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Abstract:Aquaculture in offshore structures is an effective method for developing ocean ranching and meeting the increasing demand for marine produce and is one of the most promising ecological breeding structures. The offshore pile-net enclosure structure has wide application prospects owing to its large aquaculture volume, and its aquaculture environment is very similar to that of the natural environment. However, the existence of the pile and net structure will have a certain impact on the wave fields inside and around the pile net enclosure. Owing to the large scale of the pile-net enclosure structure, the structure significantly changes the original wave field in passing waves. Because of the complexity of the wave field at sea, construction site selection will have a significant impact on the safety of the purse seine structure. The pile-net enclosure structure also has an effect on the contents of nitrogen, phosphorus, heavy metals, and other substances in the area. Study of the impact of the pile-net enclosure structure on the original wave field is also of great significance for addressing environmental problems. Previous studies on pile-net enclosure structures have focused on the stress of the structure, and few studies have examined the impact of the structure on the original wave field in passing waves. Therefore, the propagation tendency of the pile net enclosure structure on the wave field in the original area under the action of waves and the hydrodynamic characteristics of the pile net enclosure were studied. This is of great significance for the structural design and site selection of the entire pile net enclosure. Based on the fully nonlinear Boussinesq equation and the numerical model FUNWAVE-TVD with shock capture ability, the propagation tendency of regular waves through a pile-net enclosure structure was studied. First, the wave was simulated based on the fully nonlinear Boussinesq equation. For a structure with large porosity, the pile-net enclosure structure was simplified as a porous medium model, ignoring its linear force, considering only the drag force of the netting, and using a reasonable calculation formula to calculate the flow-blocking effect of the netting. The influence of the pile-net enclosure on the wave field was simulated by combining the two. The finite volume and finite difference methods were used in the numerical calculation. First, a physical model experiment based on the lander verified whether the model was accurate for the calculation of the wave field of the netting. The physical model experiment designed different wave conditions and three netting models with large differences in compactness. The square coefficient of the wave height was used to describe the change in wave energy after the wave passed through the pile-net enclosure structure. Compared to the results of the physical model experiments, the error was small. Subsequently, the numerical model was verified from the perspective of the cage using Bi´s physical model test data. The model used a net cage model to simulate the influence of the net cage on the wave field. A numerical flume similar to that of the physical model experiment was designed, and the wave-dissipation area was set before and after. The cage size was the same as that used in the physical model experiment. The results showed that the model could better simulate the influence of the cage structure on the wave field. Different working conditions were then designed, the effects of the wave period on the wave field characteristics of the pile-net enclosure were further studied, and the internal relationship between the wave field characteristics of the pile-net enclosure structure and these hydrodynamic factors were discussed. In this study, a numerical flume similar to the physical model experiment was designed. Then, different designed wave conditions were used to determine the effects of the wave period on the wave field characteristics of the pile-net enclosure. The results showed that the influence of the pile-net enclosure on the larger periodic wave field had a radial trend and a fan-shaped radiation area behind the pile-net enclosure structure, which increased with an increase in the wave period, indicating that the wave period increased and its influence area expanded. Within a certain range on the lee side of the pile-net enclosure, the wave height showed a recovery trend, and the attenuation gradually decreased, while the attenuation area of small periodic waves was more concentrated. The wave with a small period was significantly affected by the pile net enclosure, and its wave transmission coefficient was the smallest in this study. The results show that the numerical model can effectively simulate the wave field characteristics around a pile net enclosure, which has a reference value for the location and construction of a pile net enclosure in an ocean ranching environment.

Abstract:There is strategic demand in China to open up space for deep-sea aquaculture and develop related large-scale base-station equipment and technology. Research and development of deep-sea aquaculture cages will greatly promote the spatial expansion and production-mode transformation of aquaculture in China and improve the utilization capacity of deep-sea and fishery resources. Generally, large cages located in deep water are more vulnerable to strong currents, high waves, and typhoons than cages inshore. It is critical to analyze the hydrodynamic characteristics of such cages because of their rocking and drifting motions under heavy marine environmental loads. In this study, a numerical model of a semi-submersible cage under wave action was established based on the finite element method, and the mooring line tension and motion of the cage were calculated by simulations. The cage was modeled using the 3D modeling software Creo Elements/Pro. After model development, it was imported into ANSYS for pre-processing. The simplified cage structure model was imported into the ANSYS Workbench to divide the mesh; the hydrodynamic response and other data related to the cage structure could then be analyzed by establishing a wet surface model. The water depth calculated by numerical simulation in this study was 120 m. The cage adopts a 4-point anchoring form, and the length of the anchor rope is 360 m. The cage can reach a semi-submersible working state by adjusting the bottom pontoon. To compare the dynamic response characteristics of the cage under different draft conditions, three different working conditions were set for the semi-submersible cage: no-load (draft depth of 8.4 m), half-load (draft depth of 33.6 m), and full-load (draft depth of 43.2 m). First, the accuracy of the numerical model was verified by comparing the computer simulation values with the physical flume test values. Then, the dynamic responses of the semi-submersible cage were studied under three ballast conditions. Finally, the calculation results of the mooring line tension, heave, surge, and pitch of the cage were analyzed and compared under different wave conditions. The results show that the calculated values were in agreement with experimental values, and the relative error between them was approximately 5%. This demonstrates that the numerical model can be used to analyze the hydrodynamic characteristics of the semi-submersible deep-sea cage. When the wave height is the same, there is no obvious correlation between the mooring line tension of the cage and the wave period. On the other hand, when the wave period is constant, the mooring line tension on both sides increases with an increase in wave height. Additionally, the heave, surge, and pitch of the cage were all positively correlated with the wave height. With an increase in the draft of the semi-submersible cage, the heave, surge, and pitch values of the cage decreased. The maximum heave and surge values of the cage are 12.67 m and 10.59 m, respectively, under the three ballast conditions, and the maximum pitch value of the cage under no-load conditions is less than 15°. Based on the above work, it can be observed that the semi-submersible cage structure has good stability. Moreover, the results can provide a theoretical reference for the design of marine cages in China.

Abstract:At present, China's leading offshore aquaculture cages are enormous high-density polyethylene gravity deep water cages, generally composed of floating frame systems, netting systems, counterweight systems, and mooring systems. The cage culture space is formed by the counterweight system and the netting system, therefore, it will be significantly reduced when the environmental load is large, which will affect the expected growth of the fish, even causing extrusion and other problems. This makes the cage generally choose to be installed in the coastal areas with a slight wind and wave environmental loads, such as ports and coasts, and adopt the intensive development model. In the past 20 years, this industry has provided many high-quality animal proteins to the Chinese people, alleviated the pressure of food supply to some extent, and has indispensable importance for China's marine fisheries and economic development. However, with the rapid development of science, technology, and the economy in China in recent years, the cage industry has gradually improved. The cost of cage manufacturing has decreased annually, and an increasing number of anglers have joined. Due to the lack of space and unreasonable layout of offshore aquaculture, environmental deterioration and frequent diseases are becoming increasingly severe, greatly influencing the quality and safety of aquatic products. To obtain a vast water space and high-quality environmental conditions, developing a method to explore deep-sea aquaculture vigorously is essential. Traditional gravity cages are difficult to adapt to the harsh environment of the deep sea and cannot be placed offshore for aquaculture. As one of the critical aquaculture equipment for the development of deep-sea fishery, the deep-sea cage has integrated aquaculture management with a larger main structure. Its large-scale and intelligent characteristics can significantly reduce the cost of aquaculture and improve the interests of anglers in aquaculture. However, it should be noted that the environment faced by cages operating in the deep sea is more complex and harsher than offshore cages. In particular, the East China Sea and the South China Sea are in typhoon-prone areas. They often encounter strong wind jets, causing irreversible damage to the cages. Therefore, for this new type of large-scale intelligent aquaculture equipment, we need to understand its hydrodynamic parameters in the marine environment to provide primary theoretical guidance data for the construction, site selection, and operation of the cage and ensure the safe operation and stable output of the cage. The research object of this study is development of a new type of anti-wind wave cage with a single-point mooring system. The main body of the cage is a hybrid structure consisting of a ship truss and a floating body. The overall floating and diving off the mainframe of the cage are realized through the front and rear floating ballast water to meet the operational requirements of the aquaculture process. The single-point mooring system can adapt the cage to adjust its position during typhoons and big waves and significantly reduce the impact of wind and waves on the cage. It has the characteristics of high safety and structural stability. The research was mainly conducted through physical model tests with a model scale of 1:40. The cage's hydrodynamic characteristics (including mooring force, heave, pitch, and roll) under the wave and current were analyzed and compared. The purpose was to determine the dynamic response characteristics of the cage under the action of waves and currents and the wave resistance after diving. The cage is in a floating state during regular operation and only adjusts to a diving state when encountering strong waves and current. Therefore, three groups of tests were conducted: 1. Dynamic response characteristics of cages in floating state (7.5~12.5 cm) under general wave conditions. 2. The cage's performance in the high wave (15.0 and 17.5 cm) after diving. 3. Effect of current on the wave resistance performance of the cage under the combined action of waves and currents. The results show that under the condition of 7.5~12.5 cm wave height, the floating cage's mooring force and motion response are small, proving that it has good safety and stability and can meet aquaculture requirements under these conditions. Under severe conditions, that is, under a wave height of 15.0 and 17.5 cm in this experiment, the cage showed good wave resistance by increasing draught, in which the mooring force was reduced by more than 70%, and the motion components such as heave, pitch, and roll were also reduced by 20%~60%. In the wave combined with the current test, the current has a particular influence on the wave resistance of the cage. However, generally, it still has good wave resistance. In this study, the dynamic response characteristics of cages under wave currents were understood. The research results can provide a theoretical basis and data reference for the safe operation and daily management of single-point mooring diving cages and help the innovation of deep-sea aquaculture equipment in China to promote the development of marine fisheries.

