exposed group increased rapidly after a 3d recovery (P < 0.05), and there was no significant difference in the SOD activity between groups (Fig. 6b).

 

DISCUSSION

ROS production commonly accounts for 0.1-0.2% of the daily oxygen consumption in fish (Gorr et al., 2010). Large amounts of ROS can damage tissue structures (Costantini et al., 2010). Generally, the CAT and SOD systems are regarded as important cellular defense systems against oxidative stress.

 

We found that the air-breathing loach P. dabryanus can adjust the levels of oxidative stress it experiences when confronted with different environmental conditions. In recent studies, CAT and SOD activity in the gills, foregut, and midgut of P. dabryanus has been shown to decrease significantly when the fish are exposed to air. In contrast, in jeju (Hoplerythrinus unitaeniatus), the exposure of fish tissues to air may stimulate ROS production (Pelster et al., 2016). Under normal physiological conditions, organisms continually eliminate ROS and thus avoid damage to the body caused by oxidation. The mechanism of its elimination depends mainly on the body’s antioxidant defense system, with CAT and SOD having important roles. We found that the CAT and SOD activity in the hindgut of P. dabryanus increased in the inhibited group. Studies have shown that when fish face stress conditions, ROS are produced more than they are scavenged (Hur et al., 2001). The ROS content accumulates, which results in oxidative stress, leading to the body reaching a pathological state. Demple (1999) showed that in a body under chronic stress conditions the activity of antioxidant enzymes can increase to reduce oxidative damage.

 

When the fish were placed in normoxic conditions for 1 d, the CAT and SOD activities were significantly increased, which may be due to the feedback regulation of dissolved oxygen in the environment. Some studies of the antioxidant system have observed an increased activity in antioxidant enzymes under hypoxic stress or following a return to normoxic levels (Costantini et al., 2010; Majmudar et al., 2010). Other studies have shown that fish are accompanied by significant oxidative stress when they recover from hypoxia, and some extremely hypoxia-tolerant animals are more susceptible to oxidative stress during recovery than under hypoxia (Bickler and Buck, 2007).

With the re-introduction of oxygen, ROS may rapidly accumulate in tissues or cells. Excess ROS can cause oxidative stress and disorders of the antioxidant system (Lushchak et al., 2005). The results of this study are consistent with those of de Oliveira et al. (2005), who found that the antioxidant capacity of the body during reoxygenation may change significantly under hypoxia or stress, which may be an antioxidant strategy that organisms use to adapt to hypoxia and reoxygenation. After a 3 d resumption of normoxia, there was no significant difference in the antioxidant enzyme activity between groups. Under different environmental conditions, the fluctuation of the oxygen utilization rate in the matrix itself has an important effect on the ROS content (Welker et al., 2013). Pannunzio (1998) found that with the prolongation of hypoxia stress, the amount of oxygen in the body decreased and the SOD was reduced to O2ˉ reducing its activity in the body. When restored to normoxia for 1 d, the CAT in the air exposed group was significantly increased and was higher than that in the control group. The aerobic capacity of the hindgut in the air exposure group and the inhibition group was similar to that of the control group. Different tissues have different adaptions to overcome environmental stress. These reactions are all distinctive anti-oxidant strategies that prevent the body’s ROS from damaging itself.

 

CONCLUSIONS

Based on the above data, when P. dabryanus was exposed to the hypoxic and inhibited treatments, they had a high viability under the air-exposed condition (in anaerobic conditions), while the level of oxidative stress in the gill, foregut, and midgut clearly decreased. When exposed to normoxia conditions, P. dabryanus recovered quickly. The aquatic air-breathing fish P. dabryanus was able to regulate its physiological responses to the environmental disturbances provided in this study.

 

ACKNOWLEDGEMENTS

This work was financially supported by the Special Fund for Agro-scientific Research in the Public Interest under Grant number 201203086, Guangdong Province Natural Science Fund under Grant number 2016A030313147, Open Fund project of Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education) under Grant number Klas-2018-01. National Natural Science Foundation of China under Grant number 31870527.

 

Statement of conflict of interest

Authors have declared no conflict of interest.

 

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