Short-Term Flow Velocity Stress on the Behavioral, Physiological, and Biochemical Responses of the Large Yellow Croaker (Larimichthys crocea)

Abstract

Flow velocity is a key environmental factor that affects the behavioral strategies and physiological homeostasis of fish. To study the effects of flow velocity on the behavioral changes and blood physiology of the yellow croaker, the behavioral patterns of yellow croakers in response to flow velocity stress were obtained by analyzing changes in tail wagging frequency and amplitude. Differences in blood glucose, lactate, and cortisol were compared to determine their appropriate flow rate ranges. The juvenile stage of the large yellow croaker is crucial, as environmental changes can affect the physiology of fish. Therefore, juvenile yellow croakers were selected as the experimental subjects for this study. Twenty-four healthy and uniformly sized large yellow croakers with body weights of (90.26 ± 9.91) g and body lengths of (19.91 ± 0.69) cm were randomly assigned to one control group and three experimental groups, with five fish in each group. The experimental group was set with three flow rates, namely 1 bl/s (20 cm/s), 2 bl/s (40 cm/s), and 3 bl/s (60 cm/s), with a flow rate stress duration of 1 h. The results showed that: (1) Under different flow velocities, the fish exhibited different tail wagging patterns. At low flow velocities, their tail fins exhibited a “C”-shaped swing, while at high flow velocities, their bodies exhibited an “S”-shaped swing. (2) Oscillation frequency and amplitude both increased with increasing flow velocity. At a flow velocity of 2 bl/s, the oscillation frequency significantly increased. When the flow velocity reached 3 bl/s, the oscillation amplitude significantly increased (p < 0.05). (3) Blood physiology showed significant changes with increased flow rate, and blood glucose content continuously decreased with increased flow rate, significantly decreasing at a flow rate of 2 bl/s (p < 0.05). Lactic acid and cortisol both increased with increasing flow rate, and significantly increased at a flow rate of 3 bl/s (p < 0.05). In summary, under high-flow velocity stress, significant changes occurred in the behavior and physiology of large yellow croakers, which were consistent with physiological changes in the blood. A flow rate higher than 2 bl/s can lead to intense swimming behavior, decreased blood sugar concentration, and increased lactate accumulation and stress levels. Therefore, the short-term tolerance of yellow croakers is 2 bl/s, and a flow rate of 1 bl/s is more suitable.

1. Introduction

Due to limitations in environmental protection and ecological aquaculture, traditional marine aquaculture is undergoing a transformation. China’s marine aquaculture industry is vigorously promoting deep-sea and offshore aquaculture, with aquaculture areas shifting from nearshore to offshore. This has put forward new requirements for the equipment and objects of aquaculture, and changes in aquaculture modes have led to changes in the aquaculture environment, which undoubtedly poses a challenge for the fish being farmed.

The large yellow croaker (Larimichthys crocea) is the largest farmed marine fish in China and one of the most important commercial fish species in coastal waters [1]. The large yellow croaker has clustering and swimming characteristics, which are very suitable for deep-sea aquaculture. The large yellow croaker has become a popular species in marine aquaculture due to its high reproductive capacity, high nutritional value, and delicious meat quality [2]. In recent years, the scale of breeding yellow croakers has gradually expanded, and breeding technology has also matured, resulting in an increase in yield and economic benefits year by year. The main aquaculture methods for large yellow croakers include seawater net cage, soil pond aquaculture, deep-sea net cage, and industrial flow aquaculture. Among them, seawater net cage aquaculture is the most important aquaculture method, accounting for more than 95% of the production of large yellow croakers [3]. At present, with the continuous progress and improvement of aquaculture technology, the scale of large yellow croaker aquaculture has expanded, but it also faces some challenges and problems. The marine environment in offshore cages is complex, and the water flow situation is variable, which is undoubtedly a major challenge for the growth of large yellow croakers at a critical stage. Avoiding discomfort caused by short-term and long-term stress is the core of improving aquaculture welfare, and research in related fields is relatively extensive. Scholars have conducted extensive research on environmental stress in yellow croakers, including studies on temperature [4,5,6], nitrite [7], dissolved oxygen [8], and salinity [9,10], but there have been few studies on water flow. Related studies have shown that wild yellow croakers in higher-flow velocity environments have a thinner and longer body size compared to cultured individuals, and have higher market value [11]. Therefore, studying the effect of flow velocity on the behavior and physiological and biochemical aspects of the yellow croaker is of great significance, but currently there is a lack of research on the effect of flow velocity. This study conducted research in this area based on this.

