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Article

Stress and Energy Mobilization Responses of Climbing Perch Anabas testudineus During Terrestrial Locomotion

by
Efim D. Pavlov
1,*,
Tran Duc Dien
2 and
Ekaterina V. Ganzha
1
1
Institute of Ecology and Evolution A.N. Severtsov of the Russian Academy of Sciences, 119071 Moscow, Russia
2
Coastal Branch of Joint Vietnam-Russia Tropical Science and Technology Research Center, Nha Trang 65000, Vietnam
*
Author to whom correspondence should be addressed.
Stresses 2025, 5(3), 45; https://doi.org/10.3390/stresses5030045
Submission received: 25 June 2025 / Revised: 17 July 2025 / Accepted: 21 July 2025 / Published: 23 July 2025
(This article belongs to the Section Animal and Human Stresses)
Browse Figure
Versions Notes

Abstract

The climbing perch, Anabas testudineus, is one of the most widely distributed freshwater amphibious fishes in South and Southeast Asia, exhibiting terrestrial movements. Our experimental study aimed to investigate endocrinological and biochemical changes in the blood of climbing perch associated with their terrestrial movements. To achieve this, the fish were divided into two groups: one group was exposed to aquatic conditions for twenty minutes, while the other group was subjected to terrestrial conditions for the same duration through rapid water level decrease. In terrestrial conditions, the fish predominantly exhibit movements on land, whereas in aquatic environments, they primarily remain immobile or swim. Elevated levels of stress-induced cortisol and glucose after short-term exposure indicate a high-stress response involving both neuroendocrine and metabolic mechanisms. Changes in the activity of aspartate aminotransferase and increased concentrations of triglycerides in the blood serum suggest energy mobilization through aerobic metabolic pathways. Extreme environmental changes did not affect thyroid axis function, including deiodination, thereby maintaining essential physiological activities under new conditions. Additionally, the anaerobic metabolic pathway appears to be minimally utilized at the onset of terrestrial movement, as no significant changes in lactate dehydrogenase concentrations were observed. Overall, the terrestrial movements of the climbing perch are likely predominantly forced and associated with high stress.

1. Introduction

A wide diversity of fishes exhibits amphibious behaviors for a variety of reasons. Amphibious fishes are a sub-category of air-breathing fishes that are defined as fishes that naturally spend part of their life out of the water, and so are considered “amphibious” [1]. Such fish not only stay (sometimes for quite a long time) on land, but also move around on the ground in various ways [2,3]. The majority of studies of amphibious fishes have focused on elucidating the mechanisms that underlie breathing air, determining what triggers emersion, and understanding the physiological demands of aerial exposure. Consequently, little is known about the maximum distance that air-breathing fishes can travel over land, the behavioral variations in predisposition to emerge within or across populations, the cost of transport during terrestrial sojourns, or specific physiological features of amphibious fishes to provide movement on land [4].
The climbing perch, Anabas testudineus, is one of the most widely distributed and renowned freshwater fish in Southeast and South Asia. The species is an obligate air-breather with morphological and physiological adaptations, such as a suprabranchial (labyrinth) organ, that enable it to move overland [2,5,6,7,8]. Sustained terrestrial movements allow the climbing perch to spread among hydrologically independent bodies of water [2,6,7,9,10,11]. The ecological significance of its movements on land are interpreted as a result of specific feeding behavior [12,13], reproduction [5,14], evading predators [13], avoiding competition due to high population density in the habitat [10], or as a response to significant environmental factors, such as elevated water temperature [14] or a drastic decrease in water levels [9]. It appears that seasonality, in comparison with the high habitat heterogeneity of the climbing perch, is an important factor regulating the terrestrial movements of this species. Moreover, the life history and population structure of the climbing perch can vary in different regions of the species’ range [3], which provides flexibility in their occurrence of land movements. The terrestrial movement of climbing perch may play a crucial role in their survival and dispersal in response to changing environmental conditions, such as habitat droughts, associated with global warming in the modern world.
The climbing perch exhibits terrestrial movements through its gill covers, which are adapted to a wide range of motion in combination with the rotation of the subopercle in both ventral and lateral directions [8]. The fish can traverse difficult soils and climb steep surfaces on land [9] due to these morphological features. It is likely that performing some or all of the above functions in air for climbing perch would require more energetic expenditure than performing similar functions in water [2,11]. Additionally, movement out of water may be associated with increased stress in fish [9], leading to several changes in internal physiological processes, such as endocrine regulation and metabolism. Facultative air-breathing enables amphibious fishes to maintain aerobic metabolism when the supply of dissolved oxygen is limited, or demand is increased during bouts of activity [1]. Metabolic reorganization and alterations in intermediary metabolic pathways occur in fish to meet their increased energy demands for adaptations to environmental changes [15]. The adaptive role of these processes in Anabas testudineus during its terrestrial movements remains unclear.
In the present study, we estimated the blood concentrations of thyroid hormones, cortisol (Cort), triglycerides (TGs), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and glucose (GLU) in climbing perch both in aquatic environments and on land. The levels of these parameters characterize the changes in energy production, aerobic (AST) and anaerobic (LDH) metabolism [16,17,18]. To assess fish stress during terrestrial movements, we measured the concentrations of the stress-induced hormone cortisol [18,19]. Also, thyroid hormones could regulate the pattern and magnitude of stress response in fishes as they modify either their own actions or the actions of stress hormones [20]. We suggest that fish terrestrial locomotion is a stress-induced and energetically demanding process that activates the organism’s internal resources to access new bodies of water containing suitable habitats for the species. A comprehensive analysis of the studied biochemical parameters will enhance our understanding of the adaptability mechanisms related to the terrestrial movements of amphibious fish.
The study aimed to experimentally assess the biochemical changes in the blood of climbing perch associated with terrestrial movements.

