Frequency-Specific Responses: The Impact of an Acoustic Stimulus on Behavioral and Physiological Indices in Large Yellow Croaker

Abstract

This study assessed the impact of an acoustic stimulus on the behavioral responses and physiological states of the large yellow croaker (Larimichthys crocea). The test fish, with an average body weight of approximately 352.81 ± 70.99 g, were exposed to one hour of acoustic stimulation at seven different frequencies: 100 Hz, 125 Hz, 160 Hz, 200 Hz, 500 Hz, 630 Hz, and 800 Hz. The aim was to delineate the specific effects of acoustic stimulation on the behavior and physiological indices. The results show that acoustic stimulation significantly altered the behavioral patterns of the large yellow croaker, predominantly manifested as avoidance behavior towards the sound source. At a stimulus frequency of 630 Hz, the test fish exhibited continuous irregular motion and erratic swimming. Physiologically, one hour of exposure to acoustic stimulation notably affected the endocrine system. The levels of Epinephrine and thyroxine were significantly elevated at 200 Hz, while the cortisol levels did not show significant differences. Additionally, the lactic acid content significantly increased at 800 Hz, and the blood glucose content peaked at 630 Hz. This study discovered that sound frequencies of 200 Hz, 630 Hz, and 800 Hz led to a significant increase in the levels of Epinephrine, glucose, thyroid hormones, and lactate in large yellow croaker, consequently affecting their behavior. The changes in these physiological indicators reflect the stress response of the large yellow croaker in specific sonic environments, providing crucial insights into the physiological and behavioral responses of fish to acoustic stimuli.

1. Introduction

Hearing plays a crucial role in the life of Sciaenidae fish. They should be sensitive to underwater ambient noise and need to make timely adaptive responses to effectively improve their survival rate and reproductive success [1].

With the rapid development of the global aquaculture industry, the welfare of fish has increasingly attracted attention [2]. Among the issues being dealt with, the impact of noise has emerged as a novel research direction within fish welfare studies. As human aquatic industrial activities expand, the effects of anthropogenic noise on marine organisms have become a focal point in international academic circles [3,4,5]. These noises can induce shifts in the hearing threshold of fish, damaging not only the sensory cells of the inner ear but also disrupting communication and other biological sounds. This disturbance can reduce the foraging efficiency of fish and make them more susceptible to predation [6,7,8,9,10]. Furthermore, an increase in noise levels can be traced to significant alterations in fish behavior and physiology [11,12,13,14]. Exposure to high-intensity noise can even lead to fish mortality [15]. Consequently, noise pollution poses a significant threat to marine life, highlighting the importance of the implementation of effective measures to reduce its detrimental effects.

Jacobsen et al. [16] investigated the impact of boat noise on the behavior of native fish during human water activities. Their findings revealed that Rutilus rutilus significantly elevated its swimming speed upon the initiation of boat motor operation. Additionally, Perca fluviatilis exhibited an increased frequency of swimming episodes following the onset of ship noise, suggesting that such noise is a primary source of disturbance. Fewtrell et al. [17] demonstrated that an increase in air gun sound pressure levels can induce behavioral changes in fish and squid, affecting both their positioning in the water and their swimming speed. While numerous studies have focused on the influence of noise on fish behavior, some of the more severe effects may not be readily observable at the behavioral level. Fish may endure noise disturbances and remain in noisy environments to fulfill essential biological needs, such as foraging and reproduction. However, this tolerance does not mean that fish are unaffected by noise [18]. On the contrary, noise-induced stress responses can have detrimental effects on reproductive success, growth rates, and immune system function. Additionally, behavioral responses to stress vary depending on individuals’ unique coping styles [19]. The differences in individual response capabilities may explain why some fish can tolerate noise better, while it is harmful to others [20].

Smith et al. [21] discovered that Carassius auratus did not exhibit sustained physiological stress responses to long-term exposure to acoustic stimuli. However, the effects of short-term acoustic exposure were pronounced. Notably, following 21 days of noise exposure, it required an additional 14 days for the goldfish to fully reset their hearing levels. This indicates that fish with sensitive hearing are sensitive to stress and potential hearing loss due to acoustic exposure. Santulli et al. [22] determined that acoustic stress induced by air gun explosions leads to alterations in the blood indices of fish, including cortisol, glucose, and lactic acid levels. However, X-ray imaging revealed that the vibrations resulting from these explosions have no significant macroscopic effects on the skeletal structure. Daniel Alves et al. [23] investigated the impact of ship noise on Halobatrachus didactylus and found that it significantly influences the species’ acoustic communication and group vocalizations. This disruption could potentially impair individual reproductive success, thereby affecting the ecological adaptability of the species. Celi et al. [24] highlighted that human-generated noise negatively affected several biochemical indicators in Sparus aurata, with significant increases observed in their levels of adrenocorticotropic hormone (ACTH), cortisol, blood sugar, lactic acid, hemocrit, and 70 kDa heat shock protein in the test fish exposed to ship noise environments. This research underscores the detrimental influence of anthropogenic noise on specific physiological processes in fish. It is essential to consider and assess the potential threats of noise pollution to aquatic biodiversity and ecosystems’ health.