Blood glucose, lactate, and cortisol are the most widely used blood biochemical indicators to reflect the physiological status and stress situation of fish [12]. Peng [13] determined that the energy supply of silver pomfret (Pampus argenteus) comes from liver glycogen by analyzing the changes in blood glucose levels under transport stress. The lactate concentration in the blood of blackhead seabream (Acanthopagrus schlegelii) increases with the prolongation of exercise time, and the degree of fatigue is positively correlated with lactate concentration [14]. Wu [15] compared the anti-stress ability of rainbow trout (Oncorhynchus mykiss) and Steelhead by analyzing changes in serum cortisol content. Changes in environmental factors can lead to varying degrees of stress response in fish, resulting in altered blood glucose, lactate, and cortisol levels, ultimately leading to behavioral changes.

The purpose of this study was to use computer vision and blood sample collection methods to analyze the changes in behavior and blood physiology of yellow croakers under different flow rate stresses in order to determine the relationship between behavior and blood physiology, and ultimately determine the flow rate range that yellow croakers can tolerate. This study will help improve their living environment, enhance their welfare, and provide a theoretical basis for the deep-sea aquaculture of large yellow croaker.

2. Materials and Methods

2.1. Experimental Fish

To reduce the impact of individual differences on the experiment, 24 large yellow croakers of the same size (19.91 ± 0.69 cm, 90.26 ± 9.91 g) from Xiangshan Port Aquaculture Commercial Breeding Company (Ningbo, China) were used in this study. All the fish were temporarily kept in six 3 m³ ponds. The environmental water temperature was 25 °C, salinity was 21 ‰, dissolved oxygen was approximately 7 mg/L, and light cycle consisted of natural light (12L: 12D). The fish were fed with compound feed (Tianbang) twice a day (9:00 and 16:00).

2.2. Experimental Device

The experiment was conducted in a circulating water aquaculture tank at the pilot base of the Fisheries Machinery and Instrument Research Institute of the Chinese Academy of Fishery Sciences (Figure 1). The system consists of a push flow area, experimental area, filter, temperature control device, biofilter, UV lamp, and reservoir to ensure a clean water environment. The biological filter is equipped with an aeration device to ensure sufficient dissolved oxygen in the experiment. There is a guide plate in front of the experimental area to rectify the jet water flow. The rear of the experimental area is equipped with a fish net, which allows for free adjustment of the size of the experimental area. A camera is installed above the experimental area to record the behavior of the large yellow croaker under flow velocity stress for later observation and analysis. Fill lights are installed in the front and back to fill the test area. The required flow velocity in the experiment is provided by a water pump (Chiquan, ABB-200, Yitian Aquarium Equipment Co., Ltd., Guangzhou, China) for pushing flow. The flow velocity in the experimental area is controlled by controlling the water pump current and adjusting the throttle valve. The size of each swimming room is 30 cm × 60 cm. Multiple points in the experimental area are measured using a flow meter, and the flow velocity inside the swimming room is consistent and uniform. The pushing flow velocity in the tank is 0.1–1.2 m/s.