Abstract:Shallow sea net enclosure aquaculture (NEA) is a new ecological aquaculture model in which piles, nets, ropes, and other structures are the main components. The breeding space extends from the sea surface to the seabed and includes the natural shoreline. The general aquaculture area of NEA is approximately dozens of hectares, and the effective aquaculture water volume can be up to several million cubic meters. The vast aquaculture space provides a sufficient range of activities for farmed fish, and the quality of farmed fish is significantly better than that produced by other traditional farming modes. In recent years, more than ten NEAs have been built across the country, all of which have achieved good farming results. However, according to the field survey, almost all modern NEA facilities have different levels of damage on the net, which causes huge economic losses. After this analysis and summary, it was found that the main problem lies in the net panel. The lack of effective structural safety theory and design specifications was found to be a key factor. In this study, a numerical model of the NEA was established using the lumped mass point method. The forces on the lumped mass point of the net included gravity, buoyancy, twine force, horizontal drag, and horizontal inertia. StokesⅢorder wave theory was used to calculate water particle velocity and acceleration. Newton's second law equation was used to establish the equation of motion, and Euler´s method was used to solve it. Finally, MATLAB was used to visualize the results. In the direct connection mode, the left and right edges of the twine were tied directly to the piles, which is the common connection method. In the indirect connection mode, the left and right edges of the net were not directly tied to the piles, but were connected to the piles through horizontal ropes. In this mode, the twine was not directly connected to the piles; therefore, it can transfer the force of the rope to the twine, thereby reducing the force on the edge of the rope and protecting the net. In this study, the effect of the pile and net connection method on the maximum displacement of the net panel, maximum force of the twine and rope, and force of the joint point under the action of wave loads were investigated. The results showed that the displacement of the net panel increased with the wave height. When the wave height was less than 4 m, the maximum displacement at the wave crest and wave trough were almost the same. When the wave height was greater than 4 m, the contact area of the net panel with the wave gradually decreased at the moment of a wave trough. In the same situation, the wave force acting on the net panel was reduced. In short, as the wave moves from crest to trough, the height of the net above the water surface increases. When the wave height is 4 m, the distance from the highest point of the net to the wave trough is 4 m. When the wave height is 5 m, the distance from the highest point of the net to the wave trough is 4.5 m; when the wave height is 6 m, the distance from the highest point of the net to the wave trough is 5 m. And when the wave height was 6 m, the net is 5 m above the water surface. At the moment of the wave trough, the overall net panel was driven to move backward, and the force that can act on the net panel decreased; thus, the displacement was reduced. The indirectly connected net was not connected to the piles, resulting in all the forces at the edges being borne by the ropes. In the direct connection method, the force was shared between the twine and the rope. This caused the maximum displacement of the indirect connection to be larger than that of the direct connection. In addition, the overall movement trajectory of the net panel was asymmetrical. According to the law of wave motion, the contact area between the net and seawater differs at different times under the condition of water discharge. Consequently, the forces acting on it and the maximum displacement are different. Under the same wave height, the maximum force positions of the twine and rope were both at the connecting channel steel of the vertical ropes. The maximum force of the indirect connection was lower than that of the direct connection. The maximum force of the joint point was within 0~1 m below the horizontal plane, and the maximum force at the joint point of the indirect connection was less than that of the direct connection. During the actual construction process, special reinforcement measures should be taken for the maximum-force parts of the twine, rope, and joint points. Such construction can effectively reduce the risk of breaking the rope and twine, and avoid unnecessary construction work caused by blind reinforcement. As the wave height increased, the force of the horizontal twine increased; the force of the twine in the indirect connection method was less than that in the direct connection method. The findings of this study can be used to determine the mode of connection and safety of an NEA system.

Abstract:If damages to aquaculture nets are not found in time, they will result in the escape of fish, thereby, causing considerable losses to farmers. Therefore, it is necessary to detect whether damage to fishing net occurs. At present, the primary method for detecting damage to fishing nets is the manual inspection of staff diving into the water, but this method is labor-intensive and inefficient. This paper proposes a damage detection method based on digital twin, which uses sensor monitoring instead of manual monitoring to overcome these limitations and realize real-time monitoring of fishing nets. The research first shows that the numerical simulation data in good agreement with the physical model experimental data can be obtained through the numerical simulation of the lumped mass mechanical model. In the numerical simulation, considering a kind of damage to the fishing net, a total of 11 simulations were carried out: the tensile values of the horizontal and vertical ropes of the fishing net, nine sea conditions as training samples, and two sea conditions as test samples. The artificial neural network adopts the error backpropagation training method that takes the significant wave height Hs, the spectral peak period Tp, and the tensile value of the vertical and horizontal rope as inputs, and the complete state and damaged state of the fishing net as the outputs. After training, the recognition model recognition accuracy rates for the training and test samples were 99.21% and 95.11%, respectively. The measured actual physical sensor data were also used as test data. The recognition accuracy of the recognition model is 94.32%, which indicates the feasibility of the practical application of digital twin technology in the damage detection of the net. It can, therefore, be used as a new method of fishing net damage detection. As the wave-current environment is more complex in the actual sea area, our following research will focus on dealing with the sensing data and detection of the damage of fishing nets in a more realistic sea condition.

Abstract:Far-reaching mariculture is an important way to relieve the pressure on the ecological environment of coastal waters, expand new space for mariculture in China, optimize the layout of the marine fishery industry, and promote the green and high-quality development of aquaculture. In recent years, moving the islands and reefs far away from the continental coastline as the early development sea area and the equipment-first behavior as the development characteristics of deep-sea aquaculture, China´s far-reaching mariculture has been preliminarily developed. Combined with China´s sea areas, sea conditions, breeding varieties, and market demand, the independent and innovative large-scale engineering fence facility breeding model of Pseudosciaena crocea has been successful. In order to evaluate the sediment status in the sea area of the large-scale fenced large yellow croaker (Larimichthys crocea) in the deep-sea, the regional distribution, content change, and pollution level of Cu, Zn, organic carbon, sulfide and other indicators in the sediments of the surveyed sea area were analyzed. These parameters were evaluated through four investigations in the fenced culture area, peripheral area of the fence, peripheral area of the cage, and control area of L. crocea, in different culture periods, and the Nemerow index was used to comprehensively evaluate the quality of sediments in the surveyed sea area. The results showed that the Cu content in the sediments varied from 15~33 mg/kg; the Zn content varied from 80~137 mg/kg; the organic carbon content varied from 0.14%~1.90%; and the sulfide content varied from 0.3~128.0 mg/kg. The contents of organic carbon and sulfide in the sediments in the fenced culture area were higher than those in other areas, however, they all met the first class of marine sediment quality standards (GB18668-2002). There was no significant difference in the contents of Cu, Zn, and organic carbon in the sediments of the different investigation areas (P>0.05), and the content of sulfide in the sediments of the breeding area in the fence center was significantly higher than that in the control area (P<0.05). In this study, the Nemerow index comprehensive analysis revealed that the sediment quality of all sampling stations during the survey was of clean (classⅠ) or relatively clean (classⅡ) status, and the sediment quality of each station maintained a stable and good level during the survey time. On one hand, high breeding density is not an issue in the sea area, and the breeding layout is more scientific and reasonable, but good water exchange in the sea area can dilute and diffuse some pollutants, such as residual bait and feces; on the other hand, the deep-water network in the sea area generally occurs around December every year. Therefore, the large yellow croaker cultured in boxes and fences could be caught and sold. The large yellow croaker species can be re-invested around May of the next year. Thus, the sea area has several months for renewal of its resources in the non-breeding period. No obvious accumulation trend of Cu, Zn, and sulfide in the sediments was observed, but a slight accumulation trend of organic carbon in the sediments was observed during the study period. The research and development, and application of formula feed for the large yellow croaker should be enhanced to alleviate the current large-scale investment of frozen fish bait and reduce the bait coefficient, thus reducing the pollution of organic matter accumulation in the sedimentary environment to a certain extent. According to the breeding cycle of large-scale fenced L. crocea in the far-reaching sea, this study monitors the key characteristics and sedimentary environmental factors in the fenced breeding area and the surrounding sea areas. It comprehensively evaluates the current situation of sediment quality in order to have a basic understanding of the current situation of pollutants in large-scale fenced breeding. The results provide a basis for rational planning of the large-scale fenced breeding areas in the far-reaching sea and provide data support for disease control and sustainable development of mariculture.