2. Results

During the acclimatization period in the starting tank, the climbing perch either moved or remained motionless on the bottom. Some fish lay on the ladder near the water surface, but their bodies were always fully submerged in the water. The fish often moved toward the surface to gulp air. Additionally, they jumped out of the water.
In aquatic conditions (drain valves were closed), the fish exhibited the same behavior as observed previously. These fish remained in the water and did not move to the dry corridor.
During trials involving terrestrial conditions (drain valves were open), the fish changed their movement patterns. They more frequently rose to the water surface compared to the control group. The climbing perch predominantly moved to the corridor after the water level decreased to less than 3 cm, around the fourth to fifth minute of the trial. Additionally, fish were able to move into the corridor as early as one minute after the water level began to decrease. Climbing perches could enter the corridor in small groups of two to three individuals. The fish exhibited two types of entering the corridor: jumping into the entrance or climbing using their pectoral fins on the ladder. The first type was experienced when the water level was sufficient for jumping (>2 cm). Occasionally, individuals moved back from the corridor to the start tank during the trial. By the end of the trial, only 30% of the fish remained in the start tank.

2.1. Thyroid Hormones and Cortisol

Similar thyroid hormone concentrations were observed in fish both in aquatic and in terrestrial conditions (Table 1). Furthermore, the percentage of FT3 fraction (%FT3) and T4/T3 ratio values were comparable between these groups. Cortisol levels increased 1.9-fold in fish following terrestrial movement compared to individuals maintained in aquatic conditions (Kruskal–Wallis H test: H1;40 = 19.92; p = 8.07 × 10−6).
The concentrations of free triiodothyronine (FT3) and cortisol showed significant correlations with fish body length (FT3: rs = 0.38, p = 0.017; Cort: rs = 0.42, p = 0.007) and body weight (FT3: rs = 0.37, p = 0.018; Cort: rs = 0.35, p = 0.029). Cortisol levels were negatively correlated with the T4/T3 ratio (rs = −0.40, p = 0.010).