2.3. Protocol

2.3.1. Experimental Grouping

In previous studies, it was found that the swimming ability of fish is related to body length, so the unit bl/s (body length/second) was used to represent flow velocity in the experiment. According to Zhang’s research, a flow rate of 4 mL/s (1 m/s) was used as the anti-flow rate group for adult yellow croakers (24 cm, 300 g) [16]. However, the anti-flow ability of juvenile yellow croakers was weaker than that of adult fish, so a flow rate of 3 bl/s was used as the high flow rate group in this experiment. The fish were divided into three groups: a low flow velocity group (1 bl/s), medium flow velocity group (2 bl/s), and high flow velocity group (3 bl/s), as well as one control still water group (0 bl/s). The experimental fish were subjected to 48 h of starvation treatment and their intestines were emptied. One fish from each group at a time were placed in each swimming room and replaced after the experiment, for a total of 5 fish. During this period, water quality parameters were monitored to ensure a stable water environment. Before the experiment, the fish were placed in the swimming room to adapt to a 1 h micro water cycle, thereby reducing the pressure on the fish body. At the beginning of the experiment, the flow rate was gradually increased by 0.1 bl/s every 10 s to reach the set flow rate. During the experiment, each fish experienced a flow rate stress of 1 h, with a total of 24 fish requiring 24 h. During this period, the camera recorded the entire process of the tests. After the experiment, the recording was stopped and the pushing flow was stopped. Anesthetic (MS-222) was added to the sink and the fish were removed after complete anesthesia to reduce the impact of the operation on the fish’s body stress. After the experiment, the water in the sink was replaced to prevent the continuing influence of anesthetics.

2.3.2. Indicator Measurement

(1) Behavioral indicators. Video analysis of flow velocity stress in large yellow croakers, selecting tail wagging videos under stable swimming conditions. A 5 s tail flick video of 5 fish in each group was cut and the change in tail flick angle within 5 s was recorded. The video was split into images and the images were analyzed using ImageJ software (https://imagej.net/) (Figure 2A). The time of the maximum positive and negative tail sway angles were recorded and a graph was drawn of the changes in tail sway angles (Figure 2B). Tail swinging frequency was calculated using the following formula:

f = N_T/t

N_T is the number of tail swaying cycles for a duration of 5 s, with t being 5 s.

(2) Physiological index. The experimental fish were anesthetized and blood was collected using the tail handle blood drawing method. After collection, it was left to stand in the refrigerator for 1 h. Then, a centrifuge was used and it was centrifuged at a speed of 3500 r/min at 4 °C for 10 min. After centrifugation, a pipette was used to transfer the serum into a cryopreservation tube, which was stored in a −80 °C refrigerator for the subsequent detection of blood glucose, lactate, and cortisol. Blood glucose (GLU) concentrations were measured via the glucose oxidase method, blood lactic acid (LD) concentrations were measured via the colorimetric method, and blood cortisol (COR) was measured via the double antibody sandwich method with an enzyme-linked immunoassay kit. The above physiological indicators were detected using reagent kits and an enzyme-linked immunosorbent assay (ELISA) reader (Thermo Fisher Scientific Multiskan FC, Waltham, MA, USA). The experimental procedures were strictly carried out according to the instructions.

2.4. Statistical Analysis

Excel 2019 software was used for data organization. All statistical analyses were conducted using SPSS 26.0 software, and the graphics were drawn using Prism GraphPad 9 software. A one-way ANOVA was performed on the data obtained, followed by a Duncan multiple comparison analysis to determine whether there is a significant difference (p < 0.05). The experimental results are represented as mean ± standard deviation (mean ± SD).

3. Results

3.1. Behavioral Changes of Yellow Croakers

3.1.1. Changes in Swinging Tail Form

In the experiment, there were two ways for yellow croakers to swing their tails. One type is a C-shaped tail swing (Figure 3A), with the head moving towards the direction of water flow and utilizing the tail fin swing to maintain balance and relative stillness in flowing water. Another type is the S-shaped swing (Figure 3B), where the body swings and tail fin swings, generating significant thrust to maintain balance in turbulent water flow.

3.1.2. Tail Swing Angle Change

The large yellow croaker shows periodic swaying of its tail, with a maximum swaying angle of no more than 20° at a flow rate of 1 bl/s (Figure 4). The interval between swaying gradually increases, and the tail fin sways in a “C” shape. The large yellow croaker is able to cope with water flow of 1 bl/s calmly. At a flow rate of 2 bl/s, its maximum flapping angle approaches 20°, the flapping frequency gradually increases, and the flapping behavior becomes more pronounced. At a flow rate of 3 bl/s, the tailing behavior becomes more intense, with a maximum tailing angle approaching 40°. The fish swims against the current, presenting an “S” shape with a significantly increased frequency of tail swaying.