Abstract:To further exploit offshore fishery resources while avoiding coastal pollution issues, offshore net cages are gradually moved towards the deep sea. However, the deep ocean environment is very complex and harsh, which severely threatens the safety of offshore net cage structures. Therefore, developing a deep-water net cage with a strong ability to resist high waves and maintain structural safety is essential. Based on the structural characteristics of the jack-up platform that allows the main body to move up and down along the platform legs, a new offshore net cage has been presented. When extreme weather occurs, the net cage body can quickly submerge along the legs, providing a solution for open-sea aquaculture to resist extreme loading. Research on jack-up platforms is mainly in traditional ocean oil and gas platforms and offshore wind turbine installation facilities. It can provide helpful suggestions for this research. The jack-up net cage developed in this study is composed of the leading steel frame and eight pile legs. The bottom of the pile leg was inserted into the seabed to fix the entire net cage. The cage frame could move up and down along the eight pile legs to meet the requirements of cage operation, survival, and towing conditions. The material of the cage was Q345 steel with a yield strength of 345 MPa. In this study, the safety factor was 1.5, and the allowable stress was 230 MPa. To analyze the structural safety of the jack-up net cage under operating conditions and survival conditions, numerical models of the net cage were first created using the general finite element software ANSYS Mechanical according to the geometric and material parameters of the net cage. The element Pipe59 was used to simulate all pipe structures, including the legs and cage frame. The pile leg sleeves were simulated using Solid45. In addition, node coupling technology was used to simulate the relationship between the legs and leg sleeves. The primary environmental parameters under operating and survival conditions are known based on sea statistics. Then, the wave phase angle search code using the APDL language was developed in this study. The implementation of the proposed code successfully simulated the maximum wave-current coupling force; hence, the most dangerous loading condition under different phase angles was obtained. As the cage structure is symmetrical about the x and y-axes, only three loading directions of 0°, 45°, and 90° were considered in this study. After analyzing the reaction force variation of different pile legs with phase angle, 32 loading conditions were determined. Next, structural response analyses of the jack-up net cage under operating and survival conditions were performed. The entire structural deformation, stress distribution, and maximum von Mises stress location were obtained. When the loading direction was 90°, and the phase angle was 102°, a maximum deformation of 0.115 m was obtained under the operating conditions. At the same time, the maximum equivalent stress of 191 MPa was observed at the No.7 pile leg, which was less than the allowable stress of 230 MPa. Under the survival condition, the net cage had a maximum deformation of 0.097 m when the loading direction was 45°, and the phase angle was 322°. Under loading conditions LC18 (loading direction 0°, phase angle 162°) and LC29 (loading direction 90°, phase angle 95°), maximum equivalent stress of 190 MPa was achieved at the connection between the No.4 pile leg brace and the upper frame and the strong vertical brace of No.6 pile leg, which was also less than the allowable stress of 230 MPa. According to the results above, it can be concluded that the structural safety of the offshore net cage in this study meets the strength and stiffness requirements by analyzing the deformation of the offshore net cage and comparing the maximum stress with the allowable stress. In addition, the maximum equivalent stress was mainly found at the legs or the braces near the legs, regardless of the operating or survival conditions, which indicates that these vulnerable parts can quickly fail and require key consideration in design. Moreover, to indicate the effect of the jack-up net cage on the extreme environmental conditions, the structural deformation and stress of the net cage under operating conditions, under the same loading conditions as the survival conditions, were calculated. The results indicate that the net cage's maximum deformation and equivalent stress under diving conditions were reduced by 55% and 41.4%, respectively, compared with those of the net cage under operating conditions. This proves that this jack-up type net cage can handle extreme environmental conditions by moving down the cage body. This study provides a method for the structural safety evaluation of large-scale offshore net cages. Considering that safety evaluation is systematic work, the fatigue strength, pile leg stability, and dynamic response characteristics under towing conditions may be studied in further work.

Abstract:Offshore aquaculture net pens have the characteristics of open spaces, vast waters, and natural environments. This is one of the important ways to expand aquaculture space, create a new production base for high-quality protein, and develop deep and offshore sea aquaculture. A sudden change in ambient water temperature may significantly affect the normal physiological status of fish as they are ectotherms. In the present study, respiratory frequency, serum cortisol and glucose concentrations, the hematological index, and hepatic hsp70 and hsp90 mRNA expression were determined during various thermal durations and recovery periods to illustrate the thermal tolerance and the underlying physiological response of two sizes of spotted knifejaw. The respiratory frequency of the two sizes of spotted knifejaw first increased and then decreased subject to thermal stress at 32℃, with the highest value (P<0.05) observed at 0.3 (20 min) and 1 h in 50 and 200 g fish, respectively. The serum cortisol and glucose concentrations of the two sizes of spotted knifejaw showed similar trends to the respiratory frequency. In 50 g spotted knifejaw, the serum cortisol concentration was significantly higher at 1 h (P<0.05) and the serum glucose concentration was significantly higher at 6 h (P<0.05) than that in the other treatment groups. However, in 200 g spotted knifejaw, serum cortisol and glucose concentrations remained high under thermal stress. Hematological data of the two sizes of spotted knifejaw were rather different. Specifically, the number of white blood cells (WBCs), red blood cells (RBCs), and hemoglobin (HGB) in the blood of 50 g spotted knifejaw first increased and then decreased and were significantly higher than that in the control group at 3 h (P<0.05). In 200 g spotted knifejaw, no significant changes in WBC, RBC, and hematocrit were noted (P>0.05), although HGB showed an initial increase, followed by a decreasing trend and was significantly higher than that in the control group at 3 h (P<0.05). The hepatic hsp70 and hsp90 mRNA expression of the two sizes of spotted knifejaw increased first and then decreased. As such, hsp70 mRNA expression was the highest at 3 h, while hsp90 mRNA expression was the highest at 1 h (P<0.05). In conclusion, acute thermal stress significantly increased the respiratory rate and serum cortisol and glucose concentrations of two different sizes of spotted knifejaw and upregulated hepatic hsp70 and hsp90 mRNA expression; all aforementioned parameters gradually recovered to normal levels after treatment at 25℃ for 12 h. Meanwhile, small spotted knifejaw manifested greater thermal tolerance. These results provide useful data for spotted knifejaw culture in offshore aquaculture net pens and can strengthen our understanding of the regulatory mechanism of spotted knifejaw temperature tolerance.

Abstract:Dissolved oxygen is an important environmental factor affecting the growth and survival of aquatic organisms, including fish. Hypoxia has gradually become common in aquatic ecosystems and poses a significant challenge for fish farming. A decrease of dissolved oxygen levels in the body or a lack of oxygen will lead to a severe stress response for fish, hindering the development of the aquaculture economy. Dissolved oxygen is an important environmental factor that affects the aquaculture production of spotted knifejaw (Oplegnathus punctatus). The solubility of oxygen decreases with the increase of temperature, and the solubility of gaseous substances decreases with the increase of temperature and pressure. These show significant seasonal variation: The higher the temperature, the smaller the gap between the water molecules, and the lower the dissolved oxygen. Fish in a low-oxygen environment breathe normally and this can lead to physiological metabolic disorders, hence affecting fish behavior and physiological and biochemical indicators. However, in the process of evolution, fish have also developed different response modes and adaptive regulation mechanisms to maintain normal physiological functions. To clarify the changes in physiological and biochemical indices in blood during hypoxic tolerance and hypoxic stress, physiological and ecological methods were used in this study. The solubility of dissolved oxygen under two different specifications of critical oxygen tension (Pcrit) and loss equilibrium (LOE) were investigated, and the changes in respiration behavior under natural oxygen consumption were observed. Changes in cortisol and glucose, concentration of hemoglobin (Hb), number of red blood cells (RBC) and white blood cells (WBC), and hematocrit (HCT) during hypoxic stress and recovery were analyzed. Results showed that in a water temperature of (23.0±0.5)℃, the ammonia nitrogen concentration was less than 0.5 mg/L, nitrite concentration 0~0.05 mg/L, salinity 30 and pH 7.80; the dissolved oxygen concentrations at Pcrit were (4.05±0.09) mg/L and (3.15±0.12) mg/L for 200 g and 50 g O. punctatus, respectively. The concentrations of dissolved oxygen at LOE value were (1.16±0.08) mg/L and (0.93±0.11) mg/L. The time until the 50 g O. punctatus reached LOE (t=480 min) was longer than that of the 200 g O. punctatus (t=110 min). In addition, there were significant changes in respiration rate between hypoxia and re-oxygenation in both the 200 g and 50 g O. punctatus. In the process of natural oxygen consumption, the respiration rate of the two first increased and then decreased, and increased significantly at the Pcrit value. When the dissolved oxygen concentration decreased to 1.27 mg/L (420 min) and 1.10 mg/L (90 min), respiration rates of the 50 g and the 200 g fish reached their maximum (P<0.05). After recovery in normal dissolved oxygen for 24 h, the respiration rate did not differ significantly between the two sizes of O. punctatus nor with the control group. Plasma glucose and cortisol showed similar results to the respiratory rates; hypoxic stress led to significant increases and the highest values were obtained at LOE (P<0.05). The plasma cortisol concentration was the same in the two sizes of fish. While the values for the 50 g O. punctatus were significantly lower than those of the 200 g fish during hypoxic stress and re-oxygenation (P<0.05), the glucose and cortisol did not differ significantly from the control after recovery in normal dissolved oxygen for 24 h. Hypoxic stress had a significant effect on the blood biochemical indexes of O. punctatus. At Pcrit and LOE values, the number of WBCs, RBCs, Hbs and HCT, at 50 g were significantly higher than those in the control group (P<0.05), and the maximum values were at the Pcrit value. The concentrations of the four indexes were significantly increased in the 200 g O. punctatus compared with the control group (P<0.05). The maximum values of WBC, HCT, and Hb were reached at LOE, and the maximum values of RBC were reached at Pcrit. After recovery in normal dissolved oxygen for 24 h, the two sizes of O. punctatus showed normal swimming behavior and respiratory activities, and the above physiological and biochemical indexes did not differ significantly from those of the control group (P<0.05). Lastly, the body weight of O. punctatus differed during hypoxia-induced hematological and biochemical responses. In O. punctatus, the 50 g fish were more tolerant to hypoxic environments than the 200 g, while the 200 g fish were more sensitive to hypoxic stress. Both sizes of O. punctatus could enhance the absorption and utilization of dissolved oxygen by increasing their respiratory rate, increasing the number of WBCs and RBCs, and increasing the concentrations of cortisol, glucose, and Hb in response to hypoxia stress. In this study, the tolerance to hypoxia and the physiological responses of different sizes of O. punctatus were elucidated; their adaptability to low dissolved oxygen was explored, and their tolerance threshold to hypoxia was determined. The results provide data for land-sea relay breeding of O. punctatus, a theoretical basis for efficient land-sea relay, and an early warning range of hypoxia for breeding O. punctatus, to help reduce economic losses caused by decreased oxygen.