During the 1950s and 1960s, the utilization of the large yellow croaker’s auditory sensitivity led to unsustainable fishing methods, including reefing operations and overfishing, which nearly drove wild croaker populations to extinction. In recent years, however, there have been significant strides in the artificial breeding of this species and the advancement of progressive marine aquaculture technologies. Consequently, the annual production of farmed large yellow croaker has been on the rise, positioning it as the marine fish with the most extensive aquaculture operations and the highest yield within China’s marine fisheries. However, human-generated noise can have a range of adverse effects on the large yellow croaker within its breeding environment. By understanding the auditory characteristics of the large yellow croaker, it is possible to design a more conducive breeding environment that minimizes noise interference. This approach can enhance both the breeding efficiency and the quality of the fish. Zhang et al. [25] investigated the otolith characteristics of the large yellow croaker and observed that within the hearing frequency range of 200 to 1300 Hz, the shear stress response exhibited a trend of an initial increase followed by a decrease, peaking at 800 Hz. This finding underscores the importance of understanding the effects of acoustic exposure at various frequencies on the large yellow croaker. It forms the basis for mitigating the threats posed by human-generated noise to the species’ welfare and for developing effective farming systems.

This study integrates behavioral and physiological approaches to study the large yellow croaker’s response to sound exposure across a range of frequencies. By analyzing behavior trajectory diagrams and sequence diagrams and conducting physiological index assessments, the research aims to identify the species’ sensitive frequency range to sound. This will facilitate the development of strategies to minimize the impact of anthropogenic noise on large yellow croaker populations. Furthermore, the findings will offer a scientific foundation for refining the regulatory mechanisms of stress effects induced by acoustic stimulation. This knowledge is crucial for devising effective measures for ecological and environmental protection, safeguarding the fisheries industry, and ensuring human food security.

2. Materials and Methods

2.1. The Selection of Experimental Site and Subjects

The subject of the experiment was the man-made cultivated L. crochet in a farming facility within Fujian Province. Prior to experimentation, it was temporarily housed in a stringent indoor cement reservoir for 2 weeks (6.3 × 6.3 × 0.6 m). The marine water utilized was sand-filtered, and the water temperature was maintained at 19.0 ± 0.5 °C. An aerator was deployed to constantly supply oxygen to the temporary pool, while pellet feed was offered at 9:00 a.m. and 14:00 p.m. each day. A total of 80 viable specimens were selected in the experiment, with an average body mass of 352.81 ± 70.99 g. The test specimens were all mature 1-year-old male fish. The Use of Laboratory Animal Committee, Dalian Ocean University, approved the animal studies and all procedures.

2.2. Behavioral Response to Sound Stimulation

The experiment was conducted within a metal tank (width: 170 cm, length: 200 cm, height: 120 cm, 408 L) under conditions which maintained consistency with the transient environment (Figure 1). In this research, a function generator and power amplifier were employed to interface the underwater loudspeaker (UWS-015 type, sensitivity 105 dB re 1V/μPa, amplification angle 180 °, bandwidth 80~20 kHz) for sound dissemination. The acoustic stimulation frequency assortment was as follows: 100 Hz, 125 Hz, 160 Hz, 200 Hz, 500 Hz, 630 Hz, and 800 Hz sine wave; the sound pressure level intensity was set at 155 dB, and each stimulus duration was 1 h (Figure 2).Underwater background noise was measured using a Japanese AQH hydrophone (sensitivity: −193 dB re 1Vμ/Pa, frequency bandwidth 20 Hz–20 kHz, Japan).

As a wave, frequency and amplitude become important attributes to describe sound waves. The magnitude of the frequency corresponds to what we commonly refer to as pitch, while the amplitude affects the loudness of the sound. Sound can always be decomposed into a superposition of sine waves with different frequencies and intensities through Fourier transform [26]. Therefore, the sound exposure stimulus in this experiment uses sine waves for stimulation.