3.1.3. Frequency and Amplitude Difference of Tail Wagging

At high flow rates, in order to maintain its position, the large yellow croaker increases the frequency of wagging its tail, and its wagging amplitude also varies (Figure 5). At a flow rate of 2 bl/s, the swimming direction of the yellow croaker remains unchanged, and the tail fin swings in a “C” shape. By increasing the frequency of tail wagging, one can effectively maintain their position. At a flow rate of 3 bl/s, the swing amplitude was significantly higher than that of the 2 bl/s flow rate group and the 1 bl/s flow rate group (p < 0.05). At this time, the swimming movement of the fish body is intense, and the whole body swings in an “S” shape. In this study, the tail frequency and tail angle were fitted, and the linear fitting relationship was expressed as y = 25.33 * x − 15.93 (R² = 0.9241). As shown in the Figure 5 below, the tail swing angle increases with the increase of tail swing frequency. Not only does it increase the frequency and amplitude of tail swinging, but it also needs to constantly adjust its swimming direction to cope with higher flow velocities.

3.2. Physiological and Biochemical Responses of Yellow Croaker

3.2.1. Blood Glucose Changes

Under high flow rate stress, there was a significant change in blood glucose content (p < 0.05) (Figure 6). As the flow rate increases, the blood sugar content gradually decreases. The 2 bl/s flow rate group and 3 bl/s flow rate group showed a significant decrease compared to the control group (p < 0.05), while there was no significant difference between the 1 bl/s flow rate group and the control group (p > 0.05).

3.2.2. Lactic Acid Changes

As the flow rate increases, the lactate concentration of yellow croaker also gradually increases, showing significant changes (Figure 7). There was no significant change (p > 0.05) at a flow rate of 2 bl/s. However, when the flow rate reached 3 bl/s, the lactate concentration significantly increased (p < 0.05).

3.2.3. Cortisol Changes

The cortisol content of yellow croaker showed a trend of first decreasing and then increasing with the increase of flow rate (Figure 8). Among them, there was a significant increase in cortisol content at a flow rate of 3 bl/s (p < 0.05), and the stress level of the fish was relatively high. There was no significant change at other flow rates (p > 0.05).

4. Discussion

By controlling the flow rate, experimental fish are driven to swim, providing important behavioral information related to the fish [17]. There are three ways for fish to increase their swimming speed, namely increasing gill hole drainage, increasing tail wagging frequency, and increasing tail wagging amplitude [18]. The wagging behavior of fish is a manifestation of their movement patterns, and the frequency and amplitude of wagging can reflect the intensity of fish movement [19]. Under the impact of flow velocity, fish can maintain balance through active swimming or posture changes. In this experiment, as the water flow velocity increased, the swimming speed also increased, and the frequency and amplitude of tail swaying continuously changed, which is consistent with the research results of Cai et al. [20]. Wave swimming is the most common swimming method among fish, which controls swimming speed by changing the frequency and amplitude of tail fin oscillation. However, fish seem to be more inclined to control swimming speed by changing the frequency of tail oscillation [21]. Because the almost linear relationship between tail wagging frequency and swimming speed can further simplify speed control, fish do not need to optimize multiple parameters in the frequency and amplitude parameter space. Instead, they can simply focus on adjusting frequency and fixing tail wagging amplitude near the ideal value [22]. In this experiment, there is indeed an almost linear relationship between oscillation frequency and water flow velocity, while oscillation amplitude varied non-linearly. In this experiment, the large yellow croaker swayed its tail at an angle of around 10° under medium to low water flow, and its tail fin swayed in a C-shape. When the flow rate increased to 3 bl/s, the tailing angle increased significantly to 17°, and the whole body swung in an S-shape. When the flow velocity was at different transition points, it exhibited different swimming postures, presenting a C-shaped or S-shaped posture; that is, only tail swinging or full body swinging posture [23]. High flow rates can also increase the cost of swimming, making it difficult for fish to maintain their original direction [24]. Yuan [25] found in their research on the slender split belly fish that increasing the flow velocity within the acceptable range would increase the frequency of tail flapping, reduce the amplitude of tail flapping, improve energy utilization efficiency, and save energy consumption. When fish swim, they need to consume energy to overcome water resistance, and the frequency of tail wagging is positively correlated with flow velocity. An increase in the swing amplitude of the tail fin significantly enhances the swimming ability of the fish body, but at the same time, it also increases the resistance of the fish body in flowing water, increasing its energy consumption. Therefore, the swimming behavior strategy of fish is related to its energy consumption. Viderer’s [26] research suggests that swimming behavior is adjusted based on energy consumption. Fish will use their pectoral fins to brake at low flow rates, reducing the amplitude of their tails and saving energy, while the opposite is true at high flow rates. In this experiment, the yellow croaker only increased its tail wagging frequency to cope with water flow at an adaptive flow rate, and the amplitude did not increase, as an increase in tail wagging amplitude would significantly increase swimming energy expenditure and consume additional energy to maintain body balance. At a flow rate of 3 bl/s, the yellow croaker had to increase its tail swing, improve swimming speed, and correct its position, which can be considered as an uncomfortable manifestation.