Abstract:Seriola is a genus of Carangidae in Perciforms; they are long-distance migratory oceanic species with global distribution and inhabit temperate and subtropical marine waters worldwide. There are nine species in Genus Seriola. Three species, including S. lalandi, S. dumerili, and S. quinqueradiata, are found in China's coastal waters. Seriola fish is large, fast-growing, and highly favored by the international consumption market due to its excellent taste, nutritional quality, and economic value; furthermore, they are promising for open ocean aquaculture. In 2017, a significant breakthrough was achieved in seedlings production of S. lalandi by Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences in China, and the scaled juveniles' production technology was established in 2019. In 2020, another breakthrough was achieved in seedlings production of S. quinqueradiata in China. Since 2017, the farming industry of Seriola fish in China has entered the fast development era. Nowadays, the Seriola species are farmed in Liaoning, Fujian, Shandong provinces of China, etc., and the annual farming yield was about 20 thousands of tons in China since 2018. These three Seriola species, natively distributed in China, have similar exterior morphology and are hard to differentiate with naked eye and traditional differentiation methods, especially in case of the juveniles. In addition, an allotype phenomenon exists in Seriola fish in global oceanic waters. Therefore, it is necessary to establish a simple and efficient molecular species identification method to facilitate the species and population determination of Seriola fishes in international oceanic waters. Moreover, it also could be beneficial for discrimination, conservation, and sustainable utilization of natural fishery resources in international public waters. Many scientists have studied the germplasm and population genetic characteristics of S. lalandi, S. dumerili, and S. quinqueradiata from international waters using nuclear genes and mitochondrial markers. Still, there is no information on the genetic background of Seriola fishes in China. DNA barcodes are widely applied to species identification, systematic analysis, and population genetics of fish because of their sensitivity, accuracy, and reliability, especially for CO I, cytochrome oxidase b (Cyt b) genes, etc. Furthermore, when DNA barcodes are applied to species identification or hidden species analysis, the combination of several DNA barcodes could be more high-efficient and accurate. Thus, in the present study, to explore the applicability of Cyt b and NADH dehydrogenase subunit 1 and 2 (ND1 and ND2) to species identification and evolutionary analysis of three Seriola species, including S. lalandi, S. dumerili, and S. quinqueradiata were investigated and determined. The finclips samples were collected from three native distributed Seriola species. S. lalandi samples were also collected from Australian and Japanese populations. Genomic DNA was extracted from finclips, specific primers for Cyt b, ND1, and ND2 genes were designed. The PCR reaction system of the three genes totalled 50 μL each, Including rTaq enzyme 25 μL, template 2 μL, upstream and downstream primer 1 μL each, and ddH2O 21 μL. The results showed that the target fragments of three genes could be amplified from the genomes of three Seriola species, and 68 gene sequences were obtained. The sizes of Cyt b, ND1, and ND2 fragments were 538 bp, 673 bp, and 907 bp in length, respectively, which were consistent with the predicted results, wherein Cyt b gene sequence showed obvious A+T bias characteristics. The mutation rate of the ND2 gene was 20.52%. The genetic diversity of ND2 gene (Hd = 0.900, Pi = 0.082) was higher than that of ND1 (Hd = 0.874, Pi = 0.077) and Cyt b (Hd= 0.814, Pi = 0.061). The results indicated that Cyt b, ND1, and ND2 could be used to identify the three Seriola species natively distributed in the China oceans. In Seriola species, the interspecific genetic distances of the three genes were more than ten times the intraspecific genetic distances. Furthermore, the evolutionary tree based on Cyt b, ND1, and ND2 shows that Chinese and Japanese S. lalandi are clustered into independent branches, effectively distinguishing Chinese, Japanese, and Australian S. lalandi populations. In addition, S. lalandi and S. quinqueradiata were clustered together, while S. dumerili and S. rivoliana were clustered together, showing similar genetic relationships with each other. Based on the Cytochrome b gene, S. carpenteri and S. dumerili showed high sequence homology, were clustered on the same node, and could not be effectively distinguished. It can be seen that except for Cyt b, the maximum-likelihood tree constructed based on ND1 and ND2 genes has accurate identification ability for different fish species from the Seriola genus. In summary, Cyt b, ND1, and ND2 could be used as DNA barcodes for species identification and geographical differentiation of Seriola fishes. A variety of DNA barcodes could be combined to achieve more precise identification results. These DNA barcodes could also be applied to evaluate and conserve the genetic diversity of farmed Seriola species, which could be beneficial for sustainable utilization of the germplasm resources of Seriola species.

Abstract:Seriola fish are long-distance migratory oceanic species inhabiting temperate and subtropical waters worldwide. Nine species have been identified in the genus Seriola, of which three species, namely S. lalandi, S. dumerili, and S. quinqueradiata, are found in the coastal waters of China. Seriola fish are large and fast-growing, and their flesh is highly favored by international consumers owing to its excellent taste, nutritional quality, and economic value. Moreover, Seriola fish are promising candidates for different culture modes, including deep-sea cages and recirculatory aquaculture systems. Since 2017, China has witnessed a great breakthrough in the seedling production of S. lalandi. Subsequently, in 2020, we achieved a breakthrough in the seedling production of S. quinqueradiata. The Seriola fish farming industry was developed in Liaoning, Fujian, and Shandong provinces, with the annual farming yield reaching approximately 20 000 tons. At present, ice trash is the primary feed for Seriola. However, excess trash fish feeding can produce a severe environmental pressure on coastal waters, which is not beneficial for sustainable development of the Seriola fish farming industry. Thus, there is an urgent need to develop specific commercial feed to solve bottleneck problems, including culture inefficiency and environmental pressure. The key to developing a specific commercial feed is to understand the digestive system structure and nutritional physiological characteristics of fish. Therefore, the distribution patterns of digestive enzyme activities in Seriola species warrant immediate attention. In December 2020, 3-year-old S. dumerili, S. lalandi, and S. quinqueradiata were collected from a cage culture base in Ningde, Fujian Province. The experimental fish were fed ice trash twice daily. Fish were healthy and vigorous. The total length and body weight of four fish of each species were measured, and their visceral structures were observed and recorded. The average length and weight were respectively (62.380±0.805) cm and (3.306±0.208) kg in S. dumerili; respectively (65.400±0.351) cm and (2.906±0.082) kg in S. lalandi; and respectively (74.640±1.041) cm and (4.622±0.258) kg in S. quinqueradiata. The stomach, intestines (to remove intestinal contents), and liver were collected, and the intestine was divided into the foregut, midgut, and hindgut according to the physiological curvature. Enzyme activity kits were purchased from Nanjing Jiancheng Reagent Company. The activities and distribution of five enzymes (trypsin, lipase, amylase, alkaline phosphatase, and acid phosphatase) in the digestive system (stomach, pyloric caeca, foregut, midgut, hindgut, and liver) of the three Seriola species studied were compared. In all Seriola species, trypsin activity was mainly distributed in the pyloric caeca and liver, α-amylase activity was mainly distributed in the pyloric caeca and midgut, lipase activity was evenly distributed in all tissues, alkaline phosphatase activity was mainly distributed in the pyloric caeca and intestinal tract, and acid phosphatase activity was mainly distributed in the pyloric caeca. Compared with the other two species, S. dumerili exhibited significantly higher trypsin activity in the liver; significantly lower α-amylase activity in the stomach, pyloric caeca, foregut, and hindgut; significantly lower lipase activity in the stomach, pyloric caeca, foregut, hindgut, and liver; significantly lower alkaline phosphatase activity in the stomach, pyloric caeca, foregut, midgut, and hindgut; and significantly lower acid phosphatase activity in the pyloric caeca, foregut, and midgut. Moreover, compared with the other two species, S. lalandi exhibited significantly higher trypsin and α-amylase activities in the pyloric caeca and foregut; significantly higher lipase activity in the stomach, pyloric caeca, and midgut; significantly higher alkaline phosphatase activity in the pyloric caeca and hindgut; and significantly higher acid phosphatase activity in the pyloric caeca. Furthermore, compared with the other two species, S. quinqueradiata exhibited significantly higher α-amylase activity in the stomach, midgut, and hindgut; significantly higher lipase activity in the foregut, hindgut, and liver; significantly higher alkaline phosphatase activity in the foregut and liver; and significantly higher acid phosphatase activity in the hindgut. In conclusion, the results of the present study indicate that the activities of the five enzymes studied show similar distribution patterns in the digestive system of the three Seriola species, with the pyloric caeca being the primary target organ for digestive enzymes. Comparative analysis revealed that the activities of all digestive enzymes, except trypsin, were significantly lower in S. dumerili; the activities of digestive enzymes in the pyloric caeca and intestines were significantly higher in S. lalandi; and the activities digestive enzyme in the pyloric caeca of S. quinqueradiata were significantly lower. These results may provide a theoretical basis for revealing the digestive physiological characteristics of Seriola species and developing highly efficient specific compound feed suitable for their digestion and growth.