The frequency range for sound production and reception in large yellow croakers is primarily 130 to 2,100 Hz, with the peak spectrum falling between 550 and 800 Hertz. Therefore, sine wave frequencies of 100 Hz, 125 Hz, 160 Hz, 200 Hz, 500 Hz, 630 Hz, and 800 Hz can be chosen as effective stimulus sources for acoustic stimulation tests [27,28].

A custom behavior analysis sequence map code for the monitoring video was employed to scrutinize the entire sound stimulation procedure, including the behavioral traits of the test fish preceding and after the sound release, the reaction of the test fish to the sound stimulus, and the modification of swimming behavior.

In this operation, Toxtrac was employed to interpret the dynamics exhibited by the footage captured by the high-speed motion camera and to construct a diagrammatic representation of the dynamic trajectory of the large yellow croaker.

Toxtrac is a Windows-based software program, freely available and specifically optimized for animal tracking applications. It employs an advanced tracking algorithm and boasts robust processing capabilities, enabling the rapid and precise tracking of one or more individual animals across various environments [29,30].

In this study, we conducted an analysis of the acoustic stimulation at varying frequencies and the ambient underwater noise levels within the experimental zone. The results revealed that the peak sound pressure level of the ambient noise was measured at 95.19 ± 0.85 dB. In contrast, the peak sound pressure level induced by the noise in the experimental group was significantly higher, recorded at 133.44 ± 4.19 dB.

2.3. Physiological Response Stimulated by Sound

Following 1 h of acoustic stimulation, six large yellow croaker samples were randomly selected from a steel tank for subsequent blood collection. Each frequency was tested once, and a total of seven experiments were conducted. The fish were anesthetized using MS-222 (use concentration of 100 mg/L, MS-222, chemical name: 3-Aminobenzoic acid ethyl ester methanesulfonate, produced by Shanghai Busi Chemical Co., LTD,Shanghai, China). Once anesthesia was confirmed, blood samples were obtained via the caudal vein. These samples were refrigerated at 4 °C for 6 h prior to centrifugation at 8000 RPM for 15 min to separate and collect the serum. Subsequently, the serum levels of cortisol, Epinephrine, and thyroxine were quantified using enzyme-linked immunosorbent assay (ELISA), while the blood glucose index was assessed by colorimetric methods, and the serum lactate level was measured by means of spectrophotometry. All reagents and kits utilized for these determinations were sourced from the Nanjing Jiancheng Bioengineering Institute, and the assays were conducted strictly in accordance with the provided protocols.

2.4. Data Analysis

Statistical analysis was conducted using SPSS 26.0 software. Data are presented as mean values ± standard error (x ± SE). A one-way analysis of variance (ANOVA) was applied to assess the physiological index data across samples from each group, which were subjected to acoustic stimulation at different frequencies. Post hoc comparisons between groups were performed using the least significant difference (LSD) test. A value of p < 0.05 was considered to indicate a statistically significant difference.

3. Results

3.1. The Impact of Noise on the Behavior of the Large Yellow Croaker

Analyzing the behavioral responses of the test fish subjected to 155 dB of acoustic stimulation at varying frequencies for 1 h revealed distinct patterns. Initially, the fish exhibited a transition from exploratory behavior to adaptability in response to the acoustic stimulus. This was characterized by an approach towards the sound source, followed by a rapid retreat, under different frequency conditions. At certain stimulation frequencies, the test fish were observed to be attracted to the source, approaching it slowly or quickly, and remaining in the vicinity for a brief period. In the later stages of acoustic stimulation, particularly at 630 Hz, the test fish consistently engaged in irregular movements and swam aimlessly. Moreover, under other frequencies of stimulation, the fish demonstrated adaptability to the sound, typically swimming along the perimeter of the tank in a regular or leisurely manner, with no significant disturbance reactions observed.

The behavioral sequence spectrum for the large yellow croaker was constructed by chronologically arranging symbols representing distinct behavioral characteristics of the species (Figure 3). This spectrum illustrates the spatial and temporal distribution of the species’ responses to a spectrum of acoustic stimuli frequencies. Notably, it reveals that the test fish displayed the most complex behaviors under acoustic stimulation at 500 Hz and 630 Hz.

In Figure 3, RM represents “random motion”, SC represents “slowly close to the sound source”, KG represents “kicking–gliding”, CR represents “cruise response”, DR represents “disturb response”, SU represents “speed up”, and SR represents “stand still response”. This diagram illustrates the behavior of the experimental fish over time within 1 h of sound stimulation.