Glycogen is the main form of energy storage in the body. During exercise, fish first consume glucose to provide energy for the body. Swimming can improve liver function and protein metabolism in tilapia (Oreochromis niloticus), and promote glucose metabolism effects [27]. In this experiment, under high flow velocity (2 bl/s and 3 bl/s) stress, fish consumed blood sugar to provide energy to the body, resulting in a significant decrease in blood sugar concentration. Due to insufficient blood sugar levels to support subsequent swimming activities, muscle glycogen and liver glycogen will be broken down for energy supply, but some will undergo glycolysis to produce pyruvate, so blood sugar levels will gradually decrease. The blood glucose concentration of largemouth bass decreases with increasing flow velocity at high flow rates [28]. On the contrary, under high flow rates, the blood glucose concentration of Pletropomus Leopardus increases [29], speculatively related to its habits. At high flow rates, the leopard striped gill bass will save energy consumption through behavioral strategies such as standing, and the glucose produced by the large amount of glycogen breakdown in the liver glycogen muscle glycogen cannot be quickly stored, so the blood sugar concentration can be maintained at a high level. The swimming frequency of the long and thin loach increases with the increase of flow velocity to adapt to the surrounding flow velocity. The adaptive swimming in the early stage only relies on aerobic exercise, but when the flow velocity exceeds the adaptive range, it will engage in explosive swimming. At this time, anaerobic respiration provides energy and lactate begins to accumulate [30]. The results of this experiment are similar. At a flow rate of 3 bl/s, the concentration of lactate in the blood continuously accumulates until it reaches its peak because sudden bursts of swimming can lead to increased anaerobic exercise and the accumulation of lactate. In order to maintain muscle motility, lactate in the muscles of fish gradually diffuses into the blood. However, the pH in the blood decreases due to the increase in lactate concentration, thereby reducing the activity of various metabolic enzymes and causing certain physiological side effects [31]. The lactate concentration in the blood of black snapper increases with the prolongation of exercise time, and the degree of fatigue is positively correlated with lactate concentration [32]. Cortisol is a stress indicator of the body, and its content can indirectly reflect the stress level of the body [33]. The changes in serum cortisol content of yellow croakers at different flow rates can reflect physiological differences in the body’s stress response. When subjected to external stress, cortisol will be released into the bloodstream by activating the hypothalamic–pituitary–interrenal (HPI) axis, leading to a series of behavioral and physiological changes [34]. HPI activation leads to the mobilization of energy sources, depletion of glycogen storage, and an increase in blood sugar levels, providing energy for high-intensity muscle activity, while also accompanied by anaerobic glycolysis and an increase in plasma lactate [35]. After 1 h of stress with different flow rates, the cortisol content of yellow croakers showed certain changes, indicating that flow rate is a stress factor for yellow croakers. Yellow croakers respond to the impact of flow rate by adjusting the cortisol content in their body. When fish are under stress, their body will increase its metabolic rate and energy supply. Under the stress of a flow rate of 3 bl/s, the cortisol content of yellow croakers significantly increased, leading to enhanced gluconeogenesis and liver glycogen breakdown to supplement blood sugar. However, due to intense anaerobic exercise, glycolysis generates a large amount of lactic acid, resulting in the accumulation of lactic acid. Therefore, in this experiment, the lactate content and cortisol content of yellow croakers increased with increased flow rate. At other flow rates, no significant changes were observed. At lower flow rates, yellow croakers did not experience stress due to flow rate stress. Since the duration of flow rate stress in this experiment was only 1 h, the effect of long-term flow rate stress on the growth, behavior, physiology, and biochemistry of the large yellow croaker needs further research.