2.2. Triglycerides and Protein Exchange Parameters

Following terrestrial movement in climbing perch, triglyceride and glucose concentrations increased significantly by 1.8-fold (Kruskal–Wallis H test: H1;37 = 6.52; p = 0.011) and 1.3-fold (Kruskal–Wallis H test: H1;39 = 4.09; p = 0.043), respectively, while aspartate aminotransferase levels decreased by 1.1-fold (Kruskal–Wallis H test: H1;35 = 7.11; p = 0.008) (Table 2).
Glucose levels showed significant positive correlations with fish body length (rs = 0.35, p = 0.029) and weight (rs = 0.48, p = 0.002), but a negative correlation with free triiodothyronine (FT3) concentration (rs = −0.55, p = 0.001). Triglyceride concentrations correlated positively with both body weight (rs = 0.44, p = 0.006) and FT3 levels (rs = 0.40, p = 0.016). Aspartate aminotransferase activity was positively associated with thyroxine (T4) concentration (rs = 0.48, p = 0.004).

3. Discussion

We documented some behavioral and biochemical blood changes in climbing perch during the initiation of terrestrial movements. Our study design represents the first attempt to elucidate the complex biochemical traits associated with terrestrial movement in this species. Additionally, we recognize that in natural environments, numerous factors can influence these traits, including the feasibility of terrestrial movement. Poor water quality, as well as biotic factors such as competition, predation, terrestrial feeding, and reproductive activities, can stimulate amphibious fishes to emerge from the water [2,11,21]. Therefore, we interpret our results as one possible response of the fish to rapid environmental changes, specifically associated with decreasing water levels. For instance, the behavioral and biochemical responses observed in climbing perch could similarly occur in local ponds during the dry season, where this species frequently inhabits.
Our results indicate that climbing perch do not often leave the aquatic environment when the water levels are between 18 and 20 cm. The fish rested on the ladder or swam with occasional jumps throughout the trial under stable water conditions. Previous studies have shown that climbing perch rarely leave the aquatic environment spontaneously; they typically do so only in response to preliminary stimulation, such as water level reduction [9]. This suggests that fish experiencing less stress are less likely to exhibit terrestrial movements, while more anxious individuals tend to move more frequently. Water deficit prompts climbing perch to adopt facultative breathing behaviors and alters their movement patterns—from jumps to climbing and from swimming to terrestrial movements. However, under the same water deficit conditions, climbing perch exhibited different behavioral responses: some fish displayed increased terrestrial movement, while others remained predominantly motionless in the start tank. In natural environments, decreasing water levels similarly affected portions of fish populations: some individuals became more mobile and exited the littoral zone as inshore habitats experienced low water levels [22]. In our study, we did not differentiate between active and immobile individuals, as individuals often alternated between these behaviors during the trial. We suggest that water deficit induces a combination of behavioral and physiological changes in fish, which are also reflected in alterations of their blood biochemistry.
The studied biochemical parameters, including thyroid hormone concentrations and lactate dehydrogenase activity, did not vary significantly with environmental transitions from aquatic to terrestrial settings or with terrestrial movements of the fish. We hypothesize that the two main reasons underlie the stability of these parameters in the blood. The first pertains to the short-term duration of environmental change, which may be insufficient for the thyroid axis and lactate dehydrogenase synthesis to respond. The second reason is that the primary role of thyroid hormones involves the regulation of fundamental biological processes—such as metabolism, growth, development, and reproduction [23]—which often require stability of thyroid axis function under various stressors. The thyroid axis modulates and fine-tunes certain adaptable processes, such as osmoregulation, in conjunction with the actions of more labile hormones like cortisol and adrenaline [20]. This is consistent with data indicating that thyroid hormone concentrations remained similar in adult pink salmon Oncorhynchus gorbuscha during osmotic stress [24], while cortisol levels increased [18]. Our study also observed that four additional blood parameters in fish changed rapidly after water lowering. We hypothesize that three primary biological factors contribute to these biochemical changes: stress induced by the rapid decrease in water levels, the involvement of facultative air respiration, and terrestrial movements.