Abstract:China is one of the world’s largest marine fishery countries. In 2020, the total output of mariculture reached 21.3531 million tons, a year-on-year increase of 3.39%. Mariculture has contributed to ensuring national food safety, supporting fishermen's income, and economic development. However, for a long time, mariculture has been largely concentrated in the coastal harbor area, with extensive breeding methods and backward facilities and equipment, resulting in frequent breeding diseases, serious water pollution, hidden dangers in quality and safety, and continuous decline in benefits, which have seriously affected the healthy development of the industry. It is urgent to change the mariculture mode and adjust the structure of the mariculture industry. Mariculture's transformation and development direction are to expand new marine space and take advantage of high-quality marine resources for healthy aquaculture. The deep-sea large-scale fence aquaculture is suitable for China's sea area, characterized by a gentle continental shelf, and for breeding various unique economic fishes in China. It is an emerging aquaculture model for the structural adjustment and industrial upgrading of mariculture in China. It has the characteristics of a large breeding area, with its activity space being closer to nature and ecology, and has broad application prospects. Deep-sea large-scale fence aquaculture is an emerging ecological culture mode integrating marine engineering, new material applications, and ecological culture. It is mainly composed of "large-scale fence project + supporting culture facilities + culture technology." Many deep-sea large-scale fenced aquaculture facilities have been implemented or planned in Taizhou, Wenzhou, Zhoushan of Zhejiang Province, Laizhou of Shandong Province, and Zhanjiang of Guangdong Province. The deep-sea large-scale fence aquaculture model avoids the pollution of coastal aquaculture to a great extent and improves the quality of cultured fish, expands the development space of aquaculture, and has good ecological, economic, and social benefits. From the first construction to the subsequent gradual development of deep-sea large-scale fence facilities in the far-reaching sea, enterprises learn from each other, continuously strengthen the strength of piles, and optimize the connection technology of net clothes. However, the basic research on large-scale deep-sea fences is still relatively weak due to the relatively short development process. Some technical links and supporting equipment need to be further improved. Among them, the four most important issues are the facility's safety, including the structural damage of the pile structure, net clothing system, and net clothing pile connection system under a dynamic environment. The second is the safety of bottom sealing, whether the bottom sandbag has enough restraint on the traction of the upper buoyancy and net clothing systems to ensure the full coupling between the net clothing and the seabed anti-escape safety breeding objects. The third problem comprises the challenges in efficient fishing. The scale of fence farming is large, and there is no net bottom. It can neither raise net fishing, like cage culture, nor low tide fishing like beach Purse Seine. The fishing mode of large-scale fence farming in the far-reaching sea must adopt an innovative mode that involves new fishing equipment and technology and more systematic research on fish behavior characteristics. Fourth, there is a problem of siltation in large-scale fenced breeding areas. The endogenous excreta of cultured fish and the exogenous pollutants brought by artificially fed pellet feed, surimi, miscellaneous fish, and other bait deposits at the bottom of the fence and form sludge over time will threaten the healthy growth of cultured fish and the marine environment. With the support of national key R&D projects and local scientific and technological research projects, scientific research institutes and relevant breeding enterprises have researched key technologies such as deep-sea large-scale fence design and construction, mesh tear and wear prevention, and improved high-strength large mesh construction technology because of the characteristics of large fenced water bodies, large waves, and rapid flow in aquaculture water areas and far away from the coastline. Scientific experiments and simulation calculations were carried out to innovate the structural strength design system of fence facilities to withstand the impact of strong winds and waves, starting from the aspects of mechanical structure, power source, material and information collection and transmission, mechanized automatic control technology, R&D of large-scale fence mechanized and intelligent control equipment that can adapt to high sea conditions, feeding on large water surfaces, feed rations, pipeline transportation, collection of environmental indicators in aquaculture sea areas, safety monitoring, waste removal, underwater cleaning of net clothes, and centralized fishing of adult fish. By continuously studying and improving the signal transmission, power supply, and equipment control technologies, and we can gradually build a technical system for supporting key equipment for deep-sea large-scale fence breeding, and integrate large-scale fence aquaculture capacity evaluation technology, friendly culture environment construction technology, and multi-dimensional culture technology to build a large-scale fence intelligent ecological culture model. This study may provide a reference for constructing an intelligent ecological breeding mode for deep-sea large-scale fence aquaculture in China.

Abstract:Owing to the major issues of pollution, limited resources, and restricted space for mariculture in coastal areas, it will be a significant advantage to develop large-scale cage aquaculture in open sea areas, which have the advantages of the finest water conditions. The vigorous development of scientific and technological innovation in deep-sea aquaculture plays a positive role in upgrading and transforming aquaculture methods and expanding the mariculture space. These lead to a new pattern of development of marine aquaculture facilities. Here, we present a summary of the status of China's cage culture industry, the demand for the development of the deep-sea cage culture industry, and the corresponding scientific and technological requirements of cage culture facilities and equipment. Then, research and applications in China and abroad are introduced in detail regarding two aspects: deep-sea cage culture facilities and related equipment and technology. Subsequently, the main problems faced in the development of deep-sea cage culture engineering and equipment in China are identified. Finally, suggestions are given for scientific planning, technological guidance, standardized construction, and innovative modes, for better development of the deep-sea cage culture industry. This review provides a reference for research and development of the deep-sea cage aquaculture industry in China.

Abstract:Inshore islands are one of the most representative marine areas responsible for a significant part of the oceanic primary productivity. The most representative archipelago in northern China, the Miaodao Archipelago, is located at the confluence of the Yellow Sea and Bohai Sea. It is an important habitat and migration channel for marine life and is a hot spot for biodiversity research. However, the increasing exploitation of marine natural resources in the waters adjacent to inshore islands, such as mariculture, fishing, shipping, and energy exploitation, is putting increasing pressure on marine life. These factors have resulted in marine community disturbance to varying degrees and pose a severe challenge to biodiversity conservation. Due to human activities and climate changes, the Miaodao Archipelago biodiversity has been affected to some extent. Nonetheless, only few investigations have been performed on the bottom fisheries in this area. With the establishment of the Changdao National Wetland Nature Reserve, it is urgent to conduct systematic biodiversity research in this sea area to promote the restoration and protection of marine biodiversity. To systematically understand the diversity distribution characteristics of bottom fishery communities in the Miaodao Archipelago adjacent sea, a single-ship trawl survey was conducted monthly in September, November, and December 2020 and from March to December 2021. Based on the richness index (D), evenness index (J´), biodiversity index (H´), redundancy analysis (RDA), and abundance-biomass comparison (ABC) curves the bottom-fishery biodiversity and its influencing factors were analyzed to provide a scientific basis for biodiversity conservation in the Miaodao Archipelago adjacent sea. It provides basic data and data support for exploring the intrinsic, environmental, and human factors affecting biodiversity in typical islands. A total of 115 species were identified in the sampling survey, including 49 species of fish, 29 mollusks, 28 crustaceans, 8 echinoderms, and one species of brachiopods. The fish are mainly Pennahia argentata, Gobiidae, and other fish. Crustaceans are mainly Oratosquilla oratoria, Alpheus japonicus, and other crustaceans. The bottom fishery communities in the Miaodao Archipelago adjacent sea were primarily composed of Gobiidae, A. japonicus, Echinodermata, and Mollusca. The dominant species composition was different in distinct months. The dominant species were Chaemrichthys stigmatias, P. argentata, Palaemon gravieri, Trachypenaeus curvirostris, O. oratoria, A. japonicus, Charybdis bimaculata, Diogenes edwardsii, Octopus ocellatus, Glossaulax didyma, Rapana venosa, Temnopleurus hardwickii, Asterina pectinifera, Ophiothrix marenzelleri. From March to June, the bottom fishery community was mainly concentrated in the southeast area of the Miaodao Archipelago but changed to the southwest area in July and August. There was no apparent regional distribution in the rest of the months. This may be because, in colder March and April, the warmer seawater brought by the Yellow Sea Warm Current provided a more suitable living environment for the bottom fishery communities, which resulted in more communities gathering in the north Yellow Sea area southeast of the Miaodao Archipelago. In May and June, many migratory marine organisms migrated from the North Yellow Sea to the Bohai Strait, possibly resulting in high biodiversity in the southeast area of the Archipelago during this period. Many migratory organisms had arrived in the Laizhou Bay by July and August. With the increase of phytoplankton along the coast of the Laizhou Bay during summer, many consumer communities are attracted. In addition, the annual water and sediment transfer from the Yellow River in June-July may result in the introduction of large flows of fresh water and nutrients into the waters southwest of the Miaodao Archipelago, resulting in higher summer biomass in this region. The summer and autumn months showed higher biodiversity, while winter showed a single feature with higher abundance and biomass. There was a significant negative correlation between biomass and abundance of P. argentata and A. japonicus. The A. japonicus biomass and abundance may depend on the abundance of its predator, P. argentata. Other species with significant positive correlations may be jointly affected by environmental factors such as temperature. The main taxa and environmental factors RDA showed that water depth and temperature were the main environmental factors affecting the bottom fishery community structure, among which echinoderms and Terebratella coreanica were found in higher amounts. A. japonicus and temperature negatively correlated, while most other species positively correlated with temperature. The ABC curves indicated that the bottom fishery community structure was disturbed to a high degree as a whole, with only some seasons showing moderate disturbance, such as May and June 2021. Since implementing the summer fishing ban in May, the bottom fishery community structure disturbance level in the Miaodao Archipelago has decreased. The off-fishing season effectively protects the young fish of fishery species and dramatically reduces the fishing of other by-catch species, effectively recovering the fishery resources. In addition, from April to May every year, many migratory species began to migrate to the Bohai Sea through the Miaodao Archipelago, providing conditions for the increase of fishery species in this area. However, due to the large area of artificial breeding in the Miaodao Archipelago and the influence of ships navigating between the islands, the sea area experiences a significant degree of disturbance and is in an unstable state. This study provides a scientific foundation for biodiversity protection in the Miaodao Archipelago, exploring the intrinsic, environmental, and human factors affecting typical island biodiversity. However, the formation and evolution mechanisms of biological diversity in typical archipelago ecosystem still need to be explored in long-term and in-depth studies.