Upon reviewing the videos depicting the behavior of the fish subjected to acoustic stimulations at various frequencies, a frequency count of the different behaviors was conducted. The statistical outcomes are depicted in Figure 4 below. We found that the experimental fish exhibited the highest number of behavioral responses to the post-acceleration stimulus at 800 Hz, with 435 recorded instances. In contrast, the greatest number of responses to the post-cruise departure stimulus was observed at 630 Hz, totaling 356 occurrences. Additionally, the frequency of gradual approaches with subsequent remaining behavior peaked at 630 Hz, with 177 occurrences. Taken together, the test fish’s behavior of cruising away upon approach showed relative stability with an increase in frequency. The behavior of gradually closing in to remain was most pronounced at 630 Hz, although its overall frequency gradually decreased. Conversely, the frequency of “accelerated departure upon approach” showed a progressive increase with a rising frequency.

The term “Approach and stay” refers to the behavior where test fish gradually move towards the sound source and remain in its vicinity. “Approach and cruising away” refers to the behavior where the test fish and, upon reaching the sound source, departs at a steady speed and in a consistent direction. “Approach and accelerating away” describes a behavior where the test fish, upon nearing the sound source, will suddenly accelerate and rapidly change its swimming direction, exhibiting a pattern akin to an escape response, which can be termed “fast escape”.

ToxTrac open-source software was used to analyze the recorded videos and obtain the behavioral and motion trajectory results for large yellow croaker (Figure 5).

The trajectory diagram of large yellow croaker, as presented in Figure 5, indicates that the species’ primary area of activity is situated opposite the sound source. Upon the initiation of sound emission, the large yellow croaker begins a series of evasive maneuvers, moving away from the sound source. With an increase in the frequency of acoustic stimulation, there is a corresponding gradual transformation in the fish’s movement posture. Initially, the large yellow croaker makes attempts to approach the sound source, but these approaches are followed by a progressive retreat. The fish’s trajectory eventually shifts to circular swimming near the inner wall on the left side of the experimental tank. Additionally, the amplitude of its swimming pattern gradually diminishes, resulting in an oval-shaped trajectory.

3.2. The Impact of Noise on the Philological Indexes of the Large Yellow Croaker

As the frequency of acoustic stimulation increased (100 to 800 Hz), the lactic acid levels in the large yellow croaker exhibited a trend of an initial increase, followed by a decrease, and then another increase (Figure 6a). Notably, when the stimulation frequency was set at 800 Hz, the experimental group demonstrated the highest lactic acid levels. Specifically, the lactic acid concentration in the 800 Hz group peaked at 3.29 mmol/L. In contrast, the 100 Hz group showed the lowest lactic acid levels, at 2.34 mmol/L, with no significant difference from the control group. Furthermore, significant increases in lactic acid levels were observed in fish subjected to stimulation frequencies of 125 Hz, 160 Hz, 200 Hz, and 800 Hz (p < 0.05).

In contrast, the blood glucose levels of the large yellow croaker increased, with the exception of the 160 Hz stimulation frequency (Figure 6b). The 160 Hz group exhibited the lowest blood glucose level at 4.99 mmol/L, with no significant difference from the control group. At a stimulation frequency of 630 Hz, the experimental group’s blood glucose levels reached a maximum of 8.54 mmol/L, which was significantly higher than that of the control group (p < 0.05).

Post-acoustic stimulation, blood cortisol levels increased in all cases (Figure 6c). At a frequency of 200 Hz, the cortisol levels were at their highest, reaching 27.73 nmol/L, marking a 27.5% increase. Although the cortisol levels in the acoustic stimulation groups were consistently higher than those in the control groups throughout the experiment, no significant difference was observed (p > 0.05).

The Epinephrine levels in the blood also increased following acoustic stimulation (Figure 6d). The Epinephrine level of the 100 Hz group was the lowest at 5.10 mmol/L, with no significant difference from the control group. At a stimulation frequency of 200 Hz, the experimental group’s Epinephrine levels peaked at 6.44 mmol/L. The Epinephrine levels at 200 Hz, 630 Hz, and 800 Hz were significantly higher than those of the control group (p < 0.05).