3.1. Parameters Related to Stress

The stress response in fish can be triggered by various external factors, including environmental changes such as water level fluctuations [25]. Upon experiencing stress, the fish’s response is characterized by the activation of the hypothalamus, which initiates the neuroendocrine system and triggers a cascade of metabolic and physiological adjustments [26,27]. The primary neuroendocrine response involves the perception of an altered internal state by the central nervous system, leading to the release of stress hormones—such as cortisol, adrenaline, and epinephrine—into the bloodstream through endocrine pathways [28]. Secondary (metabolic) responses occur as a result of the action of stress hormones released during the primary response [29]. These responses induce changes in blood and tissue chemistry, such as an increase in plasma glucose levels [30,31]. This metabolic pathway generates a surge of energy—primarily in the form of glucose—to support tissue functions, including muscle activity, and to prepare the fish to respond to an emergency [32,33]. Additionally, stress-induced metabolic responses can lead to behavioral modifications [19].
Based on this sequence, we observed both primary and secondary stress responses in climbing perch. These responses were characterized by elevated levels of cortisol and glucose in the blood serum of individuals exposed to terrestrial conditions, in contrast to fish maintained in aquatic conditions. The acute stress response mechanism in fish appears to be non-specific and essential for mobilizing energy resources to actively counteract unfavorable environmental factors. Short-term energy mobilization may be particularly advantageous for climbing perch, as terrestrial movements in amphibious fish are likely to have a higher energetic cost than movements in an aquatic environment [11]. Additionally, air respiration in climbing perch seems less energetically efficient compared to their normal aquatic respiration.

3.2. Parameters Related to Air Respiration and Terrestrial Movements

Data regarding the internal mechanisms of air respiration in amphibious fish are limited and fragmentary. Studies on the ventilation mechanics of Protopterus aethiopicus have shown that during aestivation, abdominal muscles are used to forcibly deflate the lung—a mechanism not employed during breathing in water [34,35]. According to Damsgaard et al. [1]; therefore, it can be hypothesized that aerial respiration on land may be more energetically demanding than respiration in water.
Lipids serve as a key indicator of overall energy reserves in fish and are also the primary energy source for muscle activity during endurance swimming [36,37,38]. Fish are reported to maintain a basal fatty acid flux that exceeds the additional energy demands associated with movement [39]. Triglycerides in the blood of climbing perch may be particularly relevant, as blood serves as a partial reservoir of muscle fuel [40]. In the present study, triglyceride concentrations increased by 1.8-fold following terrestrial movement in climbing perch compared to fish maintained in aquatic conditions. This increase appears to be related to the fish’s need for greater energetic resources to support air respiration and land-based movements. Moreover, the control group fish also moved within the aquatic environment (start tank) and occasionally gulped air from the water surface. However, these movements were more restricted compared to fish exposed to terrestrial conditions, likely due to the availability of air respiration opportunities and limited water space. Our findings align with data indicating that toxic treatment (2,4-dinitrophenol) combined with exhaustive swimming in zebrafish Danio rerio stimulated a significant increase in whole-body triglycerides [40]. Interestingly, this effect was observed only following toxic exposure. It is plausible that the initial occurrence of a stress factor is a key driver for the elevation of triglyceride levels in fish, as triglyceride concentrations often decrease in polluted environments [41,42]. Similar situations may occur in habitats of climbing perch, such as the irrigation Am Chua canal, where fertilizers, pesticides, and insecticides from neighboring rice fields may enter freely.
Aspartate aminotransferase (AST) is a multifunctional enzyme essential for fish aerobic metabolism, facilitating efficient energy production, amino acid utilization, and metabolic flexibility. An increase in blood AST activity in fish may indicate enhanced amino acid metabolism, stress, infection, dietary changes, or habitat heterogeneity [17], and it can serve as an indicator of protein oxidation capacity [43]. During long-term migration in salmonids, AST levels increased in white muscle tissue, suggesting a role in supplying fuels and intermediates potentially through tissue breakdown during prolonged fasting [43]. In our study, we observed a decrease in AST activity during terrestrial movement of climbing perch. This reduction may be related to its mobilization and the subsequent supply of glucose via gluconeogenesis, as has been demonstrated for amino acids [15]. Various amino acids act as donors in deamination reactions and are utilized as primary substrates for gluconeogenesis and oxidative metabolism in fish, particularly in migratory species that may experience extended periods of starvation [44]. Further research is needed to elucidate this mechanism in climbing perch.
During exhaustive exercise, metabolism shifts toward anaerobic glycolytic pathways, with a greater reliance on white muscle tissue [36]. Lactate dehydrogenase (LDH) is a crucial enzyme in the anaerobic metabolic pathway. The activity of its two isoenzymes (LDH-A and LDH-B) is particularly important in oxygen-deprived environments [45,46]. Elevated LDH-A levels have been associated with tolerance to air deprivation and muscle development during the growth of larval snakeheads Channa punctatus, which, like climbing perch, utilize a suprabranchial organ for aerial gas exchange [16]. However, the functional (physiological) implications of these changes in LDH isozyme levels remain to be fully understood. In our study, we did not observe significant changes in LDH activity during exposure of climbing perch to either aquatic or terrestrial conditions. This suggests that the aerobic metabolic pathway plays a dominant role during short-term terrestrial movements. The activation of anaerobic metabolism in climbing perch may depend on various external and internal factors, including the individual’s physiological condition, as well as the duration and intensity of terrestrial locomotion.