Abstract:Spawning grounds are essential habitats for the early life stages of marine aquatic animals. The habitat conditions of fish spawning grounds determine the quantity and growth of eggs and larvae, which in turn have an important impact on marine fish recruitment. Historically, there was one important spawning ground of Scomber japonicus in the Yantai-Weihai offshore waters, which were considered an unstable spawning ground as it is the main breeding migration channel for most of the breeding fish stocks migrating from the Yellow Sea to the Bohai Sea in the spring, and a feeding ground when adult and juvenile fish migrate out of the Bohai Sea in summer and autumn. Therefore, the Yantai-Weihai offshore waters have not been well studied in recent years. To determine the current fish spawning ground status in this area, monthly surveys were conducted from April to September 2020. Pooled analyses of the fish eggs, larvae, and environmental data employed spatial interpolation, clustering analysis, non-metric multi-dimensional scaling, similarity analysis, and redundancy analysis (RDA). The spatio-temporal distribution patterns and monthly community turnover of the marine fish early life stages and the optimal habitat of the main species were also examined. In total, 52 fish eggs and larvae species were collected, including 337 038 eggs of 33 taxa and 2122 larvae of 28 taxa. The main spawning season was from May to June, with 21 taxa accounting for 98.32% of the total species observed during the whole year. The main spawning ground was located in northern waters from the Yantai Taozi Bay to Weihai Jiming Island. Engraulis japonicus, S. japonicus, Scomberomorus niphonius, Cleisthenes herzensteini, Seriola lalandi, Callionymus beniteguri, Sillago japonica, Acanthopagrus schlegelii, Cynoglossus joyneri, and Saurida elongata were the main spawning species. The eggs and larvae community structures presented apparent monthly variance in the Yantai-Weihai offshore waters. Apart from the end of the spawning season between August and September, the monthly species turnover rates were over 50%, with average dissimilarities between eggs and larvae communities in the adjacent months of more than 73%. In April, Pseudopleuronectes herzensteini was the dominant eggs taxon, while the dominant larvae taxon was Ammodytes personatus. In May, E. japonicus and S. japonicus were the dominant eggs species, while Sebastes schlegelii was the dominant larvae species. E. japonicus eggs and larvae were both dominant in June and July. In August, the dominant eggs taxa were C. joyneri and S. japonica, and the dominant larvae taxon was E. japonicus. There was no dominant taxon in September. From April to June, E. japonicus was the indispensable species to the community structure and contributed the most to community turnover, while S. japonicus was the second most important species. Habitat environmental factors in the Yantai-Weihai offshore waters within the survey areas from April to September 2020 varied as follows: the sea surface temperature (SST) ranged from 8.27℃ to 26.05℃, the sea bottom temperature (SBT) from 8.01℃ to 24.52℃, the sea surface salinity (SSS) from 30.87 to 33.27, and the seafloor depth from 4.4 to 82.0 m. The RDA results showed that variation in SST, SBT, SSS, and seafloor depth affected fish spawning activities significantly. The suitable spawning habitat of E. japonicus, S. japonicus, S. niphonius, S. lalandi, C. herzensteini, and C. beniteguri was the areas with low SST and SBT, and high SSS. The suitable spawning habitat of P. herzensteini and A. schlegelii was the shallow waters with low SST, while low SBT and high SSS was suitable only for P. herzensteini. The suitable spawning habitat of C. joyneri was the areas with high SST and SBT, and low SSS, and for S. elongata and Trichiurus lepturus was the medium shallow waters and areas with high SBT and low SSS, respectively. Finally, the suitable spawning habitat of S. japonica was the bottom waters with high SBT. During the peak spawning season, the species number and ecological density of fish's eggs in the Yantai-Weihai offshore waters were significantly higher than those distributed in the Liaodong Bay and Laizhou Bay, which represented the typical spawning grounds in the whole Yellow Sea and Bohai Sea. In conclusion, typical fish spawning grounds are currently distributed in the Yantai-Weihai offshore waters; therefore, research on the early life history and spawning grounds of marine fish in this area is critical. This study provides important ecological information on the early life stages of fish for conservation and formulation of spawning ground protection strategies in the Yellow Sea and Bohai Sea.

Abstract:Oratosquilla oratoria is a typical commercially exploited stomatopod species that is widely distributed in the coastal waters of the Northwest Pacific. It also plays an important role in structuring the benthic communities and can significantly affect marine nutrient cycling and energy flow through sediment bioturbation. Few studies have been published on the early life stages and recruitment of O. oratoria in natural waters, particularly in Chinese coastal waters. Moreover, despite ample research on the egg and larval development, growth, feeding, reproductivity, and spatial distribution of this species, little is known regarding the habitats required for juvenile O. oratoria when they settle down to the bottom after the planktonic larval stage. In this context, one objective of the present study was to investigate the seasonal distribution of juvenile O. oratoria and its influencing factors. The other objective was to identify the early recruitment pattern of O. oratoria in the Bohai Sea based on the seasonal pattern of its juvenile occurrence and published literature on the spawning, embryonic and larval development, and growth of this species. Data used to achieve these objectives were mainly collected from eight monthly cruises of bottom trawl surveys for fisheries resources in spring (i.e., March to June) and summer to fall (i.e., August to November) of 2017. The same pair of bottom trawlers was used in all surveys, following consistent sampling protocols. O. oratoria catches were counted and weighed in number and biomass on the spot at each station, along with the depth, geographic coordinates, date, and time at the beginning and end of each tow. In addition, 50 O. oratoria individuals were randomly sampled to measure their biological characteristics (e.g., total length, weight, sex, feeding status, and fecundity) at each station; at certain stations, all individuals were measured when fewer than 50 O. oratoria were caught. The smallest and largest individuals were selected at each station to record their total length and weight when any O. oratoria was caught at a station. Moreover, surface-to-bottom temperature and salinity profiles were obtained with a SEABIRD CTD within a few minutes of the bottom trawlers moving away from the end of trawling at each station. The occurrence of juvenile O. oratoria (<60 mm total length) was determined based on the total length of the smallest individuals at each station. Subsequently, their occurrence over space, occurrence probability, and sample size were analyzed by season and month. Generalized additive models (GAMs) were used to identify significant environmental factors affecting the occurrence of juveniles separately in spring and summer to fall. Environmental factors considered in the model included depth, bottom temperature (BT), bottom salinity (BS), and five sediment-related variables. Depth, BT, and BS were measured simultaneously with the bottom trawl surveys. The five sediment variables were classified into two groups: two content variables separately representing the percentages of sand and silt and the other three representing statistical parameters of grain size distribution (i.e., mean grain size, skewness, and kurtosis) in the surface substrates (0–3 cm). The spatial distribution of these sediment factors was derived from recent literature on the grain-size distribution of surface sediments in the Bohai Sea (Yuan et al, 2020). A pseudo-stepwise procedure was used to identify significant environmental factors for optimal GAMs separately for spring and summer to fall based on the Akaike Information Criterion (AIC). Furthermore, the early recruitment pattern of O. oratoria in the Bohai Sea was elucidated in terms of the timing of early life stages and recruitment through combined analysis of historical literature on the seasonal patterns of juvenile occurrence and published literature on the spawning, embryonic and larval development, and growth of this species. Spring occurrences were severely limited by bottom temperature and sediment mean grain size and skewness; most of the juveniles occurred in Laizhou Bay and its adjacent waters, with a few others observed in the Liaodong Bay and coastal waters northwest of Dalian City. Juvenile occurrence frequency was the highest in April, followed by May, during spring. Additionally, juvenile O. oratoria occurred throughout the survey area within the Bohai Sea in September and October, with the highest juvenile sample size recorded in November, following the same sampling protocol. The occurrence distribution in fall was shaped by bottom temperature, bottom salinity, and sediment sand content, although these three factors only explained 15.5% variance in fall occurrence. Therefore, temperature and sediment elements have greater effects on juvenile O. oratoria occurrence in spring than in summer. Furthermore, the recruitments of O. oratoria tended to be multi-seasonal in the Bohai Sea. The spawning season may last from May to September. Fertilized eggs released from May to July would develop into juveniles from July to September and then grow to 50 mm in total length from September to November. Eggs released in August and September would develop into juveniles from October to November, stop growing in December to March when the temperature drops below 8℃, and then grow to large juveniles in April and May. The validation of such timings requires further studies of the monthly or seasonal trends of O. oratoria larval abundance and distribution in the Bohai Sea.

Abstract:To investigate the composition of the reproductive population and changes in the physiological characteristics of Eriocheir sinensis before and after mating in the Yangtze River estuary, a survey was conducted in December 2020. The size composition, sex ratio, and reproductive status of female crabs and changes in hepatopancreas index (HSI), gonadosomatic index (GSI), and Fulton´s condition index (K) before and after mating were analyzed. Results showed that in the reproductive population of E. sinensis in the Yangtze River estuary, the overall size of female crabs was smaller than that of male crabs, and the ratio of females to males was 0.39 : 1. The number of male crabs was significantly higher than that of female crabs (χ2=101.87, P<0.05). In the comparison of physiological characteristics, there was a significant difference in the HSI among female crabs of different reproductive statuses (P<0.05), among which the highest was in the egg-bearing status, and the lowest was in the mated status. The GSI and K index of female crabs in the mated status were higher than those in the unmated status (P>0.05), and the K condition index of females in the egg-bearing status was the lowest (P<0.05). The GSI of male crabs was significantly lower than that of females (P<0.05), while the K condition index was significantly higher than that of females (P<0.05). A highly significant negative correlation was found between the HSI, GSI, and carapace width in male crabs (P<0.01), while a highly significant negative correlation was only found between the HSI and GSI in females (P<0.01). These results enrich our understanding of the physiological characteristics of E. sinensis during the reproductive mating period and provide basic data for the conservation and management of E. sinensis resources.