Compared with the control group, the thyroxine level of large yellow croaker in the experimental group increased under different frequencies of acoustic stimulation for 1h (Figure 6e). The lowest thyroxine content was 66.72 mol/L in the 160 Hz test group, which was not significantly different from the control group. When the frequency of the acoustic stimulation was 200 Hz, the maximum thyroxine level of the experimental group was 79.95 mol/L. The thyroxine of Larimichthys crocea with stimulation frequencies of 100 Hz, 200 Hz, 500 Hz, and 630 Hz was significantly higher than that of the control group (p < 0.05).

Following 1 h of acoustic stimulation, all five stress-related physiological indices in the plasma of large yellow croaker demonstrated varying degrees of increases, with notable increases in the Epinephrine, thyroxine, and lactic acid levels (Figure 6). The levels of lactic acid, Epinephrine, and thyroxine in the large yellow croaker exposed to 200 Hz acoustic stimulation were significantly elevated compared to the control group (p < 0.05). Similarly, under 630 Hz acoustic stimulation, the plasma levels of blood glucose, Epinephrine, and thyroxine were also significantly higher than in the control group (p < 0.05). These results indicate that the large yellow croaker is particularly sensitive to acoustic frequencies of 200 Hz and 630 Hz.

4. Discussion

The large yellow croaker accelerated escape behaviors after approaching the sound source, and the trajectory changed significantly. The trajectory of the large yellow croaker stimulated by 100 Hz sound was similar to that of the normally cruising large yellow croaker after approaching the sound source. The trajectory of the large yellow croaker was affected at the position close to the sound source, with a small trajectory offset (towards the left side of the experimental flume) and accelerated away from the sound source area. As the frequency of acoustic stimulation increased to 160 Hz, the amplitude of the trajectory deviation increased, and the phenomenon of accelerated escape was more obvious. When the frequency of acoustic stimulation increased to 630 Hz, the amplitude of the trajectory deviation and reverse escape was the largest, and the range of the whole motion process deviated from the position of the sound source, which was close to the left side of the experimental tank. The initial emission of acoustic stimulation caused the large yellow croaker to be frightened, which caused the large yellow croaker to have a frightened reaction, but then some large yellow croaker adapted to the signal and were no longer disturbed by the appearance of the signal; after approaching the sound source, the normally cruising large yellow croaker did repeated circle swimming in the experimental tank, and the trajectory did not change significantly. It may be because the adaptation of fish’s hearing to sound stimuli with constant frequencies and intensities is very rapid [31].

Fish are stimulated by the outside world, and the body will secrete cortisol, which can reflect the stress status of fish, and the concentration will rise rapidly after being stimulated [32]. In this study, we found that there was no significant difference in the cortisol concentration in the blood of large yellow croaker during the experimental exposure period, which is consistent with the findings of Smith et al. [33]. After 10 min of acoustic stimulation, the cortisol concentration in the blood of the experimental group was three times that of the control group, and after 1 h of stimulation, the concentration returned to the normal value. This indicates its adaptive regulation in response to acoustic exposure stimulation. Shi Huixiong et al. [34] studied the effect of ship noise on the cortisol level in the blood of perch and large yellow croaker and found that after the Lateolabrax japonicus and large yellow croaker were stimulated by noise, the cortisol level in the blood first increased, then decreased, and finally returned to the original level. This may be due to the fact that fish have adapted to these stimuli following 1h of stimulation, resulting in a negative feedback regulation of the Hypothalamic–pituitary–adrenal (HPA) axis, and prolonged stimulation increases the tolerance to cortisol [35]. Yarahmadi [36] observed that rainbow trout had significantly increased cortisol levels under crowding stress. To ensure the accuracy of our findings and to avoid spurious results, we also advocate for the prior standardization of the organism in question before conducting any stress-related experiments.

A significant physiological response to stressors involves the release of adrenocortical hormones, which subsequently induce a secondary metabolic change characterized by an increase in plasma glucose levels [37]. This response generates a substantial amount of energy for the metabolic pathway as a whole, thereby equipping the fish with the necessary resources to manage emergency situations [38]. Studies have shown that fish can affect the glucose metabolism by changing the activity of glycogen phosphorylase under environmental stress conditions, which leads to an increase in the blood glucose concentration [39,40,41,42,43,44]. In this study, the blood glucose level of the large yellow croaker in the acoustic stimulation frequency of 630 Hz group was significantly higher than that of the control group (p < 0.05), indicating that after 1 h of acoustic stimulation, the large yellow croaker showed an obvious stress phenomenon, which further confirmed the sensitivity of the large yellow croaker to 630 Hz acoustic stimulation. At other frequencies, the plasma blood sugar levels in the fish did not exhibit significant changes, which may be attributed to the rapid consumption of the energy substrate (glucose) by fish under stress conditions. West’s [45] research has demonstrated that rainbow trout can increase their glucose utilization by nearly 30-fold during periods of peak activity. These findings imply that acoustic stimulation at 630 Hz is particularly impactful on large yellow croakers. Furthermore, fish subjected to chronic stress may experience substrate depletion, leading to reduced plasma glucose levels.