4. Materials and Methods

4.1. Animals and Housing

The study was carried out in February 2022. Wild fish capturing was performed in the Am Chua canal (12°17′26″ N, 109°06′04″ E), which flows into the Cai River near Nha Trang city (Central Vietnam). The canal flows from the Am Chua Reservoir and is primarily used for agriculture. The distance between the banks varies from 1 to 12 m, with a maximum depth of 2.5 m. The main canal is more than 17 km long, with at least two significant inflows.
Adult climbing perch Anabas testudineus were caught using a cast net measuring 5 m in length and 5 m in diameter, with a mesh size of 10 mm. Forty captured fish, with a total length (TL) of 8.0–13.7 cm and a weight of 8.8–42.7 g, were transferred in 20 L tanks to the laboratory within 30 min of capture.
In the laboratory, tap fresh water was preconditioned by settling and aeration for two weeks in a 2000 L pool. Dissolved oxygen levels in water were 7.0–7.2 mg/L (Pro Dissolved oxygen meter MW600, Milwaukee, WI, USA) and mineralization was 200 ppm (Xiaomi TDS Pen, Beijing, China). The fish were acclimatized to the new water by gradually adding it to the transport tanks until the water was fully replaced. During the study, the stability of the water’s chemical parameters was periodically checked using Sera Aqua-test kits (Sera GmbH, Heinsberg, Germany).
Single 80 L (56 × 38 × 38 (width × height × depth) cm) aquaria were used to maintain the fish stock. The aquarium was filled with 50 L of water, and the water temperature was 25–27 °C (HI 98509 Checktemp 1, Hanna instruments, Nușfalău, Romania). The sides of the aquaria were translucent white to reduce wild fish disturbance. Aquarium was covered with a white plastic sheet. The cover had a rectangular hole for the automatic feeder (Eheim Auto Feeder, EHEIM GmbH, Deizisau, Germany). Fish were fed twice daily, at 7:00 and 17:00 (GMT+7), with Humpy Head dry pellets (Yi Hu Fish Farm Trading, Singapore) at 6.5 g pellets per tank. The feed ration was ~2% of the average fish weight. Fish began consuming the feed pellets within the first three days after being transferred to the laboratory. Morning feeding was skipped in the stock tank from which individuals were taken for behavioral testing, ensuring that only unfed fish were used in the trials.
External filters (Eheim Classic 150, EHEIM GmbH, Deizisau, Germany) were used to filter the water in stock aquarium. The filters were cleaned once every five days. Illumination was natural (through laboratory windows) and varied throughout the day from 0 to 100 Lx (Lutron LX-1102 light meter, Lutron Electronic Enterprise Co., Ltd., Taipei, Taiwan). Illuminance in the stock tanks was on average comparable to the natural illuminance in the turbid water of the Am Chua canal, which ranged from 1 Lx (near the bottom) to 250 Lx (near the surface) at 12:00 (GMT+7) (modified Lutron LX-1102 light meter with waterproof sensor).
We followed the protocol of the Organization for Economic Cooperation and Development (OECD) [47] for fish acclimatization before the beginning of experiments, according to the following statement: 48 h settling-in + 7 days acclimatization = 9 days; mortality of <5% of population in seven days before the start of the test acceptance of batch.