Abstract:Presently, aquaculture production is increasing to meet the increasing demand for protein, and the total area of aquaculture ponds worldwide was 5.4 million hectares in 2016. However, pond aquaculture causes serious environmental problems. Animals generally use only 20%~30% of the input nutrients in feed, and the majority of the remainder are dispersed in the aquaculture system, leading to an increasing waste load inside the aquaculture pond system. The conventional way to improve the aquaculture pond environment is by changing water, however, wastewater drainage is not in accordance with the water shortage situation in China. Additionally, aquaculture wastewater discharge affects the surrounding environment. It showed that China’s seawater ponds discharge 4.77×104 and 3.75×103 tons of nitrogen and phosphorus, respectively, into surrounding seas annually. According to our previous research results, it is estimated that China’s freshwater aquaculture ponds discharge 2.79×105 and 2.89×104 tons of nitrogen and phosphorus, respectively, into the surrounding waters annually. To reduce the impact of aquaculture wastewater on the environment, our research team built a zero-water exchange aquaculture mode based on the polyculture system of grass carp (Ctenopharyngodon idella), bighead carp (Aristichthys nobilis), and crucian carp (Carassius carassius). At present, this mode has achieved zero-water exchange for four years, and the annual yield is 96 000 kg/hm2. However, the mechanism of zero-water exchange is unclear. In aquaculture ponds, the organic carbon, nitrogen, and phosphorus contents can directly reflect changes in the aquaculture environment, and the budget of organic carbon, nitrogen, and phosphorus can reflect the nutrient accumulation and utilization efficiency of nutrients by aquaculture animals. Quantifying the budget of organic carbon, nitrogen, and phosphorus can aid the understanding of the system from the material cycle and energy flow, and is of considerable significance for the management and optimization of the system at the ecosystem level. Therefore, this study used the zero-water exchange pond of grass, bighead, and crucian carp as the experimental group, and a common pond for grass, bighead, and crucian carp as the control group. The carbon, nitrogen, and phosphorus budget characteristics of these two groups were studied to provide a theoretical reference for optimizing the zero-water exchange culture mode. This study was conducted between January, 2017 and November, 2018. The results showed that feed was the main source of organic carbon, nitrogen, and phosphorus in the experimental and control groups, contributing 77.06%, 92.08%, and 94.18% in the experimental group, and 81.00%, 92.77%, and 95.63% in the control group, respectively. The main output of organic carbon, nitrogen, and phosphorus was sediment accumulation, which accounted for 43.32%, 61.40%, and 78.71% of the input nutrients in the experimental group and 22.10%, 52.82%, and 79.58% of that in the control group, respectively. Harvesting the fish in the experimental and control groups accounted for 10.08% and 13.05% of input carbon, 21.00% and 25.57% of input nitrogen, and 15.41% and 18.60% of input phosphorus, respectively. The water accumulation amount and accumulation rates of carbon, nitrogen, and phosphorus in the zero-water exchange ponds were significantly lower than those in the common pond (P<0.05), and the water accumulation rates decreased by 92.91%, 88.52%, and 87.12%, respectively. The sediment accumulation of nitrogen and phosphorus in the zero-water exchange pond was significantly higher than that in the common pond; however, the sediment accumulation rates of carbon and nitrogen in the zero-water exchange pond were significantly lower than those in the common pond (P<0.05), decreasing by 48.99% and 13.97%, respectively. The carbon, nitrogen, and phosphorus utilization rates of the zero-water exchange pond were significantly higher than those of the normal pond (P<0.05), increasing by 29.49%, 21.72%, and 20.65%, respectively. These results indicate that the zero-water exchange mode can effectively reduce nutrient accumulation and improve the material utilization rate, representing a green and efficient aquaculture mode. Considering the nutrient emissions of Chinese aquaculture, the zero-water exchange mode has a good application potential.

Abstract:This study aimed to explore the morphological adaptation and evolution characteristics of Triplophysa yarkandensis, an endemic fish in the Tarim River Basin, and further develop the proliferation and protection of native fish resources in the Tarim River Basin. This study is based on multivariate morphological metrics, and multivariate statistical analysis was conducted on 11 traditional indices and 22 framework indices of 509 samples from three geographical T. yarkandensis populations. A non-parametric test showed that, among the 31 proportional traits of the three geographic T. yarkandensis populations, 4–9/body length had significant differences (P<0.05), and 26 proportional traits such as body depth/body length had extremely significant differences (P<0.01). Principal component analysis revealed that the contribution rates of the first three principal components were 25.00%, 14.00% and 9.30%, with a cumulative contribution rate of 48.31%. The differences were primarily in the head and in front of the trunk. Cluster analysis and principal component analysis results were essentially identical. The Yarkand River and the Tarim River groups first converged into one branch that then converged into a branch with the Hotan River group. Specifically, the difference between the Yarkand River and Tarim River is small, and the difference between the Hotan River and the other two groups is large. During the verification of the built discriminant equations, the discriminant success rates were 88.48%, 88.89%, 87.73%, and the comprehensive discriminant rate was 88.37%. It is suggested that the nine morphological and proportional characters selected by discriminant analysis are feasible for the preliminary determination of different geographical populations of T. yarkandensis. The results showed that the special characteristics of the Tarim River system resulted in the morphological differences of T. yarkandensis in different river habitats, which could be distinguished effectively by multivariate analysis. However, the differences in habitat environment might be the predominant reason for the morphological differences or adaptation evolution of the fish.

Abstract:Shrimp production has expanded rapidly over the last few decades in China. With the increasing production, water pollution has become increasingly serious. An increase in nutrients in water results in eutrophication, characterized by low oxygen, high ammonia, high phosphorus, and high frequency of algal blooms, which in turn influence shrimp growth and yield. Phytoplankton is an important component of aquaculture ecosystems. It is a direct or indirect food source for cultured organisms, and can remove nitrogen and phosphorus and maintain water quality. Constructing a benign phytoplankton community can improve the nitrogen pollutant absorption efficiency, improve the environment, and reduce the environmental pollution caused by cultivation. Certain phytoplankton groups, such as diatoms and green algae, are desirable for their high nutritional value and contribution to water quality. The use of phytoplankton to purify and regulate aquaculture water quality could reduce the negative impacts of aquaculture, which is an environmental protection option with a low cost, low energy consumption, high benefit, and considerable development potential. Microalgal growth is significantly affected by factors such as temperature, light, and nutrient conditions. Therefore, the environmental adaptability of microalgae is the primary consideration for selecting and cultivating algal species. In situ isolation and screening of algal species can reduce the stress response of microalgae, which is conducive to their normal ecological function of regulating water quality. In an indoor industrial aquaculture system for Litopenaeus vannamei, one diatom species was dominant for a significant amount of time in the middle and late stages of aquaculture in 2019. This alga was small and covered with a silica membrane layer, which was difficult to identify with a light microscope. The shrimps effectively grew in the ponds where this alga was the dominant species. To identify the algae and explore its application in water quality regulation in aquaculture, the algal strain was isolated and purified from the indoor industrial aquaculture system, and identified by optical microscopy, electron microscopy, and 18S rRNA sequence analysis. The results identified this algal strain as Actinocyclus exiguous HY01. The cell diameter was approximately (11.4±1.0) μm. At present, A. exiguous is the smallest individual species in the genus Actinocyclus. There are numerous small pores on the mantle that were not visible under a light microscope. The pore density at the mantle center was less than that at the mantle margin. There is a pseudonodulus at the valve margin, and 3~5 labiate processes. Nitrogen is an indispensable element for the growth and metabolism of phytoplankton, and is the main component of nucleic acids, proteins, and chlorophyll in cells. Ammonia and nitrate are the two main forms of inorganic nitrogen in aquaculture water, which can be directly absorbed and utilized by microalgae. The addition of nitrogen nutrients can promote microalgal growth. However, the microalgae cell density did not increase as the nitrogen concentration increased. Excessive ammonia content is inconducive to algae growth, and even affects the absorption and transformation of other nitrogen sources. In this study, ammonia and nitrate of different concentrations were used as nitrogen sources to culture A. exiguous HY01. The results showed that both ammonia and nitrate could be used for A. exiguous HY01 growth. The optimum growth concentration of ammonia was 600 μmol/L. Under these conditions, the cell density, specific growth rate, and protein content of A. exiguous HY01 were the highest, at 4.54×10⁸ cells/L, 0.36±0.07, and 4.45 mg/g, respectively. When the ammonia concentration was 882 μmol/L, the cell density and specific growth rate of A. exiguous HY01 were the lowest, at 2.11×10⁸ cells/L and 0.27±0.05, respectively. When the ammonia concentration was 50 μmol/L, the A. exiguous HY01 protein content was the lowest at 1.38 mg/g. The optimum growth concentration of nitrate was 882 μmol/L. Under these conditions, the cell density, specific growth rate, and protein content of A. exiguous HY01 were the highest, at 5.92×10⁸ cells/L, 0.40±0.01, and 11.97 mg/g, respectively. When the nitrate concentration was 100 μmol/L, the cell density, specific growth rate, and protein content of A. exiguous HY01 were the lowest. In summary, the maximum cell density, maximum specific growth rate, and protein content of A. exiguous HY01 were lower in the medium containing ammonia-nitrogen than those in the medium with nitrate-nitrogen. We speculated that A. exiguous HY01 might prefer nitrate to ammonia, but has greater tolerance to high ammonia concentrations. This might be one reason why A. exiguous HY01 was dominant for a long time in the indoor industrial aquaculture system of L. vannamei in 2019. The results are expected to provide a reference for the targeted cultivation of this alga and its application in water quality regulation in aquaculture.