Several different characteristics have been proven in fish’s muscle fibers. The slow oxidation of muscle fibers and rapid glycolysis of muscle fiber tissue are the most important in exercise [46]. Slowly oxidized muscle fibers are used for aerobic exercise, while rapidly glycolyzed muscle fiber tissues are used for anaerobic exercise [47,48]. Anaerobic exercise provides energy during long-term swimming behavior and causes fish to enter a fatigue state, resulting in glucose being converted into lactic acid, which in turn increases the lactic acid level. In this study, we observed a significant increase in the lactic acid levels of large yellow croaker following acoustic stimulation at frequencies of 125 Hz, 160 Hz, 200 Hz, and 800 Hz (p < 0.05). This elevation in lactic acid suggests that it may be a consequence of the anaerobic exercise behavior exhibited by the fish post-stress, which is known to enhance muscle metabolism. Similar metabolic responses have been documented in spotted grouper under high-density culture conditions and in large yellow croaker who are subjected to transport stress [49,50]. An increase in swimming activity can elevate metabolic demands, potentially leading to lactic acid accumulation. This buildup may subsequently interfere with other essential biological processes, such as feeding and reproductive behaviors in fish [51]. Furthermore, noise exposure could have a significant impact on muscle metabolism.

In response to acute stress, the immune system is modulated by a complex interplay of stress hormones, with Epinephrine being a primary hormone responsible for immune enhancement within the hypothalamic–pituitary–adrenal axis [52,53]. Epinephrine exerts a significant influence on the central nervous system, enhancing the alertness and reaction speed of fish, which enables them to evade potential threats swiftly. In this study, we observed a significant increase in Epinephrine levels in large yellow croakers subjected to acoustic stimulation at frequencies of 200 Hz, 630 Hz, and 800 Hz. This increase indicates that the fish experienced stress at these frequencies. It is widely accepted that stress factors elicit a sympathetic chromaffin tissue system response, characterized by elevated Epinephrine concentrations in fish [54]. This response aligns with the observed behavior of large yellow croakers, who tend to become easily alarmed and exhibit erratic flight when exposed to sudden sounds. By the conclusion of the experiment, the swimming patterns of the croakers had transitioned to a gradual narrowing, adopting an oval shape, which may be attributed to fatigue and reduced mobility resulting from prolonged exposure to high Epinephrine levels.

Multiple lines of evidence suggest that the hypothalamic and pituitary hormones associated with the thyroid and inter-renal axes can interact, potentially regulating actions mediated by thyroid hormones (THs) and cortisol. Although these interactions are challenging to define precisely, the magnitude of the stress response in fish has been demonstrated to be influenced by changes in their TH status. This indicates a functional relationship with the endocrine stress axes, particularly the inter-renal axis [55]. In the present study, we observed that large yellow croakers exposed to acoustic stimulation at frequencies of 100 Hz, 200 Hz, 500 Hz, and 630 Hz exhibited significantly higher thyroxine levels compared to the control group (p < 0.05). Frederique Bau [56] has also observed a similar phenomenon, reporting an increase in plasma thyroid hormone levels in fish subjected to capture stress using gillnets, including Sander lucioperca, perch, and bream. Thus, elevated thyroid hormone levels may assist fish in meeting the physiological demands that arise during periods of stress.

Behavioral responses in fish are closely intertwined with physiological processes, such as the release of stress hormones that can elicit a fear response and consequently lead to an increase in swimming speed [57]. These studies provide insights into the physiological underpinnings of fish’s behavior, enabling us to predict their behavioral patterns in fluctuating environments. The findings have significant implications for the conservation of fish species, the sustainable management of aquatic resources, and the assessment of ecosystem health.

5. Conclusions

Our research indicates that the large yellow croaker exhibits the most pronounced behavioral responses to an acoustic stimulus at a frequency of 630 Hz. Physiologically, the fish demonstrate the most significant stress reactions to sound frequencies of 200, 630, and 800 Hz. Therefore, we believe that large yellow croakers are most sensitive to 630 Hz and advocate for the avoidance of noise stimulation at these specific frequencies during the breeding process to enhance the welfare of the fish.