4.2. Experimental Apparatus

The experimental apparatus was used to estimate the differences in biochemical parameters of climbing perch in aquatic environments and during terrestrial movements (Figure 1). The apparatus was a reduced version of a dry labyrinth [9] and consisted of two adjacent opaque white plastic tanks: a start tank and a dry corridor. The start tank measured 40 × 40 × 60 cm (length × width × height) and had a 20 × 20 cm entrance to the dry corridor, positioned 20 cm above the bottom. A ladder extended across the entire bottom of the start tank, leading to the entrance at a 27° angle. A guillotine door closed the entrance from the start tank to the dry corridor. A drain valve near the bottom of the start tank was used for water control. The dry corridor measured 80 × 20 × 20 cm (length × width × height). The ladder and the bottom of the corridor were covered with yellowish fabric to increase adhesion for the terrestrial movement of the climbing perch. A video camera (GoPro HERO 8 Black, GoPro Inc., San Mateo, CA, USA) with remote control was mounted above the apparatus to enable real-time monitoring of the fish without visual contact. We propose that the corridor section in the apparatus is essential for providing fish with a stimulus and serving as a signal for terrestrial movement, which would be difficult to detect in a single-section setup (start tank).

4.3. Behavioral Tests

The trials were conducted from 8:00 to 12:00 (GMT+7) on a single day, with an average illumination of 100 Lx in the start tank and 200 Lx in the dry corridor. Throughout the experiment, both the air and water temperatures inside the apparatus remained similar, at approximately 27 °C. Preliminary experiments (unpublished data), including in situ tests, indicate that climbing perch tend to move toward brighter areas both in water and on the ground. We suggest that the higher illumination in the dry corridor served as a stimulus, encouraging more movement of the fish in that direction.
Thirty liters of water were poured into the start tank. Before each session, ten fish were randomly selected from the stock aquarium, gently placed in the start tank, and allowed to acclimate for 20 min. The use of ten fish simultaneously in each trial was justified by climbing perch’s natural group behavior, as solitary maintenance induces stress-related behavioral artifacts [48,49]. After acclimation, the fish were tested under one of two trial conditions: in one, the guillotine door was open (control), and in the other, both the guillotine door and drain valve were open. When the drain valve was opened, the water depth in the start tank decreased to 1.0 cm after five minutes, and all tested fish exhibited facultative air respiration. The first condition maintained a regular aquatic environment, while the second condition simulated rapid water level decline, creating conditions analogous to a terrestrial habitat. The decrease in water level served as one of the major stimuli for terrestrial movements of the climbing perch, as mentioned earlier [9]. Each trial was limited to twenty minutes to record terrestrial movements of the fish. The trial duration of 20 min was chosen because this time is sufficient to observe terrestrial movements in most climbing perch individuals. Additionally, this duration does not significantly affect the fish’s health, as all fish survived after the treatment [9]. At the end of the trial, the guillotine door was closed, and the number of fish in the start tank and the dry corridor was counted. A total of forty fish were used across four trials: twenty fish were tested under stable water levels, and twenty fish were tested under rapid water level decline. Each fish was used in the experiment only once.

4.4. Blood Sampling

Individual blood sampling was conducted after each trial. Fish were carefully transferred from the apparatus using a dip net. One person handled the fish with wet textile gloves, while another collected blood through a single puncture of the caudal vessel using a disposable 1 mL syringe. The sampling procedure took less than one minute per fish. No anesthetics were used before blood sampling, in accordance with several protocols [18,50] due to the need for quick manipulations. After blood sampling, the body length (TL) and weight of each fish were measured, and the fish were transferred to a separate tank with water. Fish subjected to stable (aquatic) and declining water (terrestrial) conditions had similar length and weight (Mann–Whitney U test: p > 0.73). All fish remained alive after 48 h of manipulation. Following a visual assessment of fish health, they were released back into their natural environment.
Each individual blood sample, with a total volume of 50–100 μL, was poured into 0.5 mL tubes. One hour later, serum was separated by centrifugation at 2000 rpm for five minutes. The individual serum samples were transferred to new tubes, labeled, stored, and transported to the laboratory at −20 °C.