Abstract:Culter alburnus is a common freshwater aquaculture fish in China. It is popular because of its tender meat, rich nutrition, and high-protein and low-fat contents. With the breakthrough in compound feed and artificial breeding technology, the scale of C. alburnus cultivation is increasing, and it is cultivated in more than 10 provinces and many cities in China, including Anhui, Hubei, Jiangsu, Zhejiang, and Chongqing. However, with the increase in culturing scale and density of C. alburnus, the water quality is continuously deteriorating, leading to decreased immunity and spread of diseases, especially various bacterial, fungal and parasitic diseases, among which bacterial diseases are the most serious, causing huge economic losses. In August 2020, C. alburnus died in a farm, and the cumulative death rate was 20% in Rongchang District, Chongqing. The main clinical symptoms was loose scales, bleeding at the base of the fin, a large amount of ascites in the abdominal cavity, and hepatosplenomegaly. In order to identify the pathogen responsible for the death of C. alburnus, the pathogenic mechanism was investigated and appropriate drugs to treat the disease were screened. A dominant strain named CQ200825 was isolated from the liver of the diseased fishes. An artificial infection experiment was performed to verify pathogenicity of the strain CQ200825. This was combined with the morphological, physiological, and biochemical analysis of the bacteria for identification; 16S rRNA sequence and gyrB sequence analysis was also done. Several analyses were performed for accurate identification of the species, virulence gene detection, histopathological observation, and to probe its virulence factor and tissue pathological changes caused after infection. Finally, effective drugs were screened by drug susceptibility test. The results showed that naturally occurring fishes swam slowly, had decreased appetite, or did not feed at all. Their scales were partially detached and the eyeballs were protruding. An obvious hyperemia and hemorrhage around the orbit, gill cover, and base of the fin was also observed. Autopsy revealed a large amount of ascites in the abdominal cavity and the liver and spleen were swollen. The bacterium grew at 28℃ for 24 h on BHI medium, and its colony diameter was 1.0~1.5 mm, which had smooth surface and rounded edges. Strain CQ200825 was gram negative, and its size was about 0.5~1.0×1.2~2.4 μm; the strain was short rod-shaped, and most single permutation. The physiological and biochemical characterization of the strain CQ200825 showed that it could produce indole but not H2S. The M.R test and V-P test results were positive. The test results for the production of lysine decarboxylase and oxidase were positive, whereas those for arginine hydrolase, phenylamine deaminase, ornithine decarboxylase, and urease were negative. The strain could utilize D-mannitol, D-glucose, and cellobiose, but could not utilize arabinose, salicin, sorbitol, and inositol. The evolutionary tree constructed by 16S rRNA and gyrB gene sequences of the strain showed that it clustered with Aeromonas veronii, and the similarity was 99.60% and 99.63%, respectively. Based on morphology and physiological and biochemical properties, the strain CQ200825 was identified as A. veronii. Both the artificially infected and the naturally pathogenetic fishes showed symptoms of hemorrhage at the base of the fin, hepatosplenomegaly, and ascites. Meanwhile, a dominant strain was isolated from the artificially infected dying fishes, which was the same as the strain CQ200825 based on its physicochemical and molecular properties, indicating that the strain CQ200825 was the pathogen of the diseased C. alburnus. The LD50 value of strain CQ200825 against C. alburnus was 2.7×106 CFU/mL. Four virulence genes were found in the strain CQ200825, including the outer membrane protein (ompA), haemolysin (hlyA), cytotonic heat-stable enterotoxin (ast), and cytotoxic enterotoxin (act), and sequencing analysis revealed that the lengths of these genes were 903 bp, 592 bp, 504 bp, and 249 bp, respectively. Pathological analysis revealed that the liver, spleen, kidney, and intestine of the diseased C. alburnus had different degrees of pathological changes. Liver cells of the diseased fish were swollen and necrotic, and hemocytes agglutinated in blood vessels; splenic tissue was necrotic and the cells were abnormal, the boundary of red and white pulp was unclear, and the nucleus had slight swelling; the epithelial cells of renal tubules showed granulosa degeneration and glomerular atrophy; and the intestinal structure was severely damaged, serous membrane was removed, most of the intestinal villus were necrotic and exfoliated, and goblet cells epithelial cells were necrotic. The results of the drug susceptibility test revealed that the strain CQ200825 was highly sensitive to 17 antibacterial drugs, namely doxycycline, enrofloxacin, florfenicol, ceftazidime, kanamycin, cefoperazone, streptomycin, ciprofloxacin, and pipemidic acid; moderately sensitive to four antibacterial drugs, including erythromycin, cotrimoxazole, sulfisoxazole, and neomycin; and resistant to 12 antibacterial drugs namely penicillin, amoxicillin, gentamicin, midecamycin, kitasamycin, carbenicillin, cefazolin, and lincolmensin. This study can provide reference for the diagnosis and prevention of A. veronii infection in C. alburnus.

Abstract:In this study, a high-yielding siderophore strain, SDVGH3, was screened from the gut of Litopenaeus vannamei using a combination of the CAS plate method and liquid-phase detection. Strain SDVGH3 is a Pantoea sp. gram-negative, facultative anaerobe. The SDVGH3 colony was cream-yellow and round. The strain was non-hemolytic and sensitive to 18 antibiotics. The siderophore extracted by XAD-2 large-aperture resin adsorption belongs to the isohydroxamic acid type, and its yield reached 74.87%. The filter-paper method was used to test the antibacterial characteristics of the strain. The degree of inhibition of the five pathogenic bacteria of L. vannamei was as follows: Vibrio parahaemolyticus > V. harveyi > V. anguillarum > Staphylococcus aureus > V. alginolyticus. The optimal culture conditions of SDVGH3 are as follows: 10 mL of culture medium in a 50 mL triangle flask; concentration of Fe3+ in the medium of 0.01 mmol/L; salinity of 0; initial pH value of 7.5; and culture temperature of 28℃. This study investigated the effects of SDVGH3 bacterial solution at concentrations of 1010 and 107 CFU/g on growth performance, immune defense indices, and intestinal microflora of L. vannamei. The results show that feed supplemented with 1010 CFU/g SDVGH3 bacterial solution significantly promoted growth and enhanced the immune capacity of L. vannamei. The feed supplemented with 107 CFU/g SDVGH3 bacterial solution significantly increased the intestinal floral diversity of L. vannamei, and significantly decreased the proportion of Vibrio in the intestinal flora. The strain SDVGH3 screened in this study is part of the indigenous probiotics in the intestinal tract of L. vannamei and has an inhibitory effect on the main pathogenic bacteria of L. vannamei. The supplementation of feed with strain SDVGH3 can promote the growth of L. vannamei, improve its immune capacity, and improve its diversity of intestinal flora, providing a theoretical basis for the application of high-yielding siderophore strains in the culture of L. vannamei.

Abstract:Apostichopus japonicus is highly edible and economically valuable. However, because of their self-dissolving characteristics, live sea cucumbers must be processed immediately after being caught. This species produces by-products totaling approximately 30% of the specimen, including sea cucumber intestines, gonads, respiratory trees, and other tissues. At present, the processing and utilization degree of these by-products is still very inadequate, and some are directly discarded, resulting in environmental pollution. According to some studies, sea cucumber gonads are rich in high-quality proteins, which can be used to develop dietary supplement products. Sea cucumber gonads also contain sea cucumber polysaccharides, sea cucumber saponins, active lipids, and other active components. The phenols and flavonoids components in the gonads of Atlantic sea cucumbers are high and a potential source of natural antioxidants. Residents in some parts of the Pacific, such as New Zealand and the Cook Islands, regularly collect gonads from sea cucumber viscera for cooking or as a protein source for traditional meals. In Japan, sea cucumber gonads are processed into expensive functional pill and powder products. However, the development and utilization of sea cucumber gonads in China are still very low. Biological enzymatic hydrolysis is an effective way to improve the utilization of proteins in aquatic products. The enzymatic hydrolysis process degrades macromolecular proteins into small peptides, which are easy to dissolve and are conducive to human absorption. At the same time, it is often accompanied by the generation or reaction of peptides, amino acids, and volatile compounds, which in turn causes flavor changes in the enzymatic hydrolysate. The gonads of A. japonicus are rich in protein and functional components, with suitable prospects for development and utilization. To explore changes in protein and flavor during the process of enzymatic hydrolysis, neutral protease was used, and the gonads of A. japonicus were hydrolyzed using the enzymatic hydrolysis conditions established in previous research. Changes in soluble protein and amino acid nitrogen in the enzymatic hydrolysis process were detected and analyzed, and the flavor changes were studied to provide a reference for the development of related products of A. japonicus gonads. The soluble protein content was measured using the Biuret method. The amino acid nitrogen content was measured by the formaldehyde colorimetry method referring to GB/T 5009.39-2003. The hydrolysis degree was determined by Cao´s method, previously established in our laboratory. The composition of free amino acids was analyzed using an automatic amino acid analyzer. The odor characteristics of the enzymatic hydrolysate were analyzed using the PEN3 electronic nose, and the sensory evaluation mechanism was designed to describe the flavor at the same time. The relative contents of volatile components during enzymatic hydrolysis were further analyzed by gas chromatography-mass spectrometry (GC-MS). Results showed that the initial soluble protein and amino acid nitrogen contents in the gonads of A. japonicas were 1.14 g/100 g and 0.15 g/100 g, respectively. The soluble protein content increased rapidly within 30 min and then remained constant. The content of amino acid nitrogen gradually increased in the first 90 min and then decreased slightly. The degree of hydrolysis expressed by the changes in soluble protein and amino acid nitrogen during enzymatic hydrolysis reached a maximum at 90 min, up to 43.66%. With the extension of enzymatic hydrolysis time, the umami of enzymatic hydrolysis was enhanced, the fishy smell was weakened, and the sweet and bitter tastes were slightly enhanced. The fresh flavor and sweet taste of enzymatic hydrolysis were also enhanced, consistent with the high glutamate, glycine, and alanine content in free amino acids with the extension of enzymatic hydrolysis time. At the same time, the free amino acid content increased significantly after enzymatic hydrolysis (P<0.05). With the extension of the enzymatic hydrolysis time, the content of free amino acids increased gradually. Glutamic acid is a typical fresh amino acid. TAV(taste activity values) increased from 5.41 to 14.24 at 120 min of enzymatic hydrolysis, significantly contributing to the flavor of the enzymatic hydrolysate. The glutamate content was the highest, followed by glycine and alanine, which also influenced flavor. Moreover, the odor changed significantly during enzymatic hydrolysis. The results of the electronic nose analysis showed that the odor changed significantly after the enzymatic hydrolysis of the gonads of A. japonicus. Through further GC-MS analysis, it was found that the content of fishy substances represented by dimethyl sulfide decreased significantly after enzymatic hydrolysis, and the production of some hydrocarbons, alcohols, and aldehydes gave the enzymatic hydrolysis solution a pleasant smell. The types and relative content of hydrocarbons increased significantly, and the content of dimethyl sulfide decreased significantly, likely responsible for the weak fishy odor of the enzymatic hydrolysate. In this study, the changes in soluble protein and amino acid nitrogen during enzymatic hydrolysis were detected and analyzed, and the flavor changes were studied, providing a reference for the development of gonadal-related products of A. japonicus.