Swimming behavior and blood physiology undergo significant changes at higher flow rates (2 bl/s and 3 bl/s). When the frequency of tail wagging significantly increases, the blood sugar content significantly decreases. When the amplitude of tail wagging significantly increases, the content of lactate and cortisol significantly increases, and the behavior is consistent with the corresponding blood physiology. The behavior of fish is a manifestation of various physiological changes acting externally [18]. Cortisol’s response to stress may be part of a complex set of behavioral and physiological responses, and may represent different strategies or styles of coping with stress [36]. The increase in tail wagging frequency can be considered as a self adaptive behavior of fish, and when a significant change is reached, it indicates that the flow velocity has reached the threshold of its adaptive range. The change in tail swing amplitude can be considered as a result of forced adaptation, as the flow rate has exceeded its tolerance range, causing great physiological burden. Therefore, if you want to understand the appropriate flow rate range for a certain type of fish, you can first analyze it from a behavioral perspective, grasp the upper limit of its tolerance based on the rules of behavioral changes, and then conduct in-depth research on this basis. Although behavioral observation is useful as an early indicator of poor welfare and health status, caution should be exercised when explaining abnormal behavior, and corresponding physiological indicators are necessary. The measurement of physiological stress response naturally occupies an important position in welfare research. In addition, measuring the expression of stress-related genes may provide more precise information for improving their well-being. This study will help to understand the row response and physiological changes of yellow croakers under flow stress, further understand its tolerance, and consider improving aquaculture productivity as well as its welfare.

5. Conclusions

Under high flow velocity stress, there are significant changes in the behavior and physiology of yellow croakers, which show strong consistency in behavior and physiology. The frequency and amplitude of tail wagging correspond to corresponding changes in blood glucose, lactate, and cortisol. A flow rate higher than 2 bl/s can lead to intense swimming behavior, decreased blood sugar concentration, increased lactate accumulation, and increased stress levels. The short-term tolerable flow rate for the deep-sea aquaculture of yellow croakers should be around 2 bl/s, with a suitable flow rate of 1 bl/s. A flow rate of 3 bl/s has a negative impact on their swimming behavior and physiological and biochemical aspects.

Author Contributions

Methodology, C.Z. and H.L.; Validation, Z.Q. and J.X.; writing—original draft preparation, Z.Q.; writing—review and editing, M.C. and H.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

All research on animal participation in this article has passed the welfare and ethical review of experimental animals by FMIRI (FMIRI-AWE-2024-001).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available upon request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures

Figure 1. Experimental device. 1. Water pump push flow area; 2. experimental areas; 3. filters; 4. temperature control zones; 5. biofilters; 6. UV lamps; 7. reservoirs; 8. cameras.

Figure 2. Identification of swinging tail angle and period determination. (A) shows the angle formed by the trunk extension line and the caudal fin is the wagging angle. (B) shows the time required for one tail swinging cycle.

Figure 3. Changes in tail wagging shape. (A) shows the large yellow croaker swaying its tail in a C-shape, while (B) shows the large yellow croaker’s body swaying in an S-shape.

Figure 4. Changes in tailing angle at different flow velocities.

Figure 5. Tail swing amplitude and frequency variation. The “*” in the figure indicates a significant difference (p [less than] 0.05) between the two sets of data (n = 5). Data are expressed as mean ± SD.

Figure 6. Changes in blood glucose content at different flow rates. The “*” in the figure indicates a significant difference (p [less than] 0.05) between the two sets of data (n = 5). Data are expressed as mean ± SD.

Figure 7. Changes in lactate content under different flow rates. The “*” in the figure indicates a significant difference (p [less than] 0.05) between the two sets of data (n = 5). Data are expressed as mean ± SD.

Figure 8. Changes in cortisol content under different flow rates. The “*” in the figure indicates a significant difference (p [less than] 0.05) between the two sets of data (n = 5). Data are expressed as mean ± SD.

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