4.5. Biochemical Analysis

In the laboratory, serum samples were thawed at room temperature. Each sample was diluted fivefold with phosphate-buffered saline (PBS) to obtain the necessary volume for biochemical analysis. Commercial enzyme-linked immunosorbent assay (ELISA) kits from DRG International (Marburg, Germany) were used to analyze total triiodothyronine (T3), free triiodothyronine (FT3), total thyroxine (T4), and cortisol (Cort). The hormone levels in each individual sample were measured using ELISA equipment (Mindray, Shenzhen, China). The T4/T3 ratio was calculated to assess the rate of conversion of T4 to T3 (deiodination) [51,52]. Additionally, the percentage of the biologically active fraction of FT3 (%FT3) within the total T3 fraction was determined [53].
We determined four additional blood parameters using an automatic biochemistry analyzer, iMagic-S7 (Shenzhen iCubio BioMedical Technology, Shenzhen, China), with Diakon-Vet commercial biochemical kits (Pushchino, Russia). Protein metabolism was evaluated by measuring lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and glucose (GLU). Lipid metabolism was assessed through triglycerides (TGs).
Sometimes, the number of used samples was less than the number of sampled fish due to insufficient serum volume required for all studied parameters or due to equipment errors. Final values of the studied parameters were converted to absolute values based on the serum samples’ dilution factor.
Statistical data analysis was conducted using Minitab 18.1. Initial assessment of data normality using the Shapiro–Wilk test revealed non-normal distributions (p < 0.05 for all variables). We therefore implemented non-parametric analyses: Mann–Whitney U test for between-group comparisons of morphometric parameters (length and weight); Kruskal–Wallis H test for evaluating differences in biochemical blood parameters across experimental groups; and Spearman’s rank correlation coefficients to examine relationships among biochemical parameters and morphometric measurements. Statistical significance was defined as p < 0.05, with adjustment for multiple comparisons using Holm’s sequential Bonferroni procedure.

5. Conclusions

Our experimental study demonstrates that climbing perch undertake terrestrial movements primarily in response to strong environmental stressors, such as the rapid decline in water level observed in this study. However, this species may also leave the aquatic environment due to various other factors, including predation risk, high competition arising from limited food and shelter sources, spawning behaviors, and numerous additional causes. In any case, we suggest that terrestrial movements in climbing perch are often forced and carry a high risk of mortality, as the effectiveness of fish navigation on land and their ability to locate new water bodies have not been conclusively demonstrated. Nevertheless, these movements are important, especially as global warming potentially increases the risk of habitat droughts, and fish survival is highly dependent on their ability to compete for and find new habitats.
The observed increases in stress-induced cortisol and glucose levels during short-term exposure (20 min) to terrestrial conditions indicate a high-stress response, driven by both neuroendocrine and metabolic mechanisms. Changes in aspartate aminotransferase activity and elevated triglyceride concentrations in the blood serum suggest energy mobilization via aerobic metabolic pathways. Notably, extreme environmental changes did not appear to impact the thyroid axis, including deiodination processes responsible for maintaining vital functions under new conditions. Additionally, the anaerobic pathway was minimally involved at the onset of terrestrial movements, as no significant changes in lactate dehydrogenase activity were detected.
This study provides the first detailed insights into the biochemical mechanisms underlying the adaptation of amphibious fish to terrestrial locomotion. Nevertheless, further research is essential to deepen our understanding of these processes.

Author Contributions

E.D.P. and E.V.G. conceived and designed the study, performed data analyses and visualization. E.D.P. and T.D.D. conducted aquarium observations and experiments. E.D.P. conceptualized the study, interpreted the data, and wrote the original draft of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

Field work and experiments were supported by Joint Vietnam–Russia Tropical Science and Technology Research Center (Ecolan 3.2 “Taxonomic diversity, ecology and behavior of freshwater hydrobionts”, Mission 1).

Institutional Review Board Statement

All experiments were conducted in accordance with the Vietnam National Regulations on the Use of Animals in Research (Decree 32/2006/ND-CP, 2006), the Guide for the care and use of laboratory animals (Guide, 2011), the Guidelines for the treatment of animals in behavioral research and teaching (Guidelines, 2022), and the recommendations of the Bioethics Commission of the A. N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences (https://sev-in.ru/en/komissia-po-bioetike (accessed on 5 November 2021)).

Data Availability Statement

The data and materials could be available by response.

Acknowledgments

Authors would like to thank the administration and staff of Coastal Branch of the Joint Vietnam–Russia Tropical Science and Technology Research Center for their help in organizing sample collection and for kindly allowing us to use their laboratories and experimental facilities.

Conflicts of Interest

We declare the absence of competing interests.

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Stresses 05 00045 g001
Figure 1. Scheme of the experimental apparatus.
Figure 1. Scheme of the experimental apparatus.
Stresses 05 00045 g001
Table 1. Concentrations of total (T3) and free (FT3) triiodothyronine, total thyroxine (T4), cortisol (Cort) in the blood serum of Anabas testudineus and values of %FT3 and T4/T3 ratios.
Table 1. Concentrations of total (T3) and free (FT3) triiodothyronine, total thyroxine (T4), cortisol (Cort) in the blood serum of Anabas testudineus and values of %FT3 and T4/T3 ratios.
ParametersAquatic ConditionsTerrestrial Conditions
ValuenValuen
T3, ng/mL8.3 ± 2.69 (4.0–14.8)209.9 ± 2.47 (4.9–13.9)20
FT3, pg/mL10.2 ± 5.26 (4.9–22.6)2012.8 ± 8.26 (3.5–32.8)19
T4, μg/dL13.1 ± 2.92 (8.6–19.5)2013.2 ± 3.29 (8.7–22.4)20
%FT30.13 ± 0.077 (0.06–0.32)200.13 ± 0.060 (0.05–0.24)18
T4/T317.9 ± 8.9 (8.4–47.4)2014.0 ± 4.18 (7.6–25.4)20
Cort, ng/mL234 ± 58.4 (150–380)20448 ± 171.4 (214–877)20
Note. Gray rows indicate significant differences (Kruskal–Wallis H test: p < 0.05) in the values between groups. n—number of samples; before brackets are the mean value and standard deviation; in the brackets are min and max.
Table 2. Concentrations of triglycerides (TGs), glucose (GLU), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in the blood serum of Anabas testudineus.
Table 2. Concentrations of triglycerides (TGs), glucose (GLU), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in the blood serum of Anabas testudineus.
ParametersAquatic ConditionsTerrestrial Conditions
ValuenValuen
TGs, mmol/L2.5 ± 2.02 (0.3–6.8)184.4 ± 2.33 (1.1–8.5)19
GLU, mmol/L7.6 ± 3.48 (2.4–16.5)209.7 ± 3.35 (2.7–14.8)19
AST, U/L495 ± 25.6 (434–538)19455 ± 52.2 (352–542)16
LDH, U/L1211 ± 74.1 (1034–1294)191180 ± 97.1 (935–1330)19
Note. Gray rows indicate significant differences (Kruskal–Wallis H test: p < 0.05) in the values between groups. n—number of samples; before brackets are the mean value and standard deviation; in the brackets are min and max.
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Pavlov, E.D.; Dien, T.D.; Ganzha, E.V. Stress and Energy Mobilization Responses of Climbing Perch Anabas testudineus During Terrestrial Locomotion. Stresses 2025, 5, 45. https://doi.org/10.3390/stresses5030045

AMA Style

Pavlov ED, Dien TD, Ganzha EV. Stress and Energy Mobilization Responses of Climbing Perch Anabas testudineus During Terrestrial Locomotion. Stresses. 2025; 5(3):45. https://doi.org/10.3390/stresses5030045

Chicago/Turabian Style

Pavlov, Efim D., Tran Duc Dien, and Ekaterina V. Ganzha. 2025. "Stress and Energy Mobilization Responses of Climbing Perch Anabas testudineus During Terrestrial Locomotion" Stresses 5, no. 3: 45. https://doi.org/10.3390/stresses5030045

APA Style

Pavlov, E. D., Dien, T. D., & Ganzha, E. V. (2025). Stress and Energy Mobilization Responses of Climbing Perch Anabas testudineus During Terrestrial Locomotion. Stresses, 5(3), 45. https://doi.org/10.3390/stresses5030